Posts Tagged ‘Antibiotic resistance’

Hospital Acquired Infections

Larry H. Bernstein, MD, FCAP, Curator



Hospital infection control in the era of superbug outbreaks
By Leslie Small


Long before superbug outbreaks tied to a specialized medical scope sickened and killed patients across the country, infection control has been a major priority for hospitals.

But the need to curb infections has become even more pressing now that the shift from a fee-for-service model to value-based payments has led the federal government to increasingly tie reimbursements to patient outcomes. An industry trend toward transparency also has made it easier for the public to see the danger of a hospital stay.

One in 25 hospital patients acquires at least one healthcare-associated infection (HAI), according to data from the Centers for Disease Control and Prevention. There were an estimated 722,000 HAIs in U.S acute care hospitals in 2011, about 75,000 hospital patients with HAIs died during their hospitalizations, and more than half of all HAIs occurred outside of the intensive care unit.

The problem has gained more attention in recent months, when reports surfaced of patients contracting a particularly lethal antibiotic-resistant superbug, carbapenem-resistant Enterobacteriaceae (CRE), after undergoing procedures that involved a device known as a duodenoscope. A Food and Drug Administration advisory panel concluded this month what hospitals involved in the outbreak already knew–that the ability of CRE and other dangerous pathogens to stay on scopes after cleaning puts patients at a significant risk.

But while the problems posed by such outbreaks are clear, finding solutions to them–particularly in a seldom-static healthcare industry–is anything but. To help chart a way forward, this special report from FierceHealthcare examines advice from experts and hospital leaders who have learned valuable lessons from the front lines of hospital infection control.


Rise of a superbug jeopardizes patient safety

Hospital infection control in the era of superbug outbreaks
By Leslie Small

The recent carbapenem-resistant Enterobacteriaceae (CRE) outbreak burst onto the scene when reports surfaced in February that it caused two patient deaths at UCLA’s Ronald Reagan Medical Center. In addition to the California outbreak–which also sickened patients at Cedars-Sinai Medical Center in Los Angeles–cases of the superbug linked to duodenoscopes also cropped up at hospitals in North Carolina, Pittsburgh, Chicago and Seattle.

The Centers for Disease Control and Prevention (CDC) has cautioned hospitals that they must do more to mitigate the threat of CRE, which it dubbed “nightmare bacteria” due to their resistance to even last-resort antibiotics. When it reaches the bloodstream, CRE can kill up to half of all patients it infects.

The Food and Drug Administration (FDA) issued a warning in February that the complex design of duodenoscopes makes them difficult to sterilize even when hospitals follow the device manufacturers’ instructions. However, the FDA has refused to take the devices off the market because they are used for the potentially life-saving procedure known as endoscopic retrograde cholangiopancreatography (ERCP), a technique that diagnoses and treats cancers and other digestive diseases. The agency estimates that 500,000 ERCPs are performed each year. A special advisory panel recently endorsed this decision, though it urged the FDA to better protect patients from the infection risk posed by duodenoscopes,FierceHealthcare has reported.

The panel was also critical of major duodenoscope manufacturer Olympus, which declined to participate in the advisory panel’s forum but says it has supplied the FDA with data to prove that its updated cleaning instructions and new cleaning brush allow for safe reprocessing.

Indeed, news surfaced recently that the company was aware of the infection risk associated with the devices in 2013, which it communicated to European hospitals two years before the UCLA outbreak. The situation has led California lawmaker, Rep. Ted Lieu (D-Los Angeles) to call for congressional hearings into the matter. Meanwhile, Olympus faces two patient-driven lawsuits, and Virginia Mason Hospital in Seattle also has pursued legal action against the manufacturer.

The outbreaks have left many hospitals wondering what to do to make sure patients are safe and still have access to important medical devices. For its part, the FDA panel did not outright endorse any specific sterilization method.

Jackie Caynon, pictured right, a lawyer with more than 18 years of health law experience, and partner and co-chair of Mirick O’Connell’s Health Law Group, told FierceHealthcare in an exclusive interview that the answer has to come from each hospital’s unique risk management assessment.

“I’ve heard some hospitals say, you know these things are really life-saving, so if we get rid of the product we won’t be able to, obviously, save lives,” he said. But for others, he said, the risk may be too great.

“To me it just seems too risky to use it,” Caynon said. “If you’re going to do informed consent, I could see you saying to the patient ‘oh you know, this could save your life, but we won’t know until we actually go in there and look, but you run the risk of having a CRE infection because we cannot guarantee that we can properly clean this device.'”

And now that the infection risk surrounding the devices has been made public, “I think you’re going to have a lot of patients that are going to say ‘I don’t want you to use that device, period,'” he said.

Regardless of what each individual facility decides to do about the scopes, it would be a mistake to hold hospitals responsible for manufacturers’ mistakes or regulatory failures, according to Caynon.

“Holding hospitals and physicians liable here is kind of going after the wrong folks,” he said, because “the hospital is just as much of a customer as patients (are).”


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New antibiotics to address anti-microbial resistance

Larry H. Bernstein, MD, FCAP, Curator




Dr. Anthony Melvin Castro


Figure imgf000036_0001

WCK 5107

Wockhardt Limited

Useful for treating bacterial infections

CAS 1436861-97-0

disclosed in PCT International Patent Application No. PCT/IB2012/054290D

trans- sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(2S, 5R)-sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(lR,2S,5R)-l,6-Diazabicyclo [3.2.1] octane-2-carboxylic acid, 7-oxo-6-(sulfooxy)-, 2-[2-[(3R)-3-piperidinylcarbonyl]hydrazide]

trans- sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(2S, 5R)-sulphuric acid mono-[2-(N’-[(R)-piperidin-3-carbonyl]-hydrazinocarbonyl)-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl] ester

(lR,2S,5R)-l,6-Diazabicyclo [3.2.1] octane-2-carboxylic acid, 7-oxo-6-(sulfooxy)-, 2-[2-[(3R)-3 -piperidinylcarbonyl] hydrazide]


In September 2015, the drug was reported to be in phase I clinical trial.One of the family members US09132133, claims a combination of sulbactam and WCK-5107.

Bacterial infections continue to remain one of the major causes contributing towards human diseases. One of the key challenges in treatment of bacterial infections is the ability of bacteria to develop resistance to one or more antibacterial agents over time. Examples of such bacteria that have developed resistance to typical antibacterial agents include: Penicillin-resistant Streptococcus pneumoniae, Vancomycin-resistant Enterococci, and Methicillin-resistant Staphylococcus aureus. The problem of emerging drug-resistance in bacteria is often tackled by switching to newer antibacterial agents, which can be more expensive and sometimes more toxic. Additionally, this may not be a permanent solution as the bacteria often develop resistance to the newer antibacterial agents as well in due course. In general, bacteria are particularly efficient in developing resistance, because of their ability to multiply very rapidly and pass on the resistance genes as they replicate.

Treatment of infections caused by resistant bacteria remains a key challenge for the clinician community. One example of such challenging pathogen is Acinetobacter baumannii (A. baumannii), which continues to be an increasingly important and demanding species in healthcare settings. The multidrug resistant nature of this pathogen and its unpredictable susceptibility patterns make empirical and therapeutic decisions more difficult. A. baumannii is associated with infections such as pneumonia, bacteremia, wound infections, urinary tract infections and meningitis.

Therefore, there is a need for development of newer ways to treat infections that are becoming resistant to known therapies and methods. Surprisingly, it has been found that a compositions comprising cefepime and certain nitrogen containing bicyclic compounds (disclosed in PCT/IB2012/054290) exhibit unexpectedly synergistic antibacterial activity, even against highly resistant bacterial strains.






Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of WCK-5107 Alone and in Combination With Cefepime (NCT02532140) Web Site 2015, September 01, To evaluate the safety,tolerability and pharmacokinetics of single intravenous doses of WCK 5107 alone and in combination with cefepime in healthy adult human subjects.

WO2013030733A1* Aug 24, 2012 Mar 7, 2013 Wockhardt Limited 1,6- diazabicyclo [3,2,1] octan-7-one derivatives and their use in the treatment of bacterial infections
WO2014135931A1* Oct 12, 2013 Sep 12, 2014 Wockhardt Limited A process for preparation of (2s, 5r)-7-oxo-6-sulphooxy-2-[((3r)-piperidine-3-carbonyl)-hydrazino carbonyl]-1,6-diaza-bicyclo [3.2.1]- octane
IB2012054290W Title not available

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Experience with Trauma Surgery

Author: Larry H. Bernstein, MD, FCAP



In 1987, I went on vacation to Bermuda with my wife and two children.  It was a beautiful place, and the weather and the ocean were wonderful to experience.  One could travel by bus, which was very safe, which I preferred. My older daughter wanted to use a moped, which we allowed on the condition that she first be trained.  On the last day, she went to return the moped, but the station was out for lunch.  I was a photograper and wanted to photograph the white bird of Burmuda. I put my camera in the rear, but as I left the station my moped was hit by an oncoming moped that I failed to see, unaccustomed to the British style driving.  An ambulance arrived within a few minutes as I lay on the ground. My wife sent the kids home and made arrangements for my secretary to look after them.  I was impressed with the surgeon when I arrived at the hospital. He wheeled me to the bed I was to stay in. I had two blood transfusions.  He took me to the operating room, but I don’t recall any details. He had a McGill University resident who later wrote a thesis about the experience.  I was pretty knocked out, but there was another patient in the room who had fallen down his steps. He was a WWII RAF veteran who had bombed the Germans. He told me the stories about his experience.  We contacted the burn surgeon, Walter Pleban, who arranged to have me flown to Bridgeport, CT, and he arranged for the best orthopedic surgeons to admit me on arrival.  In my flight there was another patient who was dying of endstage HIV AIDS.

Herbert Hermele observed how serious this was because there were three fractures of the right tibia. The good news was that there was no need to amputate because I had the nervous innervation, but I lost a popliteal artery.  I was admitted, and at first there was only a small room. The nurse was a very competent young woman of Portuguese descent. She was able to move me as needed. I was moved when a better room became available.  It was very good when the night shift nurse came in because I was able to talk to her with some attachment.  The Vice President had me provided with good meals, as I was the director of blood bank and chemistry.  I also had visits by my supervisors and other staff.

It was not an easy time, but I was privileged because of my standing with the medical and laboratory staff.  I had a longer stay than usual because I had an infection with two gram negative resistant strains of bacteria –serratia marcesans and Enterobacter. I was put on a gram negative penicillin and the next morning I felt dizzy. When Dr. Pleban came to see me I told him that I was having a penicillin reaction because I was aware that my twin sister was allergic to penicillin. As a result, the prescription was changed and it was an improved situation.  I underwent 10 operative procedures in some weeks. Dr. Hermeles partner put an antibiotic plug into the wound and it healed.  It was only after the infection cleared that a superb reconstructive surgeon was called in and he made skin grafts to close the wound after he disconnected a tendon and pulled muscle over the wound.  I also had a call from IJ Good, University Professor of Statistics at Virginia Polytech, who had completed writing a program to analyze data that I had provided him 2 years earlier – of MB isoenzyme CK at 6 hours and 12 hours later for diagnosis of heart attack.  We published the work in the prestigious journal, Clinical Chemistry and the President of the College of American Pathologists took note of the paper. I was finally sent home, without needing excess stay to the hospital environment.  I had physical therapy at home, and my bed was made on the first floor.  When I returned to work my infection site oozed, so I went to the Chief of Infectious Disease.  He prescribed a new quinolone antibiotic that could be taken orally. The infection subsided and it has never returned.

My sister came from San Diego, California and she brought me a recording she made for imaging to heal.  It went on that I was climbing a step to the heavens and getting better and better.  She also emphases laughing.

I can only look back and recall how fortunate I was to have the attention and kindness at that time. It was in excess of what many patients experience.  I do recall that the Hungarian-Cuban music teacher my daughter had had thousands of musical pieces and thousands of stories so that she was one of the most entertaining patients ever admitted to Bridgeport Hospital.





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Genomic Pathogen Typing

Larry H. Bernstein, MD, FCAP, Curator



Genomic Pathogen Typing Using Solid-State Nanopores

Citation: Squires AH, Atas E, Meller A (2015) Genomic Pathogen Typing Using Solid-State Nanopores. PLoS ONE 10(11): e0142944.

Editor: Niyaz Ahmed, University of Hyderabad, INDIA

In clinical settings, rapid and accurate characterization of pathogens is essential for effective treatment of patients; however, subtle genetic changes in pathogens which elude traditional phenotypic typing may confer dangerous pathogenic properties such as toxicity, antibiotic resistance, or virulence. Existing options for molecular typing techniques characterize the critical genomic changes that distinguish harmful and benign strains, yet the well-established approaches, in particular those that rely on electrophoretic separation of nucleic acid fragments on a gel, have room for only incremental future improvements in speed, cost, and complexity. Solid-state nanopores are an emerging class of single-molecule sensors that can electrophoretically characterize charged biopolymers, and which offer significant advantages in terms of sample and reagent requirements, readout speed, parallelization, and automation. We present here the first application of nanopores for single-molecule molecular typing using length based “fingerprints” of critical sites in bacterial genomes. This technique is highly adaptable for detection of different types of genetic variation; as we illustrate using prototypical examples including Mycobacterium tuberculosis and methicillin-resistant Streptococcus aureus, the solid-state nanopore diagnostic platform may be used to detect large insertions or deletions, small insertions or deletions, and even single-nucleotide variations in bacterial DNA. We further show that Bayesian classification of test samples can provide highly confident pathogen typing results based on only a few tens of independent single-molecule events, making this method extremely sensitive and statistically robust.


Subtle genetic changes in bacteria can produce large variations in factors affecting pathogenicity, such as toxicity, antibiotic resistance, and virulence. These genetic variations are not only used to trace the epidemic and phylogenetic relationships among strains of bacteria, but are also critically important in clinical settings for proper patient diagnosis and treatment. Most existing approaches require sample incubation and growth over the course of multiple days prior to testing, and nearly all require expert handling of samples and interpretation of results. Traditional phenotypic typing techniques such as serotypes, biotypes, phage-types, and antibiograms lack the necessary sensitivity to distinguish between closely related pathogen strains, and therefore fail to adequately capture these critical variations for clinical applications. Gel-based techniques such as restriction fragment length polymorphism (RFLP) or cleaved amplified polymorphic sequences (CAPS) require a large amount of time and results are not easily compared or transferred among labs. Next-generation sequencing is an increasingly popular method of fully characterizing bacterial strains [1] and may be used for typing strains according to the sequences of a panel of housekeeping genes, as in multi-locus sequence typing (MLST) [2], but this approach is more commonly used to trace post hoc epidemic and phylogenetic relationships among clinical isolates. Furthermore, the complexity and quantity of sequencing data far exceeds the minimum information required to efficiently and accurately diagnose a patient. For example, bioinformatics studies suggest that a panel of just 30–50 single nucleotide variations (SNVs) could be used to uniquely identify thousands of strains of Mycobacterium tuberculosis [3, 4]. Yet SNVs are not the only source of variation among pathogens; polymorphisms from SNVs and short indels up to genetic changes as large as whole plasmids or sets of genes may be responsible for critical changes to pathogenicity. Thus there exists a clear clinical need for a novel approach to molecular typing that can quickly and simply screen patient samples for a panel of widely varying known genetic polymorphisms of dangerous pathogens.

Solid-state nanopores may be used to discriminate the lengths of unlabeled individual biopolymers such as DNA molecules across a wide range of lengths [5, 6]. Biopolymers are electrophoretically attracted and threaded through a voltage-biased nanoscale pore drilled in an ultrathin freestanding SiNx membrane [7, 8]. When a DNA molecule is threaded through a nanopore, it partially blocks the flow of ions moving through the pore, allowing real-time detection of the analyte by monitoring changes in the ion current. Nanopore sensing is biochemically simple, as it does not require labeling of the analyte with radioactive or fluorescent probes, yet it can be used to detect minute quantities of nucleic acid molecules, surpassing the sensitivity of bulk methods [8]. Moreover, nanopore sensing involves relatively simple instrumentation (primarily a current amplifier) and may be used to analyze thousands of molecules in just a few minutes, making this technique an ideal candidate for applications such as nucleic acid based diagnostics.

Here we describe and practice a novel detection scheme (Fig 1) for molecular typing of pathogens using solid-state nanopores, and demonstrate its ability to discriminate a wide range of critical genetic polymorphisms in closely related organisms with starkly different pathogenicities. In the first sensing mode of our approach (Mode I), large insertions or deletions are detected by directly classifying the length of DNA in the nanopore. In the second sensing mode (Mode II), small indels down to SNVs may be detected by sequence-specific digestion at the site of the polymorphism to produce either one or two DNA fragments, which are then detected in the nanopore. We first characterize the practical range of our nanopore system for detecting variation in DNA length, and show that fragment length differences are more readily apparent for shorter DNA lengths and for asymmetric cut sites. We then demonstrate that statistical analysis tools such as Bayesian classifiers, commonly used for automated classification, are highly effective for rapid and statistically robust discrimination among different lengths and combinations of DNA fragments translocating through a nanopore, even in cases where significant portions of these distributions overlap. We apply these techniques to demonstrate polymorphism discrimination down to the single nucleotide level in prototypical strains of Mycobacterium tuberculosis (virulent vs. avirulent) and Streptococcus aureus(methicillin-resistant vs. multi-drug resistant). This highly versatile combination of rapid length and digest discrimination, spanning several orders of magnitude of possible genomic variation size, in a single, parallelizable device, could be extended to probe a large panel of critical sites within a genome for point-of-care determination of critical pathogenic properties and sequence typing.

Fig 1.  Two Principal Modes for Nanopore Discrimination of Pathogen Genomic Variation.

Fig 1. Two Principal Modes for Nanopore Discrimination of Pathogen Genomic Variation.

Mode I: Direct length detection according to analyte translocation dwell time and depth enables discrimination of longer vs. shorter fragments; i.e: whether or not an insertion or deletion is present (left). Mode II: Prior to translocation, samples are exposed to a restriction enzyme that cuts at the site of a SNV or short indel or mutation. Detection of cleaved vs. uncleaved DNA fragments in the nanopore reveals whether or not the critical genomic variation is present.

Detection of DNA Sequence Polymorphisms in Solid-State Nanopores  

The simplest form of nanopore translocation analysis involves the measurement of the depth of each current blockade (ΔIB) and the dwell time of each molecule within the pore (tD). Both parameters have been shown to grow nonlinearly with DNA length, forming the basis for fragment length separation in the nanopore system. The statistical distributions of these independently measured quantities may be used to distinguish between analytes of different lengths, such as DNAs [5, 6, 9], or proteins having identical molecular weight but slightly different charge or 3D structure [1013]. Variation in the translocation dwell-time (tD) in solid-state nanopores measured for different DNA lengths (l), are empirically described by a power law: tDlα where α = 1.38±0.02, which has been reproduced by multiple experimental approaches [5, 9, 14]. Using a log-scale distribution of translocation times to estimate the distribution of tD, note that the difference in log(tD) for two sequences (lengths l0 and l0 + Δl) is more apparent for shorter length l0 as compared with the insertions and deletions Δl (i.e. when Δl/l0 ∼ 1) according to Eq 1:(1)

If the presence of two fragment lengths must be identified from within a single sample, it is desirable that their distributions of ΔIB or tD should be as well-separated as possible. Furthermore, if the presence of a cut sample must be distinguished from an uncut sample, then by Eq 1 the peak produced by the shorter part of a cut sample will appear farther away from the uncut peak than the longer part of a cut sample. To statistically distinguish the samples, it is desirable for the peak of the shorter part to be as dissimilar as possible from the uncut peak. Therefore, asymmetrically cut DNA pieces from a restriction digest are more readily distinguished from the original uncut length than those produced by symmetrically positioned restriction sites, provided that the shorter piece is of sufficient length to be detected by the nanopore. In cases where separation between two similar length biopolymers (Δl/l0 ∼ 1) is required, the measured histograms of either ΔIB or tD may overlap significantly, making discrimination between these molecules difficult. Combinations of multiple fragment lengths within a sample pose additional challenges, as their more complicated distributions may overlap or otherwise preclude simple contour cluster separation.

In the context of sequence typing, identification of fragments by sizing will indicate the presence of specific insertions and deletions that may enhance or reduce pathogenicity or otherwise uniquely identify a pathogenic strain. Upper bounds on Δl are set by: 1) sample preparation parameters and limitations; for example, robust and fast PCR amplification is most easily achieved for fragment lengths of ~102–103 bp [15] and 2) nanopore stability considerations; for example, nanopores are more frequently clogged by very long DNA (>20 kbp). Lower bounds on l0 are set by nanopore sensitivity; while several groups have demonstrated detection of small DNA fragments (<50 bp) [16] we find that a minimum l0 on the order of ~100 bp is more reliable since it is readily detectable in small nanopores with no additional modifications [5], producing an extremely small fraction of missed events due to the finite system bandwidth. Thus a reasonable design range for sequence typing fragments is ~100 bp minimum length forl0, ranging up to a few thousand base pairs maximum length for l0 + Δl. Many types of common genetic variations used for strain typing fall within this size range. For example, one complete IS6110 (insertion-like sequence element) insertion in M. tuberculosis is 1358 bp [17]. At the other end of this range, multi-drug resistant strains of methicillin-resistant S. aureus (MRSA) have many insertions and deletions in the range 47 bp—643 bp that affect their pathogenicity [18]. To detect the smallest indels, which fall below the minimum detectable Δl, we turn to the exquisite sequence specificity of digestion by restriction enzymes, which can identify sequence polymorphisms down to a single nucleotide variation.

Using these design principles, we present here two alternative modes of detection that illustrate the wide range of genomic variations that may be detected using a single sensor. For large insertions or deletions (Fig 1: Mode I, left panel), a nanopore may be used to discriminate the raw change in DNA length caused by the presence or absence of this sequence according to the duration of translocation events. For short indels, mutations, or single nucleotide variations (SNVs) (Fig 1: Mode II, right panel), which are more difficult to identify solely by length as discussed above, we utilize a restriction enzyme. The sample is only cut in the presence (or absence) of the critical sequence, and subsequent detection in a nanopore reveals either one or two fragments in the nanopore according to the observed durations and blockage levels of translocation events.

Event Diagram Discrimination of Sample Length and Composition

We first experimentally illustrate the practical length resolution of the nanopore platform for identifying sample length and composition. We analyzed samples containing mixtures of DNA fragments composed of one or two well-defined lengths. The resulting event diagrams create unique fingerprints that can be used to distinguish different lengths of DNA (Mode I) or whether or not a fragment of DNA has been cut (Mode II). Fig 2A–2E show event diagrams for 100 bp, 200 bp, 900 bp, 1000 bp, and 100+900 bp DNA in a single nanopore (diameter 4.8 nm, effective height 7 nm) at +300 mV bias (for additional examples, see Figs B-E in S1 File). Here, each translocation event is represented by its corresponding ion current event amplitude (ΔIB) and dwell time (tD). From comparison of Fig 2A and 2D, it is evident that insertions and deletions Δl several times larger than the base length (here: Δl:l0 = 9:1) are indeed easily distinguishable (Fig C in S1 File). Comparison of Fig 2A and 2B illustrates that Δl = 100 bp results in reasonably distinct event diagrams for l0 = 100 bp, which may be distinguished to >95% confidence with just a few events each, taking both dwell time and current amplitude into consideration (Fig D in S1 File). However, at l0 = 900 bp a minimum of several hundred events are required to confidently (>95%) differentiate l0 (Fig 2C) from l0 + Δl (1000 bp, Fig 2D), since their event diagrams overlap significantly (Fig E in S1 File). Returning to Eq 1, for Δl = 100 bp, we expect Δlog(tD) = 0.415 for l0 = 100 bp, and Δlog(tD) = 0.063 for l0 = 900 bp. For the data shown in Fig 2F, Δlog(tD) = 0.1 for l0 = 100 bp, and Δlog(tD) = 0.03 for l0 = 900 bp. The inability to easily and quickly discriminate the 900 bp DNA from the 1000 bp DNA demonstrates the practical limits set on Mode I sample identification according to the size of the insertion or deletion that must be detected.

Fig 2.  Translocation Event Diagrams Uniquely Identify DNA Fragment Lengths in a Single Nanopore.

Fig 2. Translocation Event Diagrams Uniquely Identify DNA Fragment Lengths in a Single Nanopore.

(a) 100 bp at 1 nM. (b) 200 bp at 1 nM. (c) 900 bp at 1 nM. (d) 1000 bp at 1 nM. (e) 1:1 combination of 100 bp and 900 bp, total concentration 2 nM. (f) Semilog(x) distributions of translocation dwell times for all samples (a)-(e). Translocations for all samples were collected in a single nanopore (4.8 nm diameter, effective thickness ~7 nm) with a +300 mV bias relative to trans (open pore current: 13 nA). To facilitate visualization of population density, a random white noise offset below the acquisition rate of this data (-2 μs < Δt < +2 μs, acquisition rate 250 kHz) has been added to each tD.

Fig 2E illustrates how Mode II may overcome these limitations by digesting DNA into fragments: here, a highly asymmetric ratio of lengths in a mixed sample (100+900 bp) clearly facilitates sample identification as compared to the full length 1000 bp DNA (Fig 2D). However, Mode II also presents a more challenging case for quantitative discrimination between an uncut and a cut sample. Whereas single-length samples can be identified using either their tD or Idistribution (as shown in Fig 2F), the longer fragment in a cut sample may share significant overlap with the uncut sample. This is particularly true in the case of a highly asymmetric cut site.



Fig 3. Gaussian Mixture Models for Mode II Classification of 1000 bp vs. 900+100 bp DNA Fragments.

(a) 2-D GMM for 1000 bp DNA fragment translocations. (b) 2-D GMM for 900+100 bp DNA fragment translocations. (c) Bayesian posterior estimates p(A|Θ) of correctly identifying a data set Θ as Case A, calculated for each increment of N points in Θ, repeated 1000 times (first 50 shown in gray) and averaged (blue), each using M = 1500 points in the model data set. (d) Bayesian posterior estimates p(B|Θ) of correctly identifying a data set Θ as Case B, calculated for each increment of N points in Θ, repeated 1000 times (first 50 shown in gray) and averaged (red), all using M = 1500 points in the model data set. (e) Bayesian posterior estimates p(A|Θ) for test data sets ofN points given a model based on data set size M. Each point represents the average of 1000 separate bootstrap simulations. (f) Bayesian posterior estimates p(A|Θ) for test data sets of N points given a model based on data set size M. Each point represents the average of 1000 separate bootstrap simulations. Insets: range of N for which p(A|Θ) reaches 0.95. See Methods and S1 File for complete numerical simulation details.



Fig 4. Gaussian Mixture Models of DNA Fragments for Actual Mode II Pathogen Typing at the SNV Level.

(a) Diagram of the main steps in sample preparation, detection, and classification: PCR fragments from isolated pathogens are subjected to a restriction digest, which recognizes and cuts only one genomic variant. Nanopore translocations are used to classify the pathogen according to the combination of fragment lengths detected. (b) ThemazG gene of the avirulent M. tuberculosis strain H37Ra is not cut by NaeI (942 bp), while the same gene in the closely related virulent strain H37Rv, which differs by only a single A-to-C mutation, is cut by NaeI (621bp + 321 bp). (c) Gaussian mixture model (one component) fit to translocations of mazG fragments from H37Ra. (d) Gaussian mixture model (two components) fit to translocations of mazG fragments from H37Rv. (e) Posterior probabilities for correctly identifying the H37Ra and H37Rv strains as a function of number of translocation events collected from an unknown sample, simulated using bootstrap sampling from nanopore translocation data. (f) The parC gene of the multi-drug-resistant MRSA strain FPR3757 is not cut by BseRI (886 bp) due to a single C-to-A mutation, while the closely related and less resistant strain HOU-MR is cut by BseRI (640bp + 245 bp). (g) Gaussian mixture model (one component) fit to translocations of parC fragments from FPR3757. (h) Gaussian mixture model (two components) fit to translocations of parC fragments from HOU-MR. (i) Posterior probabilities for correctly identifying the FPR3757 and HOU-MR strains as a function of number of translocation events collected from an unknown sample, simulated using bootstrap sampling from nanopore translocation data.


Solid-state nanopore based biosensing is a rapidly growing field due to its practical and conceptual simplicity, portability and versatility. To date, few reports have demonstrated the utility of the method towards clinical diagnostic applications. Yet as we have shown here, nanopores are well-suited to make statistically robust diagnostic classifications among different DNA lengths with real single-molecule data, even in cases where the distributions significantly overlap. Utilizing a Bayesian statistical model, we have demonstrated that nanopore sensing can be used to discriminate among pathogens based on well-known genomic variations. Both large indels (Mode I) or short indels and single nucleotide variations (Mode II) can be targeted using proper sequence-specific digestion with off-the-shelf restriction enzymes. Furthermore, the Bayesian classifiers indicate the statistical confidence of each classification as a function of the number of nanopore events obtained in each measurement. Even at this preliminary stage of development we find that only a few tens of events (obtained in just a few minutes using a single pore) are sufficient to produce a statistically reliable result with well-defined and small error margins.

Our method is general and can be adapted to address many different “multiple-choice” clinical questions using a nanopore biosensor or other single molecule approaches. Future extensions of this work may seek to design and implement large panels of critical sites that represent the minimum sets necessary to characterize genomic variation for various applications in healthcare and research, and to develop additional sensing modalities. Although the primary design challenge currently remains linked to the location and availability of restriction digestion sites, we expect that the ongoing development of designer restriction enzymes, for example systems based on modular zinc fingers [27], TALENs [28], or CRISPR-like proteins will provide additional design flexibility for this technique.

The nanopore fingerprinting approach presented here addresses clear needs in clinical molecular diagnostics for a rapid and simple sensor that can identify a wide range of genomic variation in pathogens to inform treatment options. We have shown here discrimination of both large and small scale genomic variations between pathogen strains, down to single SNVs. The large, flexible sample design space for lengths, cut sites, and enzyme selection at each critical locus ensures that the technique is highly customizable for different genomic variation panels that could profile pathogenicity, antibiotic resistance, or even sequence type. The inherent scalability, minimal sample requirements, speed, and simple readout of the nanopore platform would all facilitate on-site and perhaps even automated use: As successive events are recorded, an increasingly clear fingerprint of translocation times and blockage levels will permit online software to “call” the sample as soon as enough events have been accumulated. Our technique is highly portable and customizable, and the binary data would be readily transferrable among different labs.


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Mechanisms of Drug Resistance

Curator: Larry H. Bernstein, MD, FCAP

Leaders in Pharmaceutical Intelligence, CSO


Mechanisms of Drug Resistance

This discussion is a continuing discussion of matters of metabolomics and the
essential role of genomic or epigenetic mechanisms to guide the development of
proteomic driven effectors of resistance to drug therapy.
We start with the elucidation of efflux pumps in bacteria, and we conclude with
consideration of cancer cells.

Part 1. Antimicrobial Resistance

Antimicrobial resistance is the ability of microbes, such as bacteria, viruses,
parasites, or
fungi, to grow in the presence of a chemical (drug) that would normally kill it
or limit its growth.

difference between non-resistant bacteria and drug resistant bacteria

difference between non-resistant bacteria and drug resistant bacteria

Non-resistant bacteria multiply, and upon drug treatment, the bacteria die. Drug
resistant bacteria multiply as well, but upon drug treatment, the bacteria continue
to spread.

Many infectious diseases are increasingly difficult to treat because of antimicrobial-resistant organisms, including HIV infection, staphylococcal infection, tuberculosis,
influenza, gonorrhea, candida infection, and malaria.

Between 5 and 10 percent of all hospital patients develop an infection. About 90,000
of these patients die each year as a result of their infection, up from 13,300 patient
deaths in 1992.

According to the Centers for Disease Control and Prevention (April 2011), antibiotic
resistance in the United States costs an estimated $20 billion a year in excess health
care costs. In addition, a cost of $35 million in other societal costs and more than 8
million additional days that people spend in the hospital. This is because people
infected with antimicrobial-resistant organisms are more likely to have longer hospital stays and may require more complicated treatment.

Diagnostic tests designed to determine which microbe is causing infection and to
which antimicrobials the microbe might be resistant take a few days or weeks to give
results because of a requirement for the microbe to grow for it to be identified.

Part 2. Antibiotic Tolerance   
Reported By Jef Akst | June 25, 2014

Optimization of lag time underlies antibiotic tolerance in evolved bacterial

O. Fridma et al.    Nature, 2014

Populations of Escherichia coli grown in the lab develop tolerance when exposed to
repeated treatments with the antibiotic ampicillin. The bacteria evolved to stay in a
dormant “lag” phase for just longer than three-, five-, or eight-hour-long treatment
courses. Antibiotic tolerance, which allows bacteria to survive even high levels of
antibiotics by remaining dormant. Tolerance may lead to an inaccurate assumption
that an unsuccessful antibiotic treatment failed as a result of resistance, in which
the microbe has evolved to grow in the presence of the drug. Resistance is very well
known; but the issue of tolerance is much less known,” according to Tom Coenye of
the Laboratory of Pharmaceutical Microbiology (LPM) at Gent University in Belgium,
who was not involved in the research.  This is a new phenomenon, extended lag,
where mutants have a longer lag time, and that extended lag allows them to survive
an attack by antibiotics.

To gain a better understanding of how bacterial populations might evolve to tolerate
antibiotic exposure, Nathalie Q. Balaban, a microbiologist and physicist at The Hebrew
University of Jerusalem in Israel and her colleagues exposed cultures of E. coli to high
concentrations of ampicillin for three, five, or eight hours, then washed the drug away
and suspended the bacteria in fresh media to be grown overnight. The next day, the
team repeated these treatments. In 10 cycles we could see that tolerance had evolved,
” Balaban said. Indeed, while the ampicillin treatments killed more than 99.9 percent of
the E. coli, by day 10, bacterial survival had increased 100-fold.

Moreover, the bacteria were also tolerant to norfloxacin, an antibiotic with a different mechanism of action than ampicillin but also ineffective during the dormant stage,
further supporting the idea that the E. coli populations had evolved to tolerate certain
durations of antibiotic exposure. “This is characteristic of tolerance,” said Balaban.
“The bacteria that have evolved tolerance under ampicillin are also more tolerant to
this completely different class of antibiotics.” Resistance, on the other hand, is usually
class-specific, she noted.

The researchers identified three genes that seemed to play a functional role in antibiotic
tolerance. While the exact mechanism of how mutations in these genes may have
lengthened the bacteria’s lag time is not yet known, two of the genes are part of pathways
that were previously implicated in bacterial persistence, including an antitoxin in a
common toxin-antitoxin module
 that may help regulate that bacteria’s growth.

Part 3. Multidrug Resistance Perspective

Mechanisms of antibiotic resistance in salmonella: efflux pumps, genetics,
quorum sensing and biofilm formation.

Perspectives in Drug Discovery and Design 02/2011; 8:114-123.
Martins M, McCusker, Amaral, Fanning S

Multidrug resistance (MDR) to antibiotics presents a serious therapeutic problem
in the treatment of bacterial infections. The importance of this mechanism of resistance
in clinical settings is reflected in the increasing number of reports of multidrug resistant
isolates. In Salmonella enterica, the most common etiological agent of food borne
salmonellosis worldwide, MDR is becoming a major concern.

In Salmonella the main mechanisms of antibiotic resistance are mutations in target
genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as

  1. changes in the cell envelope;
  2. down regulation of membrane porins;
  3. increased lipopolysaccharide (LPS) component of the outer cell membrane;
  4. quorum sensing and
  5. biofilm formation

can also contribute to the resistance seen in this microorganism. To overcome
this problem new therapeutic approaches are urgently needed.

In the case of efflux-mediated multidrug resistant isolates, one of the treatment
options could be

  • the use of efflux pump inhibitors (EPIs)
  • in combination with the antibiotics to which the bacteria is resistant.

By blocking the efflux pumps

  • resistance is partly or wholly reversed,
  • allowing antibiotics showing no activity against the MDR strains
  • to be used to treat these infections.

Compounds that show potential as an EPI are therefore of interest, as well as new
strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation
are systems also known to be involved in antibiotic resistance. Consequently,
compounds that

  • can disrupt or inhibit these bacterial “communication systems” will be of use in
    the treatment of these infections.

Part 5. Effux pumps and S. Aureus

Multidrug Efflux Pumps in Staphylococcus aureus: an Update

SS Costa, M Viveiros, L Amaral and I Couto
1Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e
Medicina Tropical, Universidade Nova de Lisboa (IHMT, UNL), 2Centro de Recursos
Microbiológicos (CREM), UNL, Portugal,3COST ACTION BM0701 (ATENS), Brussels,
The Open Microbiology Journal 2013;(Suppl 1-M5): 59-71

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital
or in the community settings is an increasing health concern. Albeit there is no single
resistance mechanism behind multi-resistance, multidrug efflux pumps,

  • proteins that cells use to detoxify from noxious compounds,

seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria.
During the last decades, experimental data has established their contribution to low
level resistance to antimicrobials in bacteria and their

  • potential role in the appearance of MDR phenotypes, by the extrusion of multiple,
    unrelated compounds.

Recent studies suggest that

  • efflux pumps may be used by the cell as a first-line defense mechanism,

avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration
occurs, that allows survival in the presence of that agent.

In this paper we review the current knowledge on

  • MDR efflux pumps and their
  • intricate regulatory network in Staphylococcus aureus,

a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that

  • aureus MDR efflux pumps,
  • either chromosomal or plasmid-encoded, have
  • on resistance towards different antimicrobial agents and
  • on the selection of drug – resistant strains.

We will also discuss the many questions that still remain on the role of each specific
efflux pump and the need to establish appropriate methodological approaches to
address all these questions.

        Table 1. Multidrug Efflux Pumps Described for Staphylococcus aureus

Efflux Pump  Family Regulator(s) Substrate Specificity  References 
Chromosomally-encoded Efflux Systems 
NorA MFS MgrA,
Hydrophilic fluoroquinolones (ciprofloxacin,
norfloxacin) QACs (tetraphenylphosphonium,
benzalkonium chloride) Dyes (e.g. ethidium
bromide, rhodamine)
NorB MFS MgrA,
Fluoroquinolones (e.g. hydrophilic: ciprofloxacin,
norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin) Tetracycline QACs (e.g.
tetraphenylphosphonium, cetrimide) Dyes (e.g. ethidium bromide)
NorC MFS MgrA(?),
Fluoroquinolones (e.g. hydrophilic: ciprofloxacin
and hydrophobic: moxifloxacin) Dyes
(e.g. rhodamine)
MepA MATE MepR Fluoroquinolones (e.g. hydrophilic: ciprofloxacin,
norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin) Glycylcyclines (e.g. tigecycline) QACs (e.g. tetraphenylphosphonium, cetrimide, benzalkonium chloride) Dyes
(e.g. ethidium bromide)
MdeA MFS n.i. Hydrophilic fluoroquinolones (e.g. ciprofloxacin,
norfloxacin) Virginiamycin, novobiocin, mupirocin,
fusidic acid QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride, dequalinium) Dyes (e.g. ethidium bromide)
SepA n.d. n.i. QACs (e.g. benzalkonium chloride) Biguanidines
(e.g. chlorhexidine) Dyes (e.g. acriflavine)
SdrM MFS n.i. Hydrophilic fluoroquinolones (e.g. norfloxacin) Dyes (e.g. ethidium bromide, acriflavine) [42]
LmrS MFS n.i. Oxazolidinone (linezolid) Phenicols
(e.g. choramphenicol, florfenicol) Trimethoprim, erythromycin, kanamycin,
fusidic acid QACs (e.g. tetrapheny-
lphosphonium) Detergents (e.g. sodium
docecyl sulphate) Dyes (e.g. ethidium
Plasmid-encoded Efflux Systems

QacA MFS QacR QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride, dequalinium)
Biguanidines (e.g. chlorhexidine)
Diamidines (e.g. pentamidine) Dyes
(e.g. ethidium bromide,
rhodamine, acriflavine)
QacB MFS QacR QACs (e.g. tetraphenylphosphonium,
benzalkonium chloride)Dyes (e.g. ethidium bromide, rhodamine,
Smr SMR n.i. QACs (e.g. benzalkonium chloride,
cetrimide) Dyes (e.g. ethidium bromide)
QacG SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)
QacH SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)
QacJ SMR n.i. QACs (e.g. benzalkonium chloride,
cetyltrymethylammonium) Dyes
(e.g. ethidium bromide)

a n.d.: The family of transporters to which SepA belongs is not elucidated to date.
b n.i.: The transporter has no regulator identified to date.
QACs: quaternary ammonium compounds

Identification of the plasmid-encoded qacA efflux pump gene
in meticillin-resistant Staphylococcus aureus (MRSA)
strain HPV107, a representative of the MRSA Iberian clone

S.S. Costaa,b, E. Ntokouc, A. Martinsa,d, M. Viveirosa,e, S. Pournarasc,
I. Coutoa,b, L. Amarala,d,e,∗
a Unidade de Micobactérias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (IHMT, UNL), b Centro de Recursos Microbiológicos,
Universidade Nova de Lisboa (CREM, UNL), d Unidade de Parasitologia e
Microbiologia Médica (UPMM), Instituto de Higiene e Medicina Tropical, Universidade
Nova de Lisboa (IHMT, UNL), Lisbon, Portugal; e COST ACTION BM0701 (ATENS)
c Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece;
Int J Antimicrobial Agents  2010; 36: 557–561

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial
bacterium for which prevention and control measures consist mainly of

  • the application of biocides with antiseptic and disinfectant activity.

In this study, we demonstrated the presence of

  • the plasmid-located efflux pump gene qacA in MRSA strain HPV107,

a clinical isolate representative of the MRSA Iberian clone. The existence
of efflux activity in strain HPV107 due to the QacA pump was found and

  • this QacA efflux activity was linked with a phenotype of
  • reduced susceptibility towards several biocide compounds.

No association could be made with antibiotic resistance. This work
emphasises the potential of QacA pump activity in

  • the maintenance and dissemination of important MRSA strains in
    the hospital setting and, increasingly, in the community.

Efflux-mediated response of Staphylococcus aureus exposed to
ethidium bromide

I Couto1,2, S S Costa1, M Viveiros1, M Martins1,3 and L Amaral1,3*
1Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical,
Universidade Nova de Lisboa (UNL), 2Centro de Recursos Microbiolo´gicos (CREM), Faculdade de Cieˆncias e Tecnologia, UNL,3UPMM,
Instituto de Higiene e Medicina Tropical, UNL, Portugal
J Antimicrob Chemother  (2008) 62, 504–513

By adapting an antibiotic-susceptible Staphylococcus aureus strain to
increasing concentrations of ethidium bromide, a known substrate
of efflux pumps (EPs), and

  • by phenotypically and genotypically analysing the resulting progeny,
  • we characterized the molecular mechanisms of S. aureus
    adaptation to ethidium bromide.

ATCC 25923 was grown in increasing concentrations of ethidium bromide.
The MICs of representatives of eight classes of antibiotics, eight biocides
and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny
ATCC 25923EtBr were determined

  • with or without six efflux pump inhibitors (EPIs).

Efflux activity in the presence/absence of EPIs was evaluated by realtime
fluorometry. The presence and expression of eight EP genes were assayed
by PCR and quantitative RT–PCR (qRT–PCR), respectively. Mutations in
grlA, gyrA and norA promoter regions were screened by DNA sequencing.

Compared with its parental strain, ATCC 25923EtBr was

  • 32-fold more resistant to ethidium bromide and
  • also more resistant to biocides and hydrophilic fluoroquinolones.
  • Resistance to these could be reduced by the EPIs chlorpromazine,
    thioridazine and reserpine.

Increased efflux of ethidium bromide by ATCC 25923EtBr could be
inhibited by the same EPIs. qRT–PCR showed that

  • norA was 35-fold over-expressed in ATCC 25923EtBr,

whereas the remaining EP genes showed no significant increase in their

expression. Sequencing of the norA promoter region revealed

  • a 70 bp deletion in ATCC 25923EtBr.

Exposure of S. aureus to quaternary compounds such as ethidium bromide
results in decreased susceptibility of the organism to a wide variety of
compounds, including quinolones and biocides

  • through an efflux-mediated response, which
  • for strain ATCC 25923 is mainly NorA-mediated.

This altered expression may result from alterations in the norA
promoter region

Ethnic consumption of plant leaf extracts and appraisal of
their nutraceutical efficacy against multidrug resistant
staphylococcus aureus

Kaushik S1, 2*, Tomar Rs1, Shrivastav V1, Shrivastav A2 And Jain Sk3
Amity Institute of Biotechnology, Amity University Madhya Pradesh,
Gwalior (M.P.);  2: College of Life Sciences, Cancer Hospital and
Research Institute, Gwalior (M.P.); 3: Department of Microbiology,
Vikram University, Ujjain (M.P.), INDIA
IJBPAS, Feb, 2014, 3(2): 204-209

Nutraceuticals are natural bioactive chemical compounds that have
health promoting, disease preventing or medicinal properties.
Emergence of Multi Drug Resistant Staphylococci is increasing at
alarming rates and diseases caused by these strains leave patients
against multiple resistant Staphylococcus aureus.

The test bacteria were isolated and characterized by standard and
NCCLS recommended microbiological techniques. A total of eighteen
plant extracts were analysed for their antimicrobial activity. The
selection of medicinal plants was based on their traditional uses in
India. However most of these plants were not previously screened.
Antibacterial activity of these components was performed by standard
Kirby Bauer Disk Diffusion method approved by NCCLS and the
inhibitory effect was analysed by calculating Zone of inhibition.

Among the eighteen plant extracts analysed we found highest
activity in the effect of chemotherapy and as promising bio control agents

  • Guava,
  • Mango,
  • Jamun and
  • Pomengrate plant extracts,

while most of the other plants were either showing very moderate/
least activity against test bacteria. Our recent experiment indicated
that phytochemicals extracted with methanol can be utilized as
nutraceutical to lower the side.

Part 6. Efflux pumps and gram-negative organisms

Efflux Pumps that Bestow Multi-Drug Resistance of Pathogenic Gram-

Amaral L1,2*, Spengler G2, Martins A2,3 and Molnar J2
1Travel Medicine of the Centre for Malaria and Other Tropical Diseases (CMDT),
Institute of Hygiene and Tropical Medicine, Lisbon, Portugal 2Department of
Medical Microbiology and Immunobiology, Faculty of Medicine, University of
Szeged, Szeged, Hungary 3Unit of Parasitology and Medical Microbiology
(UPMM), Institute of Hygiene and Tropical Medicine, Lisbon, Portugal
Amaral et al., Biochem Pharmacol 2013; 2:3

The efflux pump

The efflux pump

Efflux pumps are integral plasma membrane protein systems that recognize and bind
noxious compounds present in the cytoplasm (toxic products produced by metabolism;
compounds that have penetrated the cell), or periplasm of the bacterial cell and extrude
it into the environment in which the bacterium resides [1].

The efflux pump machinery gives the cell additional protection to the one provided by

  • the constituents of its cell wall (example: lipopolysaccharides), and
  • provides an initial protection to noxious agents present in its
    natural environment that have penetrated into the cell (example: bile
    salts in the colon) [1].

The efflux pump machinery is divided into five superfamily classes;

  • the major facilitator (MF),
  • the ATP-binding cassette (ABC),
  • the resistance-nodulation-division (RND),
  • the small multi-drug resistance (SMR) and
  • the multi-drug and toxic compound extrusion (MATE).

With respect to Gram-negative bacteria, although they all play
important roles in the protection of the bacterium from noxious
agents present in the environment, the

  • main efflux pump of the Gram negative bacterium is a
    member of the RND superfamily, and
  • because multi-drug resistance of clinical isolates have
    been associated with the over-expression of this pump,

it has received a great deal of attention [2].

The first in vitro response of bacteria to a given noxious agent,
such as an antibiotic, is to over-express its main efflux pump [2].
If the bacterium is serially exposed in vitro to increasing
concentrations of that compound, it responds by increasing
the effective number of its main efflux pump, as well as others
that provide redundant protection [2].

However, if that “adapted” bacterium is now maintained at a
constant level of a noxious agent, the level of efflux pump
activity increases up to a maximum, followed by a gradual
return of efflux pump activity to its basal level. Concomitant
to this process, an accumulation of mutations of essential
proteins located in the plasma membrane (example penicillin
binding proteins), mutations 30 S component of the ribosome
and gyrase take place [3]. These events suggest that when
the organism is faced with an environment that contains a
constant toxic level of a compound, and the cost for
maintaining an energy consuming system, such as that
needed for the energy dependent efflux pump, is too
great a price to pay.

Therefore, in order to survive in this unchanging environment,
other mechanisms are activated. For example, activation of a
mutator master gene is thought to be an important step at this
level, which results in the mutation of genes that code for
essential proteins, reversing the over-expression of efflux-
pumps, but still conferring the bacterial resistant to the
environmental pressure via other mechanism(s), yet
to be understood [4,5].

During therapy, the level of resistance increases many fold
higher than that of the initial infecting strain. Hence, clinical
isolates from treated patients often show much higher levels
of antibiotic resistance than that of their wild type counterpart
(sometimes it can even present a 1000 fold increase) [6].
At this stage, resistance is usually related to the presence
of mutations, which reduces the survival of the resistant

  • once it is transferred to a noxious agent-free environment

that contains the competing wild type counterpart [3,4].

Depending upon when during therapy a clinical strain is isolated,
its resistance to two or more antibiotic classes (multi-drug
resistance (MDR)), may be due entirely to over-expressed
efflux pumps; to a mixture of over-expressed efflux pumps
and increasing accumulation of mutations; and only to mutations [3,4].

The degree of resistance can readily be determined with
methods that employ compounds known for their modulation
of efflux pump activity, such as

  • phenothiazines [7] or phenyl-arginine-betanaphthylamide
  • the latter which competes with the antibiotic as
    substrate of the efflux pump [8].

If in presence of such compounds,

  • the MDR bacterium is rendered fully susceptible
    to the antibiotic(s) to which it was initially resistant,
  • resistance is most likely due to its overexpressed
    efflux pump systems.
  • Contributions made by accumulated mutations
    render the organism less and less affected by the EPI.

This type of information is of great value to clinicians faced
with long-term therapy of a bacterial infection that
progresses to an MDR phenotype. It should be understood
that although the Gram-negative bacterium has essentially
one main efflux pump, such as

  • the AcrAB (Escherichia coli) or
  • the MexAB (Pseudomonas aeruginosa),

the deletion of the main efflux pump results in the over-
expression of one or more other RND efflux pumps,
such as is the case for deletion of the AcrAB, followed by

  • the over-expression of the AcrEF pump [2].

Redundancy of as many as nine RND efflux pumps [2],
provides additional protection to the organism.

The pumps belonging to the RND family form

  • a tripartite complex together with
  • the periplasmic proteins belonging to the
    membrane fusion-protein (MFP) family and
  • the outer membrane channels.

RND transporters consist of

  • a transporter protein that recognises and
    binds the noxious agent
    in the cytoplasm or periplasm and
  • transports it to the contiguous channel (TolC),
  • ending at the surface of the outer membrane.

The transporter is attached to the plasma membrane
by two or three fusion proteins, which are believed to assist the

  • extrusion of the substrate by peristaltic actions [9].

Although the actual structure of RND efflux pumps
in the cell envelop is not completely understood,

  • the structure of the transporter, TolC and fusion
    proteins are well established for major Gram-negative
    bacteria [10].

The PMF energy dependent efflux pump most likely needs the
passage of hydronium ions through its internal cavity,

  • for the release of the substrate that is
  • in turn ejected into the TolC channel via the
  • peristaltic action of the fusion proteins [11].

A low pH,

  • the concentration of hydronium ions at the surface of the cell
  • results in a pH difference of 2 or 3 pH units compared
    to that of the milieu,

the surface concentration of hydronium ions

  • provides the force for the mobility of hydronium ions
  • through porins leading to the acidification of the periplasm,
  • providing the low pH needed by the transporter
  • for the release of the substrate.

At high pH, these hydronium ions come from

  • hydrolysis of ATP by ATP synthase, and
  • are passed into the transporter, thereby
  • reducing its internal pH, so that
  • the release of the substrates can take place [11,12].

EPIs, such as the phenothiazines chlorpromazine or thioridazine,

  • exert their inhibition at pH above 6, and
  • are thought to affect hydrolysis of ATP
  • denying the efflux pump transporter hydronium ions needed

for release of the bound substrate [11,12].

The search for EPIs that are clinically useful continues, although

with respect to thioridazine, this old neuroleptic has been shown

  • to inhibit efflux pumps of pathogenic mycobacteria [13], and
  • has been successfully used to treat extensively drug resistant
    tuberculosis infections [14].

The regulation of the main efflux pump of Escherichia coli may
take place via   distinct pathways. The induced synthesis of the
transporter component of the AcrAB efflux pump, when the
organism is exposed in vitro to a noxious agent,

  1. involves the activation of the stress gene soxS,
  2. followed by the activation of the local regulator marA,
  3. then by the activation of the transporter gene acrB [8].

In the case of Salmonella spp. two component resistance
mechanisms, such as the PmrA/PmrB system, directly
activate the master efflux pump regulator ram A gene [15].
The activation of the PmrA/PmrB system takes place
readily when Salmonella spp. is phagocytosed due to
the acidic nature of the phagolysosome [15], as follows:

  1. PmrB is a sensor that self-phosphorylates, and
  2. then transfers the phosphate to PmrA.
  3. PmrA activates a nine gene operon, which
  4. codes for Lipid A introduced into the nascent
    lipopolysaccharide layer of the outer membrane.
  5. The increased presence of Lipid A renders the
    phagocytosed bacterium practically immune to
    everything, including the hydrolases of the
    phagolysososome [15].

Although some EPIs are in clinical trials, none have yet to
reach the marketplace,    mainly due to their common
toxicity against healthy mammalian cells, affecting
intrinsic mammalian efflux pumps, as for example
those of the blood brain barrier. Lastly, it should be
noted that compounds that inhibit the efflux pump
of bacteria also have the capacity to promote the
removal of plasmids that carry antibiotic resistant
genes [16,17].

  1. Nikaido H, Pages JM (2012) Broad-specificity efflux
    pumps and their role in multidrug resistance of Gram-
    negative bacteria. FEMSMicrobiol Rev 36: 340-363.
  2. Viveiros M, Jesus A, Brito M, Leandro C, Martins M,
    et al. (2005) Inducement and reversal of tetracycline
    resistance in Escherichia coli K-12 and expression of
    proton gradient-dependent multidrug efflux pump
    genes. Antimicrob Agents Chemother 49: 3578-3582.
  3. Martins A, Couto I, Aagaard L, Martins M, Viveiros M
    (2007) Prolonged exposure of methicillin-resistant
    Staphylococcus aureus (MRSA) COL strain to
    increasing concentrations of oxacillin results in a
    multidrug-resistant phenotype. Int J Antimicrob
    Agent 29: 302-305.
  4. Martins A, Spengler G, Molnar J, Amaral L (2012)
    Sequential responses of bacteria to noxious agents
    (antibiotics) leading to accumulation of mutations
    and permanent resistance. Biochem Pharmacol J
    Open Access 1: 7.

Inhibitors of efflux pumps of Gram-negative
bacteria inhibit Quorum Sensing

Leonard Amaral, Joseph Molnar
1 Grupo de Micobacterias, Unidade de Microbacterilogia,
Centro de Malaria e Doenças Tropicais (CMDT), Instituto de
Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Lisbon, Portugal; 2 Cost Action BM0701 (ATENS) of the
European Commission/European Science Foundation;
3 Department of Medical Microbiology and Immunobiology,
University of Szeged, Szeged, Hungary
Open Journal of Pharmacology, 2012, 2-2

Quorum Sensing (QS) systems of bacteria consist of

  • a producer of the QS signal and the responder.

The generation of a QS signal provides the means by which
a population can behave in a concerted manner such as

  • swarming, swimming and secretion of biofilm, etc.

Because concerted bahaviour bestows protection to the bacterial
species, and hence factors involved in the severity of an infection
such as virulence are products of QS systems, compounds that
inhibit the QS system have significant clinical relevance. Recent
evidence suggests that

  • the secretion of QS signals takes place via
  • the efflux pump system of the producer of the signal.

Interestingly, compounds such as phenothiazines and
trifluoromethyl ketones (TFs)

  • that inhibit proton motive force (PMF) activities such
    as swarming and swimming also
  • inhibit the PMF dependent efflux pump systems of
    bacteria and their QS   systems.

This review discusses the relationship between the efflux
pump, the QS system and the compounds that affect both.
Lastly, suggestions are made regarding classes of compounds
that have been shown

  • to inhibit PMF dependent efflux pumps and the need
  • to evaluate them for QS inhibitory properties.

Keywords: Quorum Sensing, QS signal, acylated hydroxyl
lactone (AHL), efflux pumps, Proton Motive Force (PMF),
inhibitors of efflux pumps, inhibitors of QS systems,
phenothiazines, Trifluormethyl Ketones (TFs), plants
sources for QS inhibitors

Efflux pumps of bacteria provide protection from noxious
agents that are present in the environment in which they
exist. Noxious agents may be naturally occurring compounds
present in environments outside and within the human.

Because over-expressed efflux pumps render antibiotic
therapy problematic, an intense search for agents that
inhibit specific efflux pumps of specific bacteria has
been conducted during the past decade [9].

Communication between bacteria of the same strain
or species and between species contributes to their
survival [11-13]. Communication involves the secretion
of signals that invoke a specific response from the responder
[11-13]. This  communication process is termed Quorum
sensing (QS). When it takes place between strains of the
same species,

  • communication is directed towards the reduction
    of population growth and
  • reducing the possibility of exceeding the nutritional
    support of the environment

Other signals may involve a population response that involves

  • the secretion of bioactive molecules that inhibit the
    replication of a competing population species [14-16]
    or even kill [biocidins) [17-21] or
  • promote a swarming effect that recruits members
    of the same species to migrate  to a specific location [22-24]
    similar to swarming by insects subsequent to signals
    indicating site of food [example bees).
  • biofilm, encase the bacteria at distances from each other
    [25-29] and within the matrix of this biofilm are
    channels used for further communication [30].

Biofilms are produced in the wild, at sites such as surfaces
of rocks which maintain the bacterial population in situ [31]
and are also produced at sites of the human colonized by
infecting bacteria [32, 33].

Agents that inhibit the QS response of the infecting bacterium
are obviously important and hence, the search for such agents
that inhibit the QS system and biofilm formation has been in
effect for the past two decades [11-13].

There is a relationship between efflux pumps (EP), QS and
biofilm (BF) secretion which has come to the forefront only
recently [13]. Control of this relationship is critical for
successful therapy of MDR bacterial infections which have
become rather commonplace. It is the intent of this review
to identify agents which may serve to interfere with the
complex system of EP-QS-BF interaction.

Proton motive force (PMF) dependent transporters obtain
their energy for function from the proton motive force. The
proton motive force is the result of cellular metabolism which
yields protons that are not used for coupling with molecular
oxygen and which are exported to the surface of the cell [43-45]
where they are distributed and bound to components of
the protective lipopolysaccharide layer that covers the cell
and constitutes a part of the outer cell wall of Gram-negative
[46] and the cell wall of Gram positive bacteria [47].

The larger the concentration of protons (hydronium ions)
on the surface of the cell with respect to their lower
concentration on the medial side of the cytoplasmic
membrane creates an electrochemical gradient that
is termed the proton motive force (PMF) [48].

Because hydronium ions cannot penetrate the cell wall
or the membrane, they may re-enter the cell only
through channels such as porins in general [49, 50].
The movement of these hydromium ions from the
surface of the cell to the periplasm or cytoplasm is
predicated upon systems that use the PMF as source
of energy-namely the resistance nodulation division
(RND) family of transporters.

E. coli has a multiplicity of efflux pumps that may
exceed 30 in number [51]. However, the main
efflux pump of this organism is the AcrAB-TolC
efflux pump [52, 53] which when deleted, its
function is replaced by the AcrEF-TolC efflux
pump [51]. Both efflux pumps are members
of the resistance nodulation division family of
transporters [51] and consist of three proteins:

  1. The transporter AcrB coded by the gene acrB and
    is intimately attached to the  plasma membrane;
  2. Two fusion proteins AcrA coded by the gene acrA
    that flank the AcrB transporter and are thought
    to assist the movement of a substrate through
    the AcrB transporter [35]; and,
  3. TolC which is also part of other tri-unit efflux pumps
    of the organism [35], is contiguous with the AcrB
    transporter and provides a conduit for the extrusion
    of the substrate [38].

Although the means for the recognition of the substrate to
be extruded appears to involve a pocket within the transporter,
it appears to be

  • defined by a phenyalanine residue [54].

Nevertheless, studies employing fluorochromes recognised by
the AcrB transporter indicate that the binding and release of
the substrate are pH dependent [55].

  • At low pH the dissociation of the substrate is high and
  • at high pH it is very slow.

In a physiological environment of ca. pH 7, if the dissociation
of the substrate is slow or not at all, then the effectiveness of
the pump to extrude a noxious agent would be nullified.
However, since the pump functions at this pH, conditions that
result in the dissociation of the substrate needed for continuous
pump action must involve a

  • decrease of the pH of the internal cavity of the pump
    to which the substrate is bound.

It has been postulated that the lowering of the pH takes place
by the generation of hydronium ions from metabolism [6] which

  • pass from the cytoplasmic side of the plasma membrane
    through the transporter.

At lower pH, there is no need for the generation of metabolically
derived  hydronium ions since these ions can be

  • diverted by the PMF from the surface of the cell
    to the periplasm via porins.

Whether hydronium ions are to be generated from the
hydrolysis of ATP at high pH or used for the synthesis
of ATP at low pH is a special

  • function of ATP synthase [56-58].

Model of the AcrAB-TolC efflux pump of a Gram-
negative bacterium

AcrAB-TolC efflux pump of a Gram-negative bacterium

AcrAB-TolC efflux pump of a Gram-negative bacterium

Hypothesis. At near neutral pH, Hydronium ions from hydrolysis of ATP
by ATP synthase pass through the AcrB

transporter, reduce the pH to a point that causes the release of the
substrate. When the hydronium ions reach the surface of the cell they
are distributed over that surface and bind to lipopolysaccharides
and basic amino acids. When there is a need for hydronium ions for
activity of the efflux pump and the pH is lower than neutral, and
the hydrolysis of ATP is not favoured, hydronium ions from the
surface of cell via the PMF mobilize through the Aqua porins
and reach the transporter where they are pushed through
the transporter by the peristaltic action caused by the fusion
proteins. Substrates bound to the transporter dissociate
when the pH is reduced by the flow of hydronium ions and
are carried out by the flow of water.

Inhibitors of bacterial efflux pumps
Inhibitors of the QS of bacteria

Because phenothiazines inhibit many energy dependent systems
of bacteria such as motility [89, 90, 95], and these phenothiazines
also inhibit efflux pumps of bacteria [6, 7, 9, 41, 51, 73, 74, 76-83],
there seems to be a correlation between an active efflux pump
system and a functional QS system. That this assumption is correct,
recent evidence has been provided showing that the efflux pumps of
the AHL responding environmental Chromobacterium violaceum
(CV026) bacterium and that of E. coli are inhibited by the phenothiazine
thioridazine (TZ) [12]. Because TZ is known to inhibit genes that
regulate and code for efflux pumps of bacteria [41, 119, 120], it is
possible that the inhibition of the responding CV0126 bacterium to
AHLs [12] involves the inhibition of genes that code and regulate
the efflux pump of the responder which is assumed to recognise the
AHL signal as an noxious agent and hence would extrude it to the
environment [12]. The inhibition of an efflux pump should manifest
itself as an inhibitor of the QS component responsible for biofilm

Since the discovery of berberine a powerful inhibitor of bacterial
efflux pumps [159], plants have become sources of inhibitors of
efflux pumps [160-164]. Given that efflux pumps and the  QS of
bacteria have an intimate relationship as described in this review,
attention has been focused on plants for potential sources of inhibitors
of efflux pumps and QS systems. Essential oils from Columbian
plants have yielded a large number of compounds that inhibit the
QS system of responding bacteria such as

  1. limonene-carvone , the
  2. citral (geranial-neral) (isolated from Lippia alba),
  3. α-pinene (from Ocotea sp.),
  4. β-pinene (from Swinglea glutinosa),
  5. cineol (from Elettaria cardamomun),
  6. α-zingiberene (from Zingiber officinale) and
  7.  pulegone (from Minthostachys mollis) [165].

Several other essential oils, in particular were shown to present
promising inhibitory properties for the short chain AHL quorum
sensing (QS) system in Escherichia coli containing the biosensor

  •  plasmid pJBA132, in  particular Lippia alba.

Citral was the only  essential oil that presented some activity for
the long chain AHL QS system in Pseudomonas putida containing

  •  the plasmid pRK-C12 [165].

The essence of this review is to correlate the relationship of the
efflux pump system to the QS system of bacteria via the use of
compounds that inhibit both systems. Simply put, inhibitors of
the efflux pump system also, when studied, inhibit the QS system
as well. Because the PMF dependent efflux pump system of Gram-
negatives that is overexpressed is responsible for the multi-drug
phenotype of the bacterium, compounds that affect the PMF of
the bacterium are candidates that will inhibit the activity of the
pump. Consequently, this inhibition will inhibit the secretion of
biofilm, and because biofilm is a deterrent to the action of antibiotics,
compounds that affect the efflux pump system are promising
candidates for clinical evaluation.

Limiting and controlling carbapenem-resistant
Klebsiella pneumonia

L Saidel-Odes, A Borer.
1Infection Control and Hospital Epidemiology Unit, 2Infectious
Diseases Institute, Soroka University Medical Center and the
Faculty of Health Sciences, Ben-Gurion University of the Negev,
Beer-Sheva, Israel
Infection and Drug Resistance 2014:7 9–14

Carbapenem-resistant Klebsiella pneumoniae (CRKP)

  • is resistant to almost all antimicrobial agents,
  • is associated with substantial morbidity and mortality, and
  • poses a serious threat to public health.

The ongoing worldwide spread of this pathogen emphasizes the
need for immediate intervention. This article reviews the global
spread and risk factors for CRKP colonization/infection, and
provides an overview of the strategy to combat CRKP dissemination

Outbreaks of CRKP that have occurred around the world have
been associated with the plasmid-encoded carbapenemase
K. pneumoniae carbapenemase (KPC),

  • a carbapenem-hydrolyzing β-lactamase.19

CRKP isolates are resistant to almost all available antimicrobials
and are susceptible

  • only to polymyxins and tigecycline;
  • a minority to the few remaining aminoglycosides,
    though resistance to these agents is increasingly reported.20,21

Several investigators have evaluated predictors for CRKP colonization.
The following summarizes various studies.

  1. In a multivariate analysis, prior use of macrolides and
    any antibiotic exposure $14 days remained the only
    independent factors associated with CRKP bacteremia
  2. Nosocomial isolation of CRKP was strongly favored by the
    selection pressure of carbapenem. In this study, prior
    treatment with fluoroquinolones was associated with
    decreased risk for the emergence of CRKP.
  3. Previous use of carbapenem and cephalosporin
  4. Nursing home residency before hospital admission, bedridden
    status, and previous antibiotic therapy
  5. exposure to fluoroquinolones
  6. the recipient of antibiotics
  7. intensive care unit (ICU) stay, and
  8. Poor functional status,
  9. Independent predictors of subsequent carbapenem-
    resistant Enterobacteriaceae (CRE) infection were
  • admission to the ICU,
  • having a central venous  catheter,
  • receipt of antibiotics, and
  • diabetes mellitus

Schwaber et al and the Israeli CRE Working Group enforced the
Israel Ministry of Health guidelines mandating physical separation
of hospitalized carriers of CRE and dedicated staffing and appointed
a professional task force charged with containment.19 The monthly
incidence of nosocomial CRE was reduced from 55.5 to 11.7 cases
per 100,000 patient days within 15 months.

Part 7.  Tuberculosis

The Mechanism by which the Phenothiazine Thioridazine
Contributes to Cure Problematic Drug-Resistant Forms
of Pulmonary Tuberculosis: Recent Patents for “New Use”

L Amaral1*, A Martins2,3, G Spengler2, A Hunyadi4 and J Molnar2
Recent Patents on Anti-Infective Drug Discovery 2013; 8(3):000-000

At this moment, over half million patients suffer from multi-drug
resistant tuberculosis (MDR-TB) according to the data from the WHO.
A large majority is terminally ill with essentially incurable pulmonary
tuberculosis. This herein mini-review provides the experimental and
observational evidence that a specific phenothiazine,

  • thioridazine,

will contribute to cure any form of drug-resistant tuberculosis. This
antipsychotic agent is no longer under patent  protection for its
initial use. The reader is informed on the recent developments

  • in patenting this compound for “new use” with a special
  • emphasis on the aspects of drug-resistance.

Given that economic motivation can stimulate the use of this drug
as an antitubercular agent, future prospects are also discussed.

Thioridazine is not the only phenothiazine that has been recommended
for therapy of pulmonary tuberculosis. In general, many phenothiazines
have been implicated for antitubercular activity [62, 80-86]. Among
these are

  • trifluoperazine [87-94],
  • methdilazine [95, 96],
  • promazine [97, 98],
  • promethazine [97, 98],
  • fluphenazin [99],
  • propiomazine [100], and
  • the methylene blue related toluidine blue [101].

There are phenothiazine compounds derived from the parental
methylene blue for therapy of pathologies unrelated to tuberculosis
that also possess

  •  antitubercular [44, 48] and/or antimalarial properties [44].

Moreover, derivatives made from any of the phenothiazines that
have in vitro activity against Mycobacterium tuberculosis are also
active [61, 67, 102, 103], suggesting ample opportunities for
patenting of new analogs developed from known, active phenothiazines
with even less side effects than those of TZ, as recently suggested by
Musuka and co-authors [104]. It is important to mention, that the
commercially available phenothiazines such as for example

  •  trifluoperazine, methdilazine, promazine, promethazine,
    fluphenazin and propiomazine

are beyond patent protection as initially intended. Nevertheless,
these compounds have been patented as adjuvants for the treatment
of MDR cancer (patent expired in 2011 [105]; and, right afterwards,
a new patent has been filed with a priority date of 28th March, 2012,
claiming combination therapy of cancer with a chemotherapeutic
agent and a dopamine receptor antagonist against Cancer stem cells (CSC).

Taking into account that intrinsic MDR is considered as one of the key
properties of CSCs [107], the subject to be covered is indeed related.
According to the MDR, XDR and TDR Mycobacterium tuberculosis,
subjects of this herein paper, the initial step for actually reaching those
in need has been made: a patent has been published in December, 2007,
for the use of TZ and its derivatives for reversing anti-microbial drug
resistance [108]. We must note, however, that, despite the six years
passed since, we were unable to find any related clinical trials, which
would certainly be of outmost importance and urgency in order to
proceed towards an effective therapy of highly resistant mycobacterial

Mechanism Of Action Of Tz: Why It Cures Multi-Drug,
Extensively Drug Resistant And Probably Totally Drug
Resistant Tuberculosis

Over-expressed efflux pumps of Mycobacterium tuberculosis render
the organism multi-drug resistant [13]. Special attention has been
given to those coded by the

  • mmpL7, p55, efpA, mmr, Rv1258c and Rv2459 genes [109].

The activity of these efflux pumps can be suppressed by

  • concentrations of TZ that have no effect on the viability of
    Mycobacterium tuberculosis
  • rendering the organism susceptible to the antibiotic to
    which it was initially resistant
  • as a consequence of the over-expression of its
    efflux pumps [109].

TZ has also been shown to inhibit the activity of the main

  • efflux pumps of bacteria belonging to other species.

TZ has strong inhibitory activity against the genes that code for
essential proteins of M. tuberculosis [122-124].  Consequently, we
may conclude that the in vitro activity of TZ involves

  • the inhibition of the efflux pumps of M. tuberculosis and that
  • the in vitro exposure of this organism to TZ renders the organism
  • susceptible to antibiotics to which it was initially resistant
  • as a consequence of over-expressed efflux pumps [21].

Phenothiazines such as CPZ, TZ, trifluoperazine, etc., also inhibit

  • the binding of calcium to calcium binding proteins such as

calmodulin in eukaryotes [125], and

  • interfere with other proteins involved in
  • the regulation of cellular activity [126].

They inhibit the transport of calcium and potassium systems

  • in eukaryotic cells [127-129] as well as in
  • mycobacteria [89, 130] and
  • E. coli [113].

In fact, in the latter case, calcium was shown essential to

  • the continuous activity of the thioridiazine sensitive
    efflux system [113].

The killing activity of the human macrophage as well as that
of the neutrophil

  • is dependent upon the retention of calcium and potassium
  • within the phagolysosome of the cell [131].

Considering this, several alternative choices are available for
patenting under “new use”, which would allow a “fresh start”
for the compound to be developed. However, the needed
experimental proof that these phenothiazine agents have
activity at the pulmonary macrophage of the alveolar unit
(the site where the causative organism of pulmonary tuberculosis
resides) is still absent.

Targeting the Human Macrophage with Combinations
of Drugs and Inhibitors of Ca2+ and K+ Transport to
Enhance the Killing of Intracellular Multi-Drug Resistant
M. tuberculosis (MDR-TB) – a Novel, Patentable Approach
to Limit the Emergence of XDR-TB

Marta Martins
UCD Centre for Food Safety, School of Agriculture, Food Science and
Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
& Unit of Mycobacteriology and UPMM; Instituto de Higiene e Medicina
Tropical, Universidade Nova de Lisboa (IHMT/UNL),  Lisbon, Portugal
Recent Patents on Anti-Infective Drug Discovery, 2011, 6, 000-000

The emergence of resistance in Tuberculosis has become a serious
problem for the control of this disease. For that reason, new therapeutic
strategies that can be implemented in the clinical setting are urgently
needed. The design of new compounds active against mycobacteria
must take into account that Tuberculosis is mainly an intracellular
infection of the alveolar macrophage and therefore must maintain
activity within the host cells.

An alternative therapeutic approach will be described in this review,
focusing on the activation of the phagocytic cell and the subsequent
killing of the internalized bacteria. This approach explores the combined
use of antibiotics and phenothiazines, or Ca2+ and K+ flux inhibitors,
in the infected macrophage.

Targeting the infected macrophage and not the internalized bacteria
could overcome the problem of bacterial multi-drug resistance. This
will potentially eliminate the appearance of new multi-drug resistant
tuberculosis (MDR-TB) cases and subsequently prevent the emergence
of extensively-drug resistant tuberculosis (XDR-TB).

Patents resulting from this novel and innovative approach could be
extremely valuable if they can be implemented in the clinical setting.
Other patents will also be discussed such as the treatment of TB
using immunomodulator compounds (for example: betaglycans).

Role of Phenothiazines and Structurally Similar
Compounds of Plant Origin in the Fight against
Infections by Drug Resistant Bacteria

SG. Dastidar 1, JE. Kristiansen 2, J Molnar 3 and L Amaral
Antibiotics 2013; 2: 58-71;

Phenothiazines have their primary effects on the plasma membranes
of prokaryotes and eukaryotes. Among the components of the
prokaryotic plasma membrane affected are

  • efflux pumps,
  • their energy sources
  • and energy providing enzymes, such as ATPase,
  • and genes that regulate and code for the permeability
    aspect of a bacterium.

The response of multidrug and extensively drug resistant
tuberculosis to phenothiazines shows an alternative therapy for the
treatment of these dreaded diseases, which are claiming more and
more lives every year throughout the world.

Many phenothiazines have shown

  • synergistic activity with several antibiotics thereby
  • lowering the doses of antibiotics administered to patients
    suffering from specific bacterial infections.

Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp)
has been found to be synergistic with penicillin and chlorpromazine
(CPZ); in addition, some antibiotics are also synergistic. Along with
the antibacterial action described in this review,

  • many phenothiazines possess plasmid curing activities, which
  • render the bacterial carrier of the plasmid sensitive to antibiotics.

Thus, simultaneous applications of a phenothiazine like TZ would not
only act as an additional antibacterial agent but also would help

  • to eliminate drug resistant plasmid from
    the infectious bacterial cells.

Part 8.  Cancer Cytotherapy

Synthesis and Structure-Activity Relationships of Novel
Dioxolanes as MDR Modulators in Cancer

A Martins 1,2,†,*, J Csábi 3,†, A Balázs 4, DKitka 1, L Amaral 5,
J Molnár 1, A Simon 6, G Tóth 6 and A Hunyadi 3,
Molecules 2013, 18, 15255-15275;

Ecdysteroids, molting hormones of insects, can exert several mild,
non-hormonal bioactivities in mammals, including humans. In a
previous study, we have found a significant effect of certain derivatives

  • on the ABCB1 transporter mediated multi-drug resistance of a
  • transfected murine leukemia cell line.

In this paper, we present a structure-activity relationship study
focused on

  • the apolar dioxolane derivatives of 20-hydroxyecdysone.

Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including
20 new compounds, is presented, supplemented with their

  • complete 1H- and 13C-NMR signal assignment

As published before [9], the 20,22-diol moiety of 20E is more reactive
than the 2,3-diol, probably due to the free rotation of the 20,22-bond
of 20E that allows the 20,22-dioxolane ring to form with less strain.

This allowed us to selectively obtain the 20,22-mono-dioxolane
derivatives 2–14, or, depending on the amount of reagent and the
reaction time, the 2,3;20,22-bis-homo-dioxolanes 17 and 21–25.

By utilizing the 20,22-monodioxolane ecdysteroids, another aldehyde
or ketone could be coupled to position 2,3, resulting in several bis-hetero-
dioxolane derivatives 26–33. For this, however, gradually decreasing
reactivity with the increase of the size of the reagent was a limiting factor:

  • larger aldehydes or ketones (mainly those containing a
    substituted aromatic ring) could not be coupled at the 2,3-position.
  • The 2,3-monodioxolane derivatives also appeared to be present as
    minor side-products of the reactions, and as a consequence of their
    low amount, only one such compound (compound 15) was isolated and studied.

To selectively obtain this kind of a compound (16) in a more reasonable
yield, another, three-step approach was successfully applied:

  • after protecting the 20,22-diol with phenylboronic acid, the
    2,3-acetonide could be prepared, and
  • removal of the 20,22 protecting group afforded the desired
    2,3-monoacetonide in a one-pot procedure.

In the case of the reactions with aldehydes or asymmetric ketones,
the new C-28 and C-29 central atoms of the dioxolane rings are
stereogenic centers and thus two possible diastereomers can be
formed at both diols. Their configuration was elucidated by two-
dimensional ROESY or selective one-dimensional ROESY experiments,
e.g., in the doubly substituted

  • dioxolane derivative 22 (R1 = R4 = n-Bu, R2 = R3 = H)
  • the unambiguous differentiation of the 1H and 13C signals of
    the two n-butyl groups was achieved in the following way
    (see Figure 2).

Assignment of the H-C(28) atoms (δ = 4.93/105.9 ppm) was supported by

  • the H-2/C-28 and H-3/C-28 HMBC correlations, and
  • that of H-C(29) (δ = 4.91/105.6 ppm) by the H-22/C-29
    cross peak, respectively.

The selective ROESY experiment irradiating at 4.93 ppm showed

  • contacts with the Hα-2 and Hα-3 atoms proving the
    α position of the R2 = H atom.

The ROESY response obtained irradiating H = R3 signal (δ = 4.91)
on H-22 (δ = 3.64 ppm) revealed their

  • cis arrangement and the R configuration around C-29.

The unambiguous assignments of the signals

  • of the two n-butyl groups R1 and R4 were achieved by
  • selective TOCSY experiments (irradiation at
  • δ = 4.93 and 4.91, respectively).

Figure 2

Stereostructure of 22. Red-ROESY proximitries. Blue- 1H. Black-1 001

Stereostructure of 22. Red-ROESY proximitries. Blue- 1H. Black-1 001

Stereostructure of 22. Red arrows indicate the detected ROESY
steric proximities, the blue numbers give the characteristic 1H,
and the black numbers the 13C chemical shifts.


Related Material

Identification of Efflux Pump-mediated Multidrug-resistant
Bacteria by the Ethidium Bromide-agar Cartwheel Method

M Martins, M Viveiros, I Couto, SS. Costa, T Pacheco, S Fanning,
Jean-Marie Pagès, and L Amaral
in vivo 2011; 25: 171-178  

Index for efflux activity of the MDR strains. The capacity to efflux EtBr
of each bacterial strain was ranked relative to the reference strain
according to the following formula:


Index for efflux activity of the MDR strains

Index for efflux activity of the MDR strains

A Simple Method for Assessment of MDR Bacteria for
Over-Expressed Efflux Pumps

M Martinsa,b*, MP. McCuskera,b, M Viveirosa,c, I Coutoc,d,
S Fanninga,b, Jean-Marie Pagès b,e, L Amaral,b,
The Open Microbiol J 2013; 7: 1-5  1874-2858/13 Bentham

Flowchart followed to test bacterial strains using the EtBr-agar
Cartwheel method.

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method.

Flowchart followed to test bacterial strains using the EtBr-agar Cartwheel method.

EtBr-agar cartwheel method applied to different bacterial species

EtBr-agar cartwheel method applied to different bacterial species

EtBr-agar cartwheel method applied to different bacterial species

The effect of selected EPIs on the resistance of the induced and
MDR Gram-positive bacteria.

Enterococcus EFC
(4×) (4×) (2×)
                                MCEtBr NOR  (mg/l) MIC NOR (mg/l)
HSEFM-E >2.5 0.125 0.125 0.125 0.125

EPI: Efflux pump inhibitor; w/o: without; TZ: thioridazine; CPZ:
chlorpromazine; PAN: phenyl arginine β-naphthylamide. Values
in bold-type correspond to a decrease of 4-fold or higher on
the MIC values in comparison to those in the absence of inhibitor.
Values in parenthesis indicate the MIC decrease relative to that
of the original culture. The concentration of each EPI used is
defined in the Materials and Methods section.

Macrocyclic diterpenes resensitizing multidrug
resistant phenotypes 

MA. Reis a, A Paterna a, RJ. Ferreira a, H Lage b,
Maria-José U. Ferreira a,⇑
a Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade
de Farmácia, Universidade de Lisboa, Lisboa, Portugal
b Charité Campus Mitte, Institute of Pathology, Berlin, Germany
Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

Herein, collateral sensitivity effect was exploited as a strategy to
select effective compounds to overcome multidrug resistance in
cancer. Thus, eleven macrocyclic diterpenes, namely jolkinol D (1),
isolated from Euphorbia piscatoria, and its derivatives (2–11) were
evaluated for their activity on three different Human cancer entities:

  • gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29)

each with a variant selected for resistance to mitoxantrone

  1. EPG85-257RN;
  2. EPP85-181RN;
  3. HT-29RN and
  • one to daunorubicin (EPG85-257RD; EPP85-181RD; HT-29RD).

Jolkinol D (1) and most of its derivatives (2–11) exhibited significant
collateral sensitivity effect towards the cell lines

  • EPG85-257RN (associated with P-glycoprotein overexpression) and
  • HT-29RD (altered topoisomerase II expression).

The benzoyl derivative, jolkinoate L (8) demonstrated ability to

  • target different cellular contexts with
  • concomitant high antiproliferative activity.

These compounds were previously assessed as
P-glycoprotein modulators,

  • at non-cytotoxic doses, on MDR1-mouse lymphoma cells.

A regression analysis between

  1. the antiproliferative activity presented herein and
  2. the previously assessed P-glycoprotein modulatory effect

showed a strong relation between the compounds that presented

  • both high P-glycoprotein modulation and cytotoxicity.

Molecular Docking Characterizes Substrate-Binding Sites
and Efflux Modulation Mechanisms within P

Ferreira,† Maria-José U. Ferreira,† and DJVA dos Santos*,†,‡
†Research Institute for Medicines and Pharmaceutical Sciences
(iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
‡REQUIMTE, Department of Chemistry & Biochemistry, Faculty of
Sciences, University of Porto, Porto, Portugal
J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

P-Glycoprotein (Pgp) is one of the best characterized ABC
, often involved

  • in the multidrug-resistance phenotype
  • overexpressed by several cancer cell lines.

Experimental studies contributed to important knowledge concerning
substrate polyspecificity, efflux mechanism, and drug binding sites.
This information is, however, scattered through different perspectives,
not existing a unifying model for the knowledge available for this transporter.
Using a previously refined structure of murine Pgp,

  • three putative drug-binding sites were hereby characterized
  • by means of molecular docking.

The modulator site (M-site) is characterized by

  • cross interactions between both Pgp halves

herein defined for the first time, having an important role in

  • impairing conformational changes leading to substrate efflux.

Two other binding sites, located next to the inner leaflet of the lipid bilayer,

  • were identified as the substrate binding H and R sites
  • by matching docking and experimental results.

A new classification model

  • with the ability to discriminate substrates from modulators

is also proposed, integrating a vast number of theoretical and experimental data.

conformational changes leading to substrate efflux

conformational changes leading to substrate efflux

conformational changes leading to substrate efflux



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Reported by: Dr. Venkat S. Karra, Ph.D.

Oral Cephalosporins No Longer a Recommended Treatment for Gonococcal Infections: an update to CDC‘s 2010 STD guidelines.

Gonorrhea is a major cause of serious reproductive complications in women and can facilitate human immunodeficiency virus (HIV) transmission (1). Effective treatment is a cornerstone of U.S. gonorrhea control efforts, but treatment of gonorrhea has been complicated by the ability of Neisseria gonorrhoeae to develop antimicrobial resistance. This report, using data from CDC’s Gonococcal Isolate Surveillance Project (GISP), describes laboratory evidence of declining cefixime susceptibility among urethral N. gonorrhoeae isolates collected in the United States during 2006–2011 and updates CDC’s current recommendations for treatment of gonorrhea (2). Based on GISP data, CDC recommends combination therapy with ceftriaxone 250 mg intramuscularly and either azithromycin 1 g orally as a single dose or doxycycline 100 mg orally twice daily for 7 days as the most reliably effective treatment for uncomplicated gonorrhea. CDC no longer recommends cefixime at any dose as a first-line regimen for treatment of gonococcal infections. If cefixime is used as an alternative agent, then the patient should return in 1 week for a test-of-cure at the site of infection.

Infection with N. gonorrhoeae is a major cause of pelvic inflammatory disease, ectopic pregnancy, and infertility, and can facilitate HIV transmission (1). In the United States, gonorrhea is the second most commonly reported notifiable infection, with >300,000 cases reported during 2011. Gonorrhea treatment has been complicated by the ability of N. gonorrhoeae to develop resistance to antimicrobials used for treatment. During the 1990s and 2000s, fluoroquinolone resistance in N. gonorrhoeae emerged in the United States, becoming prevalent in Hawaii and California and among men who have sex with men (MSM) before spreading throughout the United States. In 2007, emergence of fluoroquinolone-resistant N. gonorrhoeae in the United States prompted CDC to no longer recommend fluoroquinolones for treatment of gonorrhea, leaving cephalosporins as the only remaining recommended antimicrobial class (3). To ensure treatment of co-occurring pathogens (e.g., Chlamydia trachomatis) and reflecting concern about emerging gonococcal resistance, CDC’s 2010 sexually transmitted diseases (STDs) treatment guidelines recommended combination therapy for gonorrhea with a cephalosporin (ceftriaxone 250 mg intramuscularly or cefixime 400 mg orally) plus either azithromycin orally or doxycycline orally, even if nucleic acid amplification testing (NAAT) for C. trachomatis was negative at the time of treatment (2). From 2006 to 2010, the minimum concentrations of cefixime needed to inhibit the growth in vitro of N. gonorrhoeae strains circulating in the United States and many other countries increased, suggesting that the effectiveness of cefixime might be waning (4). Reports from Europe recently have described patients with uncomplicated gonorrhea infection not cured by treatment with cefixime 400 mg orally (5–8).

GISP is a CDC-supported sentinel surveillance system that has monitored N. gonorrhoeae antimicrobial susceptibilities since 1986, and is the only source in the United States of national and regional N. gonorrhoeae antimicrobial susceptibility data. During September–December 2011, CDC and five external GISP principal investigators, each with N. gonorrhoeae–specific expertise in surveillance, antimicrobial resistance, treatment, and antimicrobial susceptibility testing, reviewed antimicrobial susceptibility trends in GISP through August 2011 to determine whether to update CDC’s current recommendations (2) for treatment of uncomplicated gonorrhea. Each month, the first 25 gonococcal urethral isolates collected from men attending participating STD clinics (approximately 6,000 isolates each year) were submitted for antimicrobial susceptibility testing. The minimum inhibitory concentration (MIC), the lowest antimicrobial concentration that inhibits visible bacterial growth in the laboratory, is used to assess antimicrobial susceptibility. Cefixime susceptibilities were not determined during 2007–2008 because cefixime temporarily was unavailable in the United States at that time. Criteria for resistance to cefixime and ceftriaxone have not been defined by the Clinical Laboratory Standards Institute (CLSI). However, CLSI does consider isolates with cefixime or ceftriaxone MICs ≥0.5 µg/mL to have “decreased susceptibility” to these drugs (9). During 2006–2011, 15 (0.1%) isolates had decreased susceptibility to cefixime (all had MICs = 0.5 µg/mL), including nine (0.2%) in 2010 and one (0.03%) during January–August 2011; 12 of 15 were from MSM, and 12 were from the West and three from the Midwest.* No isolates exhibited decreased susceptibility to ceftriaxone. Because increasing MICs can predict the emergence of resistance, lower cephalosporin MIC breakpoints were established by GISP for surveillance purposes to provide greater sensitivity in detecting declining gonococcal susceptibility than breakpoints defined by CLSI. Cefixime MICs ≥0.25 µg/mL and ceftriaxone MICs ≥0.125 µg/mL were defined as “elevated MICs.” CLSI does not define azithromycin resistance criteria; CDC defines decreased azithromycin susceptibility as ≥2.0 µg/mL.

Evidence and Rationale

The percentage of isolates with elevated cefixime MICs (MICs ≥0.25 µg/mL) increased from 0.1% in 2006 to 1.5% during January–August 2011 (Figure). In the West, the percentage increased from 0.2% in 2006 to 3.2% in 2011 (Table). The largest increases were observed in Honolulu, Hawaii (0% in 2006 to 17.0% in 2011); Minneapolis, Minnesota (0% to 6.9%); Portland, Oregon (0% to 6.5%); and San Diego, California (0% to 6.4%). Nationally, among MSM, isolates with elevated MICs to cefixime increased from 0.2% in 2006 to 3.8% in 2011. In 2011, a higher proportion of isolates from MSM had elevated cefixime MICs than isolates from men who have sex exclusively with women (MSW), regardless of region (Table).

The percentage of isolates exhibiting elevated ceftriaxone MICs increased slightly, from 0% in 2006 to 0.4% in 2011 (Figure). The percentage increased from <0.1% in 2006 to 0.8% in 2011 in the West, and did not increase significantly in the Midwest (0% to 0.2%) or the Northeast and South (0.1% in 2006 and 2011). Among MSM, the percentage increased from 0.0% in 2006 to 1.0% in 2011.

The 2010 CDC STD treatment guidelines (2) recommend that azithromycin or doxycycline be administered with a cephalosporin as treatment for gonorrhea. The percentage of isolates exhibiting tetracycline resistance (MIC ≥2.0 µg/mL) was high but remained stable from 2006 (20.6%) to 2011 (21.6%). The percentage exhibiting decreased susceptibility to azithromycin (MIC ≥2.0 µg/mL) remained low (0.2% in 2006 to 0.3% in 2011). Among 180 isolates collected during 2006–2011 that exhibited elevated cefixime MICs, 139 (77.2%) exhibited tetracycline resistance, but only one (0.6%) had decreased susceptibility to azithromycin.

Ceftriaxone as a single intramuscular injection of 250 mg provides high and sustained bactericidal levels in the blood and is highly efficacious at all anatomic sites of infection for treatment of N. gonorrhoeae infections caused by strains currently circulating in the United States (10,11). Clinical data to support use of doses of ceftriaxone >250 mg are not available. A 400-mg oral dose of cefixime does not provide bactericidal levels as high, nor as sustained as does an intramuscular 250-mg dose of ceftriaxone, and demonstrates limited efficacy for treatment of pharyngeal gonorrhea (10,11). The significant increase in the prevalence of U.S. GISP isolates with elevated cefixime MICs, most notably in the West and among MSM, is of particular concern because the emergence of fluoroquinolone-resistant N. gonorrhoeae in the United States during the 1990s also occurred initially in the West and predominantly among MSM before spreading throughout the United States within several years. Thus, observed patterns might indicate early stages of the development of clinically significant gonococcal resistance to cephalosporins. CDC anticipates that rising cefixime MICs soon will result in declining effectiveness of cefixime for the treatment of urogenital gonorrhea. Furthermore, as cefixime becomes less effective, continued use of cefixime might hasten the development of resistance to ceftriaxone, a safe, well-tolerated, injectable cephalosporin and the last antimicrobial that is recommended and known to be highly effective in a single dose for treatment of gonorrhea at all anatomic sites of infection. Maintaining effectiveness of ceftriaxone for as long as possible is critical. Thus, CDC no longer recommends the routine use of cefixime as a first-line regimen for treatment of gonorrhea in the United States.

Based on experience with other microbes that have developed antimicrobial resistance rapidly, a theoretical basis exists for combination therapy using two antimicrobials with different mechanisms of action to improve treatment efficacy and potentially delay emergence and spread of resistance to cephalosporins. Therefore, the use of a second antimicrobial (azithromycin as a single 1-g oral dose or doxycycline 100 mg orally twice daily for 7 days) is recommended for administration with ceftriaxone. The use of azithromycin as the second antimicrobial is preferred to doxycycline because of the convenience and compliance advantages of single-dose therapy and the substantially higher prevalence of gonococcal resistance to tetracycline than to azithromycin among GISP isolates, particularly in strains with elevated cefixime MICs.


For treatment of uncomplicated urogenital, anorectal, and pharyngeal gonorrhea, CDC recommends combination therapy with a single intramuscular dose of ceftriaxone 250 mg plus either a single dose of azithromycin 1 g orally or doxycycline 100 mg orally twice daily for 7 days (Box).

Clinicians who diagnose gonorrhea in a patient with persistent infection after treatment (treatment failure) with the recommended combination therapy regimen should culture relevant clinical specimens and perform antimicrobial susceptibility testing of N. gonorrhoeae isolates. Phenotypic antimicrobial susceptibility testing should be performed using disk diffusion, Etest (BioMérieux, Durham, NC), or agar dilution. Data currently are limited on the use of NAAT-based antimicrobial susceptibility testing for genetic mutations associated with resistance in N. gonorrhoeae. The laboratory should retain the isolate for possible further testing. The treating clinician should consult an infectious disease specialist, an STD/HIV Prevention Training Center (http://www.nnptc.orgExternal Web Site Icon), or CDC (telephone: 404-639-8659) for treatment advice, and report the case to CDC through the local or state health department within 24 hours of diagnosis. A test-of-cure should be conducted 1 week after re-treatment, and clinicians should ensure that the patient’s sex partners from the preceding 60 days are evaluated promptly with culture and treated as indicated.

When ceftriaxone cannot be used for treatment of urogenital or rectal gonorrhea, two alternative options are available: cefixime 400 mg orally plus either azithromycin 1 g orally or doxycycline 100 mg twice daily orally for 7 days if ceftriaxone is not readily available, or azithromycin 2 g orally in a single dose if ceftriaxone cannot be given because of severe allergy. If a patient with gonorrhea is treated with an alternative regimen, the patient should return 1 week after treatment for a test-of-cure at the infected anatomic site. The test-of-cure ideally should be performed with culture or with a NAAT for N. gonorrhoeae if culture is not readily available. If the NAAT is positive, every effort should be made to perform a confirmatory culture. All positive cultures for test-of-cure should undergo phenotypic antimicrobial susceptibility testing. Patients who experience treatment failure after treatment with alternative regimens should be treated with ceftriaxone 250 mg as a single intramuscular dose and azithromycin 2 g orally as a single dose and should receive infectious disease consultation. The case should be reported to CDC through the local or state health department.

For all patients with gonorrhea, every effort should be made to ensure that the patients’ sex partners from the preceding 60 days are evaluated and treated for N. gonorrhoeae with a recommended regimen. If a heterosexual partner of a patient cannot be linked to evaluation and treatment in a timely fashion, then expedited partner therapy should be considered, using oral combination antimicrobial therapy for gonorrhea (cefixime 400 mg and azithromycin 1 g) delivered to the partner by the patient, a disease investigation specialist, or through a collaborating pharmacy.

The capacity of laboratories in the United States to isolate N. gonorrhoeae by culture is declining rapidly because of the widespread use of NAATs for gonorrhea diagnosis, yet it is essential that culture capacity for N. gonorrhoeae be maintained to monitor antimicrobial resistance trends and determine susceptibility to guide treatment following treatment failure. To help control gonorrhea in the United States, health-care providers must maintain the ability to collect specimens for culture and be knowledgeable of laboratories to which they can send specimens for culture. Health-care systems and health departments must support access to culture, and laboratories must maintain culture capacity or develop partnerships with laboratories that can perform culture.

Treatment of patients with gonorrhea with the most effective therapy will limit the transmission of gonorrhea, prevent complications, and likely will slow emergence of resistance. However, resistance to cephalosporins, including ceftriaxone, is expected to emerge. Reinvestment in gonorrhea prevention and control is warranted. New treatment options for gonorrhea are urgently needed.

Reported by

Carlos del Rio, MD, Rollins School of Public Health, Emory Univ, Atlanta, Georgia. Geraldine Hall, PhD, Dept of Clinical Pathology, Cleveland Clinic, Cleveland, Ohio. King Holmes, MD, Olusegun Soge, PhD, Dept of Medicine, Univ of Washington. Edward W. Hook, MD, Div of Infectious Diseases, Univ of Alabama at Birmingham. Robert D. Kirkcaldy, MD, Kimberly A. Workowski, MD, Sarah Kidd, MD, Hillard S. Weinstock, MD, John R. Papp, PhD, David Trees, PhD, Thomas A. Peterman, MD, Gail Bolan, MD, Div of Sexually Transmitted Diseases Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, CDC.Corresponding contributor: Robert D. Kirkcaldy,, 404-639-8659.


Collaborating state and local health departments. Baderinwa Offut, Emory Univ, Atlanta, Georgia. Laura Doyle, Cleveland Clinic, Ohio. Connie Lenderman, Paula Dixon, Univ of Alabama at Birmingham. Karen Winterscheid, Univ of Washington, Seattle. Tamara Baldwin, Elizabeth Delamater, Texas Dept of State Health Svcs. Alesia Harvey, Tremeka Sanders, Samera Bowers, Kevin Pettus, Div of STD Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, CDC.


  1. Fleming D, Wasserheit J. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect 1999;75:3–17.
  2. CDC. Sexually transmitted diseases treatment guidelines, 2010. MMWR 2010;59(No. RR-12).
  3. CDC. Update to CDC’s sexually transmitted diseases treatment guidelines, 2006: fluoroquinolones no longer recommended for treatment of gonococcal infections. MMWR 2007;56:332–6.
  4. CDC. Cephalosporin susceptibility among Neisseria gonorrhoeae isolates—United States, 2000–2010. MMWR 2011;60:873–7.
  5. Unemo M, Golparian D, Syversen G, Vestrheim DF, Moi H. Two cases of verified clinical failures using internationally recommended first-line cefixime for gonorrhea treatment, Norway, 2010. Euro Surveill 2010;15(47):pii:19721.
  6. Ison C, Hussey J, Sankar K, Evans J, Alexander S. Gonorrhea treatment failures to cefixime and azithromycin in England, 2010. Euro Surveill 2011;16(14):pii:19833.
  7. Unemo M, Golparian D, Stary A, Eigentler A. First Neisseria gonorrhoeae strain with resistance to cefixime causing gonorrhea treatment failure in Austria, 2011. Euro Surveill 2011;16(43):pi:19998.
  8. Unemo M, Golparian D, Nicholas R, Ohnishi M, Gallay A, Sednaoui P. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 2012;56:1273–80.
  9. National Committee for Clinical Laboratory Standards. Approved Standard M100-S20 performance standards for antimicrobial susceptibility testing; twentieth informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.
  10. Moran JS, Levine WC. Drugs of choice for the treatment of uncomplicated gonococcal infections. Clin Infect Dis 1995;20(Suppl 1):S47–65.
  11. Handsfield HH, McCormack WM, Hook EW 3rd, et al. A comparison of single-dose cefixime with ceftriaxone as treatment for uncomplicated gonorrhea. The Gonorrhea Treatment Study Group. New Engl J Med 1991;325:1337–41.

* U.S. Census regions. Northeast: Connecticut, Maine, Massachusetts, New Jersey, New Hampshire, New York, Pennsylvania, Rhode Island, and Vermont; Midwest: Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio, South Dakota, and Wisconsin; South:Alabama, Arkansas, Delaware, District of Columbia, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee, Texas, Virginia, and West Virginia; West: Alaska, Arizona, California, Colorado, Hawaii, Idaho, Montana, New Mexico, Nevada, Oregon, Utah, Washington, and Wyoming.

TABLE. Percentage of urethral Neisseria gonorrhoeae isolates with elevated cefixime MICs (≥0.25 µg/mL), by U.S. Census region and gender of sex partner — Gonococcal Isolate Surveillance Project, United States, 2006–August 2011
Region 2006 2009 2010 2011*
% (95% CI) % (95% CI) % (95% CI) % (95% CI)
West† (total) 0.2 (0.1–0.4) 1.9 (1.4–2.6) 3.3 (2.6–4.0) 3.2 (2.3–4.2)
MSM 0.1 (0.0–0.6) 2.6 (1.7–3.8) 5.0 (3.8–6.5) 4.5 (3.1–6.3)
MSW 0.2 (0.0–0.6) 1.4 (0.7–2.3) 1.3 (0.7–2.2) 1.8 (0.9–3.1)
Midwest§ (total) 0.0 (0.0–0.3) 0.5 (0.2–1.0) 0.5 (0.2–1.1) 0.6 (0.2–1.5)
MSM 0.0 (0.0–2.8) 2.3 (0.6–5.7) 3.4 (1.1–7.7) 4.9 (1.4–12.2)
MSW 0.0 (0.0–0.3) 0.3 (0.1–0.7) 0.1 (0.0–0.6) 0.0 (0.0–0.6)
Northeast and South¶ (total) 0.1 (0.0–0.3) 0.0 (0.0–0.2) 0.1 (0.0–0.4) 0.3 (0.1–0.8)
MSM 0.6 (0.0–3.0) 0.3 (0.0–1.9) 0.9 (0.2–2.5) 1.5 (0.4–3.9)
MSW 0.0 (0.0–0.2) 0.0 (0.0–0.2) 0.0 (0.0–0.2) 0.1 (0.0–0.4)
Abbreviations: CI = confidence interval; MICs = minimum inhibitory concentrations; MSM = men who have sex with men; MSW = men who have sex exclusively with women.

* January–August 2011.

† Includes data from Albuquerque, New Mexico; Denver, Colorado; Honolulu, Hawaii; Las Vegas, Nevada; Los Angeles, California; Orange County, California; Phoenix, Arizona; Portland, Oregon; San Diego, California; San Francisco, California; and Seattle, Washington.

§ Includes data from Chicago, Illinois; Cincinnati, Ohio; Cleveland, Ohio; Detroit, Michigan; Kansas City, Missouri; and Minneapolis, Minnesota.

¶ Includes data from Atlanta, Georgia; Baltimore, Maryland; Birmingham, Alabama; Dallas, Texas; Greensboro, North Carolina; Miami, Florida; New Orleans, Louisiana; New York, New York; Oklahoma City, Oklahoma; Philadelphia, Pennsylvania; and Richmond, Virginia.

FIGURE. Percentage of urethral Neisseria gonorrhoeae isolates (n = 32,794) with elevated cefixime MICs (≥0.25 µg/mL) and ceftriaxone MICs (≥0.125 µg/mL) — Gonococcal Isolate Surveillance Project, United States, 2006–August 2011

The figure shows the percentage of Neisseria gonorrhoeae isolates (n = 32,794) with elevated cefixime MICs (≥0.25 μg/mL) and ceftriaxone MICs (≥0.125 μg/mL) in the United States during 2006-August 2011, according to the Gonococcal Isolate Surveillance Project. The percentage of isolates with elevated cefixime MICs (MICs ≥0.25 μg/mL) increased from 0.1% in 2006 to 1.5% during January-August 2011.

Abbreviation: MICs = minimum inhibitory concentrations.

* Cefixime susceptibility not tested during 2007–2008.

† January–August 2011.

Alternate Text: The figure above shows the percentage of Neisseria gonorrhoeae isolates (n = 32,794) with elevated cefixime MICs (≥0.25 μg/mL) and ceftriaxone MICs (≥0.125 μg/mL) in the United States during 2006-August 2011, according to the Gonococcal Isolate Surveillance Project. The percentage of isolates with elevated cefixime MICs (MICs ≥0.25 μg/mL) increased from 0.1% in 2006 to 1.5% during January-August 2011.

BOX. Updated recommended treatment regimens for gonococcal infections
Uncomplicated gonococcal infections of the cervix, urethra, and rectum

Recommended regimen

Ceftriaxone 250 mg in a single intramuscular dose


Azithromycin 1 g orally in a single dose

or doxycycline 100 mg orally twice daily for 7 days*


Alternative regimens

If ceftriaxone is not available:

Cefixime 400 mg in a single oral dose


Azithromycin 1 g orally in a single dose

or doxycycline 100 mg orally twice daily for 7 days*


Test-of-cure in 1 week


If the patient has severe cephalosporin allergy:

Azithromycin 2 g in a single oral dose


Test-of-cure in 1 week


Uncomplicated gonococcal infections of the pharynx

Recommended regimen

Ceftriaxone 250 mg in a single intramuscular dose


Azithromycin 1 g orally in a single dose

or doxycycline 100 mg orally twice daily for 7 days*


* Because of the high prevalence of tetracycline resistance among Gonococcal Isolate Surveillance Project isolates, particularly those with elevated







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