Colistin Minimum Inhibitory Concentration Research Articles

Epidemiology and resistance phenotypes of carbapenemase-producing Klebsiella pneumoniae in Greece, 2014 to 2016

Background and aimA multicentre nationwide surveillance study was conducted in Greek hospitals to evaluate epidemiology of carbapenemase-producing Klebsiella pneumoniae clinical isolates, and their susceptibilities to last-line antibiotics. Methods: Minimum inhibitory concentrations (MICs) were evaluated by Etest, colistin MICs were also evaluated by broth microdilution SensiTest (now known as ComASP) Colistin. Carbapenemase genes were detected by PCR. Clonal relatedness was assessed by PFGE. Isolates were prospectively collected between November 2014 and April 2016, from 15 hospitals. Results: Among 394 isolates, K. pneumoniae carbepenemase (KPC) remained the most prevalent carbapenemase (66.5%). NDM was the second most prevalent (13.7%), identified in 12 hospitals, followed by VIM (8.6%). OXA-48- and double carbapenemase-producers remained rare (3.6%, 6.3%, respectively). Carbapenemase-producing K. pneumoniae isolates showed high resistance to last-line antibiotics. Gentamicin and colistin were the most active in vitro with 61.9% and 59.6% of the isolates to be inhibited at ≤ 2mg/L, followed by fosfomycin (susceptibility (S): 58.4%) and tigecycline (S: 51.5%). Ceftazidime/avibactam inhibited 99.6% of KPC and 100% of OXA-48-like-producing isolates, while temocillin was active against 58% of KPC isolates at urinary breakpoint of ≤ 32mg/L* and only 2.7% at systemic breakpoint of ≤ 8mg/L. NDM-producing isolates belonged mainly to one clone, whereas KPC, VIM, OXA-48 and double carbapenemase-producers were mainly polyclonal. Conclusions: KPC remains the predominant carbapenemase among K. pneumoniae in Greece, followed by NDM, whereas changing trends of resistance rates to last-line antimicrobials against carbapenemase-producing K. pneumoniae with the exception of ceftazidime/avibactam mandates continuing surveillance to support clinical practice.

Detection of mcr-1 Plasmids in Enterobacteriaceae Isolates From Human Specimens: Comparison With Those in Escherichia coli Isolates From Livestock in Korea.

BackgroundThe emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock.MethodsAmong 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced.ResultsTwo E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid.Conclusionsmcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.

Diverse genetic alterations responsible for post-exposure colistin resistance in populations of the same strain of Klebsiella pneumoniae

The intra- and interstrain diversity of genetic alterations in Klebsiella pneumoniae after exposure to colistin was investigated. Four colistin-susceptible K. pneumoniae strains with different sequence types were exposed to 2 mg/L colistin, the average steady-state plasma concentration. After exposure, 10 single-step colistin-resistant mutants were selected in vitro from each K. pneumoniae strain. Minimum inhibitory concentrations (MICs) were evaluated by broth microdilution. Nucleotide sequences of genes associated with colistin resistance (phoPQ, pmrAB, mgrB and crrAB) were determined, and the mRNA expression levels of pbgP, phoP, phoQ, pmrD, pmrA and pmrB were measured by quantitative real-time polymerase chain reaction. Colistin MICs for single-step mutants ranged from 4 to >64 mg/L. Diverse amino acid alterations in pmrB, phoP, phoQ and mgrB, including insertion sequences, were identified in the colistin-resistant mutants. Different mutations were found even among mutants originating from the same parental strain, and several mutants displayed no mutations in the tested genes. While most colistin-resistant mutants showed elevated expression of pbgP, seven showed no increase compared with their susceptible parental strain, and some mutants exhibited no overexpression of pmrAB, phoPQ or pbgP. These results demonstrate the diversity of genetic variations in colistin-resistant populations derived from the same parental strain, and suggest that there are still unknown mechanisms of colistin resistance in K. pneumoniae.

Prevalence of the colistin resistance gene mcr-1 in colistin-resistant Escherichia coli and Klebsiella pneumoniae isolated from humans in Thailand.

Historically, colistin has been considered a last-line therapeutic option against multidrug-resistant Gram-negative bacterial infections. However, chromosomally-encoded and plasmid-mediated colistin resistance is increasingly being reported worldwide. Spread of the plasmid-borne colistin resistance gene mcr-1 is of great concern since it can be transferred between bacteria. The aim of this study was to investigate the prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae collected from human clinical specimens in Thailand during 2014-2017. Minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution method for 317 non-duplicate Enterobacteriaceae clinical isolates (37 E. coli and 280 K. pneumoniae). All isolates were screened for the mcr-1 gene by PCR. The colistin MIC50, MIC90 and MIC range for the 37 E. coli isolates were 0.5, 8 and 0.5-32mg/L, respectively. The mcr-1 gene was detected in 11 E. coli isolates (29.7%). Escherichia coli harbouring the mcr-1 gene had a colistin MIC range of 4-32mg/L. The colistin MIC50, MIC90, and MIC range for the 280 K. pneumoniae isolates were 32, >128, and 0.25 to >128mg/L, respectively. The mcr-1 gene was detected in 4 K. pneumoniae isolates (1.4%). Klebsiella pneumoniae harbouring the mcr-1 gene had a colistin MIC range of 4-64mg/L. This is the first report on the prevalence of the mcr-1 gene in colistin-resistant E. coli and K. pneumoniae isolated from humans in Thailand. These data provide added insight into the mechanism of colistin resistance among Enterobacteriaceae pathogens.

New Class of Adjuvants Enables Lower Dosing of Colistin Against Acinetobacter baumannii

Antibiotic resistance has become increasingly prevalent over the past few decades, and this combined with a dearth in the development of new classes of antibiotics to treat multidrug resistant Gram-negative infections has led to a significant global health problem and the increased usage of colistin as the last resort antibiotic. Colistin, however, presents dose dependent toxicity in the clinic. One potential approach to combatting this problem is the use of an antibiotic adjuvant, a compound that is nontoxic to the bacteria that enhances the potency of colistin and ultimately allows for reducing dosing. Herein, we present a new urea-containing class of 2-aminoimidazole-based adjuvants that potentiates colistin activity against colistin-sensitive Acinetobacter baumannii. Lead compounds enabled 1000-fold reduction in the minimum inhibitory concentration of colistin in vitro and showed efficacy in a Galleria mellonella infection model, representing the first step toward validating the potential of employing these adjuvants to lower colistin dosage.

The effect of exposure to sub-inhibitory concentrations of hypochlorite and quaternary ammonium compounds on antimicrobial susceptibility of Pseudomonas aeruginosa

Pseudomonas is a group of medically important species that inhabit a wide range of niches, including hospital environments. Controversies have emerged about the possible link between improper use of disinfectants and the emergence of antibiotic resistance in bacteria. The aim of this study was to assess the effect of exposure of antibiotic-susceptible Pseudomonas isolates to sub-inhibitory concentrations of 2 disinfectants-didecyldimonium chloride and sodium hypochlorite-on their antibiotic susceptibility patterns. This study involved 50 Pseudomonas isolates. The antibiotic susceptibility patterns of the isolates were assessed using broth microdilution method. The minimal inhibitory concentrations (MICs) of each antibiotic were compared before and after exposure to sub-inhibitory concentrations of didecyldimonium chloride and sodium hypochlorite. After overnight incubation with sub-inhibitory concentrations of sodium hypochlorite, a statistically significant increase was observed in the MICs of colistin (P = .012), ceftazidime (P < .001), amikacin (P < .001), meropenem (P < .001), gentamicin (P < .001), piperacillin-tazobactam (P = .003), and ciprofloxacin (P = .004). In contrast, exposure to sub-inhibitory concentrations of didecyldimonium chloride showed a statistically significant increase in the MICs of amikacin (P < .001), gentamicin (P < .001), meropenem (P = .041), and ciprofloxacin (P = .008). The use of suboptimal concentrations of sodium hypochlorite and didecyldimonium chloride can lead to the evolution of antibiotic-resistant Pseudomonas strains.

Mcr-1 Gene Has No Effect on Colistin Resistance When It Coexists with Inactivated mgrB Gene in Klebsiella pneumoniae.

The inactivated mgrB gene and the mcr-1 gene are important mechanisms of colistin resistance in Klebsiella pneumoniae and they are threats to the clinical use of colistin. In this study, mcr-1 gene was cloned into K. pneumoniae strains (XH209 and KP10) and their derived strains (XH209 M and KP10 M), which showed high-level resistance to colistin. The acquisition of the mcr-1 gene led to colistin resistance in XH209 and KP10, but the addition of mcr-1 gene did not cause change of colistin minimum inhibitory concentrations in the XH209 M and KP10 M. In addition, the impact of mcr-1 gene on growth rate showed strain specific in K. pneumoniae. In conclusion, the mcr-1 gene does not cause the same level of colistin resistance as the inactivated mgrB gene in K. pneumoniae. The mcr-1 gene has no effect on colisitin resistance when it coexists with inactivated mgrB gene in K. pneumoniae.

Alkaline Peptone Water-Based Enrichment Method for mcr-3 From Acute Diarrheic Outpatient Gut Samples.

A third plasmid-mediated colistin resistance gene, mcr-3, is increasingly being reported in Enterobacteriaceae and Aeromonas spp. from animals and humans. To investigate the molecular epidemiology of mcr in the gut flora of Chinese outpatients, 152 stool specimens were randomly collected from outpatients in our hospital from May to June, 2017. Stool specimens enriched in alkaline peptone water or Luria-Bertani (LB) broth were screened for mcr-1, mcr-2, and mcr-3 using polymerase chain reaction (PCR)-based assays. Overall, 19.1% (29/152) and 5.3% (8/152) of the stool samples enriched in alkaline peptone water were PCR-positive for mcr-1 and mcr-3, respectively, while 2.7% (4/152) of samples were positive for both mcr-1 and mcr-3. Strains isolated from the samples that were both mcr-1- and mcr-3-positive were subjected to antimicrobial susceptibility testing by broth microdilution. They were also screened for the presence of other resistance genes by PCR, while multilocus sequence typing and whole-genome sequencing were used to investigate the molecular epidemiology and genetic environment, respectively, of the resistance genes. mcr-3-positive Aeromonas veronii strain 126-14, containing a mcr-3.8-mcr-3-like2 segment, and mcr-1-positive Escherichia coli strain 126-1, belonging to sequence type 1485, were isolated from the sample from a diarrheic butcher with no history of colistin treatment. A. veronii 126-14 had a colistin minimum inhibitory concentration (MIC) of 2 µg/mL and was susceptible to antibiotics in common use, while E. coli 126-1 produced TEM-1, CTX-M-55, and CTX-M-14 β-lactamases and was resistant to colistin, ceftazidime, and cefotaxime. Overall, there was a higher detection rate of mcr-3-carrying strains with low colistin MICs from the samples enriched in alkaline peptone water than from samples grown in LB broth.

Colistin versus colistin plus meropenem for severe infections Authors' reply

We appreciate Daniele Giacobbe’s Correspondence regarding the importance of colistin minimum inhibitory concentrations. A study showed that both gradient-based and semi-automated devices can overestimate susceptibility to colistin. [...]

Prospective study on human fecal carriage of Enterobacteriaceae possessing mcr-1 and mcr-2 genes in a regional hospital in Hong Kong

BackgroundHuman fecal carriage of Enterobacteriaceae possessing mobilized colistin resistance genes (mcr-1 and mcr-2) remains obscure in Hong Kong. As part of routine surveillance on emerging antibiotic resistance, we conducted a prospective study on this topic in a regional hospital in Hong Kong.MethodsFrom October 31 to November 25, 2016, all fecal specimens submitted for routine analysis were included in this surveillance study. These comprised 672 consecutive routine fecal specimens collected from 616 individuals. Fecal specimens were screened for colistin-resistant Enterobacteriaceae by culture-based method, and the presence of mcr-1 and mcr-2 genes in resistant isolates was identified by polymerase chain reaction and Sanger sequencing. Whole genome sequencing (WGS) of mcr-1-possessing Escherichia coli strains was facilitated using Illumina® MiSeq® followed by sequence analysis with appropriate bioinformatics tools.ResultsFourteen mcr-1-positive E. coli strains were isolated from 14 separate individuals (2.08% of total fecal specimens), with 9 of them being asymptomatic, healthy clients coming for health assessment. No mcr-2-possessing Enterobacteriaceae was identified. Colistin minimum inhibitory concentrations of these mcr-1-positive isolates ranged from 2 to 4 μg/mL. All these isolates were susceptible to carbapenems with 2 being extended spectrum β-lactamase producers. WGS data revealed that these isolates belonged to at least 12 different sequence types (STs) and possessed diversified plasmid replicons, virulence and acquired antibiotic resistance genes. Further study on an E. coli ST201 strain (Pasteur scheme) revealed coexistence of 47,818-bp IncP-1 and 33,309-bp IncX4 types of mcr-1 plasmids, which was a combination of stability and high transmissibility.ConclusionsTo the best of our knowledge, this is the first study on human fecal carriage of Enterobacteriaceae possessing mcr-1 and mcr-2 genes in Hong Kong. Our data further revealed asymptomatic carriage of mcr-1-possessing Enterobacteriaceae by both patients and healthy individuals. This is alarming considering wide diversity and high transmissibility of mcr-1 plasmids, which potentially facilitate emergence of pan-drug-resistant bacteria in future infection. This also highlights the importance of surveillance on emerging antibiotic resistance, especially for patients under intensive care.

Co-occurrence of mcr-1 in the chromosome and on an IncHI2 plasmid: persistence of colistin resistance in Escherichia coli

Two colistin-resistant Escherichia coli strains (FS13Z2S and FS3Z6C) possessing chromosomally encoded mcr-1 isolated from swine were characterised. Whole-genome sequencing revealed that in strain FS13Z2S mcr-1 occurred in triplicate in the chromosome with another copy encoded on a pHNSHP45-2-like IncHI2 plasmid, whereas in strain FS3Z6C only one copy mcr-1 was inserted in the chromosome. It seems likely that the triplication of chromosomal copies of mcr-1 in FS13Z2S is due to intramolecular transposition events via a composite transposon containing an mcr-1 cassette bracketed by two copies of insertion sequence ISApl1, and the pap2 gene at the insertion site was truncated by an IS1294-like element. In plasmid pFS13Z2S and the chromosome of strain FS3Z6C, only a single copy of ISApl1 was present upstream of the mcr-1 cassette. The two strains exhibited similar colistin minimum inhibitory concentrations (MICs) and featured phosphoethanolamine addition to lipid A, without regard to the copy number of mcr-1. The mcr-1-harbouring plasmid was unstable in wild-type strain FS13Z2S and was quickly lost after 7 days of passage on colistin-free Luria–Bertani broth containing 0.5% SDS, but the mcr-1 copies on the chromosome persisted. These results reveal that the single copy of mcr-1 could result in modification of lipopolysaccharide (LPS) and cause colistin resistance in E. coli. Acquisition of multiple copies of mcr-1, especially on the chromosome, would facilitate stable persistence of colistin resistance in the host strain.

322 Correlation Between Etest and Broth Microdilution for Colistin Antimicrobial Susceptibility Testing of Enterobacteriaceae

Colistin is increasingly being used to treat serious infections caused by multidrug-resistant Gram-negative bacteria. With the recent emergence of plasmid-mediated colistin resistance (mcr genes), screening for isolates with phenotypic colistin resistance has become clinically and epidemiologically relevant. However, antimicrobial susceptibility testing (AST) of colistin is associated with several technical challenges. Disk diffusion does not work because of poor diffusion of the large colistin molecule, Etest has been reported to underestimate minimum inhibitory concentration (MIC) values, and most clinical microbiology laboratories lack the capacity to perform broth microdilution (BMD). In this study, we compared the Etest method with the reference BMD method described by the Clinical and Laboratory Standards Institute (CLSI) for determining colistin MICs among Enterobacteriaceae. A total of 114 isolates of Enterobacteriaceae (51 carbapenem-resistant [CRE] and 63 carbapenem-susceptible [CSE]) were selected for colistin AST. One CRE isolate and 13 CSE isolates harbored a mcr gene. BMD testing was performed according to CLSI guidelines using a frozen reference panel with colistin concentrations ranging from 0.25 to 8 µg/mL. The Etest (BioMérieux, Durham, NC) method was performed on Mueller-Hinton agar (BBL, Becton Dickinson, Sparks, MD) according to the manufacturer’s instructions. Interpretive criteria based on CLSI epidemiological cutoff values (non-wild-type ≥4 µg/mL; wild-type ≤2 µg/mL) were used to define resistance and susceptibility to colistin, respectively. By BMD, colistin MICs ranged from ≤0.25 to >8 µg/mL and 27% were determined to be colistin-resistant. The categorical agreement between Etest and BMD was 92%. All 14 mcr-positive isolates were categorized as colistin-resistant by BMD, while Etest classified 13/14 as resistant. In conclusion, clinical microbiology laboratories lacking capacity to perform BMD could implement the Etest method with BBL Mueller-Hinton agar for epidemiological purposes to help identify candidate isolates for mcr screening. To determine whether colistin can be used clinically, BMD would still be required.

Isolation of an IncP-1 plasmid harbouring mcr-1 from a chicken isolate of Citrobacter braakii in China

The plasmid-mediated colistin resistance gene mcr-1 has been found worldwide, but the diversity of organisms harbouring this gene is unknown. In this study, 12 colistin-resistant Citrobacter spp. isolates were obtained from diseased or dead chickens in China, and PCR analysis indicated that five were positive for mcr-1. One Citrobacter braakii strain (SCC4) with a multidrug-resistant phenotype was chosen for further analysis. SCC4 was resistant or intermediate-resistant to ten of the tested antibiotics, and the colistin minimum inhibitory concentration (MIC) was >4 µg/mL. A conjugation assay demonstrated successful transfer of colistin resistance to Escherichia coli strain J53 at a frequency of 10–7 cells per recipient cell. Whole-genome sequencing revealed that SCC4 contained 13 antibiotic resistance genes in its genome, and the mcr-1 gene resided on a 44-kb self-transmissible IncP-type plasmid of a recently discovered IncP-1 clade. In addition, the mcr-1 gene was part of an insertion element (ISApl1–mcr-1–orf–ISApl1) that was excised from the plasmid as a circular intermediate form. This is the first report of mcr-1-posiitve C. braakii of animal origin and these findings highlight the fact that the mcr-1 gene can be found in normal enteric flora as part of broad-host-range plasmids.

Use of succinic & oxalic acid in reducing the dosage of colistin against New Delhi metallo-β-lactamase-1 bacteria.

Background & objectives:New Delhi metallo-β-lactamase 1 (NDM-1) cleaves the beta-lactam ring, and confers bacterial resistance against most of the beta-lactam antibiotics, except tigecycline and colistin. Among these two antibiotics, colistin is considered toxic, and therefore, its clinical use and dosage need cautious approach. In the present study, six organic acids were screened individually and in combination of two acids for their effectiveness against NDM-1 Escherichia coli and a combination of colistin and oxalic or succinic acid was tested to find out the potential of combination therapy for reducing the dose of toxic colistin.Methods:Antibacterial activity of the organic acid and their combinations was tested by disc diffusion method against NDM-1 E. coli, and minimum inhibitory concentration (MIC) was determined by broth dilution method. Synergistic effect between organic acids and colistin was tested by checkerboard method.Results:Oxalic acid showed the highest zone of inhibition (15±1 mm) followed by succinic acid, tartaric acid, fumaric acid, citric acid and malic acid. The combination of two acids did not increase the zone of inhibition significantly. MIC was found to be the lowest with oxalic acid and succinic acid (320 μg/ml). In the presence of 160 μg/ml oxalic acid or succinic acid, MIC of colistin was reduced from 8 to 4 μg/ml, indicating synergistic effect.Interpretation & conclusions:Our findings showed that combination therapy using colistin and oxalic acid or succinic acid might find safe clinical application of this antibiotic in controlling infections due to NDM-1 bacteria.

Study of mcr-1 Gene-Mediated Colistin Resistance in Enterobacteriaceae Isolated from Humans and Animals in Different Countries

In this study, we aim to characterize the genetic environment of the plasmid-mediated colistin resistance gene mcr-1 in 25 Escherichia coli and seven Klebsiella pneumoniae strains from different countries and continents. Multilocus sequence typing, conjugation experiments, plasmid typing, and the presence and location of the insertion sequence ISApl1 were investigated. Whole genome sequencing of four E. coli was performed to analyse the genetic environment of the mcr-1 gene. Colistin minimum inhibitory concentration of mcr-1 strains varied from 3 to 32 µg/mL. Six E. coli sequence types were detected: ST 4015, ST 3997, ST 10, ST 93, ST 48, and ST 648. IncHI2, IncI2, and IncP plasmid types were predominant and were unrelated to a specific country of origin. ISApl1 was found in 69% of analysed plasmids that were mainly around the mcr-1 gene. Analysis of four closed mcr-1 plasmids revealed the integration of mcr-1 into hotspots. We found that the spread of mcr-1 gene was due to the diffusion of a composite transposon and not to the diffusion of a specific plasmid or a specific bacterial clone. The ease with which the mcr-1 gene integrates into various regions facilitates its dissemination among bacteria and explains its large diffusion all over the world, both in animals and in humans.

In vitro activity of colistin mono- and combination therapy against colistin-resistant Acinetobacter baumannii, mechanism of resistance, and clinical outcomes of patients infected with colistin-resistant A. baumannii at a Thai university hospital.

PurposeColistin is a drug of last resort for treating multidrug-resistant Acinetobacter baumannii infections. Unfortunately, colistin-resistant A. baumannii (CoR-AB) has been reported. Here, we examined the in vitro effect of mono- and combined antimicrobials against CoR-AB strains and their resistance mechanism, and evaluated the clinical outcomes of CoR-AB-infected patients.Patients and methodsSeventeen clinical CoR-AB strains were isolated from patients at Phramongkutklao hospital, 2011–2015. The mono- and synergistic activities of colistin, tigecycline, sulbactam, imipenem, meropenem, amikacin, fosfomycin, and cotrimoxazole were examined by minimum inhibitory concentration (MIC) and fractional inhibitory concentration index. Clonal relationship and resistance genes were determined by repetitive extragenic palindromic polymerase chain reaction with specific primers. The effect of carbonyl cyanide 3-chlorophenylhydrazone combined with colistin was used to test efflux pump involvement. Patient treatment outcomes were also reported.ResultsThe most prevalent infection in CoR-AB patients was pneumonia (35.3%), and all patients were administered colistin combined with another agent. The 30-day mortality was 70.6%, and the colistin MIC range and MIC50 was 16–512 μg/mL and 64 μg/mL, respectively. All CoR-AB strains were sensitive to tigecycline. Sporadic isolates were susceptible to sulbactam, imipenem, meropenem, and cotrimoxazole. A synergistic or additive effect was observed for colistin plus imipenem or meropenem (16.7%), sulbactam (66.7%), or tigecycline (66.7%). The CoR-AB isolates could be divided into four different clones (A–D) with a high prevalence of group B (47.1%). Eight isolates harbored blaOXA23, blaIMP, blaKPC, and blaNDM, and one contained blaOXA23, blaIMP, and blaKPC, while the eight remaining isolates carried only blaOXA23. The MIC values of all strains were greatly reduced for colistin plus carbonyl cyanide 3-chlorophenylhydrazone.ConclusionCoR-AB clinical isolates exhibited very high colistin resistance and a high frequency of resistance genes. The mechanism of colistin resistance appears to be mediated via an efflux pump. Thus, certain antimicrobials could be used as salvage therapy for CoR-AB infection.

The Evaluation of Four In Vitro Susceptibility Testing Methods for Colistin on Carbapenenm-Resistant Acinetobacter baumannii

Background: The carbapenem resistance in Acinetobacter baumannii (CRAB) is a global health problem because of the worldwide distribution of the bacteria and a few available therapeutic options. Colistin is considered as the last resort to treat the infection. At present, there are several methods to detect the colistin susceptibility, including broth microdilution with 0.002% polysorbate 80 (BMD-P80), the E-test, broth microdilution (BMD), and agar dilution (AD). However, the differences in efficacy between the methods are not well studied. Objectives: The current study aimed at evaluating the 4 available methods to test in vitro susceptibility to colistin and observing the degree of heteroresistance in CRAB species in China. Methods: To evaluate the methods, a total of 202 CRAB species isolated from 12 hospitals in Zhejiang province, China, collected from January to December in 2010 were employed retrospectively. Colistin minimum inhibitory concentrations (MICs) were determined by the 4 different methods. Population analysis profiles (PAPs) were also conducted in 29 CRAB strains. Results: The proportions of colistin-sensitive isolates were 100%, 100%, 99.5%, and 90.6% by BMD-P80, E-test, BMD, and AD methods, respectively, according to the EUCAST breakpoints. The AD methods produced an excessive number of major errors (MEs) (9.4%), while E-test and BMD produced 0.5% MEs. Moreover, the AD method resulted in a minimum essential agreement (EA) at 9.4%, and 179 isolates obtained a higher (≥ 3 log 2) number of dilutions than the BMD-P80. Not very major errors (VMEs) were found by any of the tested methods. In 29 selected CRAB isolates, a total of 31% were heterogeneous and the rate of heteroresistance was also 31%. Conclusions: The AD method was not a prior choice of in vitro colistin susceptibility testing because it led to false colistin resistance results. E-test, BMD, and BMD-P80 may be more reliable to test the susceptibility when colistin is considered as a potential therapeutic agent. A high rate of heterogeneous and heteroresistant species were found in CRAB in China; it highlighted the importance of MICs monitoring before and through the colistin therapy period.

COLISTIN RESISTANCE IN CARBAPENEM-RESISTANT KLEBSIELLA PNEUMONIAE STRAINS

Objective: There is an increasing use of colistin consequent to increase in the infections caused by carbapenem-resistant Klebsiella pneumoniae.The present study was conducted to determine the minimum inhibitory concentration (MIC) of colistin and the resistance pattern of colistin in carbapenem-resistant K. pneumoniae (CRKP) strains in our intensive care unit (ICU).Methods: Antibiotic susceptibility testing for other antimicrobial agents was done by Kirby-Bauer disk diffusion method. MIC of colistin was determined by agar dilution method. The results of antibiotic susceptibility testing were interpreted as per Clinical Laboratory Standard Institute guidelines 2016 and MIC of colistin were interpreted as per European Committee on Antimicrobial susceptibility testing. The carbapenem resistance was phenotypically detected by modified hodge test and imipenem/imipenem ethylenediaminetetraacetic acid disk method.Results: Out of 518 K. pneumoniae, 329 were resistant to carbapenems, and 91 isolates showed resistance to colistin. The MIC of colistin ranged between 4 and &gt;512 ug/ml and MIC90 was 16 ug/L and MIC50 was 4 ug/ml. A majority of the colistin-resistant isolates were found in multidisciplinary ICU (85/91).Conclusion: The emergence of colistin-resistant strains is a major problem due to limited treatment options for infections caused by CRKP carbapenemase producing K. pneumoniae. Colistin should not be used alone, combination therapy should be preferred.

Frequency of colistin and fosfomycin resistance in carbapenem-resistant Enterobacteriaceae from a tertiary care hospital in Karachi.

IntroductionManagement of infections with carbapenem-resistant Enterobacteriaceae (CRE) is challenging. In recent times, agents such as colistin and fosfomycin have been used in combination with other antibiotics to treat such infections. In this study, we aim to seek frequency of colistin and fosfomycin resistance in CRE from Pakistan.MethodsThis study was conducted at clinical laboratories, Aga Khan University Hospital. In total, 251 CRE were included in the study. Colistin minimum inhibitory concentrations (MICs) were performed using broth microdilution (BMD) method and VITEK® 2 system, whereas fosfomycin susceptibility was performed using Kirby–Bauer method. MIC50 and MIC90 were calculated for colistin and agreement between VITEK and BMD was also calculated.ResultsOut of 251 strains colistin MIC of ≥4 µg/mL was seen in 40 (15.9%). Of these strains 20 (50%) were Klebsiella pneumoniae. Colistin MIC50 and MIC90 were found to be 0.5 and 16 µg/mL, respectively. BMD and VITEK 2 showed 100% categorical agreement. Essential agreement was 88.5% with kappa score 0.733 indicating strong agreement between VITEK and BMD. 31 out of 251 (12.3%) CREs were resistant to fosfomycin.ConclusionStudy shows frequency of colistin and fosfomycin resistance to be 15.9% and 12.3%, respectively. In countries where rate of CREs is high, emerging resistance against these last resort antibiotics is alarming as it leaves clinicians with almost no options to manage such multidrug resistant and extensively drug resistant infections.

Retrospective survey of mcr-1 and mcr-2 in German pig-fattening farms, 2011–2012

In November 2015, the first plasmid-encoded colistin resistance gene, mcr-1, was described in animals and in humans in China. Subsequently, a multitude of further studies was performed and quite recently the global spread of mcr-1 as well as the occurrence of a new gene variant, mcr-2, was reported. To obtain an overview of the occurrence of the colistin resistance genes mcr-1 and mcr-2 in German pig farms, a retrospective study, including 436 boot swab and pooled faecal samples collected from 58 pig-fattening farms throughout Germany, was performed. Whilst mcr-2 was not detected, the presence of mcr-1 was confirmed in 43 Escherichia coli isolates from 15 farms, indicating that the mcr-1 gene was present in 9.9% of the analysed samples and 25.9% of the investigated pig farms. Subsequent characterisation of the isolates showed colistin minimum inhibitory concentrations (MICs) of 4–8 µg/mL, with most isolates being resistant to several antibiotics including cephalosporins and/or fluoroquinolones. Pulsed-field gel electrophoresis (PFGE) showed great heterogeneity among the tested mcr-1-positive isolates. However, further analyses of 15 selected E. coli isolates (one per mcr-1-positive farm) indicated that the colistin resistance genes were predominantly located on IncX4 plasmids, highly similar to a plasmid initially isolated from an E. coli derived from a human patient in Brazil. The results described herein support the already expressed concern for public health and further underline the need for monitoring programmes in veterinary practice as well as in human medicine.