Comparative evaluation of susceptibility testing methods for colistin and polymyxin B on isolates of Enterobacteriaceae / 中华检验医学杂志

Objective:To provide a promising and optimal laboratory susceptibility-testing method for the clinical usage of antibiotic (polymyxin), four susceptibility-testing methods were performed and the broth microdilution (BMD) was chosen as the gold standard.Methods:A total number of eighty-eight nonduplicate clinical Enterobacteriaceae specimes were collected from January to December of 2019 in the First Affiliated Hospital, Fujian Medical University. Among the clinical specimens, of which six strains were positive for mcr-1. The minimal inhibitory concentration (MIC) of polymyxin of the clinical specimens were examined by the following methods: (1) broth microdilution, (2) colistin broth disk elution, (3) Vitek-2?, (4)BD PhoenixTM,(5)commercial broth microdilution. With BMD as reference, essential agreement (EA), categorical agreement(CA), very major error(VME) and major error (ME) of polymyxins for different methods were analyzed. The Kappa-consistency testing, paired Chi-square testing and the Spearman-rank correlation testing were used to analyze the consistency between the four antimicrobial susceptibility testing methods and the gold standard.Results:Taking broth microdilution as reference, the EA of colistin broth disk elution, Vitek-2?, BD PhoenixTM, commercial broth microdilution were 94.32% (83/88), 92.05% (81/88), 90.90% (80/88), and 96.59%(85/88), respectively. The CA of all the four methods were 100% (88/88). No VME and ME were recorded for four methods. Moreover, the consistency between four susceptibility testing methods and the gold standard is acceptable (Kappa values=1, P<0.001, McNemar test P=1 and r>0.5, P<0.05). Conclusions:In the present work, four susceptibility testing methods all met the standards recommended jointly by the Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing, of which the performance of the commercial broth microdilution and CBDE fared relatively well. Thus, these four methods could be routinely used in clinical microbiology laboratory of our hospital for colistin and polymyxin B susceptibility testing.

mcr-1-carrying Enterobacteriaceae isolated from companion animals in Brazil / Enterobacteriaceae portadora de mcr-1 em isolados clínicos de animais de companhia: primeiro relato no Brasil

Plasmid-mediated polymyxin resistance was first described in 2015, in China, in Escherichia coli carrying the mcr-1 (Mobile Colistin Resistance-1) gene. Since then, it has become a major public health challenge worldwide, representing a major threat to human and animal health. In addition, there are still few reports on the prevalence of mcr-1 in Enterobacteriaceae isolated from humans, animals and food. Therefore, the purpose of the study was to investigate the occurrence of the mcr-1 gene in bacterial isolates with phenotypic resistance to polymyxin B obtained from clinical specimens of companion animals. Phenotypic resistance to polymyxin B were determined by broth microdilution and the susceptibility profile to other antimicrobials (amikacin, amoxicillin/clavulanate, ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, doxycycline, ertapenem, gentamicin, imipenem, marbofloxacin, meropenem, phosphomycin, piperacillin/tazobactam, tetracycline, ticarcillin/clavulanate, tobramycin and trimethoprim/sulfamethoxazole) by disc-diffusion agar method. The extraction of bacterial DNA was performed via heat shock followed by spectrophotometric evaluation. To verify the presence of mcr-1, the Polymerase Chain Reaction was employed using specific primers, followed by agarose gel electrophoresis. The positive isolates had the corresponding amplicons sequenced. In this study, there were identified the first isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp. carrying the mcr-1 gene derived from specimens of companion animals in Brazil. Our results suggest the dissemination of resistance to polymyxins in the community and the environment, highlighting the need for surveillance and optimized treatment guidelines.(AU)

A resistência à polimixina mediada por plasmídeo teve sua primeira descrição em 2015, na China, em Escherichia coli portadora do gene mcr-1 (Mobile Colistin Resistance-1) e a partir de então tornou-se um grande desafio para a saúde pública em todo o mundo, constituindo uma grande ameaça à saúde humana e animal. Além disso, ainda existem poucos relatos sobre a prevalência de mcr-1 em Enterobacteriaceae isoladas de humanos, animais e alimentos. Sendo assim, o objetivo do estudo foi investigar a ocorrência do gene mcr-1 em isolados bacterianos com resistência fenotípica à polimixina B, oriundos de materiais clínicos de animais de companhia. A resistência fenotípica à polimixina B foi determinada por microdiluição em caldo e o perfil de sensibilidade aos demais antimicrobianos (amicacina, amoxicilina/clavulanato, ampicilina, ampicilina/sulbactam, aztreonam, cefazolina, cefepime, cefotaxima, cefoxitina, ceftazidima, ceftriaxona, cloranfenicol, ciprofloxacina, doxiciclina, ertapenem, gentamicina, imipinem, marbofloxacino, meropenem, fosfomicina, piperacilina/tazobactam, tetraciclina, ticarcilina/clavulanato, tobramicina sulfametoxazol/trimetoprim) foram determinados pelo método disco difusão. A extração do DNA bacteriano foi realizada via choque térmico, seguido de avaliação espectrofotométrica. Para a verificação da presença do mcr-1 foi utilizada a Reação em Cadeia da Polimerase com emprego de iniciadores específicos, seguida de eletroforese em gel de agarose. Os isolados positivos tiveram os correspondentes amplicons sequenciados. Nesse estudo foram identificados os primeiros isolados de Escherichia coli, Klebsiella spp. e Enterobacter spp. portadores do gene mcr-1 derivados de espécimes de animais de companhia no Brasil. Este estudo sugere a disseminação da resistência às polimixinas na comunidade e no meio ambiente, destacando a necessidade de vigilância e diretrizes otimizadas de tratamento.(AU)

Evaluación de la susceptibilidad a colistín y meropenem en Klebsiella pneumoniae productoras de carbapenemasas - KPC

The increase in infections caused by Enterobacterales resistant to carbapenems and other antimicrobials has limited the therapeutic alternatives which have led to the recovery of the use of colistin in clinical practices. Since 2015, a mechanism that confers resistance to colistin through plasmids related to the mcr-1 gene (Mobile Colistin Resistance) was detected, increasing the importance of its susceptibility test in the laboratory. Colistin susceptibility was evaluated by the disk elution method in 24 strains of Carbapenemase-producing type KPC Klebsiella pneumoniae, resulting 4 strains (17 %) resistant to colistin and 20 strains (83 %) intermediate. Also, in these strains, sensitivity to meropenem was evaluated by the E-test® method, finding that 10 strains (41,6 %) were within the acceptable range for their combination with colistin, 5 strains (20, 8 %) were within the uncertain range and 9 strains (37,4 %) were not appropriate for combination with colistin. For the combination of colistin with meropenem to be considered as a therapeutic alternative the MIC of colistin must be ≤ 2 µg /mL with meropenem ≤8 µg /mL, while the MIC between 12-16 µg/mL of meropenem may or not may work; and with a MIC of 32 µg/mL meropenem, the combination is not effective.

Detection of the mcr-1 gene in Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains isolated from broilers / Detecção do gene mcr-1 em estirpes de Escherichia coli Enteropatogênicas (EPEC) e Shigatoxigênicas (STEC) isoladas de frangos de corte

Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains are among the major pathotypes found in poultry and their products, which are capable of causing human enteric infections. Colistin has been claimed the drug of choice against diseases caused by multidrug-resistant Gram-negative bacteria (MDRGN) in humans. The mcr-1 gene was the first plasmidial gene that has been described to be responsible for colistin resistance and has also been detected in birds and poultry products. Our study aimed to detect the mcr-1 gene in enteropathogenic strains of E. coli in order to evaluate the resistance to colistin in broilers. The material was obtained from 240 cloacal samples and 60 broiler carcasses. The strains were isolated by the conventional bacteriological method and by the virulence genes, which characterize the enteropathogenic strains and resistance, and the samples were detected by polymerase chain reaction (PCR). Of the 213 isolated strains of E. coli, 57 (26.76%) were characterized as atypical EPEC and 35 (16.43%) as STEC. The mcr-1 gene was found in 3.5% (2/57) of the EPEC strains and 5.7% (2/35) of the STEC strains. In this study, it was possible to confirm that the mcr-1 resistance gene is already circulating in the broiler flocks studied and may be associated with the pathogenic strains.(AU)

Escherichia coli Enteropatogênica (EPEC) e Shigatoxigênica (STEC) estão entres os principais patotipos encontrados em aves e produtos avícolas que são capazes de causar doença entérica no homem. A colistina tem sido preconizada como droga de escolha para o tratamento de doenças causadas por bactérias Gram-negativas multirresistentes em humanos. O gene mcr-1 foi o primeiro gene plasmidial a ser descrito como responsável pela resistência a colistina e tem sido descrito em aves e produtos avícolas. Este estudo tem como objetivo a detecção do gene mcr-1 em estirpes de E. coli enteropatogênicas a fim de avaliar a resistência a colistina em frangos de corte. O material foi obtido a partir de 240 amostras cloacais e 60 carcaças de frango de corte. As estirpes foram isoladas pelo método bacteriológico convencional e os genes de virulência, que caracterizam as estirpes enteropatogênicas, e resistência foram detectados pela reação em cadeia pela polimerase (PCR). Das 213 estirpes de E. coli isoladas, 57 (26,76%) foram caracterizadas como EPEC atípica e 35 (16,43%) como STEC. O gene mcr-1 foi encontrado em 3,5% (2/57) das estirpes EPEC e 5,7% (2/35) das estirpes STEC. Neste estudo foi possível confirmar que o gene de resistência mcr-1 já está em circulação nos lotes de frango de corte estudados e pode estar associado às estirpes patogênicas.(AU)

Genetic analysis of the first mcr-1 positive Escherichia coli isolate collected from an outpatient in Chile

ABSTRACT Global dissemination of mcr-like genes represents a serious threat to public health since it jeopardizes the effectiveness of colistin, an antibiotic used as a last-resort treatment against highly antibiotic-resistant bacteria. In 2017, a mcr-1-positive isolate of Escherichia coli was found in Chile for the first time. Herein we report the genetic features of this strain (UCO-457) by whole-genome sequencing (WGS) and conjugation experiments. The UCO-457 strain belonged to ST4204 and carried a 285 kb IncI2-type plasmid containing the mcr-1 gene. Moreover, this plasmid was transferred by conjugation to an E. coli J53 strain at high frequency. The isolate harbored the cma, iroN, and iss virulence genes and did carry resistance genes to trimethoprim/sulfamethoxazole and fluoroquinolones. Other antibiotic resistance determinants such as β-lactamases-encoding genes were not detected, making the isolate highly susceptible to these antibiotics. Our results revealed that such susceptible isolates could be acting as platforms to disseminate plasmid-mediated colistin resistance. Based on this evidence, we consider that mcr-like prevalence deserves urgent attention and should be examined not only in highly resistant bacteria but also in susceptible isolates.

Escenario actual de la resistencia a la colistina mediada por plásmidos en América Latina / Current scenario of plasmid-mediated colistin resistance in Latin America

Colistin resistance can occur by chromosomal mutations and by acquisition of plasmid-carrying determinants, mainly mcr-1. In the recent years, we have observed the out-burst of this resistance gene in our region. Due to the risk of the rapid dissemination of mcr-1, this finding has worried and alerted different actors from the health field and has become one of the most prolific topics. Our review compiles available reports of well-documented mcr-1-positive strains of Enterobacteriaceae, obtained from different samples in Argentina and other countries of Latin America. Furthermore, it addresses the association of mcr-1 with ESBL resistance markers and outlines the platforms involved in their dissemination.

La resistencia a la colistina puede ocurrir por mutaciones cromosómicas o por la adquisición de determinantes localizados en plásmidos, el principal es mcr-1. En los últimos años hemos observado la explosiva aparición de este gen de resistencia en nuestra región. Debido al riesgo que implica la rápida diseminación de mcr-1, este hallazgo ha preocupado y alertado a los diferentes actores del área de la salud, y se ha convertido en uno de los temas de investigación más importantes. La presente revisión compila los informes de aislamientos portadores de mcr-1 debidamente documentados en Enterobacteriaceae, obtenidos de diferentes muestras en Argentina y otros países de América Latina. Además, aborda la asociación de mcr-1 con otros marcadores de resistencia, como las BLEE, y describe las plataformas involucradas en su diseminación.

Isolation and molecular epidemiology analysis of Gram-negative bacilli carrying mcr-1 gene in fecal specimens / 中华微生物学和免疫学杂志

Objective To investigate the colonization of Gram-negative bacilli carrying mcr-1 gene in intestinal tracts of inpatients and people having physical examination for further elucidating the molecular and epidemiological features of mcr-1 gene. Methods A total of 1263 and 750 fecal specimens were col-lected from inpatients in the First Affiliated Hospital of Guangzhou Medical University and people having physical examination in the Kingmed Physical Examination Centre, respectively. Drug-resistant bacteria were isolated using Maconkey agar supplemented with colistin. PCR was performed to detect the bacteria carrying mcr-1 gene. Multilocus sequence typing ( MLST) and enterobacterial repetitive intergenic consensus-PCR ( ERIC-PCR) were used for homology analysis. The transferability of mcr-1 gene was verified by plasmid transfer assays. Plasmids of mcr-1-carrying strains were typed by PCR-based replicon typing techniques. Twelve virulence-related genes were also detected by PCR. Results Ninety-two colistin-resistant strains were isolated from the 1263 samples from inpatients(7. 3％, 92/1263) and two of them were positive for mcr-1 gene ( one strain also carried the blaNDM-5 gene) . Thirty-six colistin-resistant strains were isolated from the 750 samples of physical examination group (4. 8％, 36/750) and one of them carried the mcr-1 gene. MLST analysis showed that three mcr-1-carrying Escherichia coli strains ( minimum inhibitory concentration of colistin:8 μg/ml) belonged to three different sequence types. Moreover, they exhibited different banding patterns in ERIC-PCR analysis. All of the mcr-1-carrying isolates could transfer mcr-1 gene to the recipient strains successfully. Six types of incompatibility plasmids were detected in the mcr-1-carrying isolates ( IncFⅡ, IncX2, IncHI2, IncFIB, IncX4 and IncX1). Virulence-related genes fimH, iutA and fyuA were detec-ted in all mcr-1-carrying Escherichia coli strains. Conclusions Colistin-resistant strains and mcr-1 gene are prevalent in inpatients and people having physical examination, which brings potential risk for the control of clinical infections.

Plasmid-mediated mcr-1 gene in acinetobacter baumannii and pseudomonas aeruginosa: first report from pakistan

Abstract INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.

Primera comunicación en Chile de la detección del gen mcr-1 en un aislado clínico de Escherichia coli resistente a colistín / First report in Chile of a clinical isolate of Escherichia coli resistant to colistin harbouring the mcr-1 gene

Recently it was described the plasmidial gene mcr-1 associated with colistin resistance. We screened by PCR and sequencing for gene mcr-1 in thirteen clinical isolates resistant to colistin. We observed amplification in one E. coli. To our knowledge, this is the first report of the presence of mcr-1 gene in Chile.

Resistencia plasmídica a colistin por el gen mcr-1 en Enterobacteriaceae en Paraguay / Plasmid-Mediated Colistin Resistance Gene mcr-1 in Enterobacteriaceae in Paraguay

La resistencia a las polimixinas mediada por plásmidos (gen mcr-1) representa una amenaza para la salud pública, puesto que colistina es utilizada en la práctica médica como una de las últimas alternativas para el tratamiento de gérmenes multiresistentes. Este estudio describe la circulaciónde cepas de Enterobacterias que portan este gen de resistencia, aisladas de pacientes hospitalizados, así como también de la comunidad. Los hallazgos de la Red de Vigilancia de la Resistencia a los Antimicrobianos-Paraguay fueron de casi el 5 % (4,7) en cepas remitidas con criterio de sospecha, siendo las especies involucradas Escherichiacoli, Klebsiella pneumoniae y Salmonella Schwarzengrund. Además, por métodos moleculares se confirmaron en todas ellas la portación de otros genes de resistencia (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) asociados al mcr-1. Palabras claves: Enterobacterias, resistencia, colistina, mcr-1.

Resistance to polymyxins mediated by plasmids (mcr-1 gene) represents a threat to public health, since colistin is used in medical practice, as one of the last alternatives, for the treatment of multi-resistant germs. This study describes the circulation of strains of Enterobacteria that carry this resistance gene, isolated from hospitalized patients, as well as from the community. The findings of the Red de Vigilancia de la Resistencia a los Antimicrobianos­Paraguay were almost 5% (4.7) in strains submitted with suspicion criteria; the species involved being Escherichia coli, Klebsiella pneumoniae and Salmonella Schwarzengrund. In addition, molecular methods confirmed in all of them the carrying of other resistance genes (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) associated with mcr-1. Key words: Enterobacteria, resistance, colistin, mcr-1.

Real-time quantitative PCR assay with TaqMan probe for rapid detection of colistin resistance mcr-1 gene / 中华微生物学和免疫学杂志

Objective To establish a sensitive real-time quantitative PCR assay with TaqMan probe for rapid detection of mcr-1 gene in clinical isolated strains. Methods According to the mcr-1 gene sequence, a pair of specific primers and a TaqMan probe were designed. Moreover, a recombinant plasmid with mcr-1 gene was constructed as the positive standard. TaqMan probe-based fluorescence quantitative PCR assay was used to detect the colistin resistance gene mcr-1. The sensitivity, repeatability and specificity of the assay were evaluated. Results There was a good linear relationship between the initial template amount and Ct value (R2>0. 999). The lower limit of detection was 10 copies/μL, which was 100 times more sensitive than the conventional PCR. Results of test for specificity showed that only the strains carrying the mcr-1 gene were positive, while the remaining strains were negative. Coefficients of variation of intra-and inter-group repeatability tests were less than 1％. Two out of 150 clinical isolated strains carried mcr-1 re-sistance gene and both of them were identified as Escherichia coli. Conclusion TaqMan probe-based fluo-rescence quantitative PCR for the detection of colistin resistance gene mcr-1 was established with strong spe-cificity, high sensitivity and good repeatability. It could be used for the specific detection of clinical drug-re-sistant strains positive for mcr-1 gene and provide reference for pharmacotherapy.

Progress in plasmid-mediated colistin resistance gene mcr-1 / 中华微生物学和免疫学杂志

Colistin has been revalued as the last resort for multi-drug resistant gram-negative bacte-ria infections and attracted considerable attention. The plasmid-mediated colistin resistance gene mcr-1 was first reported in 2005,which not only altered our understanding of the mechanisms of colistin resistance,but also posed a significant threat to public health. Until now,mcr-1 has been reported in more than 30 countries and regions spreading over five continents. The whole world has paid high attention to mcr-1 and taken meas-ures to control the administration of colistin in order to contain the spread and dissemination of mcr-1. This review focused on the structure,epidemiology and molecular biological characteristics of mcr-1 and measures for the prevention of mcr-1 spreading,aiming at enhancing the recognition,prevention and control of mcr-1.

Detection and epidemiology of mcr-1-positive Escherichia coli strains in Dongyang area of Zhejiang Province / 中华微生物学和免疫学杂志

Objective To investigate the prevalence of mcr-1 gene,a plasmid-mediated polymyxin resistance gene,in Escherichia coli(E.coli) strains isolated in Dongyang of Zhejiang Province and to under-stand the epidemiological characteristics of E.coli strains carrying mcr-1 gene in order to provide local clini-cians with a theoretical basis for prevention and control of the spread of mcr-1-bearing E.coli strains. Meth-ods A total of 315 E.coli strains were collected in the People′s Hospital of Dongyang, Zhejiang Province from January to December 2016. All strains were isolated from specimens of blood,urine,respiratory tract, etc. PCR was performed to detect the genes confering resistance to polymyxin (mcr-1 gene), β-lactamase and carbapenem. Minimal inhibitory concentrations (MIC) of antibiotics against mcr-1-positive strains were determined by micro-broth dilution method. Conjugation test was performed to confirm whether the mcr-1 gene was located on the transferable plasmid. Multilocus sequence typing (MLST) was used for molecular typing of mcr-1-positive strains. Results Five mcr-1-positive strains were identified from 315 E.coli strains with a positive rate of 1.6%. Two out of the five mcr-1-positive E.coli strains contained β-lactamase resist-ance genes,blaTEM-1and blaCTX-M-14. Both of them were resistant to the first, second and third generation of cephalosporins and one was also resistant to cefepime. All of the five mcr-1-positive E.coli strains were sen-sitive to ciprofloxacin and levofloxacin,but resistant to ticarcillin/clavulanic acid. No carbapenem resistance genes were detected. One transconjugant was successfully obtained by transconjugation assay. MLST analysis showed that a total of four sequence types were identified, including ST131 (two strains), ST43 (one strain),ST69 (one strain) and ST349(one strain). Conclusion Only 1.6% of all E.coli strains isolated in Dongyang area of Zhejiang Province carry mcr-1 gene,indicating that there is no epidemic of mcr-1 gene-positive E.coli infection. The coexistence of mcr-1 gene and β-lactamase resistance genes in E.coli strains isolated in Dongyang suggests that local clinicians should avoid antibiotic abuse to prevent the spread of drug-resistant E.coli.

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

BACKGROUND: The 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. METHODS: Among 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. RESULTS: Two 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. CONCLUSIONS: mcr-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.

Molecular mechanism of colistin resistance mediated by mcr-1 and pmrAB genes in Escherichia coli / 中华微生物学和免疫学杂志

Objective To investigate the molecular mechanism of colistin resistance mediated by mcr-1and pmrAB genes in clinical Escherichia coli ( E. coli) isolates. Methods A total of 1988 clinical E. coli isolates were collected from the First People′s Hospital of Shangqiu from 2010 to 2017 and screened for colistin-resistant isolates using agar dilution method. The minimum inhibitory concentrations ( MICs) of nine common clinical antibiotics were determined using broth microdilution method. PCR and sequencing analysis were performed to detect the colistin resistance genes of mcr-1 and pmrAB. Conjugation experiments were used to test the transferability of the plasmid carrying mcr-1 gene. S1-PFGE and Southern blot were used to locate the plasmid carrying mcr-1. All colistin-resistant E. coli isolates were typed by multilocus se-quence typing ( MLST) . Results Six colistin-resistant E. coli isolates were obtained by agar dilution meth-od. The results of susceptibility testing showed that all of the six isolates were multidrug resistant. PCR and sequencing analysis revealed that four out of the six strains carried mcr-1 gene, and the other two isolates both had an amino acid substitution (L167P) caused by pmrB gene mutation. The results of conjugation ex-periments, S1-PFGE and Southern hybridization analysis showed that the plasmids of four mcr-1 gene-posi-tive E. coli strains were located on a conjugative plasmid about 60 kb in length. MLST analysis classified the six isolates into six distinct sequence types ( STs) . Conclusion This study suggested that mcr-1 gene and mutations in pmrAB gene were the main mechanisms mediating the resistance of E. coli to colistin. In clinical practice, the occurrence and spread of colistin-resistant E. coli should be further monitored, and the rational use of antibiotics should be promoted to prevent the spread of colistin-resistant strains.

Prevalence of colistin-resistance gene mcr-1 in patients with bloodstream infection in 23 hospitals of Zhejiang Province / 中华微生物学和免疫学杂志

Objective To investigate the prevalence of colistin resistance and mcr-1 gene in pa-tients with bloodstream infection caused by Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneu-moniae) in Zhejiang Province. Methods A total of 869 clinical strains of the Enterobacteriaceae family, including 611 E.coli and 258 K.pneumonia strains, were isolated from patients with bloodstream infection (BSI) in Zhejiang Province from March 2014 to April 2015. Broth microdilution method and PCR were re-spectively performed to detect colistin resistance and mcr-1 gene in those stains. Susceptibilities of mcr-1-positive strains to other antibiotics were assessed by E-test. Pulsed-field gel electrophoresis(PFGE) and multilocus sequence typing(MLST) were used for molecular typing. Location of mcr-1 gene was determined by analysis of PFGE profiles of S1-digested genomic DNA and Southern blot hybridization. Plasmid transfer to E.coli recipients was investigated using filter mating test. Clinical data of the patients infected with mcr-1-positive strains was collected and analyzed. Results The minimum inhibitory concentration (MIC) values of colistin to the 869 Enterobacteriaceae strains ranged from ≤0.06 μg/ml to 16 μg/ml. Six (0.69%) E.coli strains were identified to be colistin-resistant and mcr-1-positive and the MIC values against them ranged from 8 μg/ml to 16 μg/ml. No colistin-resistant or mcr-1-positive K.pneumonia strain was identified. All mcr-1-positive strains were susceptible to carbapenems and most of them(83.33%,5/6) were suscepti-ble to tigecycline and β-lactamase inhibitor combinations tested in this study. The six mcr-1-positive strains were of different sequence types (STs) and the mcr-1 genes carried by them located on three types of plas-mids with the sizes of 33 kb,61 kb and 244.4 kb. Conclusion The prevalence of mcr-1 gene in E.coli and K.pneumonia strains isolated from patients with BSI in Zhejiang Province was relatively low, only ac-counting for 0.69% of all isolated Enterobacteriaceae strains.Those strains carrying mcr-1 gene showed low drug resistance to colistin. The mcr-1-positive strains were usually non-pathogenic clones and remained sus-ceptible to many antimicrobial agents, which was conducive to favorable outcomes. In order to clarify the clinical impact of this novel resistance gene on public health,further studies should be conducted.