Abstract Objectives To evaluate the prevalence of clinical mcr-1-positive Escherichia coli and Klebsiella pneumoniae and characterize the antimicrobial resistance profiles of mcr-1-positive E. coli and mcr-1-negative E. coli in China. Methods A total of 6264 clinical E. coli (n = 3854) and K. pneumoniae (n = 2410) were collected from hospitalized patients from 18 to 20 hospitals as part of the China Antimicrobial Resistance Surveillance Trial (CARST) between January 2007 and June 2016. PCR was used to screen for the mcr-1 gene among all isolates. Antibiotic susceptibility testing was performed using the broth microdilution method. mcr-1-positive pathogens were then characterized by MLST and minimum spanning tree analysis using the BURST algorithm for related STs. Results We examined 39 (0.62%) clinical isolates of mcr-1-positive E. coli and K. pneumoniae over a 10 year period. Resistance to antimicrobial agents was significantly more severe in mcr-1-positive isolates than mcr-1-negative isolates, particularly piperacillin (P = 0.008), amikacin (P < 0.0001), nitrofurantoin (P < 0.004) and fosfomycin (P < 0.0001). Among mcr-1-carrying isolates, ESBL production was as high as 84.6% (33 of 39) and 92.3% (36 of 39) of them displayed an MDR phenotype. STs suggested ubiquitous dissemination of mcr-1-carrying pathogens. Conclusions mcr-1-carrying E. coli and K. pneumoniae displayed a lower prevalence and abundant phylogenetic diversity in mainland China. mcr-1-positive E. coli showed significant differences in antimicrobial resistance profiles compared with mcr-1-negative E. coli strains, suggesting physicians may consider prescribing different antibiotics when faced with infections caused by mcr-1-positive pathogens. Introduction The emergence of the plasmid-mediated colistin resistance gene mcr-1 not only altered our understanding of colistin resistance mechanisms, but also posed a significant threat to public health.1,2 Colistin use in animals for years has been an important contributor for selecting colistin-resistant pathogens. In China, colistin use has become inevitable and will soon come into common use in hospitals to combat the growing number of carbapenem treatment failures,3 thereby increasing the potential risk of the dissemination of mcr-1. However, little is known about differences in the antimicrobial susceptibility patterns of mcr-1-positive and -negative bacteria in hospitals at a national level. Therefore, the treatment of infections caused by mcr-1-carrying bacteria is not backed by scientific data, meaning that further study is urgently needed. Materials and methods Bacterial isolates The China Antimicrobial Resistance Surveillance Trial (CARST) programme was established by the Ministry of Health, China, to monitor the applications of antibiotics and the trend in antimicrobial resistance among clinical isolates, providing scientific data for the treatment of clinical infection. A total of 28 tertiary teaching hospitals (20 in 2007–08; 19 in 2009–10; 18 in 2011–12; 19 in 2013–14; and 18 in 2015–16) from 24 cities located in Eastern, Central and Western China were selected as CARST monitoring sites, with two or three hospitals added or removed from this system during 2007–16. These hospitals were representative of nationwide circumstances, based on geographical distribution, medical skill and the development level of microbiological diagnostic techniques. All isolates were collected biennially by the CARST programme over five consecutive 2 year periods and were sent to a central laboratory (Institute of Clinical Pharmacology, Peking University First Hospital). All isolates were collected from clinical samples provided by hospitalized patients during routine examination without harming patients’ interests. We examined the prevalence of mcr-1-positive Escherichia coli and Klebsiella pneumoniae among clinical bacterial isolates obtained as part of the CARST programme over five consecutive 2 year periods between January 2007 and June 2016 (2007–08; 2009–10; 2011–12; 2013–14; and 2015–16). Only one isolate per species per patient was collected in this study. Species were identified by the Analytical Profile Index system and 16S rRNA analysis. All strains were recovered and cultivated on Mueller–Hinton agar; PCR and sequencing were used to detect the mcr-1 gene.2 A list of hospitals participating in the CARST programme is available as Supplementary data at JAC Online. Antimicrobial susceptibility testing MICs were determined by the broth microdilution method and interpretative breakpoint criteria were in accordance with those recommended by the CLSI guidelines,4 with the exception of colistin and tigecycline, which were in accordance with EUCAST.5 Bacterial suspensions (>104 cfu of each bacterium) were obtained by a multipoint inoculator. MDR was defined as resistant to at least one drug in a minimum of three of any of the following drug classes: β-lactam antibiotics, aminoglycosides, tetracyclines, quinolones, nitrofurantoin and fosfomycin. The double-disc synergy test was performed to identify ESBL-producing isolates among E. coli and K. pneumoniae, as recommended by CLSI (2016). E. coli ATCC 25922 and E. coli ATCC 35218 were used as quality control reference strains. Detection of other colistin resistance mechanisms We screened all mcr-1-positive isolates for the presence of chromosomal mechanisms of colistin resistance, i.e. two-component regulatory systems (pmrAB and phoPQ and its negative regulator mgrB). We amplified and sequenced pmrAB, phoPQ and mgrB using the primers described in a previous study.2 Data analysis Minimum spanning tree (MST) analysis was carried out using the BURST algorithm for related STs between different backgrounds by BioNumerics 7.5 software (Applied Maths, Belgium). All data, including patient and microbiological information, were analysed using Statistical Package for the Social Sciences 20.0 software (SPSS, Inc., Chicago, IL, USA). Binary data were analysed using Fisher’s exact tests and χ2 tests. Results with P < 0.05 were considered statistically significant using two-tailed tests. Results During 2007–16, 6264 non-duplicate E. coli (n = 3854) and K. pneumoniae (n = 2410) isolates were collected from blood (33.2%), urine (22.6%), sputum (16.5%), drainage (12.7%), body secretions (9.8%), bile (2.5%) and other specimens (2.7%, including CSF, throat swab, etc.); 53.7% of isolates were collected from females and 46.3% of isolates were collected from males. Most isolates were collected from non-ICUs (78.5%), whereas only 21.5% were from ICUs. Samples from Eastern, Central and Western China comprised 48.3%, 40.7% and 11.0% of total samples, respectively. Among the E. coli isolates, 34 were positive for mcr-1, with isolation rates of 0% (0 of 767) in 2007–08, 0% (0 of 867) in 2009–10, 1.44% (10 of 694) in 2011–12, 1.37% (11 of 805) in 2013–14 and 1.80% (13 of 721) in 2015–16. In contrast, only five mcr-1-positive K. pneumoniae isolates were detected, including one in 2007–08 (1 of 414, 0.24%), two in 2011–12 (2 of 510, 0.39%) and two in 2015–16 (2 of 563, 0.36%). These 39 strains were mainly isolated from blood samples (13 of 39, 33.3%), followed by urine samples (10 of 39, 25.6%). Of all mcr-1-positive E. coli isolates, 21 (61.8%) were from females and 13 (38.2%) were from males and statistical analysis confirmed that there was no gender bias among mcr-1-positive E. coli (P = 0.389) compared with mcr-1-negative E. coli (53.2% were isolated from females and 46.8% were isolated from males), which was different from a previous study linking mcr-1-positive strains with male sex.3 In addition, no other chromosome mutations responsible for colistin resistance were found in 39 mcr-1-carrying isolates. All mcr-1-positive isolates were resistant to colistin, with MICs in the range of 4–64 mg/L. mcr-1-positive E. coli displayed high levels of resistance to tetracycline, ciprofloxacin and some cephalosporins. Fortunately, only one mcr-1-positive E. coli isolate was resistant to carbapenems and all mcr-1-positive isolates were susceptible to tigecycline. Notably, among the 39 mcr-1-positive isolates, all but 6 E. coli isolates were ESBL producers (33 of 39, 84.6%). Moreover, 92.3% (36 of 39) isolates are MDR (Figure 1a and b and Table S1). Figure 1. View largeDownload slide Distribution and antimicrobial resistance profiles of mcr-1-positive E. coli isolates collected from hospitalized patients in mainland China between 2007 and 2016. (a) Distribution of mcr-1-positive isolates from hospitalized patients (2007–16). mcr-1-positive isolates are denoted with red stars and the provinces involved in CARST are shaded. (b) Resistance rates of mcr-1-positive E. coli isolates for tested antimicrobial agents (2011–12, 2013–14 and 2015–16). (c) Comparison of antimicrobial resistance profiles of mcr-1-positive and -negative E. coli isolates. *P < 0.05. **P < 0.01. Statistical analysis was carried out using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). PIP, piperacillin; TZP, piperacillin/tazobactam; CFZ, cefazolin; CXM, cefuroxime; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; CFP, cefoperazone; CSL, cefoperazone/sulbactam; FEP, cefepime; ATM, aztreonam; MOX, moxalactam; IPM, imipenem; MEM, meropenem; ETP, ertapenem; GEN, gentamicin; AMK, amikacin; TET, tetracycline; MIN, minocycline; TGC, tigecycline; CIP, ciprofloxacin; LVX, levofloxacin; NIT, nitrofurantoin; FOF, fosfomycin; MCRNEC, mcr-1-negative E. coli; MCRPEC, mcr-1-positive E. coli. Figure 1. View largeDownload slide Distribution and antimicrobial resistance profiles of mcr-1-positive E. coli isolates collected from hospitalized patients in mainland China between 2007 and 2016. (a) Distribution of mcr-1-positive isolates from hospitalized patients (2007–16). mcr-1-positive isolates are denoted with red stars and the provinces involved in CARST are shaded. (b) Resistance rates of mcr-1-positive E. coli isolates for tested antimicrobial agents (2011–12, 2013–14 and 2015–16). (c) Comparison of antimicrobial resistance profiles of mcr-1-positive and -negative E. coli isolates. *P < 0.05. **P < 0.01. Statistical analysis was carried out using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). PIP, piperacillin; TZP, piperacillin/tazobactam; CFZ, cefazolin; CXM, cefuroxime; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; CFP, cefoperazone; CSL, cefoperazone/sulbactam; FEP, cefepime; ATM, aztreonam; MOX, moxalactam; IPM, imipenem; MEM, meropenem; ETP, ertapenem; GEN, gentamicin; AMK, amikacin; TET, tetracycline; MIN, minocycline; TGC, tigecycline; CIP, ciprofloxacin; LVX, levofloxacin; NIT, nitrofurantoin; FOF, fosfomycin; MCRNEC, mcr-1-negative E. coli; MCRPEC, mcr-1-positive E. coli. Compared with mcr-1-negative E. coli strains, the mcr-1-positive E. coli isolates showed higher levels of resistance to colistin as well as other commonly used antibiotics in clinical practice, including cefuroxime, cefotaxime, ceftriaxone and ciprofloxacin (P < 0.05). Furthermore, extremely significant differences in the rates of resistance to piperacillin (P = 0.008), amikacin (P < 0.0001), nitrofurantoin (P = 0.004) and fosfomycin (P < 0.0001) were noted between mcr-1-positive and -negative E. coli, indicating infections caused by mcr-1-positive E. coli should be treated differently to those caused by mcr-1-negative strains (Figure 1c). MLST was carried out as previously described6 and the five K. pneumoniae were assigned to three different STs, with ST15 (n = 3) being the most common type. In contrast, the 34 mcr-1-positive E. coli isolates were assigned to 28 distinct STs, including 8 novel STs. There was no predominant ST for the mcr-1-positive E. coli isolates, with most STs only containing one isolate. Discussion This study revealed a low prevalence of mcr-1 among E. coli (0.88%, 34 of 3854) and K. pneumoniae (0.21%, 5 of 2410) in mainland China over 10 years and we did not identify any mcr-1-carrying E. coli until the third period (2011–12). ESBL production and MDR were as high as 84.6% and 92.3%, respectively. A previous study7 showed a correlation between the emergence of mcr-1 and the use of β-lactam antibiotics, which may explain the prevalence of mcr-1-positive E. coli in China, where colistin is not yet regularly used in a clinical setting, might be co-selected by the usage of β-lactam antibiotics. It might be a problem for physicians to know which antibiotics to prescribe when faced with infections caused by mcr-1-positive pathogens. Notably, our study showed significant differences in the antimicrobial resistance profiles of mcr-1-positive and -negative E. coli strains, suggesting that traditional empirical therapy may lead to treatment failure in cases of infections caused by mcr-1-carrying pathogens. Particularly for piperacillin, amikacin, nitrofurantoin and fosfomycin, the co-selection of mcr-1-carrying isolates in patients during treatment is highly possible. MLST analysis revealed high clonal diversity among the mcr-1-positive E. coli isolates. Only three isolates (one ST10 and two ST48) belonged to the ST10 clonal complex, which is associated with the spread of ESBL and quinolone resistance genes worldwide8 and is the major ST of mcr-1-positive E. coli and NDM-producing E. coli isolates from poultry. None of the E. coli isolates belonged to highly pathogenic ST131, although four isolates belonged to ST393, which is less virulent than ST131, but is also associated with clinical MDR.9 In addition, MST analysis indicated that mcr-1 in E. coli had little relationship with gender, location, time of isolation or ESBL production, further indicating that mcr-1-positive E. coli isolates display a diverse distribution across China (Figure 2 and Table S1). Figure 2. View largeDownload slide MST analysis of the 34 mcr-1-positive E. coli isolates generated from MLST data. Each ST is displayed as a circle and the size of the circle denotes the number of isolates belonging to that particular ST. Lengths of lines between each circle/ST proportionally demonstrate the number of different alleles. Each circle is labelled with the corresponding ST and number of isolates. Novel STs are identified by a black border. Different groups of isolates are identified by different colours. Isolates were divided into two or three groups according to ESBL production (a), location of isolation (b), sex of the patient (c) and year of isolation (d). Figure 2. View largeDownload slide MST analysis of the 34 mcr-1-positive E. coli isolates generated from MLST data. Each ST is displayed as a circle and the size of the circle denotes the number of isolates belonging to that particular ST. Lengths of lines between each circle/ST proportionally demonstrate the number of different alleles. Each circle is labelled with the corresponding ST and number of isolates. Novel STs are identified by a black border. Different groups of isolates are identified by different colours. Isolates were divided into two or three groups according to ESBL production (a), location of isolation (b), sex of the patient (c) and year of isolation (d). Some limitations still exist in this study. We mainly focused on the clinical isolates and did not involve the intestinal carriage of mcr-1 strains. However, mcr-1 seems to be mainly isolated from E. coli, which are regular commensals of the human gastrointestinal tract. A colonization study in China showed a higher rate of mcr-1-positive E. coli carriage of inpatients compared with healthy volunteers, suggesting that intestinal carriage may be one of the sources of clinical mcr-1-positive E. coli.3 Moreover, we only presented the antimicrobial resistance profiles and epidemiological characteristics of mcr-1-positive strains among a high number of clinical isolates over a 10 year period at a national level in China, without involving the WGS and analysis of mcr-1-positive isolates, in which the detection of additional resistance genes including ESBL genes and carbapenem resistance genes among mcr-1-carrying isolates should be taken into account. Funding This work was supported by the National Natural Science Foundation of China (grants 81572033, 31422055 and 81661138002). Transparency declarations None to declare. Supplementary data A list of hospitals participating in the CARST programme and Table S1 are available as Supplementary data at JAC Online. References 1 Olaitan AO , Morand S , Rolain JM. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria . Front Microbiol 2014 ; 5 : 643 . Google Scholar CrossRef Search ADS PubMed 2 Liu YY , Wang Y , Walsh TR et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study . Lancet Infect Dis 2016 ; 16 : 161 – 8 . Google Scholar CrossRef Search ADS PubMed 3 Wang Y , Tian GB , Zhang R et al. Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study . Lancet Infect Dis 2017 ; 17 : 390 – 9 . Google Scholar CrossRef Search ADS PubMed 4 Clinical and Laboratory Standards Institute . Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Sixth Informational Supplement M100-S26 . CLSI , Wayne, PA, USA , 2016 . 5 EUCAST . Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 8.0, 2018 . http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_8.0_Breakpoint_Tables.pdf. 6 Tartof SY , Solberg OD , Manges AR et al. Analysis of a uropathogenic Escherichia coli clonal group by multilocus sequence typing . J Clin Microbiol 2005 ; 43 : 5860 – 4 . Google Scholar CrossRef Search ADS PubMed 7 Rhouma M , Letellier A. Extended-spectrum β-lactamases, carbapenemases and the mcr-1 gene: is there a historical link? Int J Antimicrob Agents 2017 ; 49 : 269 – 71 . Google Scholar CrossRef Search ADS PubMed 8 Oteo J , Diestra K , Juan C et al. Extended-spectrum β-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/A, ST23 complex/A and ST131/B2 . Int J Antimicrob Agents 2009 ; 34 : 173 – 6 . Google Scholar CrossRef Search ADS PubMed 9 Johnson JR , Tchesnokova V , Johnston B et al. Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli . J Infect Dis 2013 ; 207 : 919 – 28 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: firstname.lastname@example.org. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Apr 17, 2018
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