Zhi Ruan

Prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae recovered from bloodstream infections in China: a multicentre longitudinal study

BACKGROUND Polymyxin antibiotics are used as last-resort therapies to treat infections caused by multidrug-resistant Gram-negative bacteria. The plasmid-mediated colistin resistance determinant MCR-1 has been identified in Enterobacteriaceae in China. We did this study to investigate the prevalence of the mcr-1 gene in clinical isolates from patients with bloodstream infections in China. METHODS Clinical isolates of Escherichia coli and Klebsiella pneumoniae were collected from patients with bloodstream infections at 28 hospitals in China, then screened for colistin resistance by broth microdilution and for the presence of the mcr-1 gene by PCR amplification. We subjected mcr-1-positive isolates to genotyping, susceptibility testing, and clinical data analysis. We established the genetic location of mcr-1 with Southern blot hybridisation, and we analysed plasmids containing mcr-1 with filter mating, electroporation, and DNA sequencing. FINDINGS 2066 isolates, consisting of 1495 E coli isolates and 571 K pneumoniae isolates were collected. Of the 1495 E coli isolates, 20 (1%) were mcr-1-positive, whereas we detected only one (<1%) mcr-1-positive isolate among the 571 K pneumoniae isolates. All mcr-1-positive E coli and K pneumoniae isolates were resistant to colistin, with minimum inhibitory concentrations values in the range of 4-32 mg/L, except for one E coli isolate that had a minimum inhibitory concentration less than or equal to 0·06 mg/L. All 21 mcr-1-positive isolates were susceptible to tigecycline and 20 isolates (95%) were susceptible to the carbapenem and β-lactamase inhibitor combination piperacillin and tazobactam. One mcr-1-positive E coli isolate also produced NDM-5, which confers resistance to beta-lactam antibiotics. The 21 mcr-1-positive isolates were clonally diverse and carried mcr-1 on two types of plasmids, a 33 kb IncX4 plasmid and a 61 kb Inc12 plasmid. The 30 day mortality of the patients with bloodstream infections caused by mcr-1-positive isolates was zero. INTERPRETATION mcr-1-positive isolates from bloodstream infections were rare, sporadic, and remained susceptible to many antimicrobial agents. E coli, rather than K pneumoniae, was the main host of the mcr-1 gene. Further studies are needed to clarify the clinical impact of this novel resistance gene. FUNDING National Natural Science Foundation of China.

Wide distribution of CC92 carbapenem-resistant and OXA-23-producing Acinetobacter baumannii in multiple provinces of China

Carbapenem-resistant Acinetobacter baumannii has spread rapidly across China and is currently reported to be a worldwide nosocomial menace. In light of its increasing clinical significance, this study aimed to investigate the molecular epidemiology of carbapenem-resistant and carbapenem-susceptible isolates obtained from multiple provinces of China. Antimicrobial susceptibility was determined by disk diffusion assay according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Multilocus sequence typing (MLST) was used to investigate the molecular epidemiology of the isolates. In addition, a minimum spanning tree algorithm was applied to cluster sequence types (STs) into clonal complexes (CCs) in order to analyse their evolutionary relatedness. Generally, the average rate of resistance to most antibiotics in carbapenem-resistant isolates was extremely high (>85%), except for minocycline (20.45%). Analysis of MLST data confirmed that the genetic background of carbapenem-resistant isolates was relatively simple, with ST92 being the most prevalent clone, occurring in every province, followed by ST138, ST75 and ST381. In contrast, carbapenem-susceptible isolates had a more diverse genetic background. Furthermore, the most widely distributed CC of carbapenem-resistant isolates was bla(OXA-23)-like-producing and predominantly CC92, which incorporate ST136 and its several single-locus variants. Interestingly, isolates belonging to CC92 possessed higher antibiotic resistance rates compared with other STs. Overall, these observations suggest a wide distribution of carbapenem-resistant and bla(OXA-23)-like-producing clone CC92, especially ST92, ST75 and ST138, as the principal reason for the rapidly increasing carbapenem resistance rate in China.

Tigecycline Susceptibility and the Role of Efflux Pumps in Tigecycline Resistance in KPC-Producing Klebsiella pneumoniae

KPC-producing Klebsiella pneumoniae isolates have emerged as important pathogens of nosocomial infections, and tigecycline is one of the antibiotics recommended for severe infections caused by KPC-producing K. pneumoniae. To identify the susceptibility profile of KPC-producing K. pneumoniae to tigecycline and investigate the role of efflux pumps in tigecycline resistance, a total of 215 KPC-producing K. pneumoniae isolates were collected. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard broth microdilution tests. Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors. Expression levels of efflux pump genes (acrB and oqxB) and their regulators (ramA, marA, soxS and rarA) were examined by real-time PCR, and the correlation between tigecycline MICs and gene expression levels were analysed. Our results show that the tigecycline resistance rate in these isolates was 11.2%. Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7% of the isolates. A statistically significant association between acrB expression and tigecycline MICs was observed, and overexpression of ramA was found in three tigecycline-resistant isolates, further analysis confirmed ramR mutations existed in these isolates. Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. These data indicate that efflux pump AcrAB, which can be up-regulated by ramR mutations and subsequent ramA activation, contributed to tigecycline resistance in K. pneumoniae clinical isolates.

Species Distribution of Clinical Acinetobacter Isolates Revealed by Different Identification Techniques

A total of 2582 non-duplicate clinical Acinetobacter spp. isolates were collected to evaluate the performance of four identification methods because it is important to identify Acinetobacter spp. accurately and survey the species distribution to determine the appropriate antimicrobial treatment. Phenotyping (VITEK 2 and VITEK MS) and genotyping (16S rRNA and rpoB gene sequencing) methods were applied for species identification, and antimicrobial susceptibility test of imipenem and meropenem was performed with a disk diffusion assay. Generally, the phenotypic identification results were quite different from the genotyping results, and their discrimination ability was unsatisfactory, whereas 16S rRNA and rpoB gene sequencing showed consistent typing results, with different resolution. Additionally, A. pittii, A. calcoaceticus and A. nosocomialis, which were phylogenetically close to A. baumannii, accounted for 85.5% of the non-A. baumannii isolates. One group, which could not be clustered with any reference strains, consisted of 11 isolates and constituted a novel Acinetobacter species that was entitled genomic species 33YU. None of the non-A. baumannii isolates harbored a bla OXA-51-like gene, and this gene was disrupted by ISAba19 in only one isolate; it continues to be appropriate as a genetic marker for A. baumannii identification. The resistance rate of non-A. baumannii isolates to imipenem and/or meropenem was only 2.6%, which was significantly lower than that of A. baumannii. Overall, rpoB gene sequencing was the most accurate identification method for Acinetobacter species. Except for A. baumannii, the most frequently isolated species from the nosocomial setting were A. pittii, A. calcoaceticus and A. nosocomialis.

BacWGSTdb, a database for genotyping and source tracking bacterial pathogens

Whole genome sequencing has become one of the routine methods in molecular epidemiological practice. In this study, we present BacWGSTdb (http://bacdb.org/BacWGSTdb), a bacterial whole genome sequence typing database which is designed for clinicians, clinical microbiologists and hospital epidemiologists. This database borrows the population structure from the current multi-locus sequence typing (MLST) scheme and adopts a hierarchical data structure: species, clonal complex and isolates. When users upload the pre-assembled genome sequences to BacWGSTdb, it offers the functionality of bacterial genotyping at both traditional MLST and whole-genome levels. More importantly, users are told which isolates in the public database are phylogenetically close to the query isolate, along with their clinical information such as host, isolation source, disease, collection time and geographical location. In this way, BacWGSTdb offers a rapid and convenient platform for worldwide users to address a variety of clinical microbiological issues such as source tracking bacterial pathogens.

Dissemination of blaOXA-23 in Acinetobacter spp. in China: Main Roles of Conjugative Plasmid pAZJ221 and Transposon Tn2009

ABSTRACT Production of the OXA-23 carbapenemase is the most common reason for the increasing carbapenem resistance in Acinetobacter spp. This study was conducted to reveal the genetic basis of blaOXA-23 dissemination in Acinetobacter spp. in China. A total of 63 carbapenem-resistant OXA-23-producing Acinetobacter sp. isolates, representing different backgrounds, were selected from 28 hospitals in 18 provinces for this study. Generally, two patterns of plasmids carrying blaOXA-23 were detected according to S1-nuclease pulsed-field gel electrophoresis and Southern blot hybridization. A ca. 78-kb plasmid, designated pAZJ221, was found in 23 Acinetobacter baumannii and three Acinetobacter nosocomialis isolates, while a novel ca. 50-kb plasmid was carried by only two other A. baumannii isolates. Three of these isolates had an additional copy of blaOXA-23 on the chromosome. Transformation of the two plasmids succeeded, but only pAZJ221 was conjugative. Plasmid pAZJ221 was sequenced completely and found to carry no previously known resistance genes except blaOXA-23. The blaOXA-23 gene of the remaining 35 isolates was chromosome borne. The blaOXA-23 genetic environments were correlated with Tn2009 in 57 isolates, Tn2008 in 5 isolates, and Tn2006 in 1 isolate. The MIC values for the carbapenems with these isolates were not significantly associated with the genomic locations or the copy numbers of blaOXA-23. Overall, these observations suggest that the plasmid pAZJ221 and Tn2009 have effectively contributed to the wide dissemination of blaOXA-23 in Acinetobacter spp. in China and that horizontal gene transfer may play an important role in dissemination of the blaOXA-23 gene.

Genome Sequences of Two Multidrug-Resistant Acinetobacter baumannii Strains Isolated from a Patient before and after Treatment with Tigecycline

Acinetobacter baumannii is a Gram-negative bacterium which emerged as a significant nosocomial pathogen worldwide. To investigate the molecular basis of the tigecycline-resistant mechanism, we determined the genome sequences of two multidrug-resistant A. baumannii strains isolated from a patient before and after treatment with tigecycline.

A glimpse into evolution and dissemination of multidrug-resistant Acinetobacter baumannii isolates in East Asia: a comparative genomics study

Clonal dissemination is characteristic of the important nosocomial pathogen Acinetobacter baumannii, as revealed by previous multi-locus sequence typing (MLST) studies. However, the disseminated phyletic unit is actually MLST sequence type instead of real bacterial clone. Here we sequenced the genomes of 13 multidrug-resistant (MDR) A. baumannii strains from Taiwan, and compared them with that of A. baumannii from other East Asian countries. Core-genome phylogenetic tree divided the analyzed strains into three major clades. Among them, one ST455 clade was a hybrid between the ST208 clade and the other ST455 clade. Several strains showed nearly identical genome sequence, but their isolation sources differed by over 2,500 km and 10 years apart, suggesting a wide dissemination of the phyletic units, which were much smaller than the sequence type. Frequent structural variation was detected even between the closely related strains in antimicrobial resistance elements such as AbaRI, class I integron, indicating strong selection pressure brought by antimicrobial use. In conclusion, wide clonal dissemination and frequent genomic variation simultaneously characterize the clinical MDR A. baumannii in East Asia.

Frequent topoisomerase IV mutations associated with fluoroquinolone resistance in Ureaplasma species

This study aimed to investigate the role of quinolone resistance-determining regions (QRDRs) of DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE) associated with fluoroquinolone resistance. A total of 114 Ureaplasma spp. strains, isolated from clinical female patients with symptomatic infection, were tested for species distribution and susceptibility to four fluoroquinolones. Moreover, we analysed the QRDRs and compared these with 14 ATCC reference strains of Ureaplasma spp. serovars to identify mutations that caused antimicrobial resistance. Our study indicated that moxifloxacin was the most effective fluoroquinolone against Ureaplasma spp. (MIC range: 0.125-32 μg ml⁻¹). However, extremely high MICs were estimated for ciprofloxacin (MIC range: 1-256 μg ml⁻¹) and ofloxacin (MIC range: 0.5-128 μg ml⁻¹), followed by levofloxacin (MIC range: 0.5-64 μg ml⁻¹). Seven amino acid substitutions were discovered in GyrB, ParC and ParE, but not in GyrA. Ser-83 → Leu/Trp (C248T/G) in ParC and Arg-448 → Lys (G1343A) in ParE, which were potentially responsible for fluoroquinolone resistance, were observed in 89 (77.2 %) and three (2.6 %) strains, respectively. Pro-462 → Ser (C1384T), Asn-481 → Ser (A1442G) and Ala-493 → Val (C1478T) in GyrB and Met-105 → Ile (G315T) in ParC seemed to be neutral polymorphisms, and were observed and occurred along with the amino acid change of Ser-83 → Leu (C248T) in ParC. Interestingly, two novel mutations of ParC and ParE were independently found in four strains. These observations suggest that amino acid mutation in topoisomerase IV appears to be the leading cause of fluoroquinolone resistance, especially the mutation of Ser-83 → Leu (C248T) in ParC. Moxifloxacin had the best activity against strains with Ser-83 → Leu mutation.

Correlation between Ureaplasma Subgroup 2 and Genitourinary Tract Disease Outcomes Revealed by an Expanded Multilocus Sequence Typing (eMLST) Scheme

The multilocus sequence typing (MLST) scheme of Ureaplasma based on four housekeeping genes (ftsH, rpL22, valS, and thrS) was described in our previous study; here we introduced an expanded MLST (eMLST) scheme with improved discriminatory power, which was developed by adding two putative virulence genes (ureG and mba-np1) to the original MLST scheme. To evaluate the discriminatory power of eMLST, a total of 14 reference strains of Ureaplasma serovars and 269 clinical strains (134 isolated from symptomatic patients and 135 obtained from asymptomatic persons) were investigated. Our study confirmed that all 14 serotype strains could successfully be differentiated into 14 eMLST STs (eSTs), while some of them could not even be differentiated by the MLST, and a total of 136 eSTs were identified among the clinical isolates we investigated. In addition, phylogenetic analysis indicated that two genetically significantly distant clusters (cluster I and II) were revealed and most clinical isolates were located in cluster I. These findings were in accordance with and further support for the concept of two well-known genetic lineages (Ureaplasma parvum and Ureaplasma urealyticum) in our previous study. Interestingly, although both clusters were associated with clinical manifestation, the sub-group 2 of cluster II had pronounced and adverse effect on patients and might be a potential risk factor for clinical outcomes. In conclusion, the eMLST scheme offers investigators a highly discriminative typing tool that is capable for precise epidemiological investigations and clinical relevance of Ureaplasma.