Congming Wu

Prevalence and Dissemination of oqxAB in Escherichia coli Isolates from Animals, Farmworkers, and the Environment

ABSTRACT OqxAB has recently been identified as one of the mechanisms of plasmid-mediated quinolone resistance (PMQR). Compared to what is observed for other PMQR determinants, there is a paucity of data with regard to the prevalence and epidemiology of OqxAB and its contribution to resistance to different antimicrobials. In this study, the prevalence and dissemination of oqxAB and other PMQR genes in Escherichia coli isolates from animals, farmworkers, and the environment in 2002 in China were investigated. Of the 172 E. coli isolates, 39.0% carried oqxA, while only 4.1%, 2.9%, and 0.6% carried qnr (1 qnrB6 isolate, 5 qnrS1 isolates, and 1 qnrD isolate), qepA, and aac(6′)-Ib-cr, respectively. Among the 33 isolates from farmworkers, 10 (30.3%) were positive for oqxA. oqxAB was associated with IS26 and was carried on the 43- to 115-kb IncF transferable plasmid. Transconjugants carrying oqxAB showed 4- to 16-fold increases in the MICs of quinolones, 16- to 64-fold increases in the MICs of quinoxalines, 8- to 32-fold increases in the MICs of chloramphenicol and trimethoprim-sulfamethoxazole, and 4- to 8-fold increases in the MICs of florfenicol compared to the levels for the recipient. The pulsed-field gel electrophoresis (PFGE) analysis showed that the high levels of prevalence and dissemination of oqxAB in E. coli in animal farms were primarily due to the transmission of plasmids carrying oqxAB, although clonal transmission between human and swine E. coli isolates was observed. It is concluded that oqxAB was widespread in animal farms in China, which may be due to the overuse of quinoxalines in animals. This study warrants the prudent use of quinoxalines in food animals.

Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study

BACKGROUNDnThe mcr-1 gene confers transferable colistin resistance. mcr-1-positive Enterobacteriaceae (MCRPE) have attracted substantial medical, media, and political attention; however, so far studies have not addressed their clinical impact. Herein, we report the prevalence of MCRPE in human infections and carriage, clinical associations of mcr-1-positive Escherichia coli (MCRPEC) infection, and risk factors for MCRPEC carriage.nnnMETHODSnWe undertook this study at two hospitals in Zhejiang and Guangdong, China. We did a retrospective cross-sectional assessment of prevalence of MCRPE infection from isolates of Gram-negative bacteria collected at the hospitals from 2007 to 2015 (prevalence study). We did a retrospective case-control study of risk factors for infection and mortality after infection, using all MCRPEC from infection isolates and a random sample of mcr-1-negative E coli infections from the retrospective collection between 2012 and 2015 (infection study). We also did a prospective case-control study to assess risk factors for carriage of MCRPEC in rectal swabs from inpatients with MCRPEC and mcr-1 negative at the hospitals and collected between May and December, 2015, compared with mcr-1-negative isolates from rectal swabs of inpatients (colonisation study). Strains were analysed for antibiotic resistance, plasmid typing, and transfer analysis, and strain relatedness.nnnFINDINGSnWe identified 21u2008621 non-duplicate isolates of Enterobacteriaceae, Acinetobacter spp, and Pseudomonas aeruginosa from 18u2008698 inpatients and 2923 healthy volunteers. Of 17u2008498 isolates associated with infection, mcr-1 was detected in 76 (1%) of 5332 E coli isolates, 13 (<1%) of 348 Klebsiella pneumoniae, one (<1%) of 890 Enterobacter cloacae, and one (1%) of 162 Enterobacter aerogenes. For the infection study, we included 76 mcr-1-positive clinical E coli isolates and 508 mcr-1-negative isolates. Overall, MCRPEC infection was associated with male sex (209 [41%] vs 47 [63%], adjusted p=0·011), immunosuppression (30 [6%] vs 11 [15%], adjusted p=0·011), and antibiotic use, particularly carbapenems (45 [9%] vs 18 [24%], adjusted p=0·002) and fluoroquinolones (95 [19%] vs 23 [30%], adjusted p=0·017), before hospital admission. For the colonisation study, we screened 2923 rectal swabs from healthy volunteers, of which 19 were MCRPEC, and 1200 rectal swabs from patients, of which 35 were MCRPEC. Antibiotic use before hospital admission (p<0·0001) was associated with MCRPEC carriage in 35 patients compared with 378 patients with mcr-1-negative E coli colonisation, whereas living next to a farm was associated with mcr-1-negative E coli colonisation (p=0·03, univariate test). mcr-1 could be transferred between bacteria at high frequencies (10-1 to 10-3), and plasmid types and MCRPEC multi-locus sequence types (MLSTs) were more variable in Guangdong than in Zhejiang and included the human pathogen ST131. MCRPEC also included 17 unreported ST clades.nnnINTERPRETATIONnIn 2017, colistin will be formally banned from animal feeds in China and switched to human therapy. Infection with MRCPEC is associated with sex, immunosuppression, and previous antibiotic exposure, while colonisation is also associated with antibiotic exposure. MLST and plasmid analysis shows that MCRPEC are diversely spread throughout China and pervasive in Chinese communities.nnnFUNDINGnNational Key Basic Research Program of China, National Natural Science Foundation of China/Zhejiang, National Key Research and Development Program, and MRC, UK.

Detection of the staphylococcal multiresistance gene cfr in Proteus vulgaris of food animal origin

OBJECTIVESnTo investigate the presence and the genetic environment of the multiresistance gene cfr in naturally occurring Gram-negative bacteria of pigs.nnnMETHODSnA total of 391 bacterial isolates with florfenicol MICs of ≥16 mg/L, obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. The species assignment of the cfr-carrying isolate was based on the results of Grams staining, colony morphology, 16S rDNA sequencing and biochemical profiling. The location of the cfr and floR genes was determined by Southern blotting and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy.nnnRESULTSnA single Proteus vulgaris isolate, which carried the genes floR and cfr, was detected in this study. A cfr-carrying segment of 7 kb with homology to a staphylococcal plasmid was found to be inserted into the chromosomal fimD gene of P. vulgaris. This segment was flanked by two IS26 elements located in the same orientation, which are believed to have played a role in this integration process. Stability testing via inverse PCR approaches showed that this integrate is not entirely stable, but the cfr-carrying centre region plus one IS26 copy can be looped out via IS26-mediated recombination.nnnCONCLUSIONSnTo the best of our knowledge, this is the first report of the cfr gene in a naturally occurring Gram-negative bacterium. Surveillance and monitoring of the cfr gene in Gram-negative bacteria are warranted with respect to food safety and consumer protection.

Detection of the staphylococcal multiresistance gene cfr in Escherichia coli of domestic-animal origin

OBJECTIVESnTo investigate the presence and the genetic environment of the multiresistance gene cfr in Escherichia coli found in domestic animals.nnnMETHODSnA total of 1230 E. coli isolates, collected from pigs, chickens and ducks, were screened by PCR for the cfr gene. The location of the cfr gene was determined by Southern blotting, the transferability of cfr gene was tested by conjugation and transformation, and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy. The location of the cfr promoter sequence was analysed by mapping the cfr transcription start site using rapid amplification of 5 cDNA ends (5 RACE).nnnRESULTSnOnly a single strain from the nasal swab of a pig harboured the cfr gene. Southern blotting indicated that the cfr gene was located on a ~110 kb plasmid, designated pEC-01. A cfr-carrying segment of 1545 bp with a sequence identical to that of the cfr-harbouring plasmid pSCFS1 was flanked by two IS26 elements in the same orientation. The IS26 transposition created a new hybrid promoter in which the -35 region was part of the left inverted repeat of IS26 while the -10-like sequence was part of the original cfr upstream region.nnnCONCLUSIONSnTo the best of our knowledge, this is the first report of the cfr gene in a naturally occurring E. coli strain. Continued surveillance of the presence of the cfr gene in Gram-negative bacteria of domestic-animal origin is warranted.

Increasing prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in food animals and the diversity of CTX-M genotypes during 2003-2012

The aim of this study was to investigate the trends and the diversity of CTX-M types of extended-spectrum β-lactamase (ESBL) in Escherichia coli isolated from food animals in China over a ten-year period. From 2003 to 2012, 2815 E. coli isolates collected from diseased animals (chickens, pigs, and waterfowl) were screened for the prevalence of CTX-M genes. CTX-M-positive isolates were tested for their susceptibilities to 10 antimicrobial agents and the clonal relationship of CTX-M-producing E. coli isolates was also assessed. Overall, 677 (20.1%) of the 2815 E. coli isolates carried CTX-M genes. Eighteen different types of CTX-M ESBLs were identified, with CTX-M-14, CTX-M-55, and CTX-M-65 being the most dominant genotypes. The occurrence of CTX-M-producing E. coli increased significantly from 5.7% in 2003-2005 to 35.3% in 2009-2012 (p<0.0001). High genetic heterogeneities were observed in the CTX-M-producing E. coli isolates. Most CTX-M-producing strains were also resistant to other classes of antimicrobials. Compared to isolates carrying CTX-M-9 subgroup of ESBLs, isolates carrying CTX-M-1 subgroup ESBLs showed significantly higher resistance rates to ceftazidime, amikacin, and fosfomycin (p<0.01). The study reported the dramatic increase of CTX-M ESBLs in E. coli isolated from animals overtime in China. The increasing incidence of CTX-M-55 with high hydrolytic activity against ceftazidime and the widely spread co-resistance in CTX-M-producing isolates alarm the serious antimicrobial resistance situation in China and highlight the need for urgent control strategies to limit the dissemination of those resistant genes in China.

Report of ribosomal RNA methylase gene erm(B) in multidrug-resistant Campylobacter coli

OBJECTIVESnCampylobacter is a major foodborne enteric pathogen and macrolides are the drug of choice for the clinical therapy of campylobacteriosis. Macrolide resistance among Campylobacter compromises clinical treatment, is associated with adverse health events and is a significant public health concern. Here, we report the first identification of a horizontally transferrable macrolide resistance mechanism in porcine Campylobacter coli ZC113 that is mediated by a ribosomal RNA methylase, Erm(B).nnnMETHODSnHorizontal transfer of a macrolide resistance determinant between C. coli and Campylobacter jejuni was performed by natural transformation. Whole-genome sequencing was initially used to identify the ribosomal methylase-encoding gene erm(B) in Campylobacter. Cloning of erm(B) into C. jejuni NCTC 11168 was performed to evaluate whether the erm(B) gene is responsible for high-level macrolide resistance in Campylobacter.nnnRESULTSnThe erm(B) gene was identified in ZC113, conferred high-level resistance to macrolides and was associated with a chromosomal multidrug-resistant genomic island (MDRGI). The MDRGI probably originated from Gram-positive bacteria and was horizontally transferred between C. coli and C. jejuni via natural transformation. Furthermore, the erm(B)-positive isolate ZC113 was resistant to all clinically important antibiotics used for treating campylobacteriosis and is essentially multidrug-resistant Campylobacter.nnnCONCLUSIONSnTo the best of our knowledge, this is the first report of a horizontally transferable macrolide resistance mechanism in thermophilic Campylobacter. Surveillance of erm(B) and its associated MDRGI in both C. coli and C. jejuni is urgently warranted.

Detection of the staphylococcal multiresistance gene cfr in Macrococcus caseolyticus and Jeotgalicoccus pinnipedialis

OBJECTIVESnTo investigate the presence and the genetic environment of the multiresistance gene cfr in Jeotgalicoccus pinnipedialis and Macrococcus caseolyticus from pigs.nnnMETHODSnA total of 391 bacterial isolates with florfenicol MICs ≥16 mg/L were obtained from nasal swabs of 557 individual pigs; of these, 75 Gram-positive isolates other than staphylococci and enterococci were screened by PCR for the presence of known florfenicol resistance genes. Species assignments of the cfr-carrying isolates were based on the results of biochemical profiling and 16S rDNA sequencing. The locations of the cfr gene were determined by Southern blotting. Regions flanking each cfr gene were sequenced by a modified random primer walking strategy, and the transferability of cfr was assessed by electrotransformation.nnnRESULTSnTwo M. caseolyticus isolates and one J. pinnipedialis isolate were cfr positive. The cfr gene was located either on a 7057 bp plasmid, pSS-03, which was widely distributed among staphylococci of pig origin, or on the ∼53 kb plasmid pJP1. The region of pJP1 that included the cfr gene and the adjacent IS21-558, showed 99.7% identity to the corresponding region of plasmid pSCFS3. In addition, the genes aadDu200a+u200aaacA-aphD, ble and erm(C), coding for aminoglycoside, bleomycin and macrolide-lincosamide-streptogramin B resistance, respectively, were also identified on plasmid pJP1.nnnCONCLUSIONSnThis study showed that plasmids carrying the multidrug resistance gene cfr are present in two new genera of commensal and environmental bacteria, Macrococcus and Jeotgalicoccus. This observation underlines the role of commensal and environmental flora in the dissemination of clinically important resistance genes, such as cfr.

Prevalence and antimicrobial resistance of Enterococcus species of food animal origin from Beijing and Shandong Province, China

To evaluate the prevalence and antimicrobial resistance of Enterococcus species from chickens and pigs in Beijing and Shandong Province, China.

Investigation of a multiresistance gene cfr that fails to mediate resistance to phenicols and oxazolidinones in Enterococcus faecalis

OBJECTIVESnTo investigate the basis of susceptibility to phenicols and oxazolidinones of the porcine Enterococcus faecalis CPPF5 despite the presence of the multiresistance gene cfr.nnnMETHODSnSouthern blotting, conjugation and transformation analyses were conducted to confirm the plasmid location and transferability of cfr in CPPF5. The genetic environment of cfr was determined by sequence analysis. Transcription and translation of cfr were examined by RT-PCR and western blotting, respectively, and modifications at A2503 within the 23S rRNA sequence were identified by primer extension.nnnRESULTSnElectrotransformation and Southern blotting indicated that CPPF5 and its transformant 5B2-3 contained two cfr-carrying plasmids ∼ 50 and ∼ 12 kb in size. The complete 12,270 bp sequence of the smaller plasmid, pCPPF5, was determined and shared 99.9% (12,269/12,270 bp) identity with the corresponding region of the cfr-carrying plasmid pEF-01 in E. faecalis of cattle origin. Moreover, the genetic environment of cfr in the ∼ 50 kb plasmid was the same as that in pCPPF5 according to sequencing results. Although cfr mRNA, Cfr protein and a modification at the A2503 site were detected, the cfr-carrying transformant 5B2-3 did not have elevated MICs of chloramphenicol, florfenicol and linezolid, indicating that cfr fails to mediate resistance to the respective antibiotics in E. faecalis.nnnCONCLUSIONSnThis is the first report of the cfr gene failing to elevate MICs of the corresponding antibiotics. Although the genetic basis for the apparent no resistance phenotype remains to be determined, this finding may have implications for surveillance studies that target the cfr gene.

Prevalence and Molecular Characterization of mcr-1-Positive Salmonella Strains Recovered from Clinical Specimens in China

ABSTRACT The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ∼30 to ∼250 kb, among which there were conjugative plasmids of ∼30 kb, ∼60 kb, and ∼250 kb and nonconjugative plasmids of ∼140 kb, ∼180 kb, and ∼240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event.