Zhangqi Shen

Novel Plasmid-Mediated Colistin Resistance Gene mcr-3 in Escherichia coli

ABSTRACT The mobile colistin resistance gene mcr-1 has attracted global attention, as it heralds the breach of polymyxins, one of the last-resort antibiotics for the treatment of severe clinical infections caused by multidrug-resistant Gram-negative bacteria. To date, six slightly different variants of mcr-1, and a second mobile colistin resistance gene, mcr-2, have been reported or annotated in the GenBank database. Here, we characterized a third mobile colistin resistance gene, mcr-3. The gene coexisted with 18 additional resistance determinants in the 261-kb IncHI2-type plasmid pWJ1 from porcine Escherichia coli. mcr-3 showed 45.0% and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively, while the deduced amino acid sequence of MCR-3 showed 99.8 to 100% and 75.6 to 94.8% identity to phosphoethanolamine transferases found in other Enterobacteriaceae species and in 10 Aeromonas species, respectively. pWJ1 was mobilized to an E. coli recipient by conjugation and contained a plasmid backbone similar to those of other mcr-1-carrying plasmids, such as pHNSHP45-2 from the original mcr-1-harboring E. coli strain. Moreover, a truncated transposon element, TnAs2, which was characterized only in Aeromonas salmonicida, was located upstream of mcr-3 in pWJ1. This ΔTnAs2-mcr-3 element was also identified in a shotgun genome sequence of a porcine E. coli isolate from Malaysia, a human Klebsiella pneumoniae isolate from Thailand, and a human Salmonella enterica serovar Typhimurium isolate from the United States. These results suggest the likelihood of a wide dissemination of the novel mobile colistin resistance gene mcr-3 among Enterobacteriaceae and aeromonads; the latter may act as a potential reservoir for mcr-3. IMPORTANCE The emergence of the plasmid-mediated colistin resistance gene mcr-1 has attracted substantial attention worldwide. Here, we examined a colistin-resistant Escherichia coli isolate that was negative for both mcr-1 and mcr-2 and discovered a novel mobile colistin resistance gene, mcr-3. The amino acid sequence of MCR-3 aligned closely with phosphoethanolamine transferases from Enterobacteriaceae and Aeromonas species originating from both clinical infections and environmental samples collected in 12 countries on four continents. Due to the ubiquitous profile of aeromonads in the environment and the potential transfer of mcr-3 between Enterobacteriaceae and Aeromonas species, the wide spread of mcr-3 may be largely underestimated. As colistin has been and still is widely used in veterinary medicine and used at increasing frequencies in human medicine, the continuous monitoring of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria is imperative for understanding and tackling the dissemination of mcr genes in both the agricultural and health care sectors. IMPORTANCE The emergence of the plasmid-mediated colistin resistance gene mcr-1 has attracted substantial attention worldwide. Here, we examined a colistin-resistant Escherichia coli isolate that was negative for both mcr-1 and mcr-2 and discovered a novel mobile colistin resistance gene, mcr-3. The amino acid sequence of MCR-3 aligned closely with phosphoethanolamine transferases from Enterobacteriaceae and Aeromonas species originating from both clinical infections and environmental samples collected in 12 countries on four continents. Due to the ubiquitous profile of aeromonads in the environment and the potential transfer of mcr-3 between Enterobacteriaceae and Aeromonas species, the wide spread of mcr-3 may be largely underestimated. As colistin has been and still is widely used in veterinary medicine and used at increasing frequencies in human medicine, the continuous monitoring of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria is imperative for understanding and tackling the dissemination of mcr genes in both the agricultural and health care sectors.

Identification of New Delhi Metallo-β-lactamase 1 in Acinetobacter lwoffii of Food Animal Origin

Background To investigate the presence of metallo-β-lactamase (MBL) genes and the genetic environment of the New Delhi metallo-β-lactamase gene bla NDM-1 in bacteria of food animal origin. Methodology/Principal Findings Gram-negative bacteria with low susceptibility to imipenem (MIC>8 µg/mL) were isolated from swab samples collected from 15 animal farms and one slaughterhouse in eastern China. These bacteria were selected for phenotypic and molecular detection of known MBL genes and antimicrobial susceptibility testing. For the bla NDM-1 positive isolate, conjugation and transformation experiments were carried out to assess plasmid transfer. Southern blotting was conducted to localize the bla NDM-1 genes, and DNA sequencing was performed to determine the sequences of bla NDM-1 and the flanking genes. In total, nine Gram-negative bacteria of four different species presented a MBL phenotype. bla NDM-1 was identified on a mobile plasmid named pAL-01 in an Acinetobacter lwoffii isolate of chicken origin. Transfer of pAL-01 from this isolate to E. coli J53 and JM109 resulted in resistance to multiple β-lactams. Sequence analysis revealed that the bla NDM-1 gene is attached to an intact insertion element ISAba125, whose right inverted repeat (IR-R) overlaps with the promoter sequence of bla NDM-1. Thus, insertion of ISAba125 likely enhances the expression of bla NDM-1. Conclusion The identification of a bla NDM-1- carrying strain of A. lwoffii in chickens suggests the potential for zoonotic transmission of bla NDM-1 and has important implications for food safety.

Early emergence of mcr-1 in Escherichia coli from food-producing animals

www.thelancet.com/infection Vol 16 March 2016 293 of the mcr-1-positive isolates would help us to understand the origin and the dissemination of this gene. We agreed that the worldwide distribution of mcr-1 might be underestimated. Considering the continuously rising trend, and now the high positive rate of mcr-1 in bacteria of animal origin, the use of colistin in veterinary practice should be urgently reconsidered.

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

BACKGROUND The 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. METHODS We 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. FINDINGS We identified 21 621 non-duplicate isolates of Enterobacteriaceae, Acinetobacter spp, and Pseudomonas aeruginosa from 18 698 inpatients and 2923 healthy volunteers. Of 17 498 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. INTERPRETATION In 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. FUNDING National Key Basic Research Program of China, National Natural Science Foundation of China/Zhejiang, National Key Research and Development Program, and MRC, UK.

Distribution of the Multidrug Resistance Gene cfr in Staphylococcus Species Isolates from Swine Farms in China

ABSTRACT A total of 149 porcine Staphylococcus isolates with florfenicol MICs of ≥16 μg/ml were screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. In total, 125 isolates carried either cfr (16 isolates), fexA (92 isolates), or both genes (17 isolates). The 33 cfr-carrying staphylococci, which included isolates of the species Staphylococcus cohnii, S. arlettae, and S. saprophyticus in which the cfr gene has not been described before, exhibited a wide variety of SmaI pulsed-field gel electrophoresis patterns. In 18 cases, the cfr gene was located on plasmids. Four different types of cfr-carrying plasmids—pSS-01 (n = 2; 40 kb), pSS-02 (n = 3; 35.4 kb), pSS-03 (n = 10; 7.1 kb), and pBS-01 (n = 3; 16.4 kb)—were differentiated on the basis of their sizes, restriction patterns, and additional resistance genes. Sequence analysis revealed that in plasmid pSS-01, the cfr gene was flanked in the upstream part by a complete aacA-aphD-carrying Tn4001-like transposon and in the downstream part by a complete fexA-carrying transposon Tn558. In plasmid pSS-02, an insertion sequence IS21-558 and the cfr gene were integrated into transposon Tn558 and thereby truncated the tnpA and tnpB genes. The smallest cfr-carrying plasmid pSS-03 carried the macrolide-lincosamide-streptogramin B resistance gene erm(C). Plasmid pBS-01, previously described in Bacillus spp., harbored a Tn917-like transposon, including the macrolide-lincosamide-streptogramin B resistance gene erm(B) in the cfr downstream region. Plasmids, which in part carry additional resistance genes, seem to play an important role in the dissemination of the gene cfr among porcine staphylococci.

Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production

By 2030, the global population will be 8.5 billion, placing pressure on international poultry production, of which China is a key producer1. From April 2017, China will implement the withdrawal of colistin as a growth promoter, removing over 8,000 tonnes per year from the Chinese farming sector2. To understand the impact of banning colistin and the epidemiology of multi-drug-resistant (MDR) Escherichia coli (using blaNDM and mcr-1 as marker genes), we sampled poultry, dogs, sewage, wild birds and flies. Here, we show that mcr-1, but not blaNDM, is prevalent in hatcheries, but blaNDM quickly contaminates flocks through dogs, flies and wild birds. We also screened samples directly for resistance genes to understand the true breadth and depth of the environmental and animal resistome. Direct sample testing for blaNDM and mcr-1 in hatcheries, commercial farms, a slaughterhouse and supermarkets revealed considerably higher levels of positive samples than the blaNDM- and mcr-1-positive E. coli, indicating a substantial segment of unseen resistome—a phenomenon we have termed the ‘phantom resistome’. Whole-genome sequencing identified common blaNDM-positive E. coli shared among farms, flies, dogs and farmers, providing direct evidence of carbapenem-resistant E. coli transmission and environmental contamination.

Campylobacter in Poultry: Ecology and Potential Interventions

SUMMARY Avian hosts constitute a natural reservoir for thermophilic Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, and poultry flocks are frequently colonized in the intestinal tract with high numbers of the organisms. Prevalence rates in poultry, especially in slaughter-age broiler flocks, could reach as high as 100% on some farms. Despite the extensive colonization, Campylobacter is essentially a commensal in birds, although limited evidence has implicated the organism as a poultry pathogen. Although Campylobacter is insignificant for poultry health, it is a leading cause of food-borne gastroenteritis in humans worldwide, and contaminated poultry meat is recognized as the main source for human exposure. Therefore, considerable research efforts have been devoted to the development of interventions to diminish Campylobacter contamination in poultry, with the intention to reduce the burden of food-borne illnesses. During the past decade, significant advance has been made in understanding Campylobacter in poultry. This review summarizes the current knowledge with an emphasis on ecology, antibiotic resistance, and potential pre- and postharvest interventions.

Transferable multiresistance plasmids carrying cfr in Enterococcus spp. from swine and farm environment.

ABSTRACT Seventy-seven porcine Enterococcus isolates with florfenicol MICs of ≥16 μg of were/ml screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. Three isolates—Enterococcus thailandicus 3-38 (from a porcine rectal swab collected at a pig farm), Enterococcus thailandicus W3, and Enterococcus faecalis W9-2 (the latter two from sewage at a different farm), carried the cfr gene. The SmaI pulsed-field gel electrophoresis patterns of the three isolates differed distinctly. In addition, E. faecalis W9-2 was assigned to a new multilocus sequence type ST469. Mating experiments and Southern blot analysis indicated that cfr is located on conjugative plasmids pW3 (∼75 kb) from E. thailandicus W3, p3-38 (∼72 kb) from E. thailandicus 3-38, and pW9-2 (∼55 kb) from E. faecalis W9-2; these plasmids differed in their sizes, additional resistance genes, and the analysis of the segments encompassing the cfr gene. Sequence analysis revealed that all plasmids harbored a 4,447-bp central region, in which cfr was bracketed by two copies of the novel insertion sequence ISEnfa4 located in the same orientation. The sequences flanking the central regions of these plasmids, including the partial tra gene regions and a ω-ε-ζ toxin-antitoxin module, exhibited >95% nucleotide sequence identity to the conjugative plasmid pAMβ1 from E. faecalis. Conjugative plasmids carrying cfr appear to play an important role in the dissemination and maintenance of the multiresistance gene cfr among enterococcal isolates and possibly other species of Gram-positive bacteria.

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

OBJECTIVES To investigate the presence and the genetic environment of the multiresistance gene cfr in naturally occurring Gram-negative bacteria of pigs. METHODS A 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. RESULTS A 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. CONCLUSIONS To 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.

Identification of a Novel Genomic Island Conferring Resistance to Multiple Aminoglycoside Antibiotics in Campylobacter coli

ABSTRACT Historically, the incidence of gentamicin resistance in Campylobacter has been very low, but recent studies reported a high prevalence of gentamicin-resistant Campylobacter isolated from food-producing animals in China. The reason for the high prevalence was unknown and was addressed in this study. PCR screening identified aminoglycoside resistance genes aphA-3 and aphA-7 and the aadE–sat4–aphA-3 cluster among 41 Campylobacter isolates from broiler chickens. Importantly, a novel genomic island carrying multiple aminoglycoside resistance genes was identified in 26 aminoglycoside resistant Campylobacter coli strains. Sequence analysis revealed that the genomic island was inserted between cadF and COO1582 on the C. coli chromosome and consists of 14 open reading frames (ORFs), including 6 genes (the aadE–sat4–aphA-3 cluster, aacA-aphD, aac, and aadE) encoding aminoglycoside-modifying enzymes. Analysis by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing indicated that the C. coli isolates carrying this unique genomic island were clonal, and the clone of PFGE subtype III and sequence type (ST) 1625 was particularly predominant among the C. coli isolates examined, suggesting that clonal expansion may be involved in dissemination of this resistance island. Additionally, we were able to transfer this genomic island from C. coli to a Campylobacter jejuni strain using natural transformation under laboratory conditions, and the transfer resulted in a drastic increase in aminoglycoside resistance in the recipient strain. These findings identify a previously undescribed genomic island that confers resistance to multiple aminoglycoside antibiotics. Since aminoglycoside antibiotics are used for treating occasional systemic infections caused by Campylobacter, the emergence and spread of this antibiotic resistance genomic island represent a potential concern for public health.