IncX3 Plasmid Research Articles

Infection of Cronobacter sakazakii ST1 Producing SHV-12 in a Premature Infant Born from Triplet Pregnancy

Cronobacter sakazakii can cause severe life-threatening invasive infections in neonates, with a high mortality rate mostly associated with powdered infant formula consumption. The study describes a fatal C. sakazakii infection in premature infant fed only with expressed human milk. Despite the identification of etiological factor from patient’s blood, the epidemiological investigation, including mother’s skin, hospital surfaces, milk expressing devices, and milk samples, did not show bacterial contamination. The infection was caused by C. sakazakii ST1, being one of the leading genotypes reported in invasive infections. The phylogenetic analysis of the international collection of the ST1 organisms allowed us to identify the isolate as a member of the main cluster. The pathogenic potential of the isolate was augmented by the presence of IncFIB-like molecule representing virulence plasmids of pESA-3 family. Isolate presented ESBL phenotype associated with blaSHV-12 gene harboured by IncX3 plasmid. The described case gave valuable information to genetics of Cronobacter, and also urges the need of wider whole-genome sequencing implementation as a part of diagnostic procedure.

Detection of a NDM-5-producing Klebsiella pneumoniae sequence type 340 (CG258) high-risk clone in swine

The emergence and rapid increase of carbapenem-resistant Enterobacteriaceae among food-producing animals poses a serious threat to public health. The aim of this study was to investigate the presence and dissemination of blaNDM-5 in porcine Klebsiella pneumoniae isolates. Of 19 meropenem-resistant K. pneumoniae isolates, 18 were blaNDM-5-positive and one carried blaNDM-1. Susceptibility testing indicated that all blaNDM-carrying K. pneumoniae showed a multiple drug resistance (MDR) profile. The blaNDM-5 gene was located on a conjugative IncX3 plasmid of ∼46-kb in all 18 blaNDM-5-carrying isolates. MLST analysis revealed that ST340 (n = 8), a member of the worldwide existing high-risk epidemic clonal group 258 (CG258), was predominant. Furthermore, whole genome sequence (WGS) analysis for one representative ST340 K. pneumoniae Kp19110124 showed a MDR profile for a wide range of antimicrobial agents, including meropenem, various cephalosporins, azteonam, gentamicin, ciprofloxacin, and florfenicol. Phylogenetic analysis exhibited that K. pneumoniae ST340 strains were clustered into one branch, which had spread across host species and across continents. Among them, K. pneumoniae Kp19110124 displayed a very close relationship with a clinical isolate collected from a patient in Canada. In conclusion, these results reveal the presence of a blaNDM-5-carrying conjugative IncX3 type plasmid into K. pneumoniae ST340 clone, which then may accelerate the dissemination of the blaNDM-5 gene in porcine K. pneumoniae isolates. The detection of high-risk blaNDM-5-carrying K. pneumoniae ST340 in food-producing animal poses a serious threat to public health.

Clonal Dissemination of Multiple Carbapenemase Genes in Carbapenem-Resistant Enterobacterales Mediated by Multiple Plasmids in China.

BackgroundCarbapenem-resistant Enterobacterales (CRE) are rapidly increasing worldwide in last two decades and lead few antibiotics for treatment. The molecular epidemiology of CRE in China was investigated to provide basis for clinical rational use of antibiotics and prevent its spread.MethodsAll CRE isolates in this study were collected from 11 hospitals from October 2015 to July 2018. The isolates were subjected to antimicrobial susceptibility tests, PCR molecular identification, pulsed-field gel electrophoresis, and multilocus sequence typing.ResultsAmong the 399 CRE isolates, 51.6% (206/399) harbored carbapenemase genes. Three carbapenemase genes were detected, namely blaKPC-2, blaNDM-1, and blaIMP at rates of 29.8% (119/399), 17.5% (70/399), and 4.0% (16/399), respectively. In Klebsiella pneumoniae (350) and Escherichia coli (26), blaKPC-2 (33.4%, 117/350) and blaNDM-1 (61.5%, 16/26) were the predominant genes. The most common genes in the CRE isolates were blaKPC (85.5%) and blaNDM-1 (76.5%) from adults and children, respectively. Particularly, ST11 K. pneumoniae with blaKPC-2 harbored by IncFII plasmids were distributed in both general and primary hospitals, suggesting a clonal transmission pattern at these sites. In addition, the clonal distribution of ST2407 K. pneumoniae with blaNDM-1 located on IncX3 plasmids and blaIMP-38-positive ST307 K. pneumoniae were detected in a children’s hospital.ConclusionThe distribution of carbapenemase genes differed among strains and age groups. Multiple carbapenemase genes in the CRE strains were clonally disseminated in the tested regions mediated by multiple plasmids. Therefore, CRE monitoring should be increased and measures should be adopted to prevent its transmission.

High prevalence of multidrug-resistant Escherichia coli and first detection of IncHI2/IncX4-plasmid carrying mcr-1 E. coli in retail ready-to-eat foods in China

Antibiotic-resistant bacteria in food pose an important threat to public health. Multidrug-resistant strains in ready-to-eat (RTE) foods can be transferred to humans through diet, which increases their health risk. This study systematically investigated antibiotic resistance and antibiotic resistance genes in E. coli isolated from retail RTE foods and characterized plasmid-mediated colistin-resistant E. coli strains. A total of 1118 RTE food samples were collected from markets in 39 cities in China, and 126 E. coli strains, >95% of which were multidrug-resistant, were isolated. The isolates showed a high prevalence of resistance to tetracycline (95.24%), ampicillin (82.54%), trimethoprim-sulfamethoxazole (77.78%), nalidixic acid (74.60%), cephalothin (72.22%), chloramphenicol (66.67%), and streptomycin (53.97%). Twenty-two extended-spectrum β-lactamase (ESBL)-producing E. coli and four colistin-resistant E. coli were identified. The resistance genes TEM, CTX-M, tetA, sul2, strA/strB, aadA, and qnrS were the most frequently detected. CTX-M-55 and CTX-M-14 were the predominant CTX-M types. All the four colistin-resistant E. coli isolates were positive for mcr-1. The mcr-1 gene can be transferred to E. coli C600 through conjugation and transformation. Whole-genome sequencing revealed that the mcr-1 genes were found in IncX4 and IncHI2 plasmids. To the best of our knowledge, this is the first report of IncHI2/IncX4 plasmid-bearing mcr-1-positive E. coli strains in RTE foods sold in markets, and the first report of the isolation of the international epidemic E. coli clone ST101 and mcr-1-carrying ESBL-producing E. coli from RTE foods. These results provide valuable information for assessing antibiotic-resistant E. coli infections and controlling antibiotic-resistant E. coli.

Emergence of IncHI2 Plasmid-Harboring blaNDM-5 from Porcine Escherichia coli Isolates in Guangdong, China.

Carbapenem resistance has posed potential harmful risks to human and animals. The objectives of this study were to understand the prevalence of blaNDM-5 in pigs and investigate the molecular characteristics of NDM-5-producing Escherichia coli isolates in Guangdong province in China. Carbapenem-resistant E. coli isolates were isolated from pigs and obtained using MacConkey plates containing 0.5 mg/L meropenem. Conjugation assay and antimicrobial susceptibility testing were conducted for the isolates and their transconjugants. Whole-genome sequence (WGS) was used to analyze the plasmid genetic feature. A total of five blaNDM-5-carrying E. coli isolates were obtained in the present investigations. They belonged to five ST types. The blaNDM-5 genes were found to be in IncX3 and IncHI2 plasmid. The IncX3 plasmid was 46,161 bp in size and identical to other reports. IncHI2 plasmid was 246,593 bp in size and similar to other IncHI2-ST3 plasmids. It consisted of a typical IncHI2 plasmid backbone region and a multiresistance region (MRR). The blaNDM-5 was closely associated with the IS3000-ISAba125-blaNDM-5-bleMBL-trpF-tat-IS26 unit. We first reported the blaNDM-5-carrying IncHI2 in E. coli isolates recovered from pigs and revealed the molecular characterization. Continued surveillance for the dissemination of blaNDM-5 among food-producing animals is required.

Antimicrobial-Resistant Escherichia coli Strains and Their Plasmids in People, Poultry, and Chicken Meat in Laos.

Antimicrobial resistant (AMR) Enterobacterales are widely distributed among the healthy population of the Indochinese peninsula, including Laos. However, the local reservoir of these pathogens are currently not known and possible sources such as agricultural settings and food have rarely been analyzed. In this work, we investigated the extended-spectrum cephalosporin- (ESC-) and colistin-resistant Escherichia coli strains (CST-R-Ec) isolated from the gut of local people, feces of poultry, and from chicken meat (60 samples each group) in Laos. Whole-genome sequencing (WGS) analysis based on both short- and long-read sequencing approaches were implemented. The following prevalence of ESC-R-Ec and CST-R-Ec were recorded, respectively: local people (70 and 15%), poultry (20 and 23.3%), and chicken meat (21.7 and 13.3%). Core-genome analysis, coupled with sequence type (ST)/core-genome ST (cgST) definitions, indicated that no common AMR-Ec clones were spreading among the different settings. ESC-R-Ec mostly possessed blaCTX–M–15 and blaCTX–M–55 associated to ISEcp1 or IS26. The majority of CST-R-Ec carried mcr-1 on IncX4, IncI2, IncP1, and IncHI1 plasmids similar or identical to those described worldwide; strains with chromosomal mcr-1 or possessing plasmid-mediated mcr-3 were also found. These results indicate a high prevalence of AMR-Ec in the local population, poultry, and chicken meat. While we did not observe the same clones among the three settings, most of the blaCTX–Ms and mcr-1/-3 were associated with mobile-genetic elements, indicating that horizontal gene transfer may play an important role in the dissemination of AMR-Ec in Laos. More studies should be planned to better understand the extent and dynamics of this phenomenon.

Antimicrobial resistant bacteria recovered from retail ground meat products in the US include a Raoultella ornithinolytica co-harboring blaKPC-2 and blaNDM-5

Retail beef and pork, including processed products, can serve as vehicles for the zoonotic foodborne transmission of pathogens and antimicrobial resistant bacteria. However, processed and seasoned products like sausages, are not often included in research and surveillance programs. The objective of this study was to investigate retail ground beef and pork, including processed products, for the presence of common foodborne pathogens and antimicrobial resistant bacteria. We purchased 763 packages of fresh and fully cooked retail meat products during 29 visits to 17 grocery stores representing seven major grocery chains located in west and central Ohio. Each package of meat was evaluated for contamination with methicillin-resistant Staphylococcus aureus (MRSA), Salmonella spp., Enterobacteriaceae expressing extended-spectrum cephalosporin resistance, and carbapenemase-producing organisms (CPO). Only 3 of the 144 (2.1%) packages of fully cooked meat products contained any of these organisms, 1 with an extended-spectrum β-lactamase-producing (ESBL) Enterobacteriaceae and 2 with CPO. Among the 619 fresh meat products, we found that 85 (13.7%) packages were contaminated with MRSA, 19 (3.1%) with Salmonella, 136 (22.0%) with Enterobacteriaceae expressing an AmpC (blaCMY) resistance genotype, 25 (4.0%) with Enterobacteriaceae expressing an ESBL (blaCTX-M) resistance genotype, and 31 (5.0%) with CPO, primarily environmental organisms expressing intrinsic carbapenem resistance. However, one CPO, a Raoultella ornithinolytica, isolated from pork sausage co-harbored both blaKPC-2 and blaNDM-5 on IncN and IncX3 plasmids, respectively. Our findings suggest that fresh retail meat, including processed products can be important vehicles for the transmission of foodborne pathogens and antimicrobial resistant bacteria, including those with epidemic carbapenemase-producing genotypes.

The Emergence and Molecular Characteristics of New Delhi Metallo β-Lactamase-Producing Escherichia coli From Ducks in Guangdong, China.

This study aimed to determine the prevalence and transmission characteristics of New Delhi metallo β-lactamase (NDM)-producing Escherichia coli from ducks in Guangdong, China. In this study, a total of 28 NDM-producing E. coli isolates were recovered from 88 unduplicated diseased duck samples (31.8%) from veterinary clinics in Guangzhou, Foshan, Qingyuan, and Huizhou. Two variants, blaNDM−1 and blaNDM−5, were detected and the latter was present in 89.6% of the isolates (25/28). Multilocus sequence typing (MLST) analysis indicated that these E. coli isolates possessed six distinct STs, and ST156 was the most prevalent followed by ST648, ST746, ST354, ST10, and ST162. In addition, phylogenomic analysis found that two of the isolates that were recovered from a single sample possessed different genomes, and the blaNDM-carrying IncX3 plasmids may be horizontal transfer between E. coli isolates in the intestinal tracts of ducks. Whole-genome sequencing (WGS) analysis further revealed that blaNDM co-existed with other 25 types of antimicrobial resistance genes (ARGs), of which 16 ARGs were highly prevalent with detection rates >50%, and a high incidence of coproducing blaNDM and mcr-1 E. coli isolates (22/88, 25.0%) was detected in ducks. This study underscores the importance of surveillance for blaNDM-harboring microbes in ducks.

Genomic Investigation of Carbapenem-Resistant Klebsiella pneumonia Colonization in an Intensive Care Unit in South Africa.

The study investigated carbapenemase-producing Klebsiella pneumoniae (CPKP) isolates of patients in an intensive care unit (ICU) in a public hospital in the KwaZulu-Natal province, South Africa using whole-genome sequencing (WGS). Ninety-seven rectal swabs, collected from all consenting adult patients (n = 31) on days 1, 3, and 7 and then weekly, were screened for carbapenemase-production using Chrome-ID selective media. Antibiotic susceptibility was determined for the fourteen positive CPKP isolates obtained using the VITEK 2 automated system. All isolates (100%) were resistant to ertapenem and meropenem, and 71.4% (n = 10) were resistant to imipenem. All CPKP isolates were subjected to ERIC/PCR, and a sub-sample of isolates was selected for WGS based on their antibiograms and clonality. All sequenced isolates harbored the blaOXA-181 carbapenemase (100%) and co-carried other β-lactamase genes such as blaOXA-1, blaCTX-M-15, blaTEM-1B, and blaSHV-1. IncF, IncX3, and Col plasmid replicons groups and class I integrons (ln191 and ln27) were detected. All isolates belonged to the same sequence type ST307 and capsular serotypes (K102, O2v2). All the isolates carried the same virulence repertoire, reflecting the epidemiological relationship between isolates. blaOXA-181 was located on a multi-replicon plasmid similar to that of E. coli p010_B-OXA181, and isolates were aligned with several South African and international clades, demonstrating horizontal and vertical transboundary distribution. The findings suggest that blaOXA-181 producing K. pneumoniae is endemic in this ICU, colonizing the patients. CRE screening and enhanced infection prevention and control measures are urgently required.

Genomic Characterization of mcr-1.1-Producing Escherichia coli Recovered From Human Infections in São Paulo, Brazil

Polymyxins are one of most important antibiotics available for multidrug-resistant Gram-negative infections. Diverse chromosomal resistance mechanisms have been described, but the polymyxin resistance phenotype is not yet completely understood. The objective of this study was to characterize colistin resistant mcr-1-producing strains isolated from human infections over one year in a hospital setting (Hospital das Clínicas, São Paulo, Brazil). We isolated 490 colistin-resistant Gram-negative rods, of which eight were mcr-1.1-positive Escherichia coli, the only species with this result, indicating a low incidence of the mcr-1 production mechanism among colistin-resistant isolates. All mcr-1.1 positive isolates showed similarly low MICs for colistin and were susceptible to most antibiotics tested. The isolates showed diversity of MLST classification. The eight mcr-1.1-positive E. coli genomes were sequenced. In seven of eight isolates the mcr-1.1 gene is located in a contig that is presumed to be a part of an IncX4 plasmid; in one isolate, it is located in a contig that is presumed to be part of an IncHI2A plasmid. Three different genomic contexts for mcr-1.1 were observed, including a genomic cassette mcr-1.1-pap2 disrupting a DUF2806 domain-containing gene in six isolates. In addition, an IS1-family transposase was found inserted next to the mcr-1.1 cassette in one isolate. An mcr-1.1-pap2 genomic cassette not disrupting any gene was identified in another isolate. Our results suggest that plasmid dissemination of hospital-resident strains took place during the study period and highlight the need for continued genomic surveillance.

The Formation of Two Hybrid Plasmids Mediated by IS26 and Tn6952 in Salmonella enterica Serotype Enteritidis.

To characterize the formation mechanism and characteristics of two cointegrate plasmids in Salmonella enterica serotype Enteritidis strain S13, plasmids from strain S13 and three corresponding transconjugants were subjected to whole genome sequencing and analyzed using bioinformatics tools. The traits of two fusion plasmids in transconjugants were characterized by stability and conjugation experiments. Sequence analysis indicated that strain S13 contained four plasmids, including mcr-1-bearing pS13-1, blaCTX–M–55-carrying pS13-2, tet(M)-bearing pS13-3, and floR-carrying pS13-4. IncN1-F33:A–:B– plasmid pS13-2, respectively, fused with IncFI:A–:B– plasmid pS13-3 and IncX1 plasmid pS13-4, which generated two cointegrate plasmids, designated pS13D and pS13F, which involved in two intermolecular replicative mechanisms mediated by IS26 and the novel transposon Tn6952 (ΔTnAS3-IS26-ΔISEcp1-ramA-ΔIS26-ΔTnAS1), respectively. This is the first report of the fusion of the IncN1-F33:A–:B– plasmid and IncFI:A–:B– plasmid mediated by IS26, and with IncX1 plasmid mediated by Tn6952. The formation and evolution of cointegrate plasmids could expand the resistance and host spectrum of fusion plasmids.

Detection of a clinical carbapenem-resistant Citrobacter portucalensis strain and the dissemination of C. portucalensis in clinical settings

A clinical carbapenem-resistant Citrobacter portucalensis was characterised by whole-genome sequencing (WGS). Strain 3839 was identified by VITEK®2.0 and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antimicrobial susceptibility testing was performed by microbroth dilution. WGS followed by bioinformatics analysis was performed. Strain 3839 was initially identified as Citrobacter freundii by VITEK and MALDI-TOF/MS, and was subsequently demonstrated to be C. portucalensis by WGS analysis. Through average nucleotide identity analysis, we detected 55 C. portucalensis genomes misidentified as C. freundii in GenBank, and at least 22 were clinically-associated, suggesting that the occurrence of C. portucalensis in the clinical setting might be underestimated by the conventional method. Strain 3839 was extensively drug-resistant and the presence of multiple resistance determinants was detected by WGS, including blaNDM-1, blaSHV-12, blaCMY-150, blaOXA-1, qnrB9, qnrA1, aac(6')-Ib-cr, aph(6)-Id_1, aph(3'')-Ib, tetA, tet34 and catB3. A total of 45 insertion sequence (IS) elements, 8 phages and 1 integron gene cassette were identified. The blaNDM-1 gene was carried by an IncX3 plasmid that was identical to a plasmid detected in a C. freundii strain. The genetic context of blaNDM-1 was IS30-blaNDM-1-bleMBL-trpF-dsbD-cutA1-groES-groEL-IS91. To our knowledge, this is the first report of carbapenem-resistant C. portucalensis isolated from a clinical sample. The blaNDM-1 gene carried by C. portucalensis may transmit among Citrobacter spp. mediated by plasmids. Together with underestimation of its clinical occurrence caused by misidentification, our study warrants the necessity of preventing the dissemination of such emerging drug-resistant species in clinical settings.

Coexistence of two blaCTX-M-14 genes in a blaNDM-5-carrying multidrug-resistant Escherichia coli strain recovered from a bloodstream infection in China

The emergence of carbapenem-resistant Enterobacteriaceae has become a serious public-health threat. Here we report the complete genome sequence of a multidrug-resistant (MDR) Escherichia coli carrying blaNDM-5 and two copies of blaCTX-M-14 recovered from a bloodstream infection in China. Whole-genome sequencing of E. coli strain 2D was performed both using Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. De novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler. In silico multilocus sequence typing (MLST), antimicrobial resistance genes and plasmid replicons were identified from the genome sequence. Core genome MLST (cgMLST) analysis between E. coli 2D and all ST354 E. coli strains retrieved from the NCBI GenBank database was performed using BacWGSTdb 2.0 server. The complete genome sequence of E. coli 2D consists of six contigs comprising 5 363 300 bp, including one chromosome and five plasmids, and was assigned to ST354. Six antimicrobial resistance genes were identified, including blaNDM-5 located in a 46 161-bp IncX3 plasmid and two copies of the blaCTX-M-14 gene located both in the chromosome and in a 96 499-bp IncB plasmid. The closest relative of E. coli 2D was another isolate recovered from Lebanon, which differed by 162 cgMLST loci. This study reports the first genome sequence of a MDR E. coli carrying blaNDM-5 and two copies of blaCTX-M-14 in China. These data may help to understand the antimicrobial resistance mechanisms and transmission dynamics of carbapenem-resistant Enterobacteriaceae in clinical settings.

OXA-484, an OXA-48-Type Carbapenem-Hydrolyzing Class D β-Lactamase From Escherichia coli.

OXA-48-like carbapenemases are among the most frequent carbapenemases in Gram-negative Enterobacterales worldwide with the highest prevalence in the Middle East, North Africa and Europe. Here, we investigated the so far uncharacterized carbapenemase OXA-484 from a clinical E. coli isolate belonging to the high-risk clone ST410 regarding antibiotic resistance pattern, horizontal gene transfer (HGT) and genetic support. OXA-484 differs by the amino acid substitution 214G compared to the most closely related variants OXA-181 (214R) and OXA-232 (214S). The blaOXA–484 was carried on a self-transmissible 51.5 kb IncX3 plasmid (pOXA-484) showing high sequence similarity with plasmids harboring blaOXA–181. Intraspecies and intergenus HGT of pOXA-484 to different recipients occurred at low frequencies of 1.4 × 10–7 to 2.1 × 10–6. OXA-484 increased MICs of temocillin and carbapenems similar to OXA-232 and OXA-244, but lower compared with OXA-48 and OXA-181. Hence, OXA-484 combines properties of OXA-181-like plasmid support and transferability as well as β-lactamase activity of OXA-232.

First Report of blaNDM-1 Bearing IncX3 Plasmid in Clinically Isolated ST11 Klebsiella pneumoniae from Pakistan.

The New Delhi Metallo-β-lactamase (NDM) is among the most threatening forms of carbapenemases produced by K. pneumoniae, well-known to cause severe worldwide infections. The molecular epidemiology of blaNDM-1-harboring K. pneumoniae is not well elucidated in Pakistan. Herein, we aim to determine the antibiotics-resistance profile, genes type, molecular type, and plasmid analysis of 125 clinically isolated K. pneumoniae strains from urine samples during July 2018 to January 2019 in Pakistan. A total of 34 (27.2%) K. pneumoniae isolates were carbapenemases producers, and 23 (18.4%) harbored the blaNDM-1 gene. The other carbapenemases encoding genes, i.e., blaIMP-1 (7.2%), blaVIM-1 (3.2%), and blaOXA-48 (2.4%) were also detected. The Multi Locus Sequence Typing (MLST) results revealed that all blaNDM-1-harboring isolates were ST11. The other sequence types detected were ST1, ST37, and ST105. The cluster analysis of Xbal Pulsed Field Gel Electrophoresis (PFGE) revealed variation amongst the clusters of the identical sequence type isolates. The blaNDM-1 gene in all of the isolates was located on a 45-kb IncX3 plasmid, successfully transconjugated. For the first time, blaNDM-1-bearing IncX3 plasmids were identified from Pakistan, and this might be a new primary vehicle for disseminating blaNDM-1 in Enterobacteriaceae as it has a high rate of transferability.

Genomic Characterization of Salmonella enterica Isolates From Retail Meat in Beijing, China.

Salmonella enterica remains one of the leading causes of foodborne bacterial disease. Retail meat is a major source of human salmonellosis. However, comparative genomic analyses of S. enterica isolates from retail meat from different sources in China are lacking. A total of 341 S. enterica strains were isolated from retail meat in sixteen districts of Beijing, China, at three different time points (January 1st, May 1st, and October 1st) in 2017. Comparative genomics was performed to investigate the genetic diversity, virulence and antimicrobial resistance gene (ARG) profiles of these isolates. The most common serotype was S. Enteritidis (203/341, 59.5%), which dominated among isolates from three different time points during the year. Laboratory retesting confirmed the accuracy of the serotyping results predicted by the Salmonella In Silico Typing Resource (SISTR) (96.5%). The pangenome of the 341 S. enterica isolates contained 13,931 genes, and the core genome contained 3,635 genes. Higher Salmonella phage 118970 sal3 (219/341, 64.2%) and Gifsy-2 (206/341, 60.4%) prevalence contributed to the diversity of the accessory genes, especially those with unknown functions. IncFII(S), IncX1, and IncFIB(S) plasmid replicons were more common in these isolates and were major sources of horizontally acquired foreign genes. The virulence gene profile showed fewer virulence genes associated with type III secretion systems in certain isolates from chicken. A total of 88 different ARGs were found in the 341 isolates. Three beta-lactamases, namely, blaCTX–M–55 (n = 15), blaCTX–M–14 (n = 11), and blaCTX–M–65 (n = 11), were more prevalent in retail meats. The emergence of qnrE1 and blaCTX–M–123 indicated a potential increase in the prevalence of retail meats. After the prohibition of colistin in China, three and four isolates were positive for the colistin resistance genes mcr-1.1 and mcr-9, respectively. Thus, we explored the evolution and genomic features of S. enterica isolates from retail meats in Beijing, China. The diverse ARGs of these isolates compromise food security and are a clinical threat.

Genomic Diversity and Virulence Potential of ESBL- and AmpC-β-Lactamase-Producing Escherichia coli Strains From Healthy Food Animals Across Europe.

The role of livestock animals as a putative source of ESBL/pAmpC E. coli for humans is a central issue of research. In a large-scale pan-European surveillance, 2,993 commensal Escherichia spp. isolates were recovered from randomly collected fecal samples of healthy cattle, pigs and chickens in various abattoirs. One-hundred Escherichia spp. isolates (0.5% from cattle, 1.3% pigs, 8.0% chickens) fulfilled the criteria for cefotaxime and ceftazidime non-wildtype (EUCAST). In silico screening of WGS data of 99 isolates (98 E. coli and 1 E. fergusonii) revealed blaSHV–12 (32.3%), blaCTX–M–1 (24.2%), and blaCMY–2 (22.2%) as predominant ESBL/pAmpC types. Other types were blaSHV–2 (1.0%), blaCTX–M–2/–14/–15 (1.0/6.1/1.0%), and blaTEM–52 (5.1%). Six isolates revealed AmpC-promoter mutations (position −42 (C > T) and one carried mcr-1. The majority (91.3%) of ESBL/pAmpC genes were located on plasmids. SHV-12 was mainly (50%) encoded on IncI1α plasmids (pST-3/-26/-95), followed by IncX3 (12.5%) and IncK2 (3.1%). The blaTEM–52 genes were located on IncI1α-pST-36 (60%) and IncX1 plasmids (20%). The dominant plasmid lineage among CTX-M-1 isolates was IncI1α (pST-3/-295/-317) (87.5%), followed by IncN-pST-1 (8.3%). CMY-2 was mostly identified on IncI1α (pST-12/-2) (54.5%) and IncK2 (31.8%) plasmids. Several plasmids revealed high similarity to published plasmids from human and animal Enterobacteriaceae. The isolates were assigned to phylogroups A/C (34.7/7.1%), B1 (27.6%), B2 (3.1%), D/F (9.2/10.2%), E (5.1%), and to E. clades (3.0%). With 51 known and 2 novel MLST types, a wide variety of STs was found, including STs previously observed in human isolates (ST10/38/117/131/648). ESBL/AmpC types or STs were rarely correlated with the geographic origin of the isolates or animal species. Virulence gene typing identified extraintestinal pathogenic E. coli (ExPEC; 2.0%), avian pathogenic E. coli (APEC; 51.5%), and atypical enteropathogenic E. coli (EPEC; 6.1%). In conclusion, the high diversity of STs and phylogenetic groups provides hardly any hint for clonal spread of single lineages but hints toward the dissemination of cephalosporin resistance genes in livestock via distinct, globally successful plasmid lineages. Even though a number of isolates could not be assigned to a distinct pathotype, our finding of combined multidrug-resistance and virulence in this facultative pathogen should be considered an additional threat to public health.

A Large Spatial Survey of Colistin-Resistant Gene mcr-1-Carrying E. coli in Rivers across Taiwan.

Background: Colistin is one of the last-line antimicrobial agents against life-threatening infections. The distribution of the colistin resistance gene mcr-1 has been reported worldwide. However, most studies have focused on the distribution of mcr-1-positive bacteria in humans, animals, food, and sewage; few have focused on their distribution in natural environments. Method: We conducted a large spatial survey of mcr-1-positive Escherichia coli at 119 sites in 48 rivers, covering the entire island of Taiwan. We investigated the relationship between the livestock or poultry density in the surveyed riverine area and the number of mcr-1-positive E. coli in the river water. We then sequenced and characterized the isolated mcr-1-positive plasmids. Results: Seven mcr-1 positive E. coli were isolated from 5.9% of the sampling sites. The mcr-1-positive sites correlated with high chicken and pig stocking densities but not human population density or other river parameters. Four of the mcr-1-positive E. coli strains harbored epidemic IncX4 plasmids, and three of them exhibited identical sequences with a size of 33,309 bp. One of the plasmids contained identical 33,309 bp sequences but carried an additional 5711-bp transposon (Tn3 family). To our knowledge, this is the first demonstration that mcr-1-carrying IncX4 plasmids can contain an insertion of such transposons. All mcr-1-positive isolates belonged to phylogenetic group A and harbored few known virulence genes. Conclusion: This study showed a positive relationship between the number of mcr-1-positive sites and high livestock and poultry density. The sequencing analyses indicated that the epidemic plasmid in the mcr-1 isolates circulates not only in humans, animals, and food but also in the associated environments or natural habitats in Taiwan, suggesting that the surveillance of antibiotics-resistance genes for livestock or poultry farm quality control should include their associated environments.

Genetic relatedness of multidrug resistant Escherichia coli isolated from humans, chickens and poultry environments

BackgroundInappropriate use of antimicrobial agents in animal production has led to the development of antimicrobial resistance (AMR) in foodborne pathogens. Transmission of AMR foodborne pathogens from reservoirs, particularly chickens to the human population does occur. Recently, we reported that occupational exposure was a risk factor for multidrug-resistant (MDR) Escherichia coli (E. coli) among poultry-workers. Here we determined the prevalence and genetic relatedness among MDR E. coli isolated from poultry-workers, chickens, and poultry environments in Abuja, Nigeria. This study was conducted to address the gaps identified by the Nigerian AMR situation analysis.MethodsWe conducted a cross-sectional study among poultry-workers, chickens, and poultry farm/live bird market (LBM) environments. The isolates were tested phenotypically for their antimicrobial susceptibility profiles, genotypically characterized using whole-genome sequencing (WGS) and in silico multilocus sequence types (MLST). We conducted a phylogenetic single nucleotide polymorphism (SNPs) analysis to determine relatedness and clonality among the isolates.ResultsA total of 115 (26.8%) out of 429 samples were positive for E. coli. Of these, 110 isolates were viable for phenotypic and genotypic characterization. The selection comprised 47 (42.7%) isolates from poultry-workers, 36 (32.7%) from chickens, and 27 (24.5%) from poultry-farm or LBM environments. Overall, 101 (91.8%) of the isolates were MDR conferring resistance to at least three drug classes. High frequency of resistance was observed for tetracycline (n = 102; 92.7%), trimethoprim/sulfamethoxazole (n = 93; 84.5%), streptomycin (n = 87; 79.1%) and ampicillin (n = 88; 80%). Two plasmid-mediated colistin genes—mcr-1.1 harboured on IncX4 plasmids were detected in environmental isolates. The most prevalent sequence types (ST) were ST-155 (n = 8), ST-48 (n = 8) and ST-10 (n = 6). Two isolates of human and environmental sources with a SNPs difference of 6161 originating from the same farm shared a novel ST. The isolates had similar AMR genes and plasmid replicons.ConclusionMDR E.coli isolates were prevalent amongst poultry-workers, poultry, and the poultry farm/LBM environment. The emergence of MDR E. coli with novel ST in two isolates may be plasmid-mediated. Competent authorities should enforce AMR regulations to ensure prudent use of antimicrobials to limit the risk of transmission along the food chain.

Draft genome sequences and resistome analysis of multidrug-resistant mcr-1-harbouring Escherichia coli isolated from pre-harvest poultry in Lebanon

Antibiotic resistance poses a serious risk in Lebanon, a country with challenges in infrastructure and antibiotic stewardship. Recently, a high prevalence of multidrug-resistant (MDR) Escherichia coli harbouring the mobile colistin resistance gene mcr-1 was reported in faecal matter of poultry in Lebanon. Some of the isolates also exhibited resistance to important antibiotics, including cephalosporins and carbapenems. We performed whole-genome sequencing (WGS) to analyse the genetic determinants of resistance and the sequence types (ST) of five of the isolates. WGS was performed using a MiSeq sequencer. Draft genomes were assembled using SPAdes v.3.9.0. ResFinder v.3.0 and PlasmidFinder v.1.3 databases were used to analyse the acquired resistome and plasmid types, respectively. STs were determined using MLST software. WGS analysis revealed that the strains harboured 9-19 antibiotic resistance genes, including mcr-1.1 and other genes encoding resistance to important antibiotics used in agriculture and human medicine. In each strain, mcr-1.1 was located on the same contig with IncX4 plasmid sequences. Furthermore, the strains belonged to different STs, including those associated with (i) zoonotic transmission from poultry to humans, (ii) food contamination and (iii) clinical samples. Our analysis revealed diverse genetic resistance determinants in MDR mcr-1-positive E. coli from poultry in Lebanon. Furthermore, these strains belonged to STs associated with transmission to humans. Taken together, the occurrence of strains harbouring resistance to critical antibiotics in Lebanese poultry and their potential transmission to humans emphasize an urgent need to mitigate the dissemination of antibiotic resistance on poultry farms in Lebanon.