Plasmid Incompatibility Types Research Articles

Genomic insights into antimicrobial resistant Salmonella in internationally traded chicken meat: First baseline findings in the United Arab Emirates

Non-typhoidal Salmonella is among the most prevalent foodborne zoonoses, challenging food safety and One Health worldwide. A dearth of knowledge exists regarding non-typhoidal Salmonella prevalence and genomic features within the United Arab Emirates (UAE), one of the top markets in chicken meat consumption worldwide. In this study, Salmonella was detected in 16 out of 254 (6.30 %, 95 % confidence interval: 3.64 %, 10.03 %) samples of imported frozen chicken carcasses sampled from UAE retails. Among the recovered serotypes, S. Minnesota; ST 548 (10/16) emerged as the most prevalent, trailed by S. Heidelberg; ST 15 (4/16), and then S. Kentucky; ST 198, and ST 152 (2/16). Antimicrobial resistance (AMR) was most prevalent against tetracycline (93.7 %), followed by ampicillin (68.7 %), and extended-spectrum cephalosporins (ceftriaxone and cefoxitin) (56.2 %); with 68.7 % (11/16) of the isolates classified as multidrug resistant (MDR). Whole-genome sequencing (WGS) analysis revealed the existence of 14 AMR genes, among which the blaCMY-2, an AmpC cephalosporinases resistance gene, presented in 10 of the 16 isolates. Among the ten out of the eleven MDR Salmonella isolates, both IncC and Col(pHAD28) plasmid incompatibility types were concurrently featured. The range of virulence genes varied from 149 to 165 genes, with an average of 168 genes per isolate. Except for one isolate, all other isolates possessed type III secretion system (TTSS) related genes known to be encoded by the Salmonella pathogenicity island-1 (SPI-1). This study contributes to our global understanding of Salmonella epidemiology, specifically focusing on the Middle East. The insights gained from this study are significant in shaping import risk analyses aimed at mitigating Salmonella exposure risks through globally traded chicken. The research underscores the value of WGS as a crucial tool for substantiating evidence-based food safety hazard assessment.

Situation Report on mcr-Carrying Colistin-Resistant Clones of Enterobacterales: A Global Update Through Human-Animal-Environment Interfaces.

In the twenty-first century, antibiotic resistance (ABR) is one of the acute medical emergencies around the globe, overwhelming human-animal-environmental interfaces. Hit-or-mis use of antibiotics exacerbates the crisis of ABR, dispersing transferable resistance traits and challenging treatment regimens based on life-saving drugs such as colistin. Colistin is the highest priority critically important antimicrobials for human medicine, but its long use as a growth promoter in animal husbandry reduces clinical efficacy. Since 2015, the emergence and spread of mobile colistin resistance (mcr)-carrying colistin-resistant clones of Enterobacterales have been markedly sustained in both humans and animals, especially in developing countries. Hospital and community transmissions of mcr clones pose a high risk for infection prevention and outbreaks at the national and international levels. Several public health and limited one health studies have highlighted the genomic insights of mcr clones, clarifying the chromosomal sequence types (STs) and plasmid incompatibility (Inc) types. But this information is segregated into humans and animals, and rarely are environmental sectors complicating the understanding of possibly intercontinental and sectoral transmission of these clones. India is the hotspot for superbugs, including mcr-carrying colistin-resistant isolates that threaten cross-border transmission. The current review provided an up-to-date worldwide scenario of mcr-carrying STs and plasmid Inc types among the Gram-negative bacilli of Enterobacterales across human-animal-environmental interfaces and correlated with the available information from India.

Investigation of multidrug-resistant plasmids from carbapenemase-producing Klebsiella pneumoniae clinical isolates from Pakistan.

The study aim was to investigate multidrug-resistant (MDR) plasmids from a collection of 10 carbapenemase-producing Klebsiella pneumoniae clinical isolates identified within the same healthcare institution in Pakistan. Full characterization of the MDR plasmids including structure, typing characteristics, and AMR content as well as determination of their plasmid-based antimicrobial susceptibility profiles were carried out. Plasmids were isolated from 10 clinical isolates of Klebsiella pneumoniae, and from a corresponding set of Escherichia coli transconjugants, then sequenced using Nanopore/Illumina technology to generate plasmid hybrid assemblies. Full characterization of MDR plasmids, including determination of next generation sequencing (NGS)-based AMR profiles, plasmid incompatibility groups, and types, was carried out. The structure of MDR plasmids was analyzed using the Galileo AMR platform. For E. coli transconjugants, the NGS-based AMR profiles were compared to NGS-predicted AMR phenotypes and conventional broth microdilution (BMD) antimicrobial susceptibility testing (AST) results. All carbapenemase-producing K. pneumoniae isolates (carrying either blaNDM-1, or/and blaOXA-48) carried multiple AMR plasmids encoding 34 antimicrobial resistance genes (ARGs) conferring resistance to antimicrobials from 6 different classes. The plasmid incompatibility groups and types identified were: IncC (types 1 and 3), IncFIA (type 26) IncFIB, IncFII (types K1, K2, K7, and K9), IncHI1B, and IncL. None of the blaNDM-1 and blaESBL-plasmids identified in this study were previously described. Most blaNDM-1-plasmids shared identical AMR regions suggesting potential genetic material/plasmid exchange between K. pneumoniae isolates of this collection. The majority of NGS-based AMR profiles from the E. coli transconjugants correlated well with both NGS-based predicted and conventional AST results. This study highlights the complexity and diversity of the plasmid-based genetic background of carbapenemase-producing clinical isolates from Pakistan. This study emphasizes the need for characterization of MDR plasmids to determine their complete molecular background and monitor AMR through plasmid transmission between multi-resistant bacterial pathogens.

Genomic characterisation of a blaKPC-2- and mcr-10-co-harbouring Enterobacter kobei isolate with high-level resistance to colistin and carbapenems

The emergence and spread of colistin resistance in carbapenem-resistant Enterobacteriaceae pose a serious threat to human and animal health. This work aimed to characterize the genetic features of antimicrobial resistance of a carbapenem and colistin resistant Enterobacter kobei SCLZS19 from hospital sewage by using whole genome sequencing. Antimicrobial susceptibility tests were performed by using the disk diffusion method. Whole genome sequencing of SCLZS19 was carried out on the HiSeq 2000 combined with PacBio RSII platforms. Sequence type, plasmid incompatibility types, resistance genes, and insertion elements were identified using the MLST, PlasmidFinder, ResFinder, and ISfinder, respectively. Conjugation assays were performed using both broth- and filter-based methods with the azide-resistant Escherichia coli J53 as the recipient. The function of the mcr-9-like variant was determined by gene cloning. E. kobei SCLZS19 has a 4,862,177-bp circular chromosome, and nine circular plasmids ranging in size from 4,120 bp to 282,472 bp. It carries 11 antibiotic resistance genes (ARGs), and 10 of them are located on plasmids. The colistin resistance gene mcr-10 is located on a 118,766-bp non-transferable IncF (Y3:A-:B-) plasmid. The carbapenemase gene blaKPC-2 is carried by a self-transmissible IncP6 plasmid which is epidemic in China. In addition, SCLZS19 also carries a mcr-9-like variant on a IncHI2 (ST1) plasmid. Cloning assay shows that the mcr-9-like variant does not mediate colistin resistance in E. coli DH5α. The findings highlight that carbapenem- and colistin-resistant Enterobacterales from water environment may serve as a reservoir for clinically significant ARGs, and continuous surveillance is required.

Enumeration, antimicrobial resistance and genomic characterization of extended-spectrum β-lactamases producing Escherichia coli from supermarket chicken meat in the United Arab Emirates

The occurrence and counts of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in retail chicken sold in the United Arab Emirates (UAE) were investigated in this study. Results indicated that 79.68 % of chicken carcasses (251/315) sampled from UAE supermarkets harbored ESBL-producing E. coli. About half (51.75 % [163/315]) of the tested samples had an ESBL-producing E. coli count range between ≥3 log10 and < 5 log10 CFU/g. The antimicrobial resistance profiles of a subset of 100 isolates showed high rates of non-susceptibility to clinically significant antibiotics, particularly ciprofloxacin (80 %) and cefepime (46 %). Moreover, 7 % of the isolates exhibited resistance to colistin, with PCR-based screening revealing the presence of the mcr-1 gene in all colistin-resistant isolates. Multiplex PCR screening identified blaCTX-M and blaTEM genes as the most frequently presented genes among the phenotypically confirmed ESBL-producing E. coli. Further whole-genome sequencing and bioinformatic analysis of 27 ESBL-producing E. coli isolates showed that the gene family blaCTX group 1 was the most prevalent, notably CTX-M-55 (55.55 % [15/27]), followed by CTX-M-15 (22.22 % [6/27]). The most common sequence types (STs) were ST359 and ST1011, with three evident clusters identified based on phylogenomic analysis, aligned with isolates from specific production companies. Analysis of plasmid incompatibility types revealed IncFIB, IncFII, Incl2, and IncX1 as the most commonly featured plasmids. The findings of this study indicate a noticeable prevalence and high counts of ESBL-producing E. coli in chicken sampled from supermarkets in the UAE. The high rates of antimicrobial resistance to clinically important antibiotics highlight the potential public health risk associated with consuming chicken contaminated with ESBL-producing E. coli. Overall, this study emphasizes the importance of continued antimicrobial resistance monitoring in the UAE food chain and calls for further exposure risk assessment of the consumption of ESBL-producing E. coli via chicken meat.

Characterization of resistance genes and plasmids from sick children caused by Salmonella enterica resistance to azithromycin in Shenzhen, China.

Samonella is 1 of 4 key global causes of diarrhoeal diseases, sometimes it can be serious, especially for yong children. Due to the extensive resistance of salmonella serotypes to conventional first-line drugs, macrolides (such as azithromycin) have been designated as the most important antibiotics for the treatment of salmonella. Antimicrobial resistance is a major public health problem in the world, and the mechanism of azithromycin resistance is rarely studied. This study determined the azithromycin resistance and plasmids of Salmonella enterica isolates from children attending the Shenzhen Children's Hospital. The susceptibility of ampicillin (AMP), ciprofloxacin (CIP), ceftriaxone (CRO), sulfamethoxazole (SMZ), chloramphenicol (CL), and azithromycin (AZM) were detected and the genes and plasmids from azithromycin-resistant Salmonella were detected by Illumina hi-seq and Nanopore MinIone whole genome sequencing (WGS) using a map-based method, and the genomic background of these factors was evaluated using various bioinformatics tools. In total, 15 strains of nontyphoid Salmonella strains that were isolated (including S. typhimurium, S.London, S. Goldcoast, and S.Stanley) demonstrated resistance to azithromycin (minimum inhibitory concentration,MIC from 32 to >256 µg/mL), and the resistance rate was 3.08% (15/487). The sensitivity test to other antibiotics demonstrated 100% resistance to AMP, and the resistance to SMZ and CL was 86.7% and 80.0%, respectively. Through WGS analysis, all isolates were positive for a plasmid-encoded mphA gene. Plasmid incompatibility typing identified five IncFIB(K), five IncHI2/HI2A/Q1, two IncC, one IncHI2/HI2A/N, one IncR, one IncFII and one IncHI2/HI2A plasmids. Sequence analyses of plasmids revealed extensive homology to various plasmids or transposons in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. mphA is the main gene involved in azithromycin, a macrolide, and resistance to Salmonella. It is usually located on plasmids and easily spreads, hence posing a great threat to the current treatment of Salmonella infection. The plasmid sequence similarities suggest that the plasmids acquired resistance genes from a variety of enterica bacteria and underscore the importance of a further understanding of horizontal gene transfer among enterica bacteria.

Coexistence of bla NDM-5 and tet(X4) in international high-risk Escherichia coli clone ST648 of human origin in China.

The emergence of pathogens is conferring resistance to last-resort therapies such as tigecycline, colistin, and carbapenems, limiting the therapeutic options, and raising concerns about the emergence of new "superbugs." This study reports the first incident of a bla NDM-5 and tet(X4) co-harboring Escherichia coli with resistance to carbapenem and tigecycline recovered as the causative agent of a urinary tract infection in a 94-year-old patient. The E. coli strain ECCL209 carries multiple resistance genes [i.e., bla TEM-1B , bla NDM-5, bla CMY-2, aadA22, florR, erm(B), mph(A), erm(42), lnuG, qnrS1, and sul2] and exhibits resistance to almost all clinically used antibiotics. MLST analysis found that the strain belongs to ST648, considered a worldwide high-risk pandemic clone. Moreover, multiple plasmid incompatibility types were detected, i.e., IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Genetic analysis revealed that bla NDM-5 and tet(X4) genes were localized on two hybrid plasmids with multiple replicons. Continuous monitoring studies are suggested to quantify the antimicrobial resistance and assess the dissemination of such superbugs into a human healthcare setting.

CRISPR-cas heterogeneity and plasmid incompatibility types in relation to virulence determinants of Shigella.

Introduction. Virulence factors (VFs) are the most potent weapon in the molecular armoury of Shigella. In bacteria, the mobile genetic elements (MGEs) are contributors to the evolution of different types of clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-cas) variants and plasmid incompatibility types. The present study explored the virulence potential of Shigella in relation to the CRISPR-cas pattern and incompatibility types among the isolates.Hypothesis/Gap Statement. The profile of the CRISPR-cas systems among clinical isolates of Shigella in India has not been reported earlier. Limited knowledge is available on the pattern of plasmid incompatibility groups among clinical isolates Shigella. The bias is always towards studying the genetic elements associated with AMR, but the present study highlights CRISPR-cas and incompatibility types among Shigella in association with virulence.Aim. We aimed to investigate the distribution of virulence factors, CRISPR-cas pattern followed by plasmid incompatibility types among Shigella isolates.Methodology. Between 2012-2017, a total of 187 isolates of Shigella were included in the study. The virulence genes' distribution was carried out. CRISPR-cas profiling followed by analysis of the repeats and spacers was carried out. PCR-based replicon typing was used to determine the incompatibility types. The interplay was statistically determined using STATA.Results. The distribution of virulence genes showed varied pattern with ipaH present in all the isolates followed by ompA (93.6 %), virF (66.8 %), ial and sen (60.4 %), set1A (39.6 %) and set1B (39 %). CRISPR 1, CRISPR 3 and Cas6-Cas5 region were dominantly conserved. Twenty-two types of spacers were identified. The CRISPR3 repeat appeared to have a highly conserved sequence. CRISPR2 being the least common CRISPR type showed a strong association with an array of virulence genes (ial-set1A-set1B-virF) while CRISPR1 being the most dominant showed the least association with virulence genes (sen-virF). The dominant plasmids were found to be belonging to the inc FII group. The incompatibility groups FII, IncIγ, U, FIIS, FIIK, K, A/C, I1alpha was found to be associated with a greater number of virulence genes.Conclusion. The isolates showed increasing diversity in their gene content that contributes to increasing heterogeneity among the isolates, which is a known virulence strategy among pathogens.

Genomic characterization of a carbapenem-resistant Citrobacter freundii cocarrying blaKPC-2 and blaNDM-1

Citrobacter freundii is an important opportunistic pathogen, and carbapenem-resistant strains pose a significant challenge to public health. Here we report the genetic features of antimicrobial resistance genes of a carbapenem-resistant C. freundii SCLZS47 from hospital sewage by using whole genome sequencing. Antimicrobial susceptibility was determined by the broth microdilution method. Whole genomic sequences of SCLZS47 were obtained by using the HiSeq 2000 combined with PacBio RSII platforms. Plasmid incompatibility types, resistance genes, and insertion elements were identified using the PlasmidFinder, ResFinder, and ISfinder, respectively. SCLZS47 has a circular chromosome and three resistance plasmids, and it carries 23 known ARGs. Among them, blaCMY-135 and three copies of blaCTX-M-14 are located on the chromosome. Sixteen ARGs are clustered in two accessory modules of a multidrug resistance (MDR) plasmid, and homologous recombination and transposition events contribute to the formation of these MDR regions. Carbapenemase genes blaKPC-2 and blaNDM-1 are carried by a pCKPC18-1-like plasmid and a pNDM-HN380-like plasmid, respectively. Conjugation experiments indicated that both KPC-2- and NDM-1-encoding plasmids are transmissible. Analysis of the genetic features of resistance genes would help to better understand their transmission mechanisms and dynamics in bacterial community, which has significant clinical implications.

Using whole-genome sequence data to examine the epidemiology of antimicrobial resistance in Escherichia coli from wild meso-mammals and environmental sources on swine farms, conservation areas, and the Grand River watershed in southern Ontario, Canada.

Antimicrobial resistance (AMR) threatens the health of humans and animals and has repeatedly been detected in wild animal species across the world. This cross-sectional study integrates whole-genome sequence data from Escherichia coli isolates with demonstrated phenotypic resistance that originated from a previous longitudinal wildlife study in southern Ontario, as well as phenotypically resistant E. coli water isolates previously collected as part of a public health surveillance program. The objective of this work was to assess for evidence of possible transmission of antimicrobial resistance determinants between wild meso-mammals, swine manure pits, and environmental sources on a broad scale in the Grand River watershed, and at a local scale-for the subset of samples collected on both swine farms and conservation areas in the previous wildlife study. Logistic regression models were used to assess potential associations between sampling source, location type (swine farm vs. conservation area), and the occurrence of select resistance genes and predicted plasmids. In total, 200 isolates from the following sources were included: water (n = 20), wildlife (n = 73), swine manure pit (n = 31), soil (n = 73), and dumpsters (n = 3). Several genes and plasmid incompatibility types were significantly more likely to be identified on swine farms compared to conservation areas. Conversely, internationally distributed sequence types (e.g., ST131), extended-spectrum beta-lactamase- and AmpC-producing E. coli were isolated in lower prevalences (<10%) and were almost exclusively identified in water sources, or in raccoon and soil isolates obtained from conservation areas. Differences in the odds of detecting resistance genes and predicted plasmids among various sources and location types suggest different primary sources for individual AMR determinants, but, broadly, our findings suggest that raccoons, skunks and opossums in this region may be exposed to AMR pollution via water and agricultural sources, as well as anthropogenic sources in conservation areas.

Rural Raccoons (Procyon lotor) Not Likely to Be a Major Driver of Antimicrobial Resistant Human Salmonella Cases in Southern Ontario, Canada: A One Health Epidemiological Assessment Using Whole-Genome Sequence Data.

Non-typhoidal Salmonella infections represent a substantial burden of illness in humans, and the increasing prevalence of antimicrobial resistance among these infections is a growing concern. Using a combination of Salmonella isolate short-read whole-genome sequence data from select human cases, raccoons, livestock and environmental sources, and an epidemiological framework, our objective was to determine if there was evidence for potential transmission of Salmonella and associated antimicrobial resistance determinants between these different sources in the Grand River watershed in Ontario, Canada. Logistic regression models were used to assess the potential associations between source type and the presence of select resistance genes and plasmid incompatibility types. A total of 608 isolates were obtained from the following sources: humans (n = 58), raccoons (n = 92), livestock (n = 329), and environmental samples (n = 129). Resistance genes of public health importance, including blaCMY−2, were identified in humans, livestock, and environmental sources, but not in raccoons. Most resistance genes analyzed were significantly more likely to be identified in livestock and/or human isolates than in raccoon isolates. Based on a 3,002-loci core genome multi-locus sequence typing (cgMLST) scheme, human Salmonella isolates were often more similar to isolates from livestock and environmental sources, than with those from raccoons. Rare instances of serovars S. Heidelberg and S. Enteritidis in raccoons likely represent incidental infections and highlight possible acquisition and dissemination of predominantly poultry-associated Salmonella by raccoons within these ecosystems. Raccoon-predominant serovars were either not identified among human isolates (S. Agona, S. Thompson) or differed by more than 350 cgMLST loci (S. Newport). Collectively, our findings suggest that the rural population of raccoons on swine farms in the Grand River watershed are unlikely to be major contributors to antimicrobial resistant human Salmonella cases in this region.

Using whole-genome sequence data to examine the epidemiology of Salmonella, Escherichia coli and associated antimicrobial resistance in raccoons (Procyon lotor), swine manure pits, and soil samples on swine farms in southern Ontario, Canada.

To better understand the contribution of wildlife to the dissemination of Salmonella and antimicrobial resistance in Salmonella and Escherichia coli, we examined whole-genome sequence data from Salmonella and E. coli isolates collected from raccoons (Procyon lotor) and environmental sources on farms in southern Ontario. All Salmonella and phenotypically resistant E. coli collected from raccoons, soil, and manure pits on five swine farms as part of a previous study were included. We assessed for evidence of potential transmission of these organisms between different sources and farms utilizing a combination of population structure assessments (using core-genome multi-locus sequence typing), direct comparisons of multi-drug resistant isolates, and epidemiological modeling of antimicrobial resistance (AMR) genes and plasmid incompatibility (Inc) types. Univariable logistic regression models were fit to assess the impact of source type, farm location, and sampling year on the occurrence of select resistance genes and Inc types. A total of 159 Salmonella and 96 resistant E. coli isolates were included. A diversity of Salmonella serovars and sequence types were identified, and, in some cases, we found similar or identical Salmonella isolates and resistance genes between raccoons, soil, and swine manure pits. Certain Inc types and resistance genes associated with source type were consistently more likely to be identified in isolates from raccoons than swine manure pits, suggesting that manure pits are not likely a primary source of those particular resistance determinants for raccoons. Overall, our data suggest that transmission of Salmonella and AMR determinants between raccoons and swine manure pits is uncommon, but soil-raccoon transmission appears to be occurring frequently. More comprehensive sampling of farms, and assessment of farms with other livestock species, as well as additional environmental sources (e.g., rivers) may help to further elucidate the movement of resistance genes between these various sources.

Propagation of mcr-1 through Inc FIA in Escherichia coli ST5162 in a tertiary referral hospital of North-East India

Colistin is regarded as the ultimate choice of antibiotic to use against gram-negative bacteria which are resistant to multiple antibiotics. The plasmid-mediated colistin resistance gene, mcr-1, which can transfer horizontally poses a notable risk to public health. Therefore, this study aimed at investigating the occurrence of mcr-1 harboring colistin-resistant Escherichia coli isolates in a tertiary referral hospital in the North-Eastern part of India. A total of 126 E. coli isolates were obtained from different wards of Silchar Medical College and Hospital during August 2018 to February 2019. Phenotypic resistance to colistin was determined by the broth microdilution method and polymyxin NP test. Genotypic characterization was done by PCR assay followed by sequencing. Co-occurrence of other resistance determinants was determined. Transferability was determined by transformation and conjugation assay. PCR- based plasmid incompatibility typing and Multilocus sequence typing (MLST) were performed. 29 E. coli isolates were screened positive for colistin resistance. The mcr-1 gene was found in two E. coli isolates belonging to sequence type ST5162. Co-occurrence of other antibiotic resistance genes namely aph(4)Ia, ant(2″)-Ia, aac(3)-I, aac(3)-IIc, sul1, and qepA were observed. The resistance determinants were found to be horizontally transferable and located within the IncFIA plasmid. This study revealed the propagation and adaptation of colistin resistance through IncFIA in a single clone of E. coli within a clinical setting which is of great concern for infection control and therapeutic choices. Therefore, urgent need for investigation is required to track down their source and continuance within hospital settings to minimize more lateral transfer.

Propagation of blaKPC-2 within two sequence types of Escherichia coli in a tertiary referral hospital of northeast India

KPC enzyme encoded by blaKPC gene has become a potential source of carbapenem resistance and a challenge to clinicians' worldwide and has been gradually emerging in India. The study investigated Escherichia coli isolates of clinical origin for the carriage of blaKPC-2 along with their resistance profile, carriers, co-existing resistance determinants and clonal types. E. coli isolates with non-susceptibility to ertapenem were collected and were investigated for the presence of blaKPC-2 and co-existence of other carbapenemases, ESBLs, aminoglycoside modifying enzymes genes, 16S methyltransferase genes, sulphonamides and quinolone resistance using PCR assay. Transformation and conjugation assay were performed to check horizontal transferability. Plasmid incompatibility typing and multilocus sequence typing was also performed. Two E. coli isolates belonging to two sequence types ST9582 and ST6171 were identified with the carriage of blaKPC-2. The isolates were multidrug resistant, however, were found were susceptible to colistin, nitrofurantoin and tigecycline. In addition to blaKPC-2, the isolate co-harboured a number of resistance genes including blaNDM-1, blaCTX-M-15, ant(2″)-Ia, aac(3)-I, aac(3)-IIc, aac(6′)-Ib and sul3. The conjugative Inc FIC plasmid was identified in our study with the carriage of blaKPC-2 gene in this study centre. The carriage of blaKPC-2 gene within conjugatively transferable Inc FIC plasmid warrants close monitoring to control inter and intra species spread.

Multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli in rescued Sloth bears (Melursus ursinus), India.

The study reports the multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli (CRE) isolated from rescued sloth bear (Melursus ursinus), India. Non-duplicate faecal samples from 21 adult rescued sloth bears were collected at once during 2015-2016 and processed for isolation of E. coli and antibacterial susceptibility pattern. From 21 samples, 45 E. coli were isolated and on phenotypic screening, 23 were MDR, 17 were ESBL producers, and five were carbapenem-resistant (CR). Three E. coli isolates (6.67%, 3/45) showed no resistance, however 42 isolates (93.33%, 42/45) exhibited resistant to at least one antibiotics. The MDR isolates carried beta-lactamase, chloramphenicol, aminoglycosides, tetracycline, fluroquinolone, and sulphadimidine resistance genes. All the phenotypic ESBL producing isolates harbored blaCTX-M genes. On genotypic screening, three CRE (60.0%, 3/5) were positive for blaNDM carbapenemase gene and efflux pump-mediated carbapenem resistance was detected in two CRE isolates (40.0%, 2/5) which were negative for carbapenemase genes. The CRE isolates (n = 5) also co-harbored AMR genes like blaTEM-1, blaAmpC, qnrA, qnrB, qnrS, tetA, tetB and sulI. Virulence screening of the resistant isolates detected the presence of Stx1(n = 1), Stx2 (n = 3), eaeA (n = 4) and hlyA (n = 3) genes. Plasmid incompatibility (Inc) typing revealed that two isolates harboured blaNDM-5 gene on Incl1 and one isolate on IncF plasmid. Apart from the NDM gene, the plasmids also carried tetracycline, beta-lactamase and quinolone resistance genes. The plasmid multilocus sequence typing (pMLST) of the E. coli Incl1 plasmid showed the Sequence Type (ST) 297. This appears to be the first report of MDR, ESBL producing and blaNDM-5 genes on Incl1 and IncF plasmids from rescued sloth bear.

Dissemination of carbapenem resistance and plasmids encoding carbapenemases in Gram-negative bacteria isolated in India.

BackgroundCarbapenem resistance in Gram-negative bacteria is an ongoing public health problem of global dimensions leaving very few treatment options for infected patients.ObjectivesTo study the dissemination of plasmid-borne carbapenemase genes in Gram-negative bacteria from a diagnostic centre in Tamil Nadu, India.MethodsA total of 151 non-repetitive isolates belonging to 10 genera were collected between January 2015 and December 2016 from a diagnostic centre in Tamil Nadu. The isolates included Escherichia coli (n = 57), Klebsiella pneumoniae (n = 45), Pseudomonas aeruginosa (n = 10), Salmonella Typhi (n = 8), Enterobacter cloacae (n = 8), Acinetobacter baumannii (n = 7), Serratia marcescens (n = 5), Achromobacter xylosoxidans (n = 5), Proteus mirabilis (n = 5), Klebsiella oxytoca (n = 5) and Elizabethkingia meningoseptica (n = 1).ResultsOf the 151 isolates, 71% (n = 107) and 68% (n = 103) were found to be resistant to meropenem and imipenem, respectively. The most prevalent β-lactamase gene was blaNDM-1 (n = 22), followed by blaOXA-181 (n = 21), blaGES-1 (n = 11), blaOXA-51 (n = 9), blaGES-9 (n = 8), blaOXA-23 (n = 7) and blaIMP-1 (n = 3). We also observed blaOXA-23 in E. coli (n = 4), and three K. pneumoniae were positive for both, blaOXA-23 and blaOXA-51. Plasmid incompatibility (inc/rep) typing results showed that the resistance genes (n = 11) were present in the isolates carrying plasmid-types IncX, IncA/C, IncFIA-FIB and IncFIIA. The plasmid-borne resistance genes in E. coli and K. pneumoniae were transferred to susceptible E. coli AB1157.ConclusionsThis study highlights the prevalence of carbapenem resistance and the acquisition of plasmid-borne carbapenemase genes in Gram-negative bacteria isolated at this centre.

Coexistence of the Oxazolidinone Resistance-Associated Genes cfr and optrA in Enterococcus faecalis From a Healthy Piglet in Brazil.

Oxazolidinones are one of the most important antimicrobials potentially active against glycopeptide- and β-lactam-resistant Gram-positive pathogens. Linezolid—the first oxazolidinone to be approved for clinical use in 2000 by the US Food and Drug Administration—and the newer molecule in the class, tedizolid, inhibit protein synthesis by suppressing the formation of the 70S ribosomal complex in bacteria. Over the past two decades, transferable oxazolidinone resistance genes, in particular cfr and optrA, have been identified in Firmicutes isolated from healthcare-related infections, livestock, and the environment. Our goals in this study were to investigate the genetic contexts and the transferability of the cfr and optrA genes and examine genomic features, such as antimicrobial resistance genes, plasmid incompatibility types, and CRISPR-Cas defenses of a linezolid-resistant Enterococcus faecalis isolated in feces from a healthy pig during an antimicrobial surveillance program for animal production in Brazil. The cfr gene was found to be integrated into a transposon-like structure of 7,759 nt flanked by IS1216E and capable of excising and circularizing, distinguishing it from known genetic contexts for cfr in Enterococcus spp., while optrA was inserted into an Inc18 broad host-range plasmid of >58 kb. Conjugal transfer of cfr and optrA was shown by filter mating. The coexistence of cfr and optrA in an E. faecalis isolated from a healthy nursery pig highlights the need for monitoring the use of antibiotics in the Brazilian swine production system for controlling spread and proliferation of antibiotic resistance.

Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India

BackgroundThis study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre.MethodsA total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with β-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed.ResultsA total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906.ConclusionThe present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.

Whole-Genome Sequencing-Based Characteristics in Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolated from Retail Meats in Korea.

The spread of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) has posed a critical health risk to both humans and animals, because resistance to beta-lactam antibiotics makes treatment for commonly infectious diseases more complicated. In this study, we report the prevalence and genetic characteristics of ESBL-ECs isolated from retail meat samples in Korea. A total of 1205 E. coli strains were isolated from 3234 raw meat samples, purchased from nationwide retail stores between 2015 and 2018. Antimicrobial susceptibility testing was performed for all isolates by a broth microdilution method, and the ESBL phenotype was determined according to the Clinical and Laboratory Standards Institute (CLSI) confirmatory method. All ESBL-EC isolates (n = 29) were subjected to whole-genome sequencing (WGS). The antimicrobial resistance genes, plasmid incompatibility types, E. coli phylogroups, and phylogenetic relations were investigated based on the WGS data. The prevalence of ESBL-ECs in chicken was significantly higher than that in other meat samples. The results in this study demonstrate that clonally diverse ESBL-ECs with a multidrug resistance phenotype were distributed nationwide, although their prevalence from retail meat was 0.9%. The dissemination of ESBL-ECs from retail meat poses a potential risk to consumers and food-handlers, suggesting that the continuous surveillance of ESBL-ECs in retail meat should be conducted at the national level.

Biofilm-Forming by Carbapenem Resistant Enterobacteriaceae May Contribute to the Blood Stream Infection.

Bloodstream infection (BSI) due to carbapenem-resistant Enterobacteriaceae (CRE) has a high mortality rate and is a serious threat worldwide. Ten CRE strains (eight Enterobacter cloacae, one Klebsiella pneumoniae and one Citrobacter freundii) were isolated from the blood of nine patients, a percentage of whom had been treated with indwelling devices. The steps taken to establish cause included minimum inhibitory concentration (MIC) tests, a pulsed-field gel electrophoresis (PFGE), biofilm study, a multiplex PCR for resistant genes of carbapenemases and extended-spectrum beta-lactamases (ESBLs), and plasmid incompatibility typing. All strains showed a tendency toward resistance to multiple antibiotics, including carbapenems. Frequently isolated genes of ESBLs and carbapenemases include blaTEM-1 (four strains), blaSHV-12 (four strains) and blaIMP-1 (six strains). A molecular analysis by PFGE was used to divide the XbaI-digested genomic DNAs of 10 CRE strains into eight patterns, and the analysis showed that three E. cloacae strains detected from two patients were either identical or closely related. The biofilm production of all CRE strains was examined using a microtiter biofilm assay, and biofilm growth in continuous flow chambers was observed via the use of a confocal laser scanning microscope. Our study indicates that biofilm formation on indwelling devices may pose a risk of BSI due to CRE.