The Molecular Biological Characteristics of Vibrio vulnificus Isolated from Wenzhou, China

ABSTRACTProphages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage. We extracted the complete genome sequences of seven pathogens, including ESKAPE bacteria and Escherichia coli from a public database, and examined the distribution of both the AMR and VF genes in prophage-like regions. We found that the ratios of AMR and VF genes greatly varied among the seven species. More than 70% of Enterobacter cloacae strains had VF genes, but only 1.2% of Klebsiella pneumoniae strains had VF genes from prophages. AMR and VF genes are unlikely to exist together in the same prophage region except in E. coli and Staphylococcus aureus, and the distribution patterns of prophage types containing AMR genes are distinct from those of VF gene-carrying prophage types. AMR genes in the prophage were located near transposase and/or integrase. The prophage containing class 1 integrase possessed a significantly greater number of AMR genes than did prophages with no class 1 integrase. The results of this study present a comprehensive picture of AMR and VF genes present within, or close to, prophage-like elements and different prophage patterns between AMR- or VF-encoding prophage-like elements.IMPORTANCE Although we believe phages play an important role in horizontal gene transfer in exchanging genetic material, we do not know the distribution of the antimicrobial resistance (AMR) and/or virulence factor (VF) genes in prophages. We collected different prophage elements from the complete genome sequences of seven species—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli—and characterized the distribution of antimicrobial resistance and virulence genes located in the prophage region. While virulence genes in prophage were species specific, antimicrobial resistance genes in prophages were highly conserved in various species. An integron structure was detected within specific prophage regions such as P1-like prophage element. Maximum of 10 antimicrobial resistance genes were found in a single prophage region, suggesting that prophages act as a reservoir for antimicrobial resistance genes. The results of this study show the different characteristic structures between AMR- or VF-encoding prophages.

BackgroundThe presence of antimicrobial resistance and virulence genes in Escherichia coli allows them to survive and cause infections. The close contact between humans and pets can reinforce the risk of transmitting resistant and virulent bacteria between them.ObjectivesThis study aims to compare the patterns of the presence of tetracycline and streptomycin resistance genes, as well as important virulence genes in E. coli isolated from faeces of healthy dogs and their owners.MethodsPolymerase chain reactions were performed for detection of antimicrobial resistance (tetA, tetB, tetC, tetD, strA and strB) and virulence (fimH, iss, sitA and malX) genes in 144 faecal E. coli isolates from 28 dog–owner pairs and 16 humans who did not keep any pets as controls.ResultsAmong the investigated antimicrobial resistance and virulence genes, tetA (52.1%) and fimH (86.8%) genes had the highest prevalence. No statistically significant difference was found between the prevalence of antimicrobial resistance and virulence genes in isolates of dogs and their owners. In total, 46.4% of dog–owner pairs had the same patterns of presence or absence of six antimicrobial resistance genes, 50.0% had the same patterns of presence or absence of four virulence genes and 25.0% had the same patterns of presence or absence of all 10 tested genes.ConclusionThe presence of antimicrobial‐resistant virulent E. coli in humans and pets may predispose them to infections that are hard to cure with conventional antibiotics. Notable frequency of dogs’ and their owners’ E. coli isolates with similar patterns of antimicrobial resistance and virulence genes may indicate the possibility of sharing virulent antimicrobial resistant E. coli between them.

Simple SummaryWorldwide, antimicrobial resistance (AMR) is of major concern for human and animal health since infections with multidrug-resistant bacteria are often more challenging and costly. In the family Staphyloccocaceae, the species Staphylococcusaureus in particular was reported to cause severe infections. Although most of the other Staphylococcaceae members were not shown to cause severe illnesses, the transmission of AMR genes to harmful species might take place. Therefore, the monitoring of AMR potential in different environments is of high relevance. Mammaliicocci on dairy farms might represent such an AMR gene reservoir. Thus, in this study, the AMR potential of mammaliicocci isolates from German dairy farms was investigated. Whole-genome sequencing (WGS) of the isolates was conducted to evaluate the phylogenetic relationship of the isolates and analyze AMR genes. In addition, antimicrobial susceptibility testing was performed to compare the AMR genotype with the phenotype. It turned out that mammaliicocci may harbor large numbers of different AMR genes and exhibit phenotypic resistance to various antibiotics. Since some AMR genes are likely located on mobile genetic elements, such as plasmids, AMR gene transmission between members of the Staphylococcaceae family might occur.Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae.

The transmissible locus of stress tolerance (tLST) confers resistance to multiple stresses in E. coli. Utilizing 18,959 E. coli genomes available in the NCBI database, we investigated the prevalence, phylogenetic distribution, and configuration patterns of tLST, and correlations between tLST, and virulence and antimicrobial resistance (AMR) genes in E. coli. Four tLST variants were found in 2.7% of E. coli, with the most prevalent (77.1%) variant being tLST1 followed by tLST2 (8.3%), tLST3b (8.3%) and tLST3a (6.3%). The majority (93%) of those tLST were in E. coli belonging to phylogroup A in which the prevalence was 10.4%. tLST was also found in phylogroup B1 (0.5%) and C (0.5%) but not found in B2 or D-G. An additional 1% of the 18,959 E. coli genomes harbored tLST fragments to various extent. Phylogenetic analysis revealed both intra- and interspecies transmission of both chromosomal and plasmid-borne tLST, with E. coli showing a preference of chromosomal over plasmid-borne tLST. The presence of tLST and virulence genes in E. coli was overall negatively correlated, but tLST was found in all genomes of a subgroup of enterotoxigenic E. coli (ST2332). Of note, no Shiga toxin-producing E. coli (n = 3,492) harbored tLST. The prevalence of tLST and AMR genes showed different temporal trends over the period 1985 to 2019. However, a substantial fraction of tLST positive E. coli harbor AMR genes, posing a threat to public health. In conclusion, this study improves our understanding of the genetic characteristics of tLST and E. coli harboring tLST. IMPORTANCE This study, through a large-scale genomic analysis, demonstrated that the genomic island tLST related to multiple stress resistance (such as extreme heat resistance and oxidative stress tolerance) in E. coli is differentially present in subgroups of E. coli and is strongly associated with certain phylogenetic background of the host strain. The study also shows the transmission mechanisms of tLST in E. coli and other bacterial species. The overall negative association of tLST, and virulence genes and antimicrobial (AMR) genes suggest the selective pressures for the acquisition and transmission of these traits likely differ. Even so, the high prevalence of tLST in the enterotoxigenic E. coli clone ST2332 and co-occurrence of tLST and AMR genes in E. coli are concerning. Thus, the findings better our understanding of tLST evolution and provide information for risk assessment of tLST harboring bacteria.

We report for the first time whole-genome sequencing of four multidrug-resistant sequence type (ST) 307 Klebsiella pneumoniae recovered from patients in two hospitals in Armenia. Comparative genomic analysis revealed that the isolates were closely related, with a maximum of 39 single nucleotide polymorphism (SNP) differences in the core genome. All Armenian isolates carried the integrative and conjugative element ICEKp4, which bears the yersiniabactin locus, and shared a common evolutionary origin, diverging around 2005 (95% CI: 1999 to 2011). Antibiotic susceptibility testing showed resistance to several antibiotics, including ampicillin, amoxicillin-clavulanic acid, cefepime, ceftazidime, norfloxacin, levofloxacin, and chloramphenicol. Specifically, isolates designated as ARM03 and ARM06 were resistant to piperacillin-tazobactam, ARM04 and ARM05 had intermediate resistance to both piperacillin-tazobactam and imipenem, and ARM03 showed intermediate resistance to amikacin. We further identified antimicrobial resistance (AMR) genes in four Armenian isolates, including blaOXA-1, blaTEM-1D, blaSHV-28, dfrA14, tet(A), sul2, qnrB1, aac(6´)-Ib-cr, strA, strB and the extended-spectrum β-lactamase gene blaCTX-M-15. Additionally, ARM03 and ARM06 also obtained dfrA5, sul1, sul3, cmlA1, mphA, aph3-Ia and the unique colistin resistance gene mcr-8.1, which was absent in all other publicly available ST307 isolates. These two isolates also acquired aerobactin siderophore-encoding gene clusters (iucABCD-iutA) and the hypermucoidy locus rmpADC (ARM06 had rmpA fragment). ARM04 and ARM05, as well as ARM03 and ARM06, had nearly identical AMR and virulence genes, along with similar plasmid replicon profiles, respectively. Our findings suggest that a transmission event occurred between the two hospitals in Armenia, likely facilitated by patients or community members, during which K. pneumoniae ST307 isolates acquired plasmids carrying AMR and virulence genes.IMPORTANCEMultidrug-resistant (MDR) Klebsiella pneumoniae sequence type (ST) 307 has emerged as a high-risk clone associated with hospital- and community-acquired infections, posing a major threat to global public health. We report in-depth comparative genomics analyses of K. pneumoniae ST307 isolates recovered from patients in Armenia. The unique colistin resistance gene mcr-8.1 identified in ARM03 and ARM06 was absent in all other ST307 isolates obtained from the publicly available data sets. ARM03 and ARM06 also acquired aerobactin siderophore-encoding gene clusters (iucABCD-iutA) and the hypermucoidy locus rmpADC (ARM06 possessed incomplete rmpA fragment). Our findings suggest that a transmission event has occurred between two hospitals in Armenia either through patients or community members. In addition, the Armenian isolates obtained plasmids carrying virulence and AMR genes during the transmission event. Our study emphasises the importance of genomic surveillance of this emerging MDR-hypervirulent pathogen to provide early interventions.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Relationship between virulence factors and antimicrobial resistance genes of pathogenic Escherichia coli from diarrheic weaned piglets

Klebsiella sp. are responsible for a multitude of infectious diseases in both humans and animals. In this study, phylogenetic relationships, virulence and antimicrobial resistance gene properties of 16 Klebsiella sp. isolated from 49 pet turtles were investigated. The isolates including Klebsiella oxytoca (n=13) and Klebsiella pneumoniae (n=3) were identified using 16S rRNA gene sequencing and each species formed distinct clusters in the neighbour-joining phylogenetic tree. The prevalence of virulence genes including ureC (100%) and kfu (68·75%) was observed among the isolates usingPolymerase chain reaction (PCR) assay. The fimH, mrkD and rmpA genes were detected in all K. pneumoniae while these were absent in every K. oxytoca isolate. In antimicrobial susceptibility testing, high resistance rates were observed against ampicillin (100%) and cephalothin (62·50%). The resistance rates against imipenem, tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid and ciprofloxacin were 12·50, 12·50, 12·50, 6·25 and 6·25% respectively. The presence of antimicrobial resistance genes such as plasmid-mediated quinolone resistance (PMQR) [qnrB (37·50%), qnrA (31·25%), qnrS (12·50%) and aac(6')-Ib-cr (12·50%)], extended-spectrum β-lactamase (ESBL) [blaCTX-M (18·75%)], β-lactamase [blaSHV-1 (18·75%)] and tetracycline resistance [tetE (12·50%)] was observed. The results revealed that pet turtle-borne Klebsiella sp. may carry different types of virulence and antimicrobial resistance genes which represents a potential threat to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: Klebsiella sp. are nonmotile Gram-negative bacteria that are found in different environments. The virulence and antimicrobial resistance properties of pet turtle-borne Klebsiella sp. have not been studied before. Phylogenetic relationships, virulence traits and antimicrobial resistance profiles of pet turtle-borne Klebsiella sp. were characterized for the first time in Korea. Multiple virulence and antimicrobial resistance genes were observed among the isolates. The occurrence of virulence and antimicrobial resistance determinants in Klebsiella sp. may represent a potential threat to public health.

Antimicrobial resistance (AMR) is a major public health threat. The issue has disproportionately affected in low- and middle-income countries including Thailand and its neighboring counties where high rates of resistant Salmonella have been continuously reported in pigs, pork, and humans. Recognizing the significant role of R plasmids in AMR distribution, knowledge on virulence genes associated plasmids is still limited. The frequency and patterns of virulence factors are still not routinely tracked. This study aimed to characterize Salmonella enterica isolates carrying plasmid-associated virulence and AMR genes from pig, pork, and human isolates in border provinces of Thailand, Lao PDR, Cambodia, and Myanmar. A total of 366 isolates were collected from pigs (n=265), pork (n=69), and humans (n=32). Among these isolates, multidrug resistance was common (69.4%). The highest resistancerates was observed for ampicillin (73.2%) followed by tetracycline (70.2%), and sulfamethoxazole (62.8%). Five isolates (1.4%) carried plasmid-associated virulence genes including spvBCD, pefABCD, rck, and mig-5. Whole genome sequencing of these S. Enteritidis ST11 isolates from diarrheal patients, identified two plasmid patterns including IncFIB-IncFII-IncX1 (n=3/5) and IncFIB-IncFII (n=2/5). The IncFIB/IncFII plasmids were virulence plasmids (pSEVs) carrying plasmid-associated virulence genes. The pSEVs were closely related to pSLT from S. Typhimurium LT2 and pSCV50 from S. Choleraesuis SC-B67. IncX1 plasmids were R plasmids carrying blaTEM. These Salmonella harboured both R and virulence plasmids simultaneously. The co-existence of virulence plasmids and AMR genes could increase the severity of infection and complicated the antibiotic therapy. The results emphasizes the need for surveillance programs to track plasmid-associated virulence and AMR genes in humans and animals. Such comprehensive approach could improve strategies to prevent and manage resistant Salmonella infections.

A DNA microarray for identification of virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Co-occurrence of antimicrobial and metal resistance genes in pig feces and agricultural fields fertilized with slurry

Aeromonas hydrophila has been isolated from various fish species in Egypt and is known to carry virulence and antimicrobial resistance genes, which pose a risk for public health. The aim of the present study is to report, for the first time, the infection of mullet (Mugil cephalus) with A. hydrophila and to clarify the potential association between antimicrobial resistance and virulence traits encoded in A. hydrophila. In this study, the occurrence of A. hydrophila in marketed mullet and the antimicrobial resistance phenotypes of these isolates were determined. Aeromonas hydrophila isolates were screened for the presence of virulence and β-lactam resistance genes; the correlation between both gene groups was also investigated. The infection rate of examined mullet with A. hydrophila was 37% (50/135). The highest antimicrobial resistance was detected to cefoxitin (100%), followed by ampicillin (84%), ceftazidime (56%) and cefotaxime (40%). Only 4% of the isolates were resistant to erythromycin; 6% were resistant to both gentamicin and kanamycin with no resistance to ciprofloxacin. Variable frequencies of virulence and β-lactam resistance genes were evident from PCR, where aerA and blaTEM predominated. The study also indicated a general weak positive correlation (R=0·3) between both virulence and β-lactam resistance genes. Some of the studied virulence genes (e.g. aerA:hlyA and hlyA:ast) were found to correlate positively. The presence of virulence and resistance genes in A. hydrophila from food sources poses a serious threat to public health. To our knowledge, this is the first report describing the occurrence of A. hydrophila in mullet and highlighting the coexistence of virulence and β-lactam resistance genes encoded by these bacteria. These data provide insights into the potential association of antimicrobial resistance and virulence genes in A. hydrophila from marketed mullet in Egypt, which could pose threats to humans even if a weak positive correlation exists between both genes.

Detection of plasmids in Salmonella from poultry and investigating the potential horizontal transfer of antimicrobial resistance and virulence genes: PLASMID TRANSFER OF RESISTANCE AND VIRULENCE.