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September 22, 2019  |  

Antibiotic-resistant indicator bacteria in irrigation water: High prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli.

Irrigation water is a major source of fresh produce contamination with undesired microorganisms including antibiotic-resistant bacteria (ARB), and contaminated fresh produce can transfer ARB to the consumer especially when consumed raw. Nevertheless, no legal guidelines exist so far regulating quality of irrigation water with respect to ARB. We therefore examined irrigation water from major vegetable growing areas for occurrence of antibiotic-resistant indicator bacteria Escherichia coli and Enterococcus spp., including extended-spectrum ß-lactamase (ESBL)-producing E. coli and vancomycin-resistant Enterococcus spp. Occurrence of ARB strains was compared to total numbers of the respective species. We categorized water samples according to total numbers and found that categories with higher total E. coli or Enterococcus spp. numbers generally had an increased proportion of respective ARB-positive samples. We further detected high prevalence of ESBL-producing E. coli with eight positive samples of thirty-six (22%), while two presumptive vancomycin-resistant Enterococcus spp. were vancomycin-susceptible in confirmatory tests. In disk diffusion assays all ESBL-producing E. coli were multidrug-resistant (n = 21) and whole-genome sequencing of selected strains revealed a multitude of transmissible resistance genes (ARG), with blaCTX-M-1 (4 of 11) and blaCTX-M-15 (3 of 11) as the most frequent ESBL genes. Overall, the increased occurrence of indicator ARB with increased total indicator bacteria suggests that the latter might be a suitable estimate for presence of respective ARB strains. Finally, the high prevalence of ESBL-producing E. coli with transmissible ARG emphasizes the need to establish legal critical values and monitoring guidelines for ARB in irrigation water.

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September 22, 2019  |  

Genomic surveillance of Enterococcus faecium reveals limited sharing of strains and resistance genes between livestock and humans in the United Kingdom.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause of nosocomial infection and is categorized as high priority by the World Health Organization global priority list of antibiotic-resistant bacteria. In the past, livestock have been proposed as a putative reservoir for drug-resistant E. faecium strains that infect humans, and isolates of the same lineage have been found in both reservoirs. We undertook cross-sectional surveys to isolate E. faecium (including VREfm) from livestock farms, retail meat, and wastewater treatment plants in the United Kingdom. More than 600 isolates from these sources were sequenced, and their relatedness and antibiotic resistance genes were compared with genomes of almost 800 E. faecium isolates from patients with bloodstream infection in the United Kingdom and Ireland. E. faecium was isolated from 28/29 farms; none of these isolates were VREfm, suggesting a decrease in VREfm prevalence since the last UK livestock survey in 2003. However, VREfm was isolated from 1% to 2% of retail meat products and was ubiquitous in wastewater treatment plants. Phylogenetic comparison demonstrated that the majority of human and livestock-related isolates were genetically distinct, although pig isolates from three farms were more genetically related to human isolates from 2001 to 2004 (minimum of 50?single-nucleotide polymorphisms [SNPs]). Analysis of accessory (variable) genes added further evidence for distinct niche adaptation. An analysis of acquired antibiotic resistance genes and their variants revealed limited sharing between humans and livestock. Our findings indicate that the majority of E. faecium strains infecting patients are largely distinct from those from livestock in this setting, with limited sharing of strains and resistance genes.IMPORTANCE The rise in rates of human infection caused by vancomycin-resistant Enterococcus faecium (VREfm) strains between 1988 to the 2000s in Europe was suggested to be associated with acquisition from livestock. As a result, the European Union banned the use of the glycopeptide drug avoparcin as a growth promoter in livestock feed. While some studies reported a decrease in VREfm in livestock, others reported no reduction. Here, we report the first livestock VREfm prevalence survey in the UK since 2003 and the first large-scale study using whole-genome sequencing to investigate the relationship between E. faecium strains in livestock and humans. We found a low prevalence of VREfm in retail meat and limited evidence for recent sharing of strains between livestock and humans with bloodstream infection. There was evidence for limited sharing of genes encoding antibiotic resistance between these reservoirs, a finding which requires further research. Copyright © 2018 Gouliouris et al.

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September 22, 2019  |  

Spread of the florfenicol resistance floR gene among clinical Klebsiella pneumoniae isolates in China.

Florfenicol is a derivative of chloramphenicol that is used only for the treatment of animal diseases. A key resistance gene for florfenicol, floR, can spread among bacteria of the same and different species or genera through horizontal gene transfer. To analyze the potential transmission of resistance genes between animal and human pathogens, we investigated floR in Klebsiella pneumoniae isolates from patient samples. floR in human pathogens may originate from animal pathogens and would reflect the risk to human health of using antimicrobial agents in animals.PCR was used to identify floR-positive strains. The floR genes were cloned, and the minimum inhibitory concentrations (MICs) were determined to assess the relative resistance levels of the genes and strains. Sequencing and comparative genomics methods were used to analyze floR gene-related sequence structure as well as the molecular mechanism of resistance dissemination.Of the strains evaluated, 20.42% (67/328) were resistant to florfenicol, and 86.96% (20/23) of the floR-positive strains demonstrated high resistance to florfenicol with MICs =512 µg/mL. Conjugation experiments showed that transferrable plasmids carried the floR gene in three isolates. Sequencing analysis of a plasmid approximately 125 kb in size (pKP18-125) indicated that the floR gene was flanked by multiple copies of mobile genetic elements. Comparative genomics analysis of a 9-kb transposon-like fragment of pKP18-125 showed that an approximately 2-kb sequence encoding lysR-floR-virD2 was conserved in the majority (79.01%, 83/105) of floR sequences collected from NCBI nucleotide database. Interestingly, the most similar sequence was a 7-kb fragment of plasmid pEC012 from an Escherichia coli strain isolated from a chicken.Identified on a transferable plasmid in the human pathogen K. pneumoniae, the floR gene may be disseminated through horizontal gene transfer from animal pathogens. Studies on the molecular mechanism of resistance gene dissemination in different bacterial species of animal origin could provide useful information for preventing or controlling the spread of resistance between animal and human pathogens.

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September 22, 2019  |  

Complete genome sequence of blaIMP-6-positive Metakosakonia sp. MRY16-398 isolate from the ascites of a diverticulitis patient.

A novel species of carbapenemase-producing Enterobacteriaceae (CPE) was isolated from a patient diagnosed with sigmoid colon diverticulitis. At first, laboratory testing suggested it was Klebsiella oxytoca or Pantoea sp.; however, a complete genome sequence of the isolate, MRY16-398, revealed that it could be novel species, most similar to [Kluyvera] intestini, of which taxonomic nomenclature is still under discussion. Orthologous conserved gene analysis among 42 related bacterial strains indicated that MRY16-398 was classified as the newly proposed genus Metakosakonia. Further, MRY16-398 was found to harbor the blaIMP-6 gene-positive class 1 integron (In722) in plasmid pMRY16-398_2 (IncN replicon, 47.4 kb in size). This finding implies that rare and opportunistic bacteria could be potential infectious agents. In conclusion, our results highlight the need for continuous monitoring for CPE even in nonpathogenic bacteria in the nosocomial environment.

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September 22, 2019  |  

A mcr-1-carrying conjugative IncX4 plasmid in colistin-resistant Escherichia coli ST278 strain isolated from dairy cow feces in Shanghai, China.

Enterobacteriaceae, including Escherichia coli, has been shown to acquire the colistin resistance gene mcr-1. A strain of E. coli, EC11, which is resistant to colistin, polymyxin B and trimethoprim-sulfamethoxazole, was isolated in 2016 from the feces of a dairy cow in Shanghai, China. Strain EC11 identifies with sequence type ST278 and is susceptible to 19 frequently used antibiotics. Whole genome sequencing of strain EC11 showed that this strain contains a 31-kb resistance plasmid, pEC11b, which belongs to the IncX4 group. The mcr-1 gene was shown to be inserted into a 2.6-kb mcr-1-pap2 cassette of pEC11b. Plasmid pEC11b also contained putative conjugal transfer components, including an oriT-like region, relaxase, type IV coupling protein, and type IV secretion system. We were successful in transferring pEC11b to E. coli C600 with an average transconjugation efficiency of 4.6 × 10-5. Additionally, a MLST-based analysis comparing EC11 and other reported mcr-positive E. coli populations showed high genotypic diversity. The discovery of the E. coli strain EC11 with resistance to colistin in Shanghai emphasizes the importance of vigilance in detecting new threats like mcr genes to public health. Detection of mcr genes helps in tracking, slowing, and responding to the emergence of antibiotic resistance in Chinese livestock farming.

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September 22, 2019  |  

Insights into the microbiota of Asian seabass (Lates calcarifer) with tenacibaculosis symptoms and description of sp. nov. Tenacibaculum singaporense

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the Bacteroidetes genus Tenacibaculum, most notably T. maritimum. The impact of tenacibaculosis on fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissue types of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farm fish. The microbiota of diseased farm fish was dominated by Proteobacteria (relative abundancetextpmstandard deviation, 74.5%textpm22.8%) and Bacteroidetes (18.07%textpm21.7%), the latter mainly comprised by a high abundance of Tenacibaculum species (17.6%textpm20.7%). In healthy seabass Proteobacteria had also highest relative abundance (48.04%textpm0.02%), but Firmicutes (34.2%textpm0.02%) and Fusobacteria (12.0%textpm0.03%) were the next two major constituents. Experimentally infected fish developed lesions characteristic for tenacibaculosis, but the microbiota was primarily dominated by Proteobacteria (90.4%textpm0.2%) and Firmicutes (6.2%textpm0.1%). The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in the commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One strain was isolated and is described here as sp. nov. Tenacibaculum singaporense TLL-A1T (=DSM 106434T, KCTC 62393T). The genome of T. singaporense was sequenced and compared to those of T. maritimum DSM 17995T and the newly sequenced T. mesophilum DSM 13764T.

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September 22, 2019  |  

Construction of stable fluorescent laboratory control strains for several food safety relevant Enterobacteriaceae.

Using naturally-occurring bacterial strains as positive controls in testing protocols is typically feared due to the risk of cross-contaminating samples. We have developed a collection of strains which express Green Fluorescent Protein (GFP) at high-level, permitting rapid screening of the following species on selective or non-selective plates: Escherichia coli O157:H7, Shigella sonnei, S. flexneri, Salmonella enterica subsp. Enterica serovar Gaminera, S. Mbandaka, S. Tennesse, S. Minnesota, S. Senftenberg and S. Typhimurium. These new strains fluoresce when irradiated with UV light and maintain this phenotype in absence of antibiotic selection. Recombinants were phenotypically equivalent to the parent strain, except for S. Tennessee Sal66 that appeared Lac- on Xylose Lysine Deoxycholate (XLD) agar plates and Lac+ on Mac Conkey and Hektoen Enteric agar plates. Analysis of closed whole genome sequences revealed that Sal66 had lost one lactose operon; slower rates of lactose metabolism may affect lactose fermentation on XLD agar. These fluorescent enteric control strains were challenging to develop and should provide an easy and effective means of identifying cross-contamination. Published by Elsevier Ltd.

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September 22, 2019  |  

Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio.

Companion animals are likely relevant in the transmission of antimicrobial-resistant bacteria. Enterobacter xiangfangensis sequence type 171 (ST171), a clone that has been implicated in clusters of infections in humans, was isolated from two dogs with clinical disease in Ohio. The canine isolates contained IncHI2 plasmids encoding blaKPC-4 Whole-genome sequencing was used to put the canine isolates in phylogenetic context with available human ST171 sequences, as well as to characterize their blaKPC-4 plasmids. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Cloning and characterization of short-chain N-acyl homoserine lactone-producing Enterobacter asburiae strain L1 from lettuce leaves.

In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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September 22, 2019  |  

Implications of stx loss for clinical diagnostics of Shiga toxin-producing Escherichia coli.

The dynamics related to the loss of stx genes from Shiga toxin-producing Escherichia coli remain unclear. Current diagnostic procedures have shortcomings in the detection and identification of STEC. This is partly owing to the fact that stx genes may be lost during an infection or in the laboratory. The aim of the present study was to provide new insight into in vivo and in vitro stx loss in order to improve diagnostic procedures. Results from the study support the theory that loss of stx is a strain-related phenomenon and not induced by patient factors. It was observed that one strain could lose stx both in vivo and in vitro. Whole genome comparison of stx-positive and stx-negative isolates from the same patient revealed that different genomic rearrangements, such as complete or partial loss of the parent prophage, may be factors in the loss of stx. Of diagnostic interest, it was shown that patients can be co-infected with different E. coli pathotypes. Therefore, identification of eae-positive, but stx-negative isolates should not be interpreted as “Shiga toxin-lost” E. coli without further testing. Growth and recovery of STEC were supported by different selective agar media for different strains, arguing for inclusion of several media in STEC diagnostics.

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September 22, 2019  |  

A large, refractory nosocomial outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli demonstrates carbapenemase gene outbreaks involving sink sites require novel approaches to infection control.

Carbapenem-resistant Enterobacteriaceae (CRE) represent a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly being highlighted as important potential reservoirs. We investigated a large Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli outbreak and wider CRE incidence trends in the Central Manchester University Hospital NHS Foundation Trust (CMFT) (United Kingdom) over 8 years, to determine the impact of infection prevention and control measures. Bacteriology and patient administration data (2009 to 2017) were linked, and a subset of CMFT or regional hospital KPC-producing E. coli isolates (n = 268) were sequenced. Control interventions followed international guidelines and included cohorting, rectal screening (n = 184,539 screens), environmental sampling, enhanced cleaning, and ward closure and plumbing replacement. Segmented regression of time trends for CRE detections was used to evaluate the impact of interventions on CRE incidence. Genomic analysis (n = 268 isolates) identified the spread of a KPC-producing E. coli outbreak clone (strain A, sequence type 216 [ST216]; n = 125) among patients and in the environment, particularly on 2 cardiac wards (wards 3 and 4), despite control measures. ST216 strain A had caused an antecedent outbreak and shared its KPC plasmids with other E. coli lineages and Enterobacteriaceae species. CRE acquisition incidence declined after closure of wards 3 and 4 and plumbing replacement, suggesting an environmental contribution. However, ward 3/ward 4 wastewater sites were rapidly recolonized with CRE and patient CRE acquisitions recurred, albeit at lower rates. Patient relocation and plumbing replacement were associated with control of a clonal KPC-producing E. coli outbreak; however, environmental contamination with CRE and patient CRE acquisitions recurred rapidly following this intervention. The large numbers of cases and the persistence of blaKPC in E. coli, including pathogenic lineages, are of concern. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Genomic characterization of carbapenemase-producing Klebsiella pneumoniae with chromosomally encoded blaNDM-1.

We report here Klebsiella pneumoniae strains carrying chromosomal blaNDM-1 in Thailand. The genomes of these two isolates include a 160-kbp insertion containing blaNDM-1, which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS5-mediated intermolecular transposition and Tn3 transposase-mediated homologous recombination resulted in the integration of blaNDM-1 into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant Enterobacteriaceae may threaten public health and warrants further monitoring. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Investigation of a cluster of Sphingomonas koreensis infections.

Plumbing systems are an infrequent but known reservoir for opportunistic microbial pathogens that can infect hospitalized patients. In 2016, a cluster of clinical sphingomonas infections prompted an investigation.We performed whole-genome DNA sequencing on clinical isolates of multidrug-resistant Sphingomonas koreensis identified from 2006 through 2016 at the National Institutes of Health (NIH) Clinical Center. We cultured S. koreensis from the sinks in patient rooms and performed both whole-genome and shotgun metagenomic sequencing to identify a reservoir within the infrastructure of the hospital. These isolates were compared with clinical and environmental S. koreensis isolates obtained from other institutions.The investigation showed that two isolates of S. koreensis obtained from the six patients identified in the 2016 cluster were unrelated, but four isolates shared more than 99.92% genetic similarity and were resistant to multiple antibiotic agents. Retrospective analysis of banked clinical isolates of sphingomonas from the NIH Clinical Center revealed the intermittent recovery of a clonal strain over the past decade. Unique single-nucleotide variants identified in strains of S. koreensis elucidated the existence of a reservoir in the hospital plumbing. Clinical S. koreensis isolates from other facilities were genetically distinct from the NIH isolates. Hospital remediation strategies were guided by results of microbiologic culturing and fine-scale genomic analyses.This genomic and epidemiologic investigation suggests that S. koreensis is an opportunistic human pathogen that both persisted in the NIH Clinical Center infrastructure across time and space and caused health care-associated infections. (Funded by the NIH Intramural Research Programs.).

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September 22, 2019  |  

Genome wide characterization of enterotoxigenic Escherichia coli serogroup O6 isolates from multiple outbreaks and sporadic infections from 1975-2016.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea globally, particularly among children under the age of five in developing countries. ETEC O6 is the most common ETEC serogroup, yet the genome wide population structure of isolates of this serogroup is yet to be determined. In this study, we have characterized 40 ETEC O6 isolates collected between 1975-2016 by whole genome sequencing (WGS) and by phenotypic antimicrobial susceptibility testing. To determine the relatedness of isolates, we evaluated two methods-whole genome high-quality single nucleotide polymorphism (whole genome-hqSNP) and core genome SNP analyses using Lyve-SET and Parsnp respectively. All isolates were tested for antimicrobial susceptibility using a panel of 14 antibiotics. ResFinder 2.1 and a custom quinolone resistance determinants workflow were used for resistance determinant detection. VirulenceFinder 1.5 was used for prediction of the virulence genes. Thirty-seven isolates clustered into three major clades (I, II, III) by whole genome-hqSNP and core genome SNP analyses, while three isolates included in the whole genome-hqSNP analysis only did not cluster with clades I-III by both analyses and formed a distantly related outgroup, designated clade IV. Median number of pairwise whole genome-hqSNPs in clonal ETEC O6 outbreaks ranged from 0 to 5. Of the 40 isolates tested for antimicrobial susceptibility, 18 isolates were pansusceptible. Twenty-two isolates were resistant to at least one antibiotic, nine of which were multidrug resistant. Phenotypic antimicrobial resistance (AR) correlated with AR determinants in 22 isolates. Thirty-two isolates harbored both enterotoxin virulence genes while the remaining 8 isolates had only one of the two virulence genes. In summary, whole genome-hqSNP and core genome SNP analyses from this study revealed similar evolutionary relationships and an overall diversity of ETEC O6 isolates independent of time of isolation. Less than 5 pairwise hqSNPs between ETEC O6 isolates is circumstantially indicative of an outbreak cluster. Findings from this study will be a basis for quicker outbreak detection and control by efficient subtyping by WGS.

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September 22, 2019  |  

Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle.

Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco).Thirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium.This study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.

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