RESUMO
Oxford Nanopore sequencing has been widely used to achieve complete genomes of bacterial pathogens. However, the error rates of Oxford Nanopore long reads are high. Various polishing algorithms using Illumina short reads to correct the errors in Oxford Nanopore long-read assemblies have been developed. The impact of polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads on improving genomic analyses was evaluated using both simulated and real reads. Ten species (10 strains) were selected for simulated reads, while real reads were tested on 11 species (11 strains). Oxford Nanopore long reads were assembled with Unicycler to produce a draft assembly, followed by three rounds of polishing with Illumina short reads using two polishing tools, Pilon and NextPolish. One round of NextPolish polishing generated genome completeness and accuracy parameters similar to the reference genomes, whereas two or three rounds of Pilon polishing were needed, though contiguity remained unchanged after polishing. The polished assemblies of Escherichia coli O157:H7, Salmonella Typhimurium, and Cronobacter sakazakii with simulated reads did not provide accurate plasmid identifications. One round of NextPolish polishing was needed for accurately identifying plasmids in Staphylococcus aureus and E. coli O26:H11 with real reads, whereas one and two rounds of Pilon polishing were necessary for these two strains, respectively. Polishing failed to provide an accurate antimicrobial resistance (AMR) genotype for S. aureus with real reads. One round of polishing recovered an accurate AMR genotype for Klebsiella pneumoniae with real reads. The reference genome and draft assembly of Citrobacter braakii with real reads differed, which carried blaCMY-83 and fosA6, respectively, while both genes were present after one round of polishing. However, polishing did not improve the assembly of E. coli O26:H11 with real reads to achieve numbers of virulence genes similar to the reference genome. The draft and polished assemblies showed a phylogenetic tree topology comparable with the reference genomes. For multilocus sequence typing and pan-genome analyses, one round of NextPolish polishing was sufficient to obtain accurate results, while two or three rounds of Pilon polishing were needed. Overall, NextPolish outperformed Pilon for polishing the Oxford Nanopore long-read assemblies of bacterial pathogens, though both polishing strategies improved genomic analyses compared to the draft assemblies.
Assuntos
Nanoporos , Escherichia coli , Genoma Bacteriano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Filogenia , Análise de Sequência de DNA/métodos , Staphylococcus aureusRESUMO
Enteric pathogens, including Salmonella, are capable of long-term survival after desiccation and resist heat treatments that are lethal to hydrated cells. The mechanisms of dry-heat resistance differ from those of wet-heat resistance. To elucidate the mechanisms of dry-heat resistance in Salmonella, screening of the dry-heat resistance of 108 Salmonella strains, representing 39 serotypes, identified the 22 most resistant and the 8 most sensitive strains for comparative genome analysis. A total of 289 genes of the accessory genome were differently distributed between resistant and sensitive strains. Among these genes, 28 proteins with a putative relationship to stress resistance were selected for to quantify relative gene expression before and after desiccation and expression by solid-state cultures on agar plates relative to cultures growing in liquid culture media. Of these 28 genes, 15 genes were upregulated (P < 0.05) after desiccation or by solid-state cultures on agar plates. These 15 genes were cloned into the low-copy-number vector pRK767 under the control of the lacZ promoter. The expression of 6 of these 15 genes increased (P < 0.05) resistance to dry heat and to treatment with pressure of 500 MPa. Our finding extends the knowledge of mechanisms of stress resistance in desiccated Salmonella to improve control of this bacterium in dry food. IMPORTANCE This study directly targeted an increasing threat to food safety and developed knowledge and targeted strategies that can be used by the food industry to help reduce the risk of foodborne illness in their dry products and thereby reduce the overall burden of foodborne illness. Genomic and physiological analyses have elucidated mechanisms of bacterial resistance to many food preservation technologies, including heat, pressure, disinfection chemicals, and UV light; however, information on bacterial mechanisms of resistance to dry heat is scarce. Mechanisms of tolerance to desiccation likely also contribute to resistance to dry heat, but this assumption has not been verified experimentally. It remains unclear how mechanisms of resistance to wet heat relate to dry-heat resistance. Thus, this study will fill a knowledge gap to improve the safety of dry foods.
Assuntos
Dessecação , Salmonella enterica , Ágar , Regulação Bacteriana da Expressão Gênica , Salmonella enterica/genética , Salmonella enterica/fisiologia , Estresse FisiológicoRESUMO
Shiga toxin-producing E. coli (STEC) are major foodborne pathogens. While many studies have focused on the "top-7 STEC", little is known for minor serogroups. A total of 284 non-top-7 STEC strains isolated from cattle feces were subjected to whole-genome sequencing (WGS) to determine the serotypes, the presence of virulence genes and antimicrobial resistance (AMR) determinants. Nineteen typeable and three non-typeable serotypes with novel O-antigen loci were identified. Twenty-one AMR genes and point mutations in another six genes that conferred resistance to 10 antimicrobial classes were detected, as well as 46 virulence genes. The distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (p < 0.05). Among all strains, 81.7% (n = 232) and 14.1% (n = 40) carried stx2 and stx1 only, respectively; only 4.2% (n = 12) carried both. Subtypes stx1a, stx1c, stx2a, stx2c, stx2d, and stx2g were identified. Forty-six strains carried eae and stx2a and therefore had the potential cause severe diseases; 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We were able to demonstrate the utility of WGS as a surveillance tool to characterize the novel serotypes, as well as AMR and virulence profiles of uncommon STEC that could potentially cause human illness.
Assuntos
Doenças dos Bovinos/microbiologia , Farmacorresistência Bacteriana , Infecções por Escherichia coli/veterinária , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Antibacterianos/farmacologia , Bovinos , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fezes/microbiologia , Filogenia , Sorogrupo , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Virulência , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: We benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads. RESULTS: Unicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K), while the reference genome and SPAdes and Unicycler assemblies harbored blaZ. The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads. CONCLUSIONS: Our research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.
Assuntos
Genoma Bacteriano , Genômica/métodos , Sequenciamento por Nanoporos/métodos , Benchmarking , Campylobacter jejuni , Mapeamento de Sequências Contíguas/métodos , Mapeamento de Sequências Contíguas/normas , Cronobacter sakazakii , Farmacorresistência Bacteriana , Genômica/normas , Listeria monocytogenes , Sequenciamento por Nanoporos/normas , Pseudomonas aeruginosa , Salmonella typhimurium , VirulênciaRESUMO
Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of Klebsiella variicola with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of Escherichia coli O157:H7 and K. variicola with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.
Assuntos
Bactérias/genética , Genoma Bacteriano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/patogenicidade , Biologia Computacional/métodos , Farmacorresistência Bacteriana , Tipagem de Sequências Multilocus , Filogenia , Reprodutibilidade dos Testes , Virulência/genética , Fatores de Virulência/genéticaRESUMO
This study aimed to characterize novel conjugative plasmids that encode transferable ciprofloxacin resistance in Salmonella In this study, 157 nonduplicated Salmonella isolates were recovered from food products, of which 55 were found to be resistant to ciprofloxacin. Interestingly, 37 of the 55 CiprSalmonella isolates (67%) did not harbor any mutations in the quinolone resistance-determining regions (QRDR). Six Salmonella isolates were shown to carry two novel types of conjugative plasmids that could transfer the ciprofloxacin resistance phenotype to Escherichia coli J53 (azithromycin resistant [Azir]). The first type of conjugative plasmid belonged to the â¼110-kb IncFIB-type conjugative plasmids carrying qnrB-bearing and aac(6')-Ib-cr-bearing mobile elements. Transfer of the plasmid between E. coli and Salmonella could confer a ciprofloxacin MIC of 1 to 2 µg/ml. The second type of conjugative plasmid belonged to â¼240-kb IncH1/IncF plasmids carrying a single PMQR gene, qnrS Importantly, this type of conjugative ciprofloxacin resistance plasmid could be detected in clinical Salmonella isolates. The dissemination of these conjugative plasmids that confer ciprofloxacin resistance poses serious challenges to public health and Salmonella infection control.
Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Salmonella/efeitos dos fármacos , Salmonella/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genéticaRESUMO
We characterized antibiotic resistance profiles, antibiotic resistance-associated genes, and pulsed-field gel electrophoresis (PFGE) patterns of 145 Salmonella enterica serotype Typhimurium isolates from human infections and retail foods that were possibly responsible for salmonellosis outbreaks from 2008 to 2012 in Shanghai, China. Resistance to at least three antibiotics was found in 66.7% of chicken isolates, 76.5% of duck isolates, 77.8% of pork isolates, and 80.5% of human isolates. Seven antibiotic resistance phenotypes were detected in chicken isolates, 16 in pork isolates, 17 in duck isolates, and 50 in human isolates. No significant difference (p > 0.05) was found between Salmonella isolates derived from human salmonellosis and from retail foods in terms of the percent resistance of ampicillin, amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, nalidixic acid, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, sulfisoxazole, and sulfamethoxazole/trimethoprim. PFGE using XbaI and BlnI showed that some Salmonella isolates recovered from human infections and retail foods had same or highly similar genetic profile. Same or similar antibiotic resistance profiles, antibiotic resistance associated genes (i.e., qnrA, qnrB, qnrS, aac(6')-Ib, and oqxAB), gene cassettes (i.e., aadA2, dfrA12-aadA2, and aadA1), and mutations were detected in those isolates that exhibited high genetic similarities. These findings highlighted the frequent presence of Salmonella Typhimurium in retail chicken, pork, duck, and humans.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Contaminação de Alimentos , Salmonella typhimurium/isolamento & purificação , Animais , Tipagem de Bacteriófagos , China/epidemiologia , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Microbiologia de Alimentos , Humanos , Produtos da Carne/microbiologia , Testes de Sensibilidade Microbiana , Aves Domésticas/microbiologia , Intoxicação Alimentar por Salmonella/epidemiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/classificação , SorotipagemRESUMO
Salmonella producing ß-lactamases has spread rapidly worldwide and poses a serious threat to human and animal health. In this study, we characterized 220 ceftriaxone (CRO)-resistant isolates identified among 3153 Salmonella from humans, animals, food, and water collected in Shanghai, China. They were assessed for antimicrobial susceptibility, phenotypic identification of extended-spectrum ß-lactamases (ESBLs), and ß-lactamase genes and integrons. CRO resistance in Salmonella increased from 5.0% in 2011 to 8.4% in 2013. Salmonella Enteritidis (45.5%), Salmonella Typhimurium (20.9%) from humans, and Salmonella Indiana (14.5%) from poultry represented the majority of the CRO-resistant isolates. Many isolates were also resistant to other antimicrobials, including nalidixic acid (84.5%), sulfisoxazole (70.5%), and tetracycline (61.8%). Resistance to ciprofloxacin was also found in 33.6% of the isolates. Most isolates (98.2%) were confirmed as ESBL producers. Resistance genes such as blaCTX-M, blaTEM, and blaOXA were detected in 207 (94.1%), 99 (45%), and 53 (24.1%) isolates, respectively. Three types of integron I and one type of integron II were identified in 13 (5.9%) and 2 (0.9%) isolates, respectively. The integrons encompassed 10 different genes: dfrA1/12/17/25, aadA1/2/5, sat2, orfF, and ybeA. Our study underscores concern for increasing CRO resistance, and highlights the widespread ESBL genes in Salmonella enterica.
Assuntos
Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Salmonella enterica/genética , beta-Lactamases/genética , Animais , China , Microbiologia de Alimentos , Humanos , Integrons/genética , Carne/microbiologia , Testes de Sensibilidade Microbiana , Aves Domésticas/microbiologia , Rios/microbiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/enzimologia , Suínos/microbiologiaRESUMO
Multidrug-resistant (MDR) plasmids play an important role in disseminating antimicrobial resistance genes. To elucidate the antimicrobial resistance gene compositions in A/C incompatibility complex (IncA/C) plasmids carried by animal-derived MDR Salmonella Newport, and to investigate the spread mechanism of IncA/C plasmids, this study characterizes the complete nucleotide sequences of IncA/C plasmids by comparative analysis. Complete nucleotide sequencing of plasmids and chromosomes of six MDR Salmonella Newport strains was performed using PacBio RSII. Open reading frames were assigned using prokaryotic genome annotation pipeline (PGAP). To understand genomic diversity and evolutionary relationships among Salmonella Newport IncA/C plasmids, we included three complete IncA/C plasmid sequences with similar backbones from Salmonella Newport and Escherichia coli: pSN254, pAM04528, and peH4H, and additional 200 draft chromosomes. With the exception of canine isolate CVM22462, which contained an additional IncI1 plasmid, each of the six MDR Salmonella Newport strains contained only the IncA/C plasmid. These IncA/C plasmids (including references) ranged in size from 80.1 (pCVM21538) to 176.5 kb (pSN254) and carried various resistance genes. Resistance genes floR, tetA, tetR, strA, strB, sul, and mer were identified in all IncA/C plasmids. Additionally, blaCMY-2 and sugE were present in all IncA/C plasmids, excepting pCVM21538. Plasmid pCVM22462 was capable of being transferred by conjugation. The IncI1 plasmid pCVM22462b in CVM22462 carried blaCMY-2 and sugE. Our data showed that MDR Salmonella Newport strains carrying similar IncA/C plasmids clustered together in the phylogenetic tree using chromosome sequences and the IncA/C plasmids from animal-derived Salmonella Newport contained diverse resistance genes. In the current study, we analyzed genomic diversities and phylogenetic relationships among MDR Salmonella Newport using complete plasmids and chromosome sequences and provided possible spread mechanism of IncA/C plasmids in Salmonella Newport Lineage II.
Assuntos
Doenças dos Bovinos/microbiologia , Galinhas/microbiologia , Doenças do Cão/microbiologia , Doenças das Aves Domésticas/microbiologia , Carne Vermelha/microbiologia , Salmonelose Animal/microbiologia , Salmonella/genética , Animais , Bovinos , Cães , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Filogenia , Plasmídeos/genética , Salmonella/efeitos dos fármacos , Salmonella/isolamento & purificação , Análise de Sequência de DNA/veterináriaRESUMO
We tested a diverse set of 500 isolates of nontyphoidal Salmonella enterica subsp. enterica from various animal, food, and human clinical sources for susceptibility to antimicrobials currently lacking epidemiological cutoff values (ECOFFs) set by the European Committee on Antimicrobial Susceptibility Testing. A consortium of five different laboratories each tested 100 isolates, using broth microdilution panels containing twofold dilutions of ceftriaxone, cefepime, and colistin to determine the minimum inhibitory concentrations of each drug when tested against the Salmonella isolates. Based on the resulting data, new ECOFFs of 0.25 µg/mL for ceftriaxone, 0.12 µg/mL for cefepime, and 2 µg/mL for colistin have been proposed. These thresholds will aid in the identification of Salmonella that have phenotypically detectable resistance mechanisms to these important antimicrobials.
Assuntos
Cefepima/farmacologia , Ceftriaxona/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana/normas , Salmonella enterica/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Humanos , Salmonella enterica/isolamento & purificação , Estados UnidosRESUMO
Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms.IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/isolamento & purificação , Animais , California/epidemiologia , Bovinos , Doenças dos Bovinos/epidemiologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/classificação , Escherichia coli O157/genética , Fezes/microbiologia , Genômica , FilogeniaRESUMO
One thousand four hundred ninety-one Salmonella isolates recovered from retail foods including chicken, beef, fish, pork, dumplings, and cold dishes in China in 2007, 2008, 2010, 2011, and 2012 were analyzed for distribution of serotype and antimicrobial susceptibility. A total of 129 Salmonella serotypes were detected among 1491 isolates. Salmonella Enteritidis (21.5%), Typhimurium (11.0%), Indiana (10.8%), Thompson (5.4%), Derby (5.1%), Agona (3.8%), and Shubra (3.0%) were the seven most important serotypes in 1491 isolates. For antibiotic susceptibility, except 16 (1.1%) isolates were susceptible to all tested antibiotics, 131 (8.8%) resisted 1-2 and 1344 (90.1%) resisted three or more antibiotics. One thousand forty-six (70.2%) of 1491 Salmonella isolates were identified as multidrug-resistant (MDR) isolates, which could resist three or more categories of antibiotics. Resistance to sulfisoxazole (78.1%) was most common among the tested Salmonella, followed by tetracycline (70.6%), trimethoprim/sulfamethoxazole (68.0%), and nalidixic acid (63.4%). Resistances to amikacin (20.0%), levofloxacin (18.7%), gatifloxacin (17.9%), ceftriaxone (17.7%), and cefoxitin (13.2%) were less frequently detected. Resistance to fluoroquinolones was most common among Salmonella Shubra and Indiana isolates, while resistance to cephalosporins was frequently detected among Salmonella Thompson isolates. The results highlighted the diversity of Salmonella serotypes and the high prevalence of Salmonella MDR isolates in China. Compared with Salmonella Enteritidis and Typhimurium isolates, the higher fluoroquinolones and cephalosporins resistance rates of some individual serotypes (Salmonella Shubra, Indiana, and Thompson) also provided more information for further study related to fluoroquinolones or cephalosporin-resistant Salmonella.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Salmonella/efeitos dos fármacos , Resistência às Cefalosporinas , Cefalosporinas/farmacologia , China , Fluoroquinolonas/farmacologia , Contaminação de Alimentos/prevenção & controle , Microbiologia de Alimentos , Carne/microbiologia , Salmonella/isolamento & purificaçãoRESUMO
BACKGROUND: Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India. METHODS: Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network. RESULTS: Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India. CONCLUSIONS: These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general.
Assuntos
Surtos de Doenças , Doenças Transmitidas por Alimentos/epidemiologia , Infecções por Salmonella/epidemiologia , Salmonella enterica/classificação , Salmonella enterica/isolamento & purificação , Animais , Doenças Transmitidas por Alimentos/microbiologia , Genoma Bacteriano , Genótipo , Humanos , Índia , Epidemiologia Molecular , Tipagem Molecular , Filogeografia , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Análise de Sequência de DNA , Atum/microbiologia , Estados Unidos/epidemiologiaRESUMO
UNLABELLED: Escherichia coli O26 is the second most important enterohemorrhagic E. coli (EHEC) serogroup worldwide. Serogroup O26 strains are categorized mainly into two groups: enteropathogenic (EPEC) O26, carrying a locus of enterocyte effacement (LEE) and mostly causing mild diarrhea, and Shiga-toxigenic (STEC) O26, which carries the Shiga toxin (STX) gene (stx), responsible for more severe outcomes. stx-negative O26 strains can be further split into two groups. One O26 group differs significantly from O26 EHEC, while the other O26 EHEC-like group shows all the characteristics of EHEC O26 except production of STX. In order to determine the different populations of O26 E. coli present in U.S. cattle, we sequenced 42 O26:H11 strains isolated from feedlot cattle and compared them to 37 O26:H11 genomes available in GenBank. Phylogenetic analysis by whole-genome multilocus sequence typing (wgMLST) showed that O26:H11/H(-) strains in U.S. cattle were highly diverse. Most strains were sequence type 29 (ST29). By wgMLST, two clear lineages could be distinguished among cattle strains. Lineage 1 consisted of O26:H11 EHEC-like strains (ST29) (4 strains) and O26:H11 EHEC strains (ST21) (2 strains), and lineage 2 (36 strains) consisted of O26:H11 EPEC strains (ST29). Overall, our analysis showed U.S. cattle carried pathogenic (ST21; stx1 (+) ehxA(+) toxB(+)) and also potentially pathogenic (ST29; ehxA(+) toxB(+)) O26:H11 E. coli strains. Furthermore, in silico analysis showed that 70% of the cattle strains carried at least one antimicrobial resistance gene. Our results showed that whole-genome sequence analysis is a robust and valid approach to identify and genetically characterize E. coli O26:H11, which is of importance for food safety and public health. IMPORTANCE: Escherichia coli O26 is the second most important type of enterohemorrhagic E. coli (EHEC) worldwide. Serogroup O26 strains are categorized into two groups: enteropathogenic (EPEC) carrying LEE, causing mild diarrhea, and Shiga toxigenic (STEC) carrying the stx gene, responsible for more severe outcomes. However, there are currently problems in distinguishing one group from the other. Furthermore, several O26 stx-negative strains are consistently misidentified as either EHEC-like or EPEC. The use of whole-genome sequence (WGS) analysis of O26 strains from cattle in the United States (i) allowed identification of O26 strains present in U.S. cattle, (ii) determined O26 strain diversity, (iii) solved the misidentification problem, and (iv) screened for the presence of antimicrobial resistance and virulence genes in the strains. This study provided a framework showing how to easily and rapidly use WGS information to identify and genetically characterize E. coli O26:H11, which is important for food safety and public health.
Assuntos
Infecções por Escherichia coli/veterinária , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Genes Bacterianos , Variação Genética , Genoma Bacteriano , Fatores de Virulência/análise , Animais , Bovinos , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Filogenia , Estados Unidos/epidemiologia , Fatores de Virulência/genéticaRESUMO
Terrapins and turtles are known to transmit Salmonella to humans. However, little was known about the occurrence of this pathogen in soft-shelled terrapin that is a popular delicacy in Chinese and other East Asian cuisines. We isolated and characterized 82 (24.4%) isolates of Salmonella from 336 fecal samples of soft-shelled terrapins (51 of 172; 29.7%) and pet turtles (31 of 164; 18.9%) in Shanghai. Salmonella Thompson was the most common serotype (17.1%) among others. Many isolates (84.1%) were resistant to multiple antimicrobials (≥3). Molecular analysis of Salmonella Thompson and Salmonella Typhimurium using pulsed-field gel electrophoresis unveiled a close genetic relationship between several human and terrapin isolates. Our results highlight the risk associated with the handling and consumption of turtles and their role in the spread of Salmonella in the human salmonellosis.
Assuntos
Reservatórios de Doenças/microbiologia , Intoxicação Alimentar por Salmonella/microbiologia , Intoxicação Alimentar por Salmonella/transmissão , Salmonella/isolamento & purificação , Tartarugas/microbiologia , Animais , China , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Fezes/microbiologia , Microbiologia de Alimentos , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Salmonella/classificação , Salmonella/genética , Salmonella typhimurium/isolamento & purificação , SorotipagemRESUMO
Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli O157/classificação , Escherichia coli O157/genética , Variação Genética , Fatores de Virulência/genética , Eletroforese em Gel de Campo Pulsado , Escherichia coli O157/isolamento & purificação , Evolução Molecular , Humanos , Epidemiologia Molecular , Tipagem Molecular , Sorogrupo , Estados UnidosRESUMO
Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Salmonella/isolamento & purificação , Verduras/microbiologia , Animais , Microbiologia de Alimentos , Humanos , Plantas/microbiologia , Salmonella/classificação , Salmonella/genética , Infecções por Salmonella/microbiologia , Sensibilidade e EspecificidadeRESUMO
A total of 359 non-O157 STEC isolates from food, humans and animals were examined for serotypes, Shiga toxin subtypes and intimin subtypes. Isolates solely harboring stx2 from the three sources were selected for Vero cell cytotoxicity test. stx subtypes in eae negative isolates were more diverse than in eae positive isolates primarily carrying stx2a. Four eae subtypes (eaeß,eaeε1,eaeγ1 and eaeγ2/θ) were observed and correlated with serotypes and flagella. Food isolates showed more diverse serotypes, virulence factors and cell cytotoxicities than human isolates. Some isolates from produce belonged to serotypes that have been implicated in human diseases, carried stx2a or/and stx2dact and exhibited high cell cytotoxicity similar to human isolates. This indicates that foods can be contaminated with potentially pathogenic STEC isolates that may cause human diseases. Given the increased produce consumption and growing burden of foodborne outbreaks due to produce, produce safety should be given great importance.
Assuntos
Microbiologia de Alimentos , Toxina Shiga/classificação , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Adesinas Bacterianas/classificação , Adesinas Bacterianas/genética , Animais , Chlorocebus aethiops , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Flagelos , Inocuidade dos Alimentos , Frutas/microbiologia , Genótipo , Humanos , Carne/microbiologia , Sorotipagem , Verduras/microbiologia , Células Vero , VirulênciaRESUMO
One hundred and twenty six Salmonella Enteritidis isolates recovered from 1152 retail raw poultries were characterized by antimicrobial susceptibility test, pulsed-field gel electrophoresis (PFGE), presence of quinolone resistance (Qnr) associated genes, Class I integron, extended spectrum beta-lactamases (ESBLs) encoding genes, and mutations in quinolone resistance-determining region (QRDR) of GyrA and ParC. Resistance was most frequently found to nalidixic acid (88.1%), followed by to tetracycline (65.9%), sulfisoxazole (65.1%), and ampicillin (61.9%), and a less extent to cefoxitin (8.7%), gatifloxacin (8.7%), levofloxacin (7.9%), ceftriaxone (7.1%), and ceftiofur (6.3%). One hundred and twenty three (98.4%) isolates were resistant to at least one antibiotic, and 93 (74.4%) to at least four antibiotics. aac(6')-Ib-cr, qnrB, qnrA and qnrS genes were detected in 15 (11.9%), 11 (8.7%), 6 (4.8%) and 1 (0.8%) isolates, respectively. Amino acid substitutions of Ser83Tyr, Asp87Asn, Asp87Tyr, Asp87Gly and Ser83Phe/Asp87Asn were detected in QRDR of GyrA, Arg80Ser was the unique mutation in ParC. Eight isolates were detected with amino acid substitution both in GyrA and ParC. Three isolates carried Class I integron that harboring dfrA17-aadA5, dhfR1-aadA1, and dfrA1, respectively. Five isolates were detected carrying bla(TEM)-bla(ACC) (n = 1), bla(TEM) (n = 1), bla(TEM)-bla(OxA) (n = 3), respectively. Genetic diversities (D = 0.9255) were found among isolates based on PFGE analysis.
Assuntos
Carne/microbiologia , Salmonella enteritidis/genética , Salmonella enteritidis/isolamento & purificação , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Galinhas , China , Farmacorresistência Bacteriana Múltipla , Contaminação de Alimentos/análise , Contaminação de Alimentos/economia , Carne/economia , Testes de Sensibilidade Microbiana , Salmonella enteritidis/classificação , Salmonella enteritidis/efeitos dos fármacosRESUMO
ß-Lactamases and extended-spectrum ß-lactamases (ESBLs) producing pathogenic bacteria were widely studied previously in China, but were seldom focused on foodborne Salmonella. In this study, an investigation concerning ß-lactamases and ESBLs producing Salmonella recovered from retail raw chickens was performed. Sixty of 699 foodborne Salmonella isolates were detected as ß-lactamases and ESBLs-producing ones that covered 12 Salmonella serotypes and exhibited different pulsed-field gel electrophoresis genotypes. Forty-four of 60 ß-lactamases and ESBLs-producing strains were simultaneously resistant to ampicillin, amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, and cefoxitin. The most commonly detected ß-lactamases and ESBLs-encoding gene was bla(TEM-1) (n = 44), followed by bla(OXA-1) (n = 38), bla(CMY-2) (n = 29), bla(PSE-1-like) (n = 1), bla(CTX-M-3) (n = 16), and bla(CTX-M-15) (n = 1), respectively. Fourteen, 24, 21, and 1 isolates were detected simultaneously positive for 1, 2, 3, and 4 of the detected ß-lactamases and ESBLs-encoding genes, respectively. A Salmonella strain simultaneously co-carrying bla(TEM-1), bla(OXA-1), bla(CMY-2), and bla(CTX-M-3) was first reported in the present study. Amino acid substitution of Trp244Cys/His247Leu was detected in PSE-1, Val218Asp in CMY-2, and Asp242Gly in CTX-M-15 enzymes, respectively. A difference was found among the amino acid sequences of the detected OXA-1, CMY-2, CTX-M, PSE-1, and TEM-1. The results demonstrated that ß-lactamases and ESBLs were emerging and prevalent in foodborne Salmonella.