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Contamination of poultry products by Salmonella enterica serovar Typhimurium (STm) is a major cause of foodborne infections and outbreaks. This study aimed to assess the diversity and antimicrobial resistance (AMR) carriage of STm in three chicken processing plants using genomic sequencing. It also aimed to investigate whether any particular strain types were associated with cases of human illness. Multilevel genome typing (MGT) was used to analyze 379 STm isolates from processed chicken carcasses. The diversity of chicken STm sequence types (STs) increased from MGT1 (2 STs) to MGT9 (257 STs). STs at MGT5 to MGT9 levels that were unique to one processing plant and shared among the processing plants were identified, likely reflecting the diversity of STm at their farm source. Fifteen medium resolution MGT5 STs matched those from human infections in Australia and globally. However, no STs matched between the chicken and human isolates at high resolution levels (MGT8 or MGT9), indicating the two STm populations were phylogenetically related but were unlikely to be directly epidemiologically linked. AMR genes were rare, with only a blaTEM-1 gene carried by a 95 kb IncI1 Alpha plasmid being identified in 20 isolates. In conclusion, subpopulations that were widespread in processing plants and had caused human infections were described using MGT5 STs. In this STM population, AMR was rare with only sporadic resistance to a single drug class observed. The genomic analysis of STm from chicken processing plants in this study provided insights into STm that contaminate meat chickens early in the food production chain.

ABSTRACT Prevotella copri is the dominant species of the Prevotella genus in the gut, which is genomically heterogeneous and difficult to isolate; hence, scarce research was carried out for this species. This study aimed to investigate the effect of P. copri on hyperglycemia. Thirty-nine strains were isolated from healthy individuals, and three strains (HF2123, HF1478, and HF2130) that had the highest glucose consumption were selected to evaluate the effects of P. copri supplementation on hyperglycemia. Microbiomics and non-target metabolomics were used to uncover the underlying mechanisms. Oral administration of P. copri in diabetic db/db mice increased the expression and secretion of glucagon-like peptide-1 (GLP-1), significantly improved hyperglycemia, insulin resistance, and lipid accumulation, and alleviated the pathological morphology in the pancreas, liver, and colon. P. copri changed the composition of the gut microbiota of diabetic db/db mice, which was characterized by increasing the ratio of Bacteroidetes to Firmicutes and increasing the relative abundance of genera Bacteroides, Akkermansia, and Faecalibacterium. After intervention with P. copri, fecal metabolic profiling showed that fumaric acid and homocysteine contents decreased, and glutamine contents increased. Furthermore, amino acid metabolism and cAMP/PKA signaling pathways were enriched. Our findings indicate that P. copri improved glucose metabolism abnormalities in diabetic db/db mice. Especially, one of the P. copri strains, HF2130, has shown superior performance in improving hyperglycemia, which may have the potential as a probiotic against hyperglycemia.IMPORTANCEAs a core member of the human intestinal ecosystem, Prevotelal copri has been associated with glucose metabolic homeostasis in previous studies. However, these results have often been derived from metagenomic studies, and the experimental studies have been based solely on the type of strain DSM 18205T. Therefore, more experimental evidence from additional isolates is needed to validate the results according to their high genomic heterogeneity. In this study, we isolated different branches of strains and demonstrated that P. copri could improve the metabolic profile of hyperglycemic mice by modulating microbial activity. This finding supports the causal contribution of P. copri in host glucose metabolism.

ABSTRACTCampylobacter species are typically helical shaped, Gram-negative, and non-spore-forming bacteria. Species in this genus include established foodborne and animal pathogens as well as emerging pathogens. The accumulation of genomic data from the Campylobacter genus has increased exponentially in recent years, accompanied by the discovery of putative new species. At present, the lack of a standardized species boundary complicates distinguishing established and novel species. We defined the Campylobacter genus core genome (500 loci) using publicly available Campylobacter complete genomes (n = 498) and constructed a core genome phylogeny using 2,193 publicly available Campylobacter genomes to examine inter-species diversity and species boundaries. Utilizing 8,440 Campylobacter genomes representing 33 species and 8 subspecies, we found species delineation based on an average nucleotide identity (ANI) cutoff of 94.2% is consistent with the core genome phylogeny. We identified 60 ANI genomic species that delineated Campylobacter species in concordance with previous comparative genetic studies. All pairwise ANI genomic species pairs had in silico DNA-DNA hybridization scores of less than 70%, supporting their delineation as separate species. We provide the tool Campylobacter Genomic Species typer (CampyGStyper) that assigns ANI genomic species to query genomes based on ANI similarities to medoid genomes from each ANI genomic species with an accuracy of 99.96%. The ANI genomic species definitions proposed here allow consistent species definition in the Campylobacter genus and will facilitate the detection of novel species in the future.IMPORTANCEIn recent years, Campylobacter has gained recognition as the leading cause of bacterial gastroenteritis worldwide, leading to a substantial rise in the collection of genomic data of the Campylobacter genus in public databases. Currently, a standardized Campylobacter species boundary at the genomic level is absent, leading to challenges in detecting emerging pathogens and defining putative novel species within this genus. We used a comprehensive representation of genomes of the Campylobacter genus to construct a core genome phylogenetic tree. Furthermore, we found an average nucleotide identity (ANI) of 94.2% as the optimal cutoff to define the Campylobacter species. Using this cutoff, we identified 60 ANI genomic species which provided a standardized species definition and nomenclature. Importantly, we have developed Campylobacter Genomic Species typer (CampyGStyper), which can robustly and accurately assign these ANI genomic species to Campylobacter genomes, thereby aiding pathogen surveillance and facilitating evolutionary and epidemiological studies of existing and emerging pathogens in the genus Campylobacter.

ABSTRACT Vibrio cholerae O1 has caused cholera pandemics. Non-pandemic V. cholerae O1 strains, which are genetically distinctive from the pandemic clones, have been isolated from both human infections and the environment. We aimed to better understand the non-pandemic O1 strains and their pandemic potential. We sequenced 109 non-pandemic O1 isolates from Zhejiang, China (from 1963 to 1996) and compared them with 62 publicly available non-pandemic O1 genomes. The isolates from Zhejiang can be classified into three lineages (L1–L3). All grouped together with L3 sharing the most recent common ancestor with the pandemic clones. L2 and L3 emerged in the 1960s while L1 emerged in the 1970s. L1 and L2 disappeared after the 1990s, but L3 persisted until recently. All isolates contained the type VI secretion system. The Vibrio pathogenicity island was present in all L3 isolates, whereas the type III secretion system was present in all L1 isolates. L2 did not carry any unique virulence genes. An intact CTXφ was present in only two L3 isolates. An intact Vibrio seventh pandemic island 1 was present in only three L3 isolates. The blaCARB-7 gene was identified in 96.3% of L2 isolates. Each of the non-pandemic O1 lineages has unique properties contributing to their capacity to cause disease. Our findings offer new insight into the evolution of O1 V. cholerae for cholera prevention and control. IMPORTANCE It is well recognized that only Vibrio cholerae O1 causes cholera pandemics. However, not all O1 strains cause pandemic-level disease. In this study, we analyzed non-pandemic O1 V. cholerae isolates from the 1960s to the 1990s from China and found that they fell into three lineages, one of which shared the most recent common ancestor with pandemic O1 strains. Each of these non-pandemic O1 lineages has unique properties that contribute to their capacity to cause cholera. The findings of this study enhanced our understanding of the emergence and evolution of both pandemic and non-pandemic O1 V. cholerae.

Vibrio metschnikovii is an emergent pathogen that causes human infections which may be fatal. However, the phylogenetic characteristics and pathogenicity determinants of V. metschnikovii are poorly understood. Here, the whole-genome features of 103 V. metschnikovii strains isolated from different sources are described. On phylogenetic analysis V. metschnikovii populations could be divided into two major lineages, defined as lineage 1 (L1) and 2 (L2), of which L1 was more likely to be associated with human activity. Meanwhile, we defined 29 V. metschnikovii O-genotypes (VMOg, named VMOg1–VMOg29) by analysis of the O-antigen biosynthesis gene clusters (O-AGCs). Most VMOgs (VMOg1 to VMOg28) were assembled by the Wzx/Wzy pathway, while only VMOg29 used the ABC transporter pathway. Based on the sequence variation of the wzx and wzt genes, an in silico O-genotyping system for V. metschnikovii was developed. Furthermore, nineteen virulence-associated factors involving 161 genes were identified within the V. metschnikovii genomes, including genes encoding motility, adherence, toxins, and secretion systems. In particular, V. metschnikovii was found to promote a high level of cytotoxicity through the synergistic action of the lateral flagella and T6SS. The lateral flagellar-associated flhA gene played an important role in the adhesion and colonization of V. metschnikovii during the early stages of infection. Overall, this study provides an enhanced understanding of the genomic evolution, O-AGCs diversity, and potential pathogenic features of V. metschnikovii.

Bordetella pertussis causes pertussis (or whooping cough), a severe respiratory infectious disease in infants, although it can be prevented by whole cell and acellular vaccines. The recent pertussis resurgence in industrialised countries is partly attributed to pathogen adaptation to vaccines, while emergence of antimicrobial resistance, specifically to macrolides in China, has become a concern. Surveillance of current circulating and emerging strains is therefore vital to understand the risks they pose to public health. Although the use of genomics-based typing is increasing a genomic nomenclature for this pathogen has not been well established. Here, we implemented the multilevel genome typing (MGT) system for B. pertussis with five levels of resolution, which provide targeted typing of relevant lineages and discrimination of closely related strains at the finest scale. The lower resolution levels (MGT2 and MGT3) describe the distribution of major vaccine antigen alleles including ptxP, fim3, fhaB and prn, as well as temporal and spatial trends within the B. pertussis global population. Mid-resolution levels (MGT3 and MGT4) enable typing of antibiotic-resistant lineages and Prn deficient lineages within the ptxP3 clade. The high-resolution level (MGT5) can capture finer-scale epidemiology such as outbreaks and local transmission events, with comparable resolution to existing genomic methods of strain-relatedness assessment. The scheme offers stable MGT-type assignments aiding harmonisation of typing and communication between laboratories. The scheme is available at https://mgtdb.unsw.edu.au/pertussis, is regularly updated from global data repositories and accepts public submissions. The MGT scheme provides a comprehensive, robust, and scalable system for global surveillance of B. pertussis.

ABSTRACT Aeromonas species are emerging human enteric pathogens. This study examines the isolation of Aeromonas and other enteric bacterial pathogens from patients with and without inflammatory bowel disease (IBD). This study also investigates the intestinal epithelial pathogenic mechanisms of Aeromonas veronii. The isolation rates of seven enteric bacterial pathogens from 2,279 patients with IBD and 373,276 non-IBD patients were compared. An A. veronii strain (AS1) isolated from intestinal biopsies of a patient with IBD was used for pathogenic mechanism investigation, and Escherichia coli K12 was used as a bacterial control. HT-29 cells were used as a model of human intestinal epithelium. A significantly higher isolation of Aeromonas species was found in patients with IBD as compared to non-IBD patients (P = 0.0001, odds ratio = 2.11). A. veronii upregulated 177 inflammatory genes and downregulated 52 protein-coding genes affecting chromatin assembly, multiple small nuclear RNAs, multiple nucleolar RNAs, and 55 cytoplasmic tRNAs in HT-29 cells. These downregulation effects were unique to A. veronii and not observed in HT-29 cells infected with E. coli K12. A. veronii induced intestinal epithelial apoptosis involving the intrinsic pathway. A. veronii caused epithelial microvilli shortening and damage and epithelial production of IL-8. In conclusion, this study for the first time reports the association between IBD and Aeromonas enteric infection detected by bacterial cultivation. This study also reports that A. veronii damages intestinal epithelial cells via multiple mechanisms, of which the downregulating cytoplasmic tRNA, small nuclear RNA, and small nucleolar RNA are novel bacterial pathogenic mechanisms. IMPORTANCE This study for the first time reports the association between inflammatory bowel disease (IBD) and Aeromonas enteric infection detected by bacterial pathogen cultivation, highlighting the need of clinical and public health attention. The finding that patients with IBD are more susceptible to Aeromonas enteric infection suggests that detection of Aeromonas enteric infection should be routinely performed for the diagnosis and treatment of IBD. This study also reports novel bacterial pathogenic mechanisms employed by Aeromonas veronii. Through comparative transcriptomic analysis and other techniques, this study revealed the pathogenic mechanisms by which A. veronii causes damage to intestinal epithelial cells. Among the various pathogenic mechanisms identified, the downregulating tRNA, small nuclear and nucleolar RNAs in human intestinal epithelial cells are novel bacterial pathogenic mechanisms.

Pertussis, commonly known as whooping cough is a severe respiratory disease caused by the bacterium, Bordetella pertussis. Despite widespread vaccination, pertussis resurgence has been observed globally. The development of the current acellular vaccine (ACV) has been based on planktonic studies. However, recent studies have shown that B. pertussis readily forms biofilms. A better understanding of B. pertussis biofilms is important for developing novel vaccines that can target all aspects of B. pertussis infection. This study compared the proteomic expression of biofilm and planktonic B. pertussis cells to identify key changes between the conditions. Major differences were identified in virulence factors including an upregulation of toxins (adenylate cyclase toxin and dermonecrotic toxin) and downregulation of pertactin and type III secretion system proteins in biofilm cells. To further dissect metabolic pathways that are altered during the biofilm lifestyle, the proteomic data was then incorporated into a genome scale metabolic model using the Integrative Metabolic Analysis Tool (iMAT). The generated models predicted that planktonic cells utilised the glyoxylate shunt while biofilm cells completed the full tricarboxylic acid cycle. Differences in processing aspartate, arginine and alanine were identified as well as unique export of valine out of biofilm cells which may have a role in inter-bacterial communication and regulation. Finally, increased polyhydroxybutyrate accumulation and superoxide dismutase activity in biofilm cells may contribute to increased persistence during infection. Taken together, this study modeled major proteomic and metabolic changes that occur in biofilm cells which helps lay the groundwork for further understanding B. pertussis pathogenesis.

Whooping cough (pertussis) is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis. Despite high vaccine coverage, pertussis has re-emerged in many countries including Australia and caused two large epidemics in Australia since 2007. Here, we undertook a genomic and phylogeographic study of 385 Australian B. pertussis isolates collected from 2008 to 2017. The Australian B. pertussis population was found to be composed of mostly ptxP3 strains carrying different fim3 alleles, with ptxP3-fim3A genotype expanding far more than ptxP3-fim3B. Within the former, there were six co-circulating epidemic lineages (EL1 to EL6). The multiple ELs emerged, expanded, and then declined at different time points over the two epidemics. In population genetics terms, both hard and soft selective sweeps through vaccine selection pressures have determined the population dynamics of Australian B. pertussis. Relative risk estimation suggests that once a new B. pertussis lineage emerged, it was more likely to spread locally within the first 1.5 years. However, after 1.5 years, any new lineage was likely to expand to a wider region. Phylogenetic analysis revealed the expansion of ptxP3 strains was also associated with replacement of the type III secretion system allele bscI1 with bscI3. bscI3 is associated with decreased T3SS secretion and may allow B. pertussis to reduce immune recognition. This study advanced our understanding of the epidemic population structure and spatial and temporal dynamics of B. pertussis in a highly immunized population.

The enteroinvasive bacterium Shigella flexneri is known as a highly host-adapted human pathogen. There had been no known other reservoirs reported until recently. Here 34 isolates obtained from animals (yaks, dairy cows and beef cattle) from 2016 to 2017 and 268 human S. flexneri isolates from China were sequenced to determine the relationships between animal and human isolates and infer the evolutionary history of animal-associated S. flexneri. The 18 animal isolates (15 yak and 3 beef cattle isolates) in PG1 were separated into 4 lineages, and the 16 animal isolates (1 yak, 5 beef cattle and 10 dairy cow isolates) in PG3 were clustered in 8 lineages. The most recent human isolates from China belonged to PG3 whereas Chinese isolates from the 1950s–1960s belonged to PG1. PG1 S. flexneri may has been transmitted to the yaks during PG1 circulation in the human population in China and has remained in the yak population since, while PG3 S. flexneri in animals were likely recent transmissions from the human population. Increased stability of the large virulence plasmid and acquisition of abundant antimicrobial resistance determinants may have enabled PG3 to expand globally and replaced PG1 in China. Our study confirms that animals may act as a reservoir for S. flexneri. Genomic analysis revealed the evolutionary history of multiple S. flexneri lineages in animals and humans in China. However, further studies are required to determine the public health threat of S. flexneri from animals.

ABSTRACT Salmonella enterica serovar Enteritidis is one of the leading causes of salmonellosis in Australia. In this study, a total of 568 S. Enteritidis isolates from two Australian states across two consecutive years were analyzed and compared to international strains, using the S. Enteritidis multilevel genome typing (MGT) database, which contained 40,390 publicly available genomes from 99 countries. The Australian S. Enteritidis isolates were divided into three phylogenetic clades (A, B, and C). Clades A and C represented 16.4% and 3.5% of the total isolates, respectively, and were of local origin. Clade B accounted for 80.1% of the isolates which belonged to seven previously defined lineages but was dominated by the global epidemic lineage. At the MGT5 level, three out of five top sequence types (STs) in Australia were also top STs in Asia, suggesting that a fair proportion of Australian S. Enteritidis cases may be epidemiologically linked with Asian strains. In 2018, a large egg-associated local outbreak was caused by a recently defined clade B lineage prevalent in Europe and was closely related, but not directly linked, to three European isolates. Additionally, over half (54.8%) of predicted multidrug resistance (MDR) isolates belonged to 10 MDR-associated MGT-STs, which were also frequent in Asian S. Enteritidis . Overall, this study investigated the genomic epidemiology of S. Enteritidis in Australia, including the first large local outbreak, using MGT. The open MGT platform enables a standardized and sharable nomenclature that can be effectively applied to public health for unified surveillance of S. Enteritidis nationally and globally. IMPORTANCE Salmonella enterica serovar Enteritidis is a leading cause of foodborne infections. We previously developed a genomic typing database (MGTdb) for S. Enteritidis to facilitate global surveillance of this pathogen. In this study, we examined the genomic features of Australian S. Enteritidis using the MGTdb and found that Australian S. Enteritidis is mainly epidemiologically linked with Asian strains (especially strains carrying antimicrobial resistance genes), followed by European strains. The first large-scale egg-associated local outbreak in Australia was caused by a recently defined lineage prevalent in Europe, and three European isolates in the MGTdb were closely related but not directly linked to this outbreak. In summary, the S. Enteritidis MGTdb open platform is shown to be a potentially powerful tool for national and global public health surveillance of this pathogen.

ABSTRACT Hyperlipidemia is a risk factor and key indicator for cardiovascular diseases, and the gut microbiota is highly associated with hyperlipidemia. Bacteroides vulgatus is a prevalent mutualist across human populations and confers multiple health benefits such as immunoregulation, antiobesity, and coronary artery disease intervention. However, its role in antihyperlipidemia has not been systematically characterized. This study sought to identify the effect of B. vulgatus Bv46 on hyperlipidemia. Hyperlipidemic rats were modeled by feeding them a high-fat diet for 6 weeks. The effect of B. vulgatus Bv46 supplementation was evaluated by measuring anthropometric parameters, lipid and inflammation markers, and the liver pathology. Multi-omics was used to explore the underlying mechanisms. The ability of B. vulgatus Bv46 to produce bile salt hydrolase was confirmed by gene annotation and in vitro experiments. Oral administration of B. vulgatus Bv46 in hyperlipidemic rats significantly reduced the body weight gain, food efficiency, and liver index, improved the serum lipid profile, lowered the levels of serum inflammatory cytokines, promoted the loss of fecal bile acids (BAs), and extended the fecal pool of short-chain fatty acids (SCFAs), especially propionate and butyrate. B. vulgatus Bv46 induced compositional shifts of the gut microbial community of hyperlipidemic rats, characterized by a lower ratio of Firmicutes to Bacteroidetes with an increase of genera Bacteroides and Parabacteroides. After intervention, serum metabolite profiling exhibited an adaptation in amino acids and glycerophospholipid metabolism. Transcriptomics further detected altered biological processes, including primary bile acid biosynthesis and fatty acid metabolic process. Taken together, the findings suggest that B. vulgatus Bv46 could be a promising candidate for interventions against hyperlipidemia. IMPORTANCE As a core microbe of the human gut ecosystem, Bacteroides vulgatus has been linked to multiple aspects of metabolic disorders in a collection of associative studies, which, while indicative, warrants more direct experimental evidence to verify. In this study, we experimentally demonstrated that oral administration of B. vulgatus Bv46 ameliorated the serum lipid profile and systemic inflammation of high-fat diet-induced hyperlipidemic rats in a microbiome-regulated manner, which appears to be associated with changes of bile acid metabolism, short-chain fatty acid biosynthesis, and serum metabolomic profile. This finding supports the causal contribution of B. vulgatus in host metabolism and helps to form the basis of novel therapies for the treatment of hyperlipidemia.

ABSTRACT Listeria monocytogenes is a foodborne pathogen that can cause invasive disease with high mortality in immunocompromised individuals and can survive in a variety of food-associated environments for a long time. L. monocytogenes clonal complex (CC) 87 is composed of ST87 and three other STs and has been identified as the most common subgroup associated with both foods and human clinical infections in China. Therefore, the persistence of CC87 L. monocytogenes in food-associated environments poses a significant concern for food safety. In this study, 83 draft genomes of CC87 L. monocytogenes, including 60 newly sequenced genomes, were analyzed with all isolates from our previous surveillance in Zigong, Sichuang, China. Sixty-eight of the studied isolates were isolated from one retail market (M1 market), while the others were from seven other markets (M2–M8 markets) in the same city. Whole-genome multilocus sequence typing (wg-MLST) and the whole-genome single nucleotide polymorphism (wg-SNP) analysis were performed. Three persistent contamination routes were identified in the M1 market, caused by 2 clusters (A and B) and a wgST31 type. Cluster A isolates were associated with the persistent contamination in a raw meat stall (M1-S77), while Cluster B isolates caused a persistent contamination in aquatic foods stalls. Five wgST31 isolates caused persistent contamination in a single aquatic stall (M1-S65). A pLM1686-like plasmid was found in all Cluster A isolates. A novel plasmid, pLM1692, a truncated pLM1686 plasmid without the cadmium, and other heavy metal resistance genes were conserved in all wgST31 isolates. By comparing persistent and putative non-persistent isolates, four genes that were all located in the prophage comK might be associated with persistence. These findings enhanced our understanding of the underlying mechanisms of contamination and assist in formulating targeted strategies for the prevention and control of L. monocytogenes transmission from the food processing chain to humans. IMPORTANCE Contamination of food by Listeria monocytogenes at retail level leads to potential consumption of contaminated food with high risk of human infection. Our previous study found persistent contamination of CC87 L. monocytogenes from a retail market in China through pulsed-field gel electrophoresis and multilocus sequence typing. In this study, whole-genome sequencing was used to obtain the highest resolution inference of the source and reasons for persistent contamination; meat grinders and minced meat were the major reservoir of persistent contamination in meat stalls, whereas fishponds were the major reservoir in seafood stalls, with different L. monocytogenes isolates involved. These isolates carried different properties such as plasmids and prophages, which may have contributed to their ability to survive or adapt to the different environments. Our findings suggest that whole-genome sequencing will be an effective surveillance tool to detect persistent L. monocytogenes contamination in retail food markets and to design new control strategies to improve food safety.

Shiga toxin-producing Escherichia coli (STEC) have more than 470 serotypes. The well-known STEC O157:H7 serotype is a leading cause of STEC infections in humans. However, the incidence of non-O157:H7 STEC serotypes associated with foodborne outbreaks and human infections has increased in recent years. Current detection and serotyping assays are focusing on O157 and top six (“Big six”) non-O157 STEC serogroups. In this study, we performed phylogenetic analysis of nearly 41,000 publicly available STEC genomes representing 460 different STEC serotypes and identified 19 major and 229 minor STEC clusters. STEC cluster-specific gene markers were then identified through comparative genomic analysis. We further identified serotype-specific gene markers for the top 10 most frequent non-O157:H7 STEC serotypes. The cluster or serotype specific gene markers had 99.54% accuracy and more than 97.25% specificity when tested using 38,534 STEC and 14,216 non-STEC E. coli genomes, respectively. In addition, we developed a freely available in silico serotyping pipeline named STECFinder that combined these robust gene markers with established E. coli serotype specific O and H antigen genes and stx genes for accurate identification, cluster determination and serotyping of STEC. STECFinder can assign 99.85% and 99.83% of 38,534 STEC isolates to STEC clusters using assembled genomes and Illumina reads respectively and can simultaneously predict stx subtypes and STEC serotypes. Using shotgun metagenomic sequencing reads of STEC spiked food samples from a published study, we demonstrated that STECFinder can detect the spiked STEC serotypes, accurately. The cluster/serotype-specific gene markers could also be adapted for culture independent typing, facilitating rapid STEC typing. STECFinder is available as an installable package (https://github.com/LanLab/STECFinder) and will be useful for in silico STEC cluster identification and serotyping using genome data.

ABSTRACT Since 1817, cholera, caused by Vibrio cholerae, has been characterized by seven distinct pandemics. The ongoing seventh pandemic (7P) began in 1961. In this study, we developed a Multilevel Genome Typing (MGT) tool for classifying the V. cholerae species with a focus on the 7P. MGT is based on multilocus sequence typing (MLST), but the concept has been expanded to include a series of MLST schemes that compare population structure from broad to fine resolutions. The V. cholerae MGT consists of eight levels, with the lowest, MGT1, composed of 7 loci and the highest, MGT8, consisting of the 7P core genome (3,759 loci). We used MGT to analyze 5,771 V. cholerae genomes. The genetic relationships revealed by lower MGT levels recapitulated previous findings of large-scale 7P transmission across the globe. Furthermore, the higher MGT levels provided an increased discriminatory power to differentiate subgroups within a national outbreak. Additionally, we demonstrated the usefulness of MGT for non-7P classification. In a large non-7P MGT1 type, MGT2 and MGT3 described continental and regional distributions, respectively. Finally, MGT described trends of 7P in virulence, and MGT2 to MGT3 sequence types (STs) grouped isolates of the same ctxB, tcpA, and ctxB-tcpA genotypes and characterized their trends over the pandemic. MGT offers a range of resolutions for typing V. cholerae. The MGT nomenclature is stable, transferable, and directly comparable between investigations. The MGT database (https://mgtdb.unsw.edu.au/) can accept and process newly submitted samples. MGT allows tracking of existing and new isolates and will be useful for understanding future spread of cholera. IMPORTANCE In 2017, the World Health Organization launched the “Ending Cholera” initiative to reduce cholera-related deaths by 90% by 2030. This strategy emphasized the importance of the speed and accessibility of newer technologies to contain outbreaks. Here, we present a new tool named Multilevel Genome Typing (MGT), which classifies isolates of the cholera-causing agent, Vibrio cholerae. MGT is a freely available online database that groups genetically similar V. cholerae isolates to quickly indicate the origins of outbreaks. We validated the MGT database retrospectively in an outbreak setting, showcasing rapid confirmation of the Nepalese origins for the 2010 Haiti outbreak. In the past 5 years, thousands of V. cholerae genomes have been submitted to the NCBI database, which underscores the importance of and need for proper genome data classification for cholera epidemiology. The V. cholerae MGT database can assist in early decision making that directly impacts controlling both the local and global spread of cholera.

ObjectivesThis prospective study was carried out to investigate molecular characteristics and antimicrobial susceptibility patterns of Citrobacter spp. from extraintestinal infections.MethodsForty-six clinical Citrobacter spp. isolates were isolated from hospital patients with extraintestinal infections and analyzed by multilocus sequence typing (MLST) using seven housekeeping genes. Antimicrobial susceptibility testing was performed by disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Adhesion and cytotoxicity to HEp-2 cells were assessed.ResultsThe 46 clinical Citrobacter spp. isolates were typed into 38 sequence types (STs), 9 of which belonged to four clonal complexes (CCs). None of the isolates shared the same ST or CCs with isolates from other countries or from other parts of China. Over half of the isolates were multidrug-resistant (MDR), with 17/26 C. freundii, 5/6 C. braakii, and 3/14 C. koseri isolates being MDR. Moreover, four isolates were carbapenem resistant with resistance to imipenem or meropenem. Among eight quinolone resistant C. freundii, all had a mutation in codon 59 (Thr59Ile) in quinolone resistance determining region of the gyrA gene. Only a small proportion of the isolates were found to be highly cytotoxic and adhesive with no correlation to sample sources.ConclusionsThere was a diverse range of Citrobacter isolates causing extraintestinal infections and a high prevalence of MDR.

The Bordetella genus is divided into two groups: classical and non-classical. Bordetella pertussis, Bordetella bronchiseptica and Bordetella parapertussis are known as classical bordetellae, a group of important human pathogens causing whooping cough or whooping cough-like disease and hypothesized to have evolved from environmental non-classical bordetellae. Bordetella infections have increased globally driving the need to better understand these pathogens for the development of new treatments and vaccines. One unexplored component in Bordetella is the role of serine, threonine and tyrosine phosphorylation. Therefore, this study characterized the phosphoproteome of classical bordetellae and examined its potential role in Bordetella biology and virulence. Applying strict identification of localization criteria, this study identified 70 unique phosphorylated proteins in the classical bordetellae group with a high degree of conservation. Phosphorylation was a key regulator of Bordetella metabolism with proteins involved in gluconeogenesis, TCA cycle, amino acid and nucleotide synthesis significantly enriched. Three key virulence pathways were also phosphorylated including type III secretion system, alcaligin synthesis and the BvgAS master transcriptional regulatory system for virulence genes in Bordetella. Seven new phosphosites were identified in BvgA with 6 located in the DNA binding domain. Of the 7, 4 were not present in non-classical bordetellae. This suggests that serine/threonine phosphorylation may play an important role in stabilizing/destabilizing BvgA binding to DNA for fine-tuning of virulence gene expression and that BvgA phosphorylation may be an important factor separating classical from non-classical bordetellae. This study provides the first insight into the phosphoproteome of classical Bordetella species and the role that Ser/Thr/Tyr phosphorylation may play in Bordetella biology and virulence.

ABSTRACTListeria monocytogenes is a high risk pathogen which can cause invasive diseases in humans. We previously reported that black-headed gulls from Dianchi Lake of Kunming carrying L. monocytogenes, while the characteristics of these isolates and the relationship with habitats of migratory gulls have not been explored. In this study, we investigated the prevalence and molecular characteristics of Listeria monocytogenes from black-headed gulls in Dianchi Lake, and phylogenetic analysis based on core genome SNPs was used to determine the genetic relationship of the strains from Dianchi Lake and other regions. Occurrence of L. monocytogenes in black-headed gull feces in 2016, 2017 and 2018 was 1.0%, 1.0% and 0.6% respectively. The predominant serotype of 28 isolates was 4b, while the predominant sequence types were ST145 and ST201. Based on their prevalence and genomic relationships, ST5 and ST87 were likely to be sourced locally while ST145 and ST201 were likely to be non-local. L. monocytogenes may travel along the bird migration route leading to transmission over a large geographical span carried by black-headed gull. Although the prevalence of L. monocytogenes was low, its carriage by the migratory black-headed gulls poses potential public health risks in regions where the migratory birds passage and reside.

ABSTRACTPertussis caused by Bordetella pertussis, remains a public health problem worldwide, despite high vaccine coverage in infants and children in many countries. Iran has been using whole cell vaccine for the last 50 years with more than 95% vaccination rate since 1988 and has experienced pertussis resurgence in recent years. Here, we sequenced 55 B. pertussis isolates mostly collected from three provinces with the highest number of pertussis cases in Iran, including Tehran, Mazandaran, and Eastern-Azarbayjan from the period of 2008-2016. Most isolates carried ptxP3/prn2 alleles (42/55, 76%), the same genotype as isolates circulating in acellular vaccine-administrating countries. The second most frequent genotype was ptxP3/prn9 (8/55, 14%). Only three isolates (5%) were ptxP1. Phylogenetic analysis showed that Iranian ptxP3 isolates can be divided into eight clades (Clades 1-8) with no temporal association. Most of the isolates from Tehran grouped together as one distinctive clade (Clade 8) with six unique single nucleotide polymorphisms (SNPs). In addition, the prn9 isolates were grouped together as Clade 5 with 12 clade-supporting SNPs. No pertactin deficient isolates were found among the 55 Iranian isolates. Our findings suggest that there is an ongoing adaptation and evolution of B. pertussis regardless of the types of vaccine used.

ABSTRACTMacrolides such as erythromycin are the empirical treatment of Bordetella pertussis infections. China has experienced an increase in erythromycin-resistant B. pertussis isolates since they were first reported in 2013. Here, we undertook a genomic study on Chinese B. pertussis isolates from 2012 to 2015 to elucidate the origins and phylogenetic relationships of erythromycin-resistant B. pertussis isolates in China. A total of 167 Chinese B. pertussis isolates were used for antibiotic sensitivity testing and multiple locus variable-number tandem repeat (VNTR) analysis (MLVA). All except four isolates were erythromycin-resistant and of the four erythromycin-sensitive isolates, three were non-ptxP1. MLVA types (MT), MT55, MT104 and MT195 were the predominant types. Fifty of those isolates were used for whole genome sequencing and phylogenetic analysis. Genome sequencing and phylogenetic analysis revealed three independent erythromycin-resistant lineages and all resistant isolates carried a mutation in the 23S rRNA gene. A novel fhaB3 allele was found uniquely in Chinese ptxP1 isolates and these Chinese ptxP1-ptxA1-fhaB3 had a 5-fold higher mutation rate than the global ptxP1-ptxA1 B. pertussis population. Our results suggest that the evolution of Chinese B. pertussis is likely to be driven by selection pressure from both vaccination and antibiotics. The emergence of the new non-vaccine fhaB3 allele in Chinese B. pertussis population may be a result of selection from vaccination, whereas the expansion of ptxP1-fhaB3 lineages was most likely to be the result of selection pressure from antibiotics. Further monitoring of B. pertussis in China is required to better understand the evolution of the pathogen.

Shewanella species are widely distributed in the aquatic environment and aquatic organisms. They are opportunistic human pathogens with increasing clinical infections reported in recent years. However, there is a lack of a rapid and accurate method to identify Shewanella species. We evaluated here matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of Shewanella. A peptide mass reference spectra (PMRS) database was constructed for the type strains of 36 Shewanella species. The main spectrum projection (MSP) cluster dendrogram showed that the type strains of Shewanella species can be effectively distinguished according to the different MS fingerprinting. The PMRS database was validated using 125 Shewanella test strains isolated from various sources and periods; 92.8% (n = 116) of the strains were correctly identified at the species level, compared with the results of multilocus sequence analysis (MLSA), which was previously shown to be a method for identifying Shewanella at the species level. The misidentified strains (n = 9) by MALDI-TOF MS involved five species of two groups, i.e., Shewanella algae–Shewanella chilikensis–Shewanella indica and Shewanella seohaensis–Shewanella xiamenensis. We then identified and defined species-specific biomarker peaks of the 36 species using the type strains and validated these selected biomarkers using 125 test strains. Our study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid identification of Shewanella strains at the species level.

ABSTRACT Our ability to predict evolutionary trajectories of pathogens is one of the promising leverages to fight against the pandemic disease, yet few studies have addressed this question in situ, due to the difficulty in monitoring the milestone evolutionary events for a given pathogen and in understanding the evolutionary strategies. In this study, we monitored the real-time evolution of Vibrio parahaemolyticus in response to successive antibiotic treatment in three shrimp farms in North China from 2011 to 2018 by whole-genome sequencing. Results showed that the stepwise emergence of resistance was associated with the antibiotic usage. Genomic analysis of resistant isolates showed that the acquisition of the resistant mobile genetic elements flanked by an insertion sequence (ISVal1) closely mirrored the antibiotics used in shrimp farms since 2014. Next, we also identified 50 insertion sites of ISVal1 in the chromosome, which facilitated the formation of pathogenicity islands (PAIs) and fitness islands in the following years. Further, horizontal transfers of a virulent trh-nik-ure genomic island (GI) and two GIs improving the fitness have been observed in two farms since 2016. In this case study, we proposed that the insertion sequence triggered four major evolutionary events during the outbreaks of shrimp disease in three farms, including horizontal transfer of transposon (HTT) (stage 1), the formation of resistance islands (stage 2) and the PAIs (stage 3), and horizontal transfer of the PAIs (stage 4). This study presented the first in vivo evolutionary trajectories for a given bacterial pathogen, which helps us to understand the emergence mechanisms of new genotypes. IMPORTANCE Most human infectious diseases originate from animals. Thus, how to reduce or prevent pandemic zoonoses before they emerge in people is becoming a critical issue. Continuous genomic surveillance of the evolutionary trajectories of potential human pathogens on farms is a promising strategy to realize early warning. Here, we conducted an 8-year surveillance of Vibrio parahaemolyticus in three shrimp farms. The results showed that the use of antibiotics and horizontal transfer of transposons (HTT) drove the evolution of V. parahaemolyticus, which could be divided into four stages: HTT, formation of resistance islands, formation of pathogenicity islands (PAIs), and horizontal transfer of PAIs. This study presented the first in vivo monitoring of evolutionary trajectories for a given bacterial pathogen, providing valuable information for the prevention of pandemic zoonoses.

Salmonella spp. are a major cause of foodborne illness throughout the world. Traditional serotyping by antisera agglutination has been used as a standard identification method for many years but newer nucleic acid-based tests have become available that may provide advantages in workflow and test turnaround time. In this study, we evaluated the Luminex® xMAP® Salmonella Serotyping Assay (SSA), a multiplex nucleic acid test capable of identifying 85% of the most common Salmonella serotypes, in comparison to the traditional serum agglutination test (SAT) on 4 standard strains and 255 isolates from human (224), environmental, and food (31) samples. Of the total of 259 isolates, 256 could be typed by the SSA. Of these, 197 (77.0%) were fully typed and 59 (23.0%) were partially typed. By SAT, 246 of the 259 isolates (95%) were successfully typed. Sixty isolates had discrepant results between SAT and SSA and were resolved using whole genome sequencing (WGS). By SAT, 80.0% (48/60) of the isolates were consistent with WGS while by SSA 91.7% (55/60) were partially consistent with WGS. By serovar, all 30 serovars except one tested were fully or partially typable. The workflow comparison showed that SSA provided advantages over SAT with a hands-on time (HOT) of 3.5 min and total turnaround time (TAT) of 6 h, as compared to 1 h HOT and 2–6 days TAT for SAT. Overall, this study showed that molecular serotyping is promising as a rapid method for Salmonella serotyping with good accuracy for typing most common Salmonella serovars circulating in China.

Escherichia coli sequence type 131 (ST131) is well known for its multidrug resistance profile. Carbapenems have been considered the treatment of choice for E. coli ST131 infections, and resistance to carbapenems is emerging due to the acquisition of carbapenemase-encoding genes. In this study, 45 carbapenem-resistant E. coli strains were collected in a hospital. The resistance mechanisms, plasmid profiles, and genetic relatedness of these strains were determined. Phylogenetic relationships between these strains were assessed by molecular profiling and aligned with patient clinical details. The genetic context of blaKPC–2 was analyzed to trace the potential dissemination of blaKPC–2. The 45 carbapenem-resistant E. coli ST131 strains were closely related. Initially prevalent only in a single ward, ST131 subsequently spread to other ward, resulting in a respiratory infection outbreak of carbapenem-resistant E. coli ST131. Eight of the 30 patients died within 28 days of the first isolation of E. coli ST131. The blaKPC–2-positive plasmid profiles suggest that the carbapenem resistance was due to the acquisition by E. coli ST131 of transmissible plasmids pE0272_KPC and pE0171_KPC carrying blaKPC–2. Additionally, diverse multidrug resistance elements were transferred and rearranged between these plasmids mediated by IS26. Our research indicates that clinical attention should be paid to the importance of E. coli ST131 in respiratory infections and the spread of blaKPC-carrying E. coli ST131.

ABSTRACT Vibrio parahaemolyticus is an important foodborne pathogen and has recently gained particular notoriety because it causes acute hepatopancreatic necrosis disease (AHPND) in shrimp, which has caused significant economic loss in the shrimp industry. Here, we report a whole-genome analysis of 233 V. parahaemolyticus strains isolated from humans, diseased shrimp, and environmental samples collected between 2008 and 2017, providing unprecedented insight into the historical spread of AHPND. The results show that V. parahaemolyticus is genetically diverse and can be divided into 84 sequence types (STs). However, genomic analysis of three STs of V. parahaemolyticus identified seven transmission routes in Asia since 1996, which promoted the transfer of an AHPND-associated plasmid. Notably, the insertion sequence (ISVal1) from the plasmid subsequently mediated the genetic exchange among V. parahaemolyticus STs and resulted in the deletion of an 11-kb region regulating cell mobility and the production of capsular polysaccharides. Phenotype assays confirmed that this deletion enhanced biofilm formation, providing a novel mechanism for environmental adaptation. We conclude that the transmission mode of AHPND consists of two steps, the transmission of V. parahaemolyticus and the subsequent horizontal transfer of the AHPND-associated plasmid. This plasmid allows ISVal1 to mediate genetic exchange and improve pathogen fitness in shrimp ponds. Current shrimp farming practices promoted such genetic exchanges, which highlighted a risk of the emergence of new virulent populations, with potentially devastating consequences for both aquaculture and human health. This study addressed the basic questions regarding the transmission mechanism of AHPND and provided novel insights into shrimp and human disease management. IMPORTANCE Global outbreaks of shrimp acute hepatopancreatic necrosis disease (AHPND) caused by V. parahaemolyticus represent an urgent issue for the shrimp industry. This study revealed that the transmission mode of AHPND consists of two steps, the transregional dissemination of V. parahaemolyticus and the horizontal transfer of an AHPND-associated plasmid. Surprisingly, the introduction of the AHPND-associated plasmid also offers a novel mechanism of genetic exchange mediated by insertion sequences, and it improved the fitness of V. parahaemolyticus in a harsh environment. The results presented herein suggest that current shrimp farming practices promote genetic mixture between endemic and oceanic V. parahaemolyticus populations, which introduced the plasmid and accelerated bacterial adaptation by the acquisition of ecologically important functions. This entails a risk of the emergence of new virulent populations both for shrimp and humans. This study improves our understanding of the global dissemination of the AHPND-associated plasmid and highlights the urgent need to improve biosecurity for shrimp farming. Author Video: An author video summary of this article is available.

MraW is a 16S rRNA methyltransferase and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that the deletion of mraW led to decreased motility, flagellar production and DNA methylation. Whole-genome bisulfite sequencing showed a genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant including flagellar genes. The methylation level of flagellar genes was confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated that the transcription of these genes was also affected. MraW was furtherly observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common flexible motif in differentially methylated regions (DMRs) of promoters and coding regions of the four flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA methyltransferase activity assay in vitro and repressed by DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza). In addition, the mraW mutant colonized poorer than wild type in mice. We also found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and have a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.

Listeria is ubiquitous in a variety of environments and can be isolated from a wide range of animal hosts. Rodents are capable of carrying pathogenic bacteria in their intestines, such as Listeria, and can disseminate those pathogens into the natural environment and to where human activity occurs. In this study, we investigated the occurrence and antimicrobial susceptibility of Listeria spp. isolated from wild rodents found in natural environments in China. We collected 341 intestinal fecal samples of rodents from five different regions of China, all representing different rodent habitats. The antimicrobial susceptibility of the Listeria spp. isolates obtained were firstly assessed using the Kirby–Bauer disk diffusion method. Thirty-one samples were positive for Listeria spp., of which 11 were positive for Listeria monocytogenes and seven were positive for Listeria ivanovii. Other species identified include Listeria innocua, Listeria fleischmannii and Listeria floridensis. All Listeria spp. isolates were sensitive to the majority of the antimicrobials tested, but largely resistant to oxacillin (94.1%) and cefuroxime (70.6%). All L. monocytogenes isolates were further characterized by serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). L. monocytogenes strains were grouped into three serotypes, five sequence types and five pulsotypes (PTs) by serotyping, MLST and PFGE, respectively. Almost half of the isolates (five of 11) belonged to serotype 1/2b, ST87 and PT1. This study determined that Listeria is carried in the intestinal tracts of wild rodents from multiple regions at a low rate, filling an epidemiological data gap on Listeria in natural environments in China.Emerging Microbes & Infections (2017) 6, e44; doi:http://10.1038/emi.2017.28; published online 7 June 2017

Salmonella enterica subspecies enterica is a highly diverse subspecies with more than 1500 serovars and the ability to distinguish serovars within this group is vital for surveillance. With the development of whole-genome sequencing technology, serovar prediction by traditional serotyping is being replaced by molecular serotyping. Existing in silico serovar prediction approaches utilize surface antigen encoding genes, core genome MLST and serovar-specific gene markers or DNA fragments for serotyping. However, these serovar-specific gene markers or DNA fragments only distinguished a small number of serovars. In this study, we compared 2258 Salmonella accessory genomes to identify 414 candidate serovar-specific or lineage-specific gene markers for 106 serovars which includes 24 polyphyletic serovars and the paraphyletic serovar Enteritidis. A combination of several lineage-specific gene markers can be used for the clear identification of the polyphyletic serovars and the paraphyletic serovar. We designed and evaluated an in silico serovar prediction approach by screening 1089 genomes representing 106 serovars against a set of 131 serovar-specific gene markers. The presence or absence of one or more serovar-specific gene markers was used to predict the serovar of an isolate from genomic data. We show that serovar-specific gene markers have comparable accuracy to other in silico serotyping methods with 84.8% of isolates assigned to the correct serovar with no false positives (FP) and false negatives (FN) and 10.5% of isolates assigned to a small subset of serovars containing the correct serovar with varied FP. Combined, 95.3% of genomes were correctly assigned to a serovar. This approach would be useful as diagnosis moves to culture-independent and metagenomic methods as well as providing a third alternative to confirm other genome-based analyses. The identification of a set of gene markers may also be useful in the development of more cost-effective molecular assays designed to detect specific gene markers of the all major serovars in a region. These assays would be useful in serotyping isolates where cultures are no longer obtained and traditional serotyping is therefore impossible.

Sulfate is an important modulator for virulence factor expression in Bordetella pertussis, the causative organism for whooping cough. During infection, sulfate is released when respiratory epithelial cells are damaged which can affect gene expression. The current predominant strains in Australia are found in single nucleotide polymorphism (SNP) cluster I (ptxP3/prn2). It has been reported that ptxP3 strains have higher mRNA expression of virulence genes than ptxP1 strains under intermediate sulfate-modulating conditions (5 mM MgSO4). Our previous proteomic study compared L1423 (cluster I, ptxP3) and L1191 (cluster II, ptxP1) in Thalen–IJssel (THIJS) media without sulfate modulation and identified an upregulation of transport proteins and a downregulation of immunogenic proteins. To determine whether proteomic differences exist between cluster I and cluster II strains in intermediate modulating conditions, this study compared the whole cell proteome and secretome between L1423 and L1191 grown in THIJS media with 5 mM MgSO4 using iTRAQ and high-resolution multiple reaction monitoring (MRM-hr). Two proteins (BP0200 and BP1175) in the whole cell were upregulated in L1423 [fold change (FC) >1.2, false discovery rate (FDR)

Escherichia coli is both of a widespread harmless gut commensal and a versatile pathogen of humans. Domestic animals are a well-known reservoir for pathogenic E. coli. However, studies of E. coli populations from wild animals that have been separated from human activities had been very limited. Here we obtained 580 isolates from intestinal contents of 116 wild Marmot Marmota himalayana from Qinghai–Tibet plateau, China, with five isolates per animal. We selected 125 (hereinafter referred to as strains) from the 580 isolates for genome sequencing, based on unique pulse field gel electrophoresis patterns and at least one isolate per animal. Whole genome sequence analysis revealed that all 125 strains carried at least one and the majority (79.2%) carried multiple virulence genes based on the analysis of 22 selected virulence genes. In particular, the majority of the strains carried virulence genes from different pathovars as potential 'hybrid pathogens'. The alleles of eight virulence genes from the Marmot E. coli were found to have diverged earlier than all known alleles from human and other animal E. coli. Phylogenetic analysis of the 125 Marmot E. coli genomes and 355 genomes selected from 1622 human and other E. coli strains identified two new phylogroups, G and H, both of which diverged earlier than the other phylogroups. Eight of the 12 well-known pathogenic E. coli lineages were found to share a most recent common ancestor with one or more Marmot E. coli strains. Our results suggested that the intestinal E. coli of the Marmots contained a diverse virulence gene pool and is potentially pathogenic to humans. These findings provided a new understanding of the evolutionary origin of pathogenic E. coli.Emerging Microbes & Infections (2016) 5, e122; doi:10.1038/emi.2016.122; published online 7 December 2016

Objectives:Citrobacter freundii is a frequent cause of nosocomial infections and a known cause of diarrheal infections, and has increasingly become multidrug resistant (MDR). In this study, we aimed to determine the genetic diversity, the antimicrobial resistance profiles and in vitro virulence properties of C. freundii from diarrheal patients and healthy individuals.Methods: 82 C. freundii isolates were obtained from human diarrheal outpatients and healthy individuals. Multilocus Sequence Typing (MLST) of seven housekeeping genes was performed. Antimicrobial susceptibility testing was carried out using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Adhesion and cytotoxicity to HEp-2 cells were assessed. PCR and sequencing were used to identify blaCTX−M, blaSHV, blaTEM, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, and qepA genes.Results: The 82 C. freundii isolates were divided into 76 sequence types (STs) with 65 STs being novel, displaying high genetic diversity. Phylogenetic analysis divided the 82 isolates into 5 clusters. All 82 isolates were sensitive to imipenem (IPM), but resistant to one or more other 16 antibiotics tested. Twenty-six isolates (31.7%) were multidrug resistant to three or more antibiotic classes out of the 10 distinct antibiotic classes tested. Five MDR isolates, all of which were isolated from 2014, harbored one or more of the resistance genes, blaTEM−1, blaCTX−M−9, aac(6')-Ib-cr, qnrS1, qnrB9, and qnrB13. All 11 qnrB-carrying C. freundii isolates belonged to cluster 1, and one C. freundii isolate carried a new qnrB gene (qnrB92). Six isolates showed strong cytotoxicity to HEp-2 cells, one of which was multidrug resistant.Conclusions:C. freundii isolates from human diarrheal outpatients and healthy individuals were diverse with variation in sequence types, antibiotic resistance profiles and virulence properties.

Listeria monocytogenes can contaminate various foods via food processing environments and contamination of raw materials. There is a limited understanding of L. monocytogenes transmission in retail market and the role of insects in L. monocytogenes transmission in the retail environments. To better understand the risk factors of raw pork contamination, the prevalence of L. monocytogenes was examined in raw pork, retail environments and insects in a retail market over a 6-month period from March to August in 2016 in Beijing, China. A total of 2,789 samples were collected, including 356 raw pork samples, 1,392 meat contact surface swabs (MCS), 712 non-meat contact surface swabs (NMCS) and 329 insect samples. Overall, 424 (15.20%) of the samples were found to be contaminated by L. monocytogenes. Analyzed by serotyping, multilocus sequence typing and pulsed-field gel electrophoresis, the 424 L. monocytogenes isolates were divided into three serotypes (1/2c, 1/2a and 3a), 15 pulsotypes (PTs) and nine sequence types (STs), 1/2c/PT4/ST9 (244/424, 58%) was the most prevalent type of L. monocytogenes strains. The raw pork, MCS of the environments and insects were contaminated with higher levels of L. monocytogenes than NMCS of the environments, which suggested that cross contamination of L. monocytogenes between raw pork and the environment existed in the retail market, and long-term contaminated surfaces and vector insects would act as high risk factors to transmit L. monocytogenes to raw pork. Thus more effective strategies are needed to reduce the risk of retail pork meat contamination by L. monocytogenes and prevent foodborne human listeriosis.

Objectives:Citrobacter spp. especially Citrobacter freundii, is frequently causing nosocomial infections, and increasingly becoming multi-drug resistant (MDR). In this study, we aimed to determine the genetic diversity and relationships of Citrobacter spp. from diarrheal patients and food sources, their antimicrobial resistance profiles and in vitro virulence properties.Methods: Sixty two Citrobacter isolates, including 13 C. freundii, 41 C. youngae and eight C. braakii isolates, were obtained from human diarrheal patients and food sources. Multilocus Sequence Typing (MLST) of seven housekeeping genes and antimicrobial susceptibility testing using the broth microdilution method according to CLSI recommendations were carried out. Adhesion and cytotoxicity to HEp-2 cells were performed. PCR and sequencing were used to identify blaCTX−M, blaSHV, blaTEM and qnr genes.Results: The 62 isolates were divided into 53 sequence types (STs) with all STs being novel, displaying high genetic diversity. ST39 was a predominant ST shared by 5 C. youngae strains isolated from four foods and a diarrheal patient. All isolates were resistant to cefoxitin, and sensitive to imipenem, meropenem and amikacin. The majority of Citrobacter isolates (61.3%) were MDR of three or more antibiotics out of the 22 antibiotics tested. Two C. freundii isolates each carried the blaTEM−1 gene and a variant of qnrB77. Three Citrobacter isolates each carried qnrS1 and aac(6')-Ib-cr genes. Seven isolates that showed strong cytotoxicity to HEp-2 cells were MDR.Conclusions:Citrobacter spp. from human and food sources are diverse with variation in virulence properties and antibiotic resistance profiles. Food may be an important source of Citrobacter species in transmission to humans. C. freundii and C. youngae are potential foodborne pathogens.

Shigella flexneri is a leading cause of bacterial dysentery in developing countries. Among the 15 known serotypes, four (1b, 3a, 3b and 4b) contain a group 6 epitope due to an acetyl group connected to the O-2 position of rhamnose III on the tetrasaccharide structure of the lipopolysaccharide. O-acetyltransferase encoded by a bacteriophage, Sf6, mediates the acetylation reaction. We found that the oac gene in serotype 1b strains was very different from that in serotypes 3a, 3b and 4b strains and is herein after referred to as oac1b which shares with oac 88%–89% identity at the DNA level and 85% identity at the protein level. Considering that S. flexneri strains of serotypes 1–5 share a recent common ancestry, the divergent oac1b is more likely to have been obtained from outside S. flexneri than to have undergone rapid divergence from the oac gene in the other serotypes (3a, 3b and 4b) within S. flexneri. The cloned oac1b gene was found to perform the same acetylation function as oac. Analysis of the genomic regions flanking oac1b showed that it was present in a prophage on the chromosome and the organizational structure is different from that of phage Sf6. Additionally, phage conversion assay showed that serotype 1b cannot be generated by infecting serotype 1a strains with Sf6. We conclude that oac1b was carried by a non-Sf6 phage and is uniquely present in serotype 1b.

ABSTRACT Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.

Campylobacter concisus is an oral bacterium that is associated with intestinal diseases. C. concisus was previously described as a bacterium that requires H2-enriched microaerobic conditions for growth. The level of H2 in the oral cavity is extremely low, suggesting that C. concisus is unlikely to have a microaerobic growth there. In this study, the anaerobic growth of C. concisus was investigated. The growth of fifty-seven oral C. concisus strains and six enteric C. concisus strains under various atmospheric conditions including anaerobic conditions with and without H2 was examined. The atmospheric conditions were generated using commercially available gas-generation systems. C. concisus putative virulence proteins were identified using mass spectrometry analysis. Under anaerobic conditions, 92% of the oral C. concisus strains (52/57) and all six enteric strains grew without the presence of H2 and the presence of H2 greatly increased C. concisus growth. An oral C. concisus strain was found to express a number of putative virulence proteins and the expression levels of these proteins were not affected by H2. The levels of H2 appeared to affect the optimal growth of C. concisus. This study provides useful information in understanding the natural colonization site and pathogenicity of C. concisus.

Ribotyping has been widely used to characterise the seventh pandemic clone including South American and O139 variants which appeared in 1991 and 1992 respectively. To reveal the molecular basis of ribotype variation we analysed the rrn operons and their flanking regions. All but one variation detected by BglI, the most discriminatory enzyme, was found to be due to changes within the rrn operons, resulting from recombination between operons. The recombinants are detected because of the presence of a BglI site in the 16S gene in three of the nine rrn operons and/or changes of intergenic spacer types of which four variants were identified. As the frequency of rrn recombination is high, ribotyping becomes a less useful tool for evolutionary studies and long term monitoring of the pathogenic clones of Vibrio cholerae as variation could undergo precise reversion by the same recombination event.