Search

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 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 expanded 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, likely driven by immune selection from pertussis vaccination and natural infection in addition to local and global transmission events. Both hard and soft selective sweeps through vaccine selection pressures determined the current B. pertussis population dynamics. Relative risk analysis found 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 and became no longer spatially structured across the country. Phylogenetic analysis revealed the expansion of ptxP3 strains was also associated with replacement of the type III secretion system allele bscI1 with bscI3. This study advanced our understanding of the epidemic population structure and spatial and temporal dynamics of B. pertussis in a highly immunised population.

Cholera caused by Vibrio cholerae O139 was first reported in Bangladesh and India in 1992. To determine the genomic epidemiology and origins of O139 in China, we sequenced 104 O139 isolates collected from Zhejiang Province, China, during 1994-2018 and compared them with 57 O139 genomes from other countries in Asia. Most Zhejiang isolates fell into 3 clusters (C1-C3), which probably originated in India (C1) and Thailand (C2 and C3) during the early 1990s. Different clusters harbored different antimicrobial resistance genes and IncA/C plasmids. The integrative and conjugative elements carried by Zhejiang isolates were of a new type, differing from ICEVchInd4 and SXT

The enteroinvasive bacterium

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.

Significance Enterohemorrhagic E. coli is a significant human pathogen that can cause severe disease due to the release of Shiga toxins. The toxins are encoded within lysogenic bacteriophage and controlled by antitermination of the phage late promoter, PR′. This promoter is always active, but terminated immediately downstream during lysogeny. A byproduct of antitermination regulation is transcription of a short RNA that is thought to be nonfunctional. Here we demonstrate that in Shiga toxin-encoding phages, this short RNA is a Hfq-binding regulatory small RNA. The small RNA represses toxin production threefold under lysogenic conditions and promotes high cell density growth. Lysogenic bacteriophages are highly abundant and our results suggest that antiterminated phage promoters may be a rich source of regulatory RNAs., Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR′ late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR′ immediately downstream of PR′, generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5′ UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.

Since acellular vaccines (ACV) were introduced in Australia, epidemic Bordetella pertussis strains changed from single nucleotide polymorphism (SNP) cluster II to SNP cluster I. Our previous proteomic analysis identified potential proteomic adaptations in the whole cell and secretome of SNP cluster I. Additionally, current ACVs were shown to be less efficacious against cluster I in mice models and there is a pressing need to discover new antigens to improve the ACV. One important source of novel antigens is the surfaceome. Therefore, in this study we established surface shaving in B. pertussis to compare the surfaceome of SNP cluster I (L1423) and II (L1191), and identify novel surface antigens for vaccine development. Surface shaving using 1 μg of trypsin for 5 min identified 126 proteins with the most abundant being virulence-associated and known outer membrane proteins. Cell viability counts showed minimal lysis from shaving. The proportion of immunogenic proteins was higher in the surfaceome than in the whole cell and secretome. Key differences in the surfaceome were identified between SNP cluster I and II, consistent with those identified in the whole cell proteome and secretome. These differences include unique transport proteins and decreased immunogenic proteins in L1423, and provides further evidence of proteomic adaptation in SNP cluster I. Finally, a comparison of proteins in each sub-proteome identified 22 common proteins. These included 11 virulence proteins (Prn, PtxA, FhaB, CyaA, TcfA, SphB1, Vag8, BrkA, BopD, Bsp22 and BipA) and 11 housekeeping proteins (TuF, CtpA, TsF, OmpH, GltA, SucC, SucD, FusA, GroEL, BP3330 and BP3561) which were immunogenic, essential and consistently expressed thus demonstrating their potential as future targets. This study established surface shaving in B. pertussis, confirmed key expression differences and identified unknown surface proteins which may be potential vaccine antigens.

Escherichia albertii is a recently recognized species in the genus Escherichia that causes diarrhoea. The population structure, genetic diversity and genomic features have not been fully examined. Here, 169 E. albertii isolates from different sources and regions in China were sequenced and combined with 312 publicly available genomes (from additional 14 countries) for genomic analyses. The E. albertii population was divided into two clades and eight lineages, with lineage 3 (L3), L5 and L8 more common in China. Clinical isolates were observed in all clades/lineages. Virulence genes were found to be distributed differently among lineages: subtypes of the intimin encoding gene eae and the cytolethal distending toxin gene cdtB were lineage associated, and the second type three secretion system (ETT2) island was truncated in L3 and L6. Seven new eae subtypes and one new cdtB subtype (cdtB-VI) were identified. Alarmingly, 85.9 % of the Chinese E. albertii isolates were predicted to be multidrug-resistant (MDR) with 35.9 % harbouring genes capable of conferring resistance to 10 to 14 different drug classes. The majority of the MDR isolates were of poultry source from China and belonged to four sequence types (STs) [ST4638, ST4479, ST4633 and ST4488]. Thirty-four plasmids with some carrying MDR and virulence genes, and 130 prophages were identified from 17 complete E. albertii genomes. The 130 intact prophages were clustered into five groups, with group five prophages harbouring more virulence genes. We further identified three E. albertii specific genes as markers for the identification of this species. Our findings provided fundamental insights into the population structure, virulence variation and drug resistance of E. albertii .

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.

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.

Salmonella enterica serovar Enteritidis is a major cause of foodborne Salmonella infections and outbreaks in humans. Effective surveillance and timely outbreak detection are essential for public health control. Multilevel genome typing (MGT) with multiple levels of resolution has been previously demonstrated as a promising tool for this purpose. In this study, we developed MGT with nine levels for S. Enteritidis and characterised the genomic epidemiology of S. Enteritidis in detail. We examined 26 670 publicly available S. Enteritidis genome sequences from isolates spanning 101 years from 86 countries to reveal their spatial and temporal distributions. Using the lower resolution MGT levels, globally prevalent and regionally restricted sequence types (STs) were identified; avian associated MGT4-STs were found that were common in human cases in the USA; temporal trends were observed in the UK with MGT5-STs from 2014 to 2018 revealing both long lived endemic STs and the rapid expansion of new STs. Using MGT3 to MGT6, we identified multidrug resistance (MDR) associated STs at various MGT levels, which improves precision of detection and global tracking of MDR clones. We also found that the majority of the global S. Enteritidis population fell within two predominant lineages, which had significantly different propensity of causing large scale outbreaks. An online open MGT database has been established for unified international surveillance of S. Enteritidis. We demonstrated that MGT provides a flexible and high-resolution genome typing tool for S. Enteritidis surveillance and outbreak detection.

Background Human-use probiotics have recently been associated with clinical infections and antibiotic resistance transfer, raising public concern over their safety. However, despite their extensive application in aquaculture and animal husbandry, the safety of animal-use probiotics remains poorly described. Methods We evaluated the safety of 92 animal-use probiotics from China. The pattern of spread of pathogens from probiotics and the consequent public health implications were also examined by conducting in-field genomic surveillance at 2 farms. Results A total of 123 probiotic Bacillus species isolates were obtained from 92 brands of probiotics, of which 45 isolates were resistant to antibiotics. Notably, 33.7% of probiotic products were contaminated with life-threatening pathogens such as Klebsiella pneumoniae. Genomic surveillance at a chicken farm identified an anthrax toxin–positive Bacillus cereus strain in a probiotic product used as a feed supplement, which was transferred into the groundwater and to a nearby fish farm. Following up retrospective analysis of the surveillance data during 2015–2018 in 3 provinces retrieved 2 B. cereus strains from human with intestinal anthrax symptoms and confirmed the transmission of B. cereus from farm to human. Surveillance of anthrax toxin revealed that cya was detected in 8 of 31 farms. Conclusions This study provides the first national safety survey of animal-use probiotics in China and confirms the spillover effects of probiotics from the farms to human. These results suggest that the large-scale application of pathogen-containing probiotics leads to the transfer of pathogens, with worrisome implications for public health. Good Manufacturing Practice should be implemented during the production of all probiotics. Animal-use probiotic products are frequently contaminated with viable pathogenic bacteria. This study revealed that virulent probiotic organisms and contaminating pathogens were colonized with farm animals and shed into the environment, which facilitated the transfer of pathogens to humans.

Wildlife is a reservoir of emerging infectious diseases of humans and domestic animals. Marmota himalayana mainly resides 2800–4000 m above sea level in the Qinghai-Tibetan Plateau, and is the primary animal reservoir of plague pathogen Yersinia pestis. Recently we isolated a new species, Escherichia marmotae from the faeces of M. himalayana. In this study we characterised E. marmotae by genomic analysis and in vitro virulence testing to determine its potential as a human pathogen. We sequenced the genomes of the seven E. marmotae strains and found that they contained a plasmid that carried a Shigella-like type III secretion system (T3SS) and their effectors, and shared the same O antigen gene cluster as Shigella dysenterae 8 and E. coli O38. We also showed that E. marmotae was invasive to HEp-2 cells although it was much less invasive than Shigella. Thus E. marmotae is likely to be an invasive pathogen. However, E. marmotae has a truncated IpaA invasin, and lacks the environmental response regulator VirF and the IcsA-actin based intracellular motility, rendering it far less invasive in comparison to Shigella. E. marmotae also carried a diverse set of virulence factors in addition to the T3SS, including an IS1414 encoded enterotoxin gene astA with 37 copies, E. coli virulence genes lifA/efa, cif, and epeA, and the sfp gene cluster, Yersinia T3SS effector yopJ, one Type II secretion system and two Type VI secretion systems. Therefore, E. marmotae is a potential invasive pathogen.

Macrolides 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.

The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.

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.

Campylobacter concisusis an emerging enteric pathogen that is associated with several gastrointestinal diseases, such as inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC). Currently, only three completeC. concisusgenomes are available and more completeC. concisusgenomes are needed in order to better understand the genomic features and pathogenicity of this emerging pathogen. DNA extracted from 22C.concisusstrains were subjected to Oxford Nanopore genome sequencing. Complete genome assembly was performed using Nanopore genome data in combination with previously reported short-read Illumina data. Genome features of completeC. concisusgenomes were analysed using bioinformatic tools. The enteric disease associations ofC. concisusplasmids were examined using 239C.concisusstrains and confirmed using PCRs. Proteomic analysis was used to examine T6SS secreted proteins. We successfully obtained 13 completeC. concisusgenomes in this study. Analysis of 16 completeC. concisusgenomes (3 from public databases) identified multiple novel plasmids. pSma1 plasmid was found to be associated with severe UC. Sec-SRP, Tat and T6SS were found to be the main secretion systems inC. concisusand proteomic data showed a functional T6SS despite the lack of ClpV. T4SS was found in 25% of completeC. concisusgenomes. This study also found that GS2 strains had larger genomes and higher GC content than GS1 strains and more often had plasmids. In conclusion, this study provides fundamental genomic data for understandingC. concisusplasmids, genomospecies features, evolution, secretion systems and pathogenicity.

To understand the environmental reservoirs of Vibrio cholerae and their public health significance, we surveyed freshwater samples from rivers in two cities (Jiaxing [JX] and Jiande [JD]) in Zhejiang, China. A total of 26 sampling locations were selected, and river water was sampled 456 times from 2015 to 2016 yielding 200 V. cholerae isolates, all of which were non-O1/non-O139. The average isolation rate was 47.3% and 39.1% in JX and JD, respectively. Antibiotic resistance profiles of the V. cholerae isolates were examined with nonsusceptibility to cefazolin (68.70%, 79/115) being most common, followed by ampicillin (47.83%, 55/115) and imipenem (27.83%, 32/115). Forty-two isolates (36.52%, 42/115) were defined as multidrug resistant (MDR). The presence of virulence genes was also determined, and the majority of the isolates were positive for toxR (198/200, 99%) and hlyA (196/200, 98%) with few other virulence genes observed. The population structure of the V. cholerae non-O1/non-O139 sampled was examined using multilocus sequence typing (MLST) with 200 isolates assigned to 128 STs and 6 subpopulations. The non-O1/non-O139 V. cholerae population in JX was more varied than in JD. By clonal complexes (CCs), 31 CCs that contained isolates from this study were shared with other parts of China and/or other countries, suggesting widespread presence of some non-O1/non-O139 clones. Drug resistance profiles differed between subpopulations. The findings suggest that non-O1/non-O139 V. cholerae in the freshwater environment is a potential source of human infections. Routine surveillance of non-O1/non-O139 V. cholerae in freshwater rivers will be of importance to public health.

Background Both long- and short-term epidemiology are fundamental to disease control and require accurate bacterial typing. Genomic data resulting from implementation of whole genome sequencing in many public health laboratories can potentially provide highly sensitive and accurate descriptions of strain relatedness. Previous typing efforts using these data have mainly focussed on outbreak detection. Aim We aimed to develop multilevel genome typing (MGT), using consecutive multilocus sequence typing (MLST) schemes of increasing sizes, stepping up from seven-gene MLST to core genome MLST, to allow examination of genetic relatedness at multiple resolution levels. Methods The system was applied to Salmonella enterica serovar Typhimurium. The MLST scheme used at each step (MGT level), defined a given MGT-level specific sequence type (ST). The list of STs generated from all of these increasing MGT levels, was named a genome type (GT). Using MGT, we typed 9,096 previously characterised isolates with publicly available data. Results Our approach could identify previously described S. Typhimurium populations, such as the DT104 multidrug resistance lineage (GT 19-2-11) and two invasive lineages of African isolates (GT 313-2-3 and 313-2-752). Further, we showed that MGT-derived clusters can accurately distinguish five outbreaks from each other and five background isolates. Conclusion MGT provides a universal and stable nomenclature at multiple resolutions for S. Typhimurium strains and could be implemented as an internationally standardised strain identification system. While established so far only for S. Typhimurium, the results here suggest that MGT could form the basis for typing systems in other similar microorganisms.

Summary Background Corynebacteritum straitum has been considered as an emerging multi-drug-resistant (MDR) pathogen. Isolation of MDR C. striatum as the only organism from respiratory samples from hospitalized patients is increasing in China. Aim To elucidate the genomic epidemiology and evolution of C. striatum in China. Methods A total of 260 isolates from 2016 to 2018 were collected from three hospitals in three regions of China. Antibiotic sensitivity testing was performed on all isolates. Whole-genome sequencing was applied to all isolates to assess their genomic diversity and relationships and detect the presence of antimicrobial resistance genes (ARG) and ARG cassettes. Findings Almost all isolates (96.2%, 250/260) showed multi-drug-resistance. Genome sequencing revealed four major lineages with lineage IV emerging as the epidemic lineage. Most of the diversity was developed in the last 6 years. Each hospital has its own predominant clones with potential spread between Hebei and Guangdong hospitals. Genomic analysis further revealed multiple antimicrobial resistance genes. Conclusions Our results suggested that four lineages of C. striatum have spread in parallel across China, causing persistent and extensive transmissions within hospitals. MDR C. striatum infection has become a national epidemic. Antibiotic-driven selection pressure may have played significant roles in forming persistent and predominant clones. Our data provide the basis for surveillance and prevention strategies to control the epidemic caused by MDR C. striatum.

We sequenced the genomes of 14 sequential Salmonella enterica serovar Typhimurium isolates obtained over a five year period from a patient with persistent Salmonella bacteriuria. The isolates formed five distinct lineages; two of which co-existed over four years. We inferred that the observed within-patient variation resulted from mutation events.

Shiga toxin producing Escherichia coli O157:H7 (STEC O157) is naturally found in the gastrointestinal tract of cattle and can cause severe disease in humans. There is limited understanding of the population dynamics and microevolution of STEC O157 at herd level. In this study, isolates from a closed beef herd of 23 cows were used to examine the population turnover in the herd. Of the nine STEC O157 clades previously described, clade 7 was found in 162 of the 169 isolates typed. Multiple locus variable number tandem repeat analysis (MLVA) differentiated 169 isolates into 33 unique MLVA types. Five predominant MLVA types were evident with most of the remaining types containing only a single isolate. MLVA data suggest that over time clonal replacement occurred within the herd. Genome sequencing of 18 selected isolates found that the isolates were divided into four lineages, representing four different 'clones' in the herd. Genome data confirmed clonal replacement over time and provided evidence of cross transmission of strains between cows. The findings enhanced our understanding of the population dynamics of STEC O157 in its natural host that will help developing effective control measures to prevent the spread of the pathogen to the human population.

Streptococcus suis sequence type 7 emerged and caused 2 of the largest human infection outbreaks in China in 1998 and 2005. To determine the major risk factors and source of the infections, we analyzed whole genomes of 95 outbreak-associated isolates, identified 160 single nucleotide polymorphisms, and classified them into 6 clades. Molecular clock analysis revealed that clade 1 (responsible for the 1998 outbreak) emerged in October 1997. Clades 2–6 (responsible for the 2005 outbreak) emerged separately during February 2002–August 2004. A total of 41 lineages of S. suis emerged by the end of 2004 and rapidly expanded to 68 genome types through single base mutations when the outbreak occurred in June 2005. We identified 32 identical isolates and classified them into 8 groups, which were distributed in a large geographic area with no transmission link. These findings suggest that persons were infected in parallel in respective geographic sites.

Salmonella enterica serovar Typhimurium is the leading cause of salmonellosis in Australia, and the ability to identify outbreaks and their sources is vital to public health. Here, we examined the utility of whole-genome sequencing (WGS), including complete genome sequencing with Oxford Nanopore technologies, in examining 105 isolates from an endemic multi-locus variable number tandem repeat analysis (MLVA) type over 5 years. The MLVA type was very homogeneous, with 90 % of the isolates falling into groups with a five SNP cut-off. We developed a new two-step approach for outbreak detection using WGS. The first clustering at a zero single nucleotide polymorphism (SNP) cut-off was used to detect outbreak clusters that each occurred within a 4 week window and then a second clustering with dynamically increased SNP cut-offs were used to generate outbreak investigation clusters capable of identifying all outbreak cases. This approach offered optimal specificity and sensitivity for outbreak detection and investigation, in particular of those caused by endemic MLVA types or clones with low genetic diversity. We further showed that inclusion of complete genome sequences detected no additional mutational events for genomic outbreak surveillance. Phylogenetic analysis found that the MLVA type was likely to have been derived recently from a single source that persisted over 5 years, and seeded numerous sporadic infections and outbreaks. Our findings suggest that SNP cut-offs for outbreak cluster detection and public-health surveillance should be based on the local diversity of the relevant strains over time. These findings have general applicability to outbreak detection of bacterial pathogens.

Background Neonatal listeriosis is a rare but severe disease manifesting as septicemia and central nervous system (CNS) infections with a high fatality rate of around 20 to 30%. Whole genome sequencing (WGS) is a promising technique for pathogen identification and infection source tracing with its high resolution. Case presentation A case of neonatal sepsis with listeriosis was reported with positive blood culture for Listeria monocytogenes. The case was investigated to confirm the vertical transmission of the infection and identify the potential food source of the maternal L. monocytogenes infection using WGS. L. monocytogenes was isolated from the neonate’s blood sample the day after caesarean delivery and from the mother’s genital and pudenda swab samples 5 days and 13 days after caesarean delivery. WGS showed that the isolate from the neonate was identical to the genome type of the isolates from the mother, with only one of the 4 isolates from the mother differing by one single nucleotide polymorphism (SNP). By WGS, one L. monocytogenes isolate from a ready-to-eat (RTE) meat sample in the patients’ community market shared the same sequence type but was ruled out as the cause of infection, with 57 SNP differences to the strain causing the maternal-neonatal infection. The food isolate also carried a novel plasmid pLM1686 that harbored heavy metal resistance genes. After caesarean section, the mother was treated with a third generation cephalosporin which L. monocytogenes is naturally resistant to, which may explain why genital and pudenda swabs were still culture-positive for L. monocytogenes 13 days after delivery. Conclusions Genital swab culture for L. monocytogenes had been informative in the diagnosis of maternal listeriosis in this case. The high resolution of WGS confirmed the maternal-neonatal transmission of L. monocytogenes infection and ruled out the L. monocytogenes contaminated RTE meat from the local market as the direct source of the mother’s infection.

Pertussis 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.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen that occurs primarily among immunocompromised and chronically ill patients. However, little is known about the genomic diversity of C. striatum, which contributes to its long-term persistence and transmission in hospitals., Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen that occurs primarily among immunocompromised and chronically ill patients. However, little is known about the genomic diversity of C. striatum, which contributes to its long-term persistence and transmission in hospitals. In this study, a total of 192 C. striatum isolates obtained from 14 September 2017 to 29 March 2018 in a hospital in Beijing, China, were analyzed by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was conducted on 91 isolates. Nearly all isolates (96.3%, 183/190) were MDR. The highest resistance rate was observed for ciprofloxacin (99.0%, 190/192), followed by cefotaxime (90.6%, 174/192) and erythromycin (89.1%, 171/192). PFGE separated the 192 isolates into 79 pulsotypes, and differences in core genome single-nucleotide polymorphisms (SNPs) partitioned the 91 isolates sequenced into four clades. Isolates of the same pulsotype were identical or nearly identical at the genome level, with some exceptions. Two dominant subclones, clade 3a, and clade 4a, were responsible for the hospital-wide dissemination. Genomic analysis further revealed nine resistance genes mobilized by eight unique cassettes. PFGE and whole-genome sequencing revealed that the C. striatum isolates studied were the result mainly of predominant clones spreading in the hospital. C. striatum isolates in the hospital progressively acquired resistance to antimicrobial agents, demonstrating that isolates of C. striatum may adapt rapidly through the acquisition and accumulation of resistance genes and thus evolve into dominant and persistent clones. These insights will be useful for the prevention of C. striatum infection in hospitals.

During the 2008-2012 pertussis epidemic in Australia, pertactin (Prn)-negative Bordetella pertussis emerged. We analyzed 78 isolates from the 2013-2017 epidemic and documented continued expansion of Prn-negative ptxP3 B. pertussis strains. We also detected a filamentous hemagglutinin-negative and Prn-negative B. pertussis isolate.

Changes in circulating Bordetella pertussis genotypes, including a novel pertussis toxin promoter ptxP3 allele and absence of pertactin (Prn) antigen, have been reported from several countries but limited data on relative severity are available. We compared markers of disease severity in children with B. pertussis infection due to strains of differing genotype.Culture confirmed cases presenting to tertiary paediatric hospitals in three Australian states between 2008 and 2012 were classified as severe if they required a hospital stay greater than seven days, were admitted to intensive care, or if death occurred. Associations between age, vaccination, genotype and severity were assessed.Of 199 pertussis cases, 81 (41%) were3 months, including 32/39 (82%) of severe cases. The proportion of isolates from these cases that were Prn deficient increased markedly between 2008 and 2012. Of B. pertussis isolates, the proportion considered severe was similar for Prn positive (27/128, 21%) and Prn deficient (12/71, 17%) cases but only 1/22 (4.5%) of non ptxP3 cases were severe versus 38/177 (21.4%) ptxP3 positive. Adjusting for ptxP type, vaccination status and age, disease severity was not significantly associated with Prn status (RRA: 0.95, [0.57-1.56]; p = 0.83).In children, we found no relationship between Prn status and markers of severe pertussis. An increased proportion of severe disease in isolates with the ptxP3 allele was observed.

Purpose. Both Shigella and enteroinvasive Escherichia coli (EIEC) can cause enterocolitis, but they have a distinct epidemiology and public health relevance. Current culture-independent testing (CIT) methods to identify Shigella in faecal samples rely on the ipaH gene as the target, which is also found in EIEC genomes. The aim of this study was to design an assay that can identify EIEC in cultures from CIT ipaH-positive samples. Methodology. Shigella and EIEC genomes were screened to find unique regions present in EIEC genomes using a comparative genomics approach and differentiating genetic loci that are suitable PCR targets were identified. The primers for these loci were designed and tested in 6501 and 104 genomes of Shigella and EIEC, respectively. Results. An assay with two sets of multiplex PCR reactions that differentiates Shigella and EIEC based on the presence/absence of at least two out of six loci was developed and evaluated. The majority of Shigella genomes lacked all six loci, while at least two loci were present in most EIEC genomes. This assay successfully differentiated clinical EIEC from Shigella with a limit of detection of 105 cells ml−1. The sensitivity and specificity were over 95 and 99%, respectively. The assay can further subtype EIEC genomes into their genetic lineages. Conclusion. This new highly specific assay can assist in the identification of EIEC in ipaH PCR-positive samples and augment the public health laboratory surveillance of EIEC and shigellosis.

We examined the population dynamics of Salmonella enterica serovar Typhimurium during seasonal salmonellosis epidemics in New South Wales, Australia, during 2009–2016. Of 15,626 isolates, 5%–20% consisted of novel genotypes. Seasons with salmonellosis epidemics were associated with a reduction in novel genotypes in the preceding winter and spring.

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.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC). C. concisus consists of two genomospecies (GS) and diverse strains. This study aimed to identify molecular markers to differentiate commensal and IBD-associated C. concisus strains. The genomes of 63 oral C. concisus strains isolated from patients with IBD and healthy controls were examined, of which 38 genomes were sequenced in this study. We identified a novel secreted enterotoxin B homologue, Csep1. The csep1 gene was found in 56% of GS2 C. concisus strains, presented in the plasmid pICON or the chromosome. A six-nucleotide insertion at the position 654–659 bp in csep1 (csep1-6bpi) was found. The presence of csep1-6bpi in oral C. concisus strains isolated from patients with active CD (47%, 7/15) was significantly higher than that in strains from healthy controls (0/29, P = 0.0002), and the prevalence of csep1-6bpi positive C. concisus strains was significantly higher in patients with active CD (67%, 4/6) as compared to healthy controls (0/23, P = 0.0006). Proteomics analysis detected the Csep1 protein. A csep1 gene hot spot in the chromosome of different C. concisus strains was found. The pICON plasmid was only found in GS2 strains isolated from the two relapsed CD patients with small bowel complications. This study reports a C. concisus molecular marker (csep1-6bpi) that is associated with active CD.

Salmonella enterica serovar Typhimurium is the most common Salmonella serovar causing foodborne infections in Australia and many other countries. Twenty-one S . Typhimurium strains from Salmonella reference collection A (SARA) were analyzed using Illumina high-throughput genome sequencing. Single nucleotide polymorphisms (SNPs) in 21 SARA strains ranged from 46 to 11,916 SNPs, with an average of 1,577 SNPs per strain. Together with 47 strains selected from publicly available S . Typhimurium genomes, the S . Typhimurium core genes (STCG) were determined. The STCG consist of 3,846 genes, a set that is much larger than that of the 2,882 Salmonella core genes (SCG) found previously. The STCG together with 1,576 core intergenic regions (IGRs) were defined as the S . Typhimurium core genome. Using 93 S . Typhimurium genomes from 13 epidemiologically confirmed community outbreaks, we demonstrated that typing based on the S . Typhimurium core genome (STCG plus core IGRs) provides superior resolution and higher discriminatory power than that based on SCG for outbreak investigation and molecular epidemiology of S . Typhimurium. STCG and STCG plus core IGR typing achieved 100% separation of all outbreaks compared to that of SCG typing, which failed to separate isolates from two outbreaks from background isolates. Defining the S . Typhimurium core genome allows standardization of genes/regions to be used for high-resolution epidemiological typing and genomic surveillance of S. Typhimurium.

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells. Citrobacter freundii strain CF74 has a complete T6SS genomic island (GI) that contains clpV , hcp-2 , and vgr T6SS genes. We constructed clpV , hcp-2 , vgr , and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased in clpV , hcp-2 , vgr , or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74Δ clpV , and CF74Δ hcp-2 mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role in C. freundii .

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non-ptxP3/non-prn2). Cluster I was mostly responsible for the 2008-2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high-resolution multiple reaction monitoring (MRM-hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (

Purpose. A recently identified colistin resistance gene, mcr-1, has been reported in many countries. In this study, we established a new real-time PCR method to detect it. Methodology. We used a real-time PCR method to detect the mcr-1 gene in a variety of isolates and faecal samples from 20 provinces and municipal cities in China. Results. Of the 2330 isolates (from 10 species) screened, 54 (2.3 %) isolates were positive for mcr-1. All of the mcr-1-positive isolates that were identified belonged to Escherichia coli strains, among which 9, 1, and 44 were identified as enteropathogenic E. coli, enteroadherent E. coli, and non-pathogenic E. coli, respectively. The majority of the mcr-1-positive isolates were obtained from farm animals from eight provinces and municipal cities across China. A total of 337 faecal samples, including 229 human and 108 pet animal faecal samples, were also screened for the mcr-1 gene. Of the 337 samples analyzed, six and eight human and pet animal faecal samples were positive for the mcr-1 gene, respectively. Conclusion. The data demonstrate that the mcr-1 gene is highly prevalent in human and animal populations in China. This occurrence suggests that active surveillance of the mcr-1 gene is imperative in curtailing its spread.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease. C. concisus has two major genomospecies, which appear to have different enteric pathogenic potential. Currently, no studies have compared the genomes of C. concisus strains from different genomospecies. In this study, a comparative genome analysis of 36 C. concisus strains was conducted including 27 C. concisus strains sequenced in this study and nine publically available C. concisus genomes. The C. concisus core-genome was defined and genomospecies-specific genes were identified. The C. concisus core-genome, housekeeping genes and 23S rRNA gene consistently divided the 36 strains into two genomospecies. Two novel genomic islands, CON_PiiA and CON_PiiB, were identified. CON_PiiA and CON_PiiB islands contained proteins homologous to the type IV secretion system, LepB-like and CagA-like effector proteins. CON_PiiA islands were found in 37.5% of enteric C. concisus strains (3/8) isolated from patients with enteric diseases and none of the oral strains (0/27), which was statistically significant. This study reports the findings of C. concisus genomospecies-specific genes, novel genomic islands that contain type IV secretion system and putative effector proteins, and other new genomic features. These data provide novel insights into understanding of the pathogenicity of this emerging opportunistic pathogen.

Characterization of Listeria monocytogenes isolated from human Listeriosis cases in China

Legionella pneumophila is an important human pathogen causing Legionnaires’ disease. In this study, whole genome sequencing (WGS) was used to study the characteristics and population structure of L. pneumophila strains. We sequenced and compared 53 isolates of L. pneumophila covering different serogroups and sequence-based typing (SBT) types (STs). We found that 1,896 single-copy orthologous genes were shared by all isolates and were defined as the minimum core genome (MCG) of L. pneumophila. A total of 323,224 single-nucleotide polymorphisms (SNPs) were identified among the 53 strains. After excluding 314,059 SNPs which were likely to be results of recombination, the remaining 9,165 SNPs were referred to as MCG SNPs. Population Structure analysis based on MCG divided the 53 L. pneumophila into nine MCG groups. The within-group distances were much smaller than the between-group distances, indicating considerable divergence between MCG groups. MCG groups were also supplied by phylogenetic analysis and may be considered as robust taxonomic units within L. pneumophila. Among the nine MCG groups, eight showed high intracellular growth ability while one showed low intracellular growth ability. Furthermore, MCG typing also showed high resolution in subtyping ST1 strains. The results obtained in this study provided significant insights into the evolution, population structure and pathogenicity of L. pneumophila.

We report on the isolation of 5 Shigella flexneri strains displaying a novel serotype, 1d, that shares serologic features from both S. flexneri serotypes 1a and X. The 1d strains contained serotype-converting bacteriophages SfI and SfX in tandem on the chromosome. These strains were likely originated from serotype X strains through SfI infection.

Evolutionary studies using single nucleotide polymorphisms (SNPs) have separated Bordetella pertussis isolates into six major clusters, with recent isolates forming cluster I. The expansion of cluster I isolates was characterised by changes in genes encoding antigenic components in acellular vaccines, including pertactin (Prn). Here, we determined the initial emergence of the pertussis toxin promoter allele, ptxP3, from an evolutionary perspective. This allele was previously shown in a study from the Netherlands to be associated with increased pertussis toxin production as a result of a single base mutation in the ptxP. The ptxP region of 313 worldwide isolates was sequenced, including 208 isolates from Australia collected over a 40 year period. Eight alleles were identified, of which only two predominated: ptxP1 and ptxP3. One novel allele was also found. ptxP3 was only found in SNP cluster I of B. pertussis and its emergence is concurrent with the change to the non-vaccine prn2 allele. Our results suggest that the globally distributed cluster I of B. pertussis has the ability to evade vaccine induced selection pressure.

Despite high vaccine coverage, pertussis incidence has increased substantially in recent years in many countries. A significant factor that may be contributing to this increase is adaptation to the vaccine by Bordetella pertussis, the causative agent of pertussis. In this study, we first assessed the genetic diversity of B. pertussis by microarray-based comparative genome sequencing of 10 isolates representing diverse genotypes and different years of isolation. We discovered 171 single nucleotide polymorphisms (SNPs) in a total of 1.4 Mb genome analyzed. The frequency of base changes was estimated as one per 32 kb per isolate, confirming that B. pertussis is one of the least variable bacterial pathogens. We then analyzed an international collection of 316 B. pertussis isolates using a subset of 65 of the SNPs and identified 42 distinct SNP profiles (SPs). Phylogenetic analysis grouped the SPs into six clusters. The majority of recent isolates belonged to clusters I-IV and were descendants of a single prevaccine lineage. Cluster I appeared to be a major clone with a worldwide distribution. Typing of genes encoding acellular vaccine (ACV) antigens, ptxA, prn, fhaB, fim2, and fim3 revealed the emergence and increasing incidence of non-ACV alleles occurring in clusters I and IV, which may have been driven by ACV immune selection. Our findings suggest that B. pertussis, despite its high population homogeneity, is evolving in response to vaccination pressure with recent expansion of clones carrying variants of genes encoding ACV antigens.

Shigella spp. are the causative agent of shigellosis with S higella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.

Multilocus variable-number tandem-repeat analysis (MLVA) of 316 Bordetella pertussis isolates collected over 40 years from Australia and 3 other continents identified 66 MLVA types (MTs), including 6 predominant MTs. Typing of genes encoding acellular vaccine antigens showed changes that may be vaccine driven in 2 MTs prevalent in Australia.

The genus Salmonella consists of two species S. enterica and S. bongori. S. enterica has a well defined subspecies structure with seven subspecies consistently delineated by sequence variation. Frequency of recombination between subspecies and within a subspecies is markedly different. Subspecies I undergoes frequent recombination as demonstrated recently, demystifying the long-held belief that Salmonella is a highly clonal organism. The majority of disease causing serovars are from subspecies I with the most important serovars in human health being Typhimurium and Typhi. Typhimurium has developed considerable diversity and may be a very old serovar. The majority of the isolates belong to a single clonal complex by multilocus sequence typing. Typhimurium isolates are divided into phage types and some of the phage types do not have a single origin as determined using mutational changes. Phage type DT104 is heterogeneous and represented in multiple sequence types, with its multidrug-resistant variant most successful causing epidemics in many parts of the world. Typhi, a human restricted serovar, is relatively young compared to Typhimurium, and has a low level of sequence variation. Single nucleotide polymorphisms (SNPs) have been shown to be very useful for typing and resolving relationships within Typhi. Genome sequences of 19 isolates revealed more than 1700 SNPs. The fully resolved phylogenetic tree allows one to trace the mutational changes occurred during clonal diversification. Genome wide SNPs have greatly enhanced our understanding of the evolution of Salmonella clones.

The Vibrio pathogenicity island (VPI) encodes the toxin-coregulated pilus and other virulence factors for Vibrio cholerae to colonize the human intestine to cause cholera. We assessed the level of genetic variation of VPI in nine nonpandemic isolates, and compared them with the sixth and seventh pandemic strains by sequencing c. 5 kb each from the start, middle and end regions of the VPI. Variation is similar among the three regions at around 2%, except for the tcpA gene, which has a much higher level of variation (23%). Numerous recombination segments were identified with sizes up to 2177 bp. Nearly all VPI genes sequenced have a ratio of synonymous to nonsynonymous substitutions considerably lower than that for housekeeping genes, suggesting that VPI genes are under positive selection pressure for change. The tagA gene was deleted or damaged in six isolates, which is likely to affect the efficiency of colonization of the human intestine. Two genes, orf2 and acfD, previously found to be translated differently in the sixth and seventh pandemic strains, were determined to be mutant in the seventh and sixth pandemic strains, respectively. These findings enhance our understanding of variation in the VPI, and of the pathogenic potential of VPI-positive environmental isolates.