Investigating genome reduction of Bordetella pertussis using a multiplex PCR-based reverse line blot assay (mPCR/RLB)

BACKGROUND: The genetic composition of the bacterium causing whooping cough, Bordetella pertussis, has been investigated using microarray studies in order to examine potential genetic contributors to the disease re-emergence in the past decade. Regions of difference (RDs) have been previously identified as clusters of genes flanked by insertion sequences which are variably present in different sets of isolates, and have also been shown to be potential markers of B. pertussis evolution. This study used microarray data to identify and select a panel of RDs; primers and probes for these RDs were then designed to test for the presence or absence of these regions in a novel and less expensive multiplex PCR-based reverse line blot (mPCR/RLB) assay. By comparing the presence or absence of RDs, we aimed to determine the genomic variability of a diverse collection of B. pertussis strains and how they have changed over time. RESULTS: A B. pertussis specific mPCR/RLB using 43 genes representing 30 RDs, was developed and used to characterise a set of 42 B. pertussis isolates. When mapped against the previously identified evolutionary relationships of the strains, the losses of two RDs - BP0910A - BP00930 and BP1948-BP1962 - were found to be associated with significant events in B. pertussis history: the loss of BP0910A - BP00930 coincided with introduction of whole cell vaccines in the 1950s while that of BP1948-BP1962 occurred after the introduction of acellular vaccines. The loss of BP1948-BP1962 also coincided with expansion of the most recent B. pertussis strains. CONCLUSIONS: The mPCR/RLB assay offers an inexpensive and fast method of determining the gene content of B. pertussis strains and also confirms that gene losses are an ongoing feature of B. pertussis evolution. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1756-0500-7-727) contains supplementary material, which is available to authorized users

In silico Identification of Serovar-Specific Genes for Salmonella Serotyping

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

Population structure of Helicobacter pylori among ethnic groups in Malaysia: recent acquisition of the bacterium by the Malay population

<p>Abstract</p> <p>Background</p> <p><it>Helicobacter pylori </it>is a major gastric bacterial pathogen. This pathogen has been shown to follow the routes of human migration by their geographical origin and currently the global <it>H. pylori </it>population has been divided into six ancestral populations, three from Africa, two from Asia and one from Europe. Malaysia is made up of three major ethnic populations, Malay, Chinese and Indian, providing a good population for studying recent <it>H. pylori </it>migration and admixture.</p> <p>Results</p> <p>Seventy eight <it>H. pylori </it>isolates, including 27 Chinese, 35 Indian and 16 Malay isolates from Malaysia were analysed by multilocus sequence typing (MLST) of seven housekeeping genes and compared with the global MLST data. STRUCTURE analysis assigned the isolates to previously identified <it>H. pylori </it>ancestral populations, hpEastAsia, hpAsia2 and hpEurope, and revealed a new subpopulation, hspIndia, within hpAsia2. Statistical analysis allowed us to identify population segregation sites that divide the <it>H. pylori </it>populations and the subpopulations. The majority of Malay isolates were found to be grouped together with Indian isolates.</p> <p>Conclusion</p> <p>The majority of the Malay and Indian <it>H. pylori </it>isolates share the same origin while the Malaysian Chinese <it>H. pylori </it>is distinctive. The Malay population, known to have a low infection rate of <it>H. pylori</it>, was likely to be initially <it>H. pylori </it>free and gained the pathogen only recently from cross infection from other populations.</p

Continuous genomic surveillance monitored the in vivo evolutionary trajectories of Vibrio parahaemolyticus and identified a new virulent genotype

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-nikure 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

Evolution of Seventh Cholera Pandemic and Origin of 1991 Epidemic, Latin America

Thirty single-nucleotide polymorphisms were used to track the spread of the seventh pandemic caused by Vibrio cholerae. Isolates from the 1991 epidemic in Latin America shared a profile with 1970s isolates from Africa, suggesting a possible origin in Africa. Data also showed that the observed genotypes spread easily and widely

Examination of the Anaerobic Growth of Campylobacter concisus

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

Whole genome sequencing of Salmonella Typhimurium illuminates distinct outbreaks caused by an endemic multi-locus variable number tandem repeat analysis type in Australia, 2014

Phylogeny of the outbreak A and M strains in the context of national and international STM isolates. Genome data analysed in Octavia et al. representing five STM outbreaks in Australia [25]; Kingsley et al. representing ST313 outbreak in Malawi [30]; Leekitcharoenphon et al. representing six STM outbreaks in Denmark [15] and Hawkey et al. representing STM DT135a outbreak in Australia [21] were also included as comparisons and marked as the corresponding study/outbreak. Other branches that are not labelled are background isolates from the above studies; draft genomes from Pang et al. [29] which include five diverse Australian STM isolates; Fu et al. representing Salmonella reference collection A; [28] and other fully sequenced STM genomes available from GenBank including LT2 (Accession No. NC003197), 798 (Accession No. CP003386), DT2 (Accession No. HG326213), DT104 (Accession No. HF937208), 14028S (Accession No. CP001363), SL1344 (Accession No. FQ312003), UK-1 (Accession No. CP002614), T000240 (Accession No. AP011957), U288 (Accession No. CP003836) and ST4/74 (Accession No. CP002487). Bootstrap values if greater than 50 %, are presented on the internal branches. (PPTX 74 kb

Vibrio cholerae Pathogenic Clones

We resolved the relationships between 2 pandemic clones of Vibrio cholerae. Using 26 housekeeping genes, we showed that the US Gulf clone, the Australian clone, and 3 El Tor strains isolated before the seventh pandemic were related to the seventh pandemic clone. The sixth pandemic clone was well separated from them

Integrating proteomic data with metabolic modeling provides insight into key pathways of Bordetella pertussis biofilms

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

Genesis of a novel Shigella flexneri serotype by sequential infection of serotype-converting bacteriophages SfX and SfI

<p>Abstract</p> <p>Background</p> <p><it>Shigella flexneri </it>is the major pathogen causing bacillary dysentery. Fifteen serotypes have been recognized up to now. The genesis of new <it>S. flexneri </it>serotypes is commonly mediated by serotype-converting bacteriophages. Untypeable or novel serotypes from natural infections had been reported worldwide but have not been generated in laboratory.</p> <p>Results</p> <p>A new <it>S. flexneri </it>serotype-serotype 1 d was generated when a <it>S. flexneri </it>serotype Y strain (native LPS) was sequentially infected with 2 serotype-converting bacteriophages, SfX first and then SfI. The new serotype 1 d strain agglutinated with both serotype X-specific anti-7;8 grouping serum and serotype 1a-specific anti- I typing serum, and differed from subserotypes 1a, 1b and 1c. Twenty four <it>S. flexneri </it>clinical isolates of serotype X were all converted to serotype 1 d by infection with phage SfI. PCR and sequencing revealed that SfI and SfX were integrated in tandem into the <it>proA-yaiC </it>region of the host chromosome.</p> <p>Conclusions</p> <p>These findings suggest a new <it>S. flexneri </it>serotype could be created in nature. Such a conversion may be constrained by susceptibility of a strain to infection by a given serotype-converting bacteriophage. This finding has significant implications in the emergence of new <it>S. flexneri </it>serotypes in nature.</p