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

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

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

Comparison of the Whole Cell Proteome and Secretome of Epidemic Bordetella pertussis Strains From the 2008–2012 Australian Epidemic Under Sulfate-Modulating Conditions

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) <0.05]. In the secretome, four proteins from the type III secretion system (T3SS) effectors were downregulated (FC < 0.8, FDR < 0.05) while six proteins, including two adhesins, pertactin (Prn) and tracheal colonization factor A (TcfA), were upregulated which were consistent with our previous proteomic study. The upregulation of Prn and TcfA in SNP cluster I may result in improved adhesion while the downregulation of the T3SS and other immunogenic proteins may reduce immune recognition, which may contribute to the increased fitness of cluster I B. pertussis strains

Distribution of <i>salmonella</i> serovars in humans, foods, farm animals and environment, companion and wildlife animals in Singapore

We analyzed the epidemiological distribution of Salmonella serovars in humans, foods, animals and the environment as a One-Health step towards identifying risk factors for human salmonellosis. Throughout the 2012&ndash;2016 period, Salmonella ser. Enteritidis was consistently the predominating serovar attributing to &gt;20.0% of isolates in humans. Other most common serovars in humans include Salmonella ser. Stanley, Salmonella ser. Weltevreden, Salmonella ser. Typhimurium and Salmonella ser. 4,5,12:b:-(dT+). S. Enteritidis was also the most frequent serovar found among the isolates from chicken/chicken products (28.5%) and eggs/egg products (61.5%) during the same period. In contrast, S. Typhimurium (35.2%) and Salmonella ser. Derby (18.8%) were prevalent in pork/pork products. S. Weltevreden was more frequent in seafood (19.2%) than others (&le;3.0%). Most isolates (&gt;80.0%) from farms, companion and wildlife animals belonged to serovars other than S. Enteritidis or S. Typhimurium. Findings demonstrate the significance of a One-Health investigative approach to understand the epidemiology Salmonella for more effective and integrated surveillance systems

Rapid increase in pertactin-deficient Bordetella pertussis isolates, Australia

Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008–2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (prn). Multiple mechanisms of prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing prn arose independently multiple times since 2008, rather than by expansion of a single prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.Connie Lam, Sophie Octavia, Lawrence Ricafort, Vitali Sintchenko, Gwendolyn L. Gilbert, Nicholas Wood, Peter McIntyre, Helen Marshall, Nicole Guiso, Anthony D. Keil, Andrew Lawrence, Jenny Robson, Geoff Hogg, and Ruiting La

Reducing spread of COVID-19 in closed environments: an outbreak investigation and modelling study in dormitory settings.

Starting with a handful of SARS-CoV-2 infections in dormitory residents in late March 2020, rapid tranmission in their dense living environments ensued and by October 2020, more than 50,000 acute infections were identified across various dormitories. Extensive epidemiological, serological and phylogentic investigations, supported by simulation models, helped to reveal the factors of transmission and impact of control measures in a dormitory. We find that asymptomatic cases and symptomatic cases who did not seek medical attention were major drivers of the outbreak. Furthermore, each resident has about 30 close contacts and each infected resident spread to 4.4 (IQR 3.5–5.3) others at the start of the outbreak. The final attack rate of the current outbreak was 76.2% (IQR 70.6%–98.0%) and could be reduced by further 10% under a modified dormitory housing condition. These findings are important when designing living environments in a post COVID-19 future to reduce disease spread and facilitate rapid implementation of outbreak control measures

Investigation of the Enteric Pathogenic Potential of Oral Campylobacter concisus Strains Isolated from Patients with Inflammatory Bowel Disease

BACKGROUND: Campylobacter concisus, a bacterium colonizing the human oral cavity, has been shown to be associated with inflammatory bowel disease (IBD). This study investigated if patients with IBD are colonized with specific oral C. concisus strains that have potential to cause enteric diseases. METHODOLOGY: Seventy oral and enteric C. concisus isolates obtained from eight patients with IBD and six controls were examined for housekeeping genes by multilocus sequence typing (MLST), Caco2 cell invasion by gentamicin-protection-assay, protein analysis by mass spectrometry and SDS-PAGE, and morphology by scanning electron microscopy. The whole genome sequenced C. concisus strain 13826 which was isolated from an individual with bloody diarrhea was included in MLST analysis. PRINCIPAL FINDINGS: MLST analysis showed that 87.5% of individuals whose C. concisus belonged to Cluster I had inflammatory enteric diseases (six IBD and one with bloody diarrhea), which was significantly higher than that in the remaining individuals (28.6%) (P<0.05). Enteric invasive C. concisus (EICC) oral strain was detected in 50% of patients with IBD and none of the controls. All EICC strains were in Cluster 1. The C. concisus strain colonizing intestinal tissues of patient No. 1 was closely related to the oral C. concisus strain from patient No. 6 and had gene recombination with the patient's own oral C. concisus. The oral and intestinal C. concisus strains of patient No. 3 were the same strain. Some individuals were colonized with multiple oral C. concisus strains that have undergone natural recombination. CONCLUSIONS: This study provides the first evidence that patients with IBD are colonized with specific oral C. concisus strains, with some being EICC strains. C. concisus colonizing intestinal tissues of patients with IBD at least in some instances results from an endogenous colonization of the patient's oral C. concisus and that C. concisus strains undergo natural recombination