Metatranscriptomic Sequencing of Medically Important Mosquitoes Reveals Extensive Diversity of RNA Viruses and Other Microbial Communities in Western Australia.

Mosquitoes harbor a wide diversity of microorganisms, including viruses that are human pathogens, or that are insect specific. We used metatranscriptomics, an unbiased high-throughput molecular approach, to describe the composition of viral and other microbial communities in six medically important mosquito species from across Western Australia: Aedes vigilax, Culex annulirostris, Cx. australicus, Cx. globocoxitus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. We identified 42 viral species, including 13 novel viruses, from 19 families. Culex mosquitoes exhibited a significantly higher diversity of viruses than Aedes mosquitoes, and no virus was shared between the two genera. Comparison of mosquito populations revealed a heterogenous distribution of viruses between geographical regions and between closely related species, suggesting that geography and host species may play a role in shaping virome composition. We also detected bacterial and parasitic microorganisms, among which Wolbachia bacteria were detected in three members of the Cx. pipiens complex, Cx. australicus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. In summary, our unbiased metatranscriptomics approach provides important insights into viral and other microbial diversity in Western Australian mosquitoes that vector medically important viruses.

A single-nucleotide polymorphism in Helicobacter pylori promotes gastric cancer development.

Single-nucleotide polymorphisms (SNPs) in various human genes are key factors in carcinogenesis. However, whether SNPs in bacterial pathogens are similarly crucial in cancer development is unknown. Here, we analyzed 1,043 genomes of the stomach pathogen Helicobacter pylori and pinpointed a SNP in the serine protease HtrA (position serine/leucine 171) that significantly correlates with gastric cancer. Our functional studies reveal that the 171S-to-171L mutation triggers HtrA trimer formation and enhances proteolytic activity and cleavage of epithelial junction proteins occludin and tumor-suppressor E-cadherin. 171L-type HtrA, but not 171S-HtrA-possessing H. pylori, inflicts severe epithelial damage, enhances injection of oncoprotein CagA into epithelial cells, increases NF-κB-mediated inflammation and cell proliferation through nuclear accumulation of ß-catenin, and promotes host DNA double-strand breaks, collectively triggering malignant changes. These findings highlight the 171S/L HtrA mutation as a unique bacterial cancer-associated SNP and as a potential biomarker for risk predictions in H. pylori infections.

Genome Characteristic of Bordetella parapertussis Isolated from Iran.

Pertussis also known as whooping cough is a respiratory infection in humans particularly with severe symptoms in infants and usually caused by Bordetella pertussis. However, Bordetella parapertussis can also cause a similar clinical syndrome. During 2012 to 2015, from nasal swabs sent from different provinces to the pertussis reference laboratory of Pasture Institute of Iran for pertussis confirmation, seven B. parapertussis isolates were identified by bacterial culture, biochemical tests, and the presence of IS1001 insertion in the genome. The expression of pertactin (Prn) as one the major virulence factor for bacterial adhesion was investigated using western blot. Moreover, the genomic characteristic of one recently collected isolate, IRBP134, from a seven-month infant was investigated using Illumina NextSeq sequencing protocol. The results revealed the genome with G+C content 65% and genome size 4.7 Mbp. A total of 81 single nucleotide polymorphisms and 13 short insertions and deletions were found in the genome compared to the B. parapertussis 12822 as a reference genome showing ongoing evolutionary changes. A phylogeny relationship of IRBP134 was also investigated using global B. parapertussis available genomes.

Bacterial communities in the gastrointestinal tract segments of helminth-resistant and helminth-susceptible sheep.

BACKGROUND: Helminth parasitism is a world-wide problem in livestock industries, with major impacts on health, welfare and productivity. The role of the gut microbiota in host-helminth interactions in ruminants has been extensively examined and the present study added to this body of knowledge by assessing the effects of resistance and susceptibility to helminth infection in the gastro-intestinal tract (GIT). Australian Sheep Breeding Values (ASBVs) for faecal egg count (FEC) were used to select the 10 highly helminth-susceptible (High-FEC) and 10 highly helminth-resistant (Low-FEC) sheep. FEC status was confirmed during the experiment. Using samples from the faeces and the lumen of the rumen, abomasum, duodenum, jejunum, ileum, caecum, and colon, DNA was extracted and used for 16 rRNA gene amplicon sequencing. RESULTS: The most frequent genera identified along the GIT were Eubacterium, Oscillibacter, and Ruminococcus. Intersectoral-specialization zones were identified along the GIT, with the duodenum displaying major differences between the High-FEC and Low-FEC animals in values for alpha and beta diversity. After taking all samples into account and adjusting for GIT segment, the High-FEC and Low-FEC sheep differed significantly for four genera Butyrivibrio, Mycoplasma, Lachnoclostridium and Succiniclasticum. In the duodenum, the abundances of Aminipila, Lachnoclostridium and Mogibacterium differed significantly between the High-FEC and Low-FEC sheep. In the ileum, on the other hand, the genus Mycoplasma was significantly depleted in the Low-FEC group. CONCLUSIONS: The gastro-intestinal microbial profile varies widely between helminth-resistant and helminth-susceptible sheep. Each GIT section appears to support a particular bacterial composition leading to inter-sectoral differences among the various microbial communities. The microbial populations were most rich and diverse in the duodenum of helminth-resistant sheep, comprising bacterial genera that generally ferment carbohydrates. This observation suggests that helminth-resistant sheep can reorganize the duodenal microbiome taxa which may restrict the development of parasites.

Evolutionary genomics of recent clinical Bordetella pertussis isolates from Iran: wide circulation of multiple ptxP3 lineages and report of the first ptxP3 filamentous hemagglutinin-negative B. pertussis.

Here we investigated nationwide clinical Bordetella pertussis isolated during 2005-2017 from different provinces of Iran, a country with more than 50 years whole cell vaccine immunisation history. Our results revealed the ongoing increase in the population of ptxP3/fim3-2 B. pertussis isolates in different provinces which were differentiated into nine clades. The largest clade (clade 8) which was previously found to be prevalent in Tehran was also prevalent across the country and clade 5 with ptxP3/prn9 genotype has also increased in frequency (14% of all ptxP3 isolates) in recent years. Furthermore, we detected the first ptxP3 B. pertussis isolates that does not express filamentous hemagglutinin (FhaB) as one of the major antigens of the pathogen and a key component of the acellular pertussis vaccine.

Genome diversity and evolutionary characteristics of clinical isolates of Bordetella pertussis circulating in Iran.

BACKGROUND AND OBJECTIVES: The re-emergence of pertussis still is being reported all over the world. Pathogen adaptation and antigenic divergence of circulating isolates from vaccine strains are the main reasons of infection resurgence. Waning immunity is also an important factor contributing to resurgence of pertussis. MATERIALS AND METHODS: The genetic diversity and evolutionary characteristics of circulating Iranian isolates of Bordetella pertussis during February 2015 to October 2018 was investigated by pulsed-field gel electrophoresis (PFGE) and subsequently ptxA, ptxP and fim3 alleles were characterized. The next generation genome sequencing was then used to compare the genomics of ptxP1 and ptxP3 of selected isolates from PFGE dendrogram. RESULTS: PFGE differentiated 62 clinical isolates and vaccine and reference strains into 19 PFGE profiles, indicating the higher level of heterogeneity in the population during 2015-2018. The predominant B. pertussis genotype harbored pertussis toxin promoter allele, ptxP3 and the expansion of ptxA1 isolates, were also observed in our population. CONCLUSION: No changes in allelic profile of predominant clone in recent years was observed but antigenic divergence between recently circulating isolates and the vaccine strain has been progressed and significantly was higher than previous studies. The comparative genomic analysis of the ptxP3 and ptxP1 isolates indicate that changes in ptxP3 genome structure including 32 unique SNPs and three unique indels may have contributed to the expansion of the ptxP3 clone. We compared ptxP3 and ptxP1 isolates in pathogenicity-associated genes and found five of them were specific for the ptxP3 isolates. The polymorphisms in pathogenicity-associated genes suggest structural adaptations for these virulence factors.

A novel taxon selection method, aimed at minimizing recombination, clarifies the discovery of a new sub-population of Helicobacter pylori from Australia.

We present a novel method for taxon selection, the aim being to minimize problems arising from highly recombinant species such as Helicobacter pylori. Helicobacter pylori has accompanied modern-human migration out of Africa and is marked by a phylogeographic strain distribution, which has been exploited to add an extra layer of information about human migrations to that obtained from human sources. However, H. pylori's genome has high sequence heterogeneity combined with a very high rate of recombination, causing major allelic diversification across strains. On the other hand, recombination events that have become preserved in sub-populations are a useful source of phylogenetic information. This creates a potential problem in selecting representative strains for particular genetic or phylogeographic clusters and generally ameliorating the impact on analyses of extensive low-level recombination. To address this issue, we perform multiple population structure-based analyses on core genomes to select exemplar strains, called 'quintessents', which exhibit limited recombination. In essence, quintessent strains are representative of their specific phylogenetic clades and can be used to refine the current MLST concatenation-based population structure classification system. The use of quintessents reduces the noise due to local recombination events, while preserving recombination events that have become fixed in sub-populations. We illustrate the method with an analysis of core genome concatenations from 185 H. pylori strains, which reveals a recent speciation event resulting from the recombination of strains from phylogeographic clade hpSahul, carried by Aboriginal Australians, and hpEurope, carried by some of the people who arrived in Australia over the past 200 years. The signal is much clearer when based on quintessent strains, but absent from the analysis based on MLST concatenations.

A Longitudinal, Population-Level, Big-Data Study of Helicobacter pylori-Related Disease across Western Australia.

Helicobacter pylori, responsible for chronic ulcers and most stomach cancers, infects half of the world's population. The Urea Breath Test (UBT) is one of the most accurate and reliable non-invasive methods for diagnosing active H. pylori infection. The objective was to use longitudinal, population-wide UBT data for Western Australia to look for H. pylori-related disease patterns. We collected 95,713 UBT results from 77,552 individuals for the years 2010-2015, likely representing all of the UBT samples analysed in Western Australia. Data collected also included sex, age and residential postcode. Other data reported here were inferred via a comparison with the 2011 Australian Census using a specially written Python program. While women appear to have more H. pylori-related disease than men, there is no difference in the disease rates once women's higher rates of presentation for testing are taken into account. On the other hand, while the treatment strategy for H. pylori infection is generally very effective in Western Australia, failure of the first-line treatment is significantly more common in women than men. Migrants and Aboriginal Australians have elevated rates of H. pylori-related disease, while the rate for non-Aboriginal Australian-born West Australians is very low. However, no significant associations were found with other socio-economic indicators. We conclude that, for some people, H. pylori-related disease is not a solved problem.

East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide.

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.

Genomic epidemiology of Iranian Bordetella pertussis: 50 years after the implementation of whole cell vaccine.

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.

High primary resistance to metronidazole and levofloxacin, and a moderate resistance to clarithromycin in Helicobacter pylori isolated from Karnataka patients.

BACKGROUND: Due to increased prevalence of H. pylori antimicrobial resistance worldwide and more importantly the resistance patterns vary between different geographical regions, it is important to survey local H. pylori antibiotic resistance profile to provide physicians with more informed drug choices to better treat H. pylori infection. To our knowledge, this is the first study to examine the prevalence of antimicrobial resistance of H. pylori in Karnataka state of South India. RESULTS: A total of 113 H. pylori strains were isolated from gastric biopsies and tested: 81.4% were resistant to metronidazole, 54.9% were resistant to levofloxacin, 20.4% were resistant to clarithromycin, 5.3% were resistant to tetracycline and 7.1% were resistant to amoxicillin. Multidrug resistance was detected in 59.3% of total isolated strains, among which 86.6% were resistant to at least both metronidazole and levofloxacin. In this study, 38 out of 113 H. pylori strains had been whole-genome sequenced. Based on the draft genomes, RdxA and/or FrxA inactivation mutations were found to present in 75% of metronidazole-resistant strains. Clarithromycin-resistant strains had mainly A2143G and G2224A mutations in the 23 rRNA gene. While 87.1% levofloxacin-resistant strains had amino acid substitution mutations occurring predominantly at N87 and D91 in GyrA, novel mutations in the same protein including an insertion of five amino acid residues (QDNSV), immediately after the start codon, and a substitution mutation at R295 were identified. CONCLUSION: High primary resistance to metronidazole and levofloxacin, and a modest occurrence of clarithromycin resistance were revealed in H. pylori strains isolated from Karnataka patients. Therefore metronidazole-, levofloxacin- and clarithromycin-based triple therapies are not suitable as first-line treatment in Karnataka. Both amoxicillin and tetracycline can still be used to eradicate H. pylori infection in this region. We also revealed novel mutations in GyrA protein that possibly contribute to H. pylori resistance in levofloxacin, which merit further investigations.

The complete genome and methylome of Helicobacter pylori hpNEAfrica strain HP14039.

BACKGROUND: Helicobacter pylori is a Gram-negative bacterium which mainly causes peptic ulcer disease in human, but is also the predominant cause of stomach cancer. It has been coevolving with human since 120,000 years and, according to Multi-locus sequence typing (MLST), H. pylori can be classified into seven major population types, namely, hpAfrica1, hpAfrica2, hpNEAfrica, hpEastAsia, hpAsia2, hpEurope and hpSahul. Helicobacter pylori harbours a large number of restriction-modification (R-M) systems. The methyltransferase (MTase) unit plays a significant role in gene regulation and also possibly modulates pathogenicity. The diversity in MTase can act as geomarkers to correlate strains with the phylogeographic origins. This paper describes the complete genome sequence and methylome of gastric pathogen H. pylori belonging to the population hpNEAfrica. RESULTS: In this paper, we present the complete genome sequence and the methylome profile of H. pylori hpNEAfrica strain HP14039, isolated from a patient who was born in Somalia and likely to be infected locally during early childhood prior to migration. The genome of HP14039 consists of 1,678,260 bp with 1574 coding genes and 38.7% GC content. The sequence analysis showed that this strain lacks the cag pathogenicity island. The vacA gene is of S2M2 type. We have also identified 15 methylation motifs, including WCANHNNNNTG and CTANNNNNNNTAYG that were not previously described. CONCLUSIONS: We have described the complete genome of H. pylori strain HP14039. The information regarding phylo-geography, methylome and associated metadata would help scientific community to study more about hpNEAfrica population type.

Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in Helicobacter pylori.

BACKGROUND: Metronidazole is one of the first-line drugs of choice in the standard triple therapy used to eradicate Helicobacter pylori infection. Hence, the global emergence of metronidazole resistance in Hp poses a major challenge to health professionals. Inactivation of RdxA is known to be a major mechanism of conferring metronidazole resistance in H. pylori. However, metronidazole resistance can also arise in H. pylori strains expressing functional RdxA protein, suggesting that there are other mechanisms that may confer resistance to this drug. METHODS: We performed whole-genome sequencing on 121 H. pylori clinical strains, among which 73 were metronidazole-resistant. Sequence-alignment analysis of core protein clusters derived from clinical strains containing full-length RdxA was performed. Variable sites in each alignment were statistically compared between the resistant and susceptible groups to determine candidate genes along with their respective amino-acid changes that may account for the development of metronidazole resistance in H. pylori. RESULTS: Resistance due to RdxA truncation was identified in 34% of metronidazole-resistant strains. Analysis of core protein clusters derived from the remaining 48 metronidazole-resistant strains and 48 metronidazole-susceptible identified four variable sites significantly associated with metronidazole resistance. These sites included R16H/C in RdxA, D85N in the inner-membrane protein RclC (HP0565), V265I in a biotin carboxylase protein (HP0370) and A51V/T in a putative threonylcarbamoyl-AMP synthase (HP0918). CONCLUSIONS: Our approach identified new potential mechanisms for metronidazole resistance in H. pylori that merit further investigation.

Gastric Helicobacter pylori infection perturbs human oral microbiota.

BACKGROUND: We investigated the effects of gastric Helicobacter pylori infection on the daytime and overnight human oral microbiota. METHODS: Twenty four volunteers were recruited. Ten tested positive for H. pylori infection by the Carbon-14 Urea Breath Test, and the rest were negative. Two oral swabs were collected: one immediately after waking up in the morning and before brushing teeth, and another in the evening before teeth-brushing. DNA extract acquired from each swab was subjected to Illumina sequencing of 16S rRNA gene amplicons. The microbial abundance and composition were analysed in relation to H. pylori infection status. RESULTS: Helicobacter pylori-positive individuals had significant changes in the alpha and beta diversities in the daytime samples in comparison to those who were H. pylori negative. To identify which taxa could be significantly affected within the cohorts in the daytime, we employed the LEfSe method. When compared against UBT-negative samples, significantly higher abundances were detected in both Pseudomonas and Roseomonas, while Fusobacterium, Solobacterium, Haemophilus and Streptococcus were significantly decreased in the UBT-positive samples. DISCUSSION: Our data demonstrated that H. pylori infection affects the human daytime oral microbiota. The hitherto undocumented changes of several bacterial genera due to H. pylori infection require more studies to examine their potential health effects on affected individuals.

Draft Genome Sequences of 42 Helicobacter pylori Isolates from Rural Regions of South India.

Helicobacter pylori is a successful human gastric pathogen that is associated with the development of gastric cancer. The draft genome sequences of 42 H. pylori clinical strains isolated from South Indian rural populations will provide further insights into the evolution and genetic makeup of Indian H. pylori strains.

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.