Genomic characterization of respiratory syncytial virus genotypes circulating in the paediatric population of Sydney, NSW, Australia.

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a major cause of acute lower respiratory infection (ALRI), particularly in young children, causing significant morbidity and mortality. We used pathogen genomics to characterize the population structure and genetic signatures of RSV isolates circulating in children in New South Wales between 2016 and 2018 and to understand the evolutionary dynamics of these strains in the context of publicly available RSV genomes from the region and globally. Whole-genome phylogenetic analysis demonstrated the co-circulation of a few major RSV clades in the paediatric population from Sydney. The whole-genome-based genotypes A23 (RSV-A ON1-like genotype) and B6 (RSV-B BA9-like genotype) were the predominant RSV-A and RSV-B genotypes circulating during the study period, respectively. These genotypes were characterized with high levels of diversity of predicted N- and O-linked glycosylation patterns in both the G and F glycoproteins. Interestingly, a novel 72-nucleotide triplication in the sequence that corresponds to the C-terminal region of the G gene was identified in four of the A23 genotype sequenced in this study. Consistently, the population dynamics analysis demonstrated a continuous increase in the effective population size of A23 and B6 genotypes globally. Further investigations including functional mapping of mutations and identifying the impact of sequence changes on virus fitness are highly required. This study highlights the potential impact of an integrated approach that uses WG-based phylogeny and studying selective pressure events in understanding the emergence and dissemination of RSV genotypes.

Evaluation of the rate, pattern and appropriateness of antibiotic prescription in a cohort of pilgrims suffering from upper respiratory tract infection during the 2018 Hajj season.

Hajj is associated with an increased risk of the transmission of infectious diseases including upper respiratory tract infections (URTIs). It can be a focal point for the emergence, persistence and dissemination of antimicrobial-resistant (AMR) bacteria. The overuse of antibiotics during Hajj can promote the development of antimicrobial resistance. Little information is known regarding the true appropriateness of prescribing antibiotics for treating URTIs during Hajj. Here we studied the rate, patterns and appropriateness of antibiotic prescription among a cohort of pilgrims who were treated for URTIs during the 2018 Hajj season. Adult pilgrims who sought medical services for URTIs [presenting with coryza, runny nose, nasal irritation, nasal congestion, cough, sore throat, headache or fever (even if subjective)] within the Holy sites were enrolled in this study and consented to provide swabs and medical information. A total of 121 pilgrims were enrolled, with the majority (60.3 %) originating from North African Arab countries. Most were male (89.3 %) with a median age of 45 years. Bacterial infections were detected in 7.3 % (n=9) of the URTI cases. The identified bacteria included Haemophilus influenzae (n=6, all resistant to ampicillin), Streptococcus pneumoniae (n=2), Staphylococcus aureus (n=1, resistant to oxacillin) and Moraxella catarrhalis (n=1, resistant to ampicillin and trimethoprim/sulfamethoxazole). The antibiotic prescription rate was 52.1%, most of which was amoxicillin (81 %). The data demonstrated that the proportion of appropriate practices in treating bacterial URTIs in this cohort was 45.5 %. This study highlights the need for implementing laboratory identification of the aetiological agents and related AMR profiles when treating URTIs in Hajj, rather than relying on clinical assessment alone.

New insights of bacterial communities in fermented vegetables from shotgun metagenomics and identification of antibiotic resistance genes and probiotic bacteria.

Consumption of fermented foods has grown worldwide due to the purported health benefits. It is thus critical to understand fermented foods microbiome that mainly influences the quality and safety of these foods. This study identified bacterial communities, including functional profiles of probiotics and antimicrobial resistance genes (ARGs), in pickled vegetables commonly consumed in the Middle Eastern, African, and Asian sub-continent regions. Eighteen samples from six pickled vegetables were collected from local markets in Saudi Arabia and analyzed using shotgun metagenomic sequencing. Statistical analyses revealed significant distance and separate clustering of bacterial communities among the different pickle types. Species of Levilactobacillus namurensis, Lentilactobacillus buchneri, Lentilactobacillus parafarraginis, Lactiplantibacillus pentosus, Pectobacterium carotovorum, Leuconostoc carnosum, Weissella confuse were found in a range of dominance in most of the samples. Binning revealed 33 high-quality, metagenome-assembled genomes (MAGs), including 4 MAGs representing putatively novel species of Lactobacillus, Alcanivorax, and Dichelobacter. Moreover, 285 ARGs and variants produce resistance against 20 classes of antibiotics were retrieved, mostly from Enterobacteriaceae contigs. The metagenomes harbored relatively high abundances of carbohydrate fermentation enzymes, as well as metabolic pathways for amino acid metabolism, cofactors and vitamins biosynthesis. Overall, by providing a comprehensive overview of bacterial communities and probiotic bacteria in pickled vegetables, the results suggest the need for more hygienic processing to avoid Enterobacteriaceae contamination and ARG spread.

Genome-wide transposon mutagenesis analysis of Burkholderia pseudomallei reveals essential genes for in vitro and in vivo survival.

Introduction: Burkholderia pseudomallei, a soil-dwelling microbe that infects humans and animals is the cause of the fatal disease melioidosis. The molecular mechanisms that underlie B. pseudomallei's versatility to survive within a broad range of environments are still not well defined. Methods: We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify B. pseudomallei essential genes. Transposon-flanking regions were sequenced and gene essentiality was assessed based on the frequency of transposon insertions within each gene. Transposon mutants were grown in LB and M9 minimal medium to determine conditionally essential genes required for growth under laboratory conditions. The Caenorhabditis elegans infection model was used to assess genes associated with in vivo B. pseudomallei survival. Transposon mutants were fed to the worms, recovered from worm intestines, and sequenced. Two selected mutants were constructed and evaluated for the bacteria's ability to survive and proliferate in the nematode intestinal lumen. Results: Approximately 500,000 transposon-insertion mutants of B. pseudomallei strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the B. pseudomallei R15 genome and 492 genes carrying low insertion frequencies were predicted to be essential. A total of 96 genes specifically required to support growth under nutrient-depleted conditions were identified. Genes most likely to be involved in B. pseudomallei survival and adaptation in the C. elegans intestinal lumen, were identified. When compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in C. elegans killing and showed decreased colonization in the organs of infected mice. Discussion: The B. pseudomallei conditional essential proteins should provide further insights into the bacteria's niche adaptation, pathogenesis, and virulence.

Draft genome sequence of colistin-resistant and extended-spectrum ß-lactamase (ESBL)-producing multidrug-resistant Escherichia coli isolated from poultry meat.

OBJECTIVES: The presence of multidrug-resistant bacteria in the food chain represents a major public-health concern and has significant economic repercussions. Here we describe the draft genome sequence of serotype O21:H16, multilocus sequence typing (MLST) ST3270 and ribosomal MLST rST120701, multidrug-resistant Escherichia coli Ch7_4SIAU, which was isolated from poultry meat collected from a local market in Jeddah, Saudi Arabia. METHODS: Ch7_4SIAU was grown on MacConkey agar and was identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). An Illumina MiSeq platform was used to generate 2.4 million paired-end reads of 150 bp. RESULTS: The draft genome of Ch7_4SIAU is ∼5.1 Mb with a GC content of ∼50% and contains five plasmid replicons [ColpVC, IncFII, IncHI2, IncI(Gamma) and IncX1]. The isolate is resistant to third- and fourth-generation cephalosporins. It carries the mobile colistin resistance gene mcr-1.1 and the extended-spectrum ß-lactamase (ESBL) gene blaCTXM-15 as well as the astA gene encoding enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1). CONCLUSION: This report presents the first draft genome of ESBL-positive E. coli isolated from poultry meat in Saudi Arabia. The findings can be used as a reference for genomic epidemiological studies of antimicrobial resistance in Enterobacteriaceae in the local food chain.

Functional analysis of colonization factor antigen I positive enterotoxigenic Escherichia coli identifies genes implicated in survival in water and host colonization.

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization pili CFA/I are common causes of diarrhoeal infections in humans. Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian host. ETEC persisting in water exhibit a distinct RNA expression profile from those growing in richer media. Multiple pathways were identified that contribute to water survival, including lipopolysaccharide biosynthesis and stress response regulons. The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.

Travel-Related Antimicrobial Resistance: A Systematic Review.

There is increasing evidence that human movement facilitates the global spread of resistant bacteria and antimicrobial resistance (AMR) genes. We systematically reviewed the literature on the impact of travel on the dissemination of AMR. We searched the databases Medline, EMBASE and SCOPUS from database inception until the end of June 2019. Of the 3052 titles identified, 2253 articles passed the initial screening, of which 238 met the inclusion criteria. The studies covered 30,060 drug-resistant isolates from 26 identified bacterial species. Most were enteric, accounting for 65% of the identified species and 92% of all documented isolates. High-income countries were more likely to be recipient nations for AMR originating from middle- and low-income countries. The most common origin of travellers with resistant bacteria was Asia, covering 36% of the total isolates. Beta-lactams and quinolones were the most documented drug-resistant organisms, accounting for 35% and 31% of the overall drug resistance, respectively. Medical tourism was twice as likely to be associated with multidrug-resistant organisms than general travel. International travel is a vehicle for the transmission of antimicrobial resistance globally. Health systems should identify recent travellers to ensure that adequate precautions are taken.

Genomic characterisation of Salmonella enterica serovar Wangata isolates obtained from different sources reveals low genomic diversity.

Salmonella enterica serovar Wangata is an important pathogen in New South Wales (NSW), Australia. The incidence of S. Wangata is increasing and transmission is suspected to be via a non-food source. A recent outbreak investigation of sources of S. Wangata recovered isolates from humans, domestic animals, wildlife and the environment. Here, we extend that investigation by characterising and describing the genomic determinates of these isolates. We found that Australian S. Wangata isolates from different sources exhibited similar virulence and antimicrobial resistance gene profiles. There were no major genomic differences between isolates obtained from different geographical regions within Australia or from different host species. In addition, we found evidence (low number of SNPs and identical virulence gene profiles) suggestive of an international transmission event between Australia and the United Kingdom. This study supports the hypothesis that S. Wangata is shared between different hosts in NSW, Australia and provides strong justification for the continued use of genomic surveillance of Salmonella.

The Contribution of Wastewater to the Transmission of Antimicrobial Resistance in the Environment: Implications of Mass Gathering Settings.

Antimicrobial resistance (AMR) is the major issue posing a serious global health threat. Low- and middle-income countries are likely to be the most affected, both in terms of impact on public health and economic burden. Recent studies highlighted the role of resistance networks on the transmission of AMR organisms, with this network being driven by complex interactions between clinical (e.g., human health, animal husbandry and veterinary medicine) and other components, including environmental factors (e.g., persistence of AMR in wastewater). Many studies have highlighted the role of wastewater as a significant environmental reservoir of AMR as it represents an ideal environment for AMR bacteria (ARB) and antimicrobial resistant genes (ARGs) to persist. Although the treatment process can help in removing or reducing the ARB load, it has limited impact on ARGs. ARGs are not degradable; therefore, they can be spread among microbial communities in the environment through horizontal gene transfer, which is the main resistance mechanism in most Gram-negative bacteria. Here we analysed the recent literature to highlight the contribution of wastewater to the emergence, persistence and transmission of AMR under different settings, particularly those associated with mass gathering events (e.g., Hajj and Kumbh Mela).

Pilot Survey of Knowledge, Attitudes and Perceptions of Hajj Deployed Health Care Workers on Antibiotics and Antibiotic Prescriptions for Upper Respiratory Tract Infections: Results from Two Hajj Seasons.

Antimicrobial resistance (AMR) is a global public health issue. Upper respiratory tract infections (URTIs) are common illnesses during Hajj, for which antibiotics are often inappropriately prescribed. Hajj healthcare workers' (HCW) knowledge, attitudes and perceptions (KAP) about AMR and antibiotic use for URTIs are not known. We conducted a survey among HCWs during Hajj to explore their KAP regarding antibiotic use for URTIs in pilgrims. Electronic or paper-based surveys were distributed to HCWs during the Hajj in 2016 and 2017. A total of 85 respondents aged 25 to 63 (median 40) years completed the surveys. Most participants were male (78.8%) and were physicians by profession (95.3%). Around 85% and 19% of respondents claimed to have heard about AMR and antimicrobial stewardship programs, respectively, among whom most had obtained their knowledge during their qualification. Implementation of URTI treatment guidelines was very low. In conclusion, HCWs at Hajj have significant knowledge gaps regarding AMR, often do not use standard clinical criteria to diagnose URTIs and display a tendency to prescribe antibiotics for URTIs.

Genomic characterization of NDM-1 and 5, and OXA-181 carbapenemases in uropathogenic Escherichia coli isolates from Riyadh, Saudi Arabia.

Urinary tract infections (UTIs) associated with Escherichia coli are a growing threat with an increase in the prevalence of multidrug resistant (MDR) strains, particularly ß-lactamase producers, occurring globally. We investigated the presence of carbapenem-resistant uropathogenic E. coli clones in community-acquired UTIs in Riyadh, Kingdom of Saudi Arabia (KSA) to identify the virulence and resistance structures of the resistant clones and relate the isolates to those circulating globally. A combination of comparative genomics and phenotypic approaches were used to characterize ten MDR-uropathogenic Escherichia coli isolates recovered from UTI patients in Riyadh between November 2014 and January 2015. We report the presence of NDM-1 and 5, and OXA-181 in carbapenem-resistant UPEC strains from Riyadh, KSA. Single nucleotide polymorphism analyses demonstrated that these ten isolates fell into four phylogenetically distinct clades within the UPEC phylogeny. Comparative genomic analyses indicate that these diverse clones could be distinguished according to their multilocus sequencing type (MLST), serology, and virulence and antimicrobial gene architectures. These clones include the blaNDM-1 carrying isolates of the globally predominant MDR ST131 and ST69 types, previously identified as one of the most common UPEC strains in KSA. This is in addition to clones of ST23Cplx (ST410) and ST448Cplx (ST448) that have likely evolved from common intestinal strains, carrying copies of ß-lactamase genes including blaNDM-5, blaCTX-M-15, blaTEM-1, blaCMY-42, blaOXA-1 and blaOXA-181. These data have identified an emerging public health concern and highlight the need to use comprehensive approaches to detect the structure of MDR E. coli populations associated with community-acquired UTIs in KSA.

Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model.

A Burkholderia cenocepacia infection usually leads to reduced survival and fatal cepacia syndrome in cystic fibrosis patients. The identification of B. cenocepacia essential genes for in vivo survival is key to designing new anti-infectives therapies. We used the Transposon-Directed Insertion Sequencing (TraDIS) approach to identify genes required for B. cenocepacia survival in the model infection host, Caenorhabditis elegans. A B. cenocepacia J2315 transposon pool of ∼500,000 mutants was used to infect C. elegans. We identified 178 genes as crucial for B. cenocepacia survival in the infected nematode. The majority of these genes code for proteins of unknown function, many of which are encoded by the genomic island BcenGI13, while other gene products are involved in nutrient acquisition, general stress responses and LPS O-antigen biosynthesis. Deletion of the glycosyltransferase gene wbxB and a histone-like nucleoid structuring (H-NS) protein-encoding gene (BCAL0154) reduced bacterial accumulation and attenuated virulence in C. elegans. Further analysis using quantitative RT-PCR indicated that BCAL0154 modulates B. cenocepacia pathogenesis via transcriptional regulation of motility-associated genes including fliC, fliG, flhD, and cheB1. This screen has successfully identified genes required for B. cenocepacia survival within the host-associated environment, many of which are potential targets for developing new antimicrobials.

Molecular epidemiology of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major cause of viral acute respiratory tract infections in young children. The virus is characterised by distinct seasonality that is dependent upon the latitude and its ability to cause reinfection. Respiratory syncytial virus demonstrates a complex molecular epidemiology pattern as multiple strains and/or genotypes cocirculate during a single epidemic. Previous studies have investigated the relationship between RSV genetic diversity, reinfection, and clinical features. Here, we review the evidence behind this relationship together with the impact that the advancement of whole genome sequencing will have upon our understanding and the need for reconsidering the classification of RSV genotypes.

Enteric Infections Circulating during Hajj Seasons, 2011-2013.

Hajj, the annual Muslim pilgrimage to Mecca, Saudi Arabia, is a unique mass gathering event that raises public health concerns in the host country and globally. Although gastroenteritis and diarrhea are common among Hajj pilgrims, the microbial etiologies of these infections are unknown. We collected 544 fecal samples from pilgrims with medically attended diarrheal illness from 40 countries during the 2011-2013 Hajj seasons and screened the samples for 16 pathogens commonly associated with diarrheal infections. Bacteria were the main agents detected, in 82.9% of the 228 positive samples, followed by viral (6.1%) and parasitic (5.3%) agents. Salmonella spp., Shigella/enteroinvasive Escherichia coli, and enterotoxigenic E. coli were the main pathogens associated with severe symptoms. We identified genes associated with resistance to third-generation cephalosporins ≈40% of Salmonella- and E. coli-positive samples. Hajj-associated foodborne infections pose a major public health risk through the emergence and transmission of antimicrobial drug-resistant bacteria.

Refugees and antimicrobial resistance: A systematic review.

BACKGROUND: There is a large increase in the numbers of refugees and asylum seekers worldwide and a lack of data on the carriage of antimicrobial resistance in refugee/asylum seeking groups. METHODS: This article aims to identify the impact of refugees and asylum seekers on the acquisition and transmission of antimicrobial resistance (AMR) through a literature search. The databases Embase, Medline, Pubmed, and Web of Science Core Collection were utilised and covered all articles before the 1st of October 2016. In total, 577 articles were identified, and studies were eligible if they met the selection criteria, including observational study design, English language, and AMR strains reported in absolute numbers. In total, 17 articles met the criteria, the majority were from the European region. RESULTS: Articles fitting the selection criteria exclusively reported AMR in bacterial species including Mycobacterium tuberculosis, Escherichia coli, Klebsiella pneumonia, K. oxytoca, Shigella spp., Staphylococcus aureus, Enterococcus faecium, and Acinetobacter baumannii. The analyses indicated that a high percentage of AMR strains, have been circulating among refugees and asylum seekers. CONCLUSION: The displacement of refugees and asylum seekers seem to play a key role in the transmission of AMR. Therefore, improved AMR control measures are essential. A knowledge gap was identified; further research is strongly recommended.

Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS.

Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.

Hajj vaccinations-facts, challenges, and hope.

Vaccination is an effective preventive measure that has been used in the unique Hajj pilgrimage setting to control the transmission of infectious diseases. The current vaccination policy applied during Hajj is reviewed herein, highlighting the effectiveness of the approaches applied and identifying research gaps that need to be filled in order to improve the development and dissemination of Hajj vaccination strategies.

Genomic and Phenotypic Analyses Reveal the Emergence of an Atypical Salmonella enterica Serovar Senftenberg Variant in China.

Human infections with Salmonella enterica subspecies enterica serovar Senftenberg are often associated with exposure to poultry flocks, farm environments, or contaminated food. The recent emergence of multidrug-resistant isolates has raised public health concerns. In this study, comparative genomics and phenotypic analysis were used to characterize 14 Salmonella Senftenberg clinical isolates recovered from multiple outbreaks in Shenzhen and Shanghai, China, between 2002 and 2011. Single-nucleotide polymorphism analyses identified two phylogenetically distinct clades of S Senftenberg, designated SC1 and SC2, harboring variations in Salmonella pathogenicity island 1 (SPI-1) and SPI-2 and exhibiting distinct biochemical and phenotypic signatures. Although the two variants shared the same serotype, the SC2 isolates of sequence type 14 (ST14) harbored intact SPI-1 and -2 and hence were characterized by possessing efficient invasion capabilities. In contrast, the SC1 isolates had structural deletion patterns in both SPI-1 and -2 that correlated with an impaired capacity to invade cultured human cells and also the year of their isolation. These atypical SC1 isolates also lacked the capacity to produce hydrogen sulfide. These findings highlight the emergence of atypical Salmonella Senftenberg variants in China and provide genetic validation that variants lacking SPI-1 and regions of SPI-2, which leads to impaired invasion capacity, can still cause clinical disease. These data have identified an emerging public health concern and highlight the need to strengthen surveillance to detect the prevalence and transmission of nontyphoidal Salmonella species.