Emergence and genomic insights of non-pandemic O1 Vibrio cholerae in Zhejiang, China.

IMPORTANCE: It is well recognized that only Vibrio cholerae O1 causes cholera pandemics. However, not all O1 strains cause pandemic-level disease. In this study, we analyzed non-pandemic O1 V. cholerae isolates from the 1960s to the 1990s from China and found that they fell into three lineages, one of which shared the most recent common ancestor with pandemic O1 strains. Each of these non-pandemic O1 lineages has unique properties that contribute to their capacity to cause cholera. The findings of this study enhanced our understanding of the emergence and evolution of both pandemic and non-pandemic O1 V. cholerae.

Screening for extended-spectrum beta-lactamase Escherichia coli in recreational beach waters in Singapore.

Aims: As part of Singapore's One Health antimicrobial resistance (AMR) management, this work was designed to understand the AMR burden in recreational beach waters using extended-spectrum beta-lactamase Escherichia coli (ESBL-EC) as an indicator. Materials & methods: A total of 90 water samples were collected from six different recreational beaches over three different time periods. Only 28/90 (31.3%) water samples yielded E. coli colonies ranging from 1 to 80 colony-forming units/100 ml. Results & conclusion: Screening of all colonies using CHROMID® ESBL agar and Luria-Bertani broth supplemented with ceftriaxone showed that none was ESBL-EC. Further monitoring is required to understand the prevalence of ESBL-EC spatiotemporally, contributing to the national AMR surveillance program and providing timely risk assessment for exposure to ESBL-EC.

Genomic epidemiology of human candidaemia isolates in a tertiary hospital.

Invasive candida infections are significant infections that may occur in vulnerable patients with high rates of mortality or morbidity. Drug-resistance rates also appear to be on the rise which further complicate treatment options and outcomes. The aims of this study were to describe the prevalence, molecular epidemiology, and genetic features of Candida bloodstream isolates in a hospital setting. The resistance mechanisms towards the two most commonly administered antifungals, fluconazole and anidulafungin, were determined. Blood culture isolates between 1 January 2018 and 30 June 2021 positive for Candida spp. were included. Susceptibility testing was performed using Etest. Whole-genome-sequencing was performed using Illumina NovaSeq with bioinformatics analysis performed. A total of 203 isolates were sequenced: 56 C. glabrata, 53 C. tropicalis, 44 C. albicans, 36 C. parapsilosis complex (consisting of C. parapsilosis, C. orthopsilosis, and C. metapsilosis), six C. krusei, five C. dubliniensis, and three C. auris. A single cluster of azole-resistant C. tropicalis, and four clusters of C. parapsilosis isolates were observed, suggesting possible transmission occurring over several years. We found 11.3%, and 52.7 % of C. tropicalis and C. parapsilosis, respectively, clustered with other isolates, suggesting exogenous sources may play a significant role of transmission, particularly for C. parapsilosis. The clusters spanned over several years suggesting the possibility of environmental reservoirs contributing to the spread. Limited clonality was seen for C. albicans. Several sequence types appeared to be dominant for C. glabrata, however the SNP differences varied widely, indicating absence of sustained transmission.

Genomic epidemiology and multilevel genome typing of Bordetella pertussis.

Bordetella pertussis causes pertussis (or whooping cough), a severe respiratory infectious disease in infants, although it can be prevented by whole cell and acellular vaccines. The recent pertussis resurgence in industrialised countries is partly attributed to pathogen adaptation to vaccines, while emergence of antimicrobial resistance, specifically to macrolides in China, has become a concern. Surveillance of current circulating and emerging strains is therefore vital to understand the risks they pose to public health. Although the use of genomics-based typing is increasing a genomic nomenclature for this pathogen has not been well established. Here, we implemented the multilevel genome typing (MGT) system for B. pertussis with five levels of resolution, which provide targeted typing of relevant lineages and discrimination of closely related strains at the finest scale. The lower resolution levels (MGT2 and MGT3) describe the distribution of major vaccine antigen alleles including ptxP, fim3, fhaB and prn, as well as temporal and spatial trends within the B. pertussis global population. Mid-resolution levels (MGT3 and MGT4) enable typing of antibiotic-resistant lineages and Prn deficient lineages within the ptxP3 clade. The high-resolution level (MGT5) can capture finer-scale epidemiology such as outbreaks and local transmission events, with comparable resolution to existing genomic methods of strain-relatedness assessment. The scheme offers stable MGT-type assignments aiding harmonisation of typing and communication between laboratories. The scheme is available at https://mgtdb.unsw.edu.au/pertussis, is regularly updated from global data repositories and accepts public submissions. The MGT scheme provides a comprehensive, robust, and scalable system for global surveillance of B. pertussis.

Field trial assessing the antimicrobial decontamination efficacy of gaseous ozone in a public bus setting.

The widespread COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) necessitated measures aimed at preventing the spread of SARS-CoV-2. To mitigate the risk of fomite-mediated transmission, environmental cleaning and disinfection regimes have been widely implemented. However, conventional cleaning approaches such as surface wipe downs can be laborious and more efficient and effective disinfecting technologies are needed. Gaseous ozone disinfection is one technology which has been shown to be effective in laboratory studies. Here, we evaluated its efficacy and feasibility in a public bus setting, using murine hepatitis virus (a related betacoronavirus surrogate) and the bacteria Staphylococcus aureus as test organisms. An optimal gaseous ozone regime resulted in a 3.65-log reduction of murine hepatitis virus and a 4.73-log reduction of S. aureus, and decontamination efficacy correlated with exposure duration and relative humidity in the application space. These findings demonstrated gaseous ozone disinfection in field settings which can be suitably translated to public and private fleets that share analogous characteristics.

Viable mpox virus in the environment of a patient room.

We conducted a prospective environmental surveillance study to investigate the air, surface, dust, and water contamination of a room occupied by a patient infected with mpox virus (MPXV) at various stages of the illness. The patient tested positive for MPXV from a throat swab and skin lesions. Environmental sampling was conducted in a negative pressure room with 12 unidirectional high efficiency particulate air filter (HEPA) air changes per hour and daily cleaning of the surfaces. A total of 179 environmental samples were collected on days 7, 8, 13, and 21 of illness. Among the days of sampling, air, surface, and dust contamination showed the highest contamination rates on day 7 and 8 of illness, with a gradual decline to the lowest contamination level by day 21. Viable MPXV was isolated from surfaces and dust samples and no viable virus was isolated from the air and water samples.

Genomic Epidemiology and Multilevel Genome Typing of Australian Salmonella enterica Serovar Enteritidis.

Salmonella enterica serovar Enteritidis is one of the leading causes of salmonellosis in Australia. In this study, a total of 568 S. Enteritidis isolates from two Australian states across two consecutive years were analyzed and compared to international strains, using the S. Enteritidis multilevel genome typing (MGT) database, which contained 40,390 publicly available genomes from 99 countries. The Australian S. Enteritidis isolates were divided into three phylogenetic clades (A, B, and C). Clades A and C represented 16.4% and 3.5% of the total isolates, respectively, and were of local origin. Clade B accounted for 80.1% of the isolates which belonged to seven previously defined lineages but was dominated by the global epidemic lineage. At the MGT5 level, three out of five top sequence types (STs) in Australia were also top STs in Asia, suggesting that a fair proportion of Australian S. Enteritidis cases may be epidemiologically linked with Asian strains. In 2018, a large egg-associated local outbreak was caused by a recently defined clade B lineage prevalent in Europe and was closely related, but not directly linked, to three European isolates. Additionally, over half (54.8%) of predicted multidrug resistance (MDR) isolates belonged to 10 MDR-associated MGT-STs, which were also frequent in Asian S. Enteritidis . Overall, this study investigated the genomic epidemiology of S. Enteritidis in Australia, including the first large local outbreak, using MGT. The open MGT platform enables a standardized and sharable nomenclature that can be effectively applied to public health for unified surveillance of S. Enteritidis nationally and globally. IMPORTANCE Salmonella enterica serovar Enteritidis is a leading cause of foodborne infections. We previously developed a genomic typing database (MGTdb) for S. Enteritidis to facilitate global surveillance of this pathogen. In this study, we examined the genomic features of Australian S. Enteritidis using the MGTdb and found that Australian S. Enteritidis is mainly epidemiologically linked with Asian strains (especially strains carrying antimicrobial resistance genes), followed by European strains. The first large-scale egg-associated local outbreak in Australia was caused by a recently defined lineage prevalent in Europe, and three European isolates in the MGTdb were closely related but not directly linked to this outbreak. In summary, the S. Enteritidis MGTdb open platform is shown to be a potentially powerful tool for national and global public health surveillance of this pathogen.

Genome Sequence of a Clinical Blood Isolate of Kodamaea ohmeri.

Kodamaea ohmeri is a rarely occurring yeast that can cause human infections. We describe the whole-genome sequence of a K. ohmeri clinical blood isolate.

MGTdb: a web service and database for studying the global and local genomic epidemiology of bacterial pathogens.

Multilevel genome typing (MGT) enables the genomic characterization of bacterial isolates and the relationships among them. The MGT system describes an isolate using multiple multilocus sequence typing (MLST) schemes, referred to as levels. Thus, for a new isolate, sequence types (STs) assigned at multiple precisely defined levels can be used to type isolates at multiple resolutions. The MGT designation for isolates is stable, and the assignment is faster than the existing approaches. MGT's utility has been demonstrated in multiple species. This paper presents a publicly accessible web service called MGTdb, which enables the assignment of MGT STs to isolates, along with their storage, retrieval and analysis. The MGTdb web service enables upload of genome data as sequence reads or alleles, which are processed and assigned MGT identifiers. Additionally, any newly sequenced isolates deposited in the National Center for Biotechnology Information's Sequence Read Archive are also regularly retrieved (currently daily), processed, assigned MGT identifiers and made publicly available in MGTdb. Interactive visualization tools are presented to assist analysis, along with capabilities to download publicly available isolates and assignments for use with external software. MGTdb is currently available for Salmonella enterica serovars Typhimurium and Enteritidis and Vibrio cholerae. We demonstrate the usability of MGTdb through three case studies - to study the long-term national surveillance of S. Typhimurium, the local epidemiology and outbreaks of S. Typhimurium, and the global epidemiology of V. cholerae. Thus, MGTdb enables epidemiological and microbiological investigations at multiple levels of resolution for all publicly available isolates of these pathogens. Database URL: https://mgtdb.unsw.edu.au.

Genomic dissection of the microevolution of Australian epidemic Bordetella pertussis.

ABSTRACTWhooping 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 including Australia 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 expanding 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. In population genetics terms, both hard and soft selective sweeps through vaccine selection pressures have determined the population dynamics of Australian B. pertussis. Relative risk estimation suggests 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. Phylogenetic analysis revealed the expansion of ptxP3 strains was also associated with replacement of the type III secretion system allele bscI1 with bscI3. bscI3 is associated with decreased T3SS secretion and may allow B. pertussis to reduce immune recognition. This study advanced our understanding of the epidemic population structure and spatial and temporal dynamics of B. pertussis in a highly immunized population.

Molecular epidemiology and phylogenomic analysis of Mycobacterium abscessus clinical isolates in an Asian population.

Mycobacterium abscessus comprises three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. These closely related strains are typically multi-drug-resistant and can cause difficult-to-treat infections. Dominant clusters of isolates with increased pathogenic potential have been demonstrated in pulmonary infections in the global cystic fibrosis (CF) population. An investigation was performed on isolates cultured from an Asian, predominantly non-CF population to explore the phylogenomic relationships within our population and compare it to global M. abscessus isolates. Whole-genome-sequencing was performed on M. abscessus isolates between 2017 and 2019. Bioinformatic analysis was performed to determine multi-locus-sequence-type, to establish the phylogenetic relationships between isolates, and to identify virulence and resistance determinants in these isolates. A total of 210 isolates were included, of which 68.5 % (144/210) were respiratory samples. These isolates consisted of 140 (66.6 %) M. abscessus subsp. massiliense, 67 (31.9 %) M. abscessus subsp. abscessus, and three (1.4 %) M. abscessus subsp. bolletii. Dominant sequence-types in our population were similar to those of global CF isolates, but SNP differences in our population were comparatively wider despite the isolates being from the same geographical region. ESX (ESAT-6 secretory) cluster three appeared to occur most commonly in ST4 and ST6 M. abscessus subsp. massiliense, but other virulence factors did not demonstrate an association with isolate subspecies or sample source. We demonstrate that although similar predominant sequence-types are seen in our patient population, cross-transmission is absent. The risk of patient-to-patient transmission appears to be largely limited to the vulnerable CF population, indicating infection from environmental sources remains more common than human-to-human transmission. Resistance and virulence factors are largely consistent across the subspecies with the exception of clarithromycin susceptibility and ESX-3.

Staphylococcus singaporensis sp. nov., a new member of the Staphylococcus aureus complex, isolated from human clinical specimens.

Staphylococcus argenteus and Staphylococcus schweitzeri are the newest members of the Staphylococcus aureus complex. The number of clinical reports attributed to these new S. aureus complex members is limited. In a retrospective clinical laboratory study conducted over a 4-month period investigating the prevalence of S. argenteus and S. schweitzeri, a total of 43 isolates were selected. Phylogeny based on core-gene multilocus sequence typing (MLST) analysis confirmed that 37 were S. argenteus but a genetically distinct clade of six isolates was identified. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses further supported the classification of these six isolates as a separate species. When compared to S. aureus complex reference genomes, the ANI values were ≤94 % and the dDDH values were <53 %. Based on the seven-gene S. aureus MLST scheme, the six isolates belong to five novel allelic profiles (ST6105, ST6106, ST6107, ST6108 and ST109). Their clinical infection features were similar to S. aureus. Skin and soft tissue infections presented in four out of the six cases. Routine clinical diagnostic identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and biochemical profiling does not differentiate these new members from the rest of the complex. Genotypic analysis suggests that the six isolates belong to a novel species, Staphylococcus singaporensis sp. nov. with isolate SS21T (=DSM 111408T=NCTC14419T) designated as the type strain.

Elucidation of global and national genomic epidemiology of Salmonella enterica serovar Enteritidis through multilevel genome typing.

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.

Factors influencing SARS-CoV-2 transmission and outbreak control measures in densely populated settings.

Starting with a handful of SARS-CoV-2 infections in dormitory residents in late March 2020, rapid transmission in their dense living environments ensued and by October 2020, more than 50,000 acute infections were identified across various dormitories in Singapore. The aim of the study is to identify combination of factors facilitating SARS-CoV-2 transmission and the impact of control measures in a dormitory through extensive epidemiological, serological and phylogenetic investigations, supported by simulation models. Our findings showed that asymptomatic cases and symptomatic cases who did not seek medical attention were major drivers of the outbreak. Furthermore, each resident had 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.

Genomic Characterization of Klebsiella quasipneumoniae from Clinical Specimens in Singapore.

A total of 1,281 specimens from 1,024 patients were screened. Phylogenetic analysis classified 44 of these isolates as Klebsiella quasipneumoniae subsp. similipneumoniae (44/1,281 [3.4%]) and the remaining three as K. quasipneumoniae subsp. quasipneumoniae. The most common specimen source was urine (21/47 [44.7%]) followed by blood (14/47 [29.8%]). K. quasipneumoniae isolates were nonclonal. Carbapenemase-encoding genes (blaNDM and blaOXA-181) were detected in only two isolates (2/47 [4.3%]). K. quasipneumoniae appears to cause a spectrum of infections similar to those of K. pneumoniae, although higher rates of susceptibility to many commonly tested antimicrobials and low prevalence of virulence genes were demonstrated.