The impact of antibiotics on the gut microbiota of children recovering from watery diarrhoea.

Infectious diarrhoeal diseases remain a substantial health burden in young children in low- and middle-income countries. The disease and its variable treatment options significantly alter the gut microbiome, which may affect clinical outcomes and overall gut health. Antibiotics are often prescribed, but their impact on the gut microbiome during recovery is unclear. Here, we used 16S rRNA sequencing to investigate changes in the gut microbiota in Vietnamese children with acute watery diarrhoea, and highlight the impact of antibiotic treatment on these changes. Our analyses identified that, regardless of treatment, recovery was characterised by reductions in Streptococcus and Rothia species and expansion of Bacteroides/Phocaeicola, Lachnospiraceae and Ruminococcacae taxa. Antibiotic treatment significantly delayed the temporal increases in alpha- and beta-diversity within patients, resulting in distinctive patterns of taxonomic change. These changes included a pronounced, transient overabundance of Enterococcus species and depletion of Bifidobacterium pseudocatenulatum. Our findings demonstrate that antibiotic treatment slows gut microbiota recovery in children following watery diarrhoea.

Changing epidemiology and antimicrobial susceptibility of bloodstream infections at a Vietnamese infectious diseases hospital (2010-2020).

Bloodstream infection (BSI) poses a global health problem, with diverse organisms and rising antimicrobial resistance (AMR). Here, we characterized trends in BSI prevalence, AMR, and antibiotic use at a Vietnamese infectious diseases hospital from 2010 to 2020. Among 108,303 cultured blood samples, 8.8% were positive, yielding 7995 pathogens. Of 7553 BSI cases, 86.4% were community-acquired. BSI prevalence varied from 17 to 35 cases/1000 admissions/year, highest in HIV/hepatitis wards and patients >60. The in-hospital mortality or hospice discharge outcome was 21.3%. The top three pathogens, E. coli (24%), K. pneumoniae (8.7%) and S. aureus (8.5%) exhibited increasing prevalence and multidrug resistance. Pathogens like Cryptococcus neoformans (8.4%), Talaromyces marneffei (6.7%), and Salmonella enterica (6.5%) declined. E. coli and K. pneumoniae were prevalent in older adults with community-acquired BSIs. Antibiotic use reached 842.6 DOT/1000 PD and significantly reduced after an antibiotic control policy. Enhanced surveillance and antimicrobial stewardship are crucial for managing BSIs in Vietnam.

Genomic insights unveil the plasmid transfer mechanism and epidemiology of hypervirulent Klebsiella pneumoniae in Vietnam.

Hypervirulent Klebsiella pneumoniae (hvKp) is a significant cause of severe invasive infections in Vietnam, yet data on its epidemiology, population structure and dynamics are scarce. We screened hvKp isolates from patients with bloodstream infections (BSIs) at a tertiary infectious diseases hospital in Vietnam and healthy individuals, followed by whole genome sequencing and plasmid analysis. Among 700 BSI-causing Kp strains, 100 (14.3%) were hvKp. Thirteen hvKp isolates were identified from 350 rectal swabs of healthy adults; none from 500 rectal swabs of healthy children. The hvKp isolates were genetically diverse, encompassing 17 sequence types (STs), predominantly ST23, ST86 and ST65. Among the 113 hvKp isolates, 14 (12.6%) carried at least one antimicrobial resistance (AMR) gene, largely mediated by IncFII, IncR, and IncA/C plasmids. Notably, the acquisition of AMR conjugative plasmids facilitated horizontal transfer of the non-conjugative virulence plasmid between K. pneumoniae strains. Phylogenetic analysis demonstrated hvKp isolates from BSIs and human carriage clustered together, suggesting a significant role of intestinal carriage in hvKp transmission. Enhanced surveillance is crucial to understand the factors driving intestinal carriage and hvKp transmission dynamics for informing preventive measures. Furthermore, we advocate the clinical use of our molecular assay for diagnosing hvKp infections to guide effective management.

Multidrug resistance plasmids underlie clonal expansions and international spread of Salmonella enterica serotype 1,4,[5],12:i:- ST34 in Southeast Asia.

Salmonella enterica serotype 1,4,[5],12:i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogen's epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring blaCTX-M-55, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a potential hub for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors.

The clinical features and genomic epidemiology of carbapenem-resistant Acinetobacter baumannii infections at a tertiary hospital in Vietnam.

OBJECTIVES: To characterise the clinical features of Acinetobacter baumannii infections and investigate the phylogenetic structure and transmission dynamics of A. baumannii in Vietnam. METHODS: Between 2019 and 2020, a surveillance of A. baumannii (AB) infections was conducted at a tertiary hospital in Ho Chi Minh City, Vietnam. Risk factors for in-hospital mortality were analysed using logistic regressions. Whole-genome sequence data were used to characterise genomic species, sequence types (STs), antimicrobial resistance genes, surface antigens, and phylogenetic relatedness of AB isolates. RESULTS: Eighty-four patients with AB infections were enrolled in the study, 96% of whom were hospital-acquired. Half of the AB isolates were identified from ICU-admitted patients, while the remaining isolates were from non-ICU patients. The overall in-hospital mortality was 56%, with associated risk factors including advanced age, ICU stay, exposure to mechanical ventilation/central venous catheterization, pneumonia as source of AB infection, prior use of linezolid/aminoglycosides, and AB treatment with colistin-based therapy. Nearly 91% of isolates were carbapenem-resistant; 92% were multidrug-resistant; and 6% were colistin-resistant. ST2, ST571, and ST16 were the three dominant carbapenem-resistant A. baumannii (CRAB) genotypes, exhibiting distinct AMR gene profiles. Phylogenetic analysis of CRAB ST2 isolates together with previously published ST2 collection provided evidence of intra- and inter-hospital transmission of this clone. CONCLUSIONS: Our study highlights a high prevalence of carbapenem resistance and multidrug resistance in A. baumannii and elucidates the spread of CRAB within and between hospitals. Strengthening infection control measures and routine genomic surveillance are crucial to reducing the spread of CRAB and detecting novel pan-drug-resistant variants in a timely fashion.

Genomic epidemiology, antimicrobial resistance and virulence factors of Enterobacter cloacae complex causing potential community-onset bloodstream infections in a tertiary care hospital of Nepal.

Objectives: Community-onset bloodstream infections (BSIs) caused by carbapenemase-producing Enterobacter cloacae complex (ECC) species are increasing internationally. This observation suggests that ECC are emerging pathogens, requiring for detailed understanding on their genomic epidemiology including transmission dynamics and antimicrobial resistance profiles. Patients and methods: We performed WGS on 79 Enterobacter spp. isolated from the patients with clinically significant BSIs and admitted to emergency department of a major tertiary hospital in Nepal between April 2016 and October 2017. Results: We identified 5 species and 13 STs of ECC. Enterobacter xiangfangensis ST171, one of the globally emerging carbapenem resistant ECC clones with epidemic potential, was the most prevalent (42%). Phylogenetic analysis showed a large (>19 400 SNPs) core genome SNP distance across major STs, which was minimal (<30 SNPs) among the isolates of each prevalent ST, suggesting the relatively recent importation of major STs followed by local clonal expansions. Genomic evidence for resistance to all major antimicrobial classes except for colistin and macrolides was detected. A limited number of isolates also carried bla NDM-1 (n = 2) and bla OXA-48 (n = 1) carbapenemase genes. Virulence factors encoding siderophores (24%), T6SSD (25%) and fimbriae (54%) were detected. Conclusions: Our study highlighted that MDR ECC clones are important pathogens of BSIs in community. Though of low prevalence, carbapenem resistance observed in our ECC isolates raised concern about further community dissemination, underscoring the need for community surveillance to identify MDR ECC clones with epidemic potential.

Tumour microbiomes and Fusobacterium genomics in Vietnamese colorectal cancer patients.

Perturbations in the gut microbiome have been associated with colorectal cancer (CRC), with the colonic overabundance of Fusobacterium nucleatum shown as the most consistent marker. Despite its significance in the promotion of CRC, genomic studies of Fusobacterium is limited. We enrolled 43 Vietnamese CRC patients and 25 participants with non-cancerous colorectal polyps to study the colonic microbiomes and genomic diversity of Fusobacterium in this population, using a combination of 16S rRNA gene profiling, anaerobic microbiology, and whole genome analysis. Oral bacteria, including F. nucleatum and Leptotrichia, were significantly more abundant in the tumour microbiomes. We obtained 53 Fusobacterium genomes, representing 26 strains, from the saliva, tumour and non-tumour tissues of six CRC patients. Isolates from the gut belonged to diverse F. nucleatum subspecies (nucleatum, animalis, vincentii, polymorphum) and a potential new subspecies of Fusobacterium periodonticum. The Fusobacterium population within each individual was distinct and in some cases diverse, with minimal intra-clonal variation. Phylogenetic analyses showed that within four individuals, tumour-associated Fusobacterium were clonal to those isolated from non-tumour tissues. Genes encoding major virulence factors (Fap2 and RadD) showed evidence of horizontal gene transfer. Our work provides a framework to understand the genomic diversity of Fusobacterium within the CRC patients, which can be exploited for the development of CRC diagnostic and therapeutic options targeting this oncobacterium.

The evolutionary history of Shigella flexneri serotype 6 in Asia.

Shigella flexneri serotype 6 is an understudied cause of diarrhoeal diseases in developing countries, and has been proposed as one of the major targets for vaccine development against shigellosis. Despite being named as S. flexneri, Shigella flexneri serotype 6 is phylogenetically distinct from other S. flexneri serotypes and more closely related to S. boydii. This unique phylogenetic relationship and its low sampling frequency have hampered genomic research on this pathogen. Herein, by utilizing whole genome sequencing (WGS) and analyses of Shigella flexneri serotype 6 collected from epidemiological studies (1987-2013) in four Asian countries, we revealed its population structure and evolutionary history in the region. Phylogenetic analyses supported the delineation of Asian Shigella flexneri serotype 6 into two phylogenetic groups (PG-1 and -2). Notably, temporal phylogenetic approaches showed that extant Asian S. flexneri serotype 6 could be traced back to an inferred common ancestor arising in the 18th century. The dominant lineage PG-1 likely emerged in the 1970s, which coincided with the times to most recent common ancestors (tMRCAs) inferred from other major Southeast Asian S. flexneri serotypes. Similar to other S. flexneri serotypes in the same period in Asia, genomic analyses showed that resistance to first-generation antimicrobials was widespread, while resistance to more recent first-line antimicrobials was rare. These data also showed a number of gene inactivation and gene loss events, particularly on genes related to metabolism and synthesis of cellular appendages, emphasizing the continuing role of reductive evolution in the adaptation of the pathogen to an intracellular lifestyle. Together, our findings reveal insights into the genomic evolution of the understudied Shigella flexneri serotype 6, providing a new piece in the puzzle of Shigella epidemiology and evolution.

Evolutionary histories and antimicrobial resistance in Shigella flexneri and Shigella sonnei in Southeast Asia.

Conventional disease surveillance for shigellosis in developing country settings relies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s-1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.

Genomic insights into the circulation of pandemic fluoroquinolone-resistant extra-intestinal pathogenic Escherichia coli ST1193 in Vietnam.

Extra-intestinal pathogenic Escherichia coli (ExPEC) ST1193, a globally emergent fluoroquinolone-resistant clone, has become an important cause of bloodstream infections (BSIs) associated with significant morbidity and mortality. Previous studies have reported the emergence of fluoroquinolone-resistant ExPEC ST1193 in Vietnam; however, limited data exist regarding the genetic structure, antimicrobial resistance (AMR) determinants and transmission dynamics of this pandemic clone. Here, we performed genomic and phylogenetic analyses of 46 ST1193 isolates obtained from BSIs and healthy individuals in Ho Chi Minh City, Vietnam, to investigate the pathogen population structure, molecular mechanisms of AMR and potential transmission patterns. We further examined the phylogenetic structure of ST1193 isolates in a global context. We found that the endemic E. coli ST1193 population was heterogeneous and highly dynamic, largely driven by multiple strain importations. Several well-supported phylogenetic clusters (C1-C6) were identified and associated with distinct blaCTX-M variants, including blaCTXM-27 (C1-C3, C5), blaCTXM-55 (C4) and blaCTXM-15 (C6). Most ST1193 isolates were multidrug-resistant and carried an extensive array of AMR genes. ST1193 isolates also exhibited the ability to acquire further resistance while circulating in Vietnam. There were phylogenetic links between ST1193 isolates from BSIs and healthy individuals, suggesting these organisms may both establish long-term colonization in the human intestinal tract and induce infections. Our study uncovers factors shaping the population structure and transmission dynamics of multidrug-resistant ST1193 in Vietnam, and highlights the urgent need for local One Health genomic surveillance to capture new emerging ExPEC clones and to better understand the origins and transmission patterns of these pathogens.

Emerging carbapenem-resistant Klebsiella pneumoniae sequence type 16 causing multiple outbreaks in a tertiary hospital in southern Vietnam.

The emergence of carbapenem resistance in Klebsiella pneumoniae represents a major global public health concern. Nosocomial outbreaks caused by multidrug-resistant K. pneumoniae are commonly reported to result in high morbidity and mortality due to limited treatment options. Between October 2019 and January 2020, two concurrent high-mortality nosocomial outbreaks occurred in a referral hospital in Ho Chi Minh City, Vietnam. We performed genome sequencing and phylogenetic analysis of eight K. pneumoniae isolates from infected patients and two environmental isolates for outbreak investigation. We identified two outbreaks caused by two distinct lineages of the international sequence type (ST) 16 clone, which displayed extensive drug resistance, including resistance to carbapenem and colistin. Carbapenem-resistant ST16 outbreak strains clustered tightly with previously described ST16 K. pneumoniae from other hospitals in Vietnam, suggesting local persistence and transmission of this particular clone in this setting. We found environmental isolates from a hospital bed and blood pressure cuff that were genetically linked to an outbreak case cluster, confirming the potential of high-touch surfaces as sources for nosocomial spread of K. pneumoniae. Further, we found colistin resistance caused by disruption of the mgrB gene by an ISL3-like element, and carbapenem resistance mediated by a transferable IncF/blaOXA-181 plasmid carrying the ISL3-like element. Our study highlights the importance of coordinated efforts between clinical and molecular microbiologists and infection control teams to rapidly identify, investigate and contain nosocomial outbreaks. Routine surveillance with advanced sequencing technology should be implemented to strengthen hospital infection control and prevention measures.

Colonization with Staphylococcus aureus and Klebsiella pneumoniae causes infections in a Vietnamese intensive care unit.

Pre-existing colonization with Staphylococcus aureus or Klebsiella pneumoniae has been found to increase the risk of infection in intensive care patients. We previously conducted a longitudinal study to characterize colonization of these two organisms in patients admitted to intensive care in a hospital in southern Vietnam. Here, using genomic and phylogenetic analyses, we aimed to assess the contribution these colonizing organisms made to infections. We found that in the majority of patients infected with S. aureus or K. pneumoniae, the sequence type of the disease-causing (infecting) isolate was identical to that of corresponding colonizing organisms in the respective patient. Further in-depth analysis revealed that in patients infected by S. aureus ST188 and by K. pneumoniae ST17, ST23, ST25 and ST86, the infecting isolate was closely related to and exhibited limited genetic variation relative to pre-infection colonizing isolates. Multidrug-resistant S. aureus ST188 was identified as the predominant agent of colonization and infection. Colonization and infection by K. pneumoniae were characterized by organisms with limited antimicrobial resistance profiles but extensive repertoires of virulence genes. Our findings augment the understanding of the link between bacterial colonization and infection in a low-resource setting, and could facilitate the development of novel evidence-based approaches to prevent and treat infections in high-risk patients in intensive care.

The genomic epidemiology of multi-drug resistant invasive non-typhoidal Salmonella in selected sub-Saharan African countries.

BACKGROUND: Invasive non-typhoidal Salmonella (iNTS) is one of the leading causes of bacteraemia in sub-Saharan Africa. We aimed to provide a better understanding of the genetic characteristics and transmission patterns associated with multi-drug resistant (MDR) iNTS serovars across the continent. METHODS: A total of 166 iNTS isolates collected from a multi-centre surveillance in 10 African countries (2010-2014) and a fever study in Ghana (2007-2009) were genome sequenced to investigate the geographical distribution, antimicrobial genetic determinants and population structure of iNTS serotypes-genotypes. Phylogenetic analyses were conducted in the context of the existing genomic frameworks for various iNTS serovars. Population-based incidence of MDR-iNTS disease was estimated in each study site. RESULTS: Salmonella Typhimurium sequence-type (ST) 313 and Salmonella Enteritidis ST11 were predominant, and both exhibited high frequencies of MDR; Salmonella Dublin ST10 was identified in West Africa only. Mutations in the gyrA gene (fluoroquinolone resistance) were identified in S. Enteritidis and S. Typhimurium in Ghana; an ST313 isolate carrying blaCTX-M-15 was found in Kenya. International transmission of MDR ST313 (lineage II) and MDR ST11 (West African clade) was observed between Ghana and neighbouring West African countries. The incidence of MDR-iNTS disease exceeded 100/100 000 person-years-of-observation in children aged <5 years in several West African countries. CONCLUSIONS: We identified the circulation of multiple MDR iNTS serovar STs in the sampled sub-Saharan African countries. Investment in the development and deployment of iNTS vaccines coupled with intensified antimicrobial resistance surveillance are essential to limit the impact of these pathogens in Africa.

The phylogeography and incidence of multi-drug resistant typhoid fever in sub-Saharan Africa.

There is paucity of data regarding the geographical distribution, incidence, and phylogenetics of multi-drug resistant (MDR) Salmonella Typhi in sub-Saharan Africa. Here we present a phylogenetic reconstruction of whole genome sequenced 249 contemporaneous S. Typhi isolated between 2008-2015 in 11 sub-Saharan African countries, in context of the 2,057 global S. Typhi genomic framework. Despite the broad genetic diversity, the majority of organisms (225/249; 90%) belong to only three genotypes, 4.3.1 (H58) (99/249; 40%), 3.1.1 (97/249; 39%), and 2.3.2 (29/249; 12%). Genotypes 4.3.1 and 3.1.1 are confined within East and West Africa, respectively. MDR phenotype is found in over 50% of organisms restricted within these dominant genotypes. High incidences of MDR S. Typhi are calculated in locations with a high burden of typhoid, specifically in children aged <15 years. Antimicrobial stewardship, MDR surveillance, and the introduction of typhoid conjugate vaccines will be critical for the control of MDR typhoid in Africa.

The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam.

BACKGROUND: Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges. METHODOLOGY: We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla(CTX-M) encoding plasmid. PRINCIPAL FINDINGS: We show that two different bla(CTX-M) genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla(CTX-M) gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the bla(CTX-M-24) gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids. SIGNIFICANCE: The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.