Genomic Serotyping, Clinical Manifestations, and Antimicrobial Resistance of Nontyphoidal Salmonella Gastroenteritis in Hospitalized Children in Ho Chi Minh City, Vietnam.

Nontyphoidal Salmonella (NTS) are among the most common etiological agents of diarrheal diseases worldwide and have become the most commonly detected bacterial pathogen in children hospitalized with diarrhea in Vietnam. Aiming to better understand the epidemiology, serovar distribution, antimicrobial resistance (AMR), and clinical manifestation of NTS gastroenteritis in Vietnam, we conducted a clinical genomics investigation of NTS isolated from diarrheal children admitted to one of three tertiary hospitals in Ho Chi Minh City. Between May 2014 and April 2016, 3,166 children hospitalized with dysentery were recruited into the study; 478 (∼15%) children were found to be infected with NTS by stool culture. Molecular serotyping of the 450 generated genomes identified a diverse collection of serogroups (B, C1, C2 to C3, D1, E1, G, I, K, N, O, and Q); however, Salmonella enterica serovar Typhimurium was the most predominant serovar, accounting for 41.8% (188/450) of NTS isolates. We observed a high prevalence of AMR to first-line treatments recommended by WHO, and more than half (53.8%; 242/450) of NTS isolates were multidrug resistant (MDR; resistant to ≥3 antimicrobial classes). AMR gene detection positively correlated with phenotypic AMR testing, and resistance to empirical antimicrobials was associated with a significantly longer hospitalization (0.91 days; P = 0.04). Our work shows that genome sequencing is a powerful epidemiological tool to characterize the serovar diversity and AMR profiles in NTS. We propose a revaluation of empirical antimicrobials for dysenteric diarrhea and endorse the use of whole-genome sequencing for sustained surveillance of NTS internationally.

Azithromycin Resistance in Shigella spp. in Southeast Asia.

Infection by Shigella spp. is a common cause of dysentery in Southeast Asia. Antimicrobials are thought to be beneficial for treatment; however, antimicrobial resistance in Shigella spp. is becoming widespread. We aimed to assess the frequency and mechanisms associated with decreased susceptibility to azithromycin in Southeast Asian Shigella isolates and use these data to assess appropriate susceptibility breakpoints. Shigella isolates recovered in Vietnam and Laos were screened for susceptibility to azithromycin (15 µg) by disc diffusion and MIC. Phenotypic resistance was confirmed by PCR amplification of macrolide resistance loci. We compared the genetic relationships and plasmid contents of azithromycin-resistant Shigella sonnei isolates using whole-genome sequences. From 475 available Shigella spp. isolated in Vietnam and Laos between 1994 and 2012, 6/181 S. flexneri isolates (3.3%, MIC ≥ 16 g/liter) and 16/294 S. sonnei isolates (5.4%, MIC ≥ 32 g/liter) were phenotypically resistant to azithromycin. PCR amplification confirmed a resistance mechanism in 22/475 (4.6%) isolates (mphA in 19 isolates and ermB in 3 isolates). The susceptibility data demonstrated the acceptability of the S. flexneri (MIC ≥ 16 g/liter, zone diameter ≤ 15 mm) and S. sonnei (MIC ≥ 32 g/liter, zone diameter ≤ 11 mm) breakpoints with a <3% discrepancy. Phylogenetic analysis demonstrated that decreased susceptibility has arisen sporadically in Vietnamese S. sonnei isolates on at least seven occasions between 2000 and 2009 but failed to become established. While the proposed susceptibility breakpoints may allow better recognition of resistant isolates, additional studies are required to assess the impact on the clinical outcome. The potential emergence of azithromycin resistance highlights the need for alternative options for management of Shigella infections in countries where Shigella is endemic.

Recent insights into Shigella.

PURPOSE OF REVIEW: Diarrhoea is a major global health problem, and recent studies have confirmed Shigella as a major contributor to this burden. Here, we review recent advances in Shigella research; focusing on their epidemiology, pathogenesis, antimicrobial resistance, and the role of the gut microbiome during infection. RECENT FINDINGS: Enhanced epidemiological data, combined with new generation diagnostics, has highlighted a greater burden of Shigella disease than was previously estimated, which is not restricted to vulnerable populations in low-middle income countries. As we gain an ever more detailed insight into the orchestrated mechanisms that Shigella exploit to trigger infection, we can also begin to appreciate the complex role of the gut microbiome in preventing and inducing such infections. The use of genomics, in combination with epidemiological data and laboratory investigations, has unravelled the evolution and spread of various species. Such measures have identified resistance to antimicrobials as a key contributor to the success of specific clones. SUMMARY: We need to apply novel findings towards sustainable approaches for treating and preventing Shigella infections. Vaccines and alternative treatments are under development and may offer an opportunity to reduce the burden of Shigella disease and restrict the mobility of antimicrobial resistant clones.

Clinical implications of reduced susceptibility to fluoroquinolones in paediatric Shigella sonnei and Shigella flexneri infections.

OBJECTIVES: We aimed to quantify the impact of fluoroquinolone resistance on the clinical outcome of paediatric shigellosis patients treated with fluoroquinolones in southern Vietnam. Such information is important to inform therapeutic management for infections caused by this increasingly drug-resistant pathogen, responsible for high morbidity and mortality in young children globally. METHODS: Clinical information and bacterial isolates were derived from a randomized controlled trial comparing gatifloxacin with ciprofloxacin for the treatment of paediatric shigellosis. Time-kill experiments were performed to evaluate the impact of MIC on the in vitro growth of Shigella and Cox regression modelling was used to compare clinical outcome between treatments and Shigella species. RESULTS: Shigella flexneri patients treated with gatifloxacin had significantly worse outcomes than those treated with ciprofloxacin. However, the MICs of fluoroquinolones were not significantly associated with poorer outcome. The presence of S83L and A87T mutations in the gyrA gene significantly increased MICs of fluoroquinolones. Finally, elevated MICs and the presence of the qnrS gene allowed Shigella to replicate efficiently in vitro in high concentrations of ciprofloxacin. CONCLUSIONS: We found that below the CLSI breakpoint, there was no association between MIC and clinical outcome in paediatric shigellosis infections. However, S. flexneri patients had worse clinical outcomes when treated with gatifloxacin in this study regardless of MIC. Additionally, Shigella harbouring the qnrS gene are able to replicate efficiently in high concentrations of ciprofloxacin and we hypothesize that such strains possess a competitive advantage against fluoroquinolone-susceptible strains due to enhanced shedding and transmission.

Prevalence and Associated Factors of optrA-Positive-Enterococcus faecalis in Different Reservoirs around Farms in Vietnam.

Linezolid is an antibiotic of last resort for the treatment of infections caused by Gram-positive bacteria, including vancomycin-resistant enterococci. Enterococcus faecalis, a member of enterococci, is a significant pathogen in nosocomial infections. E. faecalis resistance to linezolid is frequently related to the presence of optrA, which is often co-carried with fex, phenicol exporter genes, and erm genes encoding macrolide resistance. Therefore, the common use of antibiotics in veterinary might promote the occurrence of optrA in livestock settings. This is a cross-sectional study aiming to investigate the prevalence of optrA positive E. faecalis (OPEfs) in 6 reservoirs in farms in Ha Nam province, Vietnam, and its associated factors and to explore genetic relationships of OPEfs isolates. Among 639 collected samples, the prevalence of OPEfs was highest in flies, 46.8% (51/109), followed by chickens 37.3% (72/193), dogs 33.3% (17/51), humans 18.7% (26/139), wastewater 16.4% (11/67) and pigs 11.3%, (14/80). The total feeding area and total livestock unit of the farm were associated with the presence of OPEfs in chickens, flies, and wastewater. Among 186 OPEfs strains, 86% were resistant to linezolid. The presence of optrA was also related to the resistant phenotype against linezolid and levofloxacin of E. faecalis isolates. Close genotypic relationships identified by Pulsed Field Gel Electrophoresis between OPEfs isolates recovered from flies and other reservoirs including chickens, pigs, dogs, and wastewater suggested the role of flies in the transmission of antibiotic-resistant pathogens. These results provided warnings of linezolid resistance although it is not used in livestock.

The identification of novel immunogenic antigens as potential Shigella vaccine components.

BACKGROUND: Shigella is a major diarrheal pathogen for which there is presently no vaccine. Whole genome sequencing provides the ability to predict and derive novel antigens for use as vaccines. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen. METHODS: Using a reverse vaccinology approach, where genomic analysis informed the Shigella immunome via an antigen microarray, we aimed to identify novel immunogenic Shigella antigens. A core genome analysis of Shigella species, pathogenic and non-pathogenic Escherichia coli, led to the selection of 234 predicted immunogenic Shigella antigens. These antigens were expressed and probed with acute and convalescent serum from microbiologically confirmed Shigella infections. RESULTS: Several Shigella antigens displayed IgG and IgA seroconversion, with no difference in sero-reactivity across by sex or age. IgG sero-reactivity to key Shigella antigens was observed at birth, indicating transplacental antibody transfer. Six antigens (FepA, EmrK, FhuA, MdtA, NlpB, and CjrA) were identified in in vivo testing as capable of producing binding IgG and complement-mediated bactericidal antibody. CONCLUSIONS: These findings provide six novel immunogenic Shigella proteins that could serve as candidate vaccine antigens, species-specific carrier proteins, or targeted adjuvants.

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.

Pathogenic Escherichia coli Possess Elevated Growth Rates under Exposure to Sub-Inhibitory Concentrations of Azithromycin.

Antimicrobial resistance (AMR) has been identified by the World Health Organization (WHO) as one of the ten major threats to global health. Advances in technology, including whole-genome sequencing, have provided new insights into the origin and mechanisms of AMR. However, our understanding of the short-term impact of antimicrobial pressure and resistance on the physiology of bacterial populations is limited. We aimed to investigate morphological and physiological responses of clinical isolates of E. coli under short-term exposure to key antimicrobials. We performed whole-genome sequencing on twenty-seven E. coli isolates isolated from children with sepsis to evaluate their AMR gene content. We assessed their antimicrobial susceptibility profile and measured their growth dynamics and morphological characteristics under exposure to varying concentrations of ciprofloxacin, ceftriaxone, tetracycline, gentamicin, and azithromycin. AMR was common, with all organisms resistant to at least one antimicrobial; a total of 81.5% were multi-drug-resistant (MDR). We observed an association between resistance profile and morphological characteristics of the E. coli over a three-hour exposure to antimicrobials. Growth dynamics experiments demonstrated that resistance to tetracycline promoted the growth of E. coli under antimicrobial-free conditions, while resistance to the other antimicrobials incurred a fitness cost. Notably, antimicrobial exposure heterogeneously suppressed bacterial growth, but sub-MIC concentrations of azithromycin increased the maximum growth rate of the clinical isolates. Our results outline complex interactions between organism and antimicrobials and raise clinical concerns regarding exposure of sub-MIC concentrations of specific antimicrobials.

Assessing gut microbiota perturbations during the early phase of infectious diarrhea in Vietnamese children.

Diarrheal diseases remain the second most common cause of mortality in young children in developing countries. Efforts have been made to explore the impact of diarrhea on bacterial communities in the human gut, but a thorough understanding has been impeded by inadequate resolution in bacterial identification and the examination of only few etiological agents. Here, by profiling an extended region of the 16S rRNA gene in the fecal microbiome, we aimed to elucidate the nature of gut microbiome perturbations during the early phase of infectious diarrhea caused by various etiological agents in Vietnamese children. Fecal samples from 145 diarrheal cases with a confirmed infectious etiology before antimicrobial therapy and 54 control subjects were analyzed. We found that the diarrheal fecal microbiota could be robustly categorized into 4 microbial configurations that either generally resembled or were highly divergent from a healthy state. Factors such as age, nutritional status, breastfeeding, and the etiology of the infection were significantly associated with these microbial community structures. We observed a consistent elevation of Fusobacterium mortiferum, Escherichia, and oral microorganisms in all diarrheal fecal microbiome configurations, proposing similar mechanistic interactions, even in the absence of global dysbiosis. We additionally found that Bifidobacterium pseudocatenulatum was significantly depleted during dysenteric diarrhea regardless of the etiological agent, suggesting that further investigations into the use of this species as a dysentery-orientated probiotic therapy are warranted. Our findings contribute to the understanding of the complex influence of infectious diarrhea on gut microbiome and identify new opportunities for therapeutic interventions.

The genomic signatures of Shigella evolution, adaptation and geographical spread.

Shigella spp. are some of the key pathogens responsible for the global burden of diarrhoeal disease. These facultative intracellular bacteria belong to the family Enterobacteriaceae, together with other intestinal pathogens, such as Escherichia coli and Salmonella spp. The genus Shigella comprises four different species, each consisting of several serogroups, all of which show phenotypic similarity, including invasive pathogenicity. DNA sequencing suggests that this similarity results from the convergent evolution of different Shigella spp. founders. Here, we review the evolutionary relationships between Shigella spp. and E . coli, and we highlight how the genomic plasticity of these bacteria and their acquisition of a distinctive virulence plasmid have enabled the development of such highly specialized pathogens. Furthermore, we discuss the insights that genotyping and whole-genome sequencing have provided into the phylogenetics and intercontinental spread of Shigella spp.