Serogroup O1 Research Articles

Antibiotic resistant Pseudomonas aeruginosa in dental unit waterline: a case study in Milan (Italy)

Abstract P.aeruginosa (Pa) is one of the most serious cause of healthcare-associated bacteremia, well-known for its antibiotic resistance (AR). Pa has often been found in dental unit water representing a great risk in public health. Aims are to evaluate the presence of Pa in dental unit (DU) water and its phenotypic and genotypic properties. Pa isolates derive from a monitoring of DU water in an Italian hospital (Milan). Samples were collected on Monday and on Friday, before the beginning and after the end of activities. Resistant PA strains were characterized by phenotypic method using E-test for 6 different classes of antibiotics, and by genotypic method with Whole-genome sequencing (WGS). Resistance genes were annotated with ResFinder v. 4.5 and Sequence Type (ST) was determined using the PA MLST database. 70/272 strains were identified as Pa and 21 of them were AR to one or more antibiotics. They were detected in 10/44 (22.7%) DUs and 2/4 (50%) control sinks (1-2000UFC/100mL). WGS was obtained for 18 strains. 17 belonged to serogroup O11 and ST667 was prevalent (8/18) followed by ST395 (4/18). At the ground floor ST395 was the most prevalent, followed by ST667 and 260, while, at the first floor, ST309 and 667 were in the two controls and ST667, 3245 and 260 in the other DUs. Resistant genes blaPAO, aph(3’)-IIb, catB7 conferring resistance to β-lactams, aminoglycosides and amphenicol, respectively, were observed in all isolates. Only ST3245 strain did not show resistance to Fosfomycin. Genotypic and phenotypic analyses present some discrepancies with the presence of AR genes and no antibiotic resistance detection (4/18) or resistance to other different classes of antibiotics (6/18). The MLST analyses showed a high diversity, with the presence of strains sharing different antibiotic resistance mechanisms in DU water. Detection and analyses by WGS can help to track dissemination of genetic resistance determinants and potential emergence of high-risk clones. Key messages • Phenotypic and genotypic analyses in DU water are needed to evaluate risk for human health, in addition to routine microbial monitoring. • WGS can help to track dissemination of genetic resistance determinants and potential emergence of high-risk clones.

Longitudinal study of avian pathogenic Escherichia coli (APEC) serogroups associated with disease in Georgia poultry using molecular serology and virulence gene analysis

ABSTRACT Avian pathogenic Escherichia coli (APEC) is a significant cause of morbidity, mortality, and production loss to the poultry industry worldwide. Here, we characterized 569 E. coli isolates from avian-diagnosed colibacillosis cases from the state of Georgia, USA. In total, 339 isolates were assigned into 32 serogroups with the majority classifying as O78, O2, O25, O8, O1, O86, O18, and O15. Serogroup O25 was found to link with broilers, while broiler breeders were more often associated with serogroup O1 and pet/ hobby birds with serogroup O8. In addition, some serogroups (O1) were more prevalent in the summer and fall. Analysis for virulence-associated genes (VAGs) found 23.20% of isolates did not harbour any genes linked with the APEC pathotype, while ColV plasmid-associated genes (iroN, ompT, hlyF, iss, and aerJ) were frequently detected among most isolates (with 80–96% prevalence) and some of these genes were linked with serogroup. Phylogenetic analysis, classified isolates into phylogenetic groups B2 (34%), F (19%), A (15%), and G (9%). The phylogenetic group B2 isolates also harboured the highest number of VAGs. This study highlights that the current APEC-causing disease in birds in the State of Georgia has identified several emerging serogroups possessing several VAGs that could potentially lead to challenges in colibacillosis control. RESEARCH HIGHLIGHTS Several emerging APEC serogroups were observed in Georgia poultry populations. An association between APEC serogroups and bird type was observed. The prevalence of different APEC serogroups was influenced by season. A multiplex PCR assay targeting common serogroups of APEC in Georgia poultry was developed.

Draft genomes of 10 Vibrio cholerae isolates collected in Sudan in 2019.

This report presents the first genomes from positive cases of cholera in Sudan. Genomic analysis of 10 Vibrio cholerae isolates, profiled as serogroup O1, reveals evidence of antimicrobial resistance genes and a 139-kb IncC plasmid with 99.74% identity to the multidrug-resistant plasmid pCNRVC190243 previously reported in Yemen and Lebanon.

Challenges and Considerations in Selecting Animal Models for Evaluating a Live, Cold-Chain-Free, Dual-Use Vaccine (MyChol) for Diarrhoeal Diseases: A Comprehensive Review.

Diarrhoeal diseases are the second leading cause of death for children under 5 years old in 69 low- and middle-income countries, with an annual economic burden of US$ 4 billion and over 525,000 lives lost. Cholera and enterotoxigenic Escherichia coli (ETEC) traveller's diarrhoea are major diarrhoeal diseases caused by Vibrio cholerae (O1 and O139 serogroups) and ETEC, which have similar pathogeneses and can co-infect. There is no exclusive vaccine for ETEC, but cholera vaccines containing the cholera toxin B (CT-B) component offer short-term cross-protection. However, licensed oral cholera vaccines are expensive due to cold-chain supplies and the need for multiple doses. A cost-effective, dual-protection, live, cold-chain-free vaccine is, therefore, required for vaccination campaigns in low-resource settings, and MyChol - a prototype cold-chain-free live attenuated cholera vaccine, targeting V. cholerae O139 and ETEC H10407 - was developed in this context. The vaccine was evaluated in three animal models (Sprague Dawley [SD] rats, BALB/c mice and New Zealand white rabbits) for safety, colonisation capacity, reactogenicity and immunogenicity against challenge strains. In suckling mice, MyChol displayed high colonisation potential compared to unformulated VCUSM14P (the vaccine candidate) and wild-type O139. In the acute toxicity assessment, the SD rats with the highest MyChol dose (1 × 107 colony-forming unit [CFU]/kg) demonstrated no adverse effects or mortality. Mice vaccinated with MyChol exhibited elevated antibody levels, including anti-CT, anti-heat-labile enterotoxin (LT), anti-CT-B and anti-LT-B. Anti-CT antibodies neutralised LT toxin in ETEC H10407 in challenge studies and cross-protected against ETEC H10407 in both mice and rabbits, preventing weight loss and diarrhoea. Ileal loop experiments in rabbits and BALB/c mice showed no reactogenicity. This review, based on our previous research, therefore provides valuable insights into improving the selection of animal models to advance preclinical evaluations of diarrhoeal vaccines.

Diversity and Complexity of CTXΦ and Pre-CTXΦ Families in Vibrio cholerae from Seventh Pandemic.

CTXΦ is a lysogenic filamentous phage that carries the genes encoding cholera toxin (ctxAB), the main virulence factor of Vibrio cholerae. The toxigenic conversion of environmental V. cholerae strains through CTXΦ lysogenic infection is crucial for the emergence of new pathogenic clones. A special allelic form of CTXΦ, called pre-CTXΦ, is a precursor of CTXΦ and without ctxAB. Different members of the pre-CTXΦ and CTXΦ families are distinguished by the sequence of the transcriptional repressor-coding gene rstR. Multiple rstR alleles can coexist within a single strain, demonstrating the diverse structure and complex genomic integration patterns of CTXΦ/pre-CTXΦ prophage on the chromosome. Exploration of the diversity and co-integration patterns of CTXΦ/pre-CTXΦ prophages in V. cholerae can help to understand the evolution of this phage family. In this study, 21 V. cholerae strains, which were shown to carry the CTXΦ/pre-CTXΦ prophages as opposed to typical CTXETΦ-RS1 structure, were selected from approximately 1000 strains with diverse genomes. We identified two CTXΦ members and six pre-CTXΦ members with distinct rstR alleles, revealing complex chromosomal DNA integration patterns and arrangements of different prophages in these strains. Promoter activity assays showed that the transcriptional repressor RstR protected against CTXΦ superinfection by preventing the replication and integration of CTXΦ/pre-CTXΦ phages containing the same rstR allele, supporting the co-integration of the diverse CTXΦ/pre-CTXΦ members observed. The numbers and types of prophages and their co-integration arrangements in serogroup O139 strains were more complex than those in serogroup O1 strains. Also, these CTXΦ/pre-CTXΦ members were shown to present the bloom period of the CTXΦ/pre-CTXΦ family during wave 2 of the seventh cholera pandemic. Together, these analyses deepen our comprehension of the genetic variation of CTXΦ and pre-CTXΦ and provide insights into the evolution of the CTXΦ/pre-CTXΦ family in the seventh cholera pandemic.

Epidemiology and antimicrobial resistance profiles of pathogenic Escherichia coli from commercial swine and poultry abattoirs and farms in South Africa: A One Health approach

Pathogenic Escherichia coli (PEC) are important foodborne bacteria that can cause severe illness in humans. The PECs thrive within the intestines of humans as well as animals and may contaminate multiple ecosystems, including food and water, via faecal transmission. Abattoir and farm employees are at high risk of PEC exposure, which could translate to community risk through person-to-person contact. To determine the epidemiology and resistome of PECs in Gauteng and Limpopo provinces of South Africa, 198 swine faecal samples, 220 poultry cloacal swabs, 108 human hand swabs, 11 run-off water samples from abattoirs and farms were collected from four swine and five poultry commercial abattoirs and two swine farms. One effluent sample each was collected from four wastewater treatment plants (WWTP) and a tertiary hospital setting. Phenotypic and genotypic techniques were used including polymerase chain reaction, pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). Results showed EHEC and EPEC prevalence was 4.1 % (22/542) and 20.8 % (113/542), respectively, with the O26 serogroup detected the most in PEC isolates. According to the PFGE dendrogram, isolates from poultry, human hand swabs and run-off water clustered together. Diverse virulence factors such as the novel stx2k subtype and eae genes were detected among the 36 representative PEC isolates according to WGS. The results showed that 66.7 % (24/36) of sequenced PECs presented with multi-drug resistance (MDR) to β-lactamase 13.9 % (5/36), aminoglycoside 61.1 % (22/36), tetracycline 41.7 % (15/36) and quinolones 38.9 % (14/36). No colistin nor carbapenem resistance was detected. Sequence types (STs) associated with MDR in this study were: ST752, ST189, ST206, ST10, ST48 and ST38. The findings highlight the threat of zoonotic pathogens to close human contacts and the need for enhanced surveillance to mitigate the spread of MDR foodborne PECs.

Prevalence and antimicrobial susceptibility pattern of Vibrio cholerae isolates from cholera outbreak sites in Ethiopia

BackgroundCholera is an acute infectious disease caused by ingestion of contaminated food or water with Vibrio cholerae. Cholera remains a global threat to public health and an indicator of inequity and lack of social development. The aim of this study was to assess the prevalence and antimicrobial susceptibility pattern of V. cholerae from cholera outbreak sites in Ethiopia.MethodsAcross-sectional study was conducted from May 2022 to October 2023 across different regions in Ethiopia: Oromia National Regional State, Amhara National Regional State and Addis Ababa City Administration. A total of 415 fecal samples were collected from the three regions. Two milliliter fecal samples were collected from each study participants. The collected samples were cultured on Blood Agar, MacConkey Agar and Thiosulfate Citrate Bile Salt Sucrose Agar. A series of biochemical tests Oxidase test, String test, Motility, Indole, Citrate, Gas production, H2S production, Urease test were used to identify V. cholerae species. Both polyvalent and monovalent antisera were used for agglutination tests to identify and differentiate V. cholerae serogroup and serotypes. In addition, Kirby-Bauer Disk diffusion antibiotic susceptibility test method was done. Data were registered in epi-enfo version 7 and analyzed by Statistical Package for Social Science version 25. Descriptive statistics were used to determine the prevalence of Vibrio cholerae. Logistic regression model was fitted and p-value < 0.05 was considered as statically significant.ResultsThe prevalence of V. cholerae in the fecal samples was 30.1%. Majority of the isolates were from Oromia National Regional State 43.2% (n = 54) followed by Amhara National Regional State 31.2% (n = 39) and Addis Ababa City Administration 25.6% (n = 32). Most of the V. cholerae isolates were O1 serogroups 90.4% (n = 113) and Ogawa serotypes 86.4% (n = 108). Majority of the isolates were susceptible to ciprofloxacin 100% (n = 125), tetracycline 72% (n = 90) and gentamycin 68% (n = 85). More than half of the isolates were resistant to trimethoprim-sulfamethoxazole 62.4% (n = 78) and ampicillin 56.8% (n = 71). In this study, participants unable to read and write were about four times more at risk for V. cholerae infection (AOR: 3.8, 95% CI: 1.07–13.33). In addition, consumption of river water were about three times more at risk for V. cholerae infection (AOR: 2.8, 95% CI: 1.08–7.08).Conclusionour study revealed a high prevalence of V. cholerae from fecal samples. The predominant serogroups and serotypes were O1 and Ogawa, respectively. Fortunately, the isolates showed susceptible to most tested antibiotics. Drinking water from river were the identified associated risk factor for V. cholerae infection. Protecting the community from drinking of river water and provision of safe and treated water could reduce cholera outbreaks in the study areas.

Genomic insights into the 2022–2023Vibrio cholerae outbreak in Malawi

Malawi experienced its deadliest Vibrio cholerae (Vc) outbreak following devastating cyclones, with >58,000 cases and >1700 deaths reported between March 2022 and May 2023. Here, we use population genomics to investigate the attributes and origin of the Malawi 2022–2023 Vc outbreak isolates. Our results demonstrate the predominance of ST69 clone, also known as the seventh cholera pandemic El Tor (7PET) lineage, expressing O1 Ogawa (~ 80%) serotype followed by Inaba (~ 16%) and sporadic non-O1/non-7PET serogroups (~ 4%). Phylogenetic reconstruction revealed that the Malawi outbreak strains correspond to a recent importation from Asia into Africa (sublineage AFR15). These isolates harboured known antimicrobial resistance and virulence elements, notably the ICEGEN/ICEVchHai1/ICEVchind5 SXT/R391-like integrative conjugative elements and a CTXφ prophage with the ctxB7 genotype compared to historical Malawian Vc isolates. These data suggest that the devastating cyclones coupled with the recent importation of 7PET serogroup O1 strains, may explain the magnitude of the 2022–2023 cholera outbreak in Malawi.

Antigenicity evaluation of synthetic α-(1,3)-linked D, D-heptoglycan of Helicobacter pylori serotype O6 lipopolysaccharide

Helicobacter pylori, a gram‐negative bacterium, is known to be associated with various gastric pathologies including chronic gastritis, peptic ulcers and gastric carcinoma. Despite the emerging issue of bacterial resistance to antibiotic-based combination therapy, there is currently no vaccine available for H. pylori infection in the market. Here, we report the synthesis of α-(1,3)-D,D-heptoglycan with different chain length from the lipopolysaccharide of H. pylori serogroup O6. The [n + 1] iterative glycosylation strategy was used for heptoglycan chain elongation. The trifluoroacetimidate heptoside donor exhibited much higher efficiency than thioglycoside donor during glycosylation. An antigenicity evaluation using glycan microarrays indicated that α-(1,3)-D,D-hepto-disaccharide, pentasaccharide and hexasaccharide showed stronger binding affinity to IgG antibodies of H. pylori O6 LPS immunized rabbit serum or serum of H. pylori infected patients. These findings provide significant structure − activity relationship information for developing carbohydrate-based vaccines against H. pylori containing α-(1,3)-D,D-heptoglycan.

Seasonal Mortality of Wild Atlantic Menhaden (Brevoortia tyrannus) Is Caused by a Virulent Clone of Vibrio (Listonella) anguillarum; Implications for Biosecurity along the Atlantic Coastal United States

Atlantic menhaden are a highly migratory marine species in the Eastern United States that suffer from seasonal chronic mortality. Affected fish show neurologic signs referred to as spinning disease, including circling at the surface and erratic corkscrew swimming before death. We investigated three similar menhaden mortality events consistent with spinning disease in coastal New Jersey and New York between 2020 and 2021 to understand the cause. A unique strain of Vibrio (Listonella) anguillarum (serogroup O3) was detected regularly in high loads, particularly in the brains of moribund fish, by both metagenomics and bacterial isolation. The most common histopathological changes in moribund fish were hemorrhagic meningitis, encephalitis, pyknosis, and karyorrhexis of hematopoietic tissues in the kidney and spleen. Whole genome sequencing of isolates from moribund fish representing a wide spatial and temporal range showed that they were nearly identical clones, suggesting it to be a pathogenic strain circulating in the population. Though V. anguillarum is believed to be the main pathogen associated with spinning disease and mortality, Yersinia ruckeri (serotype O1) was isolated from smaller numbers of fish. Considering the highly migratory nature of Atlantic menhaden throughout the eastern United States and their use as bait for other fisheries, these findings identify potential biosecurity challenges that should be considered in Atlantic salmon aquaculture, fisheries, and emerging marine aquaculture in the region.

Epidemiological Situation on Cholera in the Russian Federation in 2023 and Forecast for 2024

The paper presents an analysis and assessment of risks of complication of epidemiological situation regarding cholera in the Russian Federation and a forecast for 2024. It briefly characterizes the dynamics of the epidemic process around the world during the last decade (2013–2022). The forecast of cholera importation into the Russian Federation, due to activity of the epidemic process in endemic countries, has been confirmed. In 2023, two cases of cholera importation from India were reported. It was established that the toxigenic strains isolated from patients belonged to the currently dominant in the world “post-Haitian” group, including in India and Bangladesh, with the genotype ctxB7 tcpACIRS rtxA4a VSP-IIΔ0495-0512. Furthermore, non-toxigenic Vibrio cholerae O1 strains were isolated from samples of biological material from persons arriving from India, containing preCTX prophage and distinct from the strain (ctxAB– tcpAEТ–) isolated from a patient with acute intestinal infection (Zaporozhye Region, Melitopol). Nontoxigenic NAG vibrios were isolated from 13 patients with acute intestinal infections, which is 1.9 times more than in 2022. Regardless of importations, a strain of V. cholerae O1 (ctxB1+tcpAEТ) of the “pre-Haitian” group was isolated from a water sample of a surface reservoir. 52 non-toxigenic V. сholerae O1 strains were found on the territory of 11 constituent entities of the Russian Federation, which is 18.9 % more than in 2022. In 2024, epidemiological risks of cholera importation from endemic countries to any administrative territory of the Russian Federation remain. The risks of epidemic manifestations of cholera are especially high in the territories of the constituent entities of the Russian Federation which are under ongoing emergency, due to migration processes, and the impact of other risk-forming factors that contribute to an increase in the degree of contact of the population with water bodies contaminated with cholera vibrios. If epidemic foci of cholera are to emerge in a neighboring country, there will be a high probability of spread of this infection in the stated constituent entities of the Russian Federation and in other territories of southern Russia. In the absence of epidemic manifestations of cholera, the possibility of occasional detection of toxigenic O1 serogroup strains in water samples due to unidentified introductions from cholera-affected areas cannot be ruled out.

Non-O1/non-O139 Vibrio cholerae bacteraemia and cholangitis: An unusual case in an oncological patient in Lecco Hospital, Italy

Vibrio species are curved, motile, aerobic and facultative anaerobic, Gram-negative bacilli that are widely distributed in aquatic environments, especially marine and estuarine waters. Vibrios of primary concern in human pathology are essentially represented by the species Vibrio cholerae. Classification of Vibrio cholerae is based on the O-antigen polysaccharide type, with over 200 serogroups described. Historically, only toxigenic serogroups O1 and O139 have been associated with widespread cholera epidemics. Other serogroups can cause small outbreaks that spread via contaminated raw seafood (primarily shellfish and molluscs) and seawater exposure, and they are collectively termed Non-O1/Non-O139 Vibrio Cholerae (NOVC). NOVC, occasionally detected also in the Mediterranean Sea, most often causes sporadic gastrointestinal manifestations requiring supportive therapy. However, rarely the V. cholerae non-O1/ non-O139 serotype can result in extraintestinal manifestations such as severe wound infections, soft tissue infections and sepsis in immunocompromised patients that represent a therapeutic challenge. In particular, NOVC bacteraemia is a rare event but has the highest mortality rates among NOVC infections (almost 33%) and usually occurs in certain immunosuppressed populations, such as patients with haematologic and solid malignancies, and those with underlying liver disease.

Un caso di diffusione intra-familiare di colera

Cholera is an acute diarrhoeal disease caused by Vibrio cholerae (VC) serogroups O1 and O139. Transmission is oro-faecal and it might lead to acute dehydration. The disease is still endemic in resource-poor countries, nevertheless importation cases are occasionally reported in developed countries. The paper reports a case of a 3-year-old boy admitted to the emergency room because of acute gastroenteritis with rice-water stools. Faecal samples were positive for VC. The child was managed with prompt parenteral crystalloid infusion, followed by oral rehydration solution. After a single dose of azythromicin, a progressive reduction in symptoms was documented. Cholera infection is then briefly reviewed.

Ингибиторный анализ протеаз препаратов бесклеточных фракций холерных вибрионов

One of the important areas of research into the pathogenesis of diseases caused by pathogens of especially dangerous infections is the study of the system of hydrolytic enzymes. It has been established that hydrolytic enzymes of pathogenic microorganisms enhance their pathogenicity (virulence) and participate in the processes of adaptation to unfavourable environmental factors. Cholera vibrio has previously electrophoretically detected hydrolases belonging to different subclasses and established differences in their spectrum in strains with different epidemic significance. Therefore, the study of their characterisation (type determination using activator/inhibitor assays) is a relevant area of research. The inhibitor analysis of secreted proteases of cholera vibrio strains of O1 serogroup El Tor biovar and O139 serogroup determined that the proteases include serine and metalloproteases.

Ингибиторный анализ протеаз препаратов бесклеточных фракций холерных вибрионов

One of the important areas of research into the pathogenesis of diseases caused by pathogens of especially dangerous infections is the study of the system of hydrolytic enzymes. It has been established that hydrolytic enzymes of pathogenic microorganisms enhance their pathogenicity (virulence) and participate in the processes of adaptation to unfavourable environmental factors. Cholera vibrio has previously electrophoretically detected hydrolases belonging to different subclasses and established differences in their spectrum in strains with different epidemic significance. Therefore, the study of their characterisation (type determination using activator/inhibitor assays) is a relevant area of research. The inhibitor analysis of secreted proteases of cholera vibrio strains of O1 serogroup El Tor biovar and O139 serogroup determined that the proteases include serine and metalloproteases.

Investigation of potentially pathogenic Vibrionaceae in Saint-Louis city, Senegal.

as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal. a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH. for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity. despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital.

Recombinant Salmonella gallinarum (S. gallinarum) Vaccine Candidate Expressing Avian Pathogenic Escherichia coli Type I Fimbriae Provides Protections against APEC O78 and O161 Serogroups and S. gallinarum Infection.

Avian pathogenic Escherichia coli (APEC) is one of the leading pathogens that cause devastating economic losses to the poultry industry. Type I fimbriae are essential adhesion factors of APEC, which can be targeted and developed as a vaccine candidate against multiple APEC serogroups due to their excellent immunogenicity and high homology. In this study, the recombinant strain SG102 was developed by expressing the APEC type I fimbriae gene cluster (fim) on the cell surface of an avirulent Salmonella gallinarum (S. gallinarum) vector strain using a chromosome-plasmid-balanced lethal system. The expression of APEC type I fimbriae was verified by erythrocyte hemagglutination assays and antigen-antibody agglutination tests. In vitro, the level of the SG102 strain adhering to leghorn male hepatoma (LMH) cells was significantly higher than that of the empty plasmid control strain, SG101. At two weeks after oral immunization, the SG102 strain remained detectable in the livers, spleens, and ceca of SG102-immunized chickens, while the SG101 strain was eliminated in SG101-immunized chickens. At 14 days after the secondary immunization with 5 × 109 CFU of the SG102 strain orally, highly antigen-specific humoral and mucosal immune responses against APEC type I fimbriae protein were detected in SG102-immunized chickens, with IgG and secretory IgA (sIgA) concentrations of 221.50 μg/mL and 1.68 μg/mL, respectively. The survival rates of SG102-immunized chickens were 65% (13/20) and 60% (12/20) after challenge with 50 LD50 doses of APEC virulent strains O78 and O161 serogroups, respectively. By contrast, 95% (19/20) and 100% (20/20) of SG101-immunized chickens died in challenge studies involving APEC O78 and O161 infections, respectively. In addition, the SG102 strain effectively provided protection against lethal challenges from the virulent S. gallinarum strain. These results demonstrate that the SG102 strain, which expresses APEC type I fimbriae, is a promising vaccine candidate against APEC O78 and O161 serogroups as well as S. gallinarum infections.

Toxigenic Vibrio cholerae strains in South-East Queensland, Australian river waterways.

Cholera is a major public health problem in developing and underdeveloped countries; however, it remains of concern to developed countries such as Australia as international travel-related or locally acquired cholera or diarrheal disease cases are still reported. Cholera is mainly caused by cholera toxin (CT) producing toxigenic O1 and O139 serogroup Vibrio cholerae strains. While most toxigenic V. cholerae cases in Australia are thought to be caused by international-acquired infections, Australia has its own indigenous toxigenic and non-toxigenic O1 and non-O1, non-O139 V. cholerae (NOVC) strains. In Australia, in the 1970s and again in 2012, it was reported that south-east Queensland riverways were a reservoir for toxigenic V. cholerae strains that were linked to local cases. Further surveillance on environmental reservoirs, such as riverways, has not been reported in the literature in the last 10 years. Here we present data from sites previously related to outbreaks and surveillance sampling to detect the presence of V. cholerae using PCR in conjunction with MALDI-TOF and whole-genome sequencing. In this study, we were able to detect NOVC at all 10 sites with all sites having toxigenic non-O1, non-O139 strains. Among 133 NOVC isolates, 22 were whole-genome sequenced and compared with previously sequenced Australian O1 and NOVC strains. None of the samples tested grew toxigenic or non-toxigenic O1 or O139, responsible for epidemic disease. Since NOVC can be pathogenic, continuous surveillance is required to assist in theclinical and envir rapid identification of sources of any outbreaks and to assist public health authorities in implementing control measures. IMPORTANCE Vibrio cholerae is a natural inhabitant of aquatic environments, both freshwater and seawater, in addition to its clinical significance as a causative agent of acute diarrhea and extraintestinal infections. Previously, both toxigenic and non-toxigenic, clinical, and environmental V. cholerae strains have been reported in Queensland, Australia. This study aimed to characterize recent surveillance of environmental NOVC strains isolated from Queensland River waterways to understand their virulence, antimicrobial resistance profile and to place genetic current V. cholerae strains from Australia in context with international strains. The findings from this study suggest the presence of unique toxigenic V. cholerae in Queensland river water systems that are of public health concern. Therefore, ongoing monitoring and genomic characterization of V. cholerae strains from the Queensland environment is important and would assist public health departments to track the source of cholera infection early and implement prevention strategies for future outbreaks. The genomics of environmental V. cholerae could assist us to understand the natural ecology and evolution of this bacterium in natural environments with respect to global warming and climate change.

Alkyl Sulfatase of Cholera Vibrios

The aim of the work was to study the structure of the alkyl sulfatase (asu) gene in Vibrio cholerae strains of various serogroups, as well as to compare nucleotide and amino acid sequences of alkyl sulfatases using various methods of bioinformatic analysis.Materials and methods. 483 strains of V. cholerae O1, O139 and nonO1/nonO139 serogroups were employed in the work. The search for the gene, its recurrence, and localization was carried out applying the Blast software. The nucleotide and corresponding amino acid sequences of the gene, as well as its structure, were studied using bioinformatic analysis. Sequencing was performed on the MiSeq (Illumina) platform. The enzymatic activity was detected using a medium, confirming the presence/absence of the gene by PCR in vitro and in silico.Results and discussion. Bioinformatic analysis of the nucleotide and corresponding amino acid sequences of the asu gene has been carried out and its structure investigated. Four functional domains have been identified. In the beta-lactamase domain, a conservative amino acid sequence -HAHADH- has been found in all strains of cholera vibrios, which is part of the Zn2+ binding motif. It has been established that the alkyl sulfatase of cholera vibrios belongs to the family of Zn2+-dependent β-lactamases. Blast analysis has revealed the similarity of nucleotide and amino acid sequences of alkyl sulfatases in representatives of V. cholerae O1 and O139 serogroups (ctxAB+tcpA+) and representatives of the genera Aeromonas and Pseudomonas, which is in the line with the data of 3D modeling of the amino acid sequence structures of the alkyl sulfatase enzyme in these microorganisms. The bioinformatic analysis of nucleotide and amino acid sequences of alkyl sulfatases in cholera vibrios has showed the conservativeness of these sequences in toxigenic strains and the presence of a number of single mutations in the asu gene in atoxigenic ones. The presence or absence of the asu gene has been established by PCR in vitro and in silico and confirmed by the results obtained using the Blast program. It is demonstrated that the presence/absence of the asu gene correlates with the ability/inability of O139 strains to hydrolyze SDS on the medium. These results can be used in studying mechanisms of cholera vibrios adaptation, persistence and pathogenicity.

Genomic epidemiology reveals multidrug resistant plasmid spread between Vibrio cholerae lineages in Yemen

Since 2016, Yemen has been experiencing the largest cholera outbreak in modern history. Multidrug resistance (MDR) emerged among Vibrio cholerae isolates from cholera patients in 2018. Here, to characterize circulating genotypes, we analysed 260 isolates sampled in Yemen between 2018 and 2019. Eighty-four percent of V. cholerae isolates were serogroup O1 belonging to the seventh pandemic El Tor (7PET) lineage, sub-lineage T13, whereas 16% were non-toxigenic, from divergent non-7PET lineages. Treatment of severe cholera with macrolides between 2016 and 2019 coincided with the emergence and dominance of T13 subclones carrying an incompatibility type C (IncC) plasmid harbouring an MDR pseudo-compound transposon. MDR plasmid detection also in endemic non-7PET V. cholerae lineages suggested genetic exchange with 7PET epidemic strains. Stable co-occurrence of the IncC plasmid with the SXT family of integrative and conjugative element in the 7PET background has major implications for cholera control, highlighting the importance of genomic epidemiological surveillance to limit MDR spread.