Serogroup O1 Research Articles

MSHA-like pili of non-toxigenic Vibrio cholerae strains

Aim . In this study, we set out to identify the homologues of genes from the msh-cluster in the genomes of non-toxigenic V cholerae, to perform the bioinformatics analysis of their products, as well as to study the adhesive properties of strains containing altered genes. Materials and methods . We analysed 17 clinical strains of non-O1/non-O139 V cholerae and 2 strains of the O1 serogroup isolated from water bodies. Genes belonging to the msh-cluster were identified in the whole genomes using the BLASTN 2.2.29 and BioEdit 7.2.5 programs. Gene translation, comparative analysis of their nucleotide sequences and the amino acid sequences of deduced products were performed using the Vector NTI Advance 11 (Invitrogen). Results and discusssion . In 18 out of the 19 studied genomes we identified gene clusters responsible for production of adhesion pili (mshH-Q) represented by diverse alleles, the majority of which differed from the prototype genes of the msh-cluster in nucleotide composition but had the same localization and arrangement. Only one strain had a cluster that was close to that of the prototype. A bioinformatics analysis of their deduced products indicated that the amino acid sequence of the major MshA pilus subunit is homologous to the prototype only in a short N-terminal region (1-41) while sharing no similarities with the rest of the sequence. Nevertheless, this protein, similar to VcfA described by Kuroki H. et al. (2001) and designated by us as MshA-like, retained a putative pilus domain. A similar pattern was observed in the minor subunits designated as MshC-like. Other minor subunits also retained their characteristic domains. All of the strains agglutinated human erythrocytes (group O) and chicken erythrocytes, and in isolates harboring modified mshA-like and mshC-like genes the reaction was not inhibited by mannose. Since most of the studied strains were isolated from hospitalized patients, it is possible that in non-toxigenic V. cholerae lacking the pathogenicity island VPI, MSHA-like pili may serve as a colonization factor of the human intestine, in contrast to VPI-positive strains. The obtained information provides a basis for experimental verification of this assumption.

Revisiting the Global Epidemiology of Cholera in Conjuction With the Genomics of Vibrio cholerae.

Toxigenic Vibrio cholerae is responsible for 1.4 to 4.3 million cases with about 21,000–143,000 deaths per year. Dominance of O1 and O139 serogroups, classical and El tor biotypes, alterations in CTX phages and the pathogenicity Islands are some of the major features of V. cholerae isolates that are responsible for cholera epidemics. Whole-genome sequencing (WGS) based analyses of single-nucleotide polymorphisms (SNPs) and other infrequent genetic variants provide a robust phylogenetic framework. Recent studies on the global transmission of pandemic V. cholerae O1 strains have shown the existence of eight different phyletic lineages. In these, the classical and El Tor biotype strains were separated as two distinctly evolved lineages. The frequency of SNP accumulation and the temporal and geographical distribution supports the perception that the seventh cholera pandemic (7CP) has spread from the Bay of Bengal region in three independent but overlapping waves. The 2010 Haitian outbreak shared a common ancestor with South-Asian wave-3 strains. In West Africa and East/Southern Africa, cholera epidemics are caused by single expanded lineage, which has been introduced several times since 1970. The Latin American epidemics that occurred in 1991 and 2010 were the result of introductions of two 7CP sublineages. Sublineages representing wave-3 have caused huge outbreaks in Haiti and Yemen. The Ogawa-Inaba serotype switchover in several cholera epidemics are believed to be due to the involvement of certain selection mechanism(s) rather than due to random events. V. cholerae O139 serogroup is phylogenetically related to the 7CP El Tor, and almost all these isolates belonged to the multilocus sequence type-69. Additional phenotypic and genotypic information have been generated to understand the pathogenicity of classical and El Tor vibrios. Presence of integrative conjugative elements (ICE) with antibiotic resistance gene cassettes, clustered regularly interspaced short palindromic repeats-associated protein system and ctxAB promoter based ToxRS expression of cholera toxin (CT) separates classical and El Tor biotypes. With the availability of WGS information, several important applications including, molecular typing, antimicrobial resistance, new diagnostics, and vaccination strategies could be generated.

Nonhemolysis of epidemic El Tor biotype strains of Vibrio cholerae is related to multiple functional deficiencies of hemolysin A

BackgroundHemolysis of bacteria is an important phenotype used for typing and characterizing strains with specific biomarkers and even a virulence factor in bacterial pathogenesis. In Vibrio cholerae, hemolysin HlyA is responsible for hemolysis of sheep red blood cells, and this hemolytic phenotype is used as a biotyping indicator and considered one of the virulence factors. At the beginning of the seventh cholera pandemic, the El Tor biotype strains of serogroup O1 were distinguished by hemolysis from the sixth pandemic O1 classical biotype strains, whereas during the following epidemics, nonhemolytic El Tor strains appeared, suggesting phenotypic and genetic variations in these strains. This study aimed to investigate the possible mechanisms involved in nonhemolysis of El Tor strains.ResultsFive sequence types of hlyA genes were found in the studied O1 El Tor strains isolated during the seventh pandemic. A 4-base deletion in hlyA caused the HlyA protein mutation and non-hemolytic phenotype. Some strains carry wildtype hlyA genes but are still non-hemolytic, and greatly reduced hlyA transcription and blocked secretion of hemolysin were observed in hemolysis tests of the subcellular components and transcription/expression analysis of hlyA.ConclusionsMechanisms responsible for nonhemolysis of the epidemic O1 El Tor strains are complex and not only confined to gene mutation but also deficiencies of transcription and extracellular transport of HlyA. Mutations in gene regulation and protein secretion systems of HlyA in the nonhemolytic V. cholerae strains should be areas of concern in future studies.

Structural and Serological Studies of the O6-Related Antigen of Aeromonas veronii bv. sobria Strain K557 Isolated from Cyprinus carpio on a Polish Fish Farm, which Contains l-perosamine (4-amino-4,6-dideoxy-l-mannose), a Unique Sugar Characteristic for Aeromonas Serogroup O6

Amongst Aeromonas spp. strains that are pathogenic to fish in Polish aquacultures, serogroup O6 was one of the five most commonly identified immunotypes especially among carp isolates. Here, we report immunochemical studies of the lipopolysaccharide (LPS) including the O-specific polysaccharide (O-antigen) of A. veronii bv. sobria strain K557, serogroup O6, isolated from a common carp during an outbreak of motile aeromonad septicemia (MAS) on a Polish fish farm. The O-polysaccharide was obtained by mild acid degradation of the LPS and studied by chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc). The following structures of the O-polysaccharides (O-PS 1 and O-PS 2) were established: O-PS 1: →2)-α-l-Rhap4NAc-(1→; O-PS 2: →2)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→. Western blotting and an enzyme-linked immunosorbent assay (ELISA) showed that the cross-reactivity between the LPS of A. veronii bv. sobria K557 and the A. hydrophila JCM 3968 O6 antiserum, and vice versa, is caused by the occurrence of common α-l-Rhap4NAc-(1→2)-α-l-Rhap4NAc and α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc disaccharides, whereas an additional →4)-α-d-GalpNAc-associated epitope defines the specificity of the O6 reference antiserum. Investigations of the serological and structural similarities and differences in the O-antigens provide knowledge of the immunospecificity of Aeromonas bacteria and are relevant in epidemiological studies and for the elucidation of the routes of transmission and relationships with pathogenicity.

Identification of the Mechanisms of Acetoin Production Change in Genetically Altered Vibrio cholerae Strains O1 Biovar El Tor

Objective of the study was to determine the mechanisms of acetoin biosynthesis change in genetically altered El Tor V. cholerae strains in Voges-Proskauer test.Materials and methods. We used nine genetically altered V. cholerae O1 strains, biovar El Tor, imported in the territory of the Russian Federation and Ukraine between 1993–2011, and four typical strains isolated in 1970–1972. When assessing acetoin production in V. cholerae strains in Voges-Proskauer test, the strain V. cholerae 569B O1 serogroup, classical biovar served as negative control of the assay. Relative gene expression was studied using real-time RT-PCR. Protein model construction was performed by means of automated server SWISS – MODEL.Results and discussion. It has been demonstrated that diagnostically significant feature – VogesProskauer reaction, utilized for V. cholerae O1 biovar differentiation, is changed in all investigated genetically altered strains of cholera agent, isolated in different periods of the current seventh pandemic (66.7 % of the strains show weakly positive reaction, 33.3 % – negative one). The data obtained testify to the reduction or absence of acetoin production in the investigated strains. Analysis of four structural genes of als operon, as well as expression of regulatory genes alsR and аphA, responsible for acetoin biosynthesis, has revealed that changes in acetoin production in the genovariants stem from the deletion of a single nucleotide (T in the position 315) in the structural gene alsD, encoding acetolaktat decarboxylase, and also from high levels of negative acetoin biosynthesis regulator expression – аphA gene. Modeling of the spatial (3-D) structure of AlsD protein in the genovariant M1293 and the reference-strain N16961 has shown that AlsD protein of the genovariant is, indeed, considerably reduced. However, spontaneous decarboxylation is possible in the absence of acetolaktat decarboxylase, which phenotypically manifests itself in borderline positive Voges-Proskauer test.

Utilization of Small RNA Genes to Distinguish Vibrio cholerae Biotypes via Multiplex Polymerase Chain Reaction.

The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiate even the closely related biotypes of V. cholerae. Oligonucleotides for selective amplification of small RNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, as well as the differentiation of V. cholerae-O1 classical, O1 El Tor, and O139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.

Convergent Synthesis of Oligosaccharides Structurally Related to Galactan I and Galactan II of Klebsiella Pneumoniae and their Use in Screening of Antibody Specificity

Klebsiella pneumoniae Is a Gram‐negative pathogenic bacterium that emerges a challenge for modern medicine due to intensively rising multi‐drug resistance. Specific LPS O‐chains represent promising targets for development of vaccines for immunoprophylaxis or immunotherapy of K. pneumoniae‐caused diseases. Herein, the synthesis of di‐, tetra‐, and hexasaccharides which represent structural fragments of both galactan I and galactan II of K. pneumoniae LPS O‐chains is described. The galactofuranoside building block used for assembling the galactan I chains was prepared by pyranoside‐into‐furanoside (PIF) rearrangement of the corresponding galactopyranoside precursor bearing an acid‐labile temporary p‐methoxybenzyl protecting group at O(3). The synthesized set of K. pneumoniae antigenic oligosaccharides in the form of their biotinylated derivatives was used to create a glycoarray which was applied for the screening of antibodies to galactan I and galactan II in anti‐K. pneumoniae sera. The obtained results demonstrate the applicability of such a glycoarray‐based assay for typing antibodies to Klebsiella strains belonging to most frequent O1 serogroup.

Expanding dynamics of the virulence-related gene variations in the toxigenic Vibrio cholerae serogroup O1

BackgroundToxigenic Vibrio cholerae serogroup O1 is the causative pathogen in the sixth and seventh cholera pandemics. Cholera toxin is the major virulent factor but other virulence and virulence-related factors play certain roles in the pathogenesis and survival in the host. Along with the evolution of the epidemic strains, the virulence-related genes also experience variation, gain and loss, and lead to genetic divergence in different strains.ResultsIn this study, we analyzed the virulence-related gene profiles in the toxigenic serogroup O1 strains isolated from 1923 to 2015, the genomes of which were publicly available. The virulence-related genes of the V. cholerae O1 strains were annotated based on the Virulence Factors Database (VFDB). An average of 230.1 virulence-related genes per strain were identified; significant differences in the average numbers were found between the classical and El Tor biotypes, and increasing trends in the number of virulence-related genes along with the isolation years were observed in the El Tor biotype strains. A total of 176 homologs of virulence-related genes were found from these strains, of which 25 belonged to the core genes, suggesting their conservative and necessary roles in V. cholerae pathogenesis. We described the diversities of the homologs by defining gene sequence type, and illustrated its association with gene duplication; we found that gene duplication clearly increased the complexity of the gene sequence types in the core virulence-related genes. In addition, we provided virulence-related gene profiles whose genetic characteristic depend on the isolation years from the view of gene gain and loss, variation, gene duplication and gene sequence type number.ConclusionsOur study reveals the comprehensive variation dynamics of the virulence-related genes in toxigenic V. cholerae serogroup O1 during epidemics. The increasing trend for the virulence-related genes may suggest the evolutional advantage of strains by gaining virulence-related genes with diverse functional categories.

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6.

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1→3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→O-PS2→2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

Environmental and Clinical Strains of Vibrio cholerae Non-O1, Non-O139 From Germany Possess Similar Virulence Gene Profiles.

Vibrio cholerae is a natural inhabitant of aquatic ecosystems globally. Strains of the serogroups O1 and O139 cause the epidemic diarrheal disease cholera. In Northern European waters, V. cholerae bacteria belonging to other serogroups (designated non-O1, non-O139) are present, of which some strains have been associated with gastrointestinal infections or extraintestinal infections, like wound infections or otitis. For this study, environmental strains from the German coastal waters of the North Sea and the Baltic Sea were selected (100 strains) and compared to clinical strains (10 isolates) that were from patients who contracted the infections in the same geographical region. The strains were characterized by MLST and examined by PCR for the presence of virulence genes encoding the cholera toxin, the toxin-coregulated pilus (TCP), and other virulence-associated accessory factors. The latter group comprised hemolysins, RTX toxins, cholix toxin, pandemic islands, and type III secretion system (TTSS). Phenotypic assays for hemolytic activity against human and sheep erythrocytes were also performed. The results of the MLST analysis revealed a considerable heterogeneity of sequence types (in total 74 STs). The presence of virulence genes was also variable and 30 profiles were obtained by PCR. One profile was found in 38 environmental strains and six clinical strains. Whole genome sequencing (WGS) was performed on 15 environmental and 7 clinical strains that were ST locus variants in one, two, or three alleles. Comparison of WGS results revealed that a set of virulence genes found in some clinical strains is also present in most environmental strains irrespective of the ST. In few strains, more virulence factors are acquired through horizontal gene transfer (i.e., TTSS, genomic islands). A distinction between clinical and environmental strains based on virulence gene profiles is not possible for our strains. Probably, many virulence traits of V. cholerae evolved in response to biotic and abiotic pressure and serve adaptation purposes in the natural aquatic environment, but provide a prerequisite for infection of susceptible human hosts. These findings indicate the need for surveillance of Vibrio spp. in Germany, as due to global warming abundance of Vibrio will rise and infections are predicted to increase.

High quality reference genomes for toxigenic and non-toxigenic Vibrio cholerae serogroup O139

Toxigenic Vibrio cholerae of the O139 serogroup have been responsible for several large cholera epidemics in South Asia, and continue to be of clinical and historical significance today. This serogroup was initially feared to represent a new, emerging V. cholerae clone that would lead to an eighth cholera pandemic. However, these concerns were ultimately unfounded. The majority of clinically relevant V. cholerae O139 isolates are closely related to serogroup O1, biotype El Tor V. cholerae, and comprise a single sublineage of the seventh pandemic El Tor lineage. Although related, these V. cholerae serogroups differ in several fundamental ways, in terms of their O-antigen, capsulation phenotype, and the genomic islands found on their chromosomes. Here, we present four complete, high-quality genomes for V. cholerae O139, obtained using long-read sequencing. Three of these sequences are from toxigenic V. cholerae, and one is from a bacterium which, although classified serologically as V. cholerae O139, lacks the CTXφ bacteriophage and the ability to produce cholera toxin. We highlight fundamental genomic differences between these isolates, the V. cholerae O1 reference strain N16961, and the prototypical O139 strain MO10. These sequences are an important resource for the scientific community, and will improve greatly our ability to perform genomic analyses of non-O1 V. cholerae in the future. These genomes also offer new insights into the biology of a V. cholerae serogroup that, from a genomic perspective, is poorly understood.

ESTIMATION OF ANTIBACTERIAL EFFECT ON VIBRIO CHOLERAE (ON THE EXAMPLE OF THE SUBSTANCE ACETYL-N- CYSTEINE–L)

We studied the effect of the substance acetyl-N-cysteine-L on cholera Vibrio. Its minimum inhibitory concentration for the strains of Vibrio cholerae El Tor O1 and O139 serogroup was 1–2.5 mg/ml in solid nutrient media - Martin's agar (pH 7.6) and LB (pH 7.2), while in Bhascaran synthetic medium, using glucose (0.1per cent) as the sole carbon source, N-acetyl-β-D-glucosamine (0.05 per cent) or colloidal chitin (0.027 per cent) it was reduced to 50–250 μg/ml. The ability of the substance acetyl-N-cysteine-L substance to suppress the activity of the purified enzyme N-acetyl-β-D-glucosaminidase (chitobiasis) was found. Antibacterial effect detected of the substance acetyl-N-cysteine-L against the strains of Vibrio cholerae El Tor O1 and O139 serogroups with different epidemic significance (presence / absence of ctxAB and tcpA genes) indicates the advisability of considering the issue on the possibility of including this substance in composition of solution components used in the rehydration therapy of diarrheal diseases cases.

Prevalence, Antibiotic Susceptibility, and Molecular Characterization of Cronobacter spp. Isolated From Edible Mushrooms in China.

Cronobacter spp. are foodborne pathogens that can infect and cause life-threatening diseases in all age groups, particularly in infants and immunocompromised elderly. This study aimed to investigate the prevalence, antibiotic susceptibility, and molecular characteristics of Cronobacter spp. isolates in edible mushrooms collected from 44 cities in China. In total, 668 edible mushrooms were collected from traditional retail markets and supermarkets and were analyzed by quantitative methods, PCR-based serotyping, multilocus sequence typing (MLST), and antibiotic susceptibility testing. Among the 668 samples tested, 89 (13.32%) were positive for Cronobacter spp., and the contamination levels exceeded the 110 most probable number (MPN)/g in 13.48% (12/89) of the samples. Flammulina velutipes samples had the highest contamination rate of 17.54% (37/211), whereas Hypsizygus marmoreus samples had the lowest contamination rate of 3.28% (2/61). Ten serotypes were identified among 115 isolates, of which the C. sakazakii serogroup O1 (n = 32) was the primary serotype. MLST indicated that there was quite high genetic diversity in Cronobacter spp. and 72 sequence types were identified, 17 of which were new. Notably, C. sakazakii ST148 (n = 10) was the most prevalent, followed by C. malonaticus ST7 (n = 5). Antibiotic susceptibility testing revealed that the majority of Cronobacter spp. strains were susceptible to the 16 antibiotics tested. However, a portion of isolates exhibited relatively high resistance to cephalothin, with resistance and intermediate rates of 93.91 and 6.09%, respectively. One isolate (cro300A) was multidrug-resistant, with resistance to five antibiotics. Overall, this large-scale study revealed the relatively high prevalence and high genetic diversity of Cronobacter spp. on edible mushrooms in China, indicating a potential public health concern. To our knowledge, this is the first large-scale and systematic study on the prevalence of Cronobacter spp. on edible mushrooms in China, and the findings can provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp on edible mushrooms during production processes.

Survey and genetic characterization of Vibrio cholerae in Apalachicola Bay, Florida (2012-2014).

A small outbreak of gastroenteritis in 2011 in Apalachicola Bay, FL was attributed to consumption of raw oysters carrying Vibrio cholerae serotype O75. To better understand possible health risks, V. cholerae was surveyed in oysters, fish and seawater, and results were compared to data for Vibrio vulnificus and Vibrioparahaemolyticus. Enrichment protocols were used to compare prevalence of V. cholerae (0, 48, 50%), V. vulnificus (89, 97, 100%) and V. parahaemolyticus (83, 83, 100%) in fish, seawater and oysters respectively. Compared to other species, Most probable number results indicated significantly (P<0·001) lower abundance of V. cholerae, which was also detected more frequently at lower salinity, near-shore sites; other species were more widely distributed throughout the bay. Genes for expression (ctxA, ctxB) and acquisition (tcpA) of cholera toxin were absent in all strains by PCR, which was confirmed by whole genome sequencing; however, other putative virulence genes (toxR, rtxA, hlyA, opmU) were common. Multi-locus sequence typing revealed 78% of isolates were genetically closer to V.cholerae O75 lineage or other non-O1 serogroups than to O1 or O139 serogroups. Resistance to amoxicillin, kanamycin, streptomycin, amikacin, tetracycline and cephalothin, as well as multidrug resistance, was noted. Results indicated minimal human health risk posed by V. cholerae, as all isolates recovered from Apalachicola Bay did not have the genetic capacity to produce cholera toxin. Vibrio cholerae was less prevalent and abundant relative to other pathogenic Vibrio species. These studies provide important baseline observations for V.cholerae virulence potential regarding: (i) genetic relatedness to V. cholerae O75, (ii) antibiotic resistance and (iii) prevalence of multiple virulence genes. These data will serve as a biomonitoring tool to better understand ecosystem status and management if bacterial densities and virulence potential are altered by environmental and climatic changes over time.

The global burden of cholera outbreaks in Niger: an analysis of the national surveillance data, 2003-2015.

Niger has experienced multiple cholera outbreaks since the occurrence of cholera in West Africa in 1970-1971. Understanding the origins of these geographic variations at different scales in the region and in health districts is an essential step in the rational and operational fight against this disease. We conducted a retrospective review of all suspected cholera cases in Niger from 2003 to 2015. Data from 2010 to 2015 were reviewed to determine the number and percentage of stool specimens tested, with distribution by serotype. We also determined the time between sampling and sending to a laboratory. From 2003 through 2015 a total of 16 328 cases of cholera, including 578 deaths, were reported to the National Surveillance Database (overall case fatality rate 3.5%). The number of cases was greater among males (63.29%). Patients >15 y of age accounted for 69.80% of cases. All regions recorded cholera cases throughout the period 2003 to 2015, except in 2009, when no outbreak was reported. All epidemics recorded were due to the subtype serogroup O1 serotype Ogawa and all strains confirmed the presence of the ctxB, rstR, tcpA, zot, ace and ctxA genes encoding the toxin of this serotype. The global burden of cholera remains high in Niger. To successfully prevent and control outbreaks, adapted guidelines and strategies should be reviewed to provide more focused information and assign clear roles and responsibilities.

A Compendium of Shigatoxigenic &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt;: Origins, Bacteriological and Clinical Data on the Serogroups, Serotypes and Untypeable Strains of &amp;lt;i&amp;gt;E. coli&amp;lt;/i&amp;gt; Reported between 1980 and 2017, Excluding O157:H7

The purpose of this paper was to create a compendium of serogroups, serotypes and untypeable strains of Shigatoxigenic/verotoxigenic (STEC/VTEC) Escherichia coli. The compendium includes the origins, bacteriological and clinical data on this important group of microbes. The collection of STEC/VTEC data extends from 1980 to 2017, and excludes STEC/VTEC O157:H7. A total of 499 papers were discovered and documented to create the compendium which includes all the serogroups and serotypes beginning with serogroup O1 through to serogroup O211 and includes O non-typeable strains and ill-defined strains including serotype O54071:H7. The paper summarises the findings of 8010 E. coli isolates and includes all details including source (human or animal), and clinical condition/disease, serogroup and serotypes, country of origin, year reported, and the author(s) with cited references. Some twenty clinical conditions were shown in association with 4320 serotypes/and untypeable strains. The compendium is a record of an important group of STEC/ VTEC and should provide a useful resource for both researchers and clinicians.

Genome wide characterization of enterotoxigenic Escherichia coli serogroup O6 isolates from multiple outbreaks and sporadic infections from 1975-2016.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea globally, particularly among children under the age of five in developing countries. ETEC O6 is the most common ETEC serogroup, yet the genome wide population structure of isolates of this serogroup is yet to be determined. In this study, we have characterized 40 ETEC O6 isolates collected between 1975–2016 by whole genome sequencing (WGS) and by phenotypic antimicrobial susceptibility testing. To determine the relatedness of isolates, we evaluated two methods—whole genome high-quality single nucleotide polymorphism (whole genome-hqSNP) and core genome SNP analyses using Lyve-SET and Parsnp respectively. All isolates were tested for antimicrobial susceptibility using a panel of 14 antibiotics. ResFinder 2.1 and a custom quinolone resistance determinants workflow were used for resistance determinant detection. VirulenceFinder 1.5 was used for prediction of the virulence genes. Thirty-seven isolates clustered into three major clades (I, II, III) by whole genome-hqSNP and core genome SNP analyses, while three isolates included in the whole genome-hqSNP analysis only did not cluster with clades I-III by both analyses and formed a distantly related outgroup, designated clade IV. Median number of pairwise whole genome-hqSNPs in clonal ETEC O6 outbreaks ranged from 0 to 5. Of the 40 isolates tested for antimicrobial susceptibility, 18 isolates were pansusceptible. Twenty-two isolates were resistant to at least one antibiotic, nine of which were multidrug resistant. Phenotypic antimicrobial resistance (AR) correlated with AR determinants in 22 isolates. Thirty-two isolates harbored both enterotoxin virulence genes while the remaining 8 isolates had only one of the two virulence genes. In summary, whole genome-hqSNP and core genome SNP analyses from this study revealed similar evolutionary relationships and an overall diversity of ETEC O6 isolates independent of time of isolation. Less than 5 pairwise hqSNPs between ETEC O6 isolates is circumstantially indicative of an outbreak cluster. Findings from this study will be a basis for quicker outbreak detection and control by efficient subtyping by WGS.

Development of a multiplex PCR assay for the detection of major virulence genes in Vibrio cholerae including non-O1 and non-O139 serogroups

Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However, non-CT producing V. cholerae from non-O1/non-O139 serogroups have been increasingly isolated from diarrheal stools and extra-intestinal infections. In this study, we have developed a multiplex PCR for the simultaneous detection of heat-stable enterotoxin (stn), type three-secretion system (vopF), and cholix toxin (chxA), along with CT (ctx) in V. cholerae strains. As other species from genus Vibrio carries homologous virulence genes, V. cholerae specific ompW was also included to differentiate V. cholerae from other vibrios. This assay was 100% specific and sensitive, and could detect homologous virulence genes like ctxA in V. mimicus and vopF in V. parahaemolyticus. This multiplex PCR assay, which can detect four major virulence genes in V. cholerae, is novel and important for epidemiologic and environmental surveillance of pathogenic V. cholerae.

Prevalence of foodborne pathogens isolated from retail freshwater fish and shellfish in China

In 2016, the nationwide food safety surveillance was conducted on fresh water fish and shellfish in China. The prevalence of Vibrio parahaemolyticus, Vibrio cholerae, Salmonella, and L. monocytogenes were 19.0% (613/3226), 3.4% (110/3226), 3.8% (124/3226), and 2.5% (80/3226), respectively. The tdh and trh genes were detected in 19 (2.6%) and 24 (3.3%) of the 718 V. parahaemolyticus isolates, respectively. All V. cholerae strains were confirmed as non-O1/O139 serogroups. The most predominant Salmonella serotypes were S. Stanley, S. Thompson, S. Litchfield, S. Typhimurium, S. Virchow, S. Carrau, and S. Infantis. The results of this comprehensive survey showed contamination by four pathogens in fresh water fish and shellfish in China for the first time. The study indicated a need for proper handling, storage, and cooking of freshwater fish and shellfish. Further risk reduction assessment and implementation are necessary to reduce foodborne disease in China.

Antimicrobial resistance of Vibrio cholerae from sub-Saharan Africa: A systematic review.

BackgroundThe World Health Assembly adopted the Global Action Plan on Antimicrobial Resistance, which includes improving the knowledge base through surveillance and research. Noteworthily, the World Health Organization has advocated a Global Antimicrobial Resistance Surveillance System to address the plan’s surveillance objective, with most African countries enrolling in or after 2017.AimThe aim of this article was to review prior data on antimicrobial resistance of Vibrio cholerae from sub-Saharan Africa with a view for future control and intervention strategies.MethodsWe used the Preferred Reporting Items for Systematic Review and Meta-Analysis (or ‘PRISMA’) guidelines to search the PubMed and African Journals Online databases, as well as additional articles provided by the Nigeria Centre for Disease Control, for articles reporting on the antibiotic susceptibility of V. cholerae between January 2000 and December 2017.ResultsWe identified 340 publications, of which only 25 (reporting from 16 countries within the sub-Saharan African region) were eligible. The majority (20; 80.0%) of the cholera toxigenic V. cholerae isolates were of the serogroup O1 of the El Tor biotype with Ogawa and Inaba serotypes predominating. Resistance was predominantly documented to trimethoprim-sulphamethoxazole (50% of the studies), ampicillin (43.3% of the studies), chloramphenicol (43.3% of the studies) and streptomycin (30% of the studies). Resistance mechanisms were reported in 40% of the studies.ConclusionOur results demonstrate a documented antimicrobial resistance of V. cholerae to multiple antibiotic classes, including cell wall active agents and antimetabolites with evidence of phenotypic/genotypic resistance to fluoroquinolones.