O-antigen Gene Research Articles

79 Microbiological effects of control measures during pork production

Abstract The objective of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and efficacy of control measures at a pork processing facility. Outbreaks of E. coli O157:H7 associated with pork, though not common globally, have been reported multiple times in recent years in Alberta. Sampling at a commercial facility processing hogs was carried out at monthly intervals for 11 mo. At the facility, the dressed carcasses are sprayed with 5% lactic acid (LA) and immediately air-chilled after the LA spray. At each sampling time, 14 different types of samples were collected: dirt from the holding pen floor (n = 2), gloves of workers (n = 5), scalding water (n = 1), tonsils (n = 5), cecal material (n = 5), five swabs each from hides of animals after bleeding (HAB), and carcasses after scalding (CAS), before evisceration (CBE), after evisceration (CAE), before LA spray (BLA), after LA spray (ALA), 1h after LA spray (ALA1), 2h after LA spray (ALA2), and after chilling (ACH). All samples were enumerated for total plate count (TPC), Enterobacteriaceae, and E. coli using specific media for each. In addition, an aliquot of each sample was enriched and tested for the presence of the O-antigen gene (rfbE) for E. coli O157 and Shiga toxin-producing gene (stx1/stx2) using real-time PCR. Cultures positive for both rfbE and stx were further plated for the isolation of E. coli O157:H7. Of the 683 enrichment cultures, 12 were positive for both marker genes, from cecal material (n = 2), dirt (n = 4) and HAB (n = 6). However, none of these samples yielded STEC O157 isolates. Of these enrichment cultures, 69, 45 and 32 were positive for stx1, stx2, and both stx genes, respectively. Positive samples were mostly associated with HAB, cecal material and dirt. HAB was most and least contaminated in August/June and December, differing by 1 log unit for TPC (P < 0.05). Enterobacteriaceae and E. coli on HAB were also affected by month of sampling (P < 0.05), but this temporal trend differed from TPC (Table). The numbers for TPC, Enterobacteriaceae, and E. coli all decreased (P < 0.05) as the stage of carcass processing progressed. The overall reduction of TPC and E. coli was 7 and 5 log units, respectively. Scalding, LA spray and air chilling all reduced (P < 0.05) the numbers of indicator bacteria on carcasses. Of the 55 samples each collected from BLA, ALA, ALA1, ALA2, and ACH, 36, 13, 2, 1 and 0 were positive for E. coli, respectively. In conclusion, pork processing environments can be contaminated with STEC from hides, feces and holding pen, but levels of STEC O157 appear to be low. Scalding, LA spray and air chilling can be effective measures to reduce the risk of bacterial contamination of pork.

Functional Identification and Genetic Analysis of O-Antigen Gene Clusters of Food-Borne Pathogen Yersinia enterocolitica O:10 and Other Uncommon Serotypes, Further Revealing Their Virulence Profiles.

Yersinia enterocolitica is a globally distributed food-borne gastrointestinal pathogen. The O-antigen variation-determined serotype is an important characteristic of Y. enterocolitica, allowing intraspecies classification for diagnosis and epidemiology purposes. Among the 11 serotypes associated with human yersiniosis, O:3, O:5,27, O:8, and O:9 are the most prevalent, and their O-antigen gene clusters have been well defined. In addition to the O-antigen, several virulence factors are involved in infection and pathogenesis of Y. enterocolitica strains, and these are closely related to their biotypes, reflecting pathogenic properties. In this study, we identified the O-AGC of a Y. enterocolitica strain WL-21 of serotype O:10, and confirmed its functionality in O-antigen synthesis. Furthermore, we analyzed in silico the putative O-AGCs of uncommon serotypes, and found that the O-AGCs of Y. enterocolitica were divided into two genetic patterns: (1) O-AGC within the hemH-gsk locus, possibly synthesizing the O-antigen via the Wzx/Wzy dependent pathway, and (2) O-AGC within the dcuC-galU-galF locus, very likely assembling the O-antigen via the ABC transporter dependent pathway. By screening the virulence genes against genomes from GenBank, we discovered that strains representing different serotypes were grouped according to different virulence gene profiles, indicating strong links between serotypes and virulence markers and implying an interaction between them and the synergistic effect in pathogenicity. Our study provides a framework for further research on the origin and evolution of O-AGCs from Y. enterocolitica, as well as on differences in virulent mechanisms among distinct serotypes.

Genome-wide association study reveals serovar-associated genetic loci in Riemerella anatipestifer

BackgroundThe disease caused by Riemerella anatipestifer (R. anatipestifer, RA) results in large economic losses to the global duck industry every year. Serovar-related genomic variation, such as the O-antigen and capsular polysaccharide (CPS) gene clusters, has been widely used for serotyping in many gram-negative bacteria. RA has been classified into at least 21 serovars based on slide agglutination, but the molecular basis of serotyping is unknown. In this study, we performed a pan-genome-wide association study (Pan-GWAS) to identify the genetic loci associated with RA serovars.ResultsThe results revealed a significant association between the putative CPS synthesis gene locus and the serological phenotype. Further characterization of the CPS gene clusters in 11 representative serovar strains indicated that they were highly diverse and serovar-specific. The CPS gene cluster contained the key genes wzx and wzy, which are involved in the Wzx/Wzy-dependent pathway of CPS synthesis. Similar CPS loci have been found in some other species within the family Weeksellaceae. We have also shown that deletion of the wzy gene in RA results in capsular defects and cross-agglutination.ConclusionsThis study indicates that the CPS synthesis gene cluster of R. anatipestifer is a serotype-specific genetic locus. Importantly, our finding provides a new perspective for the systematic analysis of the genetic basis of the R anatipestifer serovars and a potential target for establishing a complete molecular serotyping scheme.

Global emergence of a hypervirulent carbapenem-resistant Escherichia coli ST410 clone

Carbapenem-resistant Escherichia coli (CREC) ST410 has recently emerged as a major global health problem. Here, we report a shift in CREC prevalence in Chinese hospitals between 2017 and 2021 with ST410 becoming the most commonly isolated sequence type. Genomic analysis identifies a hypervirulent CREC ST410 clone, B5/H24RxC, which caused two separate outbreaks in a children’s hospital. It may have emerged from the previously characterised B4/H24RxC in 2006 and has been isolated in ten other countries from 2015 to 2021. Compared with B4/H24RxC, B5/H24RxC lacks the blaOXA-181-bearing X3 plasmid, but carries a F-type plasmid containing blaNDM-5. Most of B5/H24RxC also carry a high pathogenicity island and a novel O-antigen gene cluster. We find that B5/H24RxC grew faster in vitro and is more virulent in vivo. The identification of this newly emerged but already globally disseminated hypervirulent CREC clone, highlights the ongoing evolution of ST410 towards increased resistance and virulence.

Phage resistance mutations affecting the bacterial cell surface increase susceptibility to fungi in a model cheese community.

Diverse populations of bacteriophages infect and coevolve with their bacterial hosts. Although host recognition and infection occur within microbiomes, the molecular mechanisms underlying host-phage interactions within a community context remain poorly studied. The biofilms (rinds) of aged cheeses contain taxonomically diverse microbial communities that follow reproducible growth patterns and can be manipulated under laboratory conditions. In this study, we use cheese as a model for studying phage-microbe interactions by identifying and characterizing a tractable host-phage pair co-occurring within a model Brie-like community. We isolated a novel bacteriophage, TS33, that kills Hafnia sp. JB232, a member of the model community. TS33 is easily propagated in the lab and naturally co-occurs in the cheese community, rendering it a prime candidate for the study of host-phage interactions. We performed growth assays of the Hafnia, TS33, and the fungal community members, Geotrichum candidum and Penicillium camemberti. Employing Random Barcode Transposon Sequencing experiments, we identified candidate host factors that contribute to TS33 infectivity, many of which are homologs of bacterial O-antigen genes. Hafnia mutants in these genes exhibit decreased susceptibility to phage infection, but experience negative fitness effects in the presence of the fungi. Therefore, mutations in O-antigen biosynthesis homologs may have antagonistic pleiotropic effects in Hafnia that have major consequences for its interactions with the rest of the community. Ongoing and future studies aim to unearth the molecular mechanisms by which the O-antigen of Hafnia mediates its interactions with its viral and fungal partners.

Structure elucidation and gene cluster of the O-antigen of Shewanella xiamenensis strain DCB-2-1 containing an amide of d-glucuronic acid with d-alanine and its bonding with U, Cr and V

The aim of this work was to examine the structure and gene cluster of O-OPS of S. xiamenensis strain DCB-2-1 and survey its conceivability for chelating uranyl, chromate and vanadate ions from solution. O-polysaccharide (OPS, O-antigen) was isolated from the lipopolysaccharide of Shewanella xiamenensis DCB-2-1 and studied by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and sugar analysis. The following structure of the brunched pentasaccharide was established: [Display omitted] where d-β-GlcpA(d-Ala) is d-glucuronic acid acylated with NH group of d-Ala. The OPS structure established is unique among known bacterial polysaccharide structures. Interestingly, that dN-(d-glucuronoyl)-d-alanine derivative is not found in bacterial polysaccharides early.The O-antigen gene cluster of Shewanella xiamenensis strain DCB-2-1 has been sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the OPS structure. Based on the analysis of the IR spectra of the isolated polysaccharide DCB-2-1 and the products of its interaction with UO2(NO3)2 ∗ 6H2O, NH4VO3 and K2Cr2O7, a method of binding them can be proposed. Laboratory experiments show that the use of polysaccharide can be effective in removing uranyl, chromate and vanadate from solution.

Structural and Genetic Identification of the O-Antigen from an Escherichia coli Isolate, SD2019180, Representing a Novel Serogroup.

The O-antigen is one of the outermost surface components of Gram-negative bacteria. Its large structural variation provides the molecular basis for bacterial serological diversity. Here, we established the structure of the O-antigen from an Escherichia coli strain, SD2019180, which appeared to be completely different from the known E. coli serogroups. The O-antigen tetrasaccharide biological repeating unit was identified as → 2)-[β-d-GlcpA-(1 → 4)]-[α-d-Galp-(1 → 3)]-α-l-Fucp-(1 → 3)-α-d-GlcpNAc-(1 →. Furthermore, we analyzed the O-antigen gene cluster of SD2019180 and confirmed its role in O-antigen synthesis by using deletion and complementation experiments. Our findings indicate that SD2019180 is a novel serogroup of Escherichia coli.

Immunochemical studies and gene cluster relationships of closely related O-antigens of Aeromonas hydrophila Pt679, Aeromonas popoffii A4, and Aeromonas sobria K928 strains classified into the PGO1 serogroup dominant in Polish aquaculture of carp and rainbow trout

The present study included three Aeromonas sp. strains isolated from fish tissues during Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreaks on commercial farms, i.e.: Aeromonas hydrophila Pt679 obtained from rainbow trout as well as Aeromonas popoffii A4 (formerly Aeromonas encheleia) and Aeromonas sobria K928 both isolated from carp, which were classified into the new provisional PGO1 serogroup prevailing among aeromonads in Polish aquaculture. The structure of the O-specific polysaccharides of A4 and K928 has been previously established. Here, immunochemical studies of the O-specific polysaccharide of A. hydrophila Pt679 were undertaken. The O-specific polysaccharide was obtained from the lipopolysaccharide of A. hydrophila Pt679 after mild acid hydrolysis and separation by gel-permeation chromatography. The high-molecular-mass fraction was studied using chemical methods and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The following structure of the branched repeating unit of the O-polysaccharide from A. hydrophila Pt679 was determined:→3)[α-d-Fucp3NAc-(1→2)]-α-l-Rhap-(1→3)-β-l-Rhap-(1→4)-α-l-Rhap-(1→3)-β-d-QuipNAc-(1→2)┘The studies indicated that O-polysaccharides from A. hydrophila Pt679, A. popoffii A4 and A. sobria K928 share similarities but they also contain unique characteristics. Western blotting and an enzyme-linked immunosorbent assay revealed that the cross-reactivity of the related O-antigens is caused by the occurrence of common structural elements, whereas additional epitopes define the specificity of the O-serotypes.For genetic relationship studies, the O-antigen gene cluster was characterized in the genome of the A. hydrophila Pt679 strain and compared with the corresponding sequences of A. popoffii A4 and A. sobria K928 and with sequences available in the databases. The composition of the regions was found to be consistent with the O-antigen structures of Aeromonas strains classified into the same PGO1 serogroup.

Novel β1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides.

The efficiency of de novo synthesis of hyaluronic acid (HA) using Pasteurella multocida hyaluronate synthase (PmHAS) is limited by its low catalytic activity during the initial reaction steps when monosaccharides are the acceptor substrates. In this study, we identified and characterized a β-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant β1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-β-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, β1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (~ 12-fold) with GlcA-pNP as the acceptor, making it a better option for the initial step of de novo HA oligosaccharide synthesis. We then developed a biocatalytic approach for size-controlled HA oligosaccharide synthesis using the disaccharide produced by β1,4 EcGnT as a starting material, followed by stepwise PmHAS-catalyzed synthesis of longer oligosaccharides. Using this approach, we produced a series of HA chains of up to 10 sugar monomers. Overall, our study identifies a novel bacterial β1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides. KEY POINTS: • A novel β-1,4-N-acetylglucosaminyl-transferase (EcGnT) from E. coli O8:K48:H9. • EcGnT is superior to PmHAS for enabling de novo HA oligosaccharide synthesis. • Size-controlled HA oligosaccharide synthesis relay using EcGnT and PmHAS.

O-Antigen Diversification Masks Identification of Highly Pathogenic Shiga Toxin-Producing Escherichia coli O104:H4-Like Strains.

Shiga toxin-producing Escherichia coli (STEC) can give rise to a range of clinical outcomes from diarrhea to the life-threatening systemic condition hemolytic-uremic syndrome (HUS). Although STEC O157:H7 is the serotype most frequently associated with HUS, a major outbreak of HUS occurred in 2011 in Germany and was caused by a rare serotype, STEC O104:H4. Prior to 2011 and since the outbreak, STEC O104:H4 strains have only rarely been associated with human infections. From 2012 to 2020, intensified STEC surveillance was performed in Germany where the subtyping of ~8,000 clinical isolates by molecular methods, including whole-genome sequencing, was carried out. A rare STEC serotype, O181:H4, associated with HUS was identified, and like the STEC O104:H4 outbreak strain, this strain belongs to sequence type 678 (ST678). Genomic and virulence comparisons revealed that the two strains are phylogenetically related and differ principally in the gene cluster encoding their respective lipopolysaccharide O-antigens but exhibit similar virulence phenotypes. In addition, five other serotypes belonging to ST678 from human clinical infection, such as OX13:H4, O127:H4, OgN-RKI9:H4, O131:H4, and O69:H4, were identified from diverse locations worldwide. IMPORTANCE Our data suggest that the high-virulence ensemble of the STEC O104:H4 outbreak strain remains a global threat because genomically similar strains cause disease worldwide but that the horizontal acquisition of O-antigen gene clusters has diversified the O-antigens of strains belonging to ST678. Thus, the identification of these highly pathogenic strains is masked by diverse and rare O-antigens, thereby confounding the interpretation of their potential risk.

Structure and gene cluster annotation of the O-antigen of Aeromonas sobria strain K928 isolated from common carp and classified into the new Aeromonas PGO1 serogroup

Aeromonas sobria strain K928 was isolated from a common carp during a Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreak on a Polish fish farm and classified into the new provisional PGO1 serogroup. The lipopolysaccharide of A. sobria K928 was subjected to mild acid hydrolysis, and the O-specific polysaccharide, which was isolated by gel-permeation chromatography, was studied using sugar and methylation analyses and 1H and 13C NMR spectroscopy. The following structure of the branched O-specific polysaccharide repeating unit of A. sobria K928 was established.→2)[α-D-Fucp3NRHb-(1→3)]-α-L-Rhap-(1→3)-β-L-Rhap-(1→4)-α-L-Rhap-(1→3)-β-D-FucpNAc-(1→The O-antigen gene cluster was identified and characterized in the genome of the A. sobria K928 strain after comparison with sequences in the available databases. The composition of the O-antigen genetic region was found to be consistent with the O-polysaccharide structure, and its organization was proposed.

Unique or not unique? Comparative genetic analysis of bacterial O-antigens from the Oxalobacteraceae family.

Many plants and animals have symbiotic relationships with microorganisms, including bacteria. The interactions between bacteria and their hosts result in different outcomes for the host organism. The outcome can be neutral, harmful or have beneficial effects for participants. Remarkably, these relationships are not static, as they change throughout an organism's lifetime and on an evolutionary scale. One of the structures responsible for relationships in bacteria is O-antigen. Depending on the characteristics of its components, the bacteria can avoid the host's immune response or establish a mutualistic relationship with it. O-antigen is a key component in Gram-negative bacteria's outer membrane. This component facilitates interaction between the bacteria and host immune system or phages. The variability of the physical structure is caused by the genomic variability of genes encoding O-antigen synthesis components. The genes and pathways of O-polysaccharide (OPS) synthesis were intensively investigated mostly for Enterobacteriaceae species. Considering high genetic and molecular diversity of this structure even between strains, these findings may not have caught the entire variety possibly presented in non-model species. The current study presents a comparative analysis of genes associated with O-antigen synthesis in bacteria of the Oxalobacteraceae family. In contrast to existing studies based on PCR methods, we use a bioinformatics approach and compare O- antigens at the level of clusters rather than individual genes. We found that the O-antigen genes of these bacteria are represented by several clusters located at a distance from each other. The greatest similarity of the clusters is observed within individual bacterial genera, which is explained by the high variability of O-antigens. The study describes similarities of OPS genes inherent to the family as a whole and also considers individual unique cases of O-antigen genetic variability inherent to individual bacteria.

An ST131 clade and a phylogroup A clade bearing an O101-like O-antigen cluster predominate among bloodstream Escherichia coli isolates from South-West Nigeria hospitals.

Escherichia coli bloodstream infections are typically attributed to a limited number of lineages that carry virulence factors associated with invasiveness. In Nigeria, the identity of circulating clones is largely unknown and surveillance of their antimicrobial resistance has been limited. We verified and whole-genome sequenced 68 2016-2018 bloodstream E. coli isolates from three sentinel sites in South-Western Nigeria and susceptibility tested 67 of them. Resistance to antimicrobials commonly used in Nigeria was high, with 67 (100 %), 62 (92.5 %), 53 (79.1 %) and 37 (55.2 %) showing resistance to trimethoprim, ampicillin, ciprofloxacin and aminoglycosides, respectively. Thirty-five (51 %) isolates carried extended-spectrum β-lactamase genes and 32 (91 %) of these were multidrug resistant. All the isolates were susceptible to carbapenems and colistin. The strain set included globally disseminated high-risk clones from sequence type (ST)12 (2), ST131 (12) and ST648 (4). Twenty-three (33.8 %) of the isolates clustered within two clades. The first of these consisted of ST131 strains, comprising O16:H5 and O25:H4 sub-lineages. The second was an ST10-ST167 complex clade comprising strains carrying O-antigen and capsular genes of likely Klebsiella origin, identical to those of avian pathogenic E. coli Sanji, and serotyped in silico as O89, O101 or ONovel32, depending on the tool used. Four temporally associated ST90 strains from one sentinel were closely related enough to suggest that at least some of them represented a retrospectively detected outbreak cluster. Our data implicate a broad repertoire of E. coli isolates associated with bloodstream infections in South-West Nigeria. Continued genomic surveillance is valuable for tracking clones of importance and for outbreak identification.

Structure elucidation and gene cluster annotation of the O-antigen of Halomonas titanicae TAT1 containing three residues of 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid

A halotolerant hydrocarbon-oxidizing bacterium Halomonas titanicae strain TAT1 was isolated from a petroleum reservoir. The O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of H. titanicae TAT1 and studied by component analyses and 1D and 2D NMR spectroscopy. The following structure of the repeating linear pentasaccharide O-unit, containing only aminosugars, was established: →4)-β-d-GlcpNAc3NAcA-(1 → 4)-β-d-GlcpNAc3NAcA-(1 → 6)-α-d-GlcpNAc-(1 → 4)-β-d-GlcpNAc3NAcA-(1 → 6)-α-d-GlcpNAc-(→, where d-GlcNAc3NAcA indicates 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid. The O-antigen gene cluster was identified in the genome of H. titanicae TAT1 and compared with available database sequences. The genes revealed in the O-antigen gene cluster and the assigned functions of putative proteins were consistent with the established polysaccharide structure.

Genomic characterization of Tenacibaculum maritimum O-antigen gene cluster and development of a multiplex PCR-based serotyping scheme.

Tenacibaculum maritimum is a devastating bacterial pathogen affecting a large variety of marine fish species. It is responsible for significant economic losses in aquaculture farms worldwide. Different typing methods have been proposed to analyse bacterial diversity and population structure. Serological heterogeneity has been observed and up to four different serotypes have been described so far. However, the underlying molecular factors remain unknown. By combining conventional serotyping and genome-wide association study, we identified the genomic loci likely involved in the O-antigen biosynthesis. This finding allowed the development of a robust multiplex PCR-based serotyping scheme able to detect subgroups within each serotype and therefore performs better than conventional serotyping. This scheme was successfully applied to a large number of isolates from worldwide origin and retrieved from a large variety of fish species. No obvious correlations were observed between the mPCR-based serotype and the host species or the geographic origin of the isolates. Strikingly, the distribution of mPCR-based serotypes does not follow the core genome phylogeny. Nevertheless, this simple and cost-effective mPCR-based serotyping method could be useful for different applications such as population structure analysis, disease surveillance, vaccine formulation and efficacy follow-up.

Deficiency of exopolysaccharides and O-antigen makes Halomonas bluephagenesis self-flocculating and amenable to electrotransformation

Halomonas bluephagenesis, a haloalkaliphilic bacterium and native polyhydroxybutyrate (PHB) producer, is a non-traditional bioproduction chassis for the next generation industrial biotechnology (NGIB). A single-sgRNA CRISPR/Cas9 genome editing tool is optimized using dual-sgRNA strategy to delete large DNA genomic fragments (>50 kb) with efficiency of 12.5% for H. bluephagenesis. The non-essential or redundant gene clusters of H. bluephagenesis, including those encoding flagella, exopolysaccharides (EPSs) and O-antigen, are sequentially deleted using this improved genome editing strategy. Totally, ~3% of the genome is reduced with its rapid growth and high PHB-production ability unaffected. The deletion of EPSs and O-antigen gene clusters shows two excellent properties from industrial perspective. Firstly, the EPSs and O-antigen deleted mutant rapidly self-flocculates and precipitates within 20 min without centrifugation. Secondly, DNA transformation into the mutant using electroporation becomes feasible compared to the wild-type H. bluephagenesis. The genome-reduced H. bluephagenesis mutant reduces energy and carbon source requirement to synthesize PHB comparable to its wild type. The H. bluephagenesis chassis with a reduced genome serves as an improved version of a NGIB chassis for productions of polyhydroxyalkanoates (PHA) or other chemicals.

Structure and gene cluster of the O-antigen of Enterobacter cloacae G2559

The O-polysaccharide (OPS) was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G2559 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the branched pentasaccharide repeating unit was established. [Display omitted] The O-antigen gene cluster of Enterobacter cloacae G2559 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the O-antigen structure.

Comparative genomic analysis of hypervirulent Aeromonas hydrophila strains from striped catfish (Pangasianodon hypophthalmus) in Vietnam

Motile Aeromonad septicemia (MAS) outbreaks caused by hypervirulent Aeromonas hydrophila (vAh) strains belonging to sequence type (ST) 251, have been reported and investigated widely in China and United States (US). In this study, for the first time, two genomes of vAh strains isolated from striped catfish in Vietnam have been identified and characterized. These two vAh strains caused catfish mortalities of 51.7–86.7% post immersion challenge with 2 × 108 CFU mL−1 or intraperitoneal injection challenge with 1 × 103 CFU fish−1, while no mortalities were observed in catfish challenged with regular A. hydrophila (rAh) strains (p < 0.05). These challenge trials indicate that vAh virulence is impacted by different degrees of penetration into the fish host and suggest that disease outbreaks in production ponds are affected by other pathogens or factors that contribute to vAh invasiveness. Comparative genomic analyses of vAh and rAh strains from Vietnam, together with reference A. hydrophila strains from China and US, were performed to gain an insight into the pathogenicity of the epidemic A. hydrophila strains collected from various locations worldwide. Besides the myo-inositol utilization and mobile genetic elements identified by previous studies, this work found that attachment and motility mediated by pili /fimbriae are also key factors underlying the hypervirulent nature of vAh strains. The vAh heterogeneity was observed on the core genome phylogenetic tree and the O-antigen gene clusters, where Vietnamese vAh strains showed closer genetic relationship with vAh strains isolated from carp species in China compared to vAh strains isolated from catfish in the US. The persistent occurrence of ST251 strains has been responsible for MAS outbreaks occurring frequently in Vietnamese catfish farms since 2013. This study provides important information about the spread of vAh in farmed catfish in Vietnam, its virulence in fish and the highly clonal nature of vAh strains identified in multiple countries.

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids.

SUMMARYLipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Structural Elucidation and genetic identification of the O-antigen from a novel serogroup of Escherichia coli strain 2017LL031

The O-antigen is an important virulence factor involved in the survival, virulence and invasion of bacteria. The bacterial serological types are highly dependent on these surface-exposed and structurally unique O-antigen structures. In this work, the structure of O-antigen from an Escherichia coli strain 2017LL031 was elucidated as a hexasaccharide repeating unit: →3)-[β-D-Glcp-(1 → 2)]-α-L-Rhap-(1 → 3)-[α-D-Quip-(1 → 3)-α-D-GlcpA-(1 → 2)]-β-L-Fucp-(1 → 3)-β-D-GlcpNAc-(1→, which is completely different from all known E. coli serogroups. The O-antigen gene cluster (O-AGC) of 2017LL031 was also analyzed and correlates well to its O–Ag. Moreover, the O-AGC of 2017LL031 was deleted and its role in O–Ag biosynthesis was confirmed experimentally. Taken together, our results present that a novel E.coli serotype 2017LL031 is identified.