Wzy Gene Research Articles

Molecular and in silico analyses for detection of Shiga toxin-producing Escherichia coli (STEC) and highly pathogenic enterohemorrhagic Escherichia coli (EHEC) using genetic markers located on plasmid, O Island 57 and O Island 71

BackgroundDue to the diversity of Shiga toxin-producing Escherichia coli (STEC) isolates, detecting highly pathogenic strains in foodstuffs is challenging. Currently, reference protocols for STEC rely on the molecular detection of eae and the stx1 and/or stx2 genes, followed by the detection of serogroup-specific wzx or wzy genes related to the top 7 serogroups. However, these screening methods do not distinguish between samples in which a STEC possessing both determinants are present and those containing two or more organisms, each containing one of these genes. This study aimed to evaluate ecf1, Z2098, Z2099, and nleA genes as single markers and their combinations (ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA) as genetic markers to detect potentially pathogenic STEC by the polymerase chain reaction (PCR) in 96 animal samples, as well as in 52 whole genome sequences of human samples via in silico PCR analyses.ResultsIn animal isolates, Z2098 and Z2098/Z2099 showed a strong association with the detected top 7 isolates, with 100% and 69.2% of them testing positive, respectively. In human isolates, Z2099 was detected in 95% of the top 7 HUS isolates, while Z2098/Z2099 and ecf1/Z2099 were detected in 87.5% of the top 7 HUS isolates.ConclusionsOverall, using a single gene marker, Z2098, Z2099, and ecf1 are sensitive targets for screening the top 7 STEC isolates, and the combination of Z2098/Z2099 offers a more targeted initial screening method to detect the top 7 STEC isolates. Detecting non-top 7 STEC in both animal and human samples proved challenging due to inconsistent characteristics associated with the genetic markers studied.

Prevalence of STEC virulence markers and Salmonella as a function of abiotic factors in agricultural water in the southeastern United States.

Fresh produce can be contaminated by enteric pathogens throughout crop production, including through contact with contaminated agricultural water. The most common outbreaks and recalls in fresh produce are due to contamination by Salmonella enterica and Shiga toxin-producing E. coli (STEC). Thus, the objectives of this study were to investigate the prevalence of markers for STEC (wzy, hly, fliC, eaeA, rfbE, stx-I, stx-II) and Salmonella (invA) in surface water sources (n = 8) from produce farms in Southwest Georgia and to determine correlations among the prevalence of virulence markers for STEC, water nutrient profile, and environmental factors. Water samples (500 mL) from eight irrigation ponds were collected from February to December 2021 (n = 88). Polymerase chain reaction (PCR) was used to screen for Salmonella and STEC genes, and Salmonella samples were confirmed by culture-based methods. Positive samples for Salmonella were further serotyped. Particularly, Salmonella was detected in 6/88 (6.81%) water samples from all ponds, and the following 4 serotypes were detected: Saintpaul 3/6 (50%), Montevideo 1/6 (16.66%), Mississippi 1/6 (16.66%), and Bareilly 1/6 (16.66%). Salmonella isolates were only found in the summer months (May-Aug.). The most prevalent STEC genes were hly 77/88 (87.50%) and stx-I 75/88 (85.22%), followed by fliC 54/88 (61.63%), stx-II 41/88 (46.59%), rfbE 31/88 (35.22%), and eaeA 28/88 (31.81%). The wzy gene was not detected in any of the samples. Based on a logistic regression analysis, the odds of codetection for STEC virulence markers (stx-I, stx-II, and eaeA) were negatively correlated with calcium and relative humidity (p < 0.05). A conditional forest analysis was performed to assess predictive performance (AUC = 0.921), and the top predictors included humidity, nitrate, calcium, and solar radiation. Overall, information from this research adds to a growing body of knowledge regarding the risk that surface water sources pose to produce grown in subtropical environmental conditions and emphasizes the importance of understanding the use of abiotic factors as a holistic approach to understanding the microbial quality of water.

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.

Long-read, chromosome-scale assembly of Vitis rotundifolia cv. Carlos and its unique resistance to Xylella fastidiosa subsp. fastidiosa

BackgroundMuscadine grape (Vitis rotundifolia) is resistant to many of the pathogens that negatively impact the production of common grape (V. vinifera), including the bacterial pathogen Xylella fastidiosa subsp. fastidiosa (Xfsf), which causes Pierce’s Disease (PD). Previous studies in common grape have indicated Xfsf delays host immune response with a complex O-chain antigen produced by the wzy gene. Muscadine cultivars range from tolerant to completely resistant to Xfsf, but the mechanism is unknown.ResultsWe assembled and annotated a new, long-read genome assembly for ‘Carlos’, a cultivar of muscadine that exhibits tolerance, to build upon the existing genetic resources available for muscadine. We used these resources to construct an initial pan-genome for three cultivars of muscadine and one cultivar of common grape. This pan-genome contains a total of 34,970 synteny-constrained entries containing genes of similar structure. Comparison of resistance gene content between the ‘Carlos’ and common grape genomes indicates an expansion of resistance (R) genes in ‘Carlos.’ We further identified genes involved in Xfsf response by transcriptome sequencing ‘Carlos’ plants inoculated with Xfsf. We observed 234 differentially expressed genes with functions related to lipid catabolism, oxidation-reduction signaling, and abscisic acid (ABA) signaling as well as seven R genes. Leveraging public data from previous experiments of common grape inoculated with Xfsf, we determined that most differentially expressed genes in the muscadine response were not found in common grape, and three of the R genes identified as differentially expressed in muscadine do not have an ortholog in the common grape genome.ConclusionsOur results support the utility of a pan-genome approach to identify candidate genes for traits of interest, particularly disease resistance to Xfsf, within and between muscadine and common grape.

Isolation and Identification of Salmosamonella typhi from clinical samples with molecular detection of O-antigen encoded genes

Abstract &#x0D; Antibiotic treatment of S.typhi is difficult as compared to treatment of acute infection. Antibiotic resistance carried against S.typhi by using 6 kinds of antibiotics from different classes, their results showed that all isolates were high resistance to Ampicillin (99%), Gentamicin (98%), Amikacin (79%) and less resistances Trimethoprim (55%) , Imipenem (60%) and Ceftriaxone(66%) .&#x0D; The present study focused on the molecular detection of Wzx flippase, Wzy polymerase genes in some Salmonella typhi isolates, Samples were collected from typhoid patients by classical lab work. Antibiotics susceptibility were investigated using disc diffusion method. The DNA and molecular Wzx flippase, Wzy polymerase were implemented using specific primers. The results showed that there was 33.33% having Wzy gene. The Wzx gene didn’t observed in any Salmonella isolates. The present study concluded that there was an important of the genetic diversity of O-antigen encoded genes included Wzx and Wzy which may be effect in typhoid diagnosis and treatment types.

Sensitive and Rapid Detection of Escherichia coli O157:H7 From Beef Samples Based on Recombinase Aided Amplification Assisted CRISPR/Cas12a System.

Escherichia coli O157:H7, being the cause of hemorrhagic colitis in humans, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive, and rapid E. coli O157:H7 detection method needs to be developed since the traditional detection methods are complex, costly, and time-consuming. In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive, and rapid nucleic acid detection of E. coli O157:H7 was introduced. First, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then a total of 34 bacterial strains were used for the specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157:H7 were prepared for the sensitivity test. Third, a real-time PCR assay for detection of the specific wzy gene of E. coli O157:H7 (FDA's Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out. The developed RAA-CRISPR/Cas12a method showed high specificity, and the detection could be completed within 30 min (after 4 h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4 × 102 CFU/mL and 7.5 × 10-4 ng/μL, respectively, which exhibited higher sensitivity than the RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157:H7 in ground beef samples could be positively detected after 4 h enrichment when the initial bacterial inoculum was 9.0 CFU/25 g. The detection results of the RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157:H7 from 93 local collected ground beef samples. The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157:H7 from ground beef samples. The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive, and accurate detection of E. coli O157:H7 in foods.

Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System.

Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host for heterologous protein expression and the production of many useful chemicals. First, we engineered the K. pneumoniae strain by deleting a capsule formation-associated wzy gene. This capsule-deficient strain enabled easy collection of the cell biomass for preparing cell extracts. Then, we optimized the procedure of cell extract preparation and the reaction conditions for CFPS. Finally, the optimized CFPS system was able to synthesize a reporter protein (superfolder green fluorescent protein, sfGFP) with a maximum yield of 253 ± 15.79 μg/mL. Looking forward, our K. pneumoniae-based CFPS system will not only expand the toolkit for protein synthesis, but also provide a new platform for constructing in vitro metabolic pathways for the synthesis of high-value chemicals.

Insights into the assessment of highly pathogenic Shiga toxin-producing Escherichia coli in raw milk and raw milk cheeses by High Throughput Real-time PCR

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 serogroups in raw milk products typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy genes. As these genetic markers can also be carried by non-EHEC strains, a number of ‘false positive’ results are obtained during the screening step. The suitability of new EHEC markers (espK, espV, ureD, Z2098, and CRISPRO26:H11) were tested as candidates for a more accurate screening of EHEC in dairy products. High-throughput PCR analysis of 1451 DNA extracts from milk and raw milk cheeses positive for both stx and eae demonstrated that addition of these new markers in the detection scheme resulted in a higher selectivity with a systematic reduction of the number of presumptive positive samples that require further O-group testing and confirmation by strain isolation. This reduction is more important (26% to 52%, depending on the animal production species) in the absence of prior IMS treatment of the enriched culture for the Top7 EHEC serotypes. However, even with prior treatment of the enriched cultures by IMS, the reduction rate varied between 5% and >25%. Analysis of eae-subtype, stx-subtypes indicated strong differences in the STEC (Shiga toxin producing E. coli) flora between animal species (goat, sheep, and cow). This study also pointed toward the possible presence of EHEC O80 (a new emerging EHEC serogroup in Europe) in cow's raw milk cheeses, which warrants further investigations.

Acinetobacter baumannii K106 and K112: Two Structurally and Genetically Related 6-Deoxy-l-talose-Containing Capsular Polysaccharides.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.

Characterization of Streptococcus pneumoniae serotype 19F-variants occurring in Brazil uncovers a predominant lineage that can lead to misinterpretation in capsular typing

BackgroundStreptococcus pneumoniae (S. pneumoniae) of serogroup 19 are mainly represented by serotypes 19A and 19F, which are associated with antimicrobial resistance and disease. The wzy gene, a component of the pneumococcal capsular locus, is the target to differentiate serotypes 19A and 19F by PCR-based capsular typing. In the last decade, allelic variants of the wzy19F gene have been described, leading to misinterpretation of capsular typing results. MethodsA collection of 154 serotype 19F S. pneumoniae strains recovered from carriage and disease in Brazil was evaluated to identify and characterize wzy19F variant isolates. ResultsEleven (7%) wzy19F variant isolates were detected and identified as belonging to ST810 (n = 10) or ST13673 (n = 1; single-locus variant of ST810). They were mostly recovered from diseased patients, susceptible to the antimicrobial agents tested (except for one multidrug-resistant strain) and did not harbor pili genes. Sequences of the wzy19F gene of these variants were identical to each other and to those previously described in Brazil, but slightly different from wzy19F variants identified in other countries. ConclusionThis study indicated that wzy19F variants present a geographically driven distribution and was the first to uncover phenotypic and genetic features of a wzy19F variant lineage occurring in Brazil since 1989.

Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment.

Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Study of Various Virulence Genes, Biofilm Formation and Extended-Spectrum β-lactamase Resistance in Klebsiella pneumoniae Isolated from Urinary Tract Infections

Objective: The aims of the current study were to evaluate the capacity of K. pneumoniae isolated from hospital-acquired urinary tract infection to form biofilm, the relation of this capacity to various virulence genes and the prevalence of Extended Spectrum β-lactamases (ESBL) among these isolates by phenotypic and genotypic methods. Material and Methods: The study included 100 non-duplicate strains of K. pneumoniae isolated from 100 different urine samples from patients with hospital-acquired urinary tract infection. The isolated strains were studied for biofilm formation, ESBL production by phenotypic methods. Molecular studies were applied for the detection of ESβLs genes blaTEM, blaSHV, blaCTX-M and for detection of virulence genes fimH, uge, rmpA, mag A, wzy, kfa and aerobactin genes. Result: The majority of the isolates had the capacity to form a biofilm (81%), with ESBL prevalence rate 41%. The most prevalent gene among ESBL producing K. pneumoniae was blaCTX-M (73.2%) followed by blaSHV (53.6%) and blaTEM (51.2%). Among the virulence genes studied in K. pneumoniae isolates, the most prevalent gene was fimH (76%), uge (70%). There was significant association between ESBL production, and resistance to amikacin, cefepime, ceftazidime, gentamicin, imipenem and meropenem and biofilm production in K. pneumoniae isolates. There was significant association between blaCTX-M, blaSHV, fimH, mag, kfa, wzy, rmpA and aerobactin and biofilm production in K. pneumoniae. Conclusion: The present study highlights the prevalence of virulence genes among biofilm-forming strains of K. pneumoniae isolated from hospital-acquired urinary tract infection. Moreover, there was association between biofilm formation and ESBL production. Further studies are required to elucidate the clinical impact of the association of these different mechanisms.

The First Isolation and Molecular Characterization of Shiga Toxin-Producing Virulent Multi-Drug Resistant Atypical Enteropathogenic Escherichia coli O177 Serogroup From South African Cattle.

Atypical enteropathogenic E. coli (aEPEC) is a group of diarrhoeagenic Escherichia coli with high diversity of serogroups, which lack the bundle-forming pili (BFP) and genes encoding for shiga toxins. The aim of this study was to isolate, identify and determine virulence and antibiotic resistance profiles of aEPEC O177 strains from cattle feces. A total of 780 samples were collected from beef and dairy cattle and analyzed for the presence of E. coli O177. One thousand two hundred and seventy-two (1272) presumptive isolates were obtained and 915 were confirmed as E. coli species. Three hundred and seventy-six isolates were positively confirmed as E. coli O177 through amplification of rmlB and wzy gene sequences using multiplex PCR. None of these isolates harbored bfpA gene. A larger proportion (12.74%) of the isolates harbored hlyA gene while 11.20, 9.07, 7.25, 2.60, and 0.63% possessed stx2, stx1, eaeA, stx2a, and stx2d, respectively. Most of E. coli O177 isolates carried stx2/hlyA (9.74%). Furthermore, 7.40% of the isolates harbored stx1/stx2 while 7.09% possessed stx1/stx2/hlyA genes. Only one isolate harbored stx1/stx2/hly/eaeA/stx2a/stx2d while 5.11% of the isolates harbored all the four major virulence genes stx1/stx2/hlyA/eaeA, simultaneously. Further analysis revealed that the isolates displayed varied antimicrobial resistance to erythromycin (63.84%), ampicillin (21.54%), tetracycline (13.37%), streptomycin (17.01%), kanamycin (2.42%), chloramphenicol (1.97%), and norfloxacin (1.40%). Moreover, 20.7% of the isolates exhibited different phenotypic multi-drug resistance patterns. All 73 isolates harbored at least one antimicrobial resistance gene. The aadA, streA, streB, erm, and tetA resistance genes were detected separately and/or concurrently. In conclusion, our findings indicate that environmental isolates of aEPEC O177 strains obtained from cattle in South Africa harbored virulence and antimicrobial resistance gene determinants similar to those reported in other shiga-toxin producing E. coli strains and suggest that these determinants may contribute to the virulence of the isolates.

Acinetobacter baumannii K116 capsular polysaccharide structure is a hybrid of the K14 and revised K37 structures

The genome of Acinetobacter baumannii clinical isolate, MAR-303, recovered in Russia was sequenced and found to contain a novel gene cluster at the A. baumannii K locus for capsule biosynthesis. The gene cluster, designated KL116, included four genes for glycosyltransferases (Gtrs) and a gene for a Wzy polymerase responsible for joining oligosaccharide K units into the capsular polysaccharide (CPS). The arrangement of KL116 was a hybrid of previously described A. baumannii gene clusters, with two gtr genes and the wzy gene shared by KL37 and the two other gtr genes found in KL14. The structure of the K116 CPS was established by sugar analysis and Smith degradation, along with one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS is composed of branched pentasaccharide K units containing only neutral sugars, with three monosaccharides in the main chain and a disaccharide side chain. The K116 unit shares internal sugar linkages with the K14 and K37 units, corresponding to the presence of shared gtr genes in the gene clusters. However, the specific linkage formed by Wzy was discrepant between K116 and the previously reported K37 CPS produced by A. baumannii isolate NIPH146. The K37 structure was therefore revised in this study, and the corrected Wzy linkage found to be identical to the Wzy linkage in K116. The KL116, KL14 and KL37 gene clusters were found in genomes of a variety of A. baumannii strain backgrounds, indicating their global distribution.

The gfc operon is involved in the formation of the O antigen capsule in Aeromonas hydrophila and contributes to virulence in channel catfish

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent involved in disease outbreaks in farmed carp and catfish species in China and the Southeastern United States, respectively. To assess the role of the LPS O-antigen in vAh virulence, the O-antigen ligase (waaL) and O-antigen polymerase (wzy) genes were deleted. While neither waaL nor wzy were found to be required for vAh virulence, a waaL mutant was found to have a polar effect on an adjacent group 4 capsule (gfc) genetic operon that was predicted to play a role in capsule assembly. Mutations in the gfc operon attenuated vAh virulence in channel catfish, particularly the mutant lacking gfcD that is predicted to encode an outer membrane protein required for capsular polysaccharide export. Furthermore, the vAh gfcD mutant was found to lack significant biofilm-forming capacity or buoyancy compared to wild-type vAh and induced an adaptive immune response that protected catfish from vAh challenge. This study indicates the importance of the capsular polysaccharide assembly process in the pathogenesis of this highly virulent A. hydrophila pathotype.

K units of the K8 and K54 capsular polysaccharides produced by Acinetobacter baumannii BAL 097 and RCH52 have the same structure but contain different di-N-acyl derivatives of legionaminic acid and are linked differently

The K8 and K54 capsular polysaccharides were isolated from Acinetobacter baumannii BAL 097 and RCH52, respectively, and studied by sugar analysis, partial acid hydrolysis and selective solvolysis with CF3CO2H in the presence of 2-methyl-1-propanol, along with 1D and 2D 1H and 13C NMR spectroscopy. The following structures of related branched tetrasaccharide repeats (K units) of the polysaccharides were established: [Display omitted] where Leg indicates 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid and R indicates (R)-3-hydroxybutanoyl or acetyl in the ratio ~2.5:1. The sequences of the KL8 and KL54 capsule gene clusters were closely related. The difference in the acyl group at O-7 on the sidechain legionaminic acid is due to differences in two genes in the legionaminic acid biosynthesis cluster. The wzy genes encoding the K unit polymerases are also different and make different linkages between the K units, allowing the first sugar of both K units to be identified as d-GlcpNAc. The shared Gtr20 glycosyltransferase, also encoded in KL63, forms the α-l-FucpNAc-(1 → 3)-d-GlcpNAc linkage, and Gtr19 was predicted to form α-d-GalpNAc-(1 → 3)-l-FucpNAc. Gtr18 from KL8 is 75% identical to Gtr108 from KL54 and both would link the Leg derivative to d-GalpNAc. Hence the genes present at the K locus were consistent with the composition and structures of the K8 and K54 capsular polysaccharides.

O-Antigen Gene Clusters of Plesiomonas shigelloides Serogroups and Its Application in Development of a Molecular Serotyping Scheme.

Plesiomonas shigelloides is a Gram-negative, flagellated, rod-shaped, ubiquitous, and facultative anaerobic bacterium. It has been isolated from various sources, such as freshwater, surface water, and many wild and domestic animals. P. shigelloides is associated with diarrheal diseases of acute secretory gastroenteritis, an invasive shigellosis-like disease, and a cholera-like illness in humans. At present, 102 somatic antigens and 51 flagellar antigens of P. shigelloides have been recognized; however, very little is known about variations of O-antigens among P. shigelloides species. In this study, 12 O-antigen gene clusters of P. shigelloides, O2H1a1c (G5877), O10H41 (G5892), O12H35 (G5890), O23H1a1c (G5263), O25H3 (G5879), O26H1a1c (G5889), O32H37 (G5880), O33H38 (G5881), O34H34 (G5882), O66H3 (G5270), O75H34 (G5885), and O76H39 (G5886), were sequenced and analyzed. The genes that control O-antigen synthesis are present as chromosomal gene clusters that maps between rep and aqpZ, and most of the synthesis and translocation of OPS (O-specific polysaccharide) belongs to Wzx/Wzy pathway with the exception of O12, O25, and O66, which use the ATP-binding cassette (ABC) transporter pathway. Phylogenetic analysis of wzx and wzy show that the wzx and wzy genes are specific to individual O-antigens and can be used as targets in molecular typing. Based on the sequence data, an O-antigen specific suspension array that detects 12 distinct OPS’ has been developed. This is the first report to catalog the genetic features of P. shigelloides O-antigen variations and develop a suspension array for the molecular typing. The method has several advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for easier identification and detection of additional O-antigen in the future.

The O6 antigen of Escherichia coli strain CFT073 is a target for Myoviridae

Extraintestinal pathogenic Escherichia coli strain CFT073 is a prototypic urosepsis isolate. CFT073 is of serotype O6:K2:H1 and thus has an O-antigen. The wzy gene encodes the O-antigen polymerase Wzy, which catalyses the polymerisation of O subunits into a long chain polysaccharide. A CFT073 Δwzy mutant was constructed using the λ Red recombination system. The lack of O-antigen was confirmed by LPS purification and staining with Pro-Q Emerald 300. CFT073 Δwzy and wild-type CFT073 were tested for their bacteriophage sensitivity. ΦEB49, a member of the Myoviridae, can mediate generalised transduction in strain CFT073. CFT073 Δwzy and wild-type CFT073 mixed with the same concentrations of ΦEB49 phage were compared. Confluent growth was evident on all CFT073 Δwzy plates, whilst lysis was apparent on all CFT073 plates. The inability of ΦEB49 to lyse CFT073 Δwzy suggested that this phage binds to O antigen, hence the decreased O antigen of CFT073 Δwzy, compared to wild-type CFT073, prevented phage lysis from occurring. These data indicate that CFT073 Δwzy is not a susceptible host for ΦEB49.

The role of the wzy gene in lipopolysaccharide biosynthesis and pathogenesis of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) causes severe respiratory and systemic diseases in poultry. The wzy gene encodes the O-antigen polymerase (Wzy), which plays an important role in the synthesis of the lipopolysaccharide (LPS) of bacteria. However, the function of the wzy gene in APEC remains unclear. Hence, in this study, a strain harboring a wzy gene mutant (DE17Δwzy) was constructed and the characteristics of this strain were analyzed. The results showed that mutant of wzy changed the phenotype of the LPS and affected serum agglutination of the O-antigen. Decreased motility and biofilm formation was also observed, but the endotoxin titer of the LPS in APEC was not affected. In addition, the wzy mutation significantly decreased the adherence and invasion to DF-1 cells, especially the survival abilities in duck serum and complement. Furthermore, an LD50 assay revealed that the virulence of mutant strain DE17Δwzy was attenuated 132-fold compared with wild-type strain DE17. Moreover, the bacterial load in the blood, liver, spleen, and kidneys of ducks infected with DE17Δwzy was decreased significantly compared with wild-type strain DE17 (p < 0.0001). These results confirmed that the wzy gene is associated with LPS biosynthesis and bacterial pathogenicity in APEC.

Molecular Detection of wzx1 and wzy Genes in Multi Drugs Resistance E. coli Isolates

Escherichia coli have been genetically changes in Iraqi environments, the present study was carried out to detection E coli isolates sero-group using o-antigens gene cluster, tow genes were amplified used multiplex PCR, the results of present study show that present isolates have more than one plasmid in different size (500-700 bp). PCR results show about 100% of isolates were had a positive results of PCR products of O45 wzx1 gene (451 bp) while 25% of isolates were a positive PCR products of O45 wzy1 gene (255 bp) and about 25% of isolates have tow genes. A new finding in present study that 50% of isolates have other copy of O45 wzx1 gene, these results concluded that it may be important in Iraqi isolates identification and classification however we need more information about genes sequencing.