Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis.

Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.

Staphylococcus spp. associated with subclinical bovine mastitis in central and northeast provinces of Thailand.

BACKGROUND: Staphylococcus spp. are major cause of bovine mastitis (BM) worldwide leading to economic damage to dairy farms and public health threat. Recently, a newly emerged Staphylococcus argenteus has been found as a human and animal pathogen. Molecular characteristics, virulence and antibiotic resistant phenotypes of bacteria causing BM in Thailand are rare. This study aimed to investigated Staphylococcus spp. associated with subclinical bovine mastitis (SCM) in Thailand. METHODS: Milk samples were collected from 224 cows of 52 dairy herds in four central and northeast provinces. Total somatic cell counts (SCC) and California mastitis test (CMT) were used to identify SCM cows. Milk samples were cultured for Staphylococcus spp. Coagulase-positive isolates were subjected to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Organisms suspected as S. argenteus were verified by detecting nonribosomal peptide synthetase gene. All isolates were checked for antibiograms and the presence of various virulence genes. RESULTS: From the 224 milk samples of 224 cows, 132 (59%) were positive for SCM by SCC and CMT and 229 staphylococcal isolates were recovered. They were 32 coagulase-positive (24 S. aureus and eight S. argenteus) and 197 coagulase-negative. PFGE of the S. aureus and S. argenteus revealed 11 clusters and a non-typeable pattern. MLST of representatives of the 11 PFGE clusters, three PFGE non-typeable S. aureus isolates from different locations and S. argenteus showed 12 sequence types. The eight S. argenteus isolates belonged to ST1223 (three isolates), ST2250 (two isolates), and ST2793 (two isolates). The antimicrobial tests identified 11 (46%) methicillin-resistant S. aureus and 25 (13%) methicillin-resistant coagulase-negative isolates, while seven S. argenteus were methicillin-susceptible and one isolate was methicillin-resistant. All of the 229 isolates were multiply resistant to other antibiotics. The most prevalent virulence genes of the 24 S. aureus isolates were clfA, coa and spa (X and IgG-binding region) (100%), hla (96%), pvl (96%) and sec (79%). Six S. argenteus isolates carried one enterotoxin gene each and other virulence genes including coa, clfA, hla/hlb, spa, tsst and pvl, indicating their pathogenic potential. CONCLUSION AND PERSPECTIVE: This is the first report on the S. argenteus from cow milk samples with SCM. Data on the molecular characteristics, virulence genes and antibiograms of the Staphylococcus spp. obtained from the present study showed a wide spread and increasing trend of methicillin-resistance and multiple resistance to other antibiotics. This suggests that the "One Health" practice should be nurtured, not only at the dairy farm level, but also at the national or even the international levels through cooperation of different sectors (dairy farmers, veterinarians, medical and public health personnel and scientists) in order to effectively combat and control the spread of these pathogens.

A novel bacteriocin from Enterococcus faecalis 478 exhibits a potent activity against vancomycin-resistant enterococci.

The emergence of multidrug-resistant enterococci (MDRE) and particularly vancomycin-resistant enterococci (VRE) is considered a serious health problem worldwide, causing the need for new antimicrobials. The aim of this study was to discover and characterize bacteriocin against clinical isolates of MDRE and VRE. Over 10,000 bacterial isolates from water, environment and clinical samples were screened. E. faecalis strain 478 isolated from human feces produced the highest antibacterial activity against several MDRE and VRE strains. The optimum condition for bacteriocin production was cultivation in MRS broth at 37°C, pH 5-6 for 16 hours. The bacteriocin-like substance produced from E. faecalis strain EF478 was stable at 60°C for at least 1 hour and retained its antimicrobial activity after storage at -20°C for 1 year, at 4°C for 6 months, and at 25°C for 2 months. A nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) analysis showed that the amino acid sequences of the bacteriocin-like substance was similar to serine protease of E. faecalis, gi|488296663 (NCBI database), which has never been reported as a bacteriocin. This study reported a novel bacteriocin with high antibacterial activity against VRE and MDRE.

New facet of non-O1/non-O139 Vibrio cholerae hemolysin A: a competitive factor in the ecological niche.

Different serogroups of Vibrio cholerae may inhabit the same ecological niche. However, serogroup O1/O139 strains are rarely isolated from their ecological sources. Quite plausibly, the non-O1/non-O139 vibrios and other bacterial species suppress growth of O1/O139 strains that share the same niche. Our bacterial inhibition assay data indicated that certain non-O1/non-O139 strains used a contact-dependent type VI secretion system (T6SS) to suppress growth of the O1 El Tor, N16961 pandemic strain. Comparative proteomics of the O1 and the suppressive non-O1/non-O139 strains co-cultured in a simulated natural aquatic microcosm showed that SecB and HlyD were upregulated in the latter. The HlyD-related effective factor was subsequently found to be hemolysin A (HlyA). However, not all hlyA-positive non-O1/non-O139 strains mediated growth suppression of the N16961 V. cholerae; only strains harboring intact cluster I HlyA could exert this activity. The key feature of the HlyA is located in the ricin-like lectin domain (ß-trefoil) that plays an important role in target cell binding. In conclusion, the results of this study indicated that non-O1/non-O139 V. cholerae suppressed the growth of the O1 pandemic strain by using contact-dependent T6SS as well as by secreting the O1-detrimental hemolysin A during their co-persistence in the aquatic habitat.

Human scFvs That Counteract Bioactivities of Staphylococcus aureus TSST-1.

Some Staphylococcus aureus isolates produced toxic shock syndrome toxin-1 (TSST-1) which is a pyrogenic toxin superantigen (PTSAg). The toxin activates a large fraction of peripheral blood T lymphocytes causing the cells to proliferate and release massive amounts of pro-inflammatory cytokines leading to a life-threatening multisystem disorder: toxic shock syndrome (TSS). PTSAg-mediated-T cell stimulation circumvents the conventional antigenic peptide presentation to T cell receptor (TCR) by the antigen-presenting cell (APC). Instead, intact PTSAg binds directly to MHC-II molecule outside peptide binding cleft and simultaneously cross-links TCR-Vß region. Currently, there is neither specific TSS treatment nor drug that directly inactivates TSST-1. In this study, human single chain antibodies (HuscFvs) that bound to and neutralized bioactivities of the TSST-1 were generated using phage display technology. Three E. coli clones transfected with TSST-1-bound phages fished-out from the human scFv library using recombinant TSST-1 as bait expressed TSST-1-bound-HuscFvs that inhibited the TSST-1-mediated T cell activation and pro-inflammatory cytokine gene expressions and productions.Computerized simulation, verified by mutations of the residues of HuscFv complementarity determining regions (CDRs),predicted to involve in target binding indicated that the HuscFvs formed interface contact with the toxin residues important for immunopathogenesis. The HuscFvs have high potential for future therapeutic application.

Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (103 DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

Prevalence and Molecular Genotyping of Noroviruses in Market Oysters, Mussels, and Cockles in Bangkok, Thailand.

Noroviruses are the most common cause of acute gastroenteritis associated with bivalve shellfish consumption. This study aimed to detect and characterize noroviruses in three bivalve shellfish species: oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The virus concentration procedure (adsorption-twice elution-extraction) and a molecular method were employed to identify noroviruses in shellfish. RT-nested PCR was able to detect known norovirus GII.4 of 8.8 × 10(-2) genome copies/g of digestive tissues from oyster and cockle concentrates, whereas in mussel concentrates, the positive result was seen at 8.8 × 10(2) copies/g of digestive tissues. From August 2011 to July 2012, a total of 300 shellfish samples, including each of 100 samples from oysters, cockles, and mussels were collected and tested for noroviruses. Norovirus RNA was detected in 12.3 % of shellfish samples. Of the noroviruses, 7.7 % were of the genogroup (G) I, 2.6 % GII, and 2.0 % were mixed GI and GII. The detection rate of norovirus GI was 2.1 times higher than GII. With regards to the different shellfish species, 17 % of the oyster samples were positive, while 14.0 and 6.0 % were positive for noroviruses found in mussels and cockles, respectively. Norovirus contamination in the shellfish occurred throughout the year with the highest peak in September. Seventeen norovirus-positive PCR products were characterized upon a partial sequence analysis of the capsid gene. Based on phylogenetic analysis, five different genotypes of norovirus GI (GI.2, GI.3, GI.4, GI.5, and GI.9) and four different genotypes of GII (GII.1, GII.2, GII.3, and GII.4) were identified. These findings indicate the prevalence and distribution of noroviruses in three shellfish species. The high prevalence of noroviruses in oysters contributes to the optimization of monitoring plans to improve the preventive strategies of acute gastroenteritis.

EMERGENCY ROOM: AN UNRECOGNIZED SOURCE OF EXTENDED-SPECTRUM ß-LACTAMASE PRODUCING ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae are the leading causes of hospital-associated infections, but community-acquired cases are increasingly being reported. This study determined the prevalence of ESBL-producing E. coli and K. pneumoniae carriers, their bla genes and risk factors of 452 patients admitted to the emergency room (ER) of Ramathibodi Hospital, Mahidol University, Bangkok, Thailand between April and August 2011. Prevalence of ESBL-producing E. coli and K. pneumoniae from rectal swabs was 16.5% and 1.0%, respectively. Factors associated with ESBL-producing carriers were a previous history of hospital admission (p = 0.001) and visits to health care facilities (p = 0.002) during the previous 3 months. All ESBL-producing isolates were susceptible to imipenem, meropenem and ertapenem. The majority (78%) of ESBL-producing E. coli isolates showed very high resistance to cefotaxime and ceftriaxone (MIC50 and MIC90 > 256 µg/ml). ESBL-producing E. coli harbored chromosomal blaTEM (96%), blaCTX-M (70%) and blaSHV (1%), while 8%, 73% and 3%, respectively, were located on plasmid. The prevalence of these genes in ESBL-producing K. pneumoniae was 75%, 50% and 25%, respectively on chromosome; and 100%, 25% and 50%, respectively on plasmid. Nucleotide sequence analysis revealed that these bla genes were of the type blaTEM-1' blaTEM-116' blaCTX-M-15' blaCTX-M-161' blaSHV-12, blaSHV-28 and blaSHV-148. Detailed epidemiologic and clinical characteristics of ER patients with history of prior hospital visits should be carried out to identify the ESBL-producing organisms they have acquired in order to institute appropriate treatment for these patients as well as control measures against further dissemination of these life-threatening organisms.

Rotavirus infection in children and adults with acute gastroenteritis in Thailand.

and young children, but rotavirus gastroenteritis in adults is uncommon. In this study, 260 stool samples collected in Thailand from January 2006 to February 2007 from patients, of all ages with acute gastroenteritis, were tested for group A rotavirus and compared with rotavirus infections in children and adults. Rota- virus was detected in 42% of the patients' samples, but children (< 18 years old) have a significantly higher prevalence (57%) of rotavirus infection than adults (≥ 18 years old) (27%) (OR 3.55; 95% CI: 2.11-5.96; p < 0.001). The highest attack rate was found in the age group of < 2 years old (14%), followed by 2-4 years of age (9%), 18-59 years of age (8%), 5-17 years of age (6%) and ≥ 60 years of age (5%). The dominant genotype was G1P[8] (27%), followed by G2P[4] (7%), G3P[8] (1%), and G9P[8] (1%). The rare genotypes identified were G1P[4], G1P[6], G2P[6], G2P[8], and G3P[6]. Mixed infections mostly occurred in children, comprising G1P[4]/P[8], G1P[4]/P[6], G1P[6]/P[8], G1/G2P[4], G1/G3P[4], and G1/G3P[4]/P[8]. Rotaviruses G3, G9, and P[4] were found only in children and genotype P[6] was found in adults (75%) at a higher frequency than in children (25%) (p < 0.001). The number of rotavirus in children was 1.99x10(8)/ml and in adult patients was 7.32x10(6)/ ml. The present study highlights the higher prevalence of rotavirus infection in children compared to adults and rotavirus genetic heterogeneity. Rotaviruses are the most important cause of severe diarrhea in infants

Detection and genetic characterization of norovirus in environmental water samples in Thailand.

The aim of this study was to detect and characterize noroviruses (NoVs) in environmental water samples. One hundred and fourteen water samples were collected from a river and irrigation canals in central Thailand during 2006-2007. NoVs were detected by RT-nested PCR in 13% of the samples. The river samples (22%) contained NoVs at a higher frequency than the irrigation canal samples (4%). Among the 15 NoV-positive samples, 9 harbored genogroup (G) I, 2 samples with GII, and 4 samples with mixed GI and GII. DNA sequencing of PCR amplicons and phylogenetic analysis of partial capsid gene revealed that 5 samples were of genotype GI-2, 1 sample was GI-6, and 1 sample was a mix of GI-2 and GII-unclassified genotypes. NoVs in water samples quantified using quantitative RT-PCR were in the range of 4.91 x 10(2) -1.26 x 10(3) copies/ml for NoV GI and 3.51 x 10(3) copies/ml for NoV GII. This is the first study demonstrating the presence of NoV variants in water samples collected from a river and the adjacent canals of Thailand.

Detection of hepatitis A virus and bacterial contamination in raw oysters in Thailand.

This study was conducted to determine the presence of hepatitis A virus (HAV) in raw oysters (Crassostrea belcheri) using a virus concentration method and reverse transcription-nested polymerase chain reaction (RT-nested PCR). A total of 220 oyster samples were collected from oyster farms and local markets in Thailand. HAV was found in three oyster samples. Nested PCR products of HAV detected in oysters were characterized further by DNA sequencing of the VP1/2A region and subjected to phylogenetic analysis. All HAV sequences (168 basepairs) were associated with human HAV subgenotype IB (GIB). Fecal coliforms and Escherichia coli were determined using the multiple tube fermentation method, to assess the microbiological quality of collected oysters. Among oyster samples tested, 65% had fecal coliforms higher than the standard level for raw shellfish [< 20 Most Probable Numbers (MPN)/g]; MPN values in the range of 21.0-4.6 x 10(4)/g. Most oyster samples (85%) were contaminated with E. coli in the range of 3.0-4.6 x 10(4) MPN/g. One oyster sample with an acceptable level of fecal coliforms contained HAV GIB. E. coli was found in all HAV-positive oyster samples. The results suggest a significant presence of HAV and bacterial indicators of fecal contamination in raw oysters, which are a health risk for consumers and a source of gastrointestinal illness. Enteric viruses should also be tested to assess the microbiological quality of oysters.

Norovirus GII-4 2006b variant circulating in patients with acute gastroenteritis in Thailand during a 2006-2007 study.

Noroviruses (NoVs) are recognized as a significant cause of acute gastroenteritis in children and adults. A 14-month study, from January 2006 to February 2007, was undertaken in a hospital in Thailand to determine the prevalence and genetic characterization of NoVs in patients of all ages with acute gastroenteritis. Based on reverse transcription-nested polymerase chain reaction (RT-nested PCR), NoVs were detected in 122 of 273 (44.7%) collected stool samples. Of the 122 NoV-positive samples, 28 (23%) belonged to GI, 79 (64.8%) belonged to GII, and 15 (12.2%) were mixed infections of GI and GII strains. Three NoV GI-positive and 42 NoV GII-positive samples were characterized successfully by DNA sequencing of the RT-nested PCR products and phylogenetic analysis. For NoV GI, two genotypes were identified: GI-2 (one sample) and GI-6 (two samples). NoV GII could be classified further into five distinct genotypes: GII-2 (1 sample), GII-3 (3 samples), GII-4 (14 samples), GII-6 (3 samples), and GII-17 (2 samples), and one unclassified genotype (19 samples). All NoV GII-4 strains showed 88-98% nucleotide identity with NoV GII-4 2006b variants reported worldwide. Among genotypes of NoV characterized, one co-infected stool sample exhibited NoVs GI-6 and GII-4 2006b. This study suggests that there is an important role of NoVs as etiologic agents in patients with acute gastroenteritis. The predominant circulating genotype of NoV infections is GII-4 2006b variant.

Dissemination of class I integron in Acinetobacter baumliannii isolated from ventilator-associated pneumonia patients and their environment.

Multidrug resistant Acinetobacter baumannii has become the most common cause of health care-associated infections at Maharaj Nakhon Si Thammarat Hospital, Thailand. The objective of the study was to detect integrons using PCR-based method from 96 A. baumannii isolates from ventilator-associated pneumonia (VAP) patients and their environment. Antibiotic susceptibility was determined using a disk diffusion technique. Forty-six isolates exhibited integrase genes, with only class I and class II integron detected in 43 and 3 A. baumannii isolates, respectively. Twenty-seven of 52 clinical and 19 of 44 environmental isolates were integron-positive. Detection rate of integron-positive A. baumannii isolated from VAP patients increased from 25% to 83% over the 4 month study period. The majority (91%) of integron-positive A. baumannii showed resistance to 6 or more of 11 antibiotics tested and 72% of class I integron-positive isolates were imipenem-resistant. Thus, class I integron-positive A. baumannii had spread among the VAP patients and into hospital environment, the latter acting as reservoirs of potential pathogens possessing drug resistance genes.

Clonal spread of carbapenem resistant Acinetobacter baumannii in the patients and their environment at BMA Medical College and Vajira Hospital.

OBJECTIVE: To determine the clonal spread of carbapenem-resistant Acinetobacter baumannii (CRAB) in the patients and their environment at BMA Medical College and Vajira Hospital. MATERIAL AND METHOD: A prospective study on CRAB isolated from the clinical specimens of 30 patients and 300 from their environmental samples were carried out from September 1-15, 2008. The CRAB isolates were genotyped using PCR-based typing method. RESULTS: Twenty-six (86.7%) and 20 (66.7%) cases of 30 patients had their environment contaminated with A. baumannii and CRAB, respectively Environmental contamination rates of A. baumannii and CRAB were 18.0% (54/300) and 13.0% (39/300), respectively. The most contaminated sites with CRAB were bedside cupboards (26.7%), followed by bedrails and bed sheets (20%), BP cuffs (16.7%), over bed tables and nurse station counters (13.3% each) and push carts (10%). Four molecular types were classified among 65 CRAB isolates. Molecular type 1 was the most prevalent (90.7%) and found in all kinds of environmental samples except patient record folder and computer keyboard/mouse. About 37% of the patients had at least one of their environmental samples contaminated with CRAB clonally related with their own types. CONCLUSION: Clonal spread of CRAB was demonstrated to emphasize the important of hand hygiene, contact precaution and patient's environmental decontamination in controlling the spread of CRAB in the hospital.

Epidemiological characteristics of Acinetobacter baumannii infections at Phramongkutklao Hospital.

OBJECTIVE: To describe epidemiological characteristics of Acinetobacter baumannii infections and identify molecular patterns of A. baumannii isolated from the patients admitted in Phramongkutklao Hospital. MATERIAL AND METHOD: A retrospective study on previously isolated A. baumannii from the clinical specimens submitted to the microbiology laboratory of Phramongkutklao Hospital from January to March 2008 were carried out together with molecular typing using PCR-based method. Clinical data were obtained from IC surveillance and patients' records. RESULTS: 114 A. baumannii were isolated from 80 patients. A. baumannii was a cause of healthcare-associated infection (90%, 72 of 80 cases), colonization (7.5%), and community-acquired infection (2.5%) with mortality rate of 50%. Majority of the patients from which A. baumannii were isolated were male (58.8%), age over 60 years (56.3%), diagnosed with lower respiratory diseases (26.3%), had A. baumannii ventilator-associated pneumonia (66.7%), and admitted in medical department (57.5%) with median length of hospital stay 35 days. PDR- and MDR- A. baumannii were accounted for 67.5% and 21.1%, respectively. All isolates showed sensitive to tigecycline and colistin. Using PCR-based typing was able to distinguish 6 molecular types among 114 A. baumannii isolates. Molecular type 2 was the most common type (47.4%) and widely spread in 14 wards. Spread of clonally related isolates was found in 14 cases admitted in 8 medical wards and ICUs. CONCLUSION: Multiple clones of PDR- and MDR- A. baumannii were widely spread in the hospital. Clonally related A. baumannii infected 14 cases in 8 wards.

Bed rails and endotracheal tube connectors as possible sources for spreading Acinetobacter baumannii in ventilator-associated pneumonia patients.

This study aimed to determine molecular patterns of Acinetobacter baumannii using a PCR-based technique with REP-1, REP-2 and M13 primers to distinguish the patients' strains and the environmental strains (condensate, endotracheal tube connector, bed rail and nurses hands). There were 67 cases of ventilator-associated pneumonia (VAP) among 600 patients using mechanical ventilators in 10 wards from March to July 2006. The incidence of VAP was 11.2% or 8.9/1,000 ventilator days with a 54.5% fatality rate. Among 19 of 22 A. baumannii VAP patients, 68.4% (13/19) had their environmental samples contaminated with A. baumannii and the most common contaminated sites were bed rails and endotracheal tube connectors (36.8% each). Multidrug resistant (MDR) A. baumannii were involved in 77.3% of A. baumannii VAP. Molecular typing of 96 A. baumannii isolates was able to differentiate A. baumannii isolates into 7 types. Type 2 was the most common and found in 77.3% (17/22) of A. baumannii VAP patients admitted in 6 of 7 wards. Identical fingerprints were found in clinical isolates and their bed rails, endotracheal tube connectors and condensates of 5 patients. The results demonstrate that multiple clones of MDR A. baumannii were widely spread in the hospital. Bed rails and contaminated endotracheal tube connectors could be potential sources of A. baumannii spread.

Development of a method for concentrating and detecting rotavirus in oysters.

Identification of enteric viruses in outbreak-implicated bivalve shellfish is difficult because of low levels of contamination and natural inhibitors present in shellfish tissue. In this study, the acid adsorption-alkaline elution method developed in our laboratory was proposed for the detection of rotavirus from oyster samples. The acid adsorption-alkaline elution process included the following steps: acid adsorption at pH 4.8, elution with 2.9% tryptose phosphate broth containing 6% glycine, pH 9.0, two polyethylene glycol precipitations, chloroform extraction and reconcentration using speedVac centrifugation. Oyster concentrates were extracted for RNA and examined for rotavirus using reverse transcription-nested polymerase chain reaction (RT-nested PCR). A comparison of SuperScript One-Step RT-PCR system and RT followed by PCR before the nested PCR reaction showed the former detecting four-fold lower concentration of rotavirus (78.12 plaque forming units [PFU]/ml or 0.26 PFU/assay) than the latter (3.12 x 10(2) PFU/ml or 1.04 PFU/assay). In the seeding experiment, the developed acid adsorption-alkaline elution gave high sensitivity of rotavirus detection (125 PFU/g of oyster). From August 2005 to February 2006, 120 oyster samples (Crassostrea belcheri) were collected from local markets and oyster farms, concentrated, and tested for naturally occurring rotaviruses. Four oyster samples were group A rotavirus-positive. Based on phylogenetic analysis of rotavirus DNA sequences in those positive samples, the oyster samples contained the sequences associated with human rotavirus G9 (two samples), G3 (one sample), and G1 (one sample). The present study demonstrates the successful application of developed virus concentration method and RT-nested PCR for the detection of rotaviruses in naturally contaminated oyster samples. The method might be used as a tool for evaluating the presence of enteric viruses in shellfish for monitoring and control of public health.

Genomic analysis of the Mozambique strain of Vibrio cholerae O1 reveals the origin of El Tor strains carrying classical CTX prophage.

Cholera outbreaks in subSaharan African countries are caused by strains of the El Tor biotype of toxigenic Vibrio cholerae O1. The El Tor biotype is the causative agent of the current seventh cholera pandemic, whereas the classical biotype, which was associated with the sixth pandemic, is now extinct. Besides other genetic differences the CTX prophages encoding cholera toxin in the two biotypes of V. cholerae O1 have distinct repressor (rstR) genes. However, recent incidences of cholera in Mozambique were caused by an El Tor biotype V. cholerae O1 strain that, unusually, carries a classical type (CTX(class)) prophage. We conducted genomic analysis of the Mozambique strain and its CTX prophage together with chromosomal phage integration sites to understand the origin of this atypical strain and its evolutionary relationship with the true seventh pandemic strain. These analyses showed that the Mozambique strain carries two copies of CTX(class) prophage located on the small chromosome in a tandem array that allows excision of the prophage, but the excised phage genome was deficient in replication and did not produce CTX(class) virion. Comparative genomic microarray analysis revealed that the strain shares most of its genes with the typical El Tor strain N16961 but did not carry the TLC gene cluster, and RS1 sequence, adjacent to the CTX prophage. Our data are consistent with the Mozambique strain's having evolved from a progenitor similar to the seventh pandemic strain, involving multiple recombination events and suggest a model for origination of El Tor strains carrying the classical CTX prophage.

Genomic sequence and receptor for the Vibrio cholerae phage KSF-1phi: evolutionary divergence among filamentous vibriophages mediating lateral gene transfer.

KSF-1phi, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1phi infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1phi infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1phi. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1phi, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXphi, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.

Genomic characterization of non-O1, non-O139 Vibrio cholerae reveals genes for a type III secretion system.

Non-O1, non-O139 Vibrio cholerae can cause gastroenteritis and extraintestinal infections, but, unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content of non-O1, non-O139 strains and their phylogenetic relationship to other pathogenic V. cholerae. Comparative genomic microarray analysis of four pathogenic non-O1, non-O139 strains indicates that these strains are quite divergent from O1 and O139 strains. Genomic sequence analysis of a non-O1, non-O139 strain (AM-19226) that appeared particularly pathogenic in experimental animals suggests that this strain carries a type III secretion system (TTSS) that is related to the TTSS2 gene cluster found in a pandemic clone of Vibrio parahaemolyticus. The genes for this V. cholerae TTSS system appear to be present in many clinical and environmental non-O1, non-O139 strains, including at least one clone that is globally distributed. We hypothesize that the TTSS present in some pathogenic strains of non-O1, non-O139 V. cholerae may be involved in the virulence and environmental fitness of these strains.