A one-minute oxidase test to detect Vibrio strains isolated from cultures on thiosulphate-citrate-bile salts-sucrose (TCBS) medium.

Vibrio cholerae is oxidase positive, a primary characteristic used to differentiate it from Enterobacteriaceae. But false negative oxidase test results have been obtained with colonies from thiosulphate-citrate-bile salts-sucrose (TCBS) agar medium. A rapid oxidase test procedure is described here. This takes 1 min, avoids false negative results and the necessity to grow the bacteria in a general-purpose medium. The bacteria may be recovered after the test and used for further investigations.     

A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments

AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS). METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined. RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp. when compared with TCI (46%). SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp. from estuarine waters. Because of the public health risk presented by V. vulnificus, V. parahaemolyticus, V. cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.     

Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures

Coagulase-negative staphylococci (CoNS) are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec) type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.     

Drug resistance in Vibrio cholerae strains isolated from clinical specimens

Cholera is a serious epidemic and endemic disease caused by the Gram-negative bacterium Vibrio cholerae. SXT is an integrative conjugation element (ICE) that was isolated from a V. cholerae; it encodes resistance to the antibiotics chloramphenicol, streptomycin and sulfamethoxazole/trimethoprim. One hundred seven V. cholerae O1 strains were collected from cholera patients in Iran from 2005 to 2007 in order to study the presence of SXT constin and antibiotic resistance.The study examined 107 Vibrio cholerae strains isolated from cholera prevalent in some Iranian provinces. Bacterial isolation and identification were carried out according to standard bacteriological methods. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) to four antibiotics (chloramphenicol, streptomycin, sulfamethoxazole, and trimethoprim) were determined by broth microdilution method. PCR was employed to evaluate the presence of established antibiotic resistance genes and SXT constin using specific primer sets.The resistance of the clinical isolates to sulfamethoxazole, trimethoprime, chloramphenicol, and streptomycin was 97%, 99%, 99%, and 90%, respectively. The data obtained by PCR assay showed that the genes sulII, dfrA1, floR, strB, and sxt element were present in 95.3%, 95.3%, 81.3%, 95.3%, and 95.3% of the V. cholerae isolates.The Vibrio strains showed the typical multidrug-resistance phenotype of an SXT constin. They were resistant to sulfamethoxazole, trimethoprime, chloramphenicol, and streptomycin. The detected antibiotic resistance genes included dfrA for trimethoprim and floR, strB, sulII and int, respectively, for chloramphenicol, streptomycin, sulfamethoxazole, as well as the SXT element.     

Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish

The hydrophobicity of 44 Vibrio strains isolated from cultured, diseased gilt-head sea bream (Sparus aurata) was determined. Three different methods were used: (1) microbial adhesion to hydrocarbons (MATH), either with phosphate buffer or with phosphate urea magnesium sulfate (PUM) buffer, (2) aggregation in the presence of salt solutions (SAT), and (3) adhesion to nitrocellulose filters (NCF). The results show that experimental conditions exerted a significant influence on hydrophobicity. Thus, Kendall rank coefficients showed the presence of correlation only for SAT and NCF, and for SAT and the MATH assay with PUM buffer. Moreover, no relationships were observed between the bacterial hydrophobicity estimated with the methods mentioned above and the ability of the strains to adhere to fish mucus or cells. These results indicate that adhesion of pathogenic Vibrio strains to host surfaces is mediated mainly by specific receptor interactions, instead of by hydrophobic interactions.     

Observation on a 65-kilodalton protein isolated from kanagawa positive strains of Vibrio parahaemolyticus

Crude hemolysin from four KP+ strains of Vibrio parahaemolyticus belonging to serotype 02:K3 exhibited a major protein band (molecular weight, 65 kilodaltons (kDa] in addition to a previously known thermostable direct hemolysin band (molecular weight, 21 kDa) in SDS - polyacrylamide slab gel electrophoresis. These strains showed maximum virulence leading to 100% mouse lethality within 2-6 h. It is hypothesized that this 65-kDa protein may play a vital role in the pathogenesis of the disease caused by V. parahaemolyticus.     

Vibrio cholerae (non-O1) isolated from California coastal waters.

Nineteen strains of Vibrio cholerae non-O1 were isolated from five separate marine sites along the Santa Cruz County coast. This environmental study was initiated after a human case of non-O1 cholera-like diarrhea was acquired endemically.     

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.     

Characterization of nativeFrankia strains isolated from chilean shrubs (Rhamnaceae)

Nine nativeFrankia strains were isolated from root nodules of four chilean actinorhizal plants (Rhamnaceae). The strains were designated as ChI1, ChI2, ChI3 and ChI4 fromColletia hystrix; ReI4 and ReI6 fromRetanilla ephedra; TqI12 and TqI15 fromTalguenea quinquinervis and TtI42 fromTrevoa trinervis. By scanning electron microscopy, all the strains exhibited similar actinomycetal structures: hyphae, sporangia and vesicles. The growth patterns of the isolates in BAP medium were similar. All showed a lag phase of approximately 6–7 days, then exhibited a logarithmic phase, except the ReI4 strain which seems to follow a linear growth pattern. A common feature of all the strains was a rapid loss of biomass at the end of the growth phase. All native strains grew on BAP medium supplemented with glucose. In six out of nine strains, the glucose was the best of the carbon sources tested. However, the strains differed in their ability to use other carbon sources such as arabinose, mannitol, maltose, succinate, sucrose, pyruvate, propionate and galactose. The isolates were sensitive to six antibiotics assayed (ampicillin, penicillin G, rifampicin, chloramphenicol, erythromycin and kanamycin). Using the acetylene reduction assay, the nitrogenase activity of the strains was determined. All strains grown in BAP medium lacking a combined nitrogen source were able to reduce acetylene ‘in vitro’.     

Chemical properties of lipopolysaccharide (LPS) isolated from Vibrio anguillarum PT514

Vibrio anguillarum, one of the causative agents of fish vibriosis, is serologically and biochemically divided into three groups (A, B and C). The chemical composition and molecular architecture of lipopolysaccharide (LPS) isolated from V. anguillarum PT 514, which belongs to serogroup B, were investigated. The LPS contained glucose (Glc), fructose (Fru), L-glycero-D-mannoheptose (L-D Hep), glucosamine (GlcN) and 4-amino-4,6-dideoxyglucose as sugar constituents in molar ratios of 8.9:0.7:3.0:1.1:1.6. Sephadex G-50 gel-chromatography of a degraded polysaccharide fraction separated from the LPS by 5% acetic acid hydrolysis suggested that the O-specific polysaccharide region consists of, in average, as much as 29 moles of Glc per 3 moles of L-D Hep, while the core polysaccharide contains at least Glc, L-D Hep and GlcN in molar ratios of 3.2 : 3.0 : 0.2. Fru and 4-amino-4,6-dideoxyglucose components were released from LPS on weak-acid hydrolysis, indicating that PT 514 LPS is distinguishable from those of Vibrio anguillarum belonging to the other serogroups. 2-Keto-3-deoxyoctonate (KDO), a common sugar constituent of gram-negative bacterial LPS, was not detected by Weissbach's color reaction under the conventional hydrolysis condition, but O-phosphoryl KDO was found in the strong-acid hydrolysate (4 M HCl, 100 C, 45 min). This substance was identical, at least in high-voltage paper electrophoresis, to 5-O-phosphoryl KDO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterisation and antimicrobial resistance of Vibrio vulnificus and Vibrio alginolyticus isolated from mussels (Mytilus galloprovincialis)

Twelve Vibrio vulnificus biotype 1 and 11 Vibrio alginolyticus isolated from mussels in Italy were analysed by antimicrobial resistance, plasmid profiles, random amplification of polymorphic DNA (RAPD), and single enzyme amplified fragment length polymorphism (sAFLP). Plasmid DNA was detected in three V. vulnificus and four V. alginolyticus cultures. All isolates were resistant to at least two antimicrobial agents: all isolates were resistant to ampicillin, carbenicillin and streptomycin, except one V. alginolyticus which was sensitive to carbenicillin and two V. alginolyticus which were sensitive to streptomycin. No association was detected between the presence of plasmid DNA and antimicrobial resistance. Seven of the twelve V. vulnificus and two of the eleven V. alginolyticus cultures were susceptible to the 10 microg of the vibriostatic compound O/129; all cultures were susceptible to the 150 microg of O/129. Both RAPD and sAFLP was found to be reproducible. Ten sAFLP and seven RAPD profiles were detected amongst the 12 V. vulnificus cultures: three cultures were identified as indistinguishable by both methods. RAPD and sAFLP analysis of V. alginolyticus generated nine and seven profiles respectively, and these two methods were independent. These results demonstrate extreme variability of V. vulnificus and V. alginolyticus isolated from mussels, and both RAPD and sAFLP provided information on intraspecific differences which will be useful for molecular epidemiological or ecological studies. A combination of methods gave optimal discrimination, although a single method could provide sufficient information to characterise V. vulnificus isolates.     

Use of an isolated guinea-pig ileum assay to detect bioactive compounds in microalgal cultures

An isolated guinea-pig ileum preparation was used to screen for bioactive compounds from algae. 212 culture supernatants and methanolic extracts of randomly chosen marine and freshwater algae were tested for their effect on electrically evoked muscle contractions (recorded as a change in tension) and on the resting muscle tone. 15 out of 42 (35%) of the marine algae tested and 5 out of 64 (8%) of freshwater algae gave positive results. Of the 20 algae giving positive results, 6 had previously been shown to produce bioactive compounds (mainly toxins) but we can find no reports in the literature of bioactive compounds from the remaining 14. Of these 14 cultures, 9 were axenic and therefore production of the biological activity can be assigned unambiguously to the alga. These results confirm the usefulness of the guinea-pig ileum preparation as a screen for bioactive compounds from microbial cultures.     

Genetic diversity of Cylindrospermopsis strains (cyanobacteria) isolated from four continents

The genetic diversity of Cylindrospermopsis strains (cyanobacteria) was examined using mainly the 16S-23S internally transcribed spacer (ITS1) sequences. Strains were grouped in three clusters: (i) America, (ii) Europe, and (iii) Africa and Australia. These results suggested a recent spread of Cylindrospermopsis across the American and European continents from restricted warm refuge areas instead of exchanges between continents. On the other hand, they also suggested a recent colonization of Australia by African strains.     

Production of poly-beta-hydroxybutyrate (PHB) by Vibrio spp. isolated from marine environment

Bacteria isolated from marine sediments were screened for their ability to accumulate polyhydroxyalkanoates. Among the isolates, four Vibrio spp. (strain M11, M14, M20 and M31) were studied in detail. All synthesized intracellular lipid inclusions during growth on diverse carbon sources including acetate, glycerol, succinate, glucose and sucrose. The inclusions were identified to be poly-beta-hydroxybutyrate (PHB) using gas chromatography and nuclear magnetic resonance analysis. No other type of polyhydroxyalkanoates (PHAs) was found to be accumulated by these marine isolates, suggesting that the diversity of PHAs produced in marine environments may be not as versatile as found in other environments. Strain M11 accumulated PHB in concentrations as high as 41% of cell dry weight when grown in medium containing 4% of sodium chloride. One of the Vibrio spp. was identified to be closely related to Vibrio natriegens (98% identity) by partial 16S rDNA sequence homology. V. natriegens has the shortest generation time (9.8 min) of any bacterium and this characteristic may be an exploitable trait for the industrial production of PHB.