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Biomedical subjects

R R Colwell

Publications and source records attributed to R R Colwell.

At least 73 records · Page 4Linked to original sources
PubMed1996

Serogroup conversion of Vibrio cholerae non-O1 to Vibrio cholerae O1: effect of growth state of cells, temperature, and salinity.

Recently, we reported the occurrence of seroconversion from Vibrio cholerae non-O1 to V. cholerae O1, but little is known about the environmental and physiological factors influencing seroconversion. We investigated effects of temperature (4, 25, and 35 degrees C) and salinity ( < 0.05 and 10%0.), as well as the stage of growth of cells, on serogroup conversion. Seroconversion of V. cholerae occurred under various environmental conditions. However, the rate of seroconversion in natural water ( < 0.5% salinity) and synthetic seawater microcosms (10%0 salinity), employing cells harvested from stationary phase culture, was approximately 2 logs higher than cells harvested from cultures in the logarithmic phase (i.e., 10(5) versus 10(3) per 10(10) cells. Thus, the physiological state of the cells, and to a lesser degree, temperature and salinity, is an important factor in the conversion of V. cholerae from non-O1 to O1 serogroup.

Antigens, Bacterial↗
PubMed1995

Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation?

Vibrio cholerae cells were incubated at 4 degrees C in nutrient-limited artificial seawater (ASW) microcosms. Plate counts declined from 8 x 10(5) to less than 2 c.f.u. ml-1 in about 23 d. When samples of microcosms were shifted to 30 degrees C, plate counts increased to 2.2 x 10(5) c.f.u. ml-1 in 72 h. An experiment was performed to determine whether culturable cells obtained after temperature upshifts were the result of 'resuscitation', or outgrowth, of nonculturable cells or of cell division and growth of the few culturable cells that remained in samples. Prior to temperature upshift, samples from the microcosms were diluted 10- and 100-fold in filter-sterilized (0.1 microns) ASW from the microcosms. Undiluted, 1/10, and 1/100 diluted samples recovered culturability to about 2.2 x 10(5) c.f.u. ml-1 within 72 h of temperature upshift. If resuscitation of nonculturable cells had occurred, the resultant number of culturable cells in diluted samples would have been 1/10 and 1/100 that of undiluted samples, respectively. In microcosms where plate counts had declined to less than 1 c.f.u. ml-1, 1/100 diluted samples did not regain culturability, i.e. no culturable cells remained from which growth could occur. Our conclusions are that in the experiments reported here, recovery of culturable cells on temperature upshifts resulted from growth and that there were no growth-inhibiting factors in the spent growth medium, supported by the finding that about 10(2) recovered V. cholerae cells ml-1 inoculated into filter-sterilized microcosm ASW grew to about 6.2 x 10(5) c.f.u. ml-1 in 24 h, confirming that V. cholerae is capable of significant growth in ASW.

Colony Count, Microbial↗
PubMed1995

Decrease in culturability of Vibrio cholerae caused by glucose.

The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in determining the distribution of bacteria in marine ecosystems in situations where carbohydrates are abundant, but nitrogen and phosphorus are limiting.

Glucose↗
PubMed1995

Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.

Bacterial Toxins↗
PubMed1995

Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal.

We report on the development and testing of two monoclonal antibody-based rapid immunodiagnostic test kits, BengalScreen, a coagglutination test, and Bengal DFA, a direct fluorescent-antibody test, for direct detection of Vibrio cholerae O139 synonym Bengal in clinical and environmental specimens. The BengalScreen test requires less than 5 min to complete and can be used in the field. Bengal DFA, being more sensitive than BengalScreen, requires only one reagent and less than 20 min for detection and enumeration of V. cholerae O139 synonym Bengal. In tests for specificity, all 40 strains of V. cholerae O139 reacted with both test kits, whereas 157 strains of heterologous species examined did not, yielding 100% specificity in this study. A field trial was conducted in with both BengalScreen and Bengal DFA, and the results were compared with those obtained by conventional culture methods. BengalScreen demonstrated a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94%. Results obtained by Bengal DFA, on the other hand, were 100% sensitive and 100% specific and yielded 100% positive and negative predictive values compared with culture methods. In a second evaluation, 93 stool specimens from Mexico that were negative for V. cholerae O139 by culture were also tested with both the BengalScreen and Bengal DFA kits. None of the 93 specimens were positive for V. cholerae O139 by both tests. A concentration method was optimized for screening of environmental water samples for V. cholerae O139 synonym Bengal with rapid test kits. BengalScreen results were unequivocally positive when water samples contained at least 2.0 x 10(3) CFU/ml, whereas Bengal DFA demonstrated an unequivocally positive reaction when the water sample contained at least 1.5 x 10(2) CFU/ml. When Bengal DFA was compared with conventional culture methods for enumeration of V. cholerae O139 synonym Bengal organisms, no difference was observed.

Agglutination Tests↗
PubMed1995

Serogroup conversion of Vibrio cholerae.

Vibrio cholerae serogroup O1 can be detected in the environment in a viable but nonculturable form, whereas V. cholerae non-O1 cells can be readily cultured during interepidemic periods in geographical regions where cholera is endemic. In the present study, pure cultures of V. cholerae non-O1 cells contained O1 cells when examined by immune-fluorescence microscopy. Laboratory microcosms were used to examine the outgrowth of the O1 cells in cultures of non-O1 V. cholerae. One O1 cell per 10(6) non-O1 cells could be detected by direct fluorescent-monoclonal antibody staining but only after incubation of the non-O1 culture for 48 h. Individual O1 cells were not detected in cultures incubated less than 48 h. Hybridization study, using a polymerase chain reaction (PCR) amplified fragment of the O-antigen of V. cholerae O1 as a probe, revealed the existence of a homologous gene in a microcosm sample of V. cholerae non-O1 containing serogroup-converted cells. The mechanism by which O1 cells can occur in cultures of non-O1 V. cholerae most likely resulted from spontaneous mutation of gene(s) encoding the O-somatic properties and (or) chemical, physical, or biological changes in the environment inducing expression or repression of the controlling gene(s). These findings have important implications for the epidemiology of cholera and the environmental source(s) of toxin producing V. cholerae O1.

Base Sequence↗
PubMed1995

Future of biotechnology-based control of disease in marine invertebrates.

Infectious disease is the single most devastating problem in mollusc and shrimp aquaculture. Pathogens causing the greatest problems have been identified as viruses, prokaryotes, and protozoans. Two approaches employing methods of biotechnology have been proposed to prevent, manage, and control mollusc and shrimp diseases. The first is development of a diagnostic scheme for detection and identification of pathogens, using molecular probes. This offers the opportunity for prophylactic measures to be taken. Molecular probes have been prepared for the major pathogens of molluscs, but in the case of shrimp pathogens, only a few are available. Monoclonal antibodies have also been prepared and are used in immunodiagnosis, e.g., immunofluorescence detection. Such diagnostic tools are relatively new to aquaculture, but have enormous potential. A second approach to the control of disease in marine invertebrates, notably shrimp, involves use of genetically transformed strains resistant to specific pathogens. Pathogen-resistant transgenic animals have been developed, but such research has only just begun for molluscs and shrimp. Transfection methods applied to mollusc and shrimp embryos have been successful, with preliminary data showing efficiency of heterologous promoters in controlling expression of reporter genes. Other transformation systems also show promise, including transposable elements and densoviruses.

Animal Diseases↗
PubMed1994

Isolation of a Vibrio cholerae transposon-mutant with an altered viable but nonculturable response.

We have isolated more than 2500 mutants of Vibrio cholerae by using transposon mutagenesis. Mutants were screened under low nutrient conditions in artificial seawater for an altered viable but nonculturable response, compared to the wild-type. Mutant JR09H1 entered the viable but nonculturable state more rapidly than the wild-type at both 25 degrees C and 4 degrees C.

Bacteriological Techniques↗
PubMed1994

Colonization of professional divers by toxigenic Vibrio cholerae O1 and V. cholerae non-O1 at dive sites in the United States, Ukraine and Russia.

Vibrio cholerae, recognized as the causative agent of epidemic cholera, was isolated from healthy professional divers and from water samples collected at dive sites in the United States, Ukraine and Russia. Swabs of nose, ear and throat of divers and their tank regulators, i.e. the divers and their diving gear, were taken before and after routine dives. Blood samples were collected before and 30-60 days after each dive to measure IgG and IgA titers against the whole cell antigen of V. cholerae O1. Nine strains of V. cholerae O1 and nine strains of V. cholerae non-O1 were isolated during this study. These isolates were identified by conventional biochemical tests and indirect fluorescent antibody staining methods, using fluorescein isothiocyanate-labeled monoclonal antibody, COLTA, prepared against the 'A' antigenic factor of the lipopolysaccharide of V. cholerae O1, and serotyped by slide agglutination. Seven of the nine strains of V. cholerae O1 isolated and successfully cultured during the studies, were toxigenic by enzyme-linked immunosorbent assay and polymerase chain reaction. Analyses of IgG and IgA antibodies of the divers showed that most of the divers had prior exposure to V. cholerae O1. V. cholerae serotype non-O1 strains isolated during the study were found to be non-toxigenic.

Antibodies, Bacterial↗
PubMed1994

Cholera DFA: an improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1.

An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC). An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well as improved photographic recording of results without loss of fluorescence intensity. When tested for specificity, all 30 strains of V. cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study. A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods. Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V. cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens. The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

Antibodies, Monoclonal↗
PubMed1994

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139.

The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae, in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene (ctx) positive and ctx- strains of V. mimicus detected the presence of zot gene. An AccI-EcoRV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 of 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx- toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.

Base Sequence↗
PubMed1994

Vibrio mimicus with multiple toxin types isolated from human and environmental sources.

A collection of 13 strains of Vibrio mimicus, including both clinical and environmental isolates from different geographic regions, was examined for various toxins. One strain of environmental origin produced cholera-like toxin (CT) which was completely absorbed with anti-CT immunoglobulin G, five strains produced a haemolysin that cross-reacted with the thermostable direct haemolysin of V. parahaemolyticus and DNA from two strains hybridised with a DNA probe specific for the heat-stable enterotoxin of V. cholerae non-O1. Culture supernates of all strains produced a factor that was cytotoxic to Vero and Chinese hamster ovary cells. In this study, we were able to identify strains of V. mimicus that produced, or had the genetic potential to produce, several toxin types simultaneously. The role of these strains as genetic reservoirs is discussed.

Animals↗