SYN-004 (ribaxamase), an oral beta-lactamase, mitigates antibiotic-mediated dysbiosis in a porcine gut microbiome model.

AIM: To evaluate an antibiotic inactivation strategy to protect the gut microbiome from antibiotic-mediated damage. METHODS AND RESULTS: SYN-004 (ribaxamase) is an orally delivered beta-lactamase intended to degrade penicillins and cephalosporins within the gastrointestinal tract to protect the microbiome. Pigs (20 kg, n = 10) were treated with ceftriaxone (CRO) (IV, 50 mg kg-1 , SID) for 7 days and a cohort (n = 5) received ribaxamase (PO, 75 mg, QID) for 9 days beginning the day before antibiotic administration. Ceftriaxone serum levels were not statistically different in the antibiotic-alone and antibiotic + ribaxamase groups, indicating ribaxamase did not alter systemic antibiotic levels. Whole-genome metagenomic analyses of pig faecal DNA revealed that CRO caused significant changes to the gut microbiome and an increased frequency of antibiotic resistance genes. With ribaxamase, the gut microbiomes were not significantly different from pretreatment and antibiotic resistance gene frequency was not increased. CONCLUSION: Ribaxamase mitigated CRO-mediated gut microbiome dysbiosis and attenuated propagation of the antibiotic resistance genes in pigs. SIGNIFICANCE AND IMPACT OF THE STUDY: Damage of the microbiome can lead to overgrowth of pathogenic organisms and antibiotic exposure can promote selection for antibiotic-resistant micro-organisms. Ribaxamase has the potential to become the first therapy designed to protect the gut microbiome from antibiotic-mediated dysbiosis and reduce emergence of antibiotic resistance.

Are natural reservoirs important for cholera surveillance? The case of an outbreak in a Brazilian estuary.

UNLABELLED: Paranaguá Bay is one of the largest estuarine systems on the Southern Brazilian coast. The only recorded cholera outbreak in this region since the early 20th century occurred in 1999 and resulted in 467 cases and at least three reported deaths in a population of approx. 150 000 people. This short communication reports historical, unpublished data related to that outbreak. Water, zooplankton and bivalve samples were collected and evaluated using direct fluorescence assay to determine whether Vibrio cholerae serogroups O1 and O139 were present in the estuarine system at that time. Most of the water (83%) and zooplankton samples (75%) were positive for V. cholerae O1, while V. cholerae O139 was not detected. Shellfish (Mytella sp.) were also positive for V. cholerae O1. These results indicate that the estuary, including biological vectors such as copepods and bivalves, comprise an important reservoir of V. cholerae O1 and a probable waterborne pathway for the disease, in addition to contamination with untreated sewage. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite most of the cholera cases that occurred in Brazil during the 7th pandemic were located in the northern areas of the country, a significant outbreak in Paranaguá, an estuary in the south coast, resulted in at least three deaths in 1999. We report here the detection of Vibrio cholerae O1 in water, zooplankton and bivalve samples during the outbreak, using direct fluorescence assay as an alternative method for the traditional plate culture employed at the time by the Brazilian Sanitary Agency. Results demonstrate that aquatic natural reservoirs comprise a potential route of transmission of cholera, in addition to untreated sewage and routine monitoring is recommended.

Environmental Vibrio parahaemolyticus DNA signatures validation.

Insignia is a novel DNA computational system which uses highly efficient algorithms to compare bacterial genomes and to identify specific DNA signatures to distinguish a target bacterium, or group of bacteria, from all other known bacterial species. It is currently being validated using different bacterial groups, including Vibrio spp. In this study, the genomic analysis by Insignia was conducted on Vibrio parahaemolyticus, a halophilic gram-negative bacteria which constitutes a leading cause of seafood-borne disease. Insignia was used to identify 37 V. parahaemolyticus-specific signatures and to design PCR assays to validate the representative signature sequences by TaqMan essays. The 37 assays targeted loci distributed around the genome and detected genes coding for hypothetical proteins and for proteins involved in adhesion, starvation and virulence. A panel of V. parahaemolyticus environmental strains isolated from the North Adriatic Sea (Italy) and from the Black Sea (Georgia) was used to validate the selected signatures. The signature assays revealed both sensitive and specific and the method allowed a more accurate identification of the tested bacterial strains at the species level when compared to biochemical and PCR standard methods. Using Insignia, it was possible to distinguish two different groups among the strains previously identified as V. parahaemolyticus: most of the strains were included in a "V. parahaemolyticus-like group" showing nearly all of the signatures assayed while a small group of 10 strains contained only a few of the signatures tested. By sequencing the 16S rDNA of this latter group, it was confirmed that they were not V. parahaemolyticus but in fact belonged to other Vibrio species. No significant genome-wide differences were detected between the strains isolated in Italy and in Georgia though the very different geographical origin.

Diagnostic limitations to accurate diagnosis of cholera.

The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although more than 40% of the suspected cases failed to show cholera etiology by conventional culture methods (CMs). In the present study, suspected cholera stools collected from every 50th patient during an acute diarrheal outbreak were analyzed extensively using different microbiological and molecular tools to determine their etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1 by CMs, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay; all but three samples positive for V. cholerae O1 by CMs were also positive for V. cholerae O1 by DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V. cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct fluorescent antibody (DFA) assay, 36 (73%) amplified primers for the genes wbe O1 and ctxA by multiplex-PCR (M-PCR), and 31 (63%) showed El Tor-specific lytic phage on plaque assay (PA). Each of these methods allowed the cholera etiology to be confirmed for 97% of the stool samples. The results suggest that suspected cholera stools that fail to show etiology by CMs during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by in vivo vibriolytic action of the phage and/or nonculturability induced as a host response.

El Tor cholera with severe disease: a new threat to Asia and beyond.

During epidemics of cholera in two rural sites (Bakerganj and Mathbaria), a much higher proportion of patients came for treatment with severe dehydration than was seen in previous years. V. cholerae O1 isolated from these patients was found to be El Tor in its phenotype, but its cholera toxin (CT) was determined to be that of classical biotype. Whether the observed higher proportion of severe dehydration produced by the El Tor biotype was due to a shift from El Tor to classical CT or due to other factors is not clear. However, if cholera due to strains with increased severity spread to other areas where treatment facilities are limited, there are likely to be many more cholera deaths.

Genomic analysis of a novel integrative conjugative element in Vibrio cholerae.

Integrative conjugative elements (ICEs) are a class of self-transmissible mobile elements that mediate horizontal gene transfer in bacteria, and play an important role in bacterial evolution. Since 1992, ICEs of the SXT/R391 family have been found to be widely distributed among Vibrio cholerae strains isolated in Asian countries. Here we describe ICEVchB33, an ICE found in the genomes of two V. cholerae O1 Eltor strains, one isolated in India, 1994, and the other from Mozambique, 2004. ICEVchB33 revealed a new genetic organization, different from other ICEs of the SXT/R391 family, demonstrating the genomic plasticity of these elements.

Association of Vibrio cholerae with plankton in coastal areas of Mexico.

The El Niño event of 1997/1998 provided an opportunity to carry out a field experiment in which the relationship of sea surface temperature and the association of Vibrio cholerae with marine plankton could be assessed in Mexican coastal and estuarine areas. Plankton samples were collected from May 1997 through June 1999. Sites included the Mexican ports of Veracruz, Coatzacoalcos and Frontera in the Gulf of Mexico and Ensenada, Guaymas, Mazatlán, Manzanillo, Acapulco and Oaxaca in the Pacific Ocean. Sampling was also accomplished during two oceanographic cruises in the Yucatan channel of the Caribbean Sea. Bacteriological analyses for V. cholerae serogroups O1 and O139 were carried out. Also, the taxonomic structure of the plankton populations was determined. Vibrio cholerae O1 was detected only in Veracruz samples collected during April, May and June 1999, when La Niña climatic conditions prevailed. It is concluded that V. cholerae O1 in Mexico derives from its marine and estuarine origin and not from sewage contamination. The significant number of Acartia tonsa copepodites and V. cholerae copepodite-positive samples suggests a significant role of this copepod in the occurrence and distribution of V. cholerae in coastal areas of Mexico.

Virulence of viable but nonculturable S. Typhimurium LT2 after peracetic acid treatment.

S. Typhimurium LT2 cells suspended in sterilized sewage effluent water (SEW) and in distilled water microcosms were exposed to 0, 7, 15 and 20 mg/l peracetic acid, and tested for viability and virulence. After treatment for one hour, colony forming units decreased by at least 5 log units at peracetic acid concentration of 7 mg/l. In SEW, at peracetic acid concentration of 15 mg/l, the cells were nonculturable (VNC), but retained virulence as demonstrated by invasion assays of HeLa cells. Higher concentrations (greater than or equal to 20 mg/l) resulted in bacterial death, i.e. substrate non-responsive cells. Despite morphological alterations of the bacteria after peracetic acid treatment, visualized by transmission electronic microscopy, conservation of both adhesive and invasive capacities was confirmed by scanning electron microscopy after exposure to 0-15 mg/l peracetic acid. Public health professionals need to recognize that peracetic acid-treated Salmonella is capable of modifying its physiological characteristics, including entering and recovering from the viable but nonculturable state, and may remain virulent after a stay in SEW followed by peracetic acid treatment.

ompU genes in non-toxigenic Vibrio cholerae associated with aquaculture.

AIMS: The study was undertaken with the objective of understanding the virulence-associated genes of the CTX and TCP gene clusters in environmental isolates of Vibrio cholerae, an important human pathogen, isolated from the aquaculture environment. The involvement of the ompU gene in conferring bile resistance in these isolates was also evaluated. METHODS AND RESULTS: The V. cholerae isolates were tested by PCR and fluorescent antibody test for O1 (Ogawa and Inaba) and O139 serotypes. All isolates were found to be non-toxigenic V. cholerae confirmed by their positive PCR reaction for toxR but negative for ctx, zot and tcp gene. The hlyA gene was detected in 85% of the strains and ompU in 77%. The results on the bactericidal effect of bile salts suggest that ompU may play a role in conferring bile resistance in non-O1/non-O139 strains. CONCLUSION: The results of the study indicate that most environmental strains lacked the CTX and TCP gene clusters. However, most isolates had the hlyA gene indicating the potential of these environmental strains to cause mild gastroenteritis. It was also observed that strains lacking ompU showed less tolerance to bile salts. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on virulence factors of V. cholerae associated with aquaculture environment and products would be of value in risk assessment for human health.

Development of a hexaplex PCR assay for rapid detection of virulence and regulatory genes in Vibrio cholerae and Vibrio mimicus.

We have developed a hexaplex PCR assay for rapid detection of the virulence and regulatory genes for cholera toxin enzymatic subunit A (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and central regulatory protein ToxR (toxR) in Vibrio cholerae and Vibrio mimicus. This hexaplex PCR proved successful in screening pathogenic-toxigenic and nonpathogenic-nontoxigenic V. cholerae and V. mimicus strains from both clinical and environmental sources.

Seasonality of Chesapeake Bay bacterioplankton species.

Bacteria, gamma-subclass of Proteobacteria, Vibrio-Photobacterium, Vibrio vulnificus, Vibrio cholerae-Vibrio mimicus, and Vibrio cincinnatiensis in water samples collected from the Choptank River in Chesapeake Bay from 15 April to 16 December 1996 were enumerated using a fluorescent oligonucleotide direct-counting (FODC) procedure. FODC results obtained using a Bacteria taxon-specific probe ranged from one-third the number of to the same number as that obtained by the acridine orange direct count (AODC) procedure. The abundance of individual taxa (per liter) ranged from 0.25 x 10(10) to 2.6 x 10(10) Bacteria, 0.32 x 10(8) to 3.1 x 10(8) gamma-Proteobacteria, 0.2 x 10(8) to 2.1 x 10(8) Vibrio-Photobacterium, 0.5 x 10(7) to 10 x 10(7) V. vulnificus, 0.2 x 10(6) to 6 x 10(6) V. cholerae-V. mimicus, and 0.5 x 10(5) to 8 x 10(5) V. cincinnatiensis. The occurrence of all taxa monitored in this study was higher in summer; however, these taxa made up a larger proportion of the Bacteria when the water temperature was low. Large fluctuations in species abundance as well as in percent composition of Vibrio-Photobacterium occurred from week to week, indicating that localized blooms of these taxa occur. The cross-Choptank River transect sample profile of V. vulnificus and V. cholerae-V. mimicus varied significantly in abundance, and trans-Choptank River transect samples revealed a patchy distribution.

Bacteria of the gamma-subclass Proteobacteria associated with zooplankton in Chesapeake Bay.

The seasonal abundance of gamma-subclass Proteobacteria, Vibrio-Photobacterium, Vibrio cholerae-Vibrio mimicus, Vibrio cincinnatiensis, and Vibrio vulnificus in the Choptank River of Chesapeake Bay associated with zooplankton was monitored from April to December 1996. Large (>202- microm) and small (64- to 202- microm) size classes of zooplankton were collected, and the bacteria associated with each of the zooplankton size classes were enumerated by fluorescent oligonucleotide direct count. Large populations of bacteria were found to be associated with both the large and small size classes of zooplankton. Also, the species of bacteria associated with the zooplankton showed seasonal abundance, with the largest numbers occurring in the early spring and again in the summer, when zooplankton total numbers were correspondingly large. Approximately 0.01 to 40.0% of the total water column bacteria were associated with zooplankton, with the percentage of the total water column bacteria population associated with zooplankton varying by season. A taxonomically diverse group of bacteria was associated with zooplankton, and a larger proportion was found in and on zooplankton during the cooler months of the year, with selected taxa comprising a larger percent of the Bacteria in the summer. V. cholerae-V. mimicus and V. vulnificus comprised the bulk of the large and small zooplankton-associated Vibrio-Photobacterium species. In contrast, V. cincinnatiensis accounted for less than 0.1 to 3%. It is concluded that water column and zooplankton bacterial populations vary independently with respect to species composition since no correlation was observed between taxa occurring with highest frequency in the water column and those in association with zooplankton.

Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state.

AIMS: Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V. fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined. METHODS AND RESULTS: Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined. Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively. In contrast, V. fischeri became nonculturable at approximately 55 and 31 d in similar microcosms. Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds. Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability. CONCLUSIONS: The study confirms entry of V. harveyi and V. fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms loss of luminescence of V. harveyi and V. fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.

Occurrence of pathogenic vibrios in coastal areas of France.

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.

Antibacterial activity of black myrobalan (Terminalia chebula Retz) against Helicobacter pylori.

The effect of ether, alcoholic and water extracts of black myrobalan (Teminalia chebula Retz) on Helicobactor pylori were examined using an agar diffusion method on Columbia Agar. Water extracts of black myrobalan showed significant antibacterial activity and had a minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC) of 125 and 150 mg/l, respectively. The extract was active after autoclaving for 30 min at 121 degrees C. Plant powder (incorporated in agar) gave higher MIC and MBC values (150 and 175 mg/l, respectively). Water extracts of the black myrobalan at a concentration of 1-2.5 mg/ml inhibited urease activity of H. pylori. The results show that black myrobalan extracts contain a heat stable agent(s) with possible therapeutic potential. Other bacterial species were also inhibited by black myrobalan water extracts.

Microbiological analysis of tube-well water in a rural area of Bangladesh.

Five tube-wells in Matlab, Bangladesh, were selected for analysis of selected biophysicochemical parameters. The results showed that all tube-well water samples contained zooplankton and bacteria. Results for some of the parameters were outside the accepted limits recommended by the World Health Organization for drinking water. It is concluded that water from tube-wells should be treated if used as drinking water.

Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. nov. and emendation of description.

A polyphasic taxonomy study was undertaken of three strains of Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic relatedness was measured by DNA-DNA hybridization. Strains KMM 458T and IAM 14160T shared 99% genetic relatedness, but were only 48-49% related to the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM 12915T. The third strain, P. haloplanktis subsp. tetraodonis A-M, showed 83% genetic similarity with P. haloplanktis subsp. haloplanktis IAM 12915T and 32% with KMM 458T. From these results, it is concluded that strains KMM 458T and IAM 14160T comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas haloplanktis. Based on phenotypic and chemotaxonomic data, genomic fingerprint patterns, DNA-DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM 14160T = KMM 458T).

Genotypes associated with virulence in environmental isolates of Vibrio cholerae.

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.