El Tor Strains Research Articles

Multiply antibiotic-resistant Vibrio cholerae O1 biotype El Tor strains emerge during cholera outbreaks in Zambia.

Antibiotic resistance data, made available from laboratory records during eight cholera outbreaks between 1990 and 2004 showed Vibrio cholerae serogroup O1 to have a low level of resistance (2-3%) to tetracycline during 1990-1991. Resistance increased for tetracycline (95%), chloramphenicol (78%), doxycycline (70%) and trimethoprim-sulphamethoxazole (97%) in subsequent outbreaks. A significant drop in resistance to tetracycline and chloramphenicol followed the adoption of a national policy to replace tetracycline with erythromycin for treating cholera. Sixty-nine strains from cholera outbreaks in Zambia between 1996 and 2004, were examined for antibiotic resistance and basic molecular traits. A 140 MDa conjugative, multidrug-resistant plasmid was found to encode tetracycline resistance in strains from 1996/1997 whereas strains from 2003/2004 were resistant to furazolidone, but susceptible to tetracycline, and lacked this plasmid. PCR revealed 25 of 27 strains from 1996/1997 harboured the intl1 class 1 integron but lacked SXT, a conjugative transposon element. Similar screening of 42 strains from 2003/2004 revealed all carried SXT but not the intl1 class 1 integron. All 69 strains, except two, one lacking ctxA and the other rstR and thus presumably truncated in the CTX prophage region, were positive for important epidemic markers namely rfbO1, ctxA, rstR2, and tcpA of El Tor biotype. Effective cholera management is dependent on updated reports on culture and sensitivity to inform the choice of antibiotic. Since the emergence of antibiotic resistance may significantly influence strategies for controlling cholera, continuous monitoring of epidemic strains is crucial.

Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji

Five strains of Vibrio cholerae O1, one each from an Australian and a New Zealand tourist with gastrointestinal illness returning from an island resort in Fiji and the remaining three from water sources located in the same resort, were extensively characterized. Conventional phenotypic traits that are used for biotyping of O1 V. cholerae categorized all five strains as belonging to the El Tor biotype. Genetic screening of 11 regions that are associated with virulence in V. cholerae showed variable results. The absence of genes comprising Vibrio seventh pandemic island-I (VSP-I) and VSP-II in all the strains indicated that these strains were very similar to the pre-seventh pandemic V. cholerae O1 El Tor strains. The ctxAB genes were absent in all strains whereas orfU and zot were present in four strains, indicating that the strains were non-toxigenic. Four strains carried a truncated CTX prophage. Although epidemiological and molecular studies suggested that these strains did not cause cholera amongst tourists at the resort, their similarity to pre-seventh pandemic strains, their prior association with gastrointestinal illness and their presence in the island resort setting warrant more attention.

Transcutaneous Immunization with Toxin-Coregulated Pilin A Induces Protective Immunity against Vibrio cholerae O1 El Tor Challenge in Mice

Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 mug of TcpA or TcpA with an immunoadjuvant (cholera toxin [CT], 50 mug) on days 0, 19, and 40. Mice immunized with TcpA alone did not develop anti-TcpA responses. Mice that received transcutaneously applied TcpA and CT developed prominent anti-TcpA immunoglobulin G (IgG) serum responses but minimal anti-TcpA IgA. Transcutaneous immunization with CT induced prominent IgG and IgA anti-CT serum responses. In an infant mouse model, offspring born to dams transcutaneously immunized either with TcpA and CT or with CT alone were challenged with 10(6) CFU (one 50% lethal dose) wild-type V. cholerae O1 El Tor strain N16961. At 48 h, mice born to females transcutaneously immunized with CT alone had 36% +/- 10% (mean +/- standard error of the mean) survival, while mice born to females transcutaneously immunized with TcpA and CT had 69% +/- 6% survival (P < 0.001). Our results suggest that transcutaneous immunization with TcpA and an immunoadjuvant induces protective anti-TcpA immune responses. Anti-TcpA responses may contribute to an optimal cholera vaccine.

Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh.

We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.

Clonal Relationship among Vibrio cholerae O1 El Tor Strains Causing the Largest Cholera Epidemic in Kenya in the Late 1990s

Eighty Vibrio cholerae O1 strains selected to represent the 1998-to-1999 history of the largest cholera epidemic in Kenya were characterized by ribotyping, antimicrobial susceptibility, and random amplified polymorphic DNA patterns. Except for 19 strains from 4 local outbreaks in North Eastern Province along the Somalia border, the other 61 strains from 25 outbreaks occurring in districts scattered around the country were all ribotype B27 and resistant to chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim. The 61 strains showed similar and specific amplified DNA patterns. These findings indicate that the predominant strains that caused the Kenyan epidemic had a clonal origin and suggest that ribotype B27 strains, which first appeared in West Africa in 1994, have had a rapid spread to eastern Africa.

Antifungal and Antibacterial Activities of Mexican Tarragon (Tagetes lucida)

Mexican tarragon (Tagetes lucida Cv. Asteraceae: Campanulatae) is an important, nutritious plant and an effective herbal medicine. Seven coumarins, 7,8-dihydroxycoumarin (4), umbelliferone (7-hydroxycoumarin) (5), scoparone (6,7-dimethoxycoumarin) (7), esculetin (6,7-dihydroxycoumarin) (11), 6-hydroxy-7-methoxycoumarin (12), herniarin (7-methoxycoumarin) (13), and scopoletin (6-methoxy-7-hydroxycoumarin) (14), and three flavonoids, patuletin (18), quercetin (19), and quercetagetin (20), were isolated from CH2Cl2 and MeOH extracts from aerial parts of T. lucida. In addition, 6,7-diacetoxy coumarin (15), 6-methoxy-7-acetylcoumarin (16), and 6-acetoxy-7-methoxycoumarin (17) derivatives were synthesized. 8-Methoxypsoralen (1), 8-acetyl-7-hydroxycoumarin (2), 7,8-dihydroxy-6-meth-oxycoumarin (3), 6,7-dimethoxy-4-methylcoumarin (6), 5,7-dihydroxy-4-methylcoumarin (8), 4-hydroxycoumarin (9), 4-hydroxy-6,7-dimethylcoumarin (10), naringenin (21), glycoside-7-rhamnonaringin (22), and rutin (23) were commercially obtained (Sigma-Aldrich). All of these compounds and extracts (M1 and M2) were assayed against bacteria and fungi. The antibacterial activity was determined on Bacillus subtilis, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Salmonella typhi, Salmonella sp., Shigella boydii, Shigella sp., Enterobacter aerogenes, Enterobacter agglomerans, Sarcina lutea, Staphylococcus epidermidis, Staphylococcus aureus, Yersinia enterolitica, Vibrio cholerae (three El Tor strains, CDC-V12, clinic case, and INDRE-206, were obtained from contaminated water), and V. cholerae (NO-O1). The evaluated fungi were Aspergillus niger, Penicillium notatum, Fusarium moniliforme, Fusarium sporotrichum, Rhizoctonia solani, and Trichophyton mentagrophytes. The most active compounds against Gram-positive and -negative bacteria were the dihydroxylated coumarins 3 and 4. In addition, 2-4, 6, 7, and 11 showed an interesting activity against V. cholerae, a key bacterium in the contaminated water; 2-4 were the most active. Coumarins were the most effective compounds against Gram-negative bacteria. The extract MeOH/CH2Cl2 (1: 4) M2 at 0.4 microg/disk inhibited the growth of E. coli and P. mirabilis (40%), K. pneumoniae (31.1%), Salmonella sp. (35.5%), and Shigella sp. (0%) at 72 h of culture. The dimethoxy compounds 6 and 7 showed a strong activity against fungal strains, especially T. mentagrophytes and R. solani (100% of inhibition at 125.0 and 250.0 microg/mL, respectively).

Molecular phylogenetic analysis ofVibrio cholerae O1 El Tor strains isolated before, during and after the O139 outbreak based on the intergenomic heterogeneity of the 16S-23S rRNA intergenic spacer regions

We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eight rrn operons (rrna-rrnh) of Vibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O 139 outbreak. ISR classes 'a' and 'g' were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O 139 serogroup and post-O 139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.

Vibrio cholerae pathogenic clones.

We resolved the relationships between 2 pandemic clones of Vibrio cholerae. Using 26 housekeeping genes, we showed that the US Gulf clone, the Australian clone, and 3 El Tor strains isolated before the seventh pandemic were related to the seventh pandemic clone. The sixth pandemic clone was well separated from them.

Vibrio cholerae O139 Bengal—10 years on

A second aetiological agent of cholera, Vibrio cholerae O139 Bengal was identified in late 1992 when it caused large outbreaks of diarrhoea in India and Bangladesh. The new strain probably arose as a result of lateral transfer of genes encoding a novel somatic antigen and a capsule from an unknown bacterium to an O1 El Tor strain with the subsequent loss of genes encoding O1 somatic antigen. O139 produces a semi-rough type colony with a truncated lipopolysaccharide which contains a unique sugar, colitose. Otherwise, O139 and O1 are strikingly similar. The diseases produced by the two serogroups are indistinguishable. There is a moderate inflammatory response in cholera, and in spite of the possession of a capsule by O139, it is no more inflammatory than O1. A number of diagnostic tests have been developed for O139 that are modelled after tests for O1 including rapid tests for field use. O139 has spread to countries of south and south-east Asia, China and Russia. Currently, cholera is caused by both these serogroups in these countries with the O1 serogroup predominant. O139 continues to cause occasional local outbreaks in India and Bangladesh. Genotyping of isolates has shown circulation of multiple clones and their derivation from multiple progenitors. Although there have been changes in antibiogram with the resistance genes being carried on a constin, the strains remain susceptible to tetracycline, a preferred antibiotic for treatment. O139 carries a number of filamentous lysogenic and lytic phages and the latter have been utilized to develop a phage typing scheme. A number of environmental variables have been linked to the occurrence of cholera and lytic phages may determine the course of epidemics. Although the capsule confers serum resistance, vibriocidal antibody assay has been developed for O139. However, unlike in O1 cholera, serum vibriocidal antibody does not seem to correlate with protection against O139 infection. Promising O139 vaccines have been developed or are in the pipeline. It is too early to predict whether O139 will become the causative agent of 'the eighth pandemic of cholera'; it will require many years of monitoring and reporting from regions prone to cholera to find the answer.

Structural organization of the transfer RNA operon I ofVibrio cholerae: Differences between classical and El Tor strains

Nine major transfer RNA (tRNA) gene clusters were analysed in various Vibrio cholerae strains. Of these, only the tRNA operon I was found to differ significantly in V. cholerae classical (sixth pandemic) and El Tor (seventh pandemic) strains. Amongst the sixteen tRNA genes contained in this operon, genes for tRNA Gln3 (CAA) and tRNA Leu6 (CUA) were absent in classical strains as compared to El Tor strains. The observation strongly supported the view that the above two pandemic strains constitute two different clones.

Role of Toll-Like Receptor 4 in the Proinflammatory Response to Vibrio cholerae O1 El Tor Strains Deficient in Production of Cholera Toxin and Accessory Toxins

Following intranasal inoculation, Vibrio cholerae KFV101 (DeltactxAB DeltahapA DeltahlyA DeltartxA) colonizes and stimulates tumor necrosis factor alpha and interleukin 1beta (IL-1beta) in mice, similar to what occurs with isogenic strain P4 (DeltactxAB), but is less virulent and stimulates reduced levels of IL-6, demonstrating a role for accessory toxins in pathogenesis. Morbidity is enhanced in C3H/HeJ mice, indicating that Toll-like receptor 4 is important for infection containment.

A1.11 In vitro susceptibility testing and minimum inhibitory concentration of Vibrio cholerae 01 biotype El Tor strains associated with an outbreak of cholera in Manipal, Southern India

A1.11 In vitro susceptibility testing and minimum inhibitory concentration of Vibrio cholerae 01 biotype El Tor strains associated with an outbreak of cholera in Manipal, Southern India

Emergent Strain of Human Adenovirus Endemic in Iowa

To the Editor: We read with interest the letter by Sarkar et al. on new Vibrio cholerae phages (1). The description of new V. cholerae phages is a welcome tool for epidemiologic studies of this species. Our main concern about their work is the inaccurate picture that is presented of the cholera epidemic in Brazil. Some of the statements made in the final paragraphs are in disagreement with the official epidemiologic records and the characteristics of the Vibrio bacteria that occurred in Brazil during the 1990s epidemic (2). In 1991, the seventh cholera pandemic reached South America by the Pacific coast, spreading to Brazil in the same year (3). In Brazil, the first cholera cases were reported in the Amazon region bordering Peru; within a few months a large number of cholera cases were recorded in states facing the Atlantic Ocean in the northeastern region (2). According to the official figures of the Brazilian Ministry of Health (2), 168,598 cases of cholera caused by a V. cholerae O1 El Tor strain occurred in Brazil from 1991 to 2001. Of these, 155,363 (92.1%) occurred in the northeastern area of the country, with 2,037 deaths. From 2001 to 2003, the number of confirmed cases was 4,756, 734, and 7, respectively. Sarkar et al. (1) indicate that 60,000 cases occurred from 1991 to 2001 in Rio de Janeiro, a city localized in the southeastern region; the official records report only 349 cases. The statement that “since 1993, no cholera cases caused by O1 have been reported” is also perplexing. From 1994 to 2001, the official records report 68,583 cases of cholera in Brazil (51,324 of these in 1994, the second major year of cholera incidence). Are the authors suggesting that this number of cases was caused by non-O1 V. cholerae? The official records state that the cholera epidemic in Brazil was caused by an El Tor O1 strain (4,5).

Identification of the gene for the monomeric alkaline phosphatase of Vibrio cholerae serogroup O1 strain

Alkaline phosphatase (APase) of Vibrio cholerae is the first monomeric alkaline phosphatase reported [Roy, N.K., Ghosh, R.K., Das, J., 1982a. Monomeric alkaline phosphatase of V. cholerae. J. Bacteriol. 150, 1033–1039.]. The gene ( phoA VC ) encoding this enzyme is not identified in the published genome sequence of the V. cholerae serogroup O1 El Tor strain N16961 [Heidelberg et al., 2000, DNA sequence of both the chromosome of cholera pathogen V. cholerae. Nature 406, 477–484.]. However two genes ( phoB VC and phoR VC ) regulating the synthesis of alkaline phosphatase in this organism, equivalent to phoB and phoR of Escherichia coli, are located in tandem on chromosome I of V. cholerae. We have identified the phoA VC gene on the N16961 genome sequence by amino acid sequence analysis of the purified alkaline phosphatase of V. cholerae classical strain 569B followed by BLAST search. The gene was found to be located on the hypothetical protein locus VCA0033 of chromosome II. The identity of the gene was confirmed by expressing the cloned VCA0033 locus in phoA mutant E. coli E15 and JC9223 cells and isolating V. cholerae monomeric alkaline phosphatase. Insertional inactivation of the gene also resulted in complete loss of the phenotype. Unlike in E. coli where phoB, phoR and phoA are closely linked, phoA VC is not linked to phoB VC and phoR VC .

Cholera in Mozambique, Variant ofVibrio cholerae

Cholera in Mozambique, Variant of<i>Vibrio cholerae</i>

Survival of &lt;i&gt;Vibrio cholerae&lt;/i&gt; in industrially polluted water, with particular reference to iron concentrations

High levels of iron are often found in surface waters due to the discharge of iron-containing industrial effluents. The effect of iron as well as pH on the survival of Vibrio cholerae (non-O1, El Tor and classical strains) in water samples from 12 points, where selected industrial effluents were discharged into rivers, was studied. All three V. cholerae strains survived longest during spring. V. cholerae non -O1 survived significantly longer than El Tor and classical strains. At pH 8.0, the survival of V. cholerae increased with increase in iron concentration (1 to10 mg/&#8467;) of polluted water. At lower than pH 8.0, the effect of iron was not significant. In polluted water, at pH 8.0, the presence of iron resulted in prolonged V. cholerae survival at lower concentrations than those required for survival in distilled water. We suggest that chemical contamination of water may be one of the important factors instrumental in the subsequent ability of V. cholerae to persist, multiply and survive in the aquatic environment. Water SA Vol.30(1): 65-69

Polylysogeny and prophage induction by secondary infection in Vibrio cholerae.

Strains of Vibrio cholerae O1, biotypes El Tor and classical, were infected with a known temperate phage (PhiP15) and monitored over a 15-day period for prophage induction. Over the course of the experiment two morphologically and three genomically distinct virus-like particles were observed from the phage-infected El Tor strain by transmission electron microscopy and field inversion gel electrophoresis, respectively, whereas only one phage, PhiP15, was observed from the infected classical strain. In the uninfected El Tor culture one prophage was spontaneously induced after 6 days. No induction in either strain was observed after treatment with mitomycin C. Data indicate that El Tor biotypes of V. cholerae may be polylysogenic and that secondary infection can promote multiple prophage induction. These traits may be important in the transfer of genetic material among V. cholerae by providing an environmentally relevant route for multiple prophage propagation and transmission.

Analysis of the Vibrio pathogenicity island-encoded Mop protein suggests a pleiotropic role in the virulence of epidemic Vibrio cholerae

Epidemic Vibrio cholerae contain a large essential virulence gene cluster called the Vibrio pathogenicity island (VPI). We recently reported that no in vitro difference in virulence was found in El Tor strain N16961 containing a mutation in the VPI-encoded mop gene but this mutant was hypervirulent and reactogenic in rabbit ileal loops. In this paper, we report in vitro studies showing that independent Mop mutants of strain 3083 are significantly attenuated (∼40-fold) in cholera toxin (CT) production and have significantly increased motility and biofilm forming ability but appear to be unaffected in TcpA, hemagglutinin protease and hemolysin compared to their parent. The 3083 Mop mutant showed a 100-fold decrease in its in vivo intestinal colonization ability in the infant mouse competition assays. While reverse transcription polymerase chain reaction and phenotypic studies of a mop plasmid in both mutant and wild-type backgrounds suggest Mop is expressed by the plasmid, the differences in CT and biofilm formation could not be restored in any of the mutants. The inability to complement the Mop mutants in trans may be due either to the selection of secondary mutations or to mop possibly being part of an operon. Our findings that Mop is associated with CT, motility, biofilm formation and intestinal colonization support a hypothesis in which Mop has a pleiotropic role in the pathogenesis and persistence of epidemic V. cholerae.

Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage.

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin, which is encoded by a lysogenic filamentous bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor that is also the receptor for CTXPhi. The genes involved in the biosynthesis of TCP reside in a pathogenicity island, which has been reported to correspond to the genome of another filamentous phage (designated VPIPhi) and to encode functions necessary for the production of infectious VPIPhi particles. We examined 46 V. cholerae strains having diverse origins and carrying different genetic variants of the TCP island for the production of the VPIPhi and CTXPhi in different culture conditions, including induction of prophages with mitomycin C and UV irradiation. Although 9 of 10 V. cholerae O139 strains and 12 of 15 toxigenic El Tor strains tested produced extracellular CTXPhi, none of the 46 TCP-positive strains produced detectable VPIPhi in repeated assays, which detected as few as 10 particles of a control CTX phage per ml. These results contradict the previous report regarding VPIPhi-mediated horizontal transfer of the TCP genes and suggest that the TCP island is unable to support the production of phage particles. Further studies are necessary to understand the mechanism of horizontal transfer of the TCP island.

Class I integrons and SXT elements in El Tor strains isolated before and after 1992 Vibrio cholerae O139 outbreak, Calcutta, India.

We examined the distribution of class I integrons and SXT elements in Vibrio cholerae O1 El Tor strains, isolated in Calcutta, India, before and after the V. cholerae O139 outbreak in 1992. Class I integrons, with aadA1 gene cassette, were detected primarily in the pre-O139 strains; the SXT element was found mainly in the post-O139 strains.