Ranjan K. Nandy

Microbial Etiology of Travelers' Diarrhea in Mexico, Guatemala, and India: Importance of Enterotoxigenic Bacteroides fragilis and Arcobacter Species

ABSTRACT This study examined established enteric pathogens, Arcobacter species and enterotoxigenic Bacteroides fragilis (ETBF), in 201 U.S. and European travelers with acute diarrhea acquired in Mexico, Guatemala, and India. Arcobacter butzleri and ETBF were detected in 8% and 7% of diarrhea cases, respectively.

Intestinal Adherence of Vibrio cholerae Involves a Coordinated Interaction between Colonization Factor GbpA and Mucin

ABSTRACT The chitin-binding protein GbpA of Vibrio cholerae has been recently described as a common adherence factor for chitin and intestinal surface. Using an isogenic in-frame gbpA deletion mutant, we first show that V. cholerae O1 El Tor interacts with mouse intestinal mucus quickly, using GbpA in a specific manner. The gbpA mutant strain showed a significant decrease in intestinal adherence, leading to less colonization and fluid accumulation in a mouse in vivo model. Purified recombinant GbpA (rGbpA) specifically bound to N-acetyl-d-glucosamine residues of intestinal mucin in a dose-dependent, saturable manner with a dissociation constant of 11.2 μM. Histopathology results from infected mouse intestine indicated that GbpA binding resulted in a time-dependent increase in mucus secretion. We found that rGbpA increased the production of intestinal secretory mucins (MUC2, MUC3, and MUC5AC) in HT-29 cells through upregulation of corresponding genes. The upregulation of MUC2 and MUC5AC genes was dependent on NF-κB nuclear translocation. Interestingly, mucin could also increase GbpA expression in V. cholerae in a dose-dependent manner. Thus, we propose that there is a coordinated interaction between GbpA and mucin to upregulate each other in a cooperative manner, leading to increased levels of expression of both of these interactive factors and ultimately allowing successful intestinal colonization and pathogenesis by V. cholerae.

Classical ctxB in Vibrio cholerae O1, Kolkata, India

To the Editor: Among the 206 serogroups of Vibrio cholerae, O1 and O139 are associated with epidemic cholera. Serogroup O1 is classified into 2 biotypes, classical and El Tor. Conventionally, the 2 biotypes can be differentiated on the basis of a set of phenotypic traits. Comparative genomic analysis has shown variations in different genes between these biotypes (1). Cholera toxin (CT), the major toxin responsible for the disease cholera, has 2 epitypes or immunologic forms, CT1 and CT2 (2). Another classification recognizes 3 genotypes on the basis of the ctxB gene sequence variation (3). In the past few years, a new emerging form of V. cholerae O1, which possesses traits of both classical and El Tor biotypes, has been isolated in Bangladesh (4,5), Mozambique (6), Vietnam, Hong Kong, Japan, and Zambia (7). These strains were variously labeled as Matlab variants, hybrids, or altered El Tor strains. Our study analyzed, in chronological order, strains of V. cholerae O1 that were isolated over 17 years (1989–2005). We used strains isolated during diarrhea surveillance conducted at the Infectious Diseases Hospital, Kolkata (Calcutta), to determine precisely when the hybrid strains appeared in this region. A total of 171 strains of V. cholerae O1, which were selected to cover different months of each year, were included in this study, along with 2 reference strains for classical and El Tor biotypes. The V. cholerae strains were confirmed serologically by slide agglutination using a specific polyvalent antiserum to V. cholerae O1. We focused on the ctxB gene. The strains were examined by mismatch amplification mutation assay (MAMA)–based PCR for detecting the ctxB allele; a common forward primer was used for 2 alleles, FW-Com (5′-ACTATCTTCAGCATATGCACATGG-3′); and 2 allele-specific primers, Re-cla (5′-cctggtacttctacttgaaacg-3′) and Re-elt (5′-CCTGGTACTTCTACTTGAAACA-3′), were used for classical and El Tor biotypes, respectively (8). Results of the MAMA-PCR are summarized in the Table. All of the 123 V. cholerae O1 strains from 1995 through 2005 yielded only the classical type of ctxB, which indicates that since 1995 the classical type has completely replaced the El Tor type ctxB (Table). To reconfirm our PCR-based results, we selected 25 representative strains for DNA sequencing of the ctxB gene. The deduced amino acid sequences were aligned with the CtxB sequences of reference strains N16961 (El Tor) and O395 (classical). The deduced amino acid sequences of all 25 strains were identical to those of the classical reference strain; histidine was at position 39 and threonine was at position 68. Thus, the results from DNA sequencing of the ctxB gene confirmed the MAMA-PCR results. Table Prevalence of different types of ctxB alleles among Vibrio cholerae O1 strains, Kolkata, India, 1989–2005 Our results highlight a noteworthy event in the evolution of recent V. cholerae strains. Analysis of type ctxB that had been circulating in Kolkata for 17 years (1989–2005) showed that in 1989 only the El Tor allele of ctxB was present. Our results further indicate that classical type ctxB emerged in 1990, although El Tor type ctxB was still present in almost equal numbers during that year. During 1991, a unique event took place when the classical type became predominant, along with strains having both classical and El Tor type ctxB. In 1994, isolation of strains with El Tor ctxB became rare, and the major ctxB allele was of the classical type. V. cholerae O1 strains from 1995 onward were found to carry classical type ctxB, which totally replaced the El Tor type ctxB allele. Replacement of El Tor type ctxB by the classical allele has been reported in Bangladesh since 2001 (5), but this event seems to have occurred earlier in Kolkata. Perhaps the new type of El Tor strains arose when V. cholerae strains with typical seventh pandemic El Tor genetic background were replaced with strains having the ctxB gene, possibly driven by selective pressure to survive and adapt better in host intestines. Considering the increase in the global prevalence of cholera (9), the origin and spread of these new variants of V. cholerae strains should be tracked in the population by genome analysis. Finally, this study has described a brief period from February 1991 through December 1992 when El Tor strains had CTX prophages of both classical and El Tor types (data not shown), along with the ctxB of both biotypes. Notably, this period coincided with an unprecedented event in the history of cholera—the genesis of the O139 serogroup. After this serogroup reemerged in 1996, it harbored 2 types of CTX prophages, namely, El Tor and Calcutta (10). Furthermore, these strains with ctxB of both biotypes might also have had a pivotal role behind the emergence of El Tor strains with classical ctxB. Further studies are warranted to determine whether any distinct relationship exists between these overlapping events.

Molecular characterization of a new variant of toxin-coregulated pilus protein (TcpA) in a toxigenic non-O1/Non-O139 strain of Vibrio cholerae.

ABSTRACT A toxigenic non-O1/non-O139 strain of Vibrio cholerae(10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (tcpA) as determined by PCR and Southern hybridization experiments. Nucleotide sequence analysis data of the new tcpA gene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively. The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively. An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain. Incidentally, the tcpA sequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classicaltcpA. Further, tcpA of another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor tcpA. Analysis of these results with those already available in the literature suggests that there are at least four major variants of the tcpA gene in V. cholerae which probably evolved in parallel from a common ancestral gene. Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure.

Vibrio pathogenicity island and cholera toxin genetic element-associated virulence genes and their expression in non-O1 non-O139 strains of Vibrio cholerae.

ABSTRACT A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (aldA, tagA, tcpP/H, toxT, acfB/C, and int) and intergenic regions (tcpF to toxT and tcpH to tcpA) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30°C and pH 6.5) led to transcriptional activation of tcpP/H, toxR, toxT, and tcpA genes, but not of ctxA, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V. cholerae O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT, ctxA, and tcpA when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly (∼1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT, ctxA, or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V. cholerae strains belonging to non-O1 serogroups.

Vibrio cholerae Non-O1, Non-O139 Serogroups and Cholera-like Diarrhea, Kolkata, India

We identified 281 Vibrio cholerae non-O1, non-O139 strains from patients with diarrhea in Kolkata, India. Cholera-like diarrhea was the major symptom (66.0%); some patients (20.3%) had severe dehydration. These strains lacked the ctxA gene but many had hlyA, rtxA, and rtxC genes. Pulsed-field gel electrophoresis showed no genetic link among strains.

Trends in the epidemiology of pandemic and non-pandemic strains of Vibrio parahaemolyticus isolated from diarrheal patients in Kolkata, India.

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.

Spread of Cholera with Newer Clones of Vibrio cholerae O1 El Tor, Serotype Inaba, in India

ABSTRACT During 2004 and 2005, cholera was recorded in 15 states of India, with 7 outbreaks. The newly emerged Vibrio cholerae O1 Inaba had a different antibiogram and ribotype, different pulsotypes, and different mutations in the wbeT gene. Due to the absence of serogroup O139, the Inaba serotype may have acquired the potential to affect the population at large.

The Entner-Doudoroff Pathway Is Obligatory for Gluconate Utilization and Contributes to the Pathogenicity of Vibrio cholerae

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae, the causative agent of cholera. In this study, activation of the genes edd and eda, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (ctxA and tcpA) and their regulator (toxT). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δedd mutation [Δedd(N16961) strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.

PCR-Based Identification of Common Colonization Factor Antigens of Enterotoxigenic Escherichia coli

ABSTRACT Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli (ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes.