Pallavi Garg

Occurrence of Antibiotic Resistance Gene Cassettes aac(6′)-Ib, dfrA5, dfrA12, and ereA2 in Class I Integrons in Non-O1, Non-O139 Vibrio cholerae Strains in India

ABSTRACT Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6′)-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6′)-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.

Matrix metalloproteinases in inflammatory bowel disease: boon or a bane?

Abstract Matrix metalloproteinases (MMPs) are a family of Zn2+‐dependent extracellular matrix (ECM) degrading endopeptidases that share common functional domains, activation mechanisms, and collectively have the capacity to degrade all types of ECM proteins. In addition to playing a central role in ECM turnover, MMPs proteolytically activate or degrade a variety of nonmatrix substrates including chemokines, cytokines, growth factors, and junctional proteins. Thus, they are increasingly recognized as critical players in inflammatory response. Indeed, accumulating data from several studies indicate that they are the predominant proteases involved in the pathogenesis of inflammatory bowel disease (IBD) via their influence on the function and migration of inflammatory cells, mucosal ulceration, as well as matrix deposition and degradation. Some MMPs are constitutively expressed and play a protective role in IBD through their effect on cellular homeostasis, while others are induced during inflammation‐mediated tissue damage. This article focuses on the role of the various MMPs in IBD, discussing their physiologic and pathogenetic role in the context of intestinal defense, mucosal inflammatory response, and immune cell‐epithelial interaction. (Inflamm Bowel Dis 2007;13:97–107)

Matrix metalloproteinase-9-mediated tissue injury overrides the protective effect of matrix metalloproteinase-2 during colitis

Matrix metalloproteinases (MMP) play an important role in pathogenesis of inflammatory bowel disease (IBD). Two known gelatinases, MMP-2 and MMP-9, are upregulated during IBD. Epithelial-derived MMP-9 is an important mediator of tissue injury in colitis, whereas MMP-2 protects against tissue damage and maintains gut barrier function. It has been suggested that developing strategies to block MMP-9 activity in the gut might be of benefit to IBD. However, given that MMP-2 and MMP-9 are structurally similar, such approaches would also likely inhibit MMP-2. Thus, to gain insight into outcome of inhibiting both MMP-2 and MMP-9, MMP-2(-/-)/MMP-9(-/-) double knockout mice (dKO) lacking both MMP-2 and MMP-9 were used in this study. Three models of murine colitis were used: dextran sodium sulfate (DSS), Salmonella typhimurium (S.T.), and trinitrobenzene sulfonic acid (TNBS). Our data demonstrate that MMP-2 and MMP-9 activities were highly upregulated in wild-type (WT) mice treated with DSS, S.T., or TNBS whereas dKO mice were resistant to the development of colitis. WT mice had extensive inflammation and tissue damage compared with dKO mice as suggested by histological assessment and myeloperoxidase activity. In conclusion, these results suggest an overriding role of MMP-9 in mediating tissue injury compared with the protective role of MMP-2 in development of colitis. Thus inhibition of MMP-9 may be beneficial in treatment of colitis even if resulting in inhibition of MMP-2.

Selective Ablation of Matrix Metalloproteinase-2 Exacerbates Experimental Colitis: Contrasting Role of Gelatinases in the Pathogenesis of Colitis

The matrix metalloproteinases (MMPs), MMP-2 and MMP-9, share structural and substrate similarities and are up-regulated during human as well as animal models of inflammatory bowel disease. We recently demonstrated that epithelial-derived MMP-9 is an important mediator of inflammation and tissue damage in colitis. In this study, we examined the role of MMP-2 in acute colitis. Colitis was induced using two models, administration of dextran sodium sulfate (DSS) and Salmonella enterica subsp. serovar Typhimurium (S.T.). Bone marrow chimeras were performed using bone marrow cells from wild-type (WT) and MMP-2−/− mice. Colitis was evaluated by clinical symptoms, myeloperoxidase assay, and histology. MMP-2 protein expression and activity were up-regulated in WT mice treated with DSS or S.T. MMP-2−/− mice were highly susceptible to the development of colitis induced by DSS (or S.T.) compared with WT. During inflammation, MMP-2 expression was increased in epithelial cells as well as in the infiltrating immune cells. Bone marrow chimera demonstrated that mucosa-derived MMP-2 was required for its protective effects toward colitis. Furthermore, we demonstrate that severe colitis in MMP-2−/− is not due to a compensatory increase in MMP-9. Finally, we show that MMP-2 regulates epithelial barrier function. In contrast to MMP-9, mucosa-derived MMP-2 may be a critical host factor that is involved in the prevention or cessation of the host response to luminal pathogens or toxins, an important aspect of healing and tissue resolution. Together, our data suggest that a critical balance between the two gelatinases determines the outcome of inflammatory response during acute colitis.

Emergence of fluoroquinolone-resistant strains of Vibrio cholerae O1 biotype El Tor among hospitalized patients with cholera in Calcutta, India.

Ciprofloxacin and norfloxacin are broad-spectrum fluoroquinolones and possess excellent activity against Vibrio cholerae O1 and O139 serogroups (6). Clinical studies have shown that these drugs are effective in treatment of cholera in adults and children (2–4). For the past 11 years, we have been monitoring the incidence of antibiotic susceptibility and genotypic changes in V. cholerae isolates from cholera patients admitted at the Infectious Diseases Hospital, Calcutta, India. Here, we report the emergence of fluoroquinolone-resistant strains of V. cholerae O1 Biotype El Tor among hospitalized patients with cholera. Antimicrobial susceptibility analysis of V. cholerae strains was performed by disk diffusion (1) on Mueller-Hinton agar (Difco, Detroit, Mich.) with commercial disks (HiMedia, Mumbai, India). The following antibiotic disks were used: ciprofloxacin, 5 μg; nalidixic acid, 30 μg; norfloxacin, 10 μg; and tetracycline, 30 μg. Characterization of strains as susceptible, intermediately resistant, or resistant was based on the size of the inhibition zone according to the manufacturers instructions. These zone size interpretive criteria for susceptibility corresponded to MICs of 0.25, 0.06, and 0.06 μg/ml for nalidixic acid, norfloxacin, and ciprofloxacin, respectively. Strains showing intermediate zones of growth inhibition were classified as resistant on the basis of previous MIC studies with V. cholerae (6). We have reported considerable increases in fluoroquinolone resistance among V. cholerae strains belonging to non-O1, non-O139 serogroups during 1996 (5). All the V. cholerae strains of serogroup O1 isolated in or before 1994 are susceptible to ciprofloxacin. From 1995, we have recorded progressive increases in ciprofloxacin and norfloxacin resistance among V. cholerae O1 strains, with the highest occurrences of 38.8% in 1999 and 25% in 2000, respectively (Table ​(Table1).1). To our knowledge, this is the first report on such high incidence of fluoroquinolone resistance among toxigenic V. cholerae O1 strains. The MICs of ciprofloxacin and norfloxacin for ciprofloxacin-resistant V. cholerae strains ranged between 9 and >32 μg/ml and between 192 and >256 μg/ml, respectively, when tested with the E-test strips on Mueller-Hinton agar (AB Biodisk, Solna, Sweden). The incidence of nalidixic acid resistance among V. cholerae O1 strains was low (<10%) before 1993 and peaked during subsequent years (1999; 100%), as shown in the Table ​Table1.1. Possibly, ciprofloxacin resistance might have emerged in direct response to the selective pressure exerted by nalidixic acid coupled with disproportionate use of fluoroquinolones in the clinical settings. It is worth to mention here that the increase in the incidence of nalidixic acid-resistant strains of V. cholerae O1 (probably with a single mutation in gyrA and/or other related genes) portended a further increase in the incidence of strains with clinically significant resistance to fluoroquinolones (with two or more mutations in the gyrA gene). We are in the process of identifying the mutational “hot spots” in the quinolone resistance-determining region. TABLE 1 Resistance to quinolones among V. cholerae O1 and O139 isolates from cholera patients An interesting observation in the present report is the low incidence of quinolone resistance in the O139 serogroup. One possible elucidation is the low frequency of nalidixic acid resistance among V. cholerae O139 strains (Table ​(Table1),1), and therefore the frequency of double mutations, a prerequisite for fluoroquinolone resistance, is low, as reflected by resistance O139 strains to fluoroquinolones. In this study we encountered a higher incidence of V. cholerae O1 resistance to ciprofloxacin than to norfloxacin, which is generally less potent than ciprofloxacin. The possible explanation for this counterintuitive result is (i) since ciprofloxacin is in extensive use for all the bacterial infections in this part of the world, conditions of high selective pressure would have forced the mutant V. cholerae strains to multiply and establish themselves as the dominant population; continued selective pressure favored these progeny to have further mutations; (ii) ciprofloxacin and norfloxacin breakpoints are not comparable, at least for V. cholerae strains (the MIC for 50% of the strains [MIC50], MIC90, and MIC ranges need to be determined for both drugs to prove this hypothesis); or (iii) ciprofloxacin and norfloxacin accumulation kinetics might differ among V. cholerae O1 strains. Additional, extensive studies are needed to test these possibilities in order to determine the mechanisms responsible for heterogeneous fluoroquinolone resistance among V. cholerae O1 strains. Emergence of fluoroquinolone resistance in V. cholerae will certainly complicate the therapeutic use of these drugs, and attention must be paid to this trend. Fortunately, V. cholerae O1 and O139 strains are susceptible to tetracycline, which is an effective drug for treatment of cholera at the Infectious Disease Hospital.

Molecular epidemiology of O139 Vibrio cholerae: mutation, lateral gene transfer, and founder flush.

Vibrio cholerae in O-group 139 was first isolated in 1992 and by 1993 had been found throughout the Indian subcontinent. This epidemic expansion probably resulted from a single source after a lateral gene transfer (LGT) event that changed the serotype of an epidemic V. cholerae O1 El Tor strain to O139. However, some studies found substantial genetic diversity, perhaps caused by multiple origins. To further explore the relatedness of O139 strains, we analyzed nine sequenced loci from 96 isolates from patients at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000. We found 64 novel alleles distributed among 51 sequence types. LGT events produced three times the number of nucleotide changes compared to mutation. In contrast to the traditional concept of epidemic spread of a homogeneous clone, the establishment of variant alleles generated by LGT during the rapid expansion of a clonal bacterial population may be a paradigm in infections and epidemics.

Concomitant Infection of Enterotoxigenic Escherichia coli in an Outbreak of Cholera Caused by Vibrio cholerae O1 and O139 in Ahmedabad, India

ABSTRACT In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V. cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli(ETEC) occurred in January 2000. All of the representative V. cholerae O1 and O139 isolates examined harbored thectxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus). ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin. To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping showed that the isolates of V. cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V. cholerae O139. PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India. PFGE analysis of the V. cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta. ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes. Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid. Likewise, all of the E. coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid. This is a unique outbreak, and we believe that it is the first in which V. cholerae and ETEC were concomitantly involved.

Matrix Metalloproteinase-9 Functions as a Tumor Suppressor in Colitis-Associated Cancer

There is a well-documented association of matrix metalloproteinase-9 (MMP-9) and receptor Notch-1 overexpression in colon cancer. We recently showed that MMP-9 is also upregulated in colitis, where it modulates tissue damage and goblet cell differentiation via proteolytic cleavage of Notch-1. In this study, we investigated whether MMP-9 is critical for colitis-associated colon cancer (CAC). Mice that are wild type (WT) or MMP-9 nullizygous (MMP-9(-/-)) were used for in vivo studies and the human enterocyte cell line Caco2-BBE was used for in vitro studies. CAC was induced in mice using an established carcinogenesis protocol that involves exposure to azoxymethane followed by treatment with dextran sodium sulfate. MMP-9(-/-) mice exhibited increased susceptibility to CAC relative to WT mice. Elevations in tumor multiplicity, size, and mortality were associated with increased proliferation and decreased apoptosis. Tumors formed in MMP-9(-/-) mice exhibited expression of p21(WAF1/Cip1) and increased expression of beta-catenin relative to WT mice. In vitro studies of MMP-9 overexpression showed increased Notch-1 activation with a reciprocal decrease in beta-catenin. Notch and beta-catenin/Wnt signaling have crucial roles in determining differentiation and carcinogenesis in gut epithelia. Despite being a mediator of proinflammatory responses in colitis, MMP-9 plays a protective role and acts as a tumor suppressor in CAC by modulating Notch-1 activation, thereby resulting in activation of p21(WAF1/Cip1) and suppression of beta-catenin.

Epidemiological study of Vibrio cholerae using variable number of tandem repeats

By conventional genetic methods, including pulse-field gel electrophoresis and multilocus sequence typing, most pathogenic, cholera toxin-positive O1 and O139 isolates of Vibrio cholerae cannot be distinguished. We evaluated relationships among 173 V. cholerae isolates collected between 1992 and 2007 from different geographic areas in India by analyzing five variable number of tandem repeat (VNTR) loci. Each VNTR locus was highly variable, with between 5 and 19 alleles. eburst analysis revealed four large groups of genetically related isolates. Two groups contained genotypes of isolates with the O139 serogroup (which emerged for the first time in epidemic form in 1992), with the other two groups containing O1 strains. In subsequent analysis, it was possible to track the spread of specific genotypes across time and space. Our data highlight the utility of the methodology as an epidemiologic tool for assessing spread of isolates in both epidemic and endemic settings.

Comparison of antibiogram, virulence genes, ribotypes and DNA fingerprints of Vibrio cholerae of matching serogroups isolated from hospitalised diarrhoea cases and from the environment during 1997–1998 in Calcutta, India

This study identified 17 matching serogroups of Vibrio cholerae belonging to serogroups other than O1 and O139 isolated from human cases and from the environment during a concurrent clinical and environmental study conducted in Calcutta, a cholera endemic area. Isolates within these matching serogroups were compared by various phenotypic and genotypic traits to determine if the environment was the source of the organisms associated with the disease. Clinical strains of V. cholerae were resistant to a greater number of drugs and exhibited multi-drug resistance compared with their environmental counterparts. Except for the presence of the genes for the El Tor haemolysin and the regulatory element ToxR in most of the strains of V. cholerae examined, non-O1, non-O139 V. cholerae strains lacked most of the other known virulence traits associated with toxigenic V. cholerae O1 or O139. Restriction fragment-length polymorphism of virulence-associated genes, ribotypes and DNA fingerprints of strains of matched serogroups showed considerable diversity, although some gene polymorphisms and ribotypes of a few strains of different serogroups were similar. It is concluded that despite sharing the same serogroup, environmental and clinical isolates were genetically heterogeneous and were of different lineages.