Search

Vibrio cholerae, the Gram-negative facultative pathogen, resides in the aquatic environment and infects humans and causes diarrhoeagenic cholera. Although the environment differs drastically, V. cholerae thrives in both of these conditions aptly and chitinases play a vital role in their persistence and nutrient acquisition. Chitinases also play a role in V. cholerae pathogenesis. Chitinases and its downstream chitin utilization genes are regulated by sensor histidine kinase ChiS, which also plays a significant role in pathogenesis. Recent exploration suggests that CytR, a transcription factor of the LacI family in V. cholerae, also regulates chitinase secretion in environmental conditions. Since chitinases and chitinase regulator ChiS is involved in pathogenesis, CytR might also play a significant role in pathogenicity. However, the role of CytR in pathogenesis is yet to be known. This study explores the regulation of CytR on the activation of ChiS in the presence of mucin and its role in pathogenesis. Therefore, we created a CytR isogenic mutant strain of V. cholerae (CytR¯) and found considerably less β-hexosaminidase enzyme production, which is an indicator of ChiS activity. The CytR¯ strain greatly reduced the expression of chitinases chiA1 and chiA2 in mucin-supplemented media. Electron microscopy showed that the CytR¯ strain was aflagellate. The expression of flagellar-synthesis regulatory genes flrB , flrC and class III flagellar-synthesis genes were reduced in the CytR¯ strain. The isogenic CytR mutant showed less growth compared to the wild-type in mucin-supplemented media as well as demonstrated highly retarded motility and reduced mucin-layer penetration. The CytR mutant revealed decreased adherence to the HT-29 cell line. In animal models, reduced fluid accumulation and colonization were observed during infection with the CytR¯ strain due to reduced expression of ctxB , toxT and tcpA . Collectively these data suggest that CytR plays an important role in V. cholerae pathogenesis.

Staphylococcus aureus is a leading aetiologic agent of nosocomial- and community-acquired infectious diseases worldwide. The public health concern regarding staphylococcal infections is inflated by the increasing occurrence of multidrug-resistant strains, e.g. multidrug- and meticillin-resistant S.aureus (MDR MRSA). This study was designed to evaluate the intracellular killing, membrane-damaging and biofilm-inhibitory activities of nimbolide isolated from Azadirachta indica against MDR MRSA. In vitro antibacterial activity of nimbolide was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity was determined by membrane perturbation and scanning electron microscopy (SEM) examination. Biofilm-inhibitory activities were determined by SEM. Cellular drug accumulation and assessments of intracellular activities were performed using Vero cell culture. SEM revealed that nimbolide caused significant membrane damage and lysis of the S. aureus cells. The biofilm structure was disrupted, and the biofilm formation was greatly reduced in the presence of nimbolide as examined by SEM. The level of accumulation of nimbolide in Vero cells incubated for 24 h is relatively higher than that of ciprofloxacin and nalidixic acid (Cc/Ce for nimbolide > ciprofloxacin and nalidixic acid). The viable number of intracellular S. aureus was decreased [reduction of ~2 log10 c.f.u. (mg Vero cell protein)-1] in a time-dependent manner in the presence of nimbolide (4× MBC) that was comparable to that of tetracycline and nalidixic acid. The significant intracellular, biofilm-inhibitory and bacterial membrane-damaging activities of nimbolide demonstrated here suggested that it has potential as an effective antibacterial agent for the treatment of severe infections caused by MDR MRSA.

The composition of Escherichia coli in the neonatal gut has rarely been studied in developing countries. To gain insight into the composition of E. coli in the neonatal gut and to assess factors that could influence colonization by E. coli, analysis of the phylogenetic groups and virulence determinants of E. coli isolated from the guts of neonates in a tertiary care hospital was carried out. The distribution of the phylogroups of 124 E. coli isolates recovered showed that phylogroups A (23 %) and B1 (49 %) accounted for 72 % of the isolates. Isolates of the phylogenetic group B2 were rare (8 %). Virulence factors were also rare with the exception of aerobactin (iucC), which was detected in 45 % of the isolates and was significantly associated with phylogroup B1. Multinomial logistic regression established that colonization with phylogroup B1 was associated with a stay in the neonatal intensive care unit; phylogroup A was associated with a stay on the ward; and phylogroups B2 and D were associated with neonates delivered vaginally. Evaluation of the effect of different E. coli phylogroups, with and without identified virulence determinants, on the gut of neonatal mice showed histopathological changes in the mucosa. The severity of the changes could be correlated with the presence of virulence determinants, irrespective of the phylogroup.

Five wild-type mutant strains of Vibrio cholerae serogroup O1 that lacked the CTX virulence cassette, or contained a natural deletion of a virulence gene within the CTX virulence cassette, or possessed an additional virulence gene, along with a prototype toxigenic strain representing the El Tor classical biotypes were examined by in-vivo and in-vitro methods to determine their enterotoxic potential. The ability of whole cells and culture supernates of the strains to cause fluid accumulation in the rabbit ileal loop model revealed a pattern consistent with the presence of the various virulence gene(s), with those possessing the intact CTX virulence cassette being the most secretogenic. Culture supernates of strains without the CTX virulence cassette or the strain with an incomplete cassette were also able to evoke mild to moderate fluid accumulation in the rabbit ileal loop. Of the various media used, AKI and brain heart infusion broth appeared to support the production of a hitherto unknown secretogenic factor, because culture supernates of the non-toxigenic V. cholerae O1 strains showed higher fluid accumulation ratios when grown in these media than in the others. To confirm that the fluid accumulation elicited by these strains in the ileal loop was due to enterotoxin activity, the effect of supernate of the strains was examined in rabbit small intestine mounted on Ussing chambers. Increases in short circuit current and tissue conductance, as compared with the medium control, were observed even with the strains that did not possess the CTX virulence cassette, confirming their ability to disrupt the function of intestinal tissue. From these studies, it was concluded that strains of V. cholerae O1 devoid of the CTX virulence cassette were still able to elicit a secretory response in the ileal loop and displayed enterotoxic activity in an in-vitro experimental model.