Inhibition of growth and virulence of Vibrio cholerae by carvacrol, an essential oil component of Origanum spp.

AIMS: In the age where bacterial resistance to conventional antibiotics is increasing at an alarming rate, the use of the traditional plant, herb extracts or other bioactive constituents is gradually becoming popular as an anti-virulence agent to treat pathogenic diseases. Carvacrol, a major essential oil fraction of Oregano, possesses a wide range of bioactivities. Therefore, we aimed to study the effect of sub-inhibitory concentrations of carvacrol on major virulence traits of Vibrio cholerae. METHODS AND RESULTS: We have used in vitro as well as ex vivo models to access the anti-pathogenic role of carvacrol. We found that the sub-inhibitory concentration of carvacrol significantly repressed bacterial mucin penetrating ability. Carvacrol also reduced the adherence and fluid accumulation in the rabbit ileal loop model. Reduction in virulence is associated with the downregulated expression of tcpA, ctxB, hlyA and toxT. Furthermore, carvacrol inhibits flagellar synthesis by downregulating the expression of flrC and most of the class III genes. CONCLUSIONS: Carvacrol exhibited anti-virulence activity against V. cholerae, which involved many events including the inhibition of mucin penetration, adhesion, reduced expression of virulence-associated genes culminating in reduced fluid accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate that carvacrol possesses inhibitory activity against V. cholerae pathogenesis and might be considered as a potential bio-active therapeutic alternative to combat cholera.

Identification of common virulence factors present in enterotoxigenic Escherichia coli isolated from diarrhoeal patients in Kolkata, India.

AIMS: Development of an effective vaccine against enterotoxigenic Escherichia coli (ETEC) is largely dependent on the conscientious understanding of different virulence associated factors from diverse geographical areas. So, the objective of this study is to elucidate the distribution of enterotoxins, CF and NCVF in clinical ETEC strains isolated between 2008 and 2014 from two hospitals in Kolkata, India. METHODS AND RESULTS: Multiplex PCR method was used for detection of two enterotoxin genes, 11 common CFs and five common NCVFs. Among the 350 tested ETEC strains, 61% strains possessed est+elt genes, 25% est and 14% elt. Among 56% CF positive ETEC strains, CS21 was the prevalent one (37%) followed by CS6 (36%). NCVF genes were present in 59% of the ETEC strains; eatA was the most prevalent (65%) followed by etpA (51%). There were 29% strains negative for any CFs or NCVFs. CONCLUSIONS: We conclude that a pattern exists between CS6, eatA and toxins. We observed est with or without elt, CS6 with or without CS5 and with or without eatA were present in 24% of clinical ETEC strains (59/250) analysed. CS21 has emerged as another predominant CF but it had diverse CFs and NCVFs. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of ETEC virulence factors would help in tracking ETEC globally and suggests the need of a multivalent ETEC vaccine.

Retrospective genomic analysis of Vibrio cholerae O1 El Tor strains from different places in India reveals the presence of ctxB-7 allele found in Haitian isolates.

A total of 45 strains of Vibrio cholerae O1 isolated from 10 different places in India where they were associated with cases of cholera between the years 2007 and 2008 were examined by molecular methods. With the help of phenotypic and genotypic tests the strains were confirmed to be O1 El Tor biotype strains with classical ctxB gene. Polymerase chain reaction (PCR) analysis by double - mismatch amplification mutation assay PCR showed 16 of these strains carried the ctxB-7 allele reported in Haitian strains. Sequencing of the ctxB gene in all the 45 strains revealed that in 16 strains the histidine at the 20th amino acid position had been replaced by asparagine and this single nucleotide polymorphism did not affect cholera toxin production as revealed by beads enzyme-linked immunosorbent assay. This study shows that the new ctxB gene sequence was circulating in different places in India. Seven representatives of these 45 strains analysed by pulsed - field gel electrophoresis showed four distinct Not I digested profiles showing that multiple clones were causing cholera in 2007 and 2008.

Cross-sectional study of nutritional markers in pregnancy.

OBJECTIVES: To note the value of serum Vitamin B12, folic acid, and ferritin in normal and high-risk pregnancies (HRPs) in patients attending antenatal clinic at All India Institute of Medical Sciences (AIIMS). MATERIALS AND METHODS: This is a cross-sectional study where a total of 282 patients attending Gynaecology Outpatient Department at AIIMS, New Delhi, India were recruited. Among the 282 subjects, 251 were pregnant, and 31 were controls. The serum was tested for serum Vitamin B12, serum folic acid, and serum ferritin levels using Beckman Coulter Access 2 immunoassay. RESULTS: The median value of serum folic acid level in pregnant women was 12 pg/ml with range being 2-20 pg/ml in contrast to 8 pg/ml with range being 3-20 pg/ml in nonpregnant female. This difference was statistically significant. (P = 0.05). There was no significant difference in the median level of serum Vitamin B12 and serum ferritin in pregnant and nonpregnant group. Serum Vitamin B12 level was lower in the third trimester (127 pg/ml) than in first trimester (171 pg/ml) and the difference is statistically significant (P = 0.03). Serum ferritin levels were also significantly lower in the second trimester (16.4 pg/ml) than third trimester (24.55 pg/ml). Although the median serum folic acid level was lower in the first trimester (9.84 pg/ml) than in second trimester (10.8 pg/ml) and in the third trimester (13.18 pg/ml) but the difference was not statistically significant. There was no significant difference in Vitamin B12 level in HRPs (median value 134 pg/ml) as compared to low-risk pregnancies (149.5 pg/ml). CONCLUSION: Serum folic acid levels are significantly higher during pregnancy as compared to nonpregnant state. However, there was no significant difference in the median level of serum Vitamin B12 and serum ferritin in pregnant and nonpregnant group. Serum folic acid level and ferritin level were significantly higher in HRPs compared to low-risk pregnancies.

Cross-sectional study of serum parathyroid hormone level in high-risk pregnancies as compared to nonpregnant control.

OBJECTIVES: To note the value of serum parathyroid hormone (PTH) levels in normal and high-risk pregnancies (HRP) in patients attending antenatal visits at All India Institute of Medical Sciences (AIIMS). MATERIALS AND METHODS: This is a cross-sectional study where a total of 282 patients attending Gynecology Outpatient Department at AIIMS, New Delhi were recruited. Among the 282 subjects, 251 were pregnant, and 31 were controls. The serum was tested for serum PTH levels using Beckman coulter access 2 immunoassay. RESULTS: The median value of PTH level in pregnant women was 31.6 pg/ml with range being 0.8-505.5 pg/ml in contrast to 45.9 pg/ml with range being 19-102.7 pg/ml in nonpregnant female. This difference was statistically significant (P = 0.0012). There was no significant difference in median level of PTH in different age group. Although the median PTH levels were lower in second trimester (25.25 pg/ml) than in first trimester (35.5 pg/ml) and in third trimester (32.4 pg/ml), the difference was not statistically significant. There was no significant difference in PTH level in HRP (median value - 31.6 pg/ml) as compared to low-risk pregnancies (31.5 pg/ml). CONCLUSION: Serum PTH levels are significantly lower during pregnancy as compared to nonpregnant state. However, age, parity, and HRP did not alter PTH level during pregnancy.

Molecular characterization of high-level-cholera-toxin-producing El Tor variant Vibrio cholerae strains in the Zanzibar Archipelago of Tanzania.

Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

Culture-independent real-time PCR reveals extensive polymicrobial infections in hospitalized diarrhoea cases in Kolkata, India.

Culture-independent identification of diarrhoeal aetiological agents was performed using DNA harvested from diarrhoeal stool specimens with SYBR-Green-based real-time PCR targeting Vibrio cholerae, Vibrio parahaemolyticus, Campylobacter spp., Shigella spp. and three different pathotypes of diarrhoeagenic Escherichia coli. Conventional culture-dependent methods detected bacterial enteropathogens in 68 of 122 diarrhoeal stool specimens. Of 68 specimens, 59 (86.8%) had a single pathogen and the remaining nine (13.2%) had polymicrobial infections with multiple pathogens. Re-analysis of the 68 specimens by culture-independent real-time PCR methods showed that 25 (36.8%) specimens contained single pathogen and 43 (63.2%) specimens contained mixed infections with multiple pathogens. The prevalence of such high levels of polymicrobial infections would not have been detected without using real-time PCR. Culture-dependent analysis assigned 54 of the 122 selected archived specimens as 'no known aetiology'. However, re-analysis of these samples by real-time PCR showed the presence of single or multiple pathogens among 34 (63%) of these specimens. Estimation of relative pathogen load by real-time PCR in the stool specimens indicated that the inability of conventional culture-dependent methods to detect the pathogens was related to lower colony-forming units of the pathogen, as reflected by lower C(t) values. Detection of high levels of polymicrobial infection by real-time PCR indicates that in the settings like Kolkata and its surroundings, where cholera and other enteric diseases are endemic, the concept of one pathogen one disease might need to be re-evaluated.

Evaluation of a rapid dipstick test for identifying cholera cases during the outbreak.

BACKGROUND & OBJECTIVES: Intermittent cholera outbreaks are major problem in many of the states of India. It is essential to identify cholera at the earliest for timely mobilization of public health responses and to abort the outbreaks. The present study was a part of a diarrhoeal outbreak investigation in Secunderabad, India, during May 2009 where the usefulness of Crystal VC rapid dipstick kit was assessed for detecting the aetiologic agent of the outbreak. METHODS: Stool specimens were collected from 15 hospitalized patients with acute watery diarrhoea and analyzed for detection of cholera vibrios using Crystal VC rapid dipstick kit and the usefulness of the kit was determined by comparative analysis of the same set of specimens using both microbiological and real-time PCR (RT-PCR) based assays. RESULTS: Detection of Vibrio cholerae O1 from 10 of 15 specimens was recorded using dipstick assay. Microbiological methods detected V. cholerae O1 positivity among 11 specimens. However, RT-PCR based assay showed all 15 specimens positive for the presence of V. cholerae O1. In addition, the same assay showed that the pathogen load in the dipstick as well as RT-PCR positive specimens ranged from 10 6 colony forming units (cfu)/ml or more. INTERPRETATION & CONCLUSIONS: Crystal VC kit had the potential to identify cholera cases in 10 min in field conditions without having good laboratory support. Therefore, dipstick kit may be considered as cholera detecting tool in diarrhoeal outbreak investigations. Specimens from clinically typical cholera cases, if negative by dipstick, should be reanalyzed by culture based methods.

Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996-2004 possessing the unique VSP-II region of El Tor variant.

New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.

Emergence and progression of Vibrio cholerae O1 El Tor variants and progenitor strains of Mozambique variants in Kolkata, India.

Analysis of 75 Vibrio cholerae O1 strains isolated from hospitalized patients in Kolkata from 1989 to 1994 revealed the existence of true El Tor along with El Tor variants that possessed the classical allele of ctxB (ctxB(cl)) in strains having an El Tor backbone. Based on the existence of different combinations of ctxB and rstR alleles and their localization sites in the genome, these strains were classified into multiple genetic groups. Of 75 clinical strains, 11 were identified as non-toxigenic. These 8 strains were also devoid of pTLC, which is uncommon among the O1 strains. However, Mozambique variants isolated in 2004 were typically negative for pTLC, but these strains possessed tandemly arranged CTX prophages with ctxB(cl) in the small chromosome. Genetic manipulation studies with laboratory-generated kanamycin-tagged pCTX-Kan (derived from tandemly arranged small chromosome-localized ctxB(cl) bearing CTX prophages of 1992 VC53, a progenitor strain of the Mozambique variant) demonstrated that integration specificity of the pCTX-Kan was somewhat towards small chromosome. Such integration could be the prime step towards generation of the Mozambique variant. Based on the existence of multiple alleles of CTXϕ and their infections with non-toxigenic strains, we propose that the El Tor variant strains could have emerged following these genetic events. This study demonstrated existence of different 'intermediate strains' in a time frame that overlapped with a period of V. cholerae O139 emergence. Identification of these intermediate strains gave impetus to believe stepwise generation of the El Tor variants, and all these events profoundly influenced V. cholerae epidemiology in the following years.

Cholera toxin production by the El Tor variant of Vibrio cholerae O1 compared to prototype El Tor and classical biotypes.

Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.

Incidence, virulence factors, and clonality among clinical strains of non-O1, non-O139 Vibrio cholerae isolates from hospitalized diarrheal patients in Kolkata, India.

The incidence of Vibrio cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhea constituted 27.4% (n = 54) of the total 197 V. cholerae strains isolated from patients in Kolkata, India, in 2003. Of 197 strains, 135 were identified as O1 serotype Ogawa and 2 were identified as O139. In the same time period, six O1 background rough strains that possessed all known virulence factors were identified. Serotype analysis of the non-O1, non-O139 strains placed 42 strains into 19 serogroups, while 12 remained O nontypeable (ONT); the existing serotyping scheme involved antisera to 206 serogroups. Detection of a good number of ONT strains suggested that additional serogroups have arisen that need to be added to the current serotyping scheme. The non-O1, non-O139 strains were nontoxigenic except for an O36 strain (SC124), which regulated expression of cholera toxin as O1 classical strains did. Additionally, strain SC124 carried alleles of tcpA and toxT that were different from those of the O1 counterpart, and these were also found in five clonally related strains belonging to different serogroups. Strains carrying tcpA exhibited higher colonization in an animal model compared to those lacking tcpA. PCR-based analyses revealed remarkable variations in the distribution of other virulence factors, including hlyA, rtxA, Vibrio seventh pandemic island I (VSP-I), VSP-II, and type III secretion system (TTSS). Most strains contained hlyA (87%) and rtxA (81.5%) and secreted cytotoxic factors when grown in vitro. Approximately one-third of the strains (31.5%) contained the TTSS gene cluster, and most of these strains were more motile and hemolytic against rabbit erythrocytes. Partial nucleotide sequence analysis of the TTSS-containing strains revealed silent nucleotide mutations within vcsN2 (type III secretion cytoplasmic ATPase), indicating functional conservation of the TTSS apparatus.

Design of enclosure of a grinding machine for the noise attenuation.

Since most of the equipments are usually installed in rooms or enclosed plants, the effect of an enclosure is important. Absorptive surface of the enclosure can reduce levels to the extent of 5 dB(A) which is quite significant. Effectiveness of enclosures in sound varies with the frequency of sound and the shape, size, laying pattern of sound absorbing material, which is put on the walls of the enclosure. The sound absorbing material accomplishes little shielding if there is no acoustical absorption within it. Acoustical absorbents when applied to the interior surface of the enclosure adsorb some of the energy and the noise level within the enclosure builds up only by the energy not absorbed. This paper elucidates the procedure of developing an enclosure to study its performance in attenuating sound at varying conditions. The results found encouraging and such enclosures can be developed for plant machineries by the plant personnels without getting confused by the claims and rhetoric of advertisements of big companies engaged in developing such items.

Biotyping of Vibrio cholerae O1: time to redefine the scheme.

Considering the recent emergence of "hybrid biotype" and "El Tor variant", we propose to redefine the biotyping scheme for Vibrio cholerae O1 serogroup. The existing biotyping scheme has limitations and causes confusion as many of the hybrid biotype and El Tor variant strains have phenotypic and genetic changes. A revised biotyping scheme will play a significant role to understand the ecology, epidemiology and nature of infection of V. cholerae O1 strains in future.