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Emergence and rapid spread of multidrug-resistant (MDR) bacteria including Vibrio cholerae are a global public health issue. Much attention has been paid to natural compounds, such as spices and herbs to find novel antimicrobial compounds as they are considered to be cheaper alternatives to develop as a drug. Here, we show that methanol extract of white pepper could inhibit the growth of V. cholerae O1 El Tor variant, responsible for the recent outbreaks/epidemics. Furthermore, we demonstrate for the first time that piperine, the major component of white pepper, showed a dose-dependent bactericidal effect on V. cholerae growth irrespective of their biotypes and serogroups in the presence of 200 and 300 µg ml−1 of piperine, respectively. Piperine also inhibited the growth of MDR strains of Pseudomonas aeruginosa, Escherichia coli isolated from poultry and enterohemorrhagic/enteroaggregative E. coli O104 in the presence of 200 µg ml−1. Interestingly, we did not observe any significant inhibitory effect of piperine on E. coli strains isolated from healthy person even up to 200 µg ml−1. Our data suggest that piperine could be a novel antimicrobial agent in therapeutic and preventive applications against infections caused by pathogenic bacteria including MDR strains.

Background The Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-to-severe diarrhea (MSD) in children under 5 years old in Africa and Asia. Here, we describe the prevalence and antimicrobial susceptibility of nontyphoidal Salmonella (NTS) serovars in GEMS and examine the phylogenetics of Salmonella Typhimurium ST313 isolates. Methods Salmonella isolated from children with MSD or diarrhea-free controls were identified by classical clinical microbiology and serotyped using antisera and/or whole-genome sequence data. We evaluated antimicrobial susceptibility using the Kirby-Bauer disk-diffusion method. Salmonella Typhimurium sequence types were determined using multi-locus sequence typing, and whole-genome sequencing was performed to assess the phylogeny of ST313. Results Of 370 Salmonella-positive individuals, 190 (51.4%) were MSD cases and 180 (48.6%) were diarrhea-free controls. The most frequent Salmonella serovars identified were Salmonella Typhimurium, serogroup O:8 (C2-C3), serogroup O:6,7 (C1), Salmonella Paratyphi B Java, and serogroup O:4 (B). The prevalence of NTS was low but similar across sites, regardless of age, and was similar among both cases and controls except in Kenya, where Salmonella Typhimurium was more commonly associated with cases than controls. Phylogenetic analysis showed that these Salmonella Typhimurium isolates, all ST313, were highly genetically related to isolates from controls. Generally, Salmonella isolates from Asia were resistant to ciprofloxacin and ceftriaxone, but African isolates were susceptible to these antibiotics. Conclusions Our data confirm that NTS is prevalent, albeit at low levels, in Africa and South Asia. Our findings provide further evidence that multidrug-resistant Salmonella Typhimurium ST313 can be carried asymptomatically by humans in sub-Saharan Africa., In agreement with prior studies, we show that nontyphoidal Salmonella is prevalent in stool of children

The development of the heart follows a synergic action of several signaling pathways during gestational, pre- & postnatal stages. The current study aimed to investigate whether the myocardium experiences transcriptional changes during the transition from post-natal to adulthood stages. Herein, we used C57/Bl6/J mice at 4 (28- days; post-natal/PN) and 20 weeks (adulthood/AH) of ages and employed the next generation RNAseq (NGS) to profile the transcriptome and echocardiography analysis to monitor the structural/functional changes in the heart. NGS-based RNA-seq revealed that 1215 genes were significantly upregulated and 2549 were down regulated in the AH versus PN hearts, indicating a significant transcriptional change during this transition. A synchronized cardiac transcriptional regulation through cell cycle, growth hormones, redox homeostasis, and metabolic pathways was noticed in both PN and AH hearts. Echocardiography revealed significant structural and functional (i.e. systolic/diastolic) changes during the transition of PN to adult stage. Particularly, a progressive decline in ejection fraction and cardiac output was observed in AH hearts. These structural adaptations are in line with critical signaling pathways that drive the maturation of heart during AH. Overall, we have presented a comprehensive transcriptomic analysis along with a structural-functional relationship during the myocardial development in adult mice.

Strength and modulus of rock mass as obtained from RMR, Q and GSI have been examined with reference to modulus ratio, Mrj, for their reliability. The design parameters adopted in some case studies based on these rock mass classifications are presented. The modulus ratios in these case studies are found to be much higher than those of the corresponding values of intact rocks, even after back analyses. Based on joint factor, Jf, compressive strength, modulus, cohesion and friction angle were estimated and applied in the analyses of a few cases. The predictions of deformations agreed well with the field measurements. Based on extensive experimental data of jointed specimens of rock and rock-like materials, a joint factor, Jf, was defined as a weakness coefficient in rock mass compared to the corresponding intact rock. Jf is linked to the strength, modulus and modulus ratio of rock mass. The modulus ratio, Mrj, of rocks is less than the modulus ratio of intact rock. The Mrj concept has been adopted to present a unified classification for intact rocks and rocks masses, to define soil that rock boundary and penetration rate of TBMs.

The development of the heart follows a synergic action of several signaling pathways during gestational, pre- & postnatal stages. The current study aimed to investigate whether the myocardium experiences transcriptional changes during the transition from post-natal to adult hood stages. Herein, we used C57/Bl6/J mice at 4 (28-days; post-natal/PN) and 20 weeks (adulthood/AH) of ages and employed the next generation RNAseq (NGS) to profile the transcriptome and echocardiography analysis to monitor the structural/functional changes in the heart. NGS-based RNA-seq revealed that 1215 genes were significantly upregulated and 2549 were down regulated in the AH versus PN hearts, indicating a significant transcriptional change during this transition. A synchronized cardiac transcriptional regulation through cell cycle, growth hormones, redox homeostasis and metabolic pathways was noticed in both PN and AH hearts. Echocardiography reveals significant structural and functional (i.e. systolic/diastolic) changes during the transition of PN to adult stage. Particularly, a progressive decline in ejection fraction (EF) and cardiac output was observed in AH hearts. These structural adaptations are in line with critical signaling pathways that drive the maturation of heart during AH. Overall, we have presented a comprehensive transcriptomic analysis along with structural-functional relationship during the myocardial development in adult mice.

The human gut harbours a multifaceted and dynamic population of microorganisms, which affect human homeostasis and health. Robust network among gut microbes plays an important role in human health and disease by influencing immunity, nutrition and pathogenesis. The gut microbes are frequently exposed to antibiotics, which are used to prevent and/or treat infectious diseases. Prolonged use of antibiotics has been associated with reduced gut microbial colonization ability and their diversity. The use of antibiotics causes dysbiosis, a state that has a significant alteration in the community structure, composition and richness of the gut microbiome. Dysbiosis disrupts the gut microbial ecosystem and is associated with many diseases: diarrhoea, constipation, bloating, indigestion, irritable bowel syndrome (IBS), inflammatory bowel diseases (IBD), obesity and diabetes. The emergence of antibiotic resistance (AMR) among commensal and pathogenic bacteria has become a major threat to public health. These commensals can acquire AMR genes (ARGs) by the mobilization/transfer from the other bacteria. Several bacteria in the gut may retain these ARGs. In this review, we discuss the effects of antibiotics on the commensal gut microbiota, as well as their impact on the human host.

A newly emerged Vibrio cholerae O1 El Tor variant strain with multidrug resistance is considered a threat to public health. Recent strategies to suppress virulence factors production instead of bacterial growth may lead to less selective pressure for the emergence of resistant strains. The use of spices and their active constituents as the inhibitory agents against cholera toxin (CT) production in V. cholerae may be an alternative approach to treat cholera. In this study, we examined the potential of sweet fennel seed (Foeniculum vulgare Miller var. dulce) methanol extract to inhibit CT production in V. cholerae without affecting viability. The methanol extract of sweet fennel seeds significantly inhibited CT production in various V. cholerae strains, regardless of serogroup or biotype. Interestingly, trans-anethole and 4-allylanisole, essential oil components of sweet fennel seeds, also demonstrated similar effects. Here, we report that sub-bactericidal concentrations of sweet fennel seed methanol extract and its major components can drastically inhibit CT production in various V. cholerae strains.

The widely distributed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) has gained importance over the years in terms of its structure and function. CS6 is an afimbrial assembly in contrast to the other ETEC CFs, which are mostly fimbrial. A recent study predicted a linear fibre model for recombinant chimeric CS6 and formation of oligomers in solution. In this study, we characterized the oligomeric assembly of CS6, purified from a clinical ETEC isolate and identified its existence in the WT strain. We found that purified CS6 forms a continuous array of higher order oligomers composed of two tightly associated subunits, CssA and CssB in an equal (1:1) stoichiometry. This oligomerization occurs by formation of (CssA-CssB)n complex where 'n' increases with the concentration. The diameter of CS6 oligomers also proportionally increases with concentration. More significantly, we showed CS6 oligomers to be spherical in shape instead of being linear fibres as predicted earlier and this was further confirmed by electron microscopy. We also showed CS6 assembled on the bacterial surface in the form of an oligomeric complex. This process depends on the expression of properly folded CssA and CssB together, guided by the chaperone CssC and usher CssD. In conclusion, our results provide evidence for the existence of concentration-dependent, spherical oligomers of CS6 comprising both the structural subunits in equal stoichiometry and the CS6 oligomeric complex on the ETEC surface.

We study fermions in an electrically-probed and asymptotically anti-de Sitter Schwarzschild spacetime which interact via novel chiral symmetry-preserving interactions. Computing the dual fermion two-point correlator, we show that these bulk interactions anisotropically gap Fermi surfaces of the boundary spectrum. Consequently, the interactions we devise provide holographic models for Fermi arcs seen ubiquitously in the pseudogap regime of the cuprates. Our interactions are modifications of the chiral symmetry-breaking Pauli coupling, which has previously been proposed as the holographic realization of Mott physics. The onset of Mott insulation and pseudogap physics are respectively discussed in the context of bulk chiral and boundary parity symmetry breaking, and the Mott transition is interpreted as a deconfinement transition of non-Fermi liquid excitations., Comment: 8 pages, 2 figures

Vibrio cholerae causes fatal diarrheal disease cholera in humans due to consumption of contaminated water and food. To instigate the disease, the bacterium must evade the host intestinal innate immune system; penetrate the mucus layer of the small intestine, adhere and multiply on the surface of microvilli and produce toxin(s) through the action of virulence associated genes. V. cholerae O1 that has caused a major cholera outbreak in Haiti contained several unique genetic signatures. These novel traits are used to differentiate them from the canonical El Tor strains. Several studies reported the spread of these Haitian variant strains in different parts of the world including Asia and Africa, but there is a paucity of information on the clinical consequence of these genetic changes. To understand the impact of these changes, we undertook a study involving mice and rabbit models to evaluate the pathogenesis. The colonization ability of Haitian variant strain in comparison to canonical El Tor strain was found to be significantly more in both suckling mice and rabbit model. Adult mice also displayed the same results. Besides that, infection patterns of Haitian variant strains showed a completely different picture. Increased mucosal damaging, colonization, and inflammatory changes were observed through hematoxylin-eosin staining and transmission electron microscopy. Fluid accumulation ability was also significantly higher in rabbit model. Our study indicated that these virulence features of the Haitian variant strain may have some association with the severe clinical outcome of the cholera patients in different parts of the world.

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, has ravaged humanity from time immemorial. Although the disease can be treated using antibiotics along with administration of oral rehydration salts and controlled by good sanitation, cholera is known to have produced mayhems in ancient times when little was known about the pathogen. By the 21

Diphtheria is caused by a toxigenic bacterium Corynebacterium diphtheriae which remains as one of the important causes of illness and death among children. Globally, epidemic waves of diphtheria have killed thousands of children in early 1920s, i.e., before the vaccine era. There are four main biotypes of C. diphtheriae available namely C.d. Gravis, C.d. Intermedius, C.d. Mitis, and C.d. Belfanti. Globally, the C.d. Intermedius is most often associated with exotoxin production, although all three strains are capable of producing exotoxin. Diphtheria patients are usually prescribed for diphtheria antitoxin and antibiotics such as erythromycin/penicillin. Keeping all the pathogenic information in context, here in the present review we have accumulated all the recent updates on diphtheria and also presented a timely discussion on diagnosis and vaccination procedure, which might assist future therapeutics.

SUMMARYA total of 45 strains ofVibrio choleraeO1 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 classicalctxBgene. Polymerase chain reaction (PCR) analysis by double – mismatch amplification mutation assay PCR showed 16 of these strains carried thectxB-7allele reported in Haitian strains. Sequencing of thectxBgene 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 newctxBgene sequence was circulating in different places in India. Seven representatives of these 45 strains analysed by pulsed – field gel electrophoresis showed four distinctNot Idigested profiles showing that multiple clones were causing cholera in 2007 and 2008.

Topological semimetals are gapless states of matter which have robust surface states and interesting electromagnetic responses. In this paper, we consider the electromagnetic response of gapless phases in $3+1$-dimensions with line nodes. We show through a layering approach that an intrinsic $2$-form ${\cal{B}}_{\mu\nu}$ emerges in the effective response field theory that is determined by the geometry and energy-embedding of the nodal lines. This 2-form is shown to be simply related to the charge polarization and orbital magnetization of the sample. We conclude by discussing the relevance for recently proposed materials and heterostructures with line-node fermi-surfaces., Comment: 8 pages, 3 figures

The commonly adopted rock mass classifications, namely RMR, Q and GSI, are used to estimate compressive strength and modulus of rock masses. These values have been examined as per modulus ratio concept, M (rj) , for their reliability. The design parameters adopted in some of the recent case studies based on these classifications indicate that the M (rj) values for rock masses are higher than those of the corresponding intact rocks. The joint factor, J (f), which is defined as a weakness coefficient in rock mass suggests that modulus ratio of rock mass (M (rj) ) has to be less than the modulus ratio of the corresponding intact rock (M (ri) ), on the basis of extensive experimental evidence. With joint factor, compressive strength, elastic modulus, cohesion and friction angle were estimated and applied in the analyses of a few cases. The predictions of deformations with this approach agreed well with the field measurements by adapting equivalent continuum approach. The modulus ratio concept is considered to present a unified classification for intact rocks and rock masses. Soil-rock boundary, standup time in under ground excavations and also penetration rate of TBM estimates have been linked to M (rj) .

An outbreak of cholera struck Bihar, an Indian state, in August 2008 following a massive flood. Here we report the phenotypic and genotypic characteristics of Vibrio cholerae strains isolated from patients with diarrhea. Rectal swabs were obtained from patients with diarrhea who were admitted to medical camps or the hospital, and the strains were biochemically and serologically characterized. V. cholerae was isolated from 21 (65.6%) of 32 rectal swabs. Serological studies revealed that all the 21 isolates belonged to V. cholerae O1 Ogawa. Mismatch amplification mutation assay (MAMA)-PCR showed that the isolates belonged to El Tor variant group, and pulsed-field gel electrophoresis (PFGE) proved that these isolates were of a different lineage than the conventional El Tor variant strains. These isolates were resistant to several drugs, including ampicillin, streptomycin, tetracycline, nalidixic acid, and furazolidone. The uniqueness of the current report arises from the fact that records of cholera in Bihar are availiable for the early 1960s but not for the next 4 decades. Moreover, the present study is the first to report a cholera outbreak in Bihar that was caused by an El Tor variant strain.

We propose a one-parameter family of noninteracting lattice models for Weyl semimetals with 4 Weyl nodes and tunable Fermi arcs. These 2-band model Hamiltonians are time-reversal symmetric with $\mathrm{T}^2 = + 1$, and tuning the parameter changes the connectivity of the Fermi arcs continuously without affecting the location and chiralities of the Weyl nodes in the bulk Brillouin zone. The bulk polarization and magnetization are shown to vary with this parameter, a dependence inaccessible to the low energy effective field theory., 8 pages, 3 figures

Background & objectives: The four species of the genus Shigella, namely, S. dysenteriae , S. flexneri, S. boydii and S. sonnei cause a wide spectrum of illness from watery diarrhoea to severe dysentery. Genomes of these four species show great diversity. In this study, NotI, XbaI or I-CeuI restriction enzyme digested genomes of two Shigella dysenteriae isolates belonging to the serotypes 2 and 7 were extensively analyzed to find their relatedness, if any, with the whole genome sequenced strains of S. dysenteriae type 1 and S. flexneri type 2a. Methods: Pulsed-field gel electrophoresis (PFGE) technique was used to determine the diversity of Shigella genomes by rapid construction of physical maps. DNA end labelling, Southern hybridization and PCR techniques were also applied for mapping purposes. Results: The intron-coded enzyme I-CeuI cuts the bacterial genome specifically at its rrn operon. PFGE of I-CeuI digested S. dysenteriae genomes were found to carry seven rrn operons. However, I-CeuI profiles showed distinct restriction fragment polymorphism (RFLP) between the isolates as well as with the whole genome sequenced isolates. Further studies revealed that the genome sizes and I-CeuI linkage maps of the S. dysenteriae type 7 and type 2 isolates were similar to that of S. dysenteriae type 1 and S. flexneri type 2a genomes, respectively. Interpretation & conclusions: Our findings indicate that the type 7 and type 1 isolates of S. dysenteriae were probably evolved from a same precursor, while the type 2 and S. flexneri type 2a were probably evolved and diversified from a common progenitor.

A total of 1034 samples were collected from different sources and C. difficile was isolated from 18 (9.04%) of 199 human, 9 (4.89%) of 184 cattle, 29 (12.44%) of 233 pig, and from 23 (13.94%) of 165 poultry samples. Variations were observed on the rate of isolation according to age and clinical conditions (diarrhoea). None of the samples from cow, sheep, goat, local chicken, and wild animals yielded any C. difficile . Out of those isolates, 8, 2, 19 and 6 isolates from human, cattle, pig and poultry, respectively were toxigenic. The toxigenic isolates carried both tcd A, and tcd B (A + B + ) and most of the human and the pig isolates were also positive for binary toxin genes ( cdt A and cdt B). The A + B + isolates belonged to three different toxinotypes (0, VI and XXXIII). Human and pig A + B + isolates belonged to three (045, 126 and ACD 019) and four (046, 087, 126 and ACD 011) different ribotypes, respectively and the ribotypes of two cattle isolates were 014 and ACD 010. Six A + B + avian isolates belonged to six different ribotypes (014, 087, SLO 134, SLO 160, ACD 012, ACD 014). The non-toxigenic isolates from human, cattle, pig and poultry were grouped into 7, 4, 4 and 7 different ribotypes, respectively. PFGE analysis could not differentiate similar ribotypes/toxinotypes of toxigenic isolates. All the toxigenic isolates showed cytopathic effect on Vero and Hela cell monolayers at 1:100 dilutions of cell-free culture supernatants within 18–20 h of inoculation.

Developing countries are burdened with Shigella diarrhea. Understanding mucosal immune responses associated with natural Shigella infection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens, p an- S higella s urface p rotein antigen 1 (PSSP1) and PSSP2, common to all virulent Shigella strains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α 4 β 7 + ) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive for Shigella infection by microbiological and real-time PCR assays, respectively. The frequency of α 4 β 7 + IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of the Shigella serotype isolated from these patients. Thus, α 4 β 7 + ASC responses to Ipas may be considered an indirect marker of Shigella infection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by natural Shigella infection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude.

We study entanglement entropy for parity-violating (time-reversal breaking) quantum field theories on ${\mathbb{R}}^{1,2}$ in the presence of a domain wall between two distinct parity-odd phases. The domain wall hosts a $1+1$-dimensional conformal field theory (CFT) with nontrivial chiral central charge. Such a CFT possesses gravitational anomalies. It has been shown recently that, as a consequence, its intrinsic entanglement entropy is sensitive to Lorentz boosts around the entangling surface. Here, we show using various methods that the entanglement entropy of the three-dimensional bulk theory is also sensitive to such boosts owing to parity-violating effects, and that the bulk response to a Lorentz boost precisely cancels the contribution coming from the domain wall CFT. We argue that this can naturally be interpreted as entanglement inflow (i.e., inflow of entanglement entropy analogous to the familiar Callan-Harvey effect) between the bulk and the domain-wall, mediated by the low-lying states in the entanglement spectrum. These results can be generally applied to $2+1$-d topological phases of matter that have edge theories with gravitational anomalies, and provide a precise connection between the gravitational anomaly of the physical edge theory and the low-lying spectrum of the entanglement Hamiltonian.

A newly emerged Vibrio cholerae O1 El Tor variant strain with multidrug resistance is considered a threat to public health. Recent strategies to suppress virulence factors production instead of bacterial growth may lead to less selective pressure for the emergence of resistant strains. The use of spices and their active constituents as the inhibitory agents against cholera toxin (CT) production in V. cholerae may be an alternative approach to treat cholera. In this study, we examined the potential of sweet fennel seed (Foeniculum vulgare Miller var. dulce) methanol extract to inhibit CT production in V. cholerae without affecting viability. The methanol extract of sweet fennel seeds significantly inhibited CT production in various V. cholerae strains, regardless of serogroup or biotype. Interestingly, trans-anethole and 4-allylanisole, essential oil components of sweet fennel seeds, also demonstrated similar effects. Here, we report that sub-bactericidal concentrations of sweet fennel seed methanol extract and its major components can drastically inhibit CT production in various V. cholerae strains.

Topological crystalline insulators (TCI) are a new class of materials which have metallic surface states on select surfaces due to point group crystalline symmetries. In this letter, we consider a model for a three-dimensional (3D) topological crystalline insulator with Dirac nodes occurring on a surface that are protected by the mirror and time reversal symmetry. We demonstrate that the electromagnetic response for such a system is characterized by a $1$-form $b_{\mu}$. $b_{\mu}$ can be inferred from the locations of the surface Dirac nodes in energy-momentum space and couples to the surface Dirac nodes like a valley gauge field. From both the effective action and analytical band structure calculations, we show that the vortex core of $\vec b$ or a domain wall of a component of $\vec b$ can trap surface charges., Comment: 13 pages, 5 figures

This study examined the pattern of colonization of the neonatal gut by aerobic Gram-negative bacilli (GNB) and evaluated the association between gut colonization and sepsis in the developing world. This deserves attention because of the high incidence of sepsis and the differences in hygienic environments in developing countries compared with the developed world. The study was carried out on neonates in a tertiary-care government hospital. Serial gut samples were analysed (gastric aspirates and stool samples) for GNB. Blood samples of cases showing clinical signs of sepsis were also analysed for septic screening and culture positivity. Antibiograms, serotyping and PFGE were carried out to evaluate the relatedness of the gut and blood isolates. A diverse array of GNB was isolated from the gut of the neonates, Klebsiella pneumoniae being most common, followed by Escherichia coli. The rate of isolation of GNB was consistently higher in stool samples compared with gastric aspirate samples. Colonization was influenced by a stay in the neonatal intensive care unit and by the prolonged use of a feeding tube. GNB were the cause of sepsis in the majority of cases, with K. pneumoniae being the most frequently isolated GNB from the blood. Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae and Burkholderia cepacia were the other GNB recovered from the blood of the neonates. Neonates with GNB in the gut had a higher incidence of clinical sepsis than those without. In 50 % of cases, the genotypes of the organisms found in the blood were indistinguishable from their gut counterpart. These results show that the neonatal gut is colonized with a diverse array of GNB, and an association between gut colonization and neonatal sepsis was observed.

To study the prevalence pattern and trends in the phenotypic and genetic characteristics of shigellae, we tested 212 isolates isolated from diarrhoeal patients admitted to the Infectious Diseases Hospital, Kolkata, India, from November 2007 to October 2010. Prevalence of Shigella spp. was higher in the >5 years age group (69 %) than in children in the

Several genes including the cagA in the cag pathogenicity island (cag PAI) of Helicobacter pylori are thought to be associated with the gastroduodenal diseases and hence variation in the genetic structure of the cag PAI might be responsible for different clinical outcomes. Our study was undertaken to characterize the cag PAI of H. pylori strains from duodenal ulcer (DU) patients and asymptomatic or non-ulcer dyspepsia (NUD/AV) subjects from Kolkata, India. Strains isolated from 52 individuals (30 DU and 22 NUD/AV) were analyzed by PCR using 83 different primers for the entire cag PAI and also by dot-blot hybridization. Unlike H. pylori strains isolated from other parts of India, 82.6% of the strains used in this study had intact cag PAI, 9.6% had partially deleted cag PAI, and 7.7% of the strains lacked the entire cag PAI. Dot-blot hybridization yielded positive signals in 100% and 93.8% of PCR-negative strains for HP0522-523 and HP0532-HP0534 genes, respectively. An intact cagA promoter region was also detected in all cagA-positive strains. Furthermore, the expression of cagA mRNA was confirmed by RT-PCR for the representative strains from both DU and NUD/AV subjects indicating the active cagA promoter regions of these strains. A total of 66.7% of Kolkata strains produced a ~390-bp shorter amplicon than the standard strain 26695 for the HP0527 gene, homologue of virB10. However, sequence analyses confirmed that the deletion did not alter the reading frame of the gene, and mRNA transcripts were detected by RT-PCR analysis. The strains isolated from DU and NUD/AV express CagA protein and possess a functional type IV secretion system, as revealed by Western blot analyses. Interestingly, no significant differences in cag PAI genetic structure were found between DU and NUD/AV individuals suggesting that other bacterial virulence factors, host susceptibility, and environmental determinants also influence the disease outcome at least in certain geographical locations.

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.

SUMMARYFaecal specimens of diarrhoea cases (n=2495, collected between November 2007 and October 2009) from Infectious Diseases and Beliaghata General (ID&BG) Hospital, Kolkata, India, were screened by RT–PCR using specific primers targeting region C of the capsid gene of noroviruses (NoVs) to determine the seasonal distribution and clinical characteristics of NoVs associated with diarrhoea. NoV infection was detected in 78 cases, mostly in children aged

CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.

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.

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year-long surveillance study. A PCR-RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt-I, three for cdt-II, four for cdt-III, three for cdt-IV and four samples were positive for cdt-V, as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt-I, one cdt-II, three cdt-III, three cdt-IV and four with cdt-V. Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup O2. The other virulence genes such as astA, cnf1, eaeA, cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence-associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR-RFLP in Providencia alcalifaciens. Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.

Summary Objectives To study the phenotypic and genotypic characterization of newly emerged V. cholerae O1, Inaba strains isolated from patients with diarrhoea. Methods Bacterial characterization was made using polymerase chain reaction, ribotyping, PFGE and RFLP. Results After its first appearance in July 2004, O1 Inaba became the dominant serotype by March 2005 and totally replaced the former dominant serotype, Ogawa from May 2005. Most of the Inaba isolates belong to a new ribotype RIV. Ogawa and also some Inaba strains isolated during the same period were identified as RIII. Similarly, the majority of the Inaba isolates belong to ‘H1' pulsotype and one isolate is type ‘H', while the Ogawa isolates were mostly ‘H' pulsotype. Presence of CTX prophage was detected in a single site of the chromosome with at least two RS elements. Conclusions There has been a switch of dominant serotype from Ogawa to Inaba in the Kolkata region. This is not necessarily due to emergence of a new clone but does serve as an epidemiological marker. Further analysis at the molecular level will be required to define this trend and to monitor future spread to other regions.

SUMMARYIn this study, we have analysed the epidemiological significance of the concurrent infections caused by Vibrio cholerae and intestinal parasites among different age groups of hospitalized diarrhoeal patients in Kolkata. A total of 3556 stool samples collected during 1996–2004 were screened for vibrios and parasites. The seasonality of V. cholerae and parasitic infections were studied in detail. The detection rates for Ascaris lumbricoides and Giardia lamblia infection were more than for the hookworm, Trichuris trichiura and Entamoeba histolytica. V. cholerae O1 was identified as the predominant serogroup among diarrhoeal patients. The highest rates for V. cholerae infection were in the 2–5 years age group and combined infection of V. cholerae and parasites was recorded among children aged between 2 and 10 years.

Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 ( stx 1 + stx 2 + eae + ) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type γ. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.

During a study of microbial diversity, a bacterial strain designated HT10, was isolated from sediment collected from an unexplored sulfur spring at Athamallik, Orissa, India. Various biochemical tests and 16S rDNA sequence analysis revealed that strain HT10 is Aeromonas caviae. The growth temperature of this strain ranged from 12 to 43 degrees C and the optimum temperature was 30 degrees C. The strain HT10 showed cytotoxic and alpha-hemolytic activity. This is the first report on the isolation of Aeromonas caviae from sulfur spring.

Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei) cause bacillary dysentery (shigellosis), which is characterized by bloody mucous diarrhoea. Although a variety of antibiotics have been effective for treatment of shigellosis, options are becoming limited due to globally emerging drug resistance. In the present study, in vitro antibacterial activity of methyl gallate (MG) isolated from Terminalia chebula was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity of MG was determined by membrane perturbation and transmission electron microscopy (TEM). Cellular drug accumulation, cell infection and assessment of intracellular activities of MG and reference antibiotics were performed using HeLa cell cultures. The bactericidal activity of MG against multidrug-resistant (MDR) Shigella spp. in comparison with other commonly used drugs including fluoroquinolone was demonstrated here. TEM findings in the present study revealed that MG caused the total disintegration of inner and outer membranes, and leakage of the cytoplasmic contents of S. dysenteriae. The level of accumulation of MG and tetracycline in HeLa cells incubated for 24 h was relatively higher than that of ciprofloxacin and nalidixic acid (ratio of intracellular concentration/extracellular concentration of antibiotic for MG and tetracycline>ciprofloxacin and nalidixic acid). The viable number of intracellular S. dysenteriae was decreased in a time-dependent manner in the presence of MG (4 × MBC) and reduced to zero within 20 h. The significant intracellular activities of MG suggested that it could potentially be used as an effective antibacterial agent for the treatment of severe infections caused by MDR Shigella spp.