Ram Naresh

Bio: Ram Naresh is an academic researcher from Animal Research Institute. The author has contributed to research in topics: Brachyspira pilosicoli & Mucin. The author has an hindex of 5, co-authored 7 publications receiving 133 citations.

The Intestinal Spirochete Brachyspira pilosicoli Attaches to Cultured Caco-2 Cells and Induces Pathological Changes

Abstract: Background: Brachyspira pilosicoli is an anaerobic spirochete that has received relatively little study, partly due to its specialized culture requirements and slow growth. This bacterium colonizes the large intestine of various species, including humans; typically, a dense layer of spirochete cells may be found intimately attached by one cell end to the surface of colonic enterocytes. Colonized individuals may develop colitis, but the mechanisms involved are not understood. The current study aimed to develop an in vitro model to investigate this process. Methodology/Principal Findings: Four strains of B. pilosicoli were incubated at a high multiplicity of infection with monolayers of a human colonic adenocarcinoma cell line (Caco-2 cells). One strain isolated from a pig (95/1000) and one from a human (WesB) attached to the monolayers. Colonization increased with time, with the Caco-2 cell junctions being the initial targets of attachment. By electron microscopy, individual spirochete cells could be seen to have one cell end invaginated into the Caco-2 cell membranes, with the rest of the spirochete draped over the Caco-2 cell surface. After 6 h incubation, the monolayer was covered with a layer of spirochetes. Colonized monolayers demonstrated a time-dependent series of changes: staining with labelled phalloidin identified accumulation of actin at the cell junctions; ZO-1 staining revealed a loss of Caco-2 tight junction integrity; and Hoechst staining showed condensation and fragmentation of nuclear material consistent with apoptosis. Using quantitative reverse transcription PCR, the colonized monolayers demonstrated a significant up-regulation of interleukin-1b (IL-1b) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1β, TNF-α, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression. Conclusions/Significance: The changes induced in the Caco-2 cells provide evidence that B. pilosicoli has pathogenic potential, and give insights into the likely in vivo pathogenesis.

Attraction of Brachyspira pilosicoli to mucin

Abstract: The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species, including humans. In the colon this spirochaete can penetrate the overlying mucus layer, attach by one cell end to the underlying enterocytes, and initiate localized colitis and diarrhoea. The aim of this study was to investigate whether, as part of the colonization process, B. pilosicoli is attracted to mucin. Fifteen B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. No significant attraction was detected with 1% mucin, but some strains started to enter a 2% solution, and attraction then increased with increasing concentrations to peak at around 6-8% mucin. A similar increase was seen with B. hyodysenteriae, although this activity peaked at 6% mucin and then declined, suggesting that the two species have different affinities for mucin. These mucin concentrations were much higher than those used in previous experimental studies with Brachyspira species. The viscosities of the 6-8% mucin solutions were around 7-12 mPa s, which were similar to the measured viscosities of the mucus layer overlying the epithelium in the caecum and colon of experimental pigs. The strains varied in their motility, as assessed by their ability to enter tubes containing chemotaxis buffer, but there was no significant relationship between this motility and the extent of their ability to enter the mucin solutions. Different strains also had different propensities to enter the mucin solutions, but there were no consistent differences according to the host species of origin. B. pilosicoli strain 95/1000 was attracted towards a solution of Dserine, suggesting that chemotaxis was involved in the attraction to mucin; however, 95/1000 was also attracted to viscous solutions of polyvinylpyrrolidone (PVP), in a manner mirroring the response to mucin, and hence suggesting the involvement of viscotaxis in the attraction to mucin. B. hyodysenteriae B204 showed a similar viscotaxis to PVP. Further studies are required to determine whether the in vitro interaction of a given strain with mucin is a useful indicator of its in vivo colonization ability, and hence could be used as a potential marker for virulence.

Exposure to norepinephrine enhances Brachyspira pilosicoli growth, attraction to mucin and attachment to Caco-2 cells.

Abstract: Brachyspira pilosicoli is an anaerobic intestinal spirochaete that colonizes the large intestine of a variety of species of birds and mammals, including human beings. Colonization may result in a mild colitis and diarrhoea in a condition known as 'intestinal spirochaetosis'. The catecholamine norepinephrine (NE), which is known to influence the behaviour of many bacterial species, may be present in the colon. The purpose of the current study was to determine whether exposure of B. pilosicoli to NE would influence its in vitro behaviour in assays that may reflect in vivo colonization potential. B. pilosicoli strain 95/1000 was used in all the assays. Addition of NE at a concentration of 0.05 mM to B. pilosicoli growing in anaerobic broth significantly increased spirochaete numbers after 4 days incubation. The effect of higher concentrations of NE was not significant. Exposure to 0.05 mM NE, but not to higher concentrations, also resulted in significantly more spirochaete cells entering capillary tubes containing 4% porcine gastric mucin than occurred with untreated cultures. When NE was added to chemotaxis buffer in capillary tubes, significantly more spirochaetes were attracted to the buffer containing NE at 0.1, 0.5 and 1.0 mM than to buffer containing 0.05 mM NE, or when no NE was added. Exposure of B. pilosicoli cultures to 0.05 mM NE prior to incubation with Caco-2 monolayers resulted in more attachment to the monolayer than occurred with non-exposed cultures. These results show that at higher concentrations, NE acts as a chemoattractant for B. pilosicoli, and at 0.05 mM it increases the spirochaete's growth rate, attraction to mucin and rate of attachment to cultured enterocytes. These activities are likely to enhance the ability of B. pilosicoli to colonize, and may be induced by conditions that increase NE concentrations in the intestinal tract, such as the stresses associated with crowding.

Penicillin resistance in the intestinal spirochaete Brachyspira pilosicoli associated with OXA-136 and OXA-137, two new variants of the class D -lactamase OXA-63

Abstract: Penicillin resistance mediated by β-lactamase activity has been reported previously in the anaerobic intestinal spirochaete Brachyspira pilosicoli, and a novel class D β-lactamase (OXA-63) hydrolysing oxacillin was described recently in a resistant human strain from France. In the current study, 18 B. pilosicoli strains from Australia and Papua New Guinea were tested for ampicillin and oxacillin susceptibility, and investigated for the presence of the class D β-lactamase gene blaOXA-63 using PCR. PCR products were amplified from seven human and four porcine strains that were penicillin resistant, but not from seven penicillin-sensitive strains. Sequence analysis of the whole gene amplified from seven of the resistant strains from humans and pigs revealed only minor nucleotide differences among them, but there were significant differences compared with blaOXA-63. The predicted amino acid sequence of the enzyme from all seven strains had the same key structural motifs as the previously reported OXA-63, but two variants with 94–95 % identity with OXA-63 were identified. OXA-136 had an additional amino acid and 12 other consistent amino acid substitutions compared with OXA-63. OXA-137 had the same differences compared with OXA-63 as OXA-136, but had an additional amino acid substitution at position 16. No structures consistent with integrons or transposons were found in the nucleotide sequences in the vicinity of blaOXA-136 in partially sequenced B. pilosicoli strain 95/1000, and the GC content (25.2 mol%) of the gene was similar to that of the whole genome. The gene encoding OXA-136 from B. pilosicoli strain Cof-10 conferred penicillin resistance on Escherichia coli. This study shows that penicillin resistance in human and porcine B. pilosicoli strains from Australia is associated with the production of two variants of OXA-63, and that susceptible strains lack the genes encoding OXA-63 or the variants.

Diversity, Epidemiology, and Genetics of Class D β-Lactamases

Abstract: Class D β-lactamase-mediated resistance to β-lactams has been increasingly reported during the last decade. Those enzymes also known as oxacillinases or OXAs are widely distributed among Gram negatives. Genes encoding class D β-lactamases are known to be intrinsic in many Gram-negative rods, including Acinetobacter baumannii and Pseudomonas aeruginosa, but play a minor role in natural resistance phenotypes. The OXAs (ca. 150 variants reported so far) are characterized by an important genetic diversity and a great heterogeneity in terms of β-lactam hydrolysis spectrum. The acquired OXAs possess either a narrow spectrum or an expanded spectrum of hydrolysis, including carbapenems in several instances. Acquired class D β-lactamase genes are mostly associated to class 1 integron or to insertion sequences.

Microfluidic Organ-on-a-Chip Technology for Advancement of Drug Development and Toxicology

Abstract: In recent years, the exploitation of phenomena surrounding microfluidics has seen an increase in popularity, as researchers have found a way to use their unique properties to create superior design alternatives. One such application is representing the properties and functions of different organs on a microscale chip for the purpose of drug testing or tissue engineering. With the introduction of "organ-on-a-chip" systems, researchers have proposed various methods on various organ-on-a-chip systems to mimic their in vivo counterparts. In this article, a systematic approach is taken to review current technologies pertaining to organ-on-a-chip systems. Design processes with attention to the particular instruments, cells, and materials used are presented.

Communication between Bacteria and Their Hosts

Abstract: It is clear that a dialogue is occurring between microbes and their hosts and that chemical signals are the language of this interkingdom communication. Microbial endocrinology shows that, through their long coexistence with animals and plants, microorganisms have evolved sensors for detecting eukaryotic hormones, which the microbe uses to determine that they are within proximity of a suitable host and to optimally time the expression of genes needed for host colonisation. It has also been shown that some prokaryotic chemical communication signals are recognized by eukaryotes. Deciphering what is being said during the cross-talk between microbe and host is therefore important, as it could lead to new strategies for preventing or treating bacterial infections.