Detection of Cryptosporidium, Giardia, fecal indicator bacteria, and total bacteria in commercial jar water in Kathmandu Valley, Nepal

Introduction: Jar water is a convenient and common source of drinking water in the Kathmandu Valley. However, studies including detailed microbial analyses of this source of potable water are lacking. In this study, jar water samples were examined for the occurrence of Cryptosporidium, Giardia, fecal indicator bacteria, and total bacteria. Methods: Thirty different brands of jars were collected in September 2014. Escherichia coli and total coliforms w ere determined using a Colilert reagent. Ten of the 30 brands w ere selected to test for Cryptosporidium, Giardia, and total bacteria. Bacterial DNA extraction from water samples w as performed using the Cica Geneus DNA Extraction Kit, follow ed by quantitative polymerase chain reaction (qPCR) targeting the 16S rRNA gene of bacterial DNA. Protozoan detection was accomplished by concentrating the samples using the electronegative membrane vortex method, followed by immunomagnetic separation and fluorescent staining. Results: E. coli w as detected in 10% of the samples, with a maximum concentration of 2 most probable number (MPN)/100 mL, whereas total coliforms were detected in 97% of the samples, with a maximum and mean concentration of 7.3 × 10 2 and 3.8 × 10 1 MPN/100 mL, respectively. Total coliforms concentrations in 40% of the samples ranged from 10 2 to 10 3 MPN/100 mL. Cryptosporidium and Giardia w ere not detected in any of the tested samples. Concentrations of total bacteria in the samples ranged from 10 4 to 10 6 cells/100 mL. Conclusions: Ninety-seven percent of the jar water brands were unsuitable for drinking without proper treatment based on the guideline values of the National Drinking Water Quality Standards (NDW Q S) of Nepal. There is no guideline value for total bacteria in NDW Q S however, high concentrations can be indicative of poor control on regrowth of bacteria and recontamination or inefficient water treatment methods.Malla B, Ghaju Shrestha R, Bhandari D, Tandukar S, Shrestha S, Yoshinaga H, Inoue D, Sei K, Nishida K, Tanaka Y, Sherchand JB, Haramoto

Gut microbiome transition across a lifestyle gradient in Himalaya

Published: November 15, 2018The composition of the gut microbiome in industrialized populations differs from those living traditional lifestyles. However, it has been difficult to separate the contributions of human genetic and geographic factors from lifestyle. Whether shifts away from the foraging lifestyle that characterize much of humanity's past influence the gut microbiome, and to what degree, remains unclear. Here, we characterize the stool bacterial composition of four Himalayan populations to investigate how the gut community changes in response to shifts in traditional human lifestyles. These groups led seminomadic hunting-gathering lifestyles until transitioning to varying levels of agricultural dependence upon farming. The Tharu began farming 250-300 years ago, the Raute and Raji transitioned 30-40 years ago, and the Chepang retain many aspects of a foraging lifestyle. We assess the contributions of dietary and environmental factors on their gut-associated microbes and find that differences in the lifestyles of Himalayan foragers and farmers are strongly correlated with microbial community variation. Furthermore, the gut microbiomes of all four traditional Himalayan populations are distinct from that of the Americans, indicating that industrialization may further exacerbate differences in the gut community. The Chepang foragers harbor an elevated abundance of taxa associated with foragers around the world. Conversely, the gut microbiomes of the populations that have transitioned to farming are more similar to those of Americans, with agricultural dependence and several associated lifestyle and environmental factors correlating with the extent of microbiome divergence from the foraging population. The gut microbiomes of Raute and Raji reveal an intermediate state between the Chepang and Tharu, indicating that divergence from a stereotypical foraging microbiome can occur within a single generation. Our results also show that environmental factors such as drinking water source and solid cooking fuel are significantly associated with the gut microbiome. Despite the pronounced differences in gut bacterial composition across populations, we found little differences in alpha diversity across lifestyles. These findings in genetically similar populations living in the same geographical region establish the key role of lifestyle in determining human gut microbiome composition and point to the next challenging steps of determining how large-scale gut microbiome reconfiguration impacts human biology.Aashish R. Jha, Emily R. Davenport, Yoshina Gautam, Dinesh Bhandari, Sarmila Tandukar, Katharine M. Ng, Gabriela K. Fragiadakis, Susan Holmes, Guru Prasad Gautam, Jeff Leach, Jeevan Bahadur Sherchand, Carlos D. Bustamante, Justin L. Sonnenbur

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 × 10(-5)M) was the most effective anti-mycobacterial agent; clofazimine (1.25 × 10(-5)M and 3.13 × 10(-6)M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 × 10(-5)M) was filaricidal, but minocycline was more effective and at a lower concentration (5 × 10(-5)M and 1.25 × 10(-5)M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode

A STUDY OF MALARIA IN RELATION TO HIV AND SYPHILIS AMONG PATIENTS VISITING BHERI ZONAL HOSPITAL

ABSTRACT The study was conducted in patients, who were febrile with signs of anaemia, splenonregaly and the classic cycle of coldness, fever peaks and sweats from January to September 2000 in Bheri Zonal Hospital, Nepal. The study aimed to find out relationship of malaria with HIV and syphilis. Two hundred and twenty three blood samples collected were processed and observed using the methods of ELISA and RPR. Among total 223 suspected risk group cases, 6 individuals were detected as HIV positive and 12 individuals had syphilis. Co-infection with malaria & HIV was detected in 2 patients and malaria with syphilis in 8 patients. Similarly 2 patients were co-infected with malaria, HIV and syphilis. A small positive correlation (p<0.5) of malaria, HIV and Syphilis was detected. Key Words: Malaria, HIV, Syphilis, ELISA, RPR, Nepal

Epidemiology of Taenia solium in Nepal: is it influenced by the social characteristics of the population and the presence of Taenia asiatica?

The transmission of the zoonotic pork tapeworms Taenia solium and T. asiatica depends on a combination of specific risk factors, such as open defecation, backyard pig raising and the consumption of raw or undercooked pork and viscera. A community-based survey was conducted among 289 households in south-eastern Nepal to study the heterogeneity of these risk factor frequencies as a function of the social composition of the population. The frequency of open defecation, backyard pig raising and pork consumption differed significantly (P < 0.005) among the different coexisting caste and ethnic groups. In the same survey, the taeniosis prevalence was examined among the different groups. Tapeworm carriers were identified at a high prevalence among the Dum, one of the most disadvantaged communities of Nepal. A PCR-RFLP assay revealed that all collected tapeworm specimens were T. asiatica, a species thus far not known to occur in South Asia. These results can help to understand the epidemiology of T. solium in Nepal, which appears to be more complex than thought so far

The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of <i>Chlamydia trachomatis</i>

<div><p><i>Chlamydia trachomatis</i> is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected <i>in vitro</i> using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for <i>C</i>. <i>trachomatis</i> co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit <i>C</i>. <i>trachomatis</i> replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects <i>C</i>. <i>trachomatis</i> from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known <i>Homo sapiens</i> proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.</p></div

Influence of the tryptophan-indole-IFNγ axis on human genital Chlamydia trachomatis infection: role of vaginal co-infections

The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium’s capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxygenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis