Phenotypic and Genotypic Methods for the Identification and Quantification of Cyanobacteria in Lake Water.

Early monitoring of Microcystis, a cyanobacterium that produces microcystin, is paramount in order to confirm the presence of Microcystis spp. Both phenotypic and genotypic methods have been used. The phenotypic methods provide the presence of the microcystis but do not confirm its species type and toxin produced. Additionally, phenotypic methods cannot differentiate toxigenic from non-toxigenic Microcystis. Therefore, the current protocol also describes genetic methods based on PCR to detect toxigenic Microcystis spp. based on microcystin synthetase E (mcy E) gene and 16-23S RNA genes for species-specific identification, which can effectively comprehend distinct lineages and discrimination of potential complexity of microcystin populations. The presence of these microcystin toxins in blood, in most cases, indicates contamination of drinking water by cyanobacteria. The methods presented herein are used to identify microcystin toxins in drinking water and blood.

Hepatitis B virus genetic multiplicity and the associated HBV lamivudine resistance mutations in HBV/HIV co-infection in Western Kenya: A review article.

BACKGROUND: Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) co-infections are common as the two viruses use same routes of transmission. Studies show that HIV infection modifies the natural course of chronic HBV infection, leading to more severe and progressive liver disease, and a higher incidence of cirrhosis, liver cancer and mortality. Therefore, determining HBV status and genotypes among HIV co-infected patients would improve their therapeutic management. OBJECTIVE: This article reviewed the HBV genetic multiplicity and the associated HBV Lamivudine resistance mutations in HBV/HIV co-infection in western Kenya. METHODS: Comprehensive literature searches and analysis were performed in peer-reviewed journals in the National council for biotechnology information (NCBI), PubMed, and Web of science using key words of HIV, Hepatitis B genotypes, HBV/HIV co-infection and Lamivudine resistance. RESULTS: HBV genotype A is predominant. D and E are also present in Kenya and neighboring countries in the region. HBV polymerase rtV173L, rtL180M, and rtM204V major substitutional mutations were identified. Currently, TDF + 3TC + DTG are recommended for treatment of HBV/HIV co-infection. CONCLUSION: Evidence shows that HBV/HIV co-infection places a heavy burden to the society. Along with ART regimen, HBV genotype is a major factor determining the course of disease and treatment outcome. Treating HIV in HBV/HIV co-infection with antiretroviral agents may result in a very high prevalence of HBV 3TC-resistance mutations. Therefore, improved screening for HBV and extended follow-up of HBV/HIV co-infected individuals is needed to better understand the impact of different ART regimens on clinical outcomes.

The spleen bacteriome of wild rodents and shrews from Marigat, Baringo County, Kenya.

BACKGROUND: There is a global increase in reports of emerging diseases, some of which have emerged as spillover events from wild animals. The spleen is a major phagocytic organ and can therefore be probed for systemic microbiome. This study assessed bacterial diversity in the spleen of wild caught small mammals so as to evaluate their utility as surveillance tools for monitoring bacteria in an ecosystem shared with humans. METHODS: Fifty-four small mammals (rodents and shrews) were trapped from different sites in Marigat, Baringo County, Kenya. To characterize their bacteriome, DNA was extracted from their spleens and the V3-V4 regions of the 16S rRNA amplified and then sequenced on Illumina MiSeq. A non-target control sample was used to track laboratory contaminants. Sequence data was analyzed with Mothur v1.35, and taxomy determined using the SILVA database. The Shannon diversity index was used to estimate bacterial diversity in each animal and then aggregated to genus level before computing the means. Animal species within the rodents and shrews were identified by amplification of mitochondrial cytochrome b (cytb) gene followed by Sanger sequencing. CLC workbench was used to assemble the cytb gene sequences, after which their phylogenetic placements were determined by querying them against the GenBank nucleotide database. RESULTS: cytb gene sequences were generated for 49/54 mammalian samples: 38 rodents (Rodentia) and 11 shrews (Eulipotyphyla). Within the order Rodentia, 21 Acomys, eight Mastomys, six Arvicanthis and three Rattus were identified. In the order Eulipotyphyla, 11 Crucidura were identified. Bacteria characterization revealed 17 phyla that grouped into 182 genera. Of the phyla, Proteobacteria was the most abundant (67.9%). Other phyla included Actinobacteria (16.5%), Firmicutes (5.5%), Chlamydiae (3.8%), Chloroflexi (2.6%) and Bacteroidetes (1.3%) among others. Of the potentially pathogenic bacteria, Bartonella was the most abundant (45.6%), followed by Anaplasma (8.0%), Methylobacterium (3.5%), Delftia (3.8%), Coxiella (2.6%), Bradyrhizobium (1.6%) and Acinetobacter (1.1%). Other less abundant (<1%) and potentially pathogenic included Ehrlichia, Rickettsia, Leptospira, Borrelia, Brucella, Chlamydia and Streptococcus. By Shannon diversity index, Acomys spleens carried more diverse bacteria (mean Shannon diversity index of 2.86, p = 0.008) compared to 1.77 for Crocidura, 1.44 for Rattus, 1.40 for Arvicathis and 0.60 for Mastomys. CONCLUSION: This study examined systemic bacteria that are filtered by the spleen and the findings underscore the utility of 16S rRNA deep sequencing in characterizing complex microbiota that are potentially relevant to one health issues. An inherent problem with the V3-V4 region of 16S rRNA is the inability to classify bacteria reliably beyond the genera. Future studies should utilize the newer long read methods of 16S rRNA analysis that can delimit the species composition.

Susceptibility patterns and the role of extracellular DNA in Staphylococcus epidermidis biofilm resistance to physico-chemical stress exposure.

BACKGROUND: Over 65% of human infections are ascribed to bacterial biofilms that are often highly resistant to antibiotics and host immunity. Staphylococcus epidermidis is the predominant cause of recurrent nosocomial and biofilm-related infections. However, the susceptibility patterns of S. epidermidis biofilms to physico-chemical stress induced by commonly recommended disinfectants [(heat, sodium chloride (NaCl), sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2)] in domestic and human healthcare settings remains largely unknown. Further, the molecular mechanisms of bacterial biofilms resistance to the physico-chemical stresses remain unclear. Growing evidence demonstrates that extracellular DNA (eDNA) protects bacterial biofilms against antibiotics. However, the role of eDNA as a potential mechanism underlying S. epidermidis biofilms resistance to physico-chemical stress exposure is yet to be understood. Therefore, this study aimed to evaluate the susceptibility patterns of and eDNA release by S. epidermidis biofilm and planktonic cells to physico-chemical stress exposure. RESULTS: S. epidermidis biofilms exposed to physico-chemical stress conditions commonly recommended for disinfection [heat (60 °C), 1.72 M NaCl, solution containing 150 µL of waterguard (0.178 M NaOCl) in 1 L of water or 1.77 M H2O2] for 30 and 60 min exhibited lower log reductions of CFU/mL than the corresponding planktonic cells (p < 0.0001). The eDNA released by sub-lethal heat (50 °C)-treated S. epidermidis biofilm and planktonic cells was not statistically different (p = 0.8501). However, 50 °C-treated S. epidermidis biofilm cells released significantly increased eDNA than the untreated controls (p = 0.0098). The eDNA released by 0.8 M NaCl-treated S. epidermidis biofilm and planktonic cells was not significantly different (p = 0.9697). Conversely, 5 mM NaOCl-treated S. epidermidis biofilms exhibited significantly increased eDNA release than the corresponding planktonic cells (p = 0.0015). Further, the 50 µM H2O2-treated S. epidermidis biofilms released significantly more eDNA than the corresponding planktonic cells (p = 0.021). CONCLUSIONS: S. epidermidis biofilms were less susceptible to physico-chemical stress induced by the four commonly recommended disinfectants than the analogous planktonic cells. Further, S. epidermidis biofilms enhanced eDNA release in response to the sub-lethal heat and oxidative stress exposure than the corresponding planktonic cells suggesting a role of eDNA in biofilms resistance to the physico-chemical stresses.

Isolation of Salmonella and Shigella from fish harvested from the Winam Gulf of Lake Victoria, Kenya.

BACKGROUND: Human infections caused by pathogens transmitted from fish are quite common. The aim of this study was to isolate enteric pathogenic bacteria from fish that might be transmitted to humans after the handling or consumption of such fish. METHODOLOGY: One hundred and twenty Nile tilapia fish harvested using various fishing methods were collected from fishermen in five fish landing beaches within Winam Gulf and disinfected externally using 70% ethyl alcohol for 2 minutes then washed three times with autoclaved distilled water. Isolation of Salmonella and Shigella species from fish samples was performed using standard bacteriological procedures. Five milliliters of each fish tissue slurry was microbiologically analyzed for any Enterobacteriaceae. Twelve Nile tilapia collected from three open-air markets were analyzed for Enterobacteriaceae comparison as controls. Identification of Salmonella by using housekeeping genes and species-specific primers was performed. RESULTS: Among 120 Nile tilapia, 63 (52.5%) were infected with Enterobacteriaceae. Out of these, 25 (39.7%) were Shigella spp, 9 (14.3%) Salmonella typhimurium, 7 (11.1%) S. typhi, 4 (6.3%) S. enteritidis, 16 (25.4%) Escherichia coli, 1 (1.6%) Proteus spp. and Enterobacter aerogenes respectively. Ten fish collected from open-air markets yielded E. coli (50%), S. typhimurium (20%), S. paratyphi (10%) and S. typhi (20%). CONCLUSION: Nile tilapia within Winam Gulf are infected by human enteric pathogens. Shigella spp., Salmonella and E. coli were the most frequently isolated, an indication that the beaches may be contaminated by untreated municipal sewage, runoff, and storm-water. S. typhimurium, S. typhi and S. enteritidis were the most common Salmonella isolates.