Juliah Khayeli Akhwale

Mastitis infection remains an economically important disease in livestock. Antimicrobial resistance in caprine mastitis compromises animal welfare and smallholder livelihoods and its control is complicated by the high prevalence of resistance to existing antibiotics. Phage therapy, therefore, is considered as an alternative way of controlling mastitis infections. Lytic bacteriophages offer a narrow-spectrum alternative to antibiotics, yet the data on their application in goats remain limited. In this study, we evaluated the potential of lytic bacteriophages for the in vitro biocontrol of mastitis-associated Escherichia coli. A total of 56 milk samples from goats were screened for mastitis using California Mastitis Test (CMT) and identified for E. coli isolates by culture characteristics and 16S rRNA gene sequencing. Antimicrobial susceptibility testing (AST) was performed on Mueller-Hinton Agar using CLSI M100-2024 interpretive criteria. Nine lytic coliphages were isolated and purified from sewage samples collected in Juja Sub-County in Kiambu County. We assessed the host range, multiplicity of infection (MOI), one-step growth kinetics, stability across pH 2–10, and 20–50 °C, and growth inhibition in vitro, including a two-phage cocktail. The animal-level prevalence of subclinical mastitis was 39.3% (22/56). Among the nine confirmed E. coli isolates, 66.7% (6/9) exhibited multidrug resistance (MDR) to ≥3 antibiotic classes. Phages demonstrated optimal stability at pH 6–8 and and temperatures of 30–40 °C, retaining high infectivity within these ranges. The two-phage cocktail achieved a significant reduction in bacterial growth (p = 0.0189), demonstrating enhanced inhibitory activity (AUC = 2.15 ± 0.045, 95% CI: 2.06–2.24; 69.6% inhibition), indicating improved inhibition against multidrug-resistant caprine E. coli in vitro. Future validation in complex milk matrices and whole-genome sequencing to confirm strictly lytic lifestyles are essential next steps toward intramammary application.

Background/Objectives: Antimicrobial resistance (AMR) in Klebsiella pneumoniae poses a serious threat to healthcare, especially in sub-Saharan Africa (SSA). To complement AMR infection control in Kenya, here, clinical and environmental genomes were investigated to determine the potential roles prophages play in K. pneumoniae pathogenicity. Methods: Prophages were extracted from 89 Kenyan K. pneumoniae genomes. The intact prophages were examined for virulence genes carriage, and their phylogenetic relationships were established. Results: Eighty-eight (~99%) of the genomes encode at least a single prophage, and there is an average of four prophages and 2.8% contributory genomes per bacterial strain. From the 364 prophages identified, 250 (68.7%) were intact, while 58 (15.9%) and 57 (15.7%) were questionable and incomplete, respectively. Approximately, 30% of the intact prophages encode 38 virulence genes that are linked to iron uptake (8), regulation (6), adherence (5), secretion system (4), antiphagocytosis (4), autotransporter (4), immune modulation (3), invasion (2), toxin (1) and cell surface/capsule (1). Phylogenetic analyses revealed three distinct clades of the intact prophages irrespective of their hosts, sources and locations, which support the plasticity of the genomes and potential to mediate horizontal gene transfer. Conclusions: This study provides first evidence showing the diverse prophages that are encoded in K. pneumoniae from SSA with particular focus on Kenyan strains. This also shows the potential roles these prophages play in the pathogenicity and success of K. pneumoniae and could improve knowledge and complement control strategies in the region and across the globe. Further work is needed to show the expression of these genes through lysogenisation.

Aim: This study aimed to characterize antimicrobial resistant non-typhoidal Salmonella and other bacteria in integrated fish farming environments in Nyeri County. Study Design: Integrated fish farming systems utilizing animal manure were investigated across three sub-counties: Tetu East, Nyeri Town, and Mathira. Methodology: Samples from fish, pond water, and manure were collected and processed for microbial analysis. Fish intestinal contents, water, and manure were incubated in nutrient broth and cultured on nutrient agar. Bacterial identification was done using MALDI-ToF mass spectrometry. Non-typhoidal Salmonella (NTS) was identified using selective culture media and confirmed through 16S rDNA typing. Antimicrobial susceptibility was tested via broth micro dilution, and Plasmid DNA of resistant isolates were screened through PCR and gel electrophoresis. Results: Multiple pathogens were detected, including Escherichia coli, Shigella spp., Staphylococcus spp., Aeromonas hydrophila, Serratia. NTS was found in 42.3% of all samples. Over 88% of Salmonella isolates showed MIC ≤1 µg/ml for ciprofloxacin, while 75% had MIC ≥3.125 µg/ml for azithromycin. High resistance was observed for oxacillin (54% with MIC 300–6000 µg/ml) and streptomycin (50% with MIC ≥24,000 µg/ml). Resistance rates were 60.6% for ciprofloxacin, 100% for oxacillin and streptomycin, and 23.1% for azithromycin. Resistance genes detected included parC, gyrA, msrA, strA, and strB, though mecA was absent. Discussion and Conclusion: The findings suggest that integrated fish farming poses a risk of transmitting antimicrobial-resistant pathogens from animal manure to aquatic environments. This not only threatens aquatic animal health but also poses a serious risk to human health through potential transmission via direct contact, environmental exposure, or consumption of contaminated fish products.

The African Journal of Laboratory Medicine focuses on the role of the laboratory and its professionals in the clinical and public healthcare sectors, and is specifically based on an African frame of reference.

Multi-drug resistant (MDR) Salmonella enterica Enteritidis is one of the major causes of foodborne illnesses worldwide. This non-typhoidal Salmonella (NTS) serovar is mainly transmitted to humans through poultry products. Bacteriophages (phages) offer an alternative to antibiotics for reducing the incidence of MDR NTS in poultry farms. Phages that survive the harsh environment of the chicken gastrointestinal tract (cGIT), which have low pH, high temperatures, and several enzymes, may have a higher therapeutic or prophylactic potential. In this study, we analysed the stability of 10 different S. Enteritidis phages isolated from Kenyan poultry farms in different pH-adjusted media, incubation temperatures, as well as simulated gastric and intestinal fluids (SGF and SIF, respectively). Furthermore, their ability to persist in water sources available in Kenya, including river, borehole, rain and tap water, was assessed. All phages were relatively stable for 12 h at pHs ranging from 5 to 9 and at temperatures ranging from 25 °C to 42 °C. At pH 3, a loss in viral titre of up to three logs was observed after 3 h of incubation. In SGF, phages were stable for 20 min, after which they started losing infectivity. Phages were relatively stable in SIF for up to 2 h. The efficacy of phages to control Salmonella growth was highly reduced in pH 2- and pH 3-adjusted media and in SGF at pH 2.5, but less affected in SIF at pH 8. River water had the most significant detrimental effect on phages, while the other tested waters had a limited impact on the phages. Our data suggest that these phages may be administered to chickens through drinking water and may survive cGIT to prevent salmonellosis in poultry.

This study aimed to evaluate the genomic features of novel Kenyan virulent phage isolates infecting carbapenemase-producing Klebsiella pneumoniae and to determine the safety of their lysates using mice model in a preclinical study. The genomics showed that the Klebsiella phages vB_KpM_CPRSA and vB_KpM_CPRSB belonged to the genus Slopekvirus with a similarity index of less than 92% compared to the most closest relative species. Their genomes did not contain antimicrobial resistance and toxin genes. Then endotoxin levels in the Klebsiella phage lysates were statistically significant (p value ˃ 0.05). The serum activities of aspartate aminotransferase, alanine aminotransferase and urea in the group of balb/c mice injected with bacteriophage lysates through the intravenous route were higher compared to that of the intranasal route. Unexpectedly, there was mild congestion of the central veins of kidneys and liver without damage to renal tubules and hepatocytes and a lack of physical discomfort and pain in the mice. Our study isolated and characterised Klebsiella phages against carbapenem-resistant K. pneumoniae, which are promising therapeutic agents for the treatment of respiratory tract infections using the topical mode of administration as the preferred route of bacteriophage delivery.

Background: The development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing Klebsiella pneumoniae. Objective: This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing K. pneumoniae clinical isolate in vitro in Kenya. Methods: The study was conducted from February 2021 to October 2021 at the Institute of Primate Research, Nairobi, Kenya. Phage cocktails were formulated based on the morphology and biological properties of precipitated Klebsiella phages. The efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing K. pneumoniae (KP20). Results: The precipitated bacteriophages were members of Myoviridae, Siphoviridae and Podoviridae. Regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2φ MA) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. Moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2φ MA was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2φ MA. Conclusion: This study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing K. pneumoniae growth in vitro.

As a step towards better understanding of diversity and biology of phages and their hosts in haloalkaline Lake Elmenteita, phages were isolated from sediment samples and overlying water using indigenous bacteria as hosts. 17 seemingly different phages of diverse morphotypes with different dimensions and partly exhibiting remarkably unusual ultrastructures were revealed by transmission electron microscopy. 12 clonal phage isolates were further characterized. Infection capability of the phages was optimum at 30–35°C and in alkali condition with optimum at pH 10–12. Structural protein profiles and restriction fragment length polymorphism analyses patterns were distinct for each of the phage type. Complete nucleotide sequences of phages vB-VmeM-32, vB_EauS-123 and vB_BhaS-171 genomes varied in size from 30,926–199,912 bp and G + C content of between 36.25–47.73%. A range of 56–260 potential open reading frames were identified and annotated. The results showed that the 12 phages were distinct from each other and confirmed the presence and diversity of phages in extreme environment of haloalkaline Lake Elmenteita. The phages were deposited at the German Collection of Microorganisms and Cell Cultures and three of their genomes uploaded to NCBI GenBank.

We report complete genome sequences of eight bacteriophages isolated from Haloalkaline Lake Elmenteita found on the floor of Kenyan Rift Valley. The bacteriophages were sequenced, annotated and a comparative genomic analysis using various Bioinformatics tools carried out to determine relatedness of the bacteriophages to each other, and to those in public databases. Basic genome properties like genome size, percentage coding density, number of open reading frames, percentage GC content and gene organizations revealed the bacteriophages had no relationship to each other. Comparison to other nucleotide sequences in GenBank database showed no significant similarities hence novel. At the amino acid level, phages of our study revealed mosaicism to genes with conserved domains to already described phages. Phylogenetic analyses of large terminase gene responsible for DNA packaging and DNA polymerase gene for replication further showed diversity among the bacteriophages. Our results give insight into diversity of bacteriophages in Lake Elmenteita and provide information on their evolution. By providing primary sequence information, this study not only provides novel sequences for biotechnological exploitation, but also sets stage for future studies aimed at better understanding of virus diversity and genomes from haloalkaline lakes in the Rift Valley.