Background: Enterobacter cloacae causes nosocomial infections in 15% of patients in low- and middle-income countries with emergence of carbapenem resistance. The utilisation of bacteriophages for therapeutic purposes is crucial for eradicating these resistant bacterial strains.Objective: This study evaluated the efficacy of lytic phages on bacterial isolates of E. cloacae and determined their stability in various physicochemical conditions.Methods: Twenty-nine lytic phages were isolated from the waste water of six informal settlements in Nairobi County, Kenya, from July 2019 to December 2020 and cross-reacted with 30 anonymised clinical isolates of E. cloacae. Six phages were then selected for physicochemical property studies. Phages were described as potent upon lysing any bacterial strain in the panel.Results: Selected phages were stable at 4 °C – 50 °C with a 5.1% decrease in titre in four of six phages and a 1.8% increase in titre in two of six phages at 50 °C. The phages were efficient following two weeks incubation at 4 °C with optimal activity at human body temperature (37 °C) and an optimal pH of 7.5. Phages were active at 0.002 M and 0.015 M concentrations of Ca2+ ions. The efficiency of all phages decreased with increased exposure to ultraviolet light. All phages (n = 29) showed cross-reactivity against anonymised clinical isolates of E. cloacae strains (n = 30). The most potent phage lysed 67.0% of bacterial strains; the least potent phage lysed 27.0%.Conclusion: This study reveals the existence of therapeutic phages in Kenya that are potent enough for treatment of multi-drug resistant E. cloacae.

Non-typhoidal Salmonella (NTS) typically cause self-limiting enterocolitis, but can lead to life-threatening invasive diseases, particularly in sub-Saharan Africa. In Kenya, multidrug-resistant (MDR) NTS strains with increasing non-susceptibility to third-generation cephalosporins pose a growing public health threat. As traditional antimicrobial treatments become less effective, bacteriophages are emerging as a potential alternative. This study aimed to isolate and characterize bacteriophages targeting MDR and extended spectrum-β-lactamase (ESBL)-producing non-typhoidal Salmonella (NTS). Environmental samples were collected from seven sites in Nairobi City County, Kenya. Four NTS bacterial strains were used for phage enrichment, screening, and purification via spot tests and plaque assays. Phage efficacy was assessed in vitro by testing host range and efficiency of plating (EOP) against 12 Salmonella strains isolated in Kenya over different years. Ten selected broad-host-range phages were evaluated for thermal and pH stability and their ability to disrupt pre-formed NTS biofilms. Phage genomes were sequenced using the Illumina sequencing platform, and analyzed with bioinformatics tools to screen for antimicrobial resistance (AMR), lysogeny, virulence, and allergenic genes. The morphological characteristics of four representative phages were examined using Transmission Electron Microscopy. Thirty-one phages were isolated, with host ranges varying from lysing one strains to all 12 strains. Ten phages lysed more than 80% of the Salmonella strains and were selected for further characterization. Most phages exhibited high production EOP on at least one bacterial strain, except KE26 and KE28. All phages were stable from − 80 °C to 40 °C and pH 5 to 11, with noticeable but statistically insignificant biofilm disruption. Genome sizes ranged from 23,215 bp to 159,981 bp, and were free of known AMR, lysogeny, or virulence genes. Allergenicity screening identified no allergenic hits across most phages, with exception of KE23, which showed potential allergenic regions in its tail fiber and endolysin proteins. All phages belonged to class Caudoviricetes, with KE23, KE26, and KE28 exhibiting a myovirus-like morphotype, and KE15 displaying a siphovirus morphotype. This study identified phages with desirable safety and stability profiles for potential usage against MDR and ESBL-producing NTS infections. Further in vivo studies are recommended to evaluate their therapeutic potential. Non-typhoidal Salmonella (NTS) typically cause self-limiting enterocolitis but can lead to life-threatening invasive diseases. In Kenya, multidrug-resistant (MDR) NTS strains with increasing nonsusceptibility to third-generation cephalosporins have been reported, posing a significant public health concern that requires urgent attention. Bacteriophages are increasingly being considered as an alternative treatment for MDR bacterial infections because of the growing ineffectiveness of conventional antibiotics. Our study reports the isolation and characterization of lytic Salmonella phages devoid of detectable antimicrobial resistance (AMR) genes, lysogeny potential, allergens or virulence factors. These attributes position them as promising candidates for therapeutic interventions against MDR NTS infections. These findings highlight the potential of our study phages as a therapy for drug-resistant NTS and underscore the need for further investigation into their clinical application against MDR strains.

Hatcheries have been identified as a significant source of Salmonella within poultry production. Consequently, there is a need for effective egg disinfection methods that can reduce the pathogen burden while preserving the egg integrity and embryo. The metrics for a successful egg disinfection method are typically a reduction in Total Aerobic Count (TAC) while retaining hatching rates. In this study, a gas phase hydroxyl-radical process was validated and verified as a hatchery egg disinfection method. The process is based on applying a hydrogen peroxide mist in combination with ozone gas and UV-C to generate antimicrobial hydroxyl radicals. The treatment (2 % hydrogen peroxide, 20 ppm ozone and 19 mJ/cm² UV-C; designated as HR) for inactivating Salmonella (serotypes Enteritidis and Typhimurium) inoculated onto eggs could eliminate the pathogen (>5 log CFU/egg reduction) but left residual TAC (1.53 log CFU/egg reduction). Surface sterilization was achieved by a pre-treatment of eggs with the photo-catalyst riboflavin (13.75 mM) followed by 3 % hydrogen peroxide delivered at 70 °C prior to the hydroxyl-radical treatment (3 % hydrogen peroxide, 20 ppm ozone and 114 mJ/cm² designated HRS). The surface sterilization of eggs coincided with the removal of the cuticle layer with the HRS treatment but not HR. The cuticle layer was also compromised by formaldehyde treatment. When the different treatments were applied to fertile hatchery eggs (n=50 eggs per treatment group), there was no significant difference in hatchery rate (64-74 %), with hatch to fertility being higher for disinfected eggs (89-97 %) compared to the non-treated control (80 %). The seven-day mortality (0 - 2 birds) and feed conversion ratio (1.59 - 1.75 kg/kg feed) did not significantly differ between the treated vs controls. The HR treatment could eliminate Enterococcus faecium, Escherichia coli (>5 log CFU/egg reduction) although HRS was required to inactivate Pseudomonas aeruginosa (>5 log CFU/egg reduction) and reduce Aspergillus niger spores (3.08±2.25 log CFU reduction). The study has provided treatment options for hatchery egg disinfection and alternative to formaldehyde treatment.

Objectives: Escherichia coli and some Salmonella enterica serovars are zoonotic pathogens affecting livestock and humans. These pathogens cause significant loss of productivity in livestock, severe morbidity and mortality in humans, and have high antibiotic resistance profiles. Therefore, the exploitation of lytic phages for therapeutic purposes is important for eliminating these resistant bacterial strains. Methods: Thirty-four bacterial stock isolates comprised of 23 E. coli and 11 Salmonella spp. strains were evaluated for antimicrobial susceptibility to seven antibiotics using the Kirby-Bauer disk diffusion test. The antibiotics included Ciprofloxacin, Trimethoprim-Sulphamethoxazole, Gentamycin, Imipenem, Ceftriaxone, Cefotaxime, and Ofloxacin. Twelve (12/23) E. coli and (2/11) Salmonella spp. exhibited antimicrobial resistance. Selected six (6/12) drug-resistant E. coli strains were subjected to three different phages (PA5, EHEC005, C11S1A) for efficacy and host range assay. Similarly, two (2/2) resistant Salmonella strains were exposed to one Salmonella phage A23 for efficacy and host range assay. The E. coli (C11S1A) phage, which infected most bacterial hosts, was evaluated for optimal efficiency at various pH and temperatures. Results: E. coli isolates had the highest resistance 12/23 (52%) compared to Salmonella spp. 2/11(18%) (p<0.05). Most resistance was against Trimethoprim-Sulphamethoxazole (44%) and (9%) for E. coli and Salmonella spp., respectively. Furthermore, E. coli (C11S1A) phages killed all the Escherichia coli strains, while Salmonella phage A23 only lysed the host bacteria. The E. coli (C11S1A) phages were highly efficacious at 37 0C and pH 7.4. Conclusion: The successful isolation of novel lytic E. coli (C11S1A) phages, which killed all the E. coli strains tested, demonstrates the potential for therapeutic purposes for humans and livestock. J Microbiol Infect Dis 2021; 11(4):183-190.

The problem of antimicrobial resistance has created a new need for alternative/ complementary treatments. To this end, bacteriophages offer an exciting prospect, as they can infect and kill specific bacteria without harming the host. This survey aimed to evaluate the state of applied phage research in Africa, among the members of the Africa phage Forum (APF). This was a cross-sectional survey whereby a google form was created for the members of the Africa Phage forum to fill so as to access the stage of phage research in Africa. Data was collected between June and July 2021 using a structured questionnaire form. A total of 65 out of a total of 101 forum members completed the questionnaire. The survey indicated that a majority 68% of phage researchers in Africa were at the training stages of their career. Some available participants were limited (8%). Most of the members identified funding, lack of skill set, near absence of adequate laboratory infrastructure as major hurdles for phage research. Despite these challenges, 73.3% of APF members work with the ESKAPE group with the majority of its members carrying out research in Phage in Biocontrol (80%), whereas others perform research related to human phage therapy (60%). However, it appeared this research has not yet reached the stage of commercialization. Overall, Phage research is in its infancy in Africa. Key challenges included poor laboratory infrastructure, lack of capacity building in the phage field, and lack of local awareness on the significance of phages for policymakers and governments. APF could, therefore, play a role in creating phage awareness in Africa; mobilizing resources; enhancing networks and collaborations amongst APF members and beyond, especially with more experienced phage mentors in the Western countries, to greatly reduce the gap in knowledge and enhance phage research in Africa.

Objectives: The use of bacteriophages (phages) as an alternative treatment for multidrug-resistant bacteria has recently gained popularity. Phage cocktails have been proposed for broad-spectrum therapeutic effects against such resistant bacteria. However, the effectiveness of non-locally formulated phage cocktails for therapy on Kenyan isolates has yet to be evaluated and is a subject of investigation. This study aimed to determine the in vitro effectiveness of a non-locally made commercial pyophage cocktail on clinical Pseudomonas aeruginosa isolates from Kenya. Methodology: Forty-nine P. aeruginosa isolates from Kenya were subjected to a pyophage cocktail for efficacy studies using direct spot test (DST) and efficiency of plating (EOP). Results: The success rate of the cocktail was observed on 16.3% (8/49) isolates only and ineffective on 83.7% (41/49) isolates. Six of the eight isolates that showed cross-reactivity from DST had complete lysis with a faintly hazy background. Five of these six isolates resulted in successful and high phage progeny production in plaquing efficiency (EOP ≥ 0.5). Conclusion: Non-locally made commercial pyophage cocktail was ineffective against the 83.7% endemic clinical strains of the Kenyan P. aeruginosa isolates, demonstrating the importance of locally derived phage cocktails against endemic and multidrug-resistant isolates. J Microbiol Infect Dis 2023; 12(4):25-30.