Wesaal Khan

The study aimed to investigate the upscaled culture-based production of the predatory bacterium, Bdellovibrio bacteriovorus ( B. bacteriovorus ) PF13 (isolated from wastewater) using metabolically active and heat-inactivated Escherichia coli ( E. coli ) ATCC 417373 as prey. This approach was used to assess whether heat-treated prey cells could support the production of predatory stock lysates. Heat-inactivation was validated using resazurin assays, which showed reduced relative fluorescence units (RFU) in all heat-treated, E. coli ATCC 417373 samples in comparison to the untreated controls, with the 65 °C treatment yielding the lowest RFU. The predatory lysate was then upscaled, using suspensions of either metabolically active or heat-inactivated (65 °C) E. coli ATCC 417373, from 100 mL to 1 L, and finally to 2.5 L. For the 100 mL, 1 L and 2.5 L co-cultures, using B. bacteriovorus PF13 and metabolically active E. coli ATCC 417373, the PFU/mL and cells/mL of B. bacteriovorus PF13 increased by 2.84 and 1.12 logs, 1.40 and 0.86 logs, and 2.37 and 0.80 logs, respectively. For the 100 mL, 1 L and 2.5 L co-cultures, using B. bacteriovorus PF13 and heat-inactivated E. coli ATCC 417373, the cells/mL of B. bacteriovorus PF13 then increased by 1.27 logs, 1.23 logs, and 0.29 logs, respectively. While further optimisation is required to produce predatory lysates in shorter incubation time periods, this study demonstrated the feasibility of upscaling B. bacteriovorus production using heat-inactivated prey and culture-based methods. This strategy may potentially circumvent the need for bioreactor infrastructure, which is particularly crucial for laboratories in resource-limited countries.

The October 2023 to 2024 cholera outbreak demonstrates significant challenges related to water quality and sanitation, especially in peri-urban areas with limited access to clean water. This study assesses the presence of faecal coliforms and Escherichia coli (E. coli) in drinking water sources across five townships, identified as cholera transmission hotspots, two months post the cholera outbreak in the Lusaka District. A total of 169 water samples were collected from protected sources, treated piped water, and unprotected sources, including dams and shallow wells. Faecal coliforms and E. coli were detected across all source types. Among unprotected sources, 92.3% (12/13) of samples contained ≥100 CFU/100 mL of both faecal coliforms and E. coli. Protected sources showed variable contamination, with 18.3% exceeding ≥100 CFU/100 mL for faecal coliforms and 15.4% for E. coli. Treated water sources showed the lowest contamination, with 88.5% of samples having no detectable faecal coliforms and 90.4% having no detectable E. coli. Zero-inflated negative binomial regression showed that treated water sources were associated with substantially lower faecal coliform counts compared with protected sources (PR = 0.11, 95% CI: 0.03–0.35), while unprotected sources exhibited higher contamination intensity (PR = 1.77, 95% CI: 0.94–3.31). Treated sources were significantly more likely to be structurally free of contamination, whereas unprotected sources had an extremely low probability of yielding zero counts. These findings indicate that current water safety conditions in Lusaka’s cholera hotspot areas remain inadequate for preventing faecal-oral transmission.

Introduction. Cholera, caused by Vibrio cholerae , remains a priority public health concern, particularly in developing countries. The first cholera outbreak in Zambia was documented in the 1970s, with recurring epidemics reported since then. In 2023, a cholera outbreak affected Zambia, particularly in districts bordering Malawi, Mozambique and the Democratic Republic of Congo, with significant cases reported in these neighbouring countries. This study aims to analyse cholera cases and isolates obtained during the 2023 epidemic, focusing on geographical distribution, genetic relatedness of isolates and their antibiotic resistance profiles. Methods. Stool samples were collected from patients presenting with cholera-like symptoms across three provinces of Zambia. A total of 98 samples were cultured on thiosulphate citrate bile salts sucrose agar, resulting in 32 sequenced V. cholerae isolates. Whole-genome sequencing was performed using Oxford Nanopore Technology, and phylogenetic inference was also achieved by the analysis of SNPs. Phenotypic antimicrobial resistance testing was conducted following Clinical and Laboratory Standards Institute guidelines. The genomic data were analysed for virulence factors and antimicrobial resistance profiles. Results. Of the 98 stool samples tested, 38 confirmed cholera cases were identified. A subset of 32 confirmed V. cholerae isolates, predominantly from the Eastern Province of Zambia ( n =21), was selected for whole-genome sequencing. Genomic analysis revealed that all isolates belonged to the seventh pandemic El Tor lineage and the O1 serogroup, with two distinct clades identified corresponding to the 10th (T10) and 15th (T15) transmission events. Geographical analysis indicated a predominance of Ogawa serotypes in Eastern Province and Inaba in Northern Province. The virulence gene analysis confirmed the presence of key cholera toxin genes ( ctxA and ctxB ) and intestinal colonization factors. All isolates carried genes or mutations predicted to confer resistance to multiple antibiotics, including decreased susceptibility to ciprofloxacin, recommended for the treatment of cholera by the World Health Organization. Conclusion. The findings highlight the critical need for enhanced surveillance and targeted interventions to mitigate cholera outbreaks in Zambia. The emergence of resistant V. cholerae strains necessitates innovative strategies, including improved water sanitation, vaccination efforts and novel therapeutic approaches to combat this enduring public health threat.

Bacteriophage-antibiotic-synergy (PAS) was investigated to target Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and Enterobacter cloacae. Whole genome sequencing indicated that bacteriophage KPW17 targeting K. pneumoniae, clustered with genus Webervirus, ECSR5 targeting E. cloacae clustered with Eclunavirus, PAW33 targeting P. aeruginosa clustered with Bruynoghevirus, while ABTW1 targeting A. baumannii clustered with Vieuvirus. PAS analysis showed that the combination of ciprofloxacin (CIP) and levofloxacin (LEV) with PAW33 resulted in the synergistic eradication of all tested P. aeruginosa strains. Similarly, the combined use of doripenem (DOR) and LEV with KPW17 resulted in the synergistic eradication of the environmental and clinical K. pneumoniae strains, while the combined use of DOR and gentamicin (CN) with ECSR5 was synergistic against the clinical E. cloacae NCTC 13406. Gentamicin with ECSR5, however, only exhibited an additive effect for E. cloacae 4L, while ABTW1 with piperacillin-tazobactam (TZP) and imipenem (IPM) resulted in an indifferent interaction between the bacteriophage and tested antibiotics against the clinical A. baumannii AB3, i.e., the activity of the combination is equal to the activity of most active agent. Thus, while the observed PAS may offer an opportunity for the re-introduction or more efficient application of certain antibiotics to combat antibiotic resistance, extensive research is required to determine the optimal phage-antibiotic combinations, dosages and treatment regiments.

The study aimed to investigate the antibacterial activity, cytotoxicity, and mechanism of action of the non-ionic, cyclic lipopeptide, serrawettin W2-FL10 against Staphylococcus aureus . W2-FL10 exhibited potent activity against the Gram-positive bacteria S. aureus , Enterococcus faecalis , Enterococcus faecium , Listeria monocytogenes, and Bacillus subtilis , with minimum inhibitory concentration (MIC) values ranging from 6.3 to 31.3 μg/mL, while no activity was observed against Gram-negative bacteria. Broth microdilution assays showed that W2-FL10 interacted with key cell membrane components, such as lipid phosphatidyl glycerol and lipoteichoic acid of S. aureus . Upon membrane interaction, W2-FL10 dissipated membrane potential within 12 min and increased S. aureus membrane permeability within 28–40 min, albeit at slower rates and higher concentrations than the lytic peptide melittin. The observed membrane permeability, as detected with propidium iodide (PI), may be attributed to transmembrane pores/lesions, possibly dependent on dimer-driven lipopeptide oligomerization in the membrane. Scanning electron microscopy (SEM) imaging also visually confirmed the formation of lesions in the cell wall of one of the S. aureus strains, and cell damage within 1 h of exposure to W2-FL10, corroborating the rapid time-kill kinetics of the S. aureus strains. This bactericidal action against the S. aureus strains corresponded to membrane permeabilization by W2-FL10, indicating that self-promoted uptake into the cytosol may be part of the mode of action. Finally, this lipopeptide exhibited low to moderate cytotoxicity to the Chinese hamster ovarian (CHO) cell line in comparison to the control (emetine) with an optimal lipophilicity range (log D value of 2.5), signifying its potential as an antibiotic candidate. IMPORTANCE: Antimicrobial resistance is a major public health concern, urgently requiring antibacterial compounds exhibiting low adverse health effects. In this study, a novel antibacterial lipopeptide analog is described, serrawettin W2-FL10 (derived from Serratia marcescens ), with potent activity displayed against Staphylococcus aureus . Mechanistic studies revealed that W2-FL10 targets the cell membrane of S. aureus , causing depolarization and permeabilization because of transmembrane lesions/pores, resulting in the leakage of intracellular components, possible cytosolic uptake of W2-FL10, and ultimately cell death. This study provides the first insight into the mode of action of a non-ionic lipopeptide. The low to moderate cytotoxicity of W2-FL10 also highlights its application as a promising therapeutic agent for the treatment of bacterial infections.

Bacteria (including disinfection- and antibiotic-resistant bacteria) are abundant in the consumer water cycle, where they may cause disease, and lead to biofouling and infrastructure damage in distributions systems, subsequently resulting in significant economic losses. Bacteriophages and their associated enzymes may then offer a biological control solution for application within the water sector. Lytic bacteriophages are of particular interest as biocontrol agents as their narrow host range can be exploited for the targeted removal of specific bacteria in a designated environment. Bacteriophages can also be used to improve processes such as wastewater treatment, while bacteriophage-derived enzymes can be applied to combat biofouling based on their effectiveness against preformed biofilms. However, the host range, environmental stability, bacteriophage resistance and biosafety risks are some of the factors that need to be considered prior to the large-scale application of these bacterial viruses. Characteristics of bacteriophages that highlight their potential as biocontrol agents are thus outlined in this review, as well as the potential application of bacteriophage biocontrol throughout the consumer water cycle. Additionally, the limitations of bacteriophage biocontrol and corresponding mitigation strategies are outlined, including the use of engineered bacteriophages for improved host ranges, environmental stability and the antimicrobial re-sensitisation of bacteria. Finally, the potential public and environmental risks associated with large-scale bacteriophage biocontrol application are considered, and alternative applications of bacteriophages to enhance the functioning of the consumer water cycle, including their use as water quality or treatment indicators and microbial source tracking markers, are discussed.