Charles William Keevil

Objectives: Chemical disinfectants are critical for infection control in healthcare environments and beyond, as exemplified by their vital role during the COVID-19 pandemic. Despite research repeatedly demonstrating that bacteria can develop adaptations that mitigate the efficacy of chemical disinfectants, the underlying molecular mechanisms remain poorly characterized. This study investigates the mechanisms that underpin resistance demonstrated by disinfectant-adapted Klebsiella pneumoniae NCTC 13443 samples. Methods: Resistant samples have previously undergone long-term in vitro adaptation via serial passage in increasing concentrations of common disinfectants benzalkonium chloride (BAC), didecydimethylammonium chloride (DDAC), polyhexamethylene biguanide (PHMB), chlorocresol or bronopol. A multi-omics approach was used to conduct in-depth molecular analyses of the adaptations that contribute to resistance. Results: K. pneumoniae adaptation to BAC, DDAC and PHMB was associated with the modification of lipid A causing the reduction of the net-negative charge of the outer surface, lowering the affinity of cationic disinfectants. This mechanism is also used for polymyxin and colistin resistance, highlighting a potential cross-resistance risk. Chlorocresol-adapted K. pneumoniae samples demonstrated increased expression of efflux pumps and expression changes linked to biofilm formation. Bronopol resistance was associated with promoting biofilm formation and increased thioredoxin expression to alleviate oxidative stress. Results indicate the potential role of N-ethylmaleimide reductase NemA in bronopol resistance via enzymatic degradation. Conclusions: These findings provide novel insights into how causative pathogens of healthcare-associated infections can adapt to and mitigate the effectiveness of common chemical disinfectants that are relied on globally every day as a critical infection control measure.

Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux pump from the resistance-nodulation-cell division (RND) superfamily, is upregulated in these conditions but its molecular mechanism is unknown. Here, we report cryo-electron microscopy structures of Escherichia coli multidrug transporter MdtF within native-lipid nanodiscs, including a single-point mutant with an altered multidrug phenotype and associated substrate-bound form. Drug binding domain and channel conformational plasticity likely governs substrate polyspecificity, analogous to closely related, constitutively expressed counterpart, AcrB. Whereas we discover distinct transmembrane state transitions within MdtF, which create a more engaged proton relay network, altered drug transport allostery and an acid-responsive increase in efflux efficiency. Our findings provide mechanistic insights necessary to understand bacterial xenobiotic and toxin removal by MdtF and its role within nutrient-depleted and acid stress settings, as endured in the gastrointestinal tract.

Fresh produce-associated outbreaks of the foodborne pathogen Escherichia coli O157 are responsible for a number of disease cases, hospitalizations and deaths. In many cases, the source of contamination can be linked to the growing media of the food, although pathogen detection is problematic in these complex soil ecosystems. In this study, direct quantitative real-time PCR without pre-enrichment was used to detect 310 copies of the Tir gene, using a primer sequence specific to E. coli O157, in horticultural compost purchased from a commercial supplier. The pathogen could not be cultured on selective media but was visualized using peptide nucleic acid fluorescence in situ hybridization and cell elongation viability assay, confirming the viability. Enumeration of elongated E. coli O157 determined that there were 205 live cells per gram of compost. The nonculturability and confirmation of viability of the pathogen indicates its viable but nonculturable (VBNC) status. The detection of VBNC foodborne pathogens in environmental samples challenges current understanding of the nature of foodborne pathogen contamination.

Fresh produce-associated outbreaks of the foodborne pathogen Escherichia coli O157 are responsible for a number of disease cases, hospitalisations, and deaths. In many cases, the source of contamination can be linked to the growing media of the food, although pathogen detection is problematic in these complex soil ecosystems. In this study, direct qPCR without pre-enrichment was used to detect 310 copies of the translocated intimin receptor gene, using a primer sequence specific to E. coli O157, in horticultural compost purchased from a commercial supplier. The pathogen could not be cultured on selective media but was visualised using peptide nucleic acid fluorescence in situ hybridisation and cell elongation viability assay, confirming the viability. Enumeration of elongated E. coli O157 determined that there were 205 live cells per gram compost. The nonculturability and confirmation of viability of the pathogen indicates its viable but nonculturable (VBNC) status. The detection of VBNC foodborne pathogens in environmental samples challenges current understanding of the nature of foodborne pathogen contamination.

Fresh produce-associated outbreaks of the foodborne pathogen Escherichia coli O157 are responsible for a number of disease cases, hospitalisations, and deaths. In many cases, the source of contamination can be linked to the growing media of the food, although pathogen detection is problematic in these complex soil ecosystems. In this study, direct qPCR without pre-enrichment was used to detect 310 copies of the translocated intimin receptor gene, using a primer sequence specific to E. coli O157, in horticultural compost purchased from a commercial supplier. The pathogen could not be cultured on selective media but was visualised using peptide nucleic acid fluorescence in situ hybridisation and cell elongation viability assay, confirming the viability. Enumeration of elongated E. coli O157 determined that there were 205 live cells per gram compost. The nonculturability and confirmation of viability of the pathogen indicates its viable but nonculturable (VBNC) status. The detection of VBNC foodborne pathogens in environmental samples challenges current understanding of the nature of foodborne pathogen contamination.

Fresh produce-associated outbreaks of the foodborne pathogen Escherichia coli O157 are responsible for a number of disease cases, hospitalisations, and deaths. In many cases, the source of contamination can be linked to the growing media of the food, although pathogen detection is problematic in these complex soil ecosystems. In this study, direct qPCR without pre-enrichment was used to detect 310 copies of the translocated intimin receptor gene, using a primer sequence specific to E. coli O157, in horticultural compost purchased from a commercial supplier. The pathogen could not be cultured on selective media but was visualised using peptide nucleic acid fluorescence in situ hybridisation and cell elongation viability assay, confirming the viability. Enumeration of elongated E. coli O157 determined that there were 205 live cells per gram compost. The nonculturability and confirmation of viability of the pathogen indicates its viable but nonculturable (VBNC) status. The detection of VBNC foodborne pathogens in environmental samples challenges current understanding of the nature of foodborne pathogen contamination.

Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux pump from the resistance-nodulation-cell division (RND) superfamily, is upregulated in these conditions but its molecular mechanism is unknown. Here, we report cryo-electron microscopy structures of Escherichia coli multidrug transporter MdtF within native-lipid nanodiscs, including a single-point mutant with an altered multidrug phenotype and associated substrate-bound form. We reveal that drug binding domain and channel conformational plasticity likely governs promiscuous substrate specificity, analogous to its closely related, constitutively expressed counterpart, AcrB. Whereas we discover distinct transmembrane state transitions within MdtF, which create a more engaged proton relay network, altered drug transport allostery and an acid-responsive increase in efflux efficiency. Physiologically, this provides means of xenobiotic and metabolite disposal within remodelled cell membranes that presage encounters with acid stresses, as endured in the gastrointestinal tract.

Flagellum-mediated motility has been suggested to contribute to virulence by allowing bacteria to colonize and spread to new surfaces. In Salmonella enterica and Escherichia coli species, mutants affected by their flagellar motility have shown a reduced ability to form biofilms. While it is known that some species might act as co-aggregation factors for bacterial adhesion, studies of food-related biofilms have been limited to single-species biofilms and short biofilm formation periods. To assess the contribution of flagella and flagellum-based motility to adhesion and biofilm formation, two Salmonella and E. coli mutants with different flagellar phenotypes were produced: the fliC mutants, which do not produce flagella, and the motAB mutants, which are non-motile. The ability of wild-type and mutant strains to form biofilms was compared, and their relative fitness was determined in two-species biofilms with other foodborne pathogens. Our results showed a defective and significant behavior of E. coli in initial surface colonization (p < 0.05), which delayed single-species biofilm formation. Salmonella mutants were not affected by the ability to form biofilm (p > 0.05). Regarding the effect of motility/flagellum absence on bacterial fitness, none of the mutant strains seems to have their relative fitness affected in the presence of a competing species. Although the absence of motility may eventually delay initial colonization, this study suggests that motility is not essential for biofilm formation and does not have a strong impact on bacteria’s fitness when a competing species is present.

COVID-19, caused by the SARS-CoV-2 virus, was first reported in China in December 2019. The virus has spread rapidly around the world and is currently responsible for 500 million reported cases and over 6.4 million deaths. A risk assessment published by the Foods Standards Agency (FSA) in 2020 (Opens in a new window) concluded that it was very unlikely that you could catch coronavirus via food. This assessment included the worst-case assumption that, if food became contaminated during production, no significant inactivation of virus would occur before consumption. However, the rate of inactivation of virus on products sold at various temperatures was identified as a key uncertainty, because if inactivation does occur more rapidly in some situations, then a lower risk may be more appropriate. This project was commissioned to measure the rate of inactivation of virus on the surface of various types of food and food packaging, reducing that uncertainty. The results will be used to consider whether the assumption currently made in the risk assessment remains appropriate for food kept at a range of temperatures, or whether a lower risk is more appropriate for some. We conducted a laboratory-based study, artificially contaminating infectious SARS-CoV-2 virus onto the surfaces of foods and food packaging. We measured how the amount of infectious virus present on those surfaces declined over time, at a range of temperatures and relative humidity levels, reflecting typical storage conditions. We tested broccoli, peppers, apple, raspberry, cheddar cheese, sliced ham, olives, brine from the olives, white and brown bread crusts, croissants and pain au chocolat. The foods tested were selected as they are commonly sold loose on supermarket shelves or uncovered at deli counters or market stalls, they may be difficult to wash, and they are often consumed without any further processing i.e. cooking. The food packaging materials tested were polyethylene terephthalate (PET1) trays and bottles; aluminium cans and composite drinks cartons. These were selected as they are the most commonly used food packaging materials or consumption of the product may involve direct mouth contact with the packaging. Results showed that virus survival varied depending on the foods and food packaging examined. In several cases, infectious virus was detected for several hours and in some cases for several days, under some conditions tested. For a highly infectious agent such as SARS-CoV-2, which is thought to be transmissible by touching contaminated surfaces and then the face, this confirmation is significant. For most foods tested there was a significant drop in levels of virus contamination over the first 24 hours. However, for cheddar cheese and sliced ham, stored in refrigerated conditions and a range of relative humidity, the virus levels remained high up to a week later, when the testing period was stopped. Both cheddar cheese and sliced ham have high moisture, protein and saturated fat content, possibly offering protection to the virus. When apples and olives were tested, the virus was inactivated to the limit of detection very quickly, within an hour, when the first time point was measured. We suggest that chemicals, such as flavonoids, present in the skin of apples and olives inactivate the virus. The rate of viral decrease was rapid, within a few hours, for croissants and pain au chocolat. These pastries are both coated with a liquid egg wash, which may have an inhibitory effect on the virus. Food packaging materials tested had variable virus survival. For all food packaging, there was a significant drop in levels of virus contamination over the first 24 hours, in all relative humidity conditions and at both 6°C and 21°C; these included PET1 bottles and trays, aluminium cans and composite drinks cartons.

Precise modeling of dry surface biofilms in hospitals is critical for understanding their role in hospital-acquired infection transmission and surface contamination. Using a representative culture condition which includes a nutrient source is key to developing a phenotypically accurate biofilm community. This will enable accurate laboratory testing of cleaning products and their efficacy against dry surface biofilms.