Biology subjects

Keevil, C. W.

Publications and source records attributed to Keevil, C. W..

6 recordsLinked to original sources

bioRxiv2025

Molecular basis for multidrug efflux by an anaerobic RND transporter

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.

biochemistry↗

bioRxiv2023

Identification of diverse antibiotic resistant bacteria in agricultural soil with H218O stable isotope probing and metagenomics

BackgroundIn this study, we aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil, by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled "heavy" and the unlabelled "light" SIP fractions were sequenced. ResultsAfter incubations, an increase of the 16S rRNA copy numbers in the "heavy" fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) are highly abundant after two days of incubation with antibiotics. Several Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. On the other hand, only two ARGs from the unbinned-assembled light fractions were identified. ConclusionsThe results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil, and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.

microbiology↗

bioRxiv2021

Synergism vs Additivity - Defining the Interactions between Common Disinfectants

Many of the most common disinfectant and sanitizer products are formulations of multiple antimicrobial compounds. Products claiming to contain synergistic formulations are common, although there is often little supporting evidence. The antimicrobial interactions of all pairwise combinations of common disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol and bronopol) were classified via checkerboard and validated by time-kill analyses. Combinations were tested against Acinetobacter baumannii NCTC 12156, Enterococcus faecalis NCTC 13379, Klebsiella pneumoniae NCTC 13443 and Staphylococcus aureus NCTC 13143. Synergistic interactions were only identified between cholorocresol with benzalkonium chloride, and chlorocresol with polyhexamethylene biguanide. Synergism was not ubiquitously demonstrated against all species tested and was on the borderline of the synergism threshold. These data demonstrate that synergism between disinfectants is uncommon and circumstantial. Most of the antimicrobial interactions tested were characterised as additive. We suggest that this is due to the broad, non-specific mechanisms associated with disinfectants not providing opportunity for the combined activities of these compounds to exceed the sum of their parts. IMPORTANCEThe scarcity of observed synergistic interactions suggests that many disinfectant-based products may be misinterpreting combined mechanisms of interaction. We emphasise the need to correctly differentiate between additivity and synergism in antimicrobial formulations, as inappropriate classification may lead to unnecessary issues in the event of regulatory changes. Furthermore, we question the need to focus on synergism and disregard additivity when considering combinations of disinfectants, as the benefits that synergistic interactions provide are not necessarily relevant to the application of the final product.

microbiology↗

bioRxiv2021

Rapid inactivation of SARS-CoV-2 on copper touch surfaces determined using a cell culture infectivity assay

COVID-19, caused by SARS-CoV-2, was first reported in China in 2019 and has transmitted rapidly around the world, currently responsible for 83 million reported cases and over 1.8 million deaths. The mode of transmission is believed principally to be airborne exposure to respiratory droplets from symptomatic and asymptomatic patients but there is also a risk of the droplets contaminating fomites such as touch surfaces including door handles, stair rails etc, leading to hand pick up and transfer to eyes, nose and mouth. We have previously shown that human coronavirus 229E survives for more than 5 days on inanimate surfaces and another laboratory reproduced this for SARS-CoV-2 this year. However, we showed rapid inactivation of Hu-CoV-229E within 10 minutes on different copper surfaces while the other laboratory indicated this took 4 hours for SARS-CoV-2. So why the difference? We have repeated our work with SARS-CoV-2 and can confirm that this coronavirus can be inactivated on copper surfaces in as little as 1 minute. We discuss why the 4 hour result may be technically flawed.

microbiology↗

bioRxiv2020

Biofilm development on urinary catheters promotes the appearance of viable but non-culturable (VBNC) bacteria

Catheter-associated urinary tract infections have serious consequences, both for patients and in impacting on healthcare resources. Much work has been carried out to develop an antimicrobial catheter. Although such developments have shown promise under laboratory conditions, none have demonstrated a clear advantage in clinical trials. Using a range of microbiological and advanced microscopy techniques, a detailed laboratory study comparing biofilm development on silicone, hydrogel latex and silver alloy coated hydrogel latex catheters was carried out. Biofilm development by Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis on three commercially available catheters was tracked over time. Samples were examined with episcopic differential interference contrast (EDIC) microscopy, culture analysis and staining techniques to quantify viable but non-culturable (VBNC) bacteria. Both qualitative and quantitative assessment found biofilms to develop rapidly on all three materials. EDIC microscopy revealed the rough surface topography of the materials. Differences between culture counts and quantification of total and dead cells demonstrated the presence of VBNC populations, where bacteria retain viability but are not metabolically active. The use of non-culture based techniques showed the development of widespread VBNC populations. These VBNC populations were more evident on silver alloy coated hydrogel latex catheters, indicating a bacteriostatic effect at best. The laboratory tests reported here, that detect VBNC bacteria, allow more rigorous assessment of antimicrobial catheters offering an explanation for why there is often minimal benefit to patients. IMPORTANCESeveral antimicrobial urinary catheter materials have been developed but, although laboratory studies may show a benefit, none have significantly improved clinical outcomes. The use of poorly designed laboratory testing and lack of consideration to the impact of VBNC populations may be responsible. While the presence of VBNC populations is becoming more widely reported, there remains a lack of understanding of the clinical impact or influence of exposure to antimicrobial products. This is the first study to investigate the impact of antimicrobial surface materials and the appearance of VBNC populations. This demonstrates how improved testing is needed prior to clinical trials uptake.

microbiology↗

bioRxiv2020

Uncovering microbial populations in the lumen of neonatal enteral feeding tubes utilising 16s rRNA sequencing.

Gastrointestinal microbiome is increasingly implicated in the morbidity associated with being born preterm. Enteral tubes (ET) are essential for the nutritional care of preterm infants. Limited culture-based studies have suggested they are colonised by high densities of microorganisms. Microbial DNA was extracted from 60 ETs retrieved from infants in a tertiary neonatal unit and analysed by16s rRNA sequencing of the V4 variable region. Relative abundance analysis on dominant microorganisms demonstrated that compared to breast milk, formula significantly increased abundance of Streptococcus spp and significantly decreased Enterococcus spp and Enterobacteriaceae Vaginal birth was also associated with significantly increased relative abundance of Streptococcus. This study more accurately demonstrates the extent of microbial diversity in neonatal ETs, with feeding regime significantly influencing colonisation patterns. Colonisation with unwanted organisms, as a result of specific care regimes, could result in disruption of the fragile infant gut microbiome, with implications for long-term morbidity.

microbiology↗