Elizabeth M. Darby’s research while affiliated with University of Birmingham and other places

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Publications (11)


Staphylococcus haemolyticus is a reservoir of antibiotic resistance genes in the preterm infant gut
  • Preprint

January 2025

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6 Reads

Lisa E. Lamberte

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Elizabeth M. Darby

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Raymond K.O. Kiu

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[...]

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Willem van Schaik

Among coagulase-negative staphylococci, Staphylococcus haemolyticus is a primary cause of bloodstream infections in preterm infants, with gut colonisation being recognised as a risk factor for subsequent infection. Through a re-analysis of a 16S rRNA gene sequencing dataset (n=497 preterm infants), we found that S. haemolyticus was abundant and prevalent in the gut in the first month of life. To better understand the diversity of S. haemolyticus among preterm infants, we generated genome sequences of S. haemolyticus strains (n=140), which were isolated from 44 stool samples of 22 preterm infants from four different hospitals in the United Kingdom. Core genome phylogenetic analyses, incorporating 126 publicly available S. haemolyticus genome sequences, showed that 85/140 (60.1%) of the isolates, from three different hospitals, formed a clonal group with 79/85 (92.9%) strains being assigned to Multi-Locus Sequence Type (ST) 49. Antibiotic resistance genes were highly prevalent in the genome sequences. Using logistic regression, we found a strong association between the presence of the gene mecA and phenotypic resistance to oxacillin (odds ratio [OR]: 158.00, p<0.0001), and the aacA-aphD gene and phenotypic resistance to gentamicin aacA-aphD (OR: 162.00, p<0.001). None of the strains from the preterm infant cohort had a complete Staphylococcal Cassette Chromosome mec (SCCmec) element. The aacA-aphD gene was associated with the transposon Tn4001. Using hybrid genome assemblies, we found it to be present on the chromosome (54.5% of strains) or on diverse plasmids (27.3%). Four strains (18.2%) had Tn4001 copies on both plasmid and chromosome. Our data suggest the existence of a distinct sub-population of S. haemolyticus that has adapted to colonise the gut of preterm infants. Prevalent resistance to antibiotics is of clinical concern and the diversity of genetic contexts of mecA and Tn4001 suggests widespread horizontal gene transfer and recombination in this species.


Impact of early life antibiotic and probiotic treatment on gut microbiome and resistome of very-low-birth-weight preterm infants

December 2024

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22 Reads

Preterm infants (<37 weeks’ gestation) are often administered broad-spectrum antibiotics in hospitals due to their vulnerability to severe morbidity, including necrotising enterocolitis and sepsis. However, antibiotics can disrupt the development of early-life microbiota, potentially impairing gut immunity and colonisation resistance. Evidence shows that probiotics (e.g., certain Bifidobacterium strains) may help restore healthy gut microbiota. In this study, we examined the effects of probiotics and antibiotics on the preterm gut microbiome and resistome in two unique cohorts of 34 very-low-birth-weight, human-milk- fed preterm infants (moderate to very preterm), with one cohort receiving probiotics. Within each group, some infants were treated with antibiotics (benzylpenicillin and/or gentamicin) while others served as non-antibiotic treated controls. We performed shotgun metagenomic sequencing on 93 longitudinal faecal samples from 34 infants, generated >300 metagenome- assembled genomes, and obtained ∼90 isolate genomes through targeted culturomics, enabling analysis of the microbiome/resistome at species and strain levels. Additionally, we investigated in vitro horizontal gene transfer (HGT) capacity of preterm infant-derived multidrug-resistant (MDR) pathogen Enterococcus via neonatal gut models. Overall, probiotic supplementation significantly reduced antibiotic resistance gene prevalence, MDR pathogen load, and helped restore a typical early-life microbiota. However, the persistence of MDR pathogens like Enterococcus , with high HGT potential, highlights the need for ongoing surveillance in neonatal care. Our findings underscore the complex interactions between antibiotics, probiotics, and HGT in shaping the neonatal microbiome and support further research into probiotics for antimicrobial stewardship in preterm populations.


Figure 2. The genomic region of strains with and without eefRABCD. EasyFig alignment of the eef region in ST73 (B2), ST131 (B2), ST95 (B2), ST11 (E), ST69 (D), ST617 (A), ST10 (A). The eefRABCD operon was consistently identified between fabI
Figure 3. Diagrammatic representation of eefRABCD homology in strains that lack the operon.
Figure 4. Multi-panel representation of the Eef components. Not to scale.
Figure 5. Comparison of the predicted structures of the assembled EefABC and the experimental cryo-EM structure of AcrABZ-TolC (based on 5066.pdb; (Wang et al., 2017))
Susceptibility of E. coli to antimicrobials following loss and gain of eefABC and eefD MIC (µ
The RND efflux pump EefABC is highly conserved within lineages of E. coli commonly associated with infection
  • Preprint
  • File available

August 2024

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106 Reads

Tripartite resistance-nodulation-division (RND) efflux pumps confer multidrug resistance (MDR) in Gram-negative bacteria and are critical for many physiological functions including virulence and biofilm formation. The common laboratory strain of E. coli, K-12 MG1655 has six recognised RND transporters participating in tripartite pump formation (AcrB, AcrD, AcrF, CusA, MdtBC, and MdtF). However, by studying >20,000 E. coli genomes we show that E. coli belonging to phylogroups B2, D, E, F and G, which are commonly associated with infection, possess an additional, seventh RND transporter, EefB. It is found in a five gene operon, eefRABCD, which also encodes a TetR family transcription factor, a periplasmic adapter protein, an outer membrane factor and major facilitator superfamily pump. In contrast, E. coli from phylogroups A, B1 and C, generally containing environmental and commensal strains, do not encode the operon and instead encode an uncharacterised ORF, ycjD . In phylogroups where the eefRABCD operon is present it was very highly conserved. In fact, conservation levels were comparable to that of the major E. coli RND efflux system AcrAB-TolC, suggesting a critical biological function. Protein modelling shows that this pump is highly divergent from endogenous E. coli RND systems with unique structural features, while showing similarities to efflux systems found in Pseudomonas aeruginosa . However, unlike other major RND efflux systems, EefABC does not appear to transport antimicrobials and instead may be important for infection or survival in the host environment. Importance Efflux pumps are molecular machines that export molecules out of bacterial cells. The efflux pumps belonging to the RND family are particularly important as they export antibiotics out of Gram-negative bacterial cells, contributing to antibiotic resistance. The important human pathogen, E. coli , has been previously reported to have six RND pumps. However, we show that phylogroups of E. coli commonly associated with infection encode a seventh RND pump, EefABC which is highly conserved, suggesting an important biological function. While the function of EefABC in E. coli remains to be resolved, it does not seem to transport antimicrobial compounds. These findings are important because they reveal a new RND pump, potentially involved in virulence and survival in the host, that could represent a new therapeutic target. Additionally, it again shows that laboratory type strains of common bacterial pathogens are not representative of those that are infection causing.

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Differential development of antibiotic resistance and virulence between Acinetobacter species

April 2024

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99 Reads

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1 Citation

The two species that account for most cases of Acinetobacter-associated bacteremia in the United Kingdom are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and Acinetobacter baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii, and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30, respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defense systems and harbored a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm, and infect both in vivo and in in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesize that the fact that it only harbors a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram-negative bacteria. IMPORTANCE Acinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived bloodstream infections in England and Wales. In contrast to the well-studied and often highly drug-resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not, and could inform future novel treatment strategies.



Fig. 2 Signatures of foreign DNA acquisition. a -Number of rep genes found per whole genome sequence
Differential development of antibiotic resistance and virulence between Acinetobacter species

December 2023

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146 Reads

The two species that account for most cases of Acinetobacter-associated bacteraemia in the UK are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and A. baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii, was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30 respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defence systems and harboured a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm and infect both in vivo and in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesise that the fact that it only harbours a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram negative bacteria.


RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump

March 2023

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72 Reads

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14 Citations

Microbial Genomics

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.


Molecular mechanisms of antibiotic resistance revisited

November 2022

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1,349 Reads

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601 Citations

Nature Reviews Microbiology

Antibiotic resistance is a global health emergency, with resistance detected to all antibiotics currently in clinical use and only a few novel drugs in the pipeline. Understanding the molecular mechanisms that bacteria use to resist the action of antimicrobials is critical to recognize global patterns of resistance and to improve the use of current drugs, as well as for the design of new drugs less susceptible to resistance development and novel strategies to combat resistance. In this Review, we explore recent advances in understanding how resistance genes contribute to the biology of the host, new structural details of relevant molecular events underpinning resistance, the identification of new resistance gene families and the interactions between different resistance mechanisms. Finally, we discuss how we can use this information to develop the next generation of antimicrobial therapies. In this Review, Blair, Webber and colleagues explore our understanding of the mechanisms of antibiotic resistance, including reduced permeability, antibiotic efflux, modification or alteration of the antibiotic target, modification or destruction of the drug itself, and bypass of metabolic pathways. They also discuss how this information can aid in developing the next generation of antimicrobial therapies.


Figure 3: Venn diagrams of percentage identity between Acinetobacter RND genes
RND pumps across the Acinetobacter genus; AdeIJK is the ancestral efflux system

October 2022

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69 Reads

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1 Citation

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is extensively drug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes 6 RND efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, however the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-three species making up the Acinetobacter genus were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter , and through genomic, structural and phenotypic work we show that these genes are actually orthologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter , such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the Acinetobacter genus. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH were only found in a subset of Acinetobacter , that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter , treatments for infections can avoid efflux-mediated resistance and improve patient outcomes. Impact statement Efflux pumps extrude antibiotics from within bacterial cells directly conferring antibiotic resistance and underpinning other mechanisms of resistance. By understanding the exact complement of efflux pumps and their roles across infection-causing organisms such as those within the Acinetobacter genus, it is possible to understand how cells become resistant to antibiotics and how this might be tackled. Efflux is an attractive target for inhibition to increase susceptibility to existing drugs and therefore, knowing which pumps are present in each species is important. Furthermore, we present a novel method using conserved RND residues to predict the total number of RND proteins including currently novel systems, within bacterial genomes. Data Summary This study made use of publicly available datasets downloaded from NCBI’s GenBank. A full list of accession numbers can be found in supplementary text 3. Bioinformatics software used in this study was previously published and listed in the methods section. The BLASTp conserved residue files are in S1 text 1 and 2. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.


Expression levels (fold change) for genes in K. pneumoniae ST258 strains relative to strain 46704. Significance is indicated
The effect of the efflux pump inhibitors PaβN and CCCP on biocide susceptibility in chlorhexidine-adapted strains and their respective wild-types. Numbers highlighted in bold indicate a ≥fourfold change in MIC relative to no EPI (alone)
Contribution of the efflux pump AcrAB-TolC to the tolerance of chlorhexidine and other biocides in Klebsiella spp

March 2022

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65 Reads

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21 Citations

Journal of Medical Microbiology

Introduction. We are becoming increasingly reliant on the effectiveness of biocides to combat the spread of Gram-negative multi-drug-resistant (MDR) pathogens, including Klebsiella pneumoniae . It has been shown that chlorhexidine exposure can lead to mutations in the efflux pump repressor regulators SmvR and RamR, but the contribution of each individual efflux pump to biocide tolerance is unknown. Hypothesis. Multiple efflux pumps, including SmvA and AcrAB-TolC, are involved in increased tolerance to biocides. However, strains with upregulated AcrAB-TolC caused by biocide exposure are more problematic due to their increased MDR phenotype. Aim. To investigate the role of AcrAB-TolC in the tolerance to several biocides, including chlorhexidine, and the potential threat of cross-resistance to antibiotics through increased expression of this efflux pump. Methodology. Antimicrobial susceptibility testing was performed on K. pneumoniae isolates with ramR mutations selected for after exposure to chlorhexidine, as well as transposon mutants in components and regulators of AcrAB-TolC. RTPCR was used to detect the expression levels of this pump after biocide exposure. Strains from the globally important ST258 clade were compared for genetic differences in acrAB -TolC and its regulators and for phenotypic differences in antimicrobial susceptibility. Results. Cross-resistance to antimicrobials was observed following mutations in ramR . Exposure to chlorhexidine led to increased expression of acrA and its activator ramA , and transposon mutants in AcrAB-TolC have increased susceptibility to several biocides, including chlorhexidine. Variations in ramR within the ST258 clade led to an increase in tolerance to certain biocides, although this was strain dependent. One strain, MKP103, that had increased levels of biocide tolerance showed a unique mutation in ramR that was reflected in enhanced expression of acrA and ramA . MKP103 transposon variants were able to further enhance their tolerance to specific biocides with mutations affecting SmvA. Conclusions. Biocide tolerance in K. pneumoniae is dependent upon several components, with increased efflux through AcrAB-TolC being an important one.


Citations (5)


... Of these three, overexpression of adeAB(C) is most commonly seen in MDR clinical isolates 33,36 . Present in the core genome of all Acinetobacter species, AdeIJK is believed to be the ancestral efflux pump of this genus 22 . AdeFGH is the least studied efflux pump, with its full clinical relevance yet to be determined 40,41 . ...

Reference:

Targeting Acinetobacter baumannii resistance-nodulation-division efflux pump transcriptional regulators to combat antimicrobial resistance
RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump
  • Citing Article
  • March 2023

Microbial Genomics

... Antibiotic resistance is classified into two primary categories: natural and acquired resistance, which are either inherent within the organism or mediated through genes that are typically inactive but produce a response as antibiotic exposure, whereas acquired resistance occurs through mechanisms such as the uptake of genomic material via translation, conjugation, transposition, and mutations in the chromosomal DNA of bacteria [22]. Bacteria employ genetic mechanisms to develop antibiotic resistance by integrating exogenous DNA encoding resistance factors through horizontal gene transfer (HGT) [23]. The antimicrobial resistance (AMR) pathway can be classified into four categories: (1) drug uptake limitations, (2) drug target modifications, (3) drug inactivation, and (4) drug efflux, as illustrated in Fig. 2. ...

Molecular mechanisms of antibiotic resistance revisited
  • Citing Article
  • November 2022

Nature Reviews Microbiology

... Across the Gram-negative bacteria it is generally assumed that efflux pump sequences are conserved across a species, with genes present in common laboratory strains often taken as representatives of a whole species. This is despite several studies demonstrating that clinical isolates of Acinetobacter baumannii can lack adeB, which encodes the RND component of the AdeABC system associated with reduced antimicrobial susceptibility [32][33][34]. Here we show that while it is assumed that all E. coli isolates possess AcrB, AcrD, AcrF, CusA, MdtBC and MdtF, this is not true for the ST11 lineage. ...

RND pumps across the Acinetobacter genus; AdeIJK is the ancestral efflux system

... Interestingly, all four STEC possessed curli genes; however, strains O145 and O157 (1934) did not synthesize curli or cellulose (phenotype) when tested in-vitro on Congo red and LB agar supplemented with calcofluor white (Nan, 2023). Whole genome sequencing revealed that strains O121:H19, O157:H7 (1931), O157:H7 (R508), O45:H7, and O111:NM did carry genes coding for acrAB-TolC, emrAB-TolC and ydhC which have been linked to resistance to biocides (Figure 3-5) (Piddock, 2006;Wand et al., 2022). ...

Contribution of the efflux pump AcrAB-TolC to the tolerance of chlorhexidine and other biocides in Klebsiella spp

Journal of Medical Microbiology

... AdeFGH is normally maintained at low levels in A. baumannii 39,92 and is seemingly a minor contributor to the inherent antibiotic resistance of this microbe 39 . Even though AdeFGH and AdeL are not found ubiquitously in Acinetobacter species, most infection-causing strains express them 36,93,94 . Indeed, clinical A. baumannii isolates resistant to tigecycline and fluoroquinolones have been shown to overexpress AdeFGH 34,82,83,95 . ...

RND Efflux Pumps in Gram-Negative Bacteria; Regulation, Structure and Role in Antibiotic Resistance
  • Citing Article
  • February 2020