Adam P. Roberts

The Human Salivary Microbiome Is Shaped by Shared Environment Rather than Genetics: Evidence from a Large Family of Closely Related Individuals

ABSTRACT The human microbiome is affected by multiple factors, including the environment and host genetics. In this study, we analyzed the salivary microbiomes of an extended family of Ashkenazi Jewish individuals living in several cities and investigated associations with both shared household and host genetic similarities. We found that environmental effects dominated over genetic effects. While there was weak evidence of geographical structuring at the level of cities, we observed a large and significant effect of shared household on microbiome composition, supporting the role of the immediate shared environment in dictating the presence or absence of taxa. This effect was also seen when including adults who had grown up in the same household but moved out prior to the time of sampling, suggesting that the establishment of the salivary microbiome earlier in life may affect its long-term composition. We found weak associations between host genetic relatedness and microbiome dissimilarity when using family pedigrees as proxies for genetic similarity. However, this association disappeared when using more-accurate measures of kinship based on genome-wide genetic markers, indicating that the environment rather than host genetics is the dominant factor affecting the composition of the salivary microbiome in closely related individuals. Our results support the concept that there is a consistent core microbiome conserved across global scales but that small-scale effects due to a shared living environment significantly affect microbial community composition. IMPORTANCE Previous research shows that the salivary microbiomes of relatives are more similar than those of nonrelatives, but it remains difficult to distinguish the effects of relatedness and shared household environment. Furthermore, pedigree measures may not accurately measure host genetic similarity. In this study, we include genetic relatedness based on genome-wide single nucleotide polymorphisms (SNPs) (rather than pedigree measures) and shared environment in the same analysis. We quantify the relative importance of these factors by studying the salivary microbiomes in members of a large extended Ashkenazi Jewish family living in different locations. We find that host genetics plays no significant role and that the dominant factor is the shared environment at the household level. We also find that this effect appears to persist in individuals who have moved out of the parental household, suggesting that aspects of salivary microbiome composition established during upbringing can persist over a time scale of years. IMPORTANCE Previous research shows that the salivary microbiomes of relatives are more similar than those of nonrelatives, but it remains difficult to distinguish the effects of relatedness and shared household environment. Furthermore, pedigree measures may not accurately measure host genetic similarity. In this study, we include genetic relatedness based on genome-wide single nucleotide polymorphisms (SNPs) (rather than pedigree measures) and shared environment in the same analysis. We quantify the relative importance of these factors by studying the salivary microbiomes in members of a large extended Ashkenazi Jewish family living in different locations. We find that host genetics plays no significant role and that the dominant factor is the shared environment at the household level. We also find that this effect appears to persist in individuals who have moved out of the parental household, suggesting that aspects of salivary microbiome composition established during upbringing can persist over a time scale of years.

Transferable Plasmid-Borne mcr-1 in a Colistin-Resistant Shigella flexneri Isolate

ABSTRACT Since the initial discovery of mcr-1 in an Escherichia coli isolate from China, the gene has also been detected in Klebsiella pneumoniae and Salmonella enterica but is rarely reported in other Enterobacteriaceae. Here, we report the isolation and identification of a Shigella flexneri strain harboring mcr-1 from stool samples in a pig farm in China from 2009. The MIC of colistin for the isolate is 4 μg/ml. Conjugation assays showed that the donor S. flexneri strain has functional and transferable colistin resistance. Sequencing revealed that mcr-1 was present on a putative composite transposon flanked by inverted repeats of ISApl1. IMPORTANCE There are four species of Shigella, and Shigella flexneri is the most frequently isolated species in low- and middle-income countries (LMICs). In this study, we report a functional, transferable, plasmid-mediated mcr-1 gene in S. flexneri. We have shown that mcr-1 is located on a novel composite transposon which is flanked by inverted repeats of ISApl1. The host strain is multidrug resistant, and this multidrug resistance is also transferable. The finding of a functional mcr-1 gene in S. flexneri, a human-associated Enterobacteriaceae family member, is a cause for concern as infections due to S. flexneri are the main Shigella infections in most low- and middle-income countries.

Presence of Type I-F CRISPR/Cas systems is associated with antimicrobial susceptibility in Escherichia coli.

Background Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes are sequence-specific DNA nuclease systems found in bacteria and archaea. CRISPR/Cas systems use RNA transcripts of previously acquired DNA (spacers) to target invading genetic elements with the same sequence, including plasmids. In this research we studied the relationship between CRISPR/Cas systems and multidrug resistance in Escherichia coli . Methods The presence of Type I-E and Type I-F CRISPR systems was investigated among 82 antimicrobial-susceptible and 96 MDR clinical E. coli isolates by PCR and DNA sequencing. Phylogrouping and MLST were performed to determine relatedness of isolates. RT-PCR was performed to ascertain the expression of associated cas genes. Results Type I-F CRISPR was associated with the B2 phylogroup and was significantly overrepresented in the susceptible group (22.0%) compared with the MDR group (2.1%). The majority of CRISPR I-F-containing isolates had spacer sequences that matched IncF and IncI plasmids. RT-PCR demonstrated that Type I-F cas genes were expressed and therefore potentially functional. Conclusions The CRISPR I-F system is more likely to be found in antimicrobial-susceptible E. coli . Given that the Type I-F system is expressed in WT isolates, we suggest that this difference could be due to the CRISPR system potentially interfering with the acquisition of antimicrobial resistance plasmids, maintaining susceptibility in these isolates.

Conserved collateral susceptibility networks in diverse clinical strains of Escherichia coli.

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, is a uniquely promising strategy that enables selection against resistance during treatment. However, using CS-informed therapy depends on conserved CS networks across genetically diverse bacterial strains. We examined CS conservation in 10 clinical strains of E. coli resistant to four clinically relevant antibiotics. Collateral susceptibilities of these 40 resistant mutants were then determined against a panel of 16 antibiotics. Multivariate statistical analyses demonstrate that resistance mechanisms, in particular efflux-related mutations, as well as relative fitness were principal contributors to collateral changes. Moreover, collateral responses shifted the mutant selection window suggesting that CS-informed therapies could affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that early detection of resistance mechanisms is important to accurately predict collateral antimicrobial responses.

Carbapenemases on the move: it′s good to be on ICE

The evolution and spread of antibiotic resistance is often mediated by mobile geneticelements. Integrative and conjugative elements (ICEs) are the most abundant conjugativeelements among prokaryotes. However, the contribution of ICEs to horizontal gene transferof antibiotic resistance has been largely unexplored. Here we report that ICEs belonging tomating-pair formation (MPF) classes G and T are highly prevalent among the opportunisticpathogen Pseudomonas aeruginosa, contributing to the spread of carbapenemase-encodinggenes (CEGs). Most CEGs of the MPFG class were encoded within class I integrons, which co-harbour genes conferring resistance to other antibiotics. The majority of the integrons werelocated within Tn3-like and composite transposons. A conserved attachment site could bepredicted for the MPFGclass ICEs. MPFTclass ICEs carried the CEGs within compositetransposons which were not associated with integrons. The data presented here provides aglobal snapshot of the different CEG-harbouring ICEs and sheds light on the underappreciatedcontribution of these elements for the evolution and dissemination of antibiotic resistanceon P. aeruginosa.

Evolutionary trajectories to amoxicillin-clavulanic acid resistance in Escherichia coli are affected by growth media.

Antibiotic resistant bacterial isolates are routinely generated in the laboratory using sub-inhibitory concentrations of the antibiotics in a range of media to study various aspects of evolutionary biology. We wanted to determine whether growth in different media affects the selection of amoxicillin-clavulanic acid resistant Escherichia coli. Using a susceptible clinical isolate of E. coli resistance was selected for after 24-hour exposure to sub-MIC levels of amoxicillin-clavulanic acid in defined (M9), semi-defined (Iso-sensitest broth) and undefined (LB broth) media. Resistant colonies were isolated on LB agar containing 8 (MIC) or 16 μg/ml (2xMIC) amoxicillin-clavulanic acid. Fitness costs of three resistant lineages from each media-type were assessed using a competitive fitness assay and mutations in the ampC promoter region were identified through PCR. No resistant colonies were recovered with pre-incubation in M9, whereas Iso-sensitest and LB broth incubation resulted in growth at 8 and 16 μg/ml of amoxicillin-clavulanic acid, respectively. We observed within media-type variability in the fitness costs associated with resistance and showed that in some cases the fitness is dependent on the media in which the assay is carried out. Our data highlights the importance of media consideration when interpreting the results of evolutionary studies which will ultimately be translated into the clinic.In the face of an accelerating global antimicrobial resistance crisis, the determination of bacterial fitness following acquisition of resistance is an expanding area of research, and increased understanding of this process will be crucial to translate in vitro fitness data to successful therapies. Given that crucial clinical treatment situations are guided by in vitro diagnostic testing in an artificial environment far removed from human physiological niches, we used Escherichia coli and amoxicillin-clavulanic acid (AMC) resistance as a model to understand how such environments could affect the emergence of resistance, associated fitness costs and the predictive value of this data when strains were grown in the more physiologically relevant environments of urine and urothelial organoids. Resistant E. coli isolates were selected for following 24-hour exposure to sub-inhibitory concentrations of AMC in either M9, ISO or LB broth, followed by growth on LB agar containing AMC. No resistant colonies emerged following growth in M9, whereas resistant isolates were detected from cultures grown in ISO and LB broth. We observed both within and between media-type variability in the levels of resistance and fitness of the resistant mutants grown in LB. MICs and fitness of these resistant strains in different media (M9, ISO, LB, human urine and urothelial organoids) showed considerable variation. Media can therefore have a direct effect on the isolation of mutants that confer resistance to AMC and these mutants can exhibit unpredictable MIC and fitness profiles under different growth conditions. This study highlights the risks in relying on a single culture protocol to predict the behaviour and treatment response of bacteria in vivo and highlights the importance of developing comprehensive experimental designs to ensure effective translation of diagnostic procedures to successful clinical outcomes.

Conserved collateral antibiotic susceptibility networks in diverse clinical strains of Escherichia coli

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, might enable selection against resistance during treatment. However, the success of this approach would depend on the conservation of CS networks across genetically diverse bacterial strains. Here, we examine CS conservation across diverse Escherichia coli strains isolated from urinary tract infections. We determine collateral susceptibilities of mutants resistant to relevant antimicrobials against 16 antibiotics. Multivariate statistical analyses show that resistance mechanisms, in particular efflux-related mutations, as well as the relative fitness of resistant strains, are principal contributors to collateral responses. Moreover, collateral responses shift the mutant selection window, suggesting that CS-informed therapies may affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that rapid detection of resistance mechanisms is important to accurately predict collateral responses.Resistance to one antibiotic can in some cases increase susceptibility to other antibiotics. Here, Podnecky et al. study these collateral responses in E. coli clinical isolates and show that efflux-related resistance mechanisms and relative fitness of the strains are principal contributors to this phenomenon.

The oral microbiome

The human microbiome is receiving a great deal of attention as its role in health and disease becomes ever more apparent. The oral microbiome, perhaps due to the ease with which we can obtain samples, is arguably the most well-studied human microbiome to date. It is obvious, however, that we have only just begun to scratch the surface of the complex bacterial and bacterial–host interactions within this complex community. Here, we describe the factors which are known to influence the development of the seemingly globally conserved, core, oral microbiome and those which are likely to be responsible for the observed differences at the individual level. We discuss the paradoxical situation of maintaining a stable core microbiome which is at the same time incredibly resilient and adaptable to many different stresses encountered in the open environment of the oral cavity. Finally, we explore the interactions of the oral microbiome with the host and discuss the interactions underlying human health and disease.