Using metagenomics to investigate the impact of hospital stay and the ARK intervention on the human gut resistome

Antimicrobials are vital for modern medicine. Antimicrobial use selects for antimicrobial resistant bacteria, particularly among the gut microflora. Minimising antimicrobial resistance (AMR) selection by avoiding unnecessary antibiotic use helps combat AMR. Metagenomic analyses have the potential to provide accurate detection and quantification of AMR genes within an individual’s gut microbiome (gut ‘resistome’), allowing the impact of different types of antibiotic exposures to be evaluated and guide interventions to reduce AMR. We have developed a short-read sequencing approach to characterise the gut ‘resistome’ and piloted this in two clinical sample sets. The first consisted of paired stool samples from older hospitalised adults. 25 pairs of samples (1 to 50 days apart) showed a median AMR gene reads/kb/million total reads (RPKM) of 1841 (124 to 17,832), and a median AMR gene count of 55 (2 to 101). The second consisted of faecal discards from Clostridium difficile testing at a hospital eleven months apart. In these samples (n=21) the median AMR gene read was 923 RPKM (240 to 19,475), and the median AMR gene count was 36 (9 to 82). In both sample sets there was a trend towards an increase in AMR gene RPKM and number between the time points. dfrF, tetW and acrA were the commonest AMR genes in the first sample set, while ErmB, dfrF, CblA_1 and AHP_3 IIIa were the commonest in the second sample set. This approach is being applied to analyse the impact of an intervention (ARK-Hospital) designed to change antibiotic prescribing behaviour. The data will allow us to determine the patient-level impact of reduced antibiotic exposure on carried AMR

Klebsiella pneumonia in Sudan: multidrug resistance, polyclonal dissemination, and virulence

The emergence and global expansion of hyper-virulent and multidrug resistant (MDR) Klebsiella pneumoniae is an increasing healthcare threat worldwide. The epidemiology of MDR K. pneumoniae is under-characterized in many parts of the world, particularly Africa. In this study, K. pneumoniae isolates from hospitals in Khartoum, Sudan, have been whole-genome sequenced to investigate their molecular epidemiology, virulence, and resistome profiles. Eighty-six K. pneumoniae were recovered from patients in five hospitals in Khartoum between 2016 and 2020. Antimicrobial susceptibility was performed by disk-diffusion and broth microdilution. All isolates underwent whole genome sequencing using Illumina MiSeq; cgMLST was determined using Ridom SeqSphere+, and 7-loci MLST virulence genes and resistomes were identified. MDR was observed at 80%, with 35 isolates (41%) confirmed carbapenem-resistant. Thirty-seven sequence types were identified, and 14 transmission clusters (TC). Five of these TCs involved more than one hospital. Ybt9 was the most common virulence gene detected, in addition to some isolates harbouring iuc and rmp1. There is a diverse population of K. pneumoniae in Khartoum hospitals, harbouring multiple resistance genes, including genes coding for ESBLs, carbapenemases, and aminoglycoside-modifying enzymes, across multiple ST’s. The majority of isolates were singletons and transmissions were rare

Timing Is Everything:Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction

Mutations in the polymorphic Staphylococcus aureus agr locus responsible for quorum sensing (QS) dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Pantone-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of auto-inducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harbouring naturally occurring AgrC cytoplasmic domain mutations were identified including T247I, I311T, A343T, L245S and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. While mutations were localised in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization, ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction so reducing virulence and maintaining S. aureus in an agr-down-regulated ‘colonization’ mode

Klebsiella pneumonia in Sudan: Multidrug Resistance, Polyclonal Dissemination, and Virulence

The emergence and global expansion of hyper-virulent and multidrug resistant (MDR) Klebsiella pneumoniae is an increasing healthcare threat worldwide. The epidemiology of MDR K. pneumoniae is under-characterized in many parts of the world, particularly Africa. In this study, K. pneumoniae isolates from hospitals in Khartoum, Sudan, have been whole-genome sequenced to investigate their molecular epidemiology, virulence, and resistome profiles. Eighty-six K. pneumoniae were recovered from patients in five hospitals in Khartoum between 2016 and 2020. Antimicrobial susceptibility was performed by disk-diffusion and broth microdilution. All isolates underwent whole genome sequencing using Illumina MiSeq; cgMLST was determined using Ridom SeqSphere+, and 7-loci MLST virulence genes and resistomes were identified. MDR was observed at 80%, with 35 isolates (41%) confirmed carbapenem-resistant. Thirty-seven sequence types were identified, and 14 transmission clusters (TC). Five of these TCs involved more than one hospital. Ybt9 was the most common virulence gene detected, in addition to some isolates harbouring iuc and rmp1. There is a diverse population of K. pneumoniae in Khartoum hospitals, harbouring multiple resistance genes, including genes coding for ESBLs, carbapenemases, and aminoglycoside-modifying enzymes, across multiple ST’s. The majority of isolates were singletons and transmissions were rare

Microbial diversity and antimicrobial resistance in faecal samples from acute medical patients assessed through metagenomic sequencing

Antimicrobial resistance (AMR) is a threat to global public health. However, unsatisfactory approaches to directly measuring the AMR burden carried by individuals has hampered efforts to assess interventions aimed at reducing selection for AMR. Metagenomics can provide accurate detection and quantification of AMR genes within an individual person’s faecal flora (their gut “resistome”). Using this approach, we aimed to test the hypothesis that differences in antimicrobial use across different hospitals in the United Kingdom will result in observable differences in the resistome of individual patients. Three National Health Service acute Hospital Trusts with markedly different antibiotic use and Clostridioides difficile infection rates collected faecal samples from anonymous patients which were discarded after C. difficile testing over a period of 9 to 15 months. Metagenomic DNA was extracted from these samples and sequenced using an Illumina NovaSeq 6000 platform. The resulting sequencing reads were analysed for taxonomic composition and for the presence of AMR genes. Among 683 faecal metagenomes we found huge variation between individuals in terms of taxonomic diversity (Shannon Index range 0.10–3.99) and carriage of AMR genes (Median 1.50 genes/cell/sample overall). We found no statistically significant differences in diversity (median Shannon index 2.16 (IQR 1.71–2.56), 2.15 (IQR 1.62–2.50) and 2.26 (IQR 1.55–2.51)) or carriage of AMR genes (median 1.37 genes/cell/sample (IQR 0.70–3.24), 1.70 (IQR 0.70–4.52) and 1.43 (IQR 0.55–3.71)) at the three trusts respectively. This was also the case across the sample collection period within the trusts. While we have not demonstrated differences over place or time using metagenomic sequencing of faecal discards, other sampling frameworks may be more suitable to determine whether organisational level differences in antibiotic use are associated with individual-level differences in burden of AMR carriage

Undetected carriage explains apparent Staphylococcus aureus acquisition in a non-outbreak healthcare setting

Objectives Previous studies have been unable to identify patient or staff reservoirs for the majority of the nosocomial S. aureus acquisitions which occur in the presence of good infection control practice. We set out to establish the extent to which undetected pre-existing carriage explains apparent nosocomial S. aureus acquisition. Methods Over two years elective cardiothoracic admissions were screened for S. aureus carriage before and during hospital admission. Routine screening (nose/groin/wound sampling), was supplemented by sampling additional body sites (axilla/throat/rectum) and culture-based methods optimised to detect fastidious phenotypes (small colony variants, cell wall deficient variants) and molecular identification by PCR. Results 35% of participants (53/151) were S. aureus carriers according to routine pre-healthcare screening; increasing to 42% (63/151) when additional body sites and enhanced cultures were employed. 71% (5/7) of apparent acquisitions were explained by pre-existing carriage using augmented measures. Enhanced culture identified a minority of colonised individuals (3/151 including 1 MRSA carrier) who were undetected by routine and additional screening cultures. 4/14 (29%) participants who became culture-negative during admission had S. aureus genomic material detected at discharge.` Conclusions Conventional sampling under-estimates carriage of S. aureus and this explains the majority of apparent S. aureus acquisitions among elective cardiothoracic patients

Clonal differences in Staphylococcus aureus bacteraemia-associated mortality.

The bacterium Staphylococcus aureus is a major human pathogen for which the emergence of antibiotic resistance is a global public health concern. Infection severity, and in particular bacteraemia-associated mortality, has been attributed to several host-related factors, such as age and the presence of comorbidities. The role of the bacterium in infection severity is less well understood, as it is complicated by the multifaceted nature of bacterial virulence, which has so far prevented a robust mapping between genotype, phenotype and infection outcome. To investigate the role of bacterial factors in contributing to bacteraemia-associated mortality, we phenotyped a collection of sequenced clinical S. aureus isolates from patients with bloodstream infections, representing two globally important clonal types, CC22 and CC30. By adopting a genome-wide association study approach we identified and functionally verified several genetic loci that affect the expression of cytolytic toxicity and biofilm formation. By analysing the pooled data comprising bacterial genotype and phenotype together with clinical metadata within a machine-learning framework, we found significant clonal differences in the determinants most predictive of poor infection outcome. Whereas elevated cytolytic toxicity in combination with low levels of biofilm formation was predictive of an increased risk of mortality in infections by strains of a CC22 background, these virulence-specific factors had little influence on mortality rates associated with CC30 infections. Our results therefore suggest that different clones may have adopted different strategies to overcome host responses and cause severe pathology. Our study further demonstrates the use of a combined genomics and data analytic approach to enhance our understanding of bacterial pathogenesis at the individual level, which will be an important step towards personalized medicine and infectious disease management

Evolution and Global Transmission of a Multidrug-Resistant, Community-Associated Methicillin-Resistant Staphylococcus aureus Lineage from the Indian Subcontinent

The evolution and global transmission of antimicrobial resistance have been well documented for Gram-negative bacteria and health care-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. In this study, we traced the recent origins and global spread of a multidrug-resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole-genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data show that the clone emerged on the Indian subcontinent in the early 1960s and disseminated rapidly in the 1990s. Short-term outbreaks in community and health care settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the emergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth, and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional health care-associated clones with the epidemiological transmission of community-associated methicillin-resistant S. aureus (MRSA). Our study demonstrates the importance of whole-genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere

Evolution and Global Transmission of a Multidrug-Resistant, Community-Associated Methicillin-Resistant Staphylococcus aureus Lineage from the Indian Subcontinent.

The evolution and global transmission of antimicrobial resistance have been well documented for Gram-negative bacteria and health care-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. In this study, we traced the recent origins and global spread of a multidrug-resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole-genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data show that the clone emerged on the Indian subcontinent in the early 1960s and disseminated rapidly in the 1990s. Short-term outbreaks in community and health care settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the emergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth, and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional health care-associated clones with the epidemiological transmission of community-associated methicillin-resistant S. aureus (MRSA). Our study demonstrates the importance of whole-genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.IMPORTANCE The Bengal Bay clone (ST772) is a community-associated and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we showed that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally, resulting in small-scale community and health care outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug resistance of health care-associated S. aureus lineages. This study demonstrates the importance of whole-genome sequencing for the surveillance of highly antibiotic-resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world