Antimicrobial susceptibility prediction from genomes: a dream come true?

Genome-based diagnostics provides relevant information to guide patient treatment and support pathogen and resistance surveillance. Recently, Coll et al. introduced a curated database for predicting antimicrobial resistance (AMR) from Enterococcus faecium genomics data, offering excellent predictive values for susceptibility to important antimicrobials. Challenges to predict resistance to last-resort antimicrobials remain.

TaME-seq2: tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling.

BACKGROUND: Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA. RESULTS: Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. CONCLUSION: TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.

Phylodynamic signatures in the emergence of community-associated MRSA.

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

A tale of two STs: molecular and clinical epidemiology of MRSA t304 in Norway 2008-2016.

The purpose of this study was to investigate the epidemiological, molecular, and clinical characteristics of MRSA t304/ST8 and t304/ST6 in Norway from 2008 to 2016. Clinical and epidemiological data were collected for each case included in the study. Strains were characterized by PCR, spa typing, antimicrobial susceptibility testing, and whole genome sequencing. The overall number of cases of MRSA t304 increased from 27 in 2008 to 203 in 2016. Most MRSA t304/ST8 cases were defined as HA-MRSA (89.9%) and diagnosed in persons with Norwegian background, many of them living in nursing homes (62.3%). The number of t304/ST8 cases declined throughout the study period and it has not been reported in Norway since 2014. The increasing MRSA t304/ST6 genotype has mainly been introduced to Norway by immigration from the Middle East, but also from other parts of the world. The t304/ST6 clone is mostly classified as CA-MRSA (75.1%), does not seem to cause serious infections, is not multi-resistant, and has not yet caused outbreaks in Norway. This study provides an example of two MRSA clones with the same spa type found in different epidemiological settings. This is very unusual, but still a reminder that spa typing in some cases may have insufficient discriminatory power for surveillance of MRSA. Our results highlight the importance of active surveillance and characterization of emerging MRSA clones with high potential for spread in the community, which may potentially cause outbreaks in healthcare facilities.

Rapid nanopore-based DNA sequencing protocol of antibiotic-resistant bacteria for use in surveillance and outbreak investigation.

Outbreak investigations are essential to control and prevent the dissemination of pathogens. This study developed and validated a complete analysis protocol for faster and more accurate surveillance and outbreak investigations of antibiotic-resistant microbes based on Oxford Nanopore Technologies (ONT) DNA whole-genome sequencing. The protocol was developed using 42 methicillin-resistant Staphylococcus aureus (MRSA) isolates identified from former well-characterized outbreaks. The validation of the protocol was performed using Illumina technology (MiSeq, Illumina). Additionally, a real-time outbreak investigation of six clinical S. aureus isolates was conducted to test the ONT-based protocol. The suggested protocol includes: (1) a 20 h sequencing run; (2) identification of the sequence type (ST); (3) de novo genome assembly; (4) polishing of the draft genomes; and (5) phylogenetic analysis based on SNPs. After the sequencing run, it was possible to identify the ST in 2 h (20 min per isolate). Assemblies were achieved after 4 h (40 min per isolate) while the polishing was carried out in 7 min per isolate (42 min in total). The phylogenetic analysis took 0.6 h to confirm an outbreak. Overall, the developed protocol was able to at least discard an outbreak in 27 h (mean) after the bacterial identification and less than 33 h to confirm it. All these estimated times were calculated considering the average time for six MRSA isolates per sequencing run. During the real-time S. aureus outbreak investigation, the protocol was able to identify two outbreaks in less than 31 h. The suggested protocol enables identification of outbreaks in early stages using a portable and low-cost device along with a streamlined downstream analysis, therefore having the potential to be incorporated in routine surveillance analysis workflows. In addition, further analysis may include identification of virulence and antibiotic resistance genes for improved pathogen characterization.

Culture-Independent Genotyping, Virulence and Antimicrobial Resistance Gene Identification of Staphylococcus aureus from Orthopaedic Implant-Associated Infections.

Our culture-independent nanopore shotgun metagenomic sequencing protocol on biopsies has the potential for same-day diagnostics of orthopaedic implant-associated infections (OIAI). As OIAI are frequently caused by Staphylococcus aureus, we included S. aureus genotyping and virulence gene detection to exploit the protocol to its fullest. The aim was to evaluate S. aureus genotyping, virulence and antimicrobial resistance genes detection using the shotgun metagenomic sequencing protocol. This proof of concept study included six patients with S. aureus-associated OIAI at Akershus University Hospital, Norway. Five tissue biopsies from each patient were divided in two: (1) conventional microbiological diagnostics and genotyping, and whole genome sequencing (WGS) of S. aureus isolates; (2) shotgun metagenomic sequencing of DNA from the biopsies. Consensus sequences were analysed using spaTyper, MLST, VirulenceFinder, and ResFinder from the Center for Genomic Epidemiology (CGE). MLST was also compared using krocus. All spa-types, one CGE and four krocus MLST results matched Sanger sequencing results. Virulence gene detection matched between WGS and shotgun metagenomic sequencing. ResFinder results corresponded to resistance phenotype. S. aureus spa-typing, and identification of virulence and antimicrobial resistance genes are possible using our shotgun metagenomics protocol. MLST requires further optimization. The protocol has potential application to other species and infection types.

Rapid Diagnostics of Orthopaedic-Implant-Associated Infections Using Nanopore Shotgun Metagenomic Sequencing on Tissue Biopsies.

Conventional culture-based diagnostics of orthopaedic-implant-associated infections (OIAIs) are arduous. Hence, the aim of this study was to evaluate a culture-independent, rapid nanopore-based diagnostic protocol with regard to (a) pathogen identification, (b) time to pathogen identification, and (c) identification of antimicrobial resistance (AMR). This prospective proof-of-concept study included soft tissue biopsies from 32 patients with OIAIs undergoing first revision surgery at Akershus University Hospital, Norway. The biopsies were divided into two segments. Nanopore shotgun metagenomic sequencing and pathogen and antimicrobial resistance gene identification using the EPI2ME analysis platform (Oxford Nanopore Technologies) were performed on one segment. Conventional culture-based diagnostics were performed on the other. Microbial identification matched in 23/32 OIAI patients (72%). Sequencing detected additional microbes in 9/32 patients. Pathogens detected by culturing were identified by sequencing within a median of 1 h of sequencing start [range 1-18 h]. Phenotypic AMR was explained by the detection of resistance genes in 11/23 patients (48%). Diagnostics of OIAIs using shotgun metagenomics sequencing are possible within 24 h from biopsy using nanopore technology. Sequencing outperformed culturing with respect to speed and pathogen detection where pathogens were at sufficient concentration, whereas culture-based methods had an advantage at lower pathogen concentrations. Sequencing-based AMR detection may not yet be a suitable replacement for culture-based antibiotic susceptibility testing.

Is increased Staphylococcus aureus colonization during military service caused by specific genotypes? Molecular examination of long-term carriage in a prospective cohort study.

A 22% increase in Staphylococcus aureus colonization was observed in Norwegian recruits during first year of military service. The aim was to determine whether specific genotypes caused the increase and to examine carriage status based on genotyping. Characterization of S. aureus from nose, throat and perineum sampled at enrolment and discharge included spa typing, MLVA, detection of PVL genes and antimicrobial susceptibility testing. spa typing demonstrated high and stable genetic diversity. The three most frequent spa types were found in 15% of recruits at enrolment and in 29% at discharge. Only t084 increased significantly (p = 0.02). Subtyping revealed that t084, t065 and t002 consisted of 13, 6 and 11 different MLVA types, respectively, at discharge. The military cohort (n = 265) consisted of S. aureus carriers of identical genotype (n = 99, 38%), carriers of non-identical genotype (n = 52, 20%), intermittent carriers (n = 86, 33%) and non-carriers (n = 27, 10%). Carrier status was indefinable for one recruit due to unavailable isolates for genotyping. Antibiotic resistance towards erythromycin, fusidic acid and clindamycin increased significantly and above national surveillance levels. The observed increase in S. aureus colonization during military service was caused by many different genotypes implying many transmission routes. Genotype did not correlate with colonization niche or carriage status.

Are conventional microbiological diagnostics sufficiently expedient in the era of rapid diagnostics? Evaluation of conventional microbiological diagnostics of orthopedic implant-associated infections (OIAI).

Background and purpose - In a time when rapid diagnostics are increasingly sought, conventional procedures for detection of microbes causing orthopedic implant-associated infections (OIAI) seem extensive and time-consuming, but how extensive are they? We assessed time to (a) pathogen identification, (b) antibiotic susceptibility patterns, and (c) targeted antibiotic treatment using conventional microbiological diagnostics of OIAI in a consecutive series of patients.Patients and methods - Consecutive patients aged ≥18 years undergoing first revision surgery for acute OIAI, including prosthetic joints, fracture, and osteotomy implants, in 2017-2018 at Akershus University Hospital (Ahus), Norway were included. Information regarding microbiological diagnostics and clinical data was collected retrospectively from the hospital's diagnostic and clinical databases.Results - 123 patients fulfilled the inclusion criteria. Median time to pathogen identification was 2.5 days and to antibiotic treatment recommendations was 3.5 days. The most common pathogens were S. aureus (52%) and S. epidermidis (15%). Cultures were inconclusive in 11% of the patients. Of the 109 patients with culture-positive results, antibiotic treatment was changed in 66 (61%) patients within a median of 4 days (0-24) after the recommendation was given.Interpretation - Conventional microbiological diagnostics of OIAI is time-consuming, taking days of culturing. Same-day diagnostics would vastly improve treatment efficacy, but is dependent on rapid implementation by clinicians of the treatment recommendations given by the microbiologist.

Molecular characteristics of Staphylococcus aureus associated prosthetic joint infections after hip fractures treated with hemiarthroplasty: a retrospective genome-wide association study.

A retrospective study of Staphylococcus aureus isolates from orthopaedic patients treated between 2000 and 2017 at Akershus University Hospital, Norway was performed using a genome-wide association approach. The aim was to characterize and investigate molecular characteristics unique to S. aureus isolates from HHA associated prosthetic joint infections and potentially explain the HHA patients' elevated 1-year mortality compared to a non-HHA group. The comparison group consisted of patients with non-HHA lower-extremity implant-related S. aureus infections. S. aureus isolates from diagnostic patient samples were whole-genome sequenced. Univariate and multivariate analyses were performed to detect group-associated genetic signatures. A total of 62 HHA patients and 73 non-HHA patients were included. Median age (81 years vs. 74 years; p < 0.001) and 1-year mortality (44% vs. 15%, p < 0.001) were higher in the HHA group. A total of 20 clonal clusters (CCs) were identified; 75% of the isolates consisted of CC45, CC30, CC5, CC15, and CC1. Analyses of core and accessory genome content, including virulence, resistance genes, and k-mer analysis revealed few group-associated variants, none of which could explain the elevated 1-year mortality in HHA patients. Our findings support the premise that all S. aureus can cause invasive infections given the opportunity.

DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing.

Identification of bacteria causing tissue infections can be comprehensive and, in the cases of non- or slow-growing bacteria, near impossible with conventional methods. Performing shotgun metagenomic sequencing on bacterial DNA extracted directly from the infected tissue may improve time to diagnosis and targeted treatment considerably. However, infected tissue consists mainly of human DNA (hDNA) which hampers bacterial identification. In this proof of concept study, we present a modified version of the Ultra-Deep Microbiome Prep kit for DNA extraction procedure, removing additional human DNA. Tissue biopsies from 3 patients with orthopedic implant-related infections containing varying degrees of Staphylococcus aureus were included. Subsequent DNA shotgun metagenomic sequencing using Oxford Nanopore Technologies' (ONT) MinION platform and ONTs EPI2ME WIMP and ARMA bioinformatic workflows for microbe and antibiotic resistance genes identification, respectively. The modified DNA extraction protocol led to an additional ~10-fold reduction of human DNA while preserving S. aureus DNA. Including the DNA sequencing and bioinformatics analyses, the presented protocol has the potential of identifying the infection-causing pathogen in infected tissue within 7 hours after biopsy. However, due to low number of S. aureus reads, positive identification of antibiotic resistance genes was not possible.

Early-Stage Staphylococcus aureus Bloodstream Infection Causes Changes in the Concentrations of Lipoproteins and Acute-Phase Proteins and Is Associated with Low Antibody Titers against Bacterial Virulence Factors.

Systemic and quantitative investigations of human plasma proteins (proteomics) and Staphylococcus aureus-specific antibodies (immunoproteomics) provide complementary information and hold promise for the discovery of biomarkers in Staphylococcus aureus bloodstream infection (SABSI). Usually, data-dependent acquisition (DDA) is used for proteome analysis of serum or plasma, but data-independent acquisition (DIA) is more comprehensive and reproducible. In this prospective cohort study, we aimed to identify biomarkers associated with the early stages of SABSI using a serum DIA proteomic and immunoproteomic approach. Sera from 49 SABSI patients and 43 noninfected controls were analyzed. In total, 608 human serum proteins were identified with DIA. A total of 386 proteins could be quantified, of which 9 proteins, mainly belonging to acute-phase proteins, were significantly increased, while 7 high-density lipoproteins were lower in SABSI. In SABSI, total anti-S. aureus serum IgG was reduced compared with controls as shown by immunoproteomic quantification of IgG binding to 143 S. aureus antigens. IgG binding to 48 of these anti-S. aureus proteins was significantly lower in SABSI, while anti-Ecb IgG was the only one increased in SABSI. Serum IgG binding to autoinducing peptide MsrB, FadB, EsxA, Pbp2, FadB, SspB, or SodA was very low in SABSI. This marker panel discriminated early SABSI from controls with 95% sensitivity and 100% specificity according to random forest prediction. This holds promise for patient stratification according to their risk of S. aureus infection, underlines the protective function of the adaptive immune system, and encourages further efforts in the development of a vaccine against S. aureus IMPORTANCE S. aureus sepsis has a high complication and mortality rate. Given the limited therapeutic possibilities, effective prevention strategies, e.g., a vaccine, or the early identification of high-risk patients would be important but are not available. Our study showed an acute-phase response in patients with S. aureus bloodstream infection and evidence that lipoproteins are downregulated in plasma. Using immunoproteomics, stratification of patients appears to be achievable, since at the early stages of systemic S. aureus infection patients had low preexisting anti-S. aureus antibody levels. This strengthens the notion that a robust immune memory for S. aureus protects against infections with the pathogen.

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.

Rapid diagnostics of orthopedic implant-associated infections using Unyvero ITI implant and tissue infection application is not optimal for Staphylococcus species identification.

OBJECTIVES: This pilot study aimed to compare the commercial Unyvero ITI multiplex PCR application (U-ITI, Curetis GmbH) with conventional culturing concerning (a) detection of pathogens, (b) time to detection of pathogens and (c) time to and quality of antibiotic treatment recommendation in diagnostics of orthopedic implant-associated infections (OIAI). RESULTS: 72 tissue biopsies from 15 consecutive patients with deep OIAI infections were analyzed with conventional culturing including phenotypic antibiotic susceptibility testing and the U-ITI. U-ITI showed lower sensitivity than conventional culturing concerning detection of pathogens (73% vs 93%). 4/15 patients would have been given false negative results by U-ITI, all of which were culture-positive for Staphylococcus species. Median time to detection of pathogens was 47 h and antibiotic resistance 89 h by conventional methods compared to 13.5 h with the U-ITI. The U-ITI did not detect antibiotic resistance, whereas conventional culturing showed resistance to antibiotics covered by the U-ITI panel in 2 patients. Time to detection of pathogens was improved, but the detection limit for staphylococci was unsatisfactory. Although the time to antibiotic treatment recommendation was significantly reduced, the U-ITI would have resulted in incorrect antibiotic recommendation in 2 patients. Our data do not support use of this assay in diagnostics.

Genotypic characterization of gentamicin and cephalosporin resistant Escherichia coli isolates from blood cultures in a Norwegian university hospital 2011-2015.

Background: Cephalosporin resistance in clinical E. coli isolates is increasing internationally. The increase has been caused by virulent and often multidrug-resistant clones, especially the extended spectrum ß-lactamase (ESBL) producing E. coli clone O25b-ST131. Methods: In Norway, recommended empirical treatment of sepsis consists of gentamicin and penicillin combined, or a broad-spectrum cephalosporin. To investigate if increased gentamicin and cephalosporins resistance rates in our hospital could be caused by specific clones, we conducted a retrospective study on E. coli blood culture isolates from 2011 through 2015. All E. coli isolates non-susceptible to gentamicin and/or third-generation cephalosporins were genotyped using multiple-locus variable-number of tandem repeat analysis (MLVA) and compared with antibiotic susceptible isolates. The frequency of the most common genes causing ESBL production (blaCTX-M, blaampC) was examined by Real-Time PCR. Results: A total of 158 cephalosporin and/or gentamicin resistant and 97 control isolates were differentiated into 126 unique MLVA types. Of these, 31% of the isolates belonged to a major MLVA cluster consisting of 41% of the gentamicin resistant and 35% of the cephalosporin resistant isolates. The majority (65/80 isolates) of this MLVA cluster contained MLVA types associated with the E. coli O25b-ST131 clone. Genes encoding CTX-M enzyme phylogroups 1 and 9 occurred in 65% and 19% of cephalosporin resistant isolates, respectively, whereas blaampC-CIT was identified in 3%. Conclusion: No local E. coli bacteraemia clone was identified. Antibiotic resistance was dispersed over a variety of genotypes. However, association with the international E. coli O25b-ST131 clone was frequent and may be an important driver behind increased resistance rates. Monitoring and preventing dissemination of these resistant clones are important for continued optimal treatment.

No associations established between single nucleotide polymorphisms in human Toll-like receptor 2 and Toll-interacting protein and Staphylococcus aureus bloodstream infections.

Staphylococcus aureus bloodstream infections (SABSI) are associated with high morbidity and mortality. The Toll-like receptor 2 (TLR2) and Toll-interacting protein (TOLLIP) are important in recognition and regulation of human innate immunity response to S. aureus. Single nucleotide polymorphisms (SNPs) in the TLR2 and TOLLIP encoding genes have been associated with disease, including BSI. The aim of this study was to examine potential associations between a selection of SNPs in the genes encoding TLR2 and TOLLIP, and predisposition, severity, and outcome of SABSI. All patients ≥18 years of age with at least one S. aureus positive blood culture collected from March 2011 through February 2014 at Akershus University Hospital, Lørenskog, Norway, were considered for inclusion. Patients attending elective orthopaedic surgery (total hip and knee replacements, lumbar surgery) served as a control group. The TLR2 Arg753Gln, TLR2 Pro631His, TOLLIP rs5743942, and rs5743867 polymorphisms were analysed using TaqMan SNP Genotyping Assays. A total of 209 SABSI patients and 295 controls were included. The TLR2 Arg753Gln and TLR2 Pro631His polymorphisms were infrequent with no homozygotes and <10% heterozygotes. The included TLR2 and TOLLIP polymorphisms were not associated with susceptibility to SABSI, severity, 30-day all-cause mortality, or SABSI caused by the clonal complex 30 (CC30) genotype.