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To combat the threat to human health and biosecurity from antimicrobial resistance, an understanding of its mechanisms and drivers is needed. Emergence of antimicrobial resistance in microorganisms is a natural phenomenon, yet antimicrobial resistance selection has been driven by antimicrobial exposure in health care, agriculture, and the environment. Onward transmission is affected by standards of infection control, sanitation, access to clean water, access to assured quality antimicrobials and diagnostics, travel, and migration. Strategies to reduce antimicrobial resistance by removing antimicrobial selective pressure alone rely upon resistance imparting a fitness cost, an effect not always apparent. Minimising resistance should therefore be considered comprehensively, by resistance mechanism, microorganism, antimicrobial drug, host, and context; parallel to new drug discovery, broad ranging, multidisciplinary research is needed across these five levels, interlinked across the health-care, agriculture, and environment sectors. Intelligent, integrated approaches, mindful of potential unintended results, are needed to ensure sustained, worldwide access to effective antimicrobials.

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Å out of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site. PoxtA confers resistance to ribosome-targeting oxazolidinone (linezolid) and chloramphenicol antibiotics. Here, Crowe-McAuliffe et al. provide structural insights into how binding of PoxtA to the ribosome indirectly promotes drug dissociation.

Community-acquired urinary tract infection (CA-UTI) is the most common infection caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, but the clinical epidemiology of these infections in low prevalence countries is largely unknown. A population based case-control study was conducted to assess risk factors for CA-UTI caused by ESBL-producing E. coli or K. pneumoniae. The study was carried out in a source population in Eastern Norway, a country with a low prevalence of infections caused by ESBL-producing Enterobacteriaceae. The study population comprised 100 cases and 190 controls with CA-UTI caused by ESBL-producing and non-ESBL-producing E. coli or K. pneumoniae, respectively. The following independent risk factors of ESBL-positive UTIs were identified: Travel to Asia, The Middle East or Africa either during the past six weeks (Odds ratio (OR) = 21; 95% confidence interval (CI): 4.5-97) or during the past 6 weeks to 24 months (OR = 2.3; 95% CI: 1.1-4.4), recent use of fluoroquinolones (OR = 16; 95% CI: 3.2-80) and β-lactams (except mecillinam) (OR = 5.0; 95% CI: 2.1-12), diabetes mellitus (OR = 3.2; 95% CI: 1.0-11) and recreational freshwater swimming the past year (OR = 2.1; 95% CI: 1.0-4.0). Factors associated with decreased risk were increasing number of fish meals per week (OR = 0.68 per fish meal; 95% CI: 0.51-0.90) and age (OR = 0.89 per 5 year increase; 95% CI: 0.82-0.97). In conclusion, we have identified risk factors that elucidate mechanisms and routes for dissemination of ESBL-producing Enterobacteriaceae in a low prevalence country, which can be used to guide appropriate treatment of CA-UTI and targeted infection control measures.

Extended spectrum β-lactamase producing Escherichia coli (ESBL-EC) are excreted via effluents and sewage into the environment where they can re-contaminate humans and animals. The aim of this observational study was to detect and quantify ESBL-EC in recreational water and wastewater, and perform a genetic and phenotypic comparative analysis of the environmental strains with geographically associated human urinary ESBL-EC. Recreational fresh- and saltwater samples from four different beaches and wastewater samples from a nearby sewage plant were filtered and cultured on differential and ESBL-selective media. After antimicrobial susceptibility testing and multi-locus variable number of tandem repeats assay (MLVA), selected ESBL-EC strains from recreational water were characterized by whole genome sequencing (WGS) and compared to wastewater and human urine isolates from people living in the same area. We detected ESBL-EC in recreational water samples on 8/20 occasions (40%), representing all sites. The ratio of ESBL-EC to total number of E. coli colony forming units varied from 0 to 3.8%. ESBL-EC were present in all wastewater samples in ratios of 0.56-0.75%. ST131 was most prevalent in urine and wastewater samples, while ST10 dominated in water samples. Eight STs and identical ESBL-EC MLVA-types were detected in all compartments. Clinical ESBL-EC isolates were more likely to be multidrug-resistant (p<0.001). This study confirms that ESBL-EC, including those that are capable of causing human infection, are present in recreational waters where there is a potential for human exposure and subsequent gut colonisation and infection in bathers. Multidrug-resistant E. coli strains are present in urban aquatic environments even in countries where antibiotic consumption in both humans and animals is highly restricted.

To study the molecular characteristics of a long-term, low frequency outbreak of bla KPC-2 in a low prevalence setting involving the hospital environment. KPC-producing bacteria were screened by selective chromogenic agar and Real-Time PCR. The presence of antibiotic resistance genes was ascribed by PCRs and subsequent sequencing, and the KPC-producing isolates were phylogenetically typed using PFGE and multi-locus sequence typing. Bla KPC-2-plasmids were identified and analysed by S1-nuclease-PFGE hybridization and PCR based replicon typing. A ∼97 kb IncFII plasmid was seen to carry bla KPC-2 in all of the clinical isolates, in one of the isolates recovered from screened patients (1/136), and in the Klebsiella pneumoniae and Enterobacter asburiae isolates recovered from the environment (sinks) in one intensive care unit. The K. pneumoniae strain ST258 was identified in 6 out of 7 patients. An intergenus spread to E. asburiae and an interspecies spread to two different K. pneumoniae clones (ST27 and ST461) of the bla KPC-2 plasmid was discovered. K. pneumoniae ST258 and genetically related E. asburiae strains were found in isolates of both human and environmental origins. We document a clonal transmission of the K. pneumoniae ST258 strain, and an intergenus plasmid diffusion of the IncFII plasmid carrying bla KPC-2 in this outbreak. A major reservoir in the patient population could not be unveiled. However, the identification of a persisting environmental reservoir of strains with molecular determinants linked to human isolates, suggests a possible role of the environment in the maintenance of this long-term outbreak.

The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) is increasing worldwide. Here we present associated patient data and molecular, epidemiological and phenotypic characteristics of all CPE isolates in Norway from 2007 to 2014 confirmed at the Norwegian National Advisory Unit on Detection of Antimicrobial Resistance. All confirmed CPE isolates were characterized pheno- and genotypically, including by whole genome sequencing (WGS). Patient data were reviewed retrospectively. In total 59 CPE isolates were identified from 53 patients. Urine was the dominant clinical sample source (37%) and only 15% of the isolates were obtained from faecal screening. The majority of cases (62%) were directly associated with travel or hospitalization abroad, but both intra-hospital transmission and one inter-hospital outbreak were observed. The number of CPE cases/year was low (2-14 cases/year), but an increasing trend was observed. Klebsiella spp. (n = 38) and E. coli (n = 14) were the dominant species and blaKPC (n = 20), blaNDM (n = 19), blaOXA-48-like (n = 12) and blaVIM (n = 7) were the dominant carbapenemase gene families. The CPE isolates were genetically diverse except for K. pneumoniae where clonal group 258 associated with blaKPC dominated. All isolates were multidrug-resistant and a significant proportion (21%) were resistant to colistin. Interestingly, all blaOXA-48-like, and a large proportion of blaNDM-positive Klebsiella spp. (89%) and E. coli (83%) isolates were susceptible in vitro to mecillinam. Thus, mecillinam could have a role in the treatment of uncomplicated urinary tract infections caused by OXA-48- or NDM-producing E. coli or K. pneumoniae. In conclusion, the impact of CPE in Norway is still limited and mainly associated with travel abroad, reflected in the diversity of clones and carbapenemase genes.

Nasal carriage of Staphylococcus aureus and its antibiotic-resistant derivative, methicillin-resistant S. aureus (MRSA), is a risk factor for nosocomial S. aureus infections. Mupirocin is a topical antibiotic and a key in the decolonization of both methicillin-susceptible S. aureus (MSSA) and MRSA carriage in patients and health care personnel. Recent observations have shown a global increase in the prevalence of mupirocin-resistant MSSA and MRSA, reducing the efficacy of mupirocin in decolonization regimens. LTX-109 is a peptidomimetic synthetic compound that has shown broad-spectrum bactericidal antimicrobial activity in vitro and in animal experiments. However, the development of resistance against LTX-109 in clinical isolates of MRSA and MSSA has not been systematically examined. Background/Objectives: Here, we assess the development of spontaneous and adaptive resistance against LTX-109 in genomically diverse MRSA (n = 3) and MSSA (n = 4) strains. Methods: Adaptive and mutational resistance were examined by serial passaging strains over 60 cycles in a range of LTX-109 and mupirocin concentrations. Spontaneous resistance was examined in high-inoculum agar plates with 2–8 times the concentration above MIC. Results: Throughout serial passage, LTX-109 MICs varied less than 4-fold compared to the initial MIC of 4–8 mg/L, while mupirocin MICs increased in all susceptible strains (n = 5) from 0.25 mg/L to 16–512 mg/L. The spontaneous resistance assay demonstrated no resistance development at 4–8× MIC LTX-109 and an inoculum effect at 2× MIC. Conclusions: Our results demonstrate the novelty of LTX-109 as an antimicrobial agent with no detectable in vitro resistance development in selected clinical strains of MRSA and MSSA.

Background The success of Enterococcus faecium and E. faecalis evolving as multi-resistant nosocomial pathogens is associated with their ability to acquire and share adaptive traits, including antimicrobial resistance genes encoded by mobile genetic elements (MGEs). Here, we investigate this mobilome in successful hospital associated genetic lineages, E. faecium sequence type (ST)17 (n=10) and ST78 (n=10), E. faecalis ST6 (n=10) and ST40 (n=10) by DNA microarray analyses. Results The hybridization patterns of 272 representative targets including plasmid backbones (n=85), transposable elements (n=85), resistance determinants (n=67), prophages (n=29) and clustered regularly interspaced short palindromic repeats (CRISPR)-cas sequences (n=6) separated the strains according to species, and for E. faecalis also according to STs. RCR-, Rep_3-, RepA_N- and Inc18-family plasmids were highly prevalent and with the exception of Rep_3, evenly distributed between the species. There was a considerable difference in the replicon profile, with rep 17/pRUM , rep 2/pRE25 , rep 14/EFNP1 and rep 20/pLG1 dominating in E. faecium and rep 9/pCF10 , rep 2/pRE25 and rep 7 in E. faecalis strains. We observed an overall high correlation between the presence and absence of genes coding for resistance towards antibiotics, metals, biocides and their corresponding MGEs as well as their phenotypic antimicrobial susceptibility pattern. Although most IS families were represented in both E. faecalis and E. faecium , specific IS elements within these families were distributed in only one species. The prevalence of IS 256 -, IS 3 -, IS L3 -, IS 200 /IS 605 -, IS 110 -, IS 982 - and IS 4 -transposases was significantly higher in E. faecium than E. faecalis , and that of IS 110 -, IS 982 - and IS 1182 -transposases in E. faecalis ST6 compared to ST40. Notably, the transposases of IS 981 , IS Efm1 and IS 1678 that have only been reported in few enterococcal isolates were well represented in the E. faecium strains. E. faecalis ST40 strains harboured possible functional CRISPR-Cas systems, and still resistance and prophage sequences were generally well represented. Conclusions The targeted MGEs were highly prevalent among the selected STs, underlining their potential importance in the evolution of hospital-adapted lineages of enterococci. Although the propensity of inter-species horizontal gene transfer (HGT) must be emphasized, the considerable species-specificity of these MGEs indicates a separate vertical evolution of MGEs within each species, and for E. faecalis within each ST.

Background Epidemiological data of cephalosporin-resistant Enterobacterales in Sub-Saharan Africa is still restricted, and in particular in Mozambique. The aim of this study was to detect and characterize extended-spectrum β-lactamase (ESBL) - and plasmid-mediated AmpC (pAmpC)-producing clinical strains of Escherichia coli at Maputo Central Hospital (MCH), a 1000-bed reference hospital in Maputo, Mozambique. Methods A total of 230 clinical isolates of E. coli from urine ( n  = 199) and blood cultures ( n  = 31) were collected at MCH during August–November 2015. Antimicrobial susceptibility testing was performed by the disc diffusion method and interpreted according to EUCAST guidelines. Isolates with reduced susceptibility to 3rd generation cephalosporins were examined further; phenotypically for an ESBL−/AmpC-phenotype by combined disc methods and genetically for ESBL- and pAmpC-encoding genes by PCR and partial amplicon sequencing as well as genetic relatedness by ERIC-PCR. Results A total of 75 isolates with reduced susceptibility to cefotaxime and/or ceftazidime ( n  = 75) from urine ( n  = 58/199; 29%) and blood ( n  = 17/31; 55%) were detected. All 75 isolates were phenotypically ESBL-positive and 25/75 (33%) of those also expressed an AmpC-phenotype. ESBL-PCR and amplicon sequencing revealed a majority of bla CTX-M ( n  = 58/75; 77%) dominated by bla CTX-M-15 . All AmpC-phenotype positive isolates ( n  = 25/75; 33%) scored positive for one or more pAmpC-genes dominated by bla MOX/FOX . Multidrug resistance (resistance ≥ three antibiotic classes) was observed in all the 75 ESBL-positive isolates dominated by resistance to trimethoprim-sulfamethoxazole, ciprofloxacin and gentamicin. ERIC-PCR revealed genetic diversity among strains with minor clusters indicating intra-hospital spread. Conclusion We have observed a high prevalence of MDR pAmpC- and/or ESBL-producing clinical E. coli isolates with FOX/MOX and CTX-Ms as the major β-lactamase types, respectively. ERIC-PCR analyses revealed genetic diversity and some clusters indicating within-hospital spread. The overall findings strongly support the urgent need for accurate and rapid diagnostic services to guide antibiotic treatment and improved infection control measures.

We have performed a prospective cohort study to investigate the duration of and risk factors for prolonged fecal carriage of ESBL-producing Escherichia coli or Klebsiella pneumoniae in patients with community acquired urinary tract infection caused by these bacteria. From 2009 to 2011, 101 Norwegian patients were recruited. Stool swabs and questionnaires were collected every three months for one year and at the end of the study in 2012. Information on antibiotic prescriptions was collected from the Norwegian Prescription Database. Stool samples were cultured directly on ChromID ESBL agar as well as in an enrichment broth, and culture positive isolates were examined by blaCTX-M multiplex PCR. Isolates without blaCTX-M were investigated for alternative ESBL-determinants with a commercial microarray system. Time to fecal clearance of ESBL producing Enterobacteriaceae was also analysed using Kaplan-Meier estimates. Uni- and multivariate logistic regression was used to compare groups according to previously described risk factors. The ESBL point prevalence of fecal carriage were 61% at 4 months, 56% at 7 months, 48% at 10 months, 39% at 13 months, 19% after two years, and 15% after three years or more. We found no correlation between duration of carriage, comorbidity, antibiotic use or travel to ESBL high-prevalence countries. Prolonged carriage was associated with E. coli isolates of phylogroup B2 or D. Importantly, comparative MLST and MLVA analyses of individual paired urine and fecal E. coli isolates revealed that ESBL production commonly occurred in diverse strains within the same host. When investigating cross-transmission of ESBL producing bacteria in health care institutions, this notion should be taken into account.