Nationwide, population-based observational study of the molecular epidemiology and temporal trend of carbapenemase-producing Enterobacterales in Norway, 2015 to 2021.

IntroductionNational and regional carbapenemase-producing Enterobacterales (CPE) surveillance is essential to understand the burden of antimicrobial resistance, elucidate outbreaks, and develop infection-control or antimicrobial-treatment recommendations.AimThis study aimed to describe CPE and their epidemiology in Norway from 2015 to 2021.MethodsA nationwide, population-based observational study of all verified clinical and carriage CPE isolates submitted to the national reference laboratory was conducted. Isolates were characterised by antimicrobial susceptibility testing, whole genome sequencing (WGS) and basic metadata. Annual CPE incidences were also estimated.ResultsA total of 389 CPE isolates were identified from 332 patients of 63 years median age (range: 0-98). These corresponded to 341 cases, 184 (54%) being male. Between 2015 and 2021, the annual incidence of CPE cases increased from 0.6 to 1.1 per 100,000 person-years. For CPE-isolates with available data on colonisation/infection, 58% (226/389) were associated with colonisation and 38% (149/389) with clinical infections. WGS revealed a predominance of OXA-48-like (51%; 198/389) and NDM (34%; 134/389) carbapenemases in a diversified population of Escherichia coli and Klebsiella pneumoniae, including high-risk clones also detected globally. Most CPE isolates were travel-related (63%; 245/389). Although local outbreaks and healthcare-associated transmission occurred, no interregional spread was detected. Nevertheless, 18% (70/389) of isolates not directly related to import points towards potentially unidentified transmission routes. A decline in travel-associated cases was observed during the COVID-19 pandemic.ConclusionsThe close-to-doubling of CPE case incidence between 2015 and 2021 was associated with foreign travel and genomic diversity. To limit further transmission and outbreaks, continued screening and monitoring is essential.

Intraregional hospital outbreak of OXA-244-producing Escherichia coli ST38 in Norway, 2020.

Infections with OXA-244-carbapenemase-producing Escherichia coli with sequence type (ST)38 have recently increased in Europe. Due to its low-level activity against carbapenems, OXA-244 can be difficult to detect. Previous assessments have not revealed a clear source and route of transmission for OXA-244-producing E. coli, but there are indications of non-healthcare related sources and community spread. Here we report a hospital-associated outbreak of OXA-244-producing E. coli ST38 involving three hospitals in Western Norway in 2020. The outbreak occurred over a 5-month period and included 12 cases identified through clinical (n = 6) and screening (n = 6) samples. The transmission chain was unclear; cases were identified in several wards and there was no clear overlap of patient stay. However, all patients had been admitted to the same tertiary hospital in the region, where screening revealed an outbreak in one ward (one clinical case and five screening cases). Outbreak control measures were instigated including contact tracing, isolation, and screening; no further cases were identified in 2021. This outbreak adds another dimension to the spread of OXA-244-producing E. coli ST38, illustrating this clone's ability to establish itself in the healthcare setting. Awareness of challenges concerning OXA-244-producing E. coli diagnostic is important to prevent further spread.

Carbapenem Resistance Determinants Acquired through Novel Chromosomal Integrations in Extensively Drug-Resistant Pseudomonas aeruginosa.

Two novel blaDIM-1- or blaIMP-1-containing genomic islands (GIs) were discovered by whole-genome sequence analyses in four extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates from inpatients at a tertiary hospital in Ghana. The strains were of sequence type 234 (ST234) and formed a phylogenetic clade together with ST111, which is recognized as a global high-risk clone. Their carbapenem resistance was encoded by two Tn402-type integrons, In1592 (blaDIM-1) and In1595 (blaIMP-1), both carrying complete tni mobilization modules. In1595 was bound by conserved 25-bp inverted repeats (IRs) flanked by 5-bp direct repeats (DRs) associated with target site duplication. The integrons were embedded in two GIs that contained cognate integrases and were distinguished by a lower GC content than the chromosomal average. PAGI-97A (52.659 bp; In1592), which encoded a P4-type site-specific integrase of the tyrosine recombinase family in its 3' border, was integrated into tRNA-Pro(ggg) and bracketed by a 49-bp perfect DR created by 3'-end target duplication. GIs with the same structural features, but diverse genetic content, were identified in 41/226 completed P. aeruginosa genomes. PAGI-97B (22,636 bp; In1595), which encoded an XerC/D superfamily integrase in its 5' border, was inserted into the small RNA (sRNA) PrrF1/PrrF2 locus. Specific insertions into this highly conserved locus involved in iron-dependent regulation, all leaving PrrF1 intact, were identified in an additional six phylogenetically unrelated P. aeruginosa genomes. Our molecular analyses unveiled a hospital-associated clonal dissemination of carbapenem-resistant ST234 P. aeruginosa in which the XDR phenotype resulted from novel insertions of two GIs into specific chromosomal sites.

Alternative vanHAX promoters and increased vanA-plasmid copy number resurrect silenced glycopeptide resistance in Enterococcus faecium.

BACKGROUND: Vancomycin variable enterococci (VVE) are van-positive isolates with a susceptible phenotype that can convert to a resistant phenotype during vancomycin selection. OBJECTIVES: To describe a vancomycin-susceptible vanA-PCR positive ST203 VVE Enterococcus faecium isolate (VVESwe-S) from a liver transplantation patient in Sweden which reverted to resistant (VVESwe-R) during in vitro vancomycin exposure. METHODS: WGS analysis revealed the genetic differences between the isolates. Expression of the van-operon was investigated by qPCR. Fitness and stability of the revertant were investigated by growth measurements, competition and serial transfer. RESULTS: The VVESwe-R isolate gained high-level vancomycin (MIC >256 mg/L) and teicoplanin resistance (MIC = 8 mg/L). VVESwe-S has a 5'-truncated vanR activator sequence and the VVESwe-R has in addition acquired a 44 bp deletion upstream of vanHAX in a region containing alternative putative constitutive promoters. In VVESwe-R the vanHAX-operon is constitutively expressed at a level comparable to the non-induced prototype E. faecium BM4147 strain. The vanHAX operon of VVESwe is located on an Inc18-like plasmid, which has a 3-4-fold higher copy number in VVESwe-R compared with VVESwe-S. Resistance has a low fitness cost and the vancomycin MIC of VVESwe-R decreased during in vitro serial culture without selection. The reduction in MIC was associated with a decreased vanA-plasmid copy number. CONCLUSIONS: Our data support a mechanism by which vancomycin-susceptible VVE strains may revert to a resistant phenotype through the use of an alternative, constitutive, vanR-activator-independent promoter and a vanA-plasmid copy number increase.

Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D ß-Lactamase, OXA-436, Found in Isolates from Four Patients at Six Different Hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D ß-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Emergence of vancomycin-resistant enterococci from vancomycin-susceptible enterococci in hospitalized patients under antimicrobial therapy.

OBJECTIVES: Enterococci are opportunistic pathogens with plastic genomes that evolve, acquire, and transmit antimicrobial-resistant determinants such as vancomycin resistance clusters. While vancomycin-resistant enterococci (VRE) have emerged as successful nosocomial pathogens, the mechanism by which vancomycin-susceptible enterococci (VSE) transform to VRE in hospitalized patients remains understudied. METHODS: Genomes of Enterococcus faecium from two critically ill hospitalized patients subjected to multiple antibiotic therapies, including broad-spectrum antibiotics, were investigated. To identify mechanisms of resistance evolution, genomes of vancomycin-susceptible and -resistant isolates were compared. RESULTS: While VSE isolates were initially identified, VRE strains emerged post-vancomycin therapy. Comparative genomics revealed horizontal transmission of mobile genetic elements containing the Tn1549 transposon, which harbours the vanB-type vancomycin resistance gene cluster. This suggests that broad-spectrum antibiotic stress promoted the transfer of resistance-conferring elements, presumably from another gut inhabitant. CONCLUSION: This is one of the first studies investigating VSE and VRE isolates from the same patient. The mechanism of transmission and the within-patient evolution of vancomycin resistance via mobile genetic elements under antibiotic stress is illustrated. Our findings serve as a foundation for future studies building on this knowledge which can further elucidate the dynamics of antibiotic stress, resistance determinant transmission, and interactions within the gut microbiota.

The first vanE-type vancomycin resistant Enterococcus faecalis isolates in Norway - phenotypic and molecular characteristics.

OBJECTIVES: We aimed to characterize the vanE cluster and its genetic support in the first Norwegian vanE-type isolates and assess genetic relatedness to other vanE isolates. METHODS: Two vanE-type vancomycin resistant Enterococcus faecalis (vanE-VREfs) isolates (E1 and E2) recovered from the same patient 30 months apart were examined for antimicrobial susceptibility, genome sequence, vancomycin resistance induction, vanE transferability, genome mutation rate, and phylogenetic relationship to E. faecalis closed genomes and two vanE-VREfs from North America. RESULTS: The ST34 E1 and E2 strains expressed low-level vancomycin resistance and susceptibility to teicoplanin. Their vanE gene clusters were part of a non-transferable Tn6202. The histidine kinase part of vanSE was expressed although a premature stop codon (E1) and insertion of a transposase (E2) truncated their vanSE gene. The vancomycin resistance phenotype in E1 was inducible while constitutive in E2. E1 showed a 125-fold higher mutation rate than E2. Variant calling showed 60 variants but nearly identical chromosomal gene content and synteny between the isolates. Their genomes also showed high similarity to another ST34 vanE-VREfs from Canada. CONCLUSION: In-depth genomic analyses of the first two vanE-VREfs found in Europe identified a single chromosomal insertion site of two variants of vanE-conferring Tn6202. Single nucleotide polymorphism (SNP) and core genome multilocus sequence type (cgMLST) analyses show the genomes are different. This can be explained by the high mutation rate of E1 and acquisition of different mobile genetic elements; thus, we believe the two isolates from the same patient are genetically related. Genome similarities also suggest relatedness between the Canadian and Norwegian vanE-VREfs.

Interactions between commensal Enterococcus faecium and Enterococcus lactis and clinical isolates of Enterococcus faecium.

Enterococcus faecium (Efm) is a versatile pathogen, responsible for multidrug-resistant infections, especially in hospitalized immunocompromised patients. Its population structure has been characterized by diverse clades (A1, A2, and B (reclassified as E. lactis (Ela)), adapted to different environments, and distinguished by their resistomes and virulomes. These features only partially explain the predominance of clade A1 strains in nosocomial infections. We investigated in vitro interaction of 50 clinical isolates (clade A1 Efm) against 75 commensal faecal isolates from healthy humans (25 clade A2 Efm and 50 Ela). Only 36% of the commensal isolates inhibited clinical isolates, while 76% of the clinical isolates inhibited commensal isolates. The most apparent overall differences in inhibition patterns were presented between clades. The inhibitory activity was mainly mediated by secreted, proteinaceous, heat-stable compounds, likely indicating an involvement of bacteriocins. A custom-made database targeting 76 Bacillota bacteriocins was used to reveal bacteriocins in the genomes. Our systematic screening of the interactions between nosocomial and commensal Efm and Ela on a large scale suggests that, in a clinical setting, nosocomial strains not only have an advantage over commensal strains due to their possession of AMR genes, virulence factors, and resilience but also inhibit the growth of commensal strains.

Genomic insights into Enterococcus faecium isolates from marine bivalves highlight One Health concerns and healthcare linkages.

Enterococci, especially Enterococcus faecium, are one of today's leading causes of multidrug-resistant infections in hospital settings. The marine environment may harbour enterococci, but its role as an evolutionary niche and as a vector for the spread of enterococci is sparsely investigated. Hence, by applying enterococci in bivalves as a sentinel tool, this study aimed to describe the prevalence of enterocooci along the Norwegian coast and in addition the phylogeny of E. faecium in particular. Enterococci in batch samples of marine bivalves, harvested from 86 different locations, were quantitatively examined by a culture-dependent most probable number (MPN) method. Isolates were identified by MALDI-TOF-MS prior to antimicrobial susceptibility testing by broth microdilution. In-detail analyses of a representative selection of E. faecium isolates (n=148) were done by Illumina whole-genome sequencing, and assembled genomes were compared to closed E. faecium genomes in the public databases and to genomes from commensal and clinical isolates from Norway. Diversity among E. faecium within the same batch sample of bivalves was also explored. Enterococci were detected in 287 of the 471 examined bivalve samples, but in low concentrations with a median value of <18 MPN /100 g. From positive samples, 479 isolates of enterococci were identified belonging to ten different species, where E. faecium (n=247), Enterococcus hirae (n=114) and Enterococcus faecalis (n=66) were most frequently found. Resistance towards one or more antimicrobial agents was observed in 197 isolates (41 %), none of the isolates showed acquired resistance to vancomycin or linezolid. Phylogenetic analyses revealed high diversity among the E. faecium isolates and showed that the marine niche is dominated by strains from the non-clinical setting belonging to clade A2 (n=85) and B (E. lactis) (n=60). Only three isolates belonged to the hospital-associated clade A1 (ST80 and ST117). Two of these clustered with one isolate from a hospitalized patient and one from a non-hospitalized person. This study demonstrated a high prevalence, but low concentrations of enterococci in bivalves, and low levels of antimicrobial resistance. E. faecium genomes showed high population diversity and that very few E. faecium isolates in bivalves may have arisen from the human healthcare system. A systematic surveillance of target micro-organisms applying methods examining multiple isolates from the same bivalve sample provides important data to assess the enterococcal phylogeny, antimicrobial resistance and the level of faecal pollution in the marine environment.

The population structure of vancomycin-resistant and -susceptible Enterococcus faecium in a low-prevalence antimicrobial resistance setting is highly influenced by circulating global hospital-associated clones.

Between 2010 and 2015 the incidence of vancomycin-resistant Enterococcus faecium (VREfm) in Norway increased dramatically. Hence, we selected (1) a random subset of vancomycin-resistant enterococci (VRE) from the Norwegian Surveillance System for Communicable Diseases (2010-15; n=239) and (2) Norwegian vancomycin-susceptible E. faecium (VSEfm) bacteraemia isolates from the national surveillance system for antimicrobial resistance in microbes (2008 and 2014; n=261) for further analysis. Whole-genome sequences were collected for population structure, van gene cluster, mobile genetic element and virulome analysis, as well as antimicrobial susceptibility testing. Comparative genomic and phylogeographical analyses were performed with complete genomes of global E. faecium strains from the National Center for Biotechnology Information (NCBI) (1946-2022; n=272). All Norwegian VREfm and most of the VSEfm clustered with global hospital-associated sequence types (STs) in the phylogenetic subclade A1. The vanB2 subtype carried by chromosomal Tn1549 integrative conjugative elements was the dominant van type. The major Norwegian VREfm cluster types (CTs) were in accordance with concurrent European CTs. The dominant vanB-type VREfm CTs, ST192-CT3/26 and ST117-CT24, were mostly linked to a single hospital in Norway where the clones spread after independent chromosomal acquisition of Tn1549. The less prevalent vanA VRE were associated with more diverse CTs and vanA carrying Inc18 or RepA_N plasmids with toxin-antitoxin systems. Only 5 % of the Norwegian VRE were Enterococcus faecalis, all of which contained vanB. The Norwegian VREfm and VSEfm isolates harboured CT-specific virulence factor (VF) profiles supporting biofilm formation and colonization. The dominant VREfm CTs in general hosted more virulence determinants than VSEfm. The phylogenetic clade B VSEfm isolates (n=21), recently classified as Enterococcus lactis, harboured fewer VFs than E. faecium in general, and particularly subclade A1 isolates. In conclusion, the population structure of Norwegian E. faecium isolates mirrors the globally prevalent clones and particularly concurrent European VREfm/VSEfm CTs. Novel chromosomal acquisition of vanB2 on Tn1549 from the gut microbiota, however, formed a single major hospital VREfm outbreak. Dominant VREfm CTs contained more VFs than VSEfm.

Reversible vancomycin susceptibility within emerging ST1421 Enterococcus faecium strains is associated with rearranged vanA-gene clusters and increased vanA plasmid copy number.

Vancomycin variable enterococci (VVE) are van-positive enterococci with a vancomycin-susceptible phenotype (VVE-S) that can convert to a resistant phenotype (VVE-R) and be selected for during vancomycin exposure. VVE-R outbreaks have been reported in Canada and Scandinavian countries. The aim of this study was to examine the presence of VVE in whole genome sequenced (WGS) Australian bacteremia Enterococcus faecium (Efm) isolates collected through the Australian Group on Antimicrobial resistance (AGAR) network. Eight potential VVEAus isolates, all identified as Efm ST1421, were selected based on the presence of vanA and a vancomycin-susceptible phenotype. During vancomycin selection, two potential VVE-S harboring intact vanHAX genes, but lacking the prototypic vanRS and vanZ genes, reverted to a resistant phenotype (VVEAus-R). Spontaneous VVEAus-R reversion occurred at a frequency of 4-6 × 10-8 resistant colonies per parent cell in vitro after 48 h and led to high-level vancomycin and teicoplanin resistance. The S to R reversion was associated with a 44-bp deletion in the vanHAX promoter region and an increased vanA plasmid copy number. The deletion in the vanHAX promoter region enables an alternative constitutive promoter for the expression of vanHAX. Acquisition of vancomycin resistance was associated with a low fitness cost compared with the corresponding VVEAus-S isolate. The relative proportion of VVEAus-R vs. VVEAus-S decreased over time in serial passages without vancomycin selection. Efm ST1421 is one of the predominant VanA-Efm multilocus sequence types found across most regions of Australia, and has also been associated with a major prolonged VVE outbreak in Danish hospitals.

Molecular Epidemiological Characterisation of ESBL- and Plasmid-Mediated AmpC-Producing Escherichia coli and Klebsiella pneumoniae at Kamuzu Central Hospital, Lilongwe, Malawi.

The global rise in infections caused by multidrug resistant (MDR) Enterobacterales poses a public health problem. We have performed a molecular epidemiological characterisation of representative plasmid-mediated AmpC (pAmpC) and ESBL-positive clinical isolates of Escherichia coli (n = 38) and Klebsiella pneumoniae (n = 17) from a tertiary hospital in Malawi collected in 2017. BlaCTX-M-15 was the most prevalent ESBL-determinant in E. coli (n = 30/38) and K. pneumoniae (n = 17/17), whereas blaCMY-2 was detected in nearly all AmpC-phenotype E. coli (n = 15/17). Whole genome sequencing revealed dominant globally disseminated E. coli sequence types (STs); ST410 (n = 16), ST131 (n = 7), and ST617 (n = 6). The ST distribution in K. pneumoniae was more diverse but included ST101 (n = 2), ST14 (n = 2), and ST340 (n = 2), all considered high-risk MDR clones. The isolates expressed an MDR profile, including resistance against commonly used antibiotics, such as fluoroquinolones, aminoglycosides, and/or trimethoprim-sulfamethoxazole, and harboured corresponding resistance determinants. Clonal analyses of the major STs of E. coli revealed closely related genetic clusters within ST410, ST131, and ST617 supporting within-hospital transmission between patients and/or via a common reservoir. The overall findings add to the limited knowledge on the molecular epidemiology of MDR E. coli and K. pneumoniae in Malawi and may help health policy makers to identify areas to target when addressing this major threat of antibiotic resistance.

Comparative Genomics Reveals Factors Associated with Phenotypic Expression of Wolbachia.

Wolbachia is a widespread, vertically transmitted bacterial endosymbiont known for manipulating arthropod reproduction. Its most common form of reproductive manipulation is cytoplasmic incompatibility (CI), observed when a modification in the male sperm leads to embryonic lethality unless a compatible rescue factor is present in the female egg. CI attracts scientific attention due to its implications for host speciation and in the use of Wolbachia for controlling vector-borne diseases. However, our understanding of CI is complicated by the complexity of the phenotype, whose expression depends on both symbiont and host factors. In the present study, we perform a comparative analysis of nine complete Wolbachia genomes with known CI properties in the same genetic host background, Drosophila simulans STC. We describe genetic differences between closely related strains and uncover evidence that phages and other mobile elements contribute to the rapid evolution of both genomes and phenotypes of Wolbachia. Additionally, we identify both known and novel genes associated with the modification and rescue functions of CI. We combine our observations with published phenotypic information and discuss how variability in cif genes, novel CI-associated genes, and Wolbachia titer might contribute to poorly understood aspects of CI such as strength and bidirectional incompatibility. We speculate that high titer CI strains could be better at invading new hosts already infected with a CI Wolbachia, due to a higher rescue potential, and suggest that titer might thus be a relevant parameter to consider for future strategies using CI Wolbachia in biological control.

Shotgun-metagenomics based prediction of antibiotic resistance and virulence determinants in Staphylococcus aureus from periprosthetic tissue on blood culture bottles.

Shotgun-metagenomics may give valuable clinical information beyond the detection of potential pathogen(s). Identification of antimicrobial resistance (AMR), virulence genes and typing directly from clinical samples has been limited due to challenges arising from incomplete genome coverage. We assessed the performance of shotgun-metagenomics on positive blood culture bottles (n = 19) with periprosthetic tissue for typing and prediction of AMR and virulence profiles in Staphylococcus aureus. We used different approaches to determine if sequence data from reads provides more information than from assembled contigs. Only 0.18% of total reads was derived from human DNA. Shotgun-metagenomics results and conventional method results were consistent in detecting S. aureus in all samples. AMR and known periprosthetic joint infection virulence genes were predicted from S. aureus. Mean coverage depth, when predicting AMR genes was 209 ×. Resistance phenotypes could be explained by genes predicted in the sample in most of the cases. The choice of bioinformatic data analysis approach clearly influenced the results, i.e. read-based analysis was more accurate for pathogen identification, while contigs seemed better for AMR profiling. Our study demonstrates high genome coverage and potential for typing and prediction of AMR and virulence profiles in S. aureus from shotgun-metagenomics data.

Novel genomic islands and a new vanD-subtype in the first sporadic VanD-type vancomycin resistant enterococci in Norway.

BACKGROUND: Vancomycin-resistant enterococci (VRE) represent several types of transferable vancomycin resistance gene clusters. The vanD type, associated with moderate to high level vancomycin resistance, has only sporadically been described in clinical isolates. The aim of this study was to perform a genetic characterization of the first VanD-type VRE strains detected in Norway. METHODS: The VanD-type VRE-strains (n = 6) from two patient cases were examined by antimicrobial susceptibility testing and whole genome sequencing (WGS) to uncover Van-phenotype, strain phylogeny, the vanD gene clusters, and their genetic surroundings. The putative transferability of vanD was examined by circularization PCR and filter mating. RESULTS: The VanD-type Enterococcus faecium (n = 4) and Enterococcus casseliflavus (n = 2) strains recovered from two cases (A and B), expressed moderate to high level vancomycin resistance (MIC 64->256 mg/L) and various levels of teicoplanin susceptibility (MIC 2->256 mg/L). WGS analyses revealed phylogenetically different E. faecium strains (A1, A2, and A3 of case A and B1 from case B) as well as vanD gene clusters located on different novel genomic islands (GIs). The E. casseliflavus strains (B2 and B3 of case B) were not clonally related, but harbored nearly identical novel GIs. The vanD cluster of case B strains represents a novel vanD-subtype. All the vanD-GIs were integrated at the same chromosomal site and contained genes consistent with a Clostridiales origin. Circular forms of the vanD-GIs were detected in all strains except B1. Transfer of vanD to an E. faecium recipient was unsuccessful. CONCLUSIONS: We describe the first VanD-type E. casseliflavus strains, a novel vanD-subtype, and three novel vanD-GIs with a genetic content consistent with a Clostridiales order origin. Despite temporal occurrence, case A and B E. faecium strains were phylogenetically diverse and harbored different vanD subtypes and vanD-GIs.

Role of Horizontal Gene Transfer in the Development of Multidrug Resistance in Haemophilus influenzae.

Haemophilus influenzae colonizes the respiratory tract in humans and causes both invasive and noninvasive infections. Resistance to extended-spectrum cephalosporins in H. influenzae is rare in Europe. In this study, we defined acquired resistance gene loci and ftsI mutations in multidrug-resistant (MDR) and/or PBP3-mediated beta-lactam-resistant (rPBP3) H. influenzae strains, intending to understand the mode of spread of antibiotic resistance determinants in this species. Horizontal transfer of mobile genetic elements and transformation with resistance-conferring ftsI alleles were contributory. We found one small plasmid and three novel integrative conjugative elements (ICEs) which carry different combinations of resistance genes. Demonstration of transfer and/or ICE circular forms showed that the ICEs are functional. Two extensively MDR genetically unrelated H. influenzae strains (F and G) from the same geographical region shared an identical novel MDR ICE (Tn6686) harboring bla TEM-1, catA2-like, and tet(B). The first Nordic case of MDR H. influenzae septicemia, strain 0, originating from the same geographical area as these strains, had a similar resistance pattern but contained another ICE [Tn6687 with bla TEM-1, catP and tet(B)] with an overall structure quite similar to that of Tn6686. Comparison of the complete ftsI genes among rPBP3 strains revealed that the entire gene or certain regions of it are identical in genetically unrelated strains, indicating horizontal gene transfer. Our findings illustrate that H. influenzae is capable of acquiring resistance against a wide range of commonly used antibiotics through horizontal gene transfer, in terms of conjugative transfer of ICEs and transformation of chromosomal genes.IMPORTANCE Haemophilus influenzae colonizes the respiratory tract in humans and causes both invasive and noninvasive infections. As a threat to treatment, resistance against critically important antibiotics is on the rise in H. influenzae Identifying mechanisms for horizontal acquisition of resistance genes is important to understand how multidrug resistance develops. The present study explores the antimicrobial resistance genes and their context in beta-lactam-resistant H. influenzae with coresistance to up to four non-beta-lactam groups. The results reveal that this organism is capable of acquiring resistance to a wide range of commonly used antibiotics through conjugative transfer of mobile genetic elements and transformation of chromosomal genes, resulting in mosaic genes with a broader resistance spectrum. Strains with chromosomally mediated resistance to extended-spectrum cephalosporins, co-trimoxazole, and quinolones combined with mobile genetic elements carrying genes mediating resistance to ampicillin, tetracyclines, and chloramphenicol have been reported, and further dissemination of such strains represents a particular concern.

Horizontal Plasmid Transfer among Klebsiella pneumoniae Isolates Is the Key Factor for Dissemination of Extended-Spectrum ß-Lactamases among Children in Tanzania.

Increased knowledge about the role of horizontal gene transfer is key to improve our understanding of the spread of antimicrobial resistance (AMR) in human populations. We therefore studied the dissemination of the blaCTX-M-15 extended-spectrum-ß-lactamase (ESBL) gene in Klebsiella pneumoniae isolates obtained from stool samples from hospitalized children and healthy controls below 2 years of age in Dar es Salaam, Tanzania, from August 2010 to July 2011. We performed Illumina whole-genome sequencing (WGS) to characterize resistance genes, multilocus sequence type (MLST), plasmid incompatibility group (Inc), and plasmid MLST of 128 isolates of K. pneumoniae with blaCTX-M-15 recovered from both healthy and hospitalized children. We assessed the phylogenetic relationship using single nucleotide polymorphism (SNP)-based analysis and resolved the sequences of five reference plasmids by Oxford Nanopore technology to investigate plasmid dissemination. The WGS analyses revealed the presence of a blaCTX-M-15-positive IncFIIK5/IncR plasmid with a highly conserved backbone in 70% (90/128) of the isolates. This plasmid, harboring genes encoding resistance to most ß-lactams, aminoglycosides, trimethoprim-sulfamethoxazole, and chloramphenicol, was present in phylogenetically very diverse K. pneumoniae strains (48 different MLSTs) carried by both hospitalized and healthy children. Our data strongly suggest widespread horizontal transfer of this ESBL-carrying plasmid both in hospitals and in the general population.IMPORTANCE Horizontal spread of plasmids carrying multiple resistance genes is considered an important mechanism behind the global health problem caused by multidrug-resistant bacteria. Nevertheless, knowledge about spread of plasmids in a community is limited. Our detailed molecular analyses of K. pneumoniae isolated from hospitalized and healthy children in Tanzania disclosed an epidemic spread of a resistance plasmid. In this study population, we revealed horizontal plasmid transfer among K. pneumoniae as the key factor for dissemination of ESBLs. Traditional outbreak investigation and surveillance focus on the spread of bacterial clones, and short-read sequencing can result in erroneous plasmid composition. Our approach using long-read sequencing reveals horizontal gene transfer of antimicrobial resistance, and therefore has a potential impact on outbreak investigations and approaches to limit spread of AMR.

Genomic characterization of multidrug-resistant ESBL-producing Klebsiella pneumoniae isolated from a Ghanaian teaching hospital.

OBJECTIVES: This study delineated the clonal lineages, antibiotic resistome and plasmid replicon types in multidrug-resistant K. pneumoniae isolates from a teaching hospital in Ghana. METHODS: Identification and antibiotic susceptibility testing were done using the MALDI-TOF MS and Vitek-2 automated system. Genomic DNA extraction was carried out using the NucliSens easyMAG® (BioMérieux) kits and the DNA was subjected to whole genome sequencing (WGS) using the Illumina MiSeq platform. RESULTS: Of the 200 isolates obtained, 37 were identified as K. pneumoniae of which 9 were resistant to all second and third-generation cephalosporins. These 9 isolates selected for further genomic analysis were characterized by the presence of 8 diverse sequence types (STs), capsular polysaccharide serotypes (K types and wzi allelic types) and multiple genes encoding resistance to ß-lactams (blaCTX-M-15, blaSHV-11,blaTEM-1B,blaOXA-1), aminoglycosides (aac(3)-IIa, strB, strA, aadA16), fluoroquinolones/quinolones (qnrB66, oqxA, oqxB) and other antibiotic classes. Resistance genes were associated with plasmids, predominantly IncFIB(K) and ColRNAI. Multiple and diverse mutations in quinolone resistance-determining regions of gyrA (S83Y, D87A) and parC (S80I, N304S) in isolates resistant to ciprofloxacin (MIC ≥ 4 mg/mL) were found. Global phylogenomic analysis affirmed the diverse clonal clustering and origin of these isolates. CONCLUSIONS: The varied clonal clusters and resistome identified in the multidrug-resistant K. pneumoniae isolates is a major threat to the management of infections in Ghana. The molecular characterization of antibiotic resistance is thus imperative to inform strategies for containment.

Enterococcus faecium TIR-Domain Genes Are Part of a Gene Cluster Which Promotes Bacterial Survival in Blood.

Enterococcus faecium has undergone a transition to a multidrug-resistant nosocomial pathogen. The population structure of E. faecium is characterized by a sharp distinction of clades, where the hospital-adapted lineage is primarily responsible for bacteremia. So far, factors that were identified in hospital-adapted strains and that promoted pathogenesis of nosocomial E. faecium mainly play a role in adherence and biofilm production, while less is known about factors contributing to survival in blood. This study identified a gene cluster, which includes genes encoding bacterial Toll/interleukin-1 receptor- (TIR-) domain-containing proteins (TirEs). The cluster was found to be unique to nosocomial strains and to be located on a putative mobile genetic element of phage origin. The three genes within the cluster appeared to be expressed as an operon. Expression was detected in bacterial culture media and in the presence of human blood. TirEs are released into the bacterial supernatant, and TirE2 is associated with membrane vesicles. Furthermore, the tirE-gene cluster promotes bacterial proliferation in human blood, indicating that TirE may contribute to the pathogenesis of bacteremia.