OXA-48-Producing Uropathogenic Escherichia coli Sequence Type 127, the Netherlands, 2015-2022.

During 2015-2022, a genetic cluster of OXA-48-producing uropathogenic Escherichia coli sequence type 127 spread throughout the Netherlands. The 20 isolates we investigated originated mainly from urine, belonged to Clermont phylotype B2, and carried 18 genes encoding putative uropathogenicity factors. The isolates were susceptible to first-choice antimicrobial drugs for urinary tract infections.

Emergence of KPC-3- and OXA-181-producing ST13 and ST17 Klebsiella pneumoniae in Portugal: genomic insights on national and international dissemination.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains are of particular concern, especially strains with mobilizable carbapenemase genes such as blaKPC, blaNDM or blaOXA-48, given that carbapenems are usually the last line drugs in the ß-lactam class and, resistance to this sub-class is associated with increased mortality and frequently co-occurs with resistance to other antimicrobial classes. OBJECTIVES: To characterize the genomic diversity and international dissemination of CRKP strains from tertiary care hospitals in Lisbon, Portugal. METHODS: Twenty CRKP isolates obtained from different patients were subjected to WGS for species confirmation, typing, drug resistance gene detection and phylogenetic reconstruction. Two additional genomic datasets were included for comparative purposes: 26 isolates (ST13, ST17 and ST231) from our collection and 64 internationally available genomic assemblies (ST13). RESULTS: By imposing a 21 SNP cut-off on pairwise comparisons we identified two genomic clusters (GCs): ST13/GC1 (n = 11), all bearing blaKPC-3, and ST17/GC2 (n = 4) harbouring blaOXA-181 and blaCTX-M-15 genes. The inclusion of the additional datasets allowed the expansion of GC1/ST13/KPC-3 to 23 isolates, all exclusively from Portugal, France and the Netherlands. The phylogenetic tree reinforced the importance of the GC1/KPC-3-producing clones along with their rapid emergence and expansion across these countries. The data obtained suggest that the ST13 branch emerged over a decade ago and only more recently did it underpin a stronger pulse of transmission in the studied population. CONCLUSIONS: This study identifies an emerging OXA-181/ST17-producing strain in Portugal and highlights the ongoing international dissemination of a KPC-3/ST13-producing clone from Portugal.

Brief report: community-acquired Friedlander's pneumonia and pulmonary metastatic Klebsiella pneumoniae infection caused by hypervirulent ST23 in the Netherlands.

Infections with hypervirulent Klebsiella pneumoniae (hvKp) commonly presents with primary liver infection, bacteremia, and metastatic abscesses. Here, we present 2 cases of severe community-acquired pulmonary infections by hvKp in patients in the Netherlands without recent travel history. Both bacterial isolates are closely related to an archetype ST23 hvKp reference isolate. Based on these findings, surveillance programs on hvKp may consider to include isolates from community-acquired pneumonia by K. pneumoniae.

Phage-mediated dispersal of biofilm and distribution of bacterial virulence genes is induced by quorum sensing.

The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 µM) of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5) showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.

Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis.

Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine-aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets.

Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili: Evidence for a Novel and Heterospecific Probiotic Mechanism.

UNLABELLED: Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections. IMPORTANCE: Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria.

The current epidemic of infections caused by antibiotic-resistant gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.

Semisynthetic Lipopeptides Derived from Nisin Display Antibacterial Activity and Lipid II Binding on Par with That of the Parent Compound.

The lipid II-binding N-terminus of nisin, comprising the so-called A/B ring system, was synthetically modified to provide antibacterially active and proteolytically stable derivatives. A variety of lipids were coupled to the C-terminus of the nisin A/B ring system to generate semisynthetic constructs that display potent inhibition of bacterial growth, with activities approaching that of nisin itself. Most notable was the activity observed against clinically relevant bacterial strains including MRSA and VRE. Experiments with membrane models indicate that these constructs operate via a lipid II-mediated mode of action without causing pore formation. A lipid II-dependent mechanism of action is further supported by antagonization assays wherein the addition of lipid II was found to effectively block the antibacterial activity of the nisin-derived lipopeptides.

YfbA, a Yersinia pestis regulator required for colonization and biofilm formation in the gut of cat fleas.

For transmission to new hosts, Yersinia pestis, the causative agent of plague, replicates as biofilm in the foregut of fleas that feed on plague-infected animals or humans. Y. pestis biofilm formation has been studied in the rat flea; however, little is known about the cat flea, a species that may bridge zoonotic and anthroponotic plague cycles. Here, we show that Y. pestis infects and replicates as a biofilm in the foregut of cat fleas in a manner requiring hmsFR, two determinants for extracellular biofilm matrix. Examining a library of transposon insertion mutants, we identified the LysR-type transcriptional regulator YfbA, which is essential for Y. pestis colonization and biofilm formation in cat fleas.

YajC, a predicted membrane protein, promotes Enterococcus faecium biofilm formation in vitro and in a rat endocarditis model.

Biofilm formation is a critical step in the pathogenesis of difficult-to-treat Gram-positive bacterial infections. We identified that YajC, a conserved membrane protein in bacteria, plays a role in biofilm formation of the clinically relevant Enterococcus faecium strain E1162. Deletion of yajC conferred significantly impaired biofilm formation in vitro and was attenuated in a rat endocarditis model. Mass spectrometry analysis of supernatants of washed ΔyajC cells revealed increased amounts in cytoplasmic and cell-surface-located proteins, including biofilm-associated proteins, suggesting that proteins on the surface of the yajC mutant are only loosely attached. In Streptococcus mutans YajC has been identified in complex with proteins of two cotranslational membrane protein-insertion pathways; the signal recognition particle (SRP)-SecYEG-YajC-YidC1 and the SRP-YajC-YidC2 pathway, but its function is unknown. In S. mutans mutation of yidC1 and yidC2 resulted in impaired protein insertion in the cell membrane and secretion in the supernatant. The E. faecium genome contains all homologous genes encoding for the cotranslational membrane protein-insertion pathways. By combining the studies in S. mutans and E. faecium, we propose that YajC is involved in the stabilization of the SRP-SecYEG-YajC-YidC1 and SRP-YajC-Yid2 pathway or plays a role in retaining proteins for proper docking to the YidC insertases for translocation in and over the membrane.

Colonization of vancomycin-resistant Enterococcus faecium in human-derived colonic epithelium: unraveling the transcriptional dynamics of host-enterococcal interactions.

Enterococcus faecium is an opportunistic pathogen able to colonize the intestines of hospitalized patients. This initial colonization is an important step in the downstream pathogenesis, which includes outgrowth of the intestinal microbiota and potential infection of the host. The impact of intestinal overgrowth on host-enterococcal interactions is not well understood. We therefore applied a RNAseq approach in order to unravel the transcriptional dynamics of E. faecium upon co-culturing with human derived colonic epithelium. Co-cultures of colonic epithelium with a hospital-associated vancomycin resistant (vanA-type) E. faecium (VRE) showed that VRE resided on top of the colonic epithelium when analyzed by microscopy. RNAseq revealed that exposure to the colonic epithelium resulted in upregulation of 238 VRE genes compared to the control condition, including genes implicated in pili expression, conjugation (plasmid_2), genes related to sugar uptake, and biofilm formation (chromosome). In total, 260 were downregulated, including the vanA operon located on plasmid_3. Pathway analysis revealed an overall switch in metabolism to amino acid scavenging and reduction. In summary, our study demonstrates that co-culturing of VRE with human colonic epithelium promotes an elaborate gene response in VRE, enhancing our insight in host-E. faecium interactions, which might facilitate the design of novel anti-infectivity strategies.

Spread of carbapenemase-producing Morganella spp from 2013 to 2021: a comparative genomic study.

BACKGROUND: Morganella spp are opportunistic pathogens involved in various infections. Intrinsic resistance to multiple antibiotics (including colistin) combined with the emergence of carbapenemase producers reduces the number of active antimicrobials. The aim of this study was to characterise genetic features related to the spread of carbapenem-resistant Morganella spp. METHODS: This comparative genomic study included extensively drug-resistant Morganella spp isolates collected between Jan 1, 2013, and March 1, 2021, by the French National Reference Center (NRC; n=68) and European antimicrobial resistance reference centres in seven European countries (n=104), as well as one isolate from Canada, two reference strains from the Pasteur Institute collection (Paris, France), and two colistin-susceptible isolates from Bicêtre Hospital (Kremlin-Bicêtre, France). The isolates were characterised by whole-genome sequencing, antimicrobial susceptibility testing, and biochemical tests. Complete genomes from GenBank (n=103) were also included for genomic analysis, including phylogeny and determination of core genomes and resistomes. Genetic distance between different species or subspecies was performed using average nucleotide identity (ANI). Intrinsic resistance mechanisms to polymyxins were investigated by combining genetic analysis with mass spectrometry on lipid A. FINDINGS: Distance analysis by ANI of 275 isolates identified three groups: Morganella psychrotolerans, Morganella morganii subspecies sibonii, and M morganii subspecies morganii, and a core genome maximum likelihood phylogenetic tree showed that the M morganii isolates can be separated into four subpopulations. On the basis of these findings and of phenotypic divergences between isolates, we propose a modified taxonomy for the Morganella genus including four species, Morganella psychrotolerans, Morganella sibonii, Morganella morganii, and a new species represented by a unique environmental isolate. We propose that M morganii include two subspecies: M morganii subspecies morganii (the most prevalent) and M morganii subspecies intermedius. This modified taxonomy was supported by a difference in intrinsic resistance to tetracycline and conservation of metabolic pathways such as trehalose assimilation, both only present in M sibonii. Carbapenemase producers were mostly identified among five high-risk clones of M morganii subspecies morganii. The most prevalent carbapenemase corresponded to NDM-1, followed by KPC-2, and OXA-48. A cefepime-zidebactam combination was the most potent antimicrobial against the 172 extensively drug-resistant Morganella spp isolates in our collection from different European countries, which includes metallo-ß-lactamase producers. Lipid A analysis showed that the intrinsic resistance to colistin was associated with the presence of L-ARA4N on lipid A. INTERPRETATION: This global characterisation of, to our knowledge, the widest collection of extensively drug-resistant Morganella spp highlights the need to clarify the taxonomy and decipher intrinsic resistance mechanisms, and paves the way for further genomic comparisons. FUNDING: None.

Bacillus cereus G9241 S-layer assembly contributes to the pathogenesis of anthrax-like disease in mice.

Bacillus cereus G9241, the causative agent of anthrax-like disease, harbors virulence plasmids encoding anthrax toxins as well as hyaluronic acid (HA) and B. cereus exopolysaccharide (BPS) capsules. B. cereus G9241 also harbors S-layer genes, including homologs of Bacillus anthracis surface array protein (Sap), extractable antigen 1 (EA1), and the S-layer-associated proteins (BSLs). In B. anthracis, S-layer proteins and BSLs attach via their S-layer homology domains (SLH) to the secondary cell wall polysaccharide (SCWP) in a manner requiring csaB, a predicted ketalpyruvate transferase. Here we used a genetic approach to analyze B. cereus G9241 S-layer assembly and function. Variants lacking the csaB gene synthesized SCWP but failed to retain Sap, EA1, and BSLs in the bacterial envelope. The B. cereus G9241 csaB mutant assembled capsular polysaccharides but displayed an increase in chain length relative to the wild-type strain. This phenotype is likely due to its inability to deposit BslO murein hydrolase at divisional septa. During growth under capsule-inducing conditions, B. cereus G9241 assembled BSLs (BslA and BslO) and the Sap S-layer protein, but not EA1, in the envelope. Finally, csaB-mediated assembly of S-layer proteins and BSLs in B. cereus G9241 contributes to the pathogenesis of anthrax-like disease in mice.

Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus.

Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase-mediated cross-linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA - CNA(1), CNA(2), XNA and CNA(3) - each acquire intramolecular lysine-asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showed that mutants unable to form the intramolecular isopeptide bonds in the CNA(2) or CNA(3) domains retain the ability to form pilus bundles. A mutant lacking the CNA(1) isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X-ray crystallography revealed that the BcpA variant Asp(312) Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly-roll structure of XNA. The Asp(312) Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA(1) and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.

Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood.

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the N-terminal domain of serine glutamate repeat A (SgrA) protein from Enterococcus faecium.

Serine glutamate repeat A (SgrA) protein is an LPxTG surface adhesin of Enterococcus faecium and is the first bacterial nidogen-binding protein identified to date. It has been suggested that it binds to human nidogen, the extracellular matrix molecule of basal lamina, and plays a key role in the invasion and colonization of eukaryotic host cells. SgrA(28-288), having both a putative ligand-binding A domain and repetitive B domain, was expressed in Escherichia coli and purified using Ni-affinity and hydrophobic interaction chromatography. Further, the putative ligand-binding region, rSgrA(28-153), was subcloned, overexpressed and purified in both native and selenomethionine-derivative forms. The native rSgrA(28-153) protein crystallized in the monoclinic space group P2(1) and diffracted to 3.3 Å resolution using an in-house X-ray source, with unit-cell parameters a = 35.84, b = 56.35, c = 60.20 Å, ß = 106.5°.

Molecular characterization of MRSA collected during national surveillance between 2008 and 2019 in the Netherlands.

BACKGROUND: Although the Netherlands is a country with a low endemic level, methicillin-resistant Staphylococcus aureus (MRSA) poses a significant health care problem. Therefore, high coverage national MRSA surveillance has been in place since 1989. To monitor possible changes in the type-distribution and emergence of resistance and virulence, MRSA isolates are molecularly characterized. METHODS: All 43,321 isolates from 36,520 persons, collected 2008-2019, were typed by multiple-locus variable number tandem repeats analysis (MLVA) with simultaneous PCR detection of the mecA, mecC and lukF-PV genes, indicative for PVL. Next-generation sequencing data of 4991 isolates from 4798 persons were used for whole genome multi-locus sequence typing (wgMLST) and identification of resistance and virulence genes. RESULTS: We show temporal change in the molecular characteristics of the MRSA population with the proportion of PVL-positive isolates increasing from 15% in 2008-2010 to 25% in 2017-2019. In livestock-associated MRSA obtained from humans, PVL-positivity increases to 6% in 2017-2019 with isolates predominantly from regions with few pig farms. wgMLST reveals the presence of 35 genogroups with distinct resistance, virulence gene profiles and specimen origin. Typing shows prolonged persistent MRSA carriage with a mean carriage period of 407 days. There is a clear spatial and a weak temporal relationship between isolates that clustered in wgMLST, indicative for regional spread of MRSA strains. CONCLUSIONS: Using molecular characterization, this exceptionally large study shows genomic changes in the MRSA population at the national level. It reveals waxing and waning of types and genogroups and an increasing proportion of PVL-positive MRSA.

A group of bacteria that cause difficult-to-treat infections in humans is methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to monitor changes in the spread of MRSA, their disease causing potential and resistance to antibiotics used to treat MRSA infections. MRSA from patients and their contacts in the Netherlands were collected over a period of 12 years and characterized. This revealed new types of MRSA emerged and others disappeared. An increasing number of MRSA produces a protein called PVL toxin, enabling MRSA to cause more severe infections. Also, some people appear to carry MRSA without any disease for more than a year. These findings suggest an increasing disease potential of MRSA and possible unnoticed sources of infection. Consequently, it is important to maintain monitoring of these infections to minimize MRSA spread.

Markers of epidemiological success of methicillin-resistant Staphylococcus aureus isolates in European populations.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) infections impose a considerable burden on health systems, yet there is remarkable variation in the global incidence and epidemiology of MRSA. The MACOTRA consortium aimed to identify bacterial markers of epidemic success of MRSA isolates in Europe using a representative MRSA collection originating from France, the Netherlands and the United Kingdom. METHODS: Operational definitions of success were defined in consortium meetings to compose a balanced strain collection of successful and sporadic MRSA isolates. Isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing; genes were identified and phylogenetic trees constructed. Markers of epidemiological success were identified using genome-based time-scaled haplotypic density analysis and linear regression. Antimicrobial usage data from ESAC-Net was compared with national MRSA incidence data. RESULTS: Heterogeneity of MRSA isolate collections across countries hampered the use of a unified operational definition of success; therefore, country-specific approaches were used to establish the MACOTRA strain collection. Phenotypic antimicrobial resistance varied within related MRSA populations and across countries. In time-scaled haplotypic density analysis, fluoroquinolone, macrolide and mupirocin resistance were associated with MRSA success, whereas gentamicin, rifampicin and trimethoprim resistance were associated with sporadicity. Usage of antimicrobials across 29 European countries varied substantially, and ß-lactam, fluoroquinolone, macrolide and aminoglycoside use correlated with MRSA incidence. DISCUSSION: Our results are the strongest yet to associate MRSA antibiotic resistance profiles and antibiotic usage with the incidence of infection and successful clonal spread, which varied by country. Harmonized isolate collection, typing, resistance profiling and alignment with antimicrobial usage over time will aid comparisons and further support country-specific interventions to reduce MRSA burden.

Widespread Detection of Yersiniabactin Gene Cluster and Its Encoding Integrative Conjugative Elements (ICEKp) among Nonoutbreak OXA-48-Producing Klebsiella pneumoniae Clinical Isolates from Spain and the Netherlands.

In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages-ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27-which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages-ST11, ST101, and ST405-were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.