Development and validation of a quick, automated, and reproducible ATR FT-IR spectroscopy machine-learning model for Klebsiella pneumoniae typing.

The reliability of Fourier-transform infrared (FT-IR) spectroscopy for Klebsiella pneumoniae typing and outbreak control has been previously assessed, but issues remain in standardization and reproducibility. We developed and validated a reproducible FT-IR with attenuated total reflectance (ATR) workflow for the identification of K. pneumoniae lineages. We used 293 isolates representing multidrug-resistant K. pneumoniae lineages causing outbreaks worldwide (2002-2021) to train a random forest classification (RF) model based on capsular (KL)-type discrimination. This model was validated with 280 contemporaneous isolates (2021-2022), using wzi sequencing and whole-genome sequencing as references. Repeatability and reproducibility were tested in different culture media and instruments throughout time. Our RF model allowed the classification of 33 capsular (KL)-types and up to 36 clinically relevant K. pneumoniae lineages based on the discrimination of specific KL- and O-type combinations. We obtained high rates of accuracy (89%), sensitivity (88%), and specificity (92%), including from cultures obtained directly from the clinical sample, allowing to obtain typing information the same day bacteria are identified. The workflow was reproducible in different instruments throughout time (>98% correct predictions). Direct colony application, spectral acquisition, and automated KL prediction through Clover MS Data analysis software allow a short time-to-result (5 min/isolate). We demonstrated that FT-IR ATR spectroscopy provides meaningful, reproducible, and accurate information at a very early stage (as soon as bacterial identification) to support infection control and public health surveillance. The high robustness together with automated and flexible workflows for data analysis provide opportunities to consolidate real-time applications at a global level. IMPORTANCE We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance.

Global spread of the linezolid-resistant Enterococcus faecalis ST476 clonal lineage carrying optrA.

OBJECTIVES: To investigate the global distribution of an optrA-harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage. METHODS: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed. RESULTS: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage. CONCLUSIONS: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.

Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020.

BackgroundThe pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens.AimWe aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin.MethodsFifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics.ResultsOnly RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella. One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance (bla TEM + strA-strB + sul2 + tet(B)) and metal tolerance profiles (pco + sil + ars) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide.ConclusionSome RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.

Linezolid- and Multidrug-Resistant Enterococci in Raw Commercial Dog Food, Europe, 2019-2020.

We describe enterococci in raw-frozen dog food commercialized in Europe as a source of genes encoding resistance to the antibiotic drug linezolid and of strains and plasmids enriched in antibiotic-resistance and virulence genes in hospitalized patients. Whole-genome sequencing was fundamental to linking isolates from dog food to human cases across Europe.

Fitness cost of vancomycin-resistant Enterococcus faecium plasmids associated with hospital infection outbreaks.

BACKGROUND: Vancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. This study's aim was to analyse the effects of acquiring Tn1546-carrying plasmids with proven epidemicity in different bacterial host backgrounds. METHODS: Widespread Tn1546-carrying plasmids of different families RepA_N (n = 5), Inc18 (n = 4) and/or pHTß (n = 1), and prototype plasmids RepA_N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in the presence/absence of vancomycin. Plasmid stability was analysed (300 generations). WGS (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n = 49) was performed. SNP calling (Breseq software) of non-evolved strains was used for comparison. RESULTS: All plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-carrying plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2% to 18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9%-49%). Stability of Tn1546-carrying plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. SNPs and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. CONCLUSIONS: The stability of E. faecium Tn1546-carrying plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains.

From farm to fork: identical clones and Tn6674-like elements in linezolid-resistant Enterococcus faecalis from food-producing animals and retail meat.

OBJECTIVES: Increasing numbers of linezolid-resistant Enterococcus carrying optrA are being reported across different niches worldwide. We aimed to characterize the first optrA-carrying Enterococcus faecalis obtained from food-producing animals and retail meat samples in Tunisia. METHODS: Seven optrA-carrying E. faecalis obtained from chicken faeces (n=3, August 2017) and retail chicken meat (n=4, August 2017) in Tunisia were analysed. Antimicrobial susceptibility was determined by disc diffusion, broth microdilution and Etest against 13 antibiotics, linezolid and tedizolid, respectively (EUCAST/CLSI). optrA stability (∼600 bacterial generations), transfer (filter mating) and location (S1-PFGE/hybridization) were characterized. WGS (Illumina-HiSeq) was done for four representatives that were analysed through in silico and genomic mapping tools. RESULTS: Four MDR clones carrying different virulence genes were identified in chicken faeces (ST476) and retail meat (the same ST476 clone plus ST21 and ST859) samples. MICs of linezolid and tedizolid were stably maintained at 8 and 1-2 mg/L, respectively. optrA was located in the same transferable chromosomal Tn6674-like element in ST476 and ST21 clones, similar to isolates from pigs in Malaysia and humans in China. ST859 carried a non-conjugative plasmid of ∼40 kb with an impB-fexA-optrA segment, similar to plasmids from pigs and humans in China. CONCLUSIONS: The same chromosomal and transferable Tn6674-like element was identified in different E. faecalis clones from humans and animals. The finding of retail meat contaminated with the same linezolid-resistant E. faecalis strain obtained from a food-producing animal highlights the potential role of the food chain in the worrisome dissemination of optrA that can be stably maintained without selective pressure over generations.

Linezolid-resistant (Tn6246::fexB-poxtA) Enterococcus faecium strains colonizing humans and bovines on different continents: similarity without epidemiological link.

OBJECTIVES: poxtA is the most recently described gene conferring acquired resistance to linezolid, a relevant antibiotic for treating enterococcal infections. We retrospectively screened for poxtA in diverse enterococci and aimed to characterize its genetic/genomic contexts. METHODS: poxtA was screened by PCR in 812 enterococci from 458 samples (hospitals/healthy humans/wastewater/animals/retail food) obtained in Portugal/Angola/Tunisia (1996-2019). Antimicrobial susceptibility testing was performed for 13 antibiotics (EUCAST/CLSI). poxtA stability (∼500 generations), transfer (filter mating), clonality (SmaI-PFGE) and location (S1-PFGE/hybridization) were tested. WGS (Illumina-HiSeq) was performed for clonal representatives. RESULTS: poxtA was detected in Enterococcus faecium from six samples (1.3%): a healthy human (rectal swab) in Porto, Portugal (ST32/2001); four farm cows (milk) in Mateur, Tunisia (ST1058/2015); and a hospitalized patient (faeces) in Matosinhos, Portugal (ST1058/2015). All expressed resistance to linezolid (MIC = 8 mg/L), chloramphenicol, tetracycline and erythromycin, with variable resistance to ciprofloxacin and streptomycin. ST1058-poxtA-carrying isolates from Tunisia and Portugal differed by two SNPs and had similar plasmid content. poxtA, located in an IS1216-flanked Tn6246-like element, co-hybridized with fexB on one or more plasmids per isolate (one to three plasmids of 30-100 kb), was stable after several generations and transferred only from ST1058. ST1058 strains carried resistance/virulence genes (Efmqnr/acm) possibly induced under selective quinolone treatment. CONCLUSIONS: poxtA has been circulating in Portugal since at least 2001, corresponding to the oldest description worldwide to date. We also extend the reservoir of poxtA to bovines. The similar linezolid-resistant poxtA-carrying strains colonizing humans and livestock on different continents, and without a noticeable relationship, suggests a recent transmission event or convergent evolution of E. faecium populations in different hosts and geographic regions.

Phylogenomics of Enterococcus faecalis from wild birds: new insights into host-associated differences in core and accessory genomes of the species.

Wild birds have been suggested to be reservoirs of antimicrobial resistant and/or pathogenic Enterococcus faecalis (Efs) strains, but the scarcity of studies and available sequences limit our understanding of the population structure of the species in these hosts. Here, we analysed the clonal and plasmid diversity of 97 Efs isolates from wild migratory birds. We found a high diversity, with most sequence types (STs) being firstly described here, while others were found in other hosts including some predominant in poultry. We found that pheromone-responsive plasmids predominate in wild bird Efs while 35% of the isolates entirely lack plasmids. Then, to better understand the ecology of the species, the whole genome of fivestrains with known STs (ST82, ST170, ST16 and ST55) were sequenced and compared with all the Efs genomes available in public databases. Using several methods to analyse core and accessory genomes (AccNET, PLACNET, hierBAPS and PANINI), we detected differences in the accessory genome of some lineages (e.g. ST82) demonstrating specific associations with birds. Conversely, the genomes of other Efs lineages exhibited divergence in core and accessory genomes, reflecting different adaptive trajectories in various hosts. This pangenome divergence, horizontal gene transfer events and occasional epidemic peaks could explain the population structure of the species.

Dispersal of linezolid-resistant enterococci carrying poxtA or optrA in retail meat and food-producing animals from Tunisia.

OBJECTIVES: The epidemiology of Enterococcus resistant to priority antibiotics including linezolid has mainly been investigated in developed countries and especially in hospitals. We aimed to evaluate the contribution of different non-human reservoirs for the burden of MDR enterococci in Tunisia, where scarce data are available. METHODS: Samples (n = 287) were collected from urban wastewater (n = 57), retail meat (n = 29; poultry/bovine/ovine), milk (n = 89; bovine/ovine), farm animal faeces (n = 80; poultry/bovine/ovine) and pets (n = 32; rabbit/dogs/cats/birds) in different Tunisian regions (2014-17). They were plated onto Slanetz-Bartley agar after pre-enrichment without antibiotics. Standard methods were used for bacterial identification and characterization of antibiotic resistance and virulence genes (PCR), antibiotic susceptibility testing (disc diffusion/broth microdilution; EUCAST/CLSI) and clonality (SmaI-PFGE/MLST). RESULTS: All samples carried Enterococcus (n = 377 isolates) resistant to antibiotics considered to be critical or highly important by WHO. Even without antibiotic selection, 38% of Enterococcus faecalis (Efs) and 22% of Enterococcus faecium (Efm) were identified as MDR. Linezolid-resistant isolates (5%; MIC = 8 mg/L) comprised six poxtA-carrying Efm (cow milk), seven optrA-carrying Efs (chicken faeces/meat) and five Efm lacking cfr/optrA/poxtA (poultry/bovine/ovine/wastewater). Clinically relevant Efm clones (clade A1) were identified in animal/meat sources. Ampicillin resistance (1%) was confined to ST18/ST78-like MDR Efm clones from bovine meat/milk samples carrying relevant virulence markers (e.g. ptsD/IS16). CONCLUSIONS: This study provides evidence of the contribution of livestock and foodstuffs to the dispersal of acquired linezolid resistance genes including poxtA and optrA. We report the first poxtA-carrying Efm in Tunisia, and for the first time in bovine samples, stressing the urgent need for alternative measures to counteract the spread of linezolid-resistant enterococci globally.

2CS-CHXT Operon Signature of Chlorhexidine Tolerance among Enterococcus faecium Isolates.

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years that has recently acquired higher relevance in nosocomial infection control worldwide. Despite this, CHX tolerance among Enterococcus faecium bacteria, representing one of the leading agents causing nosocomial infections, has been poorly understood. This study provides new phenotypic and molecular data for better identification of CHX-tolerant E. faecium subpopulations in community and clinical contexts. The chlorhexidine MIC (MICCHX) distribution of 106 E. faecium isolates suggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of the 2CS-CHXT operon, identified here. It encodes glucose and amino acid-polyamine-organocation family transporters, besides the DNA-binding response regulator ChtR, with a P102H mutation previously described only in CHX-tolerant clade A1 E. faecium, and the ChtS sensor. 2CS-CHXT seems to be associated with three regulons modulating diverse bacterial biological functions. Combined data from normal MIC distribution and 2CS-CHXT operon characterization support a tentative epidemiological cutoff (ECOFF) of 8 mg/liter to CHX, which is useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds calls for the prudent use of CHX in multiple contexts.IMPORTANCE Chlorhexidine is one of the substances included in the World Health Organization's list of essential medicines, which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic in health care (skin, hands, mouthwashes, eye drops) since the 1950s, its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here, we provide a comprehensive study on chlorhexidine tolerance among strains of Enterococcus faecium, one of the leading nosocomial agents worldwide, and identify a novel 2CS-CHXT operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Our data allowed for the proposal of a tentative epidemiological cutoff of 8 mg/liter, which is useful to detect tolerant E. faecium populations in surveillance studies in community and clinical contexts. The prediction of 2CS-CHXT regulons will also facilitate the design of future experimental studies to better uncover chlorhexidine tolerance among E. faecium bacteria.

Fourier transform infrared spectroscopy: unlocking fundamentals and prospects for bacterial strain typing.

The need to identify highly related bacterial strains is ancient in clinical, industrial, or environmental microbiology. Strategies based on different phenotypic and genotypic principles have been used since the early 1930s with variable outcomes and performances, accompanying the evolution of bacterial features' knowledge as well as technologies, instruments, and data analysis tools. Today, more than ever, the implementation of bacterial typing methods that combine a high reliability and accuracy with a rapid, low-cost, and user-friendly performance is highly desirable, especially for clinical microbiology. FT-IR developments for bacterial discrimination at the infra-species level settled on the identification of bacterial groups previously defined by phenotypic or genotypic typing methods. Therefore, this review provides a brief historical overview of main bacterial strain typing methods, and a comprehensive analysis of the fundamentals and applications of Fourier transform infrared spectroscopy, a phenotypic-based method with potential for routine strain typing. The different studies on FT-IR-based strain typing of diverse Gram-negative and Gram-positive bacterial species are discussed in light of genotypic, phenotypic, and biochemical aspects, in order to definitively give this methodology credit to be widely accepted by microbiologists. Importantly, the discriminatory biochemical fingerprints observed on FT-IR spectra have been consistently correlated with sugar-based coating structures that besides reflecting strain variation are also of high relevance for the specificity in pathogen-host interactions. Thus, FT-IR-based bacterial typing might not only be useful for quick and reliable strain typing but also to help understanding the diversity, evolution, and host adaptation factors of key bacterial pathogens or subpopulations.

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

Distribution of putative virulence markers in Enterococcus faecium: towards a safety profile review.

Objectives: The criteria for identification of Enterococcus faecium (Efm) with the ability to cause human infections are currently being debated by the European Food Safety Authority (EFSA). Strains that have an MIC of ampicillin of ≤ 2 mg/L and lack IS16/esp/hyl genes should be regarded as safe for use as feed additives in animal nutrition, despite the lack of knowledge about putative virulence marker (PVM) distribution in community Efm. We analysed the distribution of major PVM and ampicillin phenotypes in large Efm collections to investigate further the safety of strains from a public health perspective. Methods: Thirty-three PVM were assessed by PCR/sequencing among clonally disparate Efm (n = 328; 1986-2015) from different origins. We analysed ampicillin susceptibility (Etest/broth microdilution) according to EUCAST guidelines, clonal relationship (MLST) and genomic location of PVM (S1-PFGE/hybridization). Results: Infection-derived Efm were more enriched in PVM and the increase in ampicillin MIC was positively correlated with an enrichment in different PVM. PVM coding for surface (esp/sgrA/ecbA/complete acm) and pili proteins, or others enhancing colonization (hyl/ptsD/orf1481) or plasticity (IS16), were strongly associated with clinical Efm (mostly clade A1), but also observed in clades A2/B at different rates. ptsD was a good marker of ampicillin-resistant Efm. ptsD, IS16, orf1481, sgrA and hospital variants of complete pili gene clusters are proposed as markers to assess the safety of Efm strains. Conclusions: Our study expands on the distribution of PVM in diverse Efm lineages and demonstrates the enrichment in infection-derived strains of PVM not previously included in EFSA's list of Efm safety criteria. The evidence of relevant Efm infection markers can impact the risk assessment of Efm strains in different public health contexts.

Detection of optrA in the African continent (Tunisia) within a mosaic Enterococcus faecalis plasmid from urban wastewaters.

OBJECTIVES: Oxazolidinone resistance is a serious limitation in the treatment of MDR Enterococcus infections. Plasmid-mediated oxazolidinone resistance has been strongly linked to animals where the use of phenicols might co-select resistance to both antibiotic families. Our goal was to assess the diversity of genes conferring phenicol/oxazolidinone resistance among diverse enterococci and to characterize the optrA genetic environment. METHODS: Chloramphenicol-resistant isolates (>16 mg/L, n = 245) from different sources (hospitals/healthy humans/wastewaters/animals) in Portugal, Angola and Tunisia (1996-2016) were selected. Phenicol (eight cat variants, fexA, fexB) or phenicol + oxazolidinone [cfr, cfr(B), optrA] resistance genes were searched for by PCR. Susceptibility (disc diffusion/microdilution), filter mating, stability of antibiotic resistance (500 bacterial generations), plasmid typing (S1-PFGE/hybridization), MLST and WGS (Illumina-HiSeq) were performed for optrA-positive isolates. RESULTS: Resistance to phenicols (n = 181, 74%) and phenicols + oxazolidinones (n = 2, 1%) was associated with the presence of cat(A-8) (40%, predominant in hospitals and swine), cat(A-7) (29%, predominant in poultry and healthy humans), cat(A-9) (2%), fexB (2%) and fexA + optrA (1%). fexA and optrA genes were co-located in a transferable plasmid (pAF379, 72 918 bp) of two ST86 MDR Tunisian Enterococcus faecalis (wastewaters) carrying several putative virulence genes. MICs of chloramphenicol, linezolid and tedizolid were stably maintained at 64, 4 and 1 mg/L, respectively. The chimeric pAF379 comprised relics of genetic elements from different Gram-positive bacteria and origins (human/porcine). CONCLUSIONS: To the best of our knowledge, we report the first detection of optrA in an African country (Tunisia) within a transferable mosaic plasmid of different origins. Its identification in isolates from environmental sources is worrisome and alerts for the need of a concerted global surveillance on the occurrence and spread of optrA.

Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012).

OBJECTIVES: Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units. METHODS: From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed. RESULTS: VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids). CONCLUSIONS: Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids.

Co-transfer of resistance to high concentrations of copper and first-line antibiotics among Enterococcus from different origins (humans, animals, the environment and foods) and clonal lineages.

OBJECTIVES: We studied the occurrence of diverse copper (Cu) tolerance genes from Gram-positive bacteria and their co-transfer with antibiotic resistance genes among Enterococcus from diverse sources. METHODS: Enterococcus (n = 922) of several species and from human, animal, environment and food samples were included. Antimicrobial and CuSO4 susceptibility and conjugation assays were performed by standard procedures, bacterial screening of Cu and antibiotic resistance genes by PCR, and clonality by PFGE/multilocus sequence typing. RESULTS: tcrB and cueO genes occurred in 15% (n = 137/922) and 14% (n = 128/922) of isolates, respectively, with the highest occurrence in piggeries (P < 0.05). They were more frequent among Enterococcus faecium (tcrB: 23% versus 8% in Enterococcus faecalis and 12% in other species; cueO: 25% versus 5% and 9%, respectively; P < 0.05). A correlation between phenotypic and genotypic assays was observed for most E. faecium (CuSO4 MIC50 = 24 mM in tcrB/cueO(+) versus CuSO4 MIC50 = 12 mM in tcrB/cueO(-)), but not for other species. Co-transfer of Cu tolerance (associated with tcrB, cueO or an unknown mechanism) with erythromycin, tetracycline, vancomycin, aminoglycosides or ampicillin resistance was demonstrated. A variety of PFGE types was detected among isolates carrying Cu tolerance mechanisms, some identified in sequence types (STs) often linked to human infections (E. faecium from ST18 and ST78 clonal lineages and E. faecalis clonal complex 2). CONCLUSIONS: Cu tolerance might contribute to the selection/maintenance of multidrug-resistant Enterococcus (including resistance to first-line antibiotics used to treat enterococcal infections) due to the use of Cu compounds (e.g. antiseptics/animal feed supplements). The distribution of the multicopper oxidase cueO and the co-transfer of ampicillin resistance along with Cu tolerance genes are described for the first time.

VirulenceFinder for Enterococcus faecium and Enterococcus lactis: an enhanced database for detection of putative virulence markers by using whole-genome sequencing data.

Enterococcus faecium (Efm) is a leading cause of hospital-associated (HA) infections, often enriched in putative virulence markers (PVMs). Recently, the Efm clade B was assigned as Enterococcus lactis (Elts), which usually lack HA-Efm infection markers. Available databases for extracting PVM are incomplete and/or present an intermix of genes from Efm and Enterococcus faecalis, with distinct virulence profiles. In this study, we constructed a new database containing 27 PVMs [acm, scm, sgrA, ecbA, fnm, sagA, hylEfm, ptsD, orf1481, fms15, fms21-fms20 (pili gene cluster 1, PGC-1), fms14-fms17-fms13 (PGC-2), empA-empB-empC (PGC-3), fms11-fms19-fms16 (PGC-4), ccpA, bepA, gls20-glsB1, and gls33-glsB] from nine reference genomes (seven Efm + two Elts). The database was validated against these reference genomes and further evaluated using a collection of well-characterized Efm (n = 43) and Elts (n = 7) control strains, by assessing PVM presence/absence and its variants together with a genomic phylogeny constructed as single-nucleotide polymorphisms. We found a high concordance between the phylogeny and in silico findings of the PVM, with Elts clustering separately and mostly carrying Elts-specific PVM gene variants. Based on our validation results, we recommend using the database with raw reads instead of assemblies to avoid missing gene variants. This newly constructed database of 27 PVMs will enable a more comprehensive characterization of Efm and Elts based on WGS data. The developed database exhibits scalability and boasts a range of applications in public health, including diagnostics, outbreak investigations, and epidemiological studies. It can be further used in risk assessment for distinguishing between safe and unsafe enterococci.IMPORTANCEThe newly constructed database, consisting of 27 putative virulence markers, is highly scalable and serves as a valuable resource for the comprehensive characterization of these closely related species using WGS data. It holds significant potential for various public health applications, including hospital outbreak investigations, surveillance, and risk assessment for probiotics and feed additives.

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.

Spread of multidrug-resistant Enterococcus to animals and humans: an underestimated role for the pig farm environment.

OBJECTIVES: The aim of this study was to discover the potential role of the pig farm environment in the spread of multidrug-resistant (MDR) Enterococcus strains, including high-risk clones, to animals and humans. METHODS: Enterococcus isolates were recovered from a variety of samples (n = 82; swine, feed/medicines/antiseptics and pig farm facilities) from six Portuguese farms, most using antibiotics. Antimicrobial susceptibility/conjugation assays were performed by standard procedures, bacterial identification/screening of antibiotic resistance genes were performed by PCR and clonality was determined using PFGE/multilocus sequence typing. RESULTS: Enterococcus isolates resistant to antibiotics (n= 473) were recovered from samples of different origin (swine, feed/antiseptics, animal residues and pig farm facilities), but only the clinically relevant species Enterococcus faecium (n = 171) and Enterococcus faecalis (n = 78) were included for further comprehensive molecular analysis. Isolates resistant to vancomycin, ampicillin, tetracyclines, erythromycin and aminoglycosides were better recovered in Slanetz-Bartley medium with these antibiotics present than in media not supplemented with antibiotics (P < 0.05). E. faecium was more frequently resistant to ampicillin, ciprofloxacin or nitrofurantoin and E. faecalis to tetracyclines, chloramphenicol or aminoglycosides (P < 0.05). Glycopeptide and erythromycin resistance rates were similar in both species. The transfer of resistance to several antibiotics, including vancomycin and ampicillin, was demonstrated. Clones associated with human infections were detected in different samples from the same farm [E. faecium from sequence type (ST) 78 lineage and E. faecalis ST16; manure, waste lagoons, faeces and drinking water] and in geographically distant farms [E. faecium clonal complex (CC) 5; E. faecalis CC21 and ST16]. CONCLUSIONS: The pig farm environment has an underestimated potential role in the transmission of MDR Enterococcus to animals and, possibly, to humans. The continuous contact of swine with MDR Enterococcus by different routes (e.g. feed, dust, air and rooms) might decrease the impact of restrictive antibiotic use policies and reinforces the need for different and preliminary interventions at the husbandry management level.