Biology subjects

Peixe, L.

Publications and source records attributed to Peixe, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Widespread occurrence of ampicillin-susceptible Enterococcus faecium and Enterococcus lactis clinical isolates with low MICs to cephalosporins from Spain and Portugal

Reduced cephalosporin resistance in Enterococcus faecium has traditionally been reported in laboratory mutants and, more recently, in a single clinical ampicillin-susceptible (AmpS) isolate. Herein, we investigated whether this phenotype is widespread by analysing 95 clinical enterococcal isolates (78 AmpS and 17 ampicillin resistant [AmpR]) collected from three hospitals in Spain and Portugal (2009-2025). Low ceftriaxone MICs ([≤]4 mg/L) were detected in 19/51 (37.3%) AmpS E. faecium and 7/27 (25.9%) E. lactis but in none of the AmpR isolates. Low ceftriaxone MICs were associated with older patient age in both species and with prior ampicillin therapy in E. faecium, but not with other clinical or epidemiological variables. Ceftaroline MICs were consistently low among AmpS isolates, while ceftriaxone and cefotaxime showed greater variability. Low-MIC isolates were distributed across multiple clonal lineages and hospitals and did not share a distinctive resistance or virulence gene profile. PBP5 phylogeny and variation at the psr-pbp5 region separated AmpS from AmpR E. faecium but did not explain variability in ceftriaxone MICs. Five AmpS isolates with reduced ceftriaxone MICs carried chromosomal deletions that included the psr-pbp5 region and genes with diverse cellular functions. Variation in other candidate resistance genes (pbpA, ponA, pbpF, croRS, stpA/stk and murAA) did not consistently explain the MIC differences. These results reveal unexpected heterogeneity in intrinsic cephalosporin resistance in clinical E. faecium and E. lactis and suggest that additional genetic or regulatory mechanisms underlie reduced susceptibility.

microbiology↗

bioRxiv2026

Multihospital expansion of vancomycin-resistant Enterococcus faecium ST117-CT7799 and transmission of linear plasmids co-carrying vanA and linezolid resistance genes, Comunitat Valenciana, Spain (2022-2024)

BackgroundVancomycin-resistant Enterococcus faecium (VREfm) is a WHO priority pathogen. In the Comunitat Valenciana (CV), Spain, VREfm prevalence has increased since 2022. We characterized the population structure, transmission patterns and resistance determinants of VREfm across eight hospitals (2021-2024). MethodsEight hospitals reported 870 VREfm cases during 2021-2024. We sequenced 254 VREfm isolates using WGS (Illumina) and inferred relatedness by MLST, cgMLST, and core-genome SNP analyses. Acquired antimicrobial resistance (AMR) genes, bacteriocins, plasmid replicases and putative virulence markers (PVMs) were identified in silico. Thirty-eight representatives underwent Nanopore long-read sequencing and hybrid assembly to resolve plasmids. ResultsThe predominant vancomycin-resistance genotype was vanB (62%; six hospitals), followed by vanA (19%; five hospitals) and vanA+vanB (17%; five hospitals). Eight sequence types (ST) and 15 clonal complexes (CT) were identified, grouped into seven main phylogenetic clades with close relatedness to publicly available genomes from other regions of Spain and Europe. A single lineage, ST117-CT7799, accounted for 198/254 (78%) isolates, persisted during 2022-2024 across seven hospitals and was enriched in the bacteriocin gene bac43 (or T8) (84%). Linezolid-resistance genes optrA and cfr(D) were present in 34% of isolates, most of which also carried vanA (32%). Hybrid assemblies revealed a diverse plasmidome including RepA_N megaplasmids with PVMs and AMR genes, and small Rep3-like plasmids harbouring bacteriocins (bac43, bac51, bacAS9); a 6 kb repA_pB82 plasmid was bac43-positive in 37/38 (97%) fully resolved carriers. Eight strains across four hospitals carried identical linear plasmids co-harbouring vanA, optrA, cfrD genes within a widespread repUS78_pZY2 background, consistent with inter-lineage plasmid transmission. ConclusionsThis first comprehensive genomic analysis of VREfm in CV indicates extensive inter-hospital spread dominated by expansion of ST117-CT7799 and highlights plasmid-mediated convergence of vancomycin and linezolid resistance via linear plasmids. Strengthened infection prevention and genomic surveillance, including long-read sequencing to track linear plasmids, are warranted.

microbiology↗

bioRxiv2026

Gardnerella fastidiominuta sp. nov. isolated from the female urinary microbiome

The genus Gardnerella comprises a group of fastidious bacteria associated with the female urogenital tract and has undergone extensive taxonomic revision in recent years. In this study, a bacterial strain, designated CCPDSM, was isolated from the female urinary microbiome and subjected to a comprehensive polyphasic taxonomic characterization. The 16S rRNA gene sequence confirmed that this strain is a member of the genus Gardnerella, and phylogenetic analyses based on cpn60 sequences, together with phylogenomic reconstruction placed strain CCPDSM within the genus Gardnerella as a distinct and well-supported lineage. Genome-based relatedness indices (ANIb, ANIm, TETRA and dDDH), demonstrated clear separation of CCPDSM from all validly published Gardnerella species. In contrast, comparisons with two publicly available closely related genomes yielded values above accepted species delineation thresholds, supporting their assignment to the same taxon. Phenotypic characterization, together with genome-based functional predictions, revealed a fastidious, fermentative metabolic profile that further differentiated CCPDSM from its closest relatives, while remaining consistent with traits characteristic of the genus. On the basis of combined phylogenetic, genomic and phenotypic evidence, strain CCPDSM is proposed as representing a novel species within the genus Gardnerella, for which the name Gardnerella fastidiominuta sp. nov. is proposed, with strain CCPDSM (=CECT 31324=CCP 588) designated as the type strain. This study expands the recognized diversity of Gardnerella and highlights the female urinary tract as a reservoir of previously uncharacterized species within this genus.

microbiology↗

bioRxiv2025

Single point mutations in global regulatory genes restore cephalosporin resistance in a low-MIC Enterococcus faecium natural isolate

Enterococcus faecium exhibits intrinsic resistance to cephalosporins (CPH), yet the genetic determinants of this phenotype remain incompletely understood. To date, E. faecium strains with low minimum inhibitory concentrations (MICs) to CPH have only been described following genetic manipulation. At Parc Tauli University Hospital, we identified a clinical isolate of ampicillin-susceptible E. faecium (Efm5) that exhibited unusually low-MICs to cefotaxime (1 mg/L), ceftriaxone (3 mg/L), and ceftaroline (0.19 mg/L). Upon single exposure to ceftriaxone (100 mg/L), Efm5 rapidly yielded variants with markedly increased MICs to ceftriaxone (>256 mg/L) and cefotaxime (>32 mg/L), while MICs to ampicillin and ceftaroline were unaffected. Whole-genome sequencing revealed that the high-MIC variants carried single nucleotide polymorphisms (SNPs) leading to non-synonymous mutations in croS, nusG or rpoB genes. Phenotypic assays confirmed that these mutations were associated with ceftriaxone resistance and immunoblots revealed increased expression of penicillin-binding protein 5 (PBP5) in all the high-MIC variants. Transcriptional profiling showed upregulation of the pbp5 operon, which includes ftsW, psr and pbp5, in the croS variants. This study provides the first evidence that E. faecium isolates with low-MICs to CPH can arise in clinical settings without laboratory manipulation, challenging the prevailing notion of absolute intrinsic CPH resistance in this species and offering a novel framework to explore previously unrecognized resistance pathways.

microbiology↗

bioRxiv2025

Unraveling the Gardnerella Pangenome: Insights into Genetic Diversity, Virulence, and Antibiotic Resistance

In recent years, the development of user-friendly and accessible bioinformatic tools has significantly advanced Gardnerella genome analysis. Despite the taxonomic reclassification of the genus in 2019, many studies continue to rely on subgroups/ clades for species identification. This leads to frequent misidentifications, hindering accurate assessments of species-specific features, such as antimicrobial resistance and pathogenic potential. This study aims to address these gaps by employing precise species-level identification to improve our understanding on Gardnerella and its clinical implications. We performed the most extensive pangenome analysis of Gardnerella to date, analyzing 153 genomes (120 from NCBI and 33 newly sequenced). G. vaginalis, G. leopoldii, G. swidsinskii, and G. pickettii species exhibited open pangenomes. The Gardnerella pangenome is highly diverse, with 90% of its genes belonging to the accessory genome. Carbohydrate transport and metabolism contributed the most for the primary genomic differences observed among Gardnerella species. Additionally, the search for antibiotic resistance genes (ARGs) revealed lsaC gene as the most prevalent ARG, potentially conferring resistance to clindamycin, a commonly prescribed antibiotic for treating bacterial vaginosis (BV). The clustering of Gardnerella genomes mimics the core genome phylogeny, pointing to species specific virulence-associated factors, highlighting important factors such as vaginolysin and sialidases. Our comprehensive pangenome analysis shows that Gardnerella species are well-adapted within the urogenital tract, with identified virulence factors and metabolic capabilities potentially enhancing the pathogenicity of some strains. Future studies should explore Gardnerellas ecological interactions and pathogenic mechanisms within the urogenital microbiome. ImportanceIn recent years, the availability of user-friendly and accessible bioinformatics tools has led to a surge in studies analyzing the Gardnerella genome. Despite the redefinition of the genus in 2019, many researchers continue to rely on subgroups or clades for Gardnerella identification. Our study builds upon the updated taxonomy of Gardnerella, offering a more comprehensive understanding of its genetic diversity. By conducting a pangenome analysis of 153 Gardnerella genomes, we uncover significant genetic variability, species-specific virulence factors, and the widespread presence of antibiotic resistance genes, highlighting the urgent need for precise species-level identification. The identification of novel genomic features and antibiotic resistance profiles not only enhances our understanding of Gardnerellas clinical implications but also sheds light on its metabolic and virulence potential. These insights help clarify the pathogenic mechanisms of Gardnerella and provide a more refined understanding of its role in the urogenital microbiome.

microbiology↗

bioRxiv2024

Real-time identification of carbapenemase-producing Klebsiella pneumoniae lineages and outbreak detection using FT-IR ATR

Expansion of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) is driven by nosocomial dissemination, and effective infection control depends on timely and reliable typing data. Here, we evaluated our previously developed Fourier-transform infrared spectroscopy (FT-IR) with attenuated total reflectance (ATR) workflow for real-time typing of Kp capsular (KL)-types and lineages to support infection control. FT-IR spectra were acquired from Columbia agar with 5% sheep blood cultures of all CP-Kp infection isolates (n=136) from hospitalized patients at a northern Portugal hospital (April 2022 - March 2023), and analyzed using automated machine-learning (ML) classification models. Typing results were confirmed by wzi sequencing, MLST and/or WGS. FT-IR typing on Columbia agar plates showed 73% sensitivity, 79% specificity and 74% accuracy. Our method correctly typed 94% of typeable isolates (78/83), from which 87% were comunicated in <24h. Sixty percent of non-typeable isolates were considered false negatives, but the majority (66%) was correctly predicted when re-tested in Mueller-Hinton agar, improving sensitivity (92%), specificity (76%) and accuracy (89%) of Kp typing. Three Kp lineages (ST147-KL64, ST15-KL19, ST268-KL20) represented 74% of the sample, with ST268-KL20 causing an outbreak in Neonatal Intensive Care unit, quickly recognized by FT-IR enabling immediate infection control measures. Epidemiological links between patiens were mostly found on medical, surgical and urology units, using EpiLinx software. Most isolates (98%) produced KPC-3. Our FT-IR ATR ML-based typing workflow demonstrated high performance standards in real-time and high adaptability to clonal dynamics. The unprecedent time-to-response (same day of species identification) represents an opportunity to implement timely and effective infection control measures. ImportanceThis study represents the first prospective and real-time evaluation of FT-IR spectroscopy to type multidrug resistant Klebsiella pneumoniae to support surveillance and infection control. We demonstrate a high sensitivity, specificity and accuracy of a previously developed workflow that allows precise identification of K. pneumoniae lineages. The adaptability to changes in clonal dynamics and bacterial typing in <24h offer significant advantages in both high- and low-income countries for a timely infection control and improvement of antimicrobial resistance management.

microbiology↗

bioRxiv2023

Bacteriocin Distribution Patterns in Enterococcus faecium and Enterococcus lactis: Bioinformatic Analysis Using a Tailored Genomics Framework

Multidrug-resistant Enterococcus faecium represent a major concern due to their ability to thrive in diverse environments and cause life-threatening infections. While antimicrobial resistance and virulence mechanisms have been extensively studied, the contribution of bacteriocins to E. faeciums adaptability remains poorly explored. E. faecium, within the Bacillota phylum, is a prominent bacteriocin producer. Here, we developed a tailored database of 76 Bacillota bacteriocins (217 sequences, including 40 novel bacteriocins) and applied it to uncover bacteriocin distribution patterns in 997 quality-filtered E. faecium and Enterococcus lactis (former E. faecium clade B) genomes. Curated using computational pipelines and literature mining, our database demonstrates superior precision versus leading public tools in identifying diverse bacteriocins. Distinct bacteriocin profiles emerged between E. faecium and E. lactis, highlighting species-specific adaptations. E. faecium strains from hospitalized patients were significantly enriched for bacteriocins as entA, bac43, bacAS5 and bacAS11. These bacteriocins strongly associated with antibiotic resistance, particularly vancomycin and ampicillin, and Inc18 rep2_pRE25-derivative plasmids, classically associated with vancomycin resistance transposons. Our integrated genomic and epidemiological analysis elucidates meaningful connections between bacteriocin determinants, antimicrobial resistance, mobile genetic elements, and ecological origins in E. faecium. This work significantly expands the knowledge on the understudied bacteriocin diversity in opportunistic enterococci, revealing their contribution to environmental adaptation. Further characterization of strain-level bacteriocin landscapes could inform strategies to combat high-risk clones. Overall, these insights provide a framework for unravelling bacteriocins therapeutic and biotechnological potential.

genomics↗

bioRxiv2022

The population-level impact of Enterococcus faecalis genetics on intestinal colonisation and extraintestinal infection

Enterococcus faecalis is a commensal pathogenic bacterium commonly found in the human gastrointestinal tract and a cause of opportunistic infections typically associated with multidrug resistance. The E. faecalis genetic changes associated with pathogenicity and extraintestinal infection, particularly through gut-to-bloodstream translocation, are poorly understood. Here, we investigate the E. faecalis genetic signatures associated with intestinal colonisation and extraintestinal infection and infection of hospitalised and non-hospitalised individuals using heritability estimation and a genome-wide association study (GWAS). We analysed 750 whole-genome sequences of faecal and bloodstream E. faecalis isolates from hospitalised patients and non-hospitalised individuals, respectively, predominantly in Europe. We found that E. faecalis infection of individuals depending on their hospitalisation status and extraintestinal infection are heritable traits and that [~]24% and [~]34% of their variation is explained by the considered genetic effects, respectively. Further, a GWAS using linear mixed models did not pinpoint any clear enrichment of individual genetic changes in isolates from different isolation sites and individuals with varying hospitalisation statuses, suggesting that these traits are highly polygenic. Altogether, our findings indicate that E. faecalis infection and extraintestinal infection are influenced by variation in genetic, host, and environmental factors, and ultimately the opportunistic pathogenic lifestyle of this versatile host generalist bacterium.

microbiology↗

bioRxiv2022

Evolution of chlorhexidine susceptibility and of the EfrEF operon among Enterococcus faecalis from diverse environments, clones and time spans

Chlorhexidine (CHX) is widely used to control the spread of pathogens (e.g. human/animal clinical settings, ambulatory care, food industry). E. faecalis, a major nosocomial pathogen, is broadly distributed in diverse hosts and environments facilitating its exposure to CHX over the years. Nevertheless, CHX activity against E. faecalis is understudied. Our goal was to assess CHX activity and the variability of ChlR-EfrEF proteins (associated with CHX tolerance) among 673 field isolates and 1784 E. faecalis genomes from PATRIC database from different sources, time spans, clonal lineages and antibiotic resistance profiles. CHX minimum inhibitory concentrations (MICCHX) and minimum bactericidal concentrations (MBCCHX) against E. faecalis presented normal distributions (0.5-64 mg/L). However, more CHX tolerant isolates were detected in the food chain and recent human infections, suggesting an adaptability of E. faecalis populations in settings where CHX is heavily used. Heterogeneity in ChlR-EfrEF sequences was identified, with isolates harboring incomplete ChlR-EfrEF proteins, particularly the EfrE identified in the ST40 clonal lineage, showing low MICCHX ([≤]1mg/L). Distinct ST40-E. faecalis subpopulations carrying truncated and non-truncated EfrE were detected, the former being predominant in human isolates. This study provides a new insight about CHX susceptibility and ChlR-EfrEF variability within diverse E. faecalis populations. The MICCHX/MBCCHX of more tolerant E. faecalis (MICCHX=8mg/L; MBCCHX=64mg/L) remain lower than in-use concentrations of CHX (>500mg/L). However, CHX increasing use combined with concentration gradients occurring in diverse environments potentially selecting multidrug-resistant strains with different CHX susceptibilities, alert to the importance of monitoring the trends of E. faecalis CHX tolerance within a One-Health approach.

microbiology↗

bioRxiv2022

Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

The recognition of microbiome inhabiting the healthy female bladder engendered the need for comprehensive characterization of the female urinary microbiome (FUM) in health and disease. Although previous studies reported FUM composition at different taxonomic levels, progress towards reliable identification at species level is highly required. The aim of this study was to comprehensively characterize bacterial species of FUM of healthy reproductive-age European women by two complementary methodologies i.e., extended culturomics and long-read third generation sequencing of near full-length 16S rRNA gene. A wide diversity of bacterial species was captured (297 species) with a median of 53 species/sample, including 16 putative uropathogens. Clustering FUM into community structure types revealed high inter-individual differences. Notably, there was not a single species common to all samples, although the Lactobacillus genus was detected in all samples. Lactobacillus crispatus, Lactobacillus iners and Lactobacillus mulieris were observed in high relative abundance in several samples as well as other species (e.g., Streptococcus agalactiae, Atopobium vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while more prevalent species were often low abundant members (e.g., Finegoldia magna). We captured remarkable richness within Corynebacterium spp. (25 species) and Lactobacillaceae (4 genera, 14 species). While amplicon sequencing allowed detection of more anaerobic species (e.g., 11 Peptoniphilus spp.), culturomics enabled the identification of recently recognized Gardnerella species and putative novel Corynebacterium species. This study provided fine-grained FUM profiling at species level and revealed detailed FUM structure, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders. IMPORTANCEDespite evidence of the resident microbial community in the female lower urinary tract, bacterial species diversity and abundance in healthy women is still unclear. This study demonstrated that complementarity between optimized culture-dependent and -independent approaches is highly beneficial for comprehensive FUM species profiling by detecting higher FUM species diversity than previously reported, including identification of unreported Lactobacillaceae species and putative novel Corynebacterium species. Although some particular species were present in high relative abundance, low-abundant members were more prevalent. FUM classification into community structure types demonstrated high inter-individual differences in urinary microbiome composition among healthy women. We also report moderate correlation between culture-dependent and -independent derived data highlighting drawbacks resulting from each methodological approach. Our findings suggest that FUM bacterial diversity reported from previous studies may be underestimated. Finally, our results contribute to the fundamental knowledge of healthy FUM required for further exploration of the urinary microbiome role in urinary tract diseases.

microbiology↗

bioRxiv2021

The darkest place is under the candlestick - healthy urogenital tract as a source of UTI-related Escherichia coli lineages

Since the discovery of the urinary microbiome, including identification of Escherichia coli in healthy hosts, its involvement in UTI development is a subject of high interest. We explored population diversity and antimicrobial resistance of E. coli from urogenital microbiome of asymptomatic and recurrent UTI (rUTI) women. We also evaluated the genomic relationship between extraintestinal pathogenic E. coli (ExPEC) strains from healthy and diseased hosts, particularly of the ST131 lineage. E. coli was highly prevalent in asymptomatic women (48%) with slightly higher prevalence in vaginal samples comparing to urine, and occasionally with multiclonal population in the same individual. B2 was the most frequent phylogenetic group, with most strains classified as ExPEC. We demonstrated that virulence associated genes profile does not allow to distinguish strains isolated from healthy and rUTI host. We identified E. coli widespread lineages e.g., sequence types (ST) 127, ST131 (asymptomatic cohort) and ST73, ST131 (rUTI), frequently resistant to at least one antibiotic. Phylogenomics of ST131 and other ExPEC lineages revealed close relatedness between healthy and diseased host. These findings demonstrate that healthy urogenital microbiome is a source of potentially pathogenic and antibiotic resistant E. coli strains, including globally spread E. coli lineages causing UTI including ST131.

microbiology↗

bioRxiv2021

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

BackgroundVancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. The studys aim was to analyse the effects of acquiring Tn1546-plasmids with proven epidemicity in different bacterial host backgrounds. MethodsWidespread Tn1546-plasmids of different families RepA_N (n=5), Inc18 (n=4) and/or pHT{beta} (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 presence/absence of vancomycin. Plasmid stability was analysed (300 generations). Whole genome sequencing (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. ResultsAll plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2%-18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9-49%). Stability of Tn1546-plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. Point mutations and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. ConclusionsThe stability of E. faecium Tn1546-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.

microbiology↗

bioRxiv2020

ICEs are the main reservoirs of the ciprofloxacin-modifying crpP gene in Pseudomonas aeruginosa

The ciprofloxacin-modifying crpP gene was recently identified in a plasmid isolated from a clinical Pseudomonas aeruginosa clinical isolate. Homologues of this gene were also identified in Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. We set out to explore the mobile genetic elements involved in the acquisition and spread of this gene in publicly available and complete genomes of Pseudomonas. The crpP gene was identified only in P. aeruginosa, in more than half of the complete chromosomes (61.9%, n=133/215) belonging to 52 sequence types, of which the high-risk clone ST111 was the most frequent. We identified 136 crpP-harboring ICEs, with 93.4% belonging to the mating-pair formation G (MPFG) family. The ICEs were integrated at the end of a tRNALys gene and were all flanked by highly conserved 45-bp direct repeats. The core ICEome contains 26 genes (2.2% of all genes), which are present in 99% or more of the crpP-harboring ICEs. The most frequently encoded traits on these ICEs include replication, transcription, intracellular trafficking and cell motility. Our work reveals that ICEs are the main vectors promoting the dissemination of the ciprofloxacin-modifying crpP gene in P. aeruginosa. Author NotesAll supporting data has been provided within the article or through supplementary data files. Supplementary material is available with the online version of this article. Impact StatementA high proportion of Pseudomonas aeruginosa clinical isolates are resistant to ciprofloxacin. Resistance to this antibiotic is often mediated by chromosomal mutations, but recently horizontally transferred genes have been identified. We assessed the repartition of the ciprofloxacin-modifying crpP gene among Pseudomonas genomes and we characterized the mobile elements associated with its acquisition. We found that this gene is prevalent in P. aeruginosa and frequently associated with integrative and conjugative elements (ICEs). Importantly, we also identified highly conserved direct repeats that can be used to accurately delimit crpP-carrying ICEs in P. aeruginosa genomes. Data SummaryAll the bacterial genomes scanned in this study have been deposited previously in the National Center for Biotechnology Information genome database and are listed on the supplementary tables. The newick files used to create the trees in Figures 1 and 4 are deposited on figshare at https://figshare.com/projects/ICEs_are_the_main_reservoirs_of_the_ciprofloxacin-modifying_crpP_gene_in_Pseudomonas_aeruginosa/79308. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/991208v2_fig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f66f4forg.highwire.dtl.DTLVardef@105b335org.highwire.dtl.DTLVardef@138882eorg.highwire.dtl.DTLVardef@74b9f9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Approximately-maximum-likelihood phylogenetic tree base on the core genome alignment of 215 complete P. aeruginosa genomes. Branches and labels from crpP-positive hits are colored blue and the branches have twice the standard width. Only bootstrap values from 0.9 to 1 are displayed. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/991208v2_fig4.gif" ALT="Figure 4"> View larger version (19K): org.highwire.dtl.DTLVardef@15dcb4borg.highwire.dtl.DTLVardef@cdba0aorg.highwire.dtl.DTLVardef@70f131org.highwire.dtl.DTLVardef@eaf032_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO Approximately-maximum-likelihood phylogenetic tree base on the alignment of the 26 core genes identified in the crpP-harboring ICEs. Genome accession numbers containing more than one crpP-harboring ICE are highlighted in orange. C_FIG

microbiology↗

bioRxiv2019

Identification of 2CS-CHXT operon signature of chlorhexidine tolerance among Enterococcus faecium

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years, recently acquiring higher relevance in nosocomial infections control worldwide. Despite of this, CHX tolerance has been poorly understood among Enterococcus faecium, one of the leading agents causing nosocomial infections. This study provides new phenotypic and molecular data for a better identification of CHX tolerant E. faecium subpopulations in community and clinical contexts. MICCHX distribution of 106 E. faecium 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 2CS-CHXT operon, here identified. It encodes a glucose and an amino-acid-polyamine-organocation family transporters, besides the DNA-binding-response-regulator ChtR with a P102H mutation previously described in only CHX tolerant clade A1 E. faecium, and the ChtS sensor. Combined data from normal MIC distribution and 2CS-CHXT operon characterization supports a tentative epidemiological cut-off (ECOFF) of 8 mg/L to CHX, useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds alerts for a prudent use of CHX in multiple contexts.\n\nImportanceChlorhexidine is one of the substances included in the World Health Organizations 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 since the 1950s in healthcare (skin, hands, mouthwashes, eye drops), its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here we provide a comprehensive study on chlorhexidine tolerance among Enterococcus faecium, one of the leading nosocomial agents worldwide, and identified a novel 2CS-CHXT operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Finally, our data allowed to propose a tentative epidemiological cut-off of 8 mg/L, useful to detect tolerant E. faecium populations in future surveillance studies in community and clinical contexts.

microbiology↗

bioRxiv2019

Megaplasmids on the rise: combining sequencing approaches to fully resolve a carbapenemase-encoding plasmid in a proposed novel Pseudomonas species

Horizontal transfer of plasmids plays a pivotal role in the dissemination of antibiotic resistance genes and emergence of multidrug-resistant bacteria. Sequencing of plasmids is thus paramount for the success of accurate epidemiological tracking strategies in the hospital setting and routine surveillance. Here, we combine Nanopore and Illumina sequencing to fully assemble a carbapenemase-encoding megaplasmid carried by a clinical isolate belonging to a putative novel Pseudomonas species. FFUP_PS_41 has a multidrug resistance phenotype and was initially identified as Pseudomonas putida, but an average nucleotide identity below the cut-off for species delineation suggests a new species related to the P. putida phylogenetic group. FFUP_PS_41 harbors a 498,516-bp untypable megaplasmid (pJBCL41) with low similarity compared with publicly available plasmids. pJBCL41 contains a full set of genes for self-transmission and genes predicted to be responsible for plasmid replication, partitioning, maintenance and heavy metal resistance. pJBCL41 carries a class 1 integron with the |aacA7|blaVIM-2|aacA4| cassette array (In103) located within a defective Tn402-like transposon that forms part of a 50,273-bp mosaic region bound by 38-bp inverted repeats typical of the Tn3 family and flanked by 5-bp direct repeats. This region is composed of different elements, including additional transposon fragments, five insertion sequences and a Tn3-Derived Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach resulted in contiguous assemblies and allowed us to fully resolve a carbapenemase-encoding megaplasmid from Pseudomonas spp. The identification of novel megaplasmids will shed a new light on the evolutionary effects of gene transfer and the selective forces driving AR.

microbiology↗

bioRxiv2019

Significant temporal shifts on clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings

OBJECTIVESTo provide detailed molecular data on clinical acquired AmpC (qAmpC)-producing Enterobacteriaceae from two different periods (2002-2008 and 2010-2013) in order to clarify the contribution of clonal and plasmid genetic platforms for the current epidemiological scenario concerning extended-spectrum beta-lactams resistance. METHODSWe analysed 1246 Enterobacteriaceae non-susceptible to third-generation cephalosporins from 2 hospitals and 1 community laboratory between 2010 and 2013. Bacterial identification, antibiotic susceptibility, identification of qAmpC and plasmid-mediated quinolone resistance genes, clonal (PFGE, MLST) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed by standard methods. WGS was performed in two ST11-K. pneumoniae isolates harbouring DHA-1. RESULTSThe occurrence of qAmpC was lower (2.6%) than that observed in a previous survey (7.4%), and varied slightly over time. Isolates produced DHA-1 (53%), CMY-2 (44%) or DHA-6 (3%), but significant epidemiological changes were observed in the two surveys. While DHA-1 persisted in different institutions by selection of a worldwide epidemic IncR plasmid in a ST11 harbouring KL105, CMY-2 rates increased over time linked to IncI1 plasmids (instead of IncK or IncA/C2) in multiple E. coli clones. CONCLUSIONSThe higher frequency of DHA-1 qAmpC in these species contrasts with the scenario of most European countries. Furthermore, the different genetic backgrounds associated with either ESBL or qAmpC in our country might have contributed to their differential expansion.

epidemiology↗

bioRxiv2019

On the front line of Klebsiella pneumoniae surface structures understanding: establishment of Fourier Transform Infrared (FT-IR) spectroscopy as a capsule typing method

Genomics-based population analysis of multidrug resistant (MDR) Klebsiella pneumoniae (Kp) motivated a renewed interest on capsule (K) types given their importance as evolutionary and virulence markers of clinically relevant strains. However, there is a gap between genotypic based predictions and information on capsular polysaccharide structure and composition. We used molecular genotypic, comparative genomics, biochemical and phenotypic data on the cps locus to support the usefulness of Fourier-Transform Infrared (FT-IR) spectroscopy as a phenotypic approach for K-type characterization and identification. The approach was validated with a collection of representative MDR Kp isolates from main lineages/Clonal Groups (CGs) involved in local or nationwide epidemics in 6 European and South American countries. FT-IR-based K-type assignments were compared with those obtained by genotypic methods and WGS (cps operon), and further complemented with data on the polysaccharide composition and structure of known K-types. We demonstrate that our FT-IR-based spectroscopy approach can discriminate all 21 K-types identified with a resolution comparable (or even higher) to that provided by WGS, considered gold-standard methodology. Besides contributing to enlighten K-type diversity among a significant MDR Kp collection, the specific associations between certain K-types and Kp lineages identified in different geographic regions over time support the usefulness of our FT-IR-based approach for strain typing. Additionally, we demonstrate that FT-IR discriminatory ability is correlated with variation on the structure/composition of known K-types and, supported on WGS data, we were able to predict the sugar composition and chemical structure of new KL-types. Our data revealed an unprecedent resolution at a quick and low-cost rate of Kp K-types at the phenotypic level. Our FT-IR spectroscopy-based approach might be extremely useful not only as a cost-effective Kp typing tool, but also to improve our understanding on sugar-based coating structures of high relevance for strain evolution and host adaptation.

developmental biology↗

bioRxiv2019

A large self-transmissible plasmid from Nigeria confers resistance to multiple antibacterials without a carrying cost

Antimicrobial resistance is rapidly expanding, in a large part due to mobile genetic elements. We screened 94 fecal fluoroquinolone-resistant Escherichia coli isolates from Nigeria for six plasmid-mediated quinolone resistance (PMQR) genes. Sixteen isolates harbored at least one of the PMQR genes and four were positive for aac-6-Ib-cr. In one strain, aac-6-Ib-cr was mapped to a 125 Kb self-transmissible IncFII plasmid, pMB2, which also bears blaCTX-M-15, seven other functional resistance genes and multiple resistance pseudogenes. We hypothesized that pMB2 had been selected by antimicrobials and that its large size would confer a growth disadvantage. However, laboratory strains carrying pMB2 grew at least as fast as isogenic strains lacking the plasmid in both rich and minimal media. We excised a 32 Kb fragment containing the sitABCD and another putative transporter, pefB, a papB homolog, and several open-reading frames of unknown function. The resulting 93 Kb mini-plasmid conferred slower growth rates and lower fitness than wildtype pMB2. Trans-complementing the deletion with the cloned sitABCD genes confirmed that they accounted for the growth advantage conferred by pMB2 in iron-depleted media. The mini-plasmid additionally conferred autoaggregation and was less transmissible and both phenotypes could be complemented with a pefB clone. pMB2 is a large plasmid with a flexible resistance region that contains multiple loci that can account for evolutionary success in the absence of antimicrobials. Ancillary functions conferred by resistance plasmids can mediate their retention and transmissibility, worsening the trajectory for antimicrobial resistance and potentially circumventing efforts to contain resistance through restricted use.

microbiology↗