Biology subjects

Novais, C.

Publications and source records attributed to Novais, C..

11 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↗

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↗

bioRxiv2024

Decoding Klebsiella pneumoniae in Poultry Chain: Unveiling Genetic Landscape, Antibiotic Resistance, and Biocide Tolerance in Non-Clinical Reservoirs

The rise of antibiotic resistance in the food chain is influenced by the use of antimicrobial agents, such as antibiotics, metals, and biocides, throughout the entire farm-to-fork continuum. Besides, non-clinical reservoirs potentially contribute to the transmission of critical pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae. However, limited knowledge exists about the population structure and genomic diversity of K. pneumoniae circulating in conventional poultry production. We conducted a comprehensive characterization of K. pneumoniae across the whole chicken production chain (flocks/environment/meat, 2019-2022), exploring factors beyond antibiotics, like copper and quaternary ammonium compounds (QACs). Clonal diversity and adaptive features of K. pneumoniae were characterized through cultural, molecular (FT-IR), and whole-genome-sequencing (WGS) approaches. All except one flock were positive for K. pneumoniae with a significant increase (p < 0.05) from early to pre-slaughter stages, most persisting in chicken meat batches. Colistin-resistant K. pneumoniae rates were low (4%), while most samples carried MDR strains (67%) and copper-tolerant isolates (63%; sil+pco clusters; MICCuSO4[≥]16mM), particularly at pre-slaughter. Benzalkonium chloride consistently exhibited activity in K. pneumoniae (MIC/MBC range=4-64mg/L) from diverse and representative strains independently of the presence/absence of genes linked to QACs tolerance. A polyclonal K. pneumoniae population, discriminated by FT-IR and WGS, included various lineages dispersed throughout the chickens lifecycle at the farm (ST29-KL124, ST11-KL106, ST15-KL19, ST1228-KL38), until the meat (ST1-KL19, ST11-KL111, ST6405-KL109, and ST6406-CG147-KL111), or over years (ST631-49 KL109, ST6651-KL107, ST6406-CG147-KL111). Notably, some lineages were identical to those from human clinical isolates. WGS also revealed F-type multireplicon plasmids carrying sil+pco (copper) co-located with qacE{Delta}1{+/-}qacF (QACs) and antibiotic resistance genes like those disseminated in humans. In conclusion, chicken farms and their derived meat are significant reservoirs for diverse K. pneumoniae clones enriched in antibiotic resistance and metal tolerance genes, some exhibiting genetic similarities with human clinical strains. Further research is imperative to unravel the factors influencing K. pneumoniae persistence and dissemination within poultry production, contributing to improved food safety risk management. This study underscores the significance of understanding the interplay between antimicrobial control strategies and non-clinical sources to effectively address the spread of antimicrobial resistance.

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↗

bioRxiv2023

From farm to fork: persistence of clinically-relevant multidrug-resistant and copper-tolerant Klebsiella pneumoniae long after colistin withdrawal in poultry production.

The concern of colistin-resistant bacteria in animal-food-environmental-human ecosystems prompted the poultry sector to implement colistin restrictions and explore alternative trace metals/copper feed supplementation. The impact of these strategies on the selection and persistence of colistin-resistant Klebsiella pneumoniae (Kp) in the whole poultry-production chain needs clarification. We assessed colistin-resistant and copper-tolerant Kp occurrence in chicken raised with inorganic and organic copper-formulas from one-day-old chicks to meat (7 farms/2019-2020), after long-term colistin withdrawal (>2-years). Clonal diversity and Kp adaptive features were characterized by cultural, molecular, and whole-genome-sequencing (WGS) approaches. Most chicken-flocks (75%) carried Kp at early+pre-slaughter stages, with a significant decrease (p<0.05) in meat batches (17%) and sporadic water/feed contamination. High rates (>50%) of colistin-resistant/mcr-negative Kp were observed among faecal samples, independently of feed. Most samples carried multidrug-resistant (90%) and copper-tolerant isolates (81%; pco+sil/MICCuSO4 [≥]16mM). WGS revealed accumulation of colistin resistance associated mutations and F-type multireplicon plasmids carrying antibiotic resistance and metal/copper-tolerance genes. The Kp population was polyclonal, with various lineages dispersed throughout poultry production. ST15-KL19, ST15-KL146 and ST392-KL27, and IncF plasmids were similar to those from global human clinical isolates, suggesting chicken-production as a reservoir/source of clinically-relevant Kp lineages and genes with potential risk to humans through food and/or environmental exposure. Despite long-term colistin ban limited mcr spread, it was ineffective in controlling colistin-resistant/mcr-negative Kp, regardless of feed. This study provides crucial insights into the persistence of clinically-relevant Kp in the poultry-production chain and highlights the need for continued surveillance and proactive food safety actions within a One-Health perspective. IMPORTANCEThe spread of bacteria resistant to last-resort antibiotics such as colistin throughout the food chain is a serious concern for public health. The poultry sector has responded by restricting colistin use and exploring alternative trace metals/copper feed supplements. However, it is unclear how and to which extent these changes impact the selection and persistence of clinically-relevant Klebsiella pneumoniae (Kp) throughout poultry chain. We found a high occurrence of copper-tolerant and colistin-resistant/mcr-negative Kp in chicken flocks, regardless of inorganic and organic copper-formulas and long-term colistin ban. Despite the high Kp diversity, the occurrence of identical lineages and plasmids across samples and/or clinical isolates suggests poultry as a potential source of human Kp exposure. This study highlights the need for continued surveillance and proactive farm-to-fork actions to mitigate the risks to public health, relevant for stakeholders involved in food industry and policymakers tasked with regulating food safety.

microbiology↗

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↗

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↗

bioRxiv2019

Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers common genetic features and a chromosomal hotspot for optrA acquisition

Linezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we still lack a comprehensive analysis of human and animal optrA-LREfs strains. We investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA-containing platforms of 28 publicly available optrA-positive E. faecalis genomes from different hosts in 7 countries. In the genome-level analysis, in house databases with 57 virulence and 391 plasmid replication genes were tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera, mainly but not exclusive from Firmicutes. The content in AMR and virulence genes was highly identical in contrast to the diversity of phages and plasmids observed. Epidemiologically unrelated clones (ST476-like and ST21-like) obtained from human clinical and animal hosts in different continents over 5 years (2012-2017) were phylogenetically related (3-122 SNPs difference). They also exhibited identical AMR and virulence profiles, highlighting a global spread of optrA-positive strains with relevant adaptive traits in livestock and that they might originate from an animal reservoir. optrA was located on the chromosome within a Tn6674-like element (n=9) or on medium-size plasmids (30-60 kb; n=14) belonging to main plasmid families (RepA_N/Inc18/Rep_3). In most cases, the immediate gene vicinity of optrA was identical in chromosomal (Tn6674) and plasmid (impB-fexA-optrA) backbones. Tn6674 was always inserted in the same {Delta}radC integration site and embedded in a 32 kb chromosomal platform common to diverse strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012-2018. This platform is conserved among hundreds of E. faecalis genomes and we here propose a conserved chromosomal hotspot for optrA integration. The finding of optrA in strains sharing identical adaptive features and genetic backgrounds across different hosts and countries suggest the occurrence of common and independent genetic events occurring in distant regions, and might explain the easy de novo generation of optrA-positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact in the efficacy of linezolid for the treatment of Gram-positive infections.

genomics↗

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↗