Genomic Characterization and Phylogenetic Analysis of Linezolid-Resistant Enterococcus from the Nostrils of Healthy Hosts Identifies Zoonotic Transmission.

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZDR) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZDR enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZDR-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZDR enterococci in all ecological niches and body parts to direct appropriate control strategies.

Decay kinetics of HIV-1-RNA in seminal plasma with dolutegravir/lamivudine versus dolutegravir plus emtricitabine/tenofovir alafenamide in treatment-naive people living with HIV.

BACKGROUND: This was a substudy of a Phase IV, randomized clinical trial (ClinicalTrials.gov identifier: NCT04295460) aiming to compare the activity of dolutegravir/lamivudine versus dolutegravir plus tenofovir alafenamide/emtricitabine (DTG + TAF/FTC) in the male genital tract. METHODS: Participants were asymptomatic adults without sexually transmitted diseases, treatment-naive people living with HIV (PLWH), with CD4+ T cell counts >200 cells/mm3 and plasma HIV-1-RNA levels >5000 and <500 000 copies/mL, randomized (1:1) to DTG + TAF/FTC or dolutegravir/lamivudine. Blood plasma (BP) and seminal plasma (SP) were collected at baseline and Weeks 4, 8, 12 and 24. HIV-1-RNA was measured in BP and SP using the Cobas 6800 system (Roche Diagnostics) with a lower detection limit of 20 copies/mL. The primary efficacy endpoint was the proportion of subjects with undetectable SP HIV-1-RNA at Week 12 by intention-to-treat analysis. RESULTS: Fifteen participants in the DTG + TAF/FTC and 16 in the dolutegravir/lamivudine arms were analysed, with basal SP viral load of 4.81 (4.30-5.43) and 4.76 (4.09-5.23), P = 0.469, respectively. At Week 12, only one participant in each treatment arm had a detectable SP HIV-1-RNA (DTG + TAF/FTC, 141 copies/mL; dolutegravir/lamivudine, 61 copies/mL). Based on the estimated means, there was no significant difference in the decay of HIV-1-RNA in both BP and SP over time between the two arms of treatment (F = 0.452, P = 0.662, and F = 1.147, P = 0.185, respectively). CONCLUSIONS: After 12 weeks of treatment, there were no differences in the percentage of undetectable SP HIV-1-RNA in naive PLWH who started dolutegravir/lamivudine compared with DTG + TAF/FTC.

Nasotracheal enterococcal carriage and resistomes: detection of optrA-, poxtA- and cfrD-carrying strains in migratory birds, livestock, pets, and in-contact humans in Spain.

This study determined the carriage rates and antimicrobial resistance (AMR) genes of enterococci from nasotracheal samples of three healthy animal species and in-contact humans. Nasal samples were collected from 27 dog-owning households (34 dogs, 41 humans) and 4 pig-farms (40 pigs, 10 pig-farmers), and they were processed for enterococci recovery (MALDI-TOF-MS identification). Also, a collection of 144 enterococci previously recovered of tracheal/nasal samples from 87 white stork nestlings were characterized. The AMR phenotypes were determined in all enterococci and AMR genes were studied by PCR/sequencing. MultiLocus-Sequence-Typing was performed for selected isolates. About 72.5% and 60% of the pigs and pig-farmers, and 29.4% and 4.9%, of healthy dogs and owners were enterococci nasal carriers, respectively. In storks, 43.5% of tracheal and 69.2% of nasal samples had enterococci carriages. Enterococci carrying multidrug-resistance phenotype was identified in 72.5%/40.0%/50.0%/23.5%/1.1% of pigs/pig-farmers/dogs/dogs' owners/storks, respectively. Of special relevance was the detection of linezolid-resistant enterococci (LRE) in (a) 33.3% of pigs (E. faecalis-carrying optrA and/or cfrD of ST59, ST330 or ST474 lineages; E. casseliflavus-carrying optrA and cfrD); (b) 10% of pig farmers (E. faecalis-ST330-carrying optrA); (c) 2.9% of dogs (E. faecalis-ST585-carrying optrA); and (d) 1.7% of storks (E. faecium-ST1736-carrying poxtA). The fexA gene was found in all optrA-positive E. faecalis and E. casseliflavus isolates, while fexB was detected in the poxtA-positive E. faecium isolate. The enterococci diversity and AMR rates from the four hosts reflect differences in antimicrobial selection pressure. The detection of LRE carrying acquired and transferable genes in all the hosts emphasizes the need to monitor LRE using a One-Health approach.

Staphylococcus aureus Carriage in the Nasotracheal Cavities of White Stork Nestlings (Ciconia ciconia) in Spain: Genetic Diversity, Resistomes and Virulence Factors.

The molecular ecology of Staphylococcus aureus in migratory birds (such as white storks) is necessary to understand their relevance in the "One Health" ecosystems. This study determined the nasotracheal carriage rates of S. aureus from white storks in Southern Spain and genetically characterized the within-host diversity. A collection of 67 S. aureus strains, previously obtained from 87 white stork nestlings (52 nasal and 85 tracheal samples) fed by their parents with food foraged in natural and landfill habitats, were tested for their antimicrobial resistance (AMR) phenotypes. Moreover, the AMR genotypes, immune evasion cluster (IEC), virulence genes and the detection of CC398 lineage were studied by PCR. The spa types and multilocus-sequencing-typing (MLST) were also determined by PCR and sequencing. Staphylococcus aureus carriage was found in 31% of storks (36.5%/11.9% in nasal/tracheal samples). All isolates were methicillin-susceptible (MSSA) and 8.8% of them were also susceptible to all tested antibiotics. The AMR phenotype/percentage/genes detected were as follows: penicillin/79.1%/blaZ; erythromycin-clindamycin-inducible/19.1%/ermA, ermT; tetracycline/11.9%/tetK; clindamycin/4.5%/lnuA and ciprofloxacin/4.5%. Twenty-one different spa types, including 2 new ones (t7778-ST15-CC15 and t18009-ST26-CC25), were detected and ascribed to 11 clonal complexes (CCs). MSSA-CC398 (8.2%), MSSA-CC15 (7.1%) and MSSA-ST291 (5.9%) were the most prevalent lineages in storks. Moreover, tst-positive (MSSA-CC22-t223 and MSSA-CC30-t1654), eta-positive (MSSA-CC9-t209) and etb-positive strains (MSSA-CC45-t015) were detected in four storks. The 18.5% of storks harboured distinct MSSA strains (with different lineages and/or AMR genes). Nestlings of storks foraging in landfills (10 CCs) had more diverse S. aureus strains than those of parents foraging in natural habitats (3 CCs). Low level of AMR was demonstrated among S. aureus strains. The predominance of MSSA-CC398 (an emergent clade) and toxigenic MSSA strains in stork nestlings highlight the need for continuous surveillance of S. aureus in wild birds.

Higher plasma levels of thymosin-α1 are associated with a lower waning of humoral response after COVID-19 vaccination: an eight months follow-up study in a nursing home.

BACKGROUND: Older people achieve lower levels of antibody titers than younger populations after Covid-19 vaccination and show a marked waning humoral immunity over time, likely due to the senescence of the immune system. Nevertheless, age-related predictive factors of the waning humoral immune response to the vaccine have been scarcely explored. In a cohort of residents and healthcare workers from a nursing home that had received two doses of the BNT162b2 vaccine, we measured specific anti-S antibodies one (T1), four (T4), and eight (T8) months after receiving the second dose. Thymic-related functional markers, including thymic output, relative telomere length, and plasma thymosin-α1 levels, as well as immune cellular subsets, and biochemical and inflammatory biomarkers, were determined at T1, and tested for their associations with the magnitude of the vaccine response (T1) and the durability of such response both, at the short- (T1-T4) and the long-term (T1-T8). We aimed to identify age-related factors potentially associated with the magnitude and persistence of specific anti-S immunoglobulin G (IgG)-antibodies after COVID-19 vaccination in older people. RESULTS: Participants (100% men, n = 98), were subdivided into three groups: young (< 50 years-old), middle-age (50-65 years-old), and older (≥65 years-old). Older participants achieved lower antibody titers at T1 and experienced higher decreases in both the short- and long-term. In the entire cohort, while the magnitude of the initial response was mainly associated with the levels of homocysteine [ß (95% CI); - 0.155 (- 0.241 to - 0.068); p = 0.001], the durability of such response at both, the short-term and the long-term were predicted by the levels of thymosin-α1 [- 0.168 (- 0.305 to - 0.031); p = 0.017, and - 0.123 (- 0.212 to - 0.034); p = 0.008, respectively]. CONCLUSIONS: Higher plasma levels of thymosin-α1 were associated with a lower waning of anti-S IgG antibodies along the time. Our results suggest that plasma levels of thymosin-α1 could be used as a biomarker for predicting the durability of the responses after COVID-19 vaccination, possibly allowing to personalize the administration of vaccine boosters.

Nasal Staphylococcus aureus and S. pseudintermedius carriage in healthy dogs and cats: a systematic review of their antibiotic resistance, virulence and genetic lineages of zoonotic relevance.

The molecular ecology of Staphylococcus aureus, Staphylococcus pseudintermedius and their methicillin-resistant strains in healthy dogs and cats could serve as good models to understand the concept of bacterial zoonosis due to animal companionship. This study aims to provide insights into pooled prevalence, genetic lineages, virulence and antimicrobial resistance (AMR) among healthy dogs and cats. Original research and brief communication articles published from 2001 to 2021 that reported the nasal detection of S. aureus and S. pseudintermedius in healthy dogs and cats in the community, homes and outside veterinary clinics were examined and analysed. Forty-nine studies were eligible and included in this systematic review. The pooled prevalence of nasal carriage of S. aureus/methicillin-resistant S. aureus (MRSA) in healthy dogs and cats were 10.9% (95% CI: 10.1-11.9)/2.8% (95% CI: 2.4-3.2) and 3.2% (95% CI: 1.9-4.8)/0.5% (95% CI: 0.0-1.1), respectively. Conversely, the pooled prevalence of S. pseudintermedius/methicillin-resistant S. pseudintermedius (MRSP) in healthy dogs and cats were 18.3% (95% CI: 17.1-19.7)/3.1% (95% CI: 2.5-3.7) and 1.3% (95% CI: 0.6-2.4)/1.2% (95% CI: 0.6-2.3), respectively. Although highly diverse genetic lineages of S. aureus were detected in healthy dogs and cats, MSSA-CC1/CC5/CC22/CC45/CC121/CC398 and MRSA-CC5/CC93/CC22/CC30 were mostly reported in dogs; and MSSA-CC5/CC8/CC15/CC48 and MRSA-CC22/CC30/CC80 in cats. Of note, MSSA-CC398 isolates (spa-types t034 and t5883) were detected in dogs. Genetic lineages often associated with MSSP/MRSP were ST20/ST71, highlighting the frequent detection of the epidemic European MRSP-ST71 clone in dogs. S. aureus isolates carrying the luk-S/F-PV, tst, eta, etb and etd genes were seldomly detected in dogs, and luk-S/F-PV was the unique virulence factor reported in isolates of cats. S. pseudintermedius isolates harbouring the luk-S/F-I, seint and expA genes were frequently found, especially in dogs. High and diverse rates of AMR were noted, especially among MRSA/MRSP isolates. There is a need for additional studies on the molecular characterization of isolates from countries with under-studied nasal staphylococci isolates.

Penicillin susceptibility among invasive MSSA infections: a multicentre study in 16 Spanish hospitals.

OBJECTIVES: To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible MSSA (MSSA-PENS) isolates. METHODS: A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018-19 (6-12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type. RESULTS: The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton-Valentine leucocidin ('PVL') positive. CONCLUSIONS: A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis.

Antibody response in patients admitted to the hospital with suspected SARS-CoV-2 infection: results from a multicenter study across Spain.

AIM: To evaluate the serological response against SARS-CoV-2 in a multicenter study representative of the Spanish COVID pandemic. METHODS: IgG and IgM + IgA responses were measured on 1466 samples from 1236 Spanish COVID-19 patients admitted to the hospital, two commercial ELISA kits (Vircell SL, Spain) based on the detection of antibodies against the viral spike protein and nucleoprotein, were used. RESULTS: Approximately half of the patients presented antibodies (56.8% were IgM + IgA positive and 43.0% were IgG positive) as soon as 2 days after the first positive PCR result. Serological test positivity increased with time from the PCR test, and 10 days after the first PCR result, 91.5% and 88.0% of the patients presented IgM + IgA and IgG antibodies, respectively. CONCLUSION: The high values of sensitivity attained in the present study from a relatively early period of time after hospitalization support the use of the evaluated serological assays as supplementary diagnostic tests for the clinical management of COVID-19.

Occurrence of Pseudomonas spp. in Raw Vegetables: Molecular and Phenotypical Analysis of Their Antimicrobial Resistance and Virulence-Related Traits.

Pseudomonas is characterized by its great capacity to colonize different ecological niches, but also by its antimicrobial resistance and pathogenicity, causing human, animal, or plant diseases. Raw and undercooked food is a potential carrier of foodborne disease. The aim of this study was to determine the occurrence of Pseudomonas spp. among raw vegetables, analysing their antimicrobial resistance, virulence, and molecular typing. A total of 163 Pseudomonas spp. isolates (12 different species) were recovered from 77 of the 145 analysed samples (53.1%) and were classified into 139 different pulsed-field gel electrophoresis patterns. Low antimicrobial resistance levels, but one multidrug-resistant isolate, were found. Among the 37 recovered P. aeruginosa strains, 28 sequence-types and nine serotypes were detected. Eleven OprD patterns and an insertion sequence (ISPa1635) truncating the oprD gene of one imipenem-resistant strain were found. Ten virulotypes were observed, including four exoU-positive and thirty-one exoS-positive strains. The lasR gene was absent in three ST155 strains and was truncated by different insertion sequences (ISPre2, IS1411, and ISPst7) in other three strains. High biofilm, motility, pigment, elastase, and rhamnolipid production were detected. Our study demonstrated a low occurrence of P. aeruginosa (18%) and low antimicrobial resistance, but a high number of virulence-related traits in these P. aeruginosa strains, highlighting their pathological importance.

A survey of tools for analysing DNA fingerprints.

DNA fingerprinting is a genetic typing technique that allows the analysis of the genomic relatedness between samples, and the comparison of DNA patterns. This technique has multiple applications in different fields (medical diagnosis, forensic science, parentage testing, food industry, agriculture and many others). An important task in molecular epidemiology of infectious diseases is the analysis and comparison of pulsed-field gel electrophoresis (PFGE) patterns. This is applied to determine the clonal diversity of bacteria in the follow-up of outbreaks or for tracking specific clones of special relevance. The resulting images produced by DNA fingerprinting are sometimes difficult to interpret, and multiple tools have been developed to simplify this task. In this article, we present a survey of tools for analysing DNA fingerprints. In particular, we compare 33 tools using a set of predefined criteria. The comparison was carried out by hands-on experiences-whenever possible-and inspecting the documentation of the tools. As no system is preferred in all the possible scenarios, we have created a spreadsheet that can be customized by researchers to determine the best system for their needs.

Improved CD4 T cell profile in HIV-infected subjects on maraviroc-containing therapy is associated with better responsiveness to HBV vaccination.

BACKGROUND: Maraviroc-containing combined antiretroviral therapy (MVC-cART) improved the response to the hepatitis B virus (HBV) vaccine in HIV-infected subjects younger than 50 years old. We aimed here to explore the effect of this antiretroviral therapy on different immunological parameters that could account for this effect. METHODS: We analysed baseline samples of vaccinated subjects under 50 years old (n = 41). We characterized the maturational subsets and the expression of activation, senescence and prone-to-apoptosis markers on CD4 T-cells; we also quantified T-regulatory cells (Treg) and dendritic cell (DC) subsets. We used binary logistic regression to evaluate the immunological impact of MVC-cART, correlation with MVC exposure and linear regression for association with the magnitude of the HBV vaccine response. RESULTS: HIV-infected subjects on MVC-cART prior to vaccination showed increased recent thymic emigrants levels and reduced myeloid-DC levels. A longer exposure to MVC-cART was associated with lower frequencies of Tregs and activated and proliferating CD4 T-cells. Furthermore, the frequencies of activated and proliferating CD4 T-cells were inversely associated with the magnitude of the HBV vaccine response. CONCLUSION: The beneficial effect of MVC-cART in the HBV vaccine response in subjects below 50 years old could be partially mediated by its reducing effect on the frequencies of activated and proliferating CD4 T-cells prior to vaccination.

[Update on antibiotic resistance in Gram-positive bacteria]. / Actualización en la resistencia antibiótica en Gram positivos.

Antimicrobial resistance among Gram-positive bacteria, especially in Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, and Streptococcus pneumoniae, is a serious threat to public health. These microorganisms have multiple resistance mechanisms to agents currently used in clinical practice. Many of these resistance mechanisms are common to all 4 of these bacterial species, but other mechanisms seem to be more specific. The prevalence and dissemination of these mechanisms varies considerably, depending on the microorganism. This review discusses the resistance mechanisms to the most clinically relevant antibiotics, with particular emphasis on the new mechanisms described for widely used antibiotics and for newer agents such as lipopeptides, lipoglycopeptides, glycylcyclines and oxazolidinones.

Unusual presence of the immune evasion gene cluster in livestock-associated MRSA of lineage CC398 causing peridural and psoas abscesses in a poultry farmer.

OBJECTIVE: To characterize a methicillin-resistant Staphylococcus aureus (MRSA) isolate responsible for an aggressive infection (peridural and psoas abscess secondary to haematogenous septic arthritis) in a poultry farmer. METHODS: Molecular characterization was performed, including spa- and multilocus sequence typing of the isolate, assessment of its resistance phenotype and detection of tetracycline resistance and of virulence and immune evasion cluster (IEC) genes were performed. RESULTS: The MRSA isolate was tetracycline- and fluorquinolone-resistant, and was ascribed to CC398, spa-t1451. The isolate harboured tet(M) (distinctive of livestock-associated (LA) MRSA-CC398 clade) and IEC-type B system (characteristic of the methicillin-susceptible human lineage, but typically absent in LA-MRSA-CC398 strains), and lacked toxin-coding genes lukF/lukS-PV, tsst-1, eta and etb. CONCLUSION: IEC re-acquisition by LA-MRSA-CC398-LA strains is an unusual finding, but could constitute an emerging public health problem. It would represent an evolutionary step towards LA-MRSA-CC398's adaptation to human hosts, and might enhance its invasiveness and ability to be transmitted to humans.

Species distribution, antibiotic resistance and virulence traits in canine and feline enterococci in Tunisia.

In order to investigate the possible role of dogs and cats in the carriage and potential dissemination of resistant enterococci, seventy faecal samples from dogs and cats were tested for enterococci. Fifty-eight enterococci were recovered. Isolates were identified as Enterococcus faecium (n = 31) and E. faecalis (n = 14) E. durans (n = 6), E. casseliflavus (n = 2), E. hirae and E. gallinarum (2 isolates each). Enterococcal isolates showed resistance to ciprofloxacin (n = 35), erythromycin (n = 31), tetracycline (n = 25), kanamycin (n = 15), streptomycin (n = 13), pristinamycin (n = 11), gentamicin (n = 10), chloramphenicol (n = 8), and linezolid (n = 6). The gene erm(B) was detected in 22 out of 31 erythromycin-resistant enterococci. All tetracycline-resistant enterococci carried tet(M) and/or tet(L) genes. The gene aac(6')-Ie-aph(2″)-Ia was identified in five of high-level gentamicin-resistant isolates, the genes aph(3')-IIIa and/or aac(6')-Ie-aph(2″)-Ia in eleven high-level kanamycin-resistant isolates and the gene ant(6)-Ia in eleven high-level streptomycin-resistant isolates. Only one strain harboured cat(A) gene, and five strains contained vat(E) or vat(D) genes. Virulence genes gel(E) (21 strains), esp (11 strains) and cylA/cylB (5 strains) were detected. High genetic diversity was demonstrated among E. faecium isolates by pulsed-field gel electrophoresis (PFGE). Dogs and cats can be carriers of antibiotic-resistant enterococci in their faeces that could shed into the household environment.

Detection of MRSA ST3061-t843-mecC and ST398-t011-mecA in white stork nestlings exposed to human residues.

OBJECTIVES: The objective of this study was to analyse the prevalence of tracheal carriage of Staphylococcus aureus/MRSA in storks and to study the resistance and virulence genes in the obtained isolates. METHODS: Tracheal samples from 92 stork nestlings of two landfill-associated and two natural-habitat colonies were inoculated in specific media for S. aureus and MRSA recovery. Antimicrobial susceptibility was tested, and the presence of resistance, virulence and immune evasion cluster (IEC) genes was analysed by PCR. S. aureus isolates were characterized by spa and agr typing. Staphylococcal cassette chromosome (SCC) mec type was determined for mecC-positive isolates, and MLST was performed for 17 selected S. aureus isolates. RESULTS: S. aureus isolates were identified in 32/92 samples (34.8%), and 38 isolates were recovered. The prevalence of S. aureus was higher in nestlings from landfills (24/43, 55.8%) than in those from natural habitats (8/49, 16.3%). Three birds from landfill-associated colonies carried MRSA, two with mecA-positive strains [clonal complex (CC) 5-spa-t002 and CC398-spa-t011] and one with a mecC-positive strain [sequence type (ST) 3061-CC130-spa-t843-agr-III-SCCmecXI). None of the MRSA isolates presented IEC genes. Thirty-five MSSA isolates, which showed 18 different spa types (ascribed to CC5, CC7, CC22, CC30, CC45, CC59, CC133 and CC398), were obtained. The agr types detected were I (63%), II (29%) and III (8%). Resistance and virulence genes identified in MSSA were blaZ (n = 25), erm(T) (n = 9), erm(A) (n = 1), tet(M) (n = 2), fexA (n = 3), str (n = 2), tst (n = 2), eta (n = 1) and cna (n = 15). The IEC types B, C, D and G were found in MSSA isolates, and two new STs were identified (ST3060 and ST3061). CONCLUSIONS: White storks are frequently tracheal carriers of S. aureus, including ST398 isolates. MRSA isolates of lineages CC398-mecA and CC130-mecC were detected in storks from landfill-associated colonies exposed to human residues.

Characterization of fecal vancomycin-resistant enterococci with acquired and intrinsic resistance mechanisms in wild animals, Spain.

The objectives were to evaluate the presence of vancomycin-resistant enterococci with acquired (VRE-a) and intrinsic (VRE-i) resistance mechanisms in fecal samples from different wild animals, and analyze their phenotypes and genotypes of antimicrobial resistance. A total of 348 cloacal/rectal samples from red-legged partridges (127), white storks (81), red kites (59), and wild boars (81) (June 2014/February 2015) were inoculated in Slanetz-Bartley agar supplemented with vancomycin (4 µg/mL). We investigated the susceptibility to 12 antimicrobials and the presence of 19 antimicrobial resistance and five virulence genes. In addition, we performed multilocus sequence typing, detection of IS16 and studied Tn1546 structure. One VRE-a isolate was identified in one wild boar. This isolate was identified as Enterococcus faecium, harbored vanA gene included into Tn1546 (truncated with IS1542/IS1216), and belonged to the new ST993. This isolate contained the erm(A), erm(B), tet(M), dfrG, and dfrK genes. Neither element IS16 nor the studied virulence genes were detected. Ninety-six VRE-i isolates were identified (89 Enterococcus gallinarum and seven Enterococcus casseliflavus), with the following prevalence: red kites (71.2 %), white storks (46.9 %), red-legged partridges (7.9 %), and wild boars (4.9 %). Most E. gallinarum isolates showed resistance to tetracycline (66.3 %) and/or erythromycin (46.1 %). High-level resistance to aminoglycosides was present among our VRE-i isolates: kanamycin (22.9 %), streptomycin (11.5 %), and gentamicin (9.4 %). In general, VRE-i isolates of red kites showed higher rates of resistance for non-glycopeptide agents than those of other animal species. The dissemination of acquired resistance mechanisms in natural environments could have implications in the global spread of resistance with public health implications.

Molecular characterization of Staphylococcus aureus isolated from humans related to a livestock farm in Spain, with detection of MRSA-CC130 carrying mecC gene: A zoonotic case?

OBJECTIVES: To conduct a study of Staphylococcus aureus carriage in members of a livestock-farmer's family with different degrees of animal contact, and to characterize the recovered isolates. METHODS: Nasal samples from 11 members of the family were taken in three sampling periods (every six months) (n=31), and 9 skin samples from superficial lesions were also obtained in 5 of them. Samples were analyzed for S. aureus susceptible (MSSA) and resistant to methicillin (MRSA). S. aureus isolates were tested for antibiotic-resistance phenotype and genotype and for the detection of virulence and IEC-system genes. Molecular typing of isolates was also performed (spa- and multilocus-sequence typing). RESULTS: Eighteen S. aureus isolates were recovered (1 MRSA and 17 MSSA) in the 40 samples analyzed. S. aureus was detected in nasal and skin samples of 7/11 and 4/5 of tested humans, respectively. The MRSA strain was detected in the skin lesion of a farmer with high animal contact, and carried the mecC gene, and was typed as ST130-CC130-t843. The 17 MSSA isolates were ascribed to 9 different spa-types and sequence types included in the clonal complexes CC22, CC30, CC45, CC121, and in the livestock-associated lineages CC9 and CC133. Six strains harbored eta or tsst-1 genes. Three of 18 strains lacked the immune-evasion-cluster (IEC) genes (MRSA-ST130, MSSA-ST1333, and MSSA-ST133), and the remaining isolates were ascribed to IEC type-A or -B. CONCLUSIONS: Animal-associated S. aureus lineages were detected in samples of the farmer's family, highlighting the detection of MSSA-CC133 and mecC-MRSA-ST130.

GelJ--a tool for analyzing DNA fingerprint gel images.

BACKGROUND: DNA fingerprinting is a technique for comparing DNA patterns that has applications in a wide variety of contexts. Several commercial and freely-available tools can be used to analyze DNA fingerprint gel images; however, commercial tools are expensive and usually difficult to use; and, free tools support the basic functionality for DNA fingerprint analysis, but lack some instrumental features to obtain accurate results. RESULTS: In this paper, we present GelJ, a feather-weight, user-friendly, platform-independent, open-source and free tool for analyzing DNA fingerprint gel images. Some of the outstanding features of GelJ are mechanisms for accurate lane- and band-detection, several options for computing migration models, a number of band- and curve-based similarity methods, different techniques for generating dendrograms, comparison of banding patterns from different experiments, and database support. CONCLUSIONS: GelJ is an easy to use tool for analyzing DNA fingerprint gel images. It combines the best characteristics of both free and commercial tools: GelJ is light and simple to use (as free programs), but it also includes the necessary features to obtain precise results (as commercial programs). In addition, GelJ incorporates new functionality that is not supported by any other tool.

Nasal carriage of coagulase positive staphylococci in patients of a Primary-Healthcare-Center: genetic lineages and resistance and virulence genes.

INTRODUCTION: Staphylococcus aureus and Staphylococcus pseudintermedius are highly important due to their capacity for producing diseases in humans and animals, respectively. The aim of the study was to investigate and characterize the coagulase positive Staphylococcus (CoPS) carriage in a Primary Healthcare Center population. METHODS: Nasal swabs were obtained from 281 non-infectious patients. The CoPS isolates recovered were typed, and their resistance phenotype and genotype, as well as their virulence profiles, were analyzed. RESULTS: CoPS isolates were recovered from 56/281 patients (19.9%). Fifty-five were S. aureus (19.6%), 54 were methicillin susceptible (MSSA) and one was methicillin resistant (MRSA). The remaining isolate was S. pseudintermedius (0.4%). A high diversity of spa-types (n=40) was detected, with 6 of them being new ones. The multi-locus-sequence-typing of 13 MSSA and one MRSA selected isolates was performed and the STs detected were: ST8, ST15, ST30, ST34, ST121, ST146, ST398, ST554, ST942, ST2499, and ST2500 (the last two STs being new). One MSSA isolate was typed as t1197-ST398-(Clonal complex)CC398. The MRSA isolate was typed as t002-ST146-CC5-SCCmec-IVc, and exhibited a multiresistance phenotype. The detected resistances were: penicillin (76%), macrolides (7%), tetracycline (7%), trimethoprim-sulfamethoxazole (7%), quinolones (7%), and lincosamides (5%). Five isolates contained lukF/lukS-PV genes, 17 tst gene, one eta gene, and two etb gene. The S. pseudintermedius isolate presented a new spa-type (t57) (belonging to a new ST180) and the genes lukS/F-I, siet, se-int, and expB. CONCLUSIONS: A high genetic diversity of S. aureus was detected. Mention must be made of the identification of MSSA CC398 and S. pseudintermedius isolates in two patients, one of them with animal contact. The detection of the genes lukF/lukS-PV and tst should be noted.

Characterization of Staphylococcus aureus from Raw Meat Samples in Tunisia: Detection of Clonal Lineage ST398 from the African Continent.

Livestock-associated Staphylococcus aureus isolates, and especially those belonging to ST398, have been increasingly described in colonized and infected animals and humans, and also in food samples in several countries. The purpose of this study was to determine the frequency of S. aureus and methicillin-resistant S. aureus (MRSA) isolates in raw meat samples destined for food consumption in Tunisia, and to characterize the recovered isolates. One hundred sixty-nine food samples of animal origin were collected. Samples were inoculated onto selective mediums for S. aureus and MRSA recovery. Different molecular typing methods were implemented (pulsed-field gel electrophoresis [PFGE], multilocus sequence typing, spa-, agr-, and SCCmec typing). MRSA was detected in 2 of these 169 samples (1.2%), both of which were of chicken origin. The two MRSA isolates (one/sample) were typed as ST30-CC30-t012-agrIII-SCCmecV and ST398-CC398-t4358-agrI-SCCmecIVa. The MRSA ST398 strain presented resistance, in addition to ß-lactams, to tetracycline (tet[M]) and erythromycin (erm[C]) and harbored the sen, hla, hlg, and hlgv virulence genes. Methicillin-susceptible S. aureus (MSSA) isolates were recovered from 42 of the 169 tested samples (24.8%). A high diversity of spa types (n=21) and SmaI-PFGE patterns (27 different profiles; 4 nontypeable) were detected among MSSA isolates. Four MSSA isolates were typed as ST398/CC398. The percentage of antimicrobial resistance and detected genes in MSSA isolates were as follows: tetracycline (28.6% tet[K] and tet[L]), kanamycin (9.5%, aph[3']-IIIa), tobramycin (2.4%, ant[4']-Ia), erythromycin (14.3%, erm[A], erm[C], msr[A]), and penicillin (95%). The genes lukS-lukF were detected in two MSSA isolates (4.5%), the gene tst in one isolate, and the gene eta in five isolates. To our knowledge, this is the first detection of MRSA and MSSA ST398 in food in an African country. The risk of transmission of S. aureus and MRSA carrying different antimicrobial resistance and virulence genes through the food chain cannot be ignored.