Bioinformatics study of expression from genomes of epidemiologically related MRSA CC398 isolates from human and wild animal samples.

One of the most important livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) genetic lineages is the clonal complex (CC) 398, which can cause typical S. aureus-associated infections in people. In this work, whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics were applied to study the genetic characteristics of three MRSA CC398 isolates recovered from humans (strains C5621 and C9017), and from an animal (strain OR418). Of the three strains, C9017 presented the broadest resistance genotype, including resistance to fluroquinolone, clindamycin, tiamulin, macrolide and aminoglycoside antimicrobial classes. The scn, sak, and chp genes of the immune evasion cluster system were solely detected in OR418. Pangenome analysis showed a total of 288 strain-specific genes, most of which are hypothetical or phage-related proteins. OR418 had the most pronounced genetic differences. RNAIII (δ-hemolysin) gene was clearly the most expressed gene in OR418 and C5621, but it was not detected in C9017. Significant differences in the proteome profiles were found between strains. For example, the immunoglobulin-binding protein Sbi was more abundant in OR418. Considering that Sbi is a multifunctional immune evasion factor in S. aureus, the results point to OR418 strain having high zoonotic potential. Overall, multiomics biomarker signatures can assume an important role to advance precision medicine in the years to come. SIGNIFICANCE: MRSA is one of the most representative drug-resistant pathogens and its dissemination is increasing due to MRSA capability of establishing new reservoirs. LA-MRSA is considered an emerging problem worldwide and CC398 is one of the most important genetic lineages. In this study, three MRSA CC398 isolates recovered from humans and from a wild animal were analyzed through whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics in order to gather systems-wide omics data and better understand the genetic characteristics of this lineage to identify distinctive markers and genomic features of relevance to public health.

Picolylamine-functionalized benz[e]indole squaraine dyes: Synthetic approach, characterization and in vitro efficacy as potential anticancer phototherapeutic agents.

Squaraine dyes are a family of compounds known for their relevant photophysical and photochemical properties potentially useful as photosensitizing agents. Since pyridines have been introduced into the skeleton of several families of compounds to enhance their pharmacological activity, and this approach had not yet been performed on squaraines, novel dyes derived from benz[e]indole functionalized with picolyl- and dipicolylamine and N-ethyl and -hexyl chains were designed and synthesized. After being fully characterized, their interaction with human albumin was in vitro and in silico evaluated. Dyes were further assessed for their phototoxicity activity, and the most interesting ones were studied regarding cell localization and induction of morphological cell changes, genotoxicity, apoptosis and cell cycle arrest. The molecules with N-ethyl chains showed the greatest in vitro light-dependent cytotoxic effects, particularly the zwitterionic squaraine dye and the one bearing a single pyridine unit, which also exhibited a more significant interaction with human albumin. Phenotypically, the cells incubated with these squaraines became smaller and rounded after irradiation, the effects varying with the tested concentration. Genotoxic effects were observed even without irradiation, being more evident for the N-ethyl picolylamine-derived dye. The fluorescence emitted by Rhodamine 123 largely coincided with that emitted by the dyes, suggesting that they are found preferentially in mitochondria. After irradiation, an increase in the subG1 population was verified by propidium iodide-staining analysis by flow cytometry, indicative of cell death by apoptosis.

The Dynamics of Patient Visits to a Public Hospital Pediatric Emergency Department: A Time-Series Model.

BACKGROUND: The overcrowding of emergency departments (EDs) is an increasingly relevant public health problem. The main aims of this study were to identify and analyze temporal periodicities of a self-referred pediatric ED (PED), correlate them with meteorological and calendar variables and build a robust forecasting model. METHODS: An 8-year administrative data set (2010-2017) of the daily number of admissions to the PED of a public hospital in Lisbon, Portugal, was used (n = 670,379). A time-series model of the daily number of visits was built, including temporal periodicities, the Portuguese school calendar, and a meteorological comfort index (humidex). RESULTS: Several temporal cycles were identified: 1 year (peak in January/February related to respiratory infections in younger children and infants), 6 months (peaks in May and October with an increase in the admissions of older children and adolescents with trauma, gastrointestinal infections and atopic symptoms), 4 months (related to annual school vacations), 1 week (lower admission values on Saturday), and half a week (low from Friday to Monday morning). School calendar and humidex were significantly correlated with daily admissions. The model yielded a mean absolute percentage error of 10.7% ± 1.10% when cross-validation was performed for the full data set. CONCLUSION: Although PED visits are multifactorial, they may be predicted and explained by a relatively small number of variables. Such a model may be easily reproduced in different settings and represents a relevant tool to improve quality in EDs through correctly adapting human resources to ED demand.

Regul-A: A Technological Application for Sensory Regulation of Children with Autism Spectrum Disorder in the Home Context.

(1) Background: Sensory processing disorder is now recognised as a core feature of autism spectrum disorder that influences children's adaptive behaviours, which, in turn, may interfere with their participation in life situations. This study describes the process of developing a technological platform, in the form of an app, to help families regulate children with ASD, aged 3-6 years old, by applying sensory strategies to improve the child's participation in daily routines in the home context. (2) Methods: A focus group formed by four specialised occupational therapists who intervene with children with ASD was selected in order to understand and discuss content that should be included in the app. At a later stage, a group of three was involved to ensure quality and veracity in technological platform elaboration. (3) Results: The purpose of the app, named Regul-A, is to help parents regulate children with ASD regarding their participation in home routines. The sensory strategies provided by the focus group in the three major occupations of the child were the first results obtained, followed by the development of the app structure. (4) Conclusions: The next phase of the study will be the use of the platform by families of children with ASD and occupational therapists. It is believed that, in the future, Regul-A will be used as a tool to gather, analyse and manage data on the occupational performance of children with ASD in the home context, particularly for activities of daily living, sleep, rest and play, facilitating the implementation of strategies and the sharing of information between parents and occupational therapists.

Staphylococci among Wild European Rabbits from the Azores: A Potential Zoonotic Issue?

ABSTRACT: The prevalence and diversity of Staphylococcus species from wild European rabbits (Oryctolagus cuniculus) in the Azores were investigated, and the antibiotic resistance phenotype and genotype of the isolates were determined. Nasal samples from 77 wild European rabbits from São Jorge and São Miguel islands in Azores were examined. Antibiotic susceptibility of the isolates was determined using the Kirby-Bauer disk diffusion method, and the presence of antimicrobial resistance genes and virulence factors was determined by PCR. The genetic lineages of S. aureus isolates were characterized by spa typing and multilocus sequence typing. A total of 49 staphylococci were obtained from 35 of the 77 wild rabbits. Both coagulase-positive (8.2%) and coagulase-negative (91.8%) staphylococci were detected: 4 S. aureus, 17 S. fleurettii, 13 S. sciuri, 7 S. xylosus, 4 S. epidermidis, and 1 each of S. simulans, S. saprophyticus, S. succinus, and S. equorum. The four S. aureus isolates showed methicillin susceptibility and were characterized as spa type t272/CC121, Panton-Valentine leukocidin negative, and hlB positive. Most of the coagulase-negative staphylococci showed resistance to fusidic acid and beta-lactams, and multidrug resistance was identified especially among S. epidermidis isolates. The mecA gene was detected in 20 isolates of the species S. fleurettii and S. epidermidis, associated with the blaZ gene in one S. epidermidis isolate. Five antimicrobial resistance genes were detected in one S. epidermidis isolate (mecA,dfrA,dfrG,aac6'-aph2'', and ant4). Our results highlight that wild rabbits are reservoirs or "temporary hosts" of Staphylococcus species with zoonotic potential, some of them carrying relevant antimicrobial resistances.

MRSA CC398 recovered from wild boar harboring new SCCmec type IV J3 variant.

A methicillin-resistant Staphylococcus aureus CC398 was recovered from a wild female boar (Sus scrofa) in the north of Portugal, in 2013 (Sousa et al. 2017). Whole genome sequencing (WGS) revealed this strain carries a new variant of a mecA-containing staphylococcal chromosomal gene cassette (SCCmec) type IV with an uncommon J3 region. WGS studies can facilitate surveillance and provide more detailed characterization of bacterial clones circulating in the wild, reinforcing the need for a one health perspective to better understand and control antimicrobial resistance.

Methicillin-Resistant Staphylococcus aureus CC398 in Purulent Lesions of Piglets and Fattening Pigs in Portugal.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is an important clinical problem. In 2005, a livestock-associated MRSA clone was described, named CC398, being mostly associated with pigs, and causing colonization and infection in pigs and in related humans. The prevalence of these strains in food-producing pigs raised concerns about the possibility of MRSA-CC398 being a foodborne pathogen. The objective of this study was to investigate the presence of S. aureus and MRSA in 141 carcasses of pigs at three slaughterhouses of Portugal, discarded from the food chain by signs of infection, and to characterize the recovered isolates. Methods: S. aureus isolates were identified by matrix-assisted laser desorption/ionization time-of-flight and they were typed (spa, CC398-clone, and SCCmec). The study of antibiotic resistance and virulence genes, and the detection of immune evasion cluster genes and prophages were performed by PCR and sequencing. Results: Twenty-eight S. aureus were obtained from 141 samples (one/sample, 19.9%), being 22 MRSA and 6 methicillin-susceptible S. aureus (MSSA). All MRSA strains were typed as CC398 and were ascribed to three spa types (t011, t108, and t1451). The SCCmec detected differed according to the spa types of MRSA isolates (SCCmecV: t011 and t108; SCCmecIVa: t1451). The MSSA strains were classified as spa-t1491-ST1-CC1. All the strains contained a wide range of antimicrobial resistance genes, the resistance to tetracycline being the prevalent one. In contrast, the strains contained only a few virulence genes. Among the 6 integrases of phages tested, three were detected: SΦ1, SΦ2, and SΦ7, with variations between MRSA and MSSA strains. Conclusions: MRSA-CC398 is not only a habitual pig colonizer but also an opportunistic pathogen in these animals, and must be controlled at the level of producers and slaughterhouses because of its impact on public health.

Polynomial-Time Algorithm for Learning Optimal BFS-Consistent Dynamic Bayesian Networks.

Dynamic Bayesian networks (DBN) are powerful probabilistic representations that model stochastic processes. They consist of a prior network, representing the distribution over the initial variables, and a set of transition networks, representing the transition distribution between variables over time. It was shown that learning complex transition networks, considering both intra- and inter-slice connections, is NP-hard. Therefore, the community has searched for the largest subclass of DBNs for which there is an efficient learning algorithm. We introduce a new polynomial-time algorithm for learning optimal DBNs consistent with a breadth-first search (BFS) order, named bcDBN. The proposed algorithm considers the set of networks such that each transition network has a bounded in-degree, allowing for p edges from past time slices (inter-slice connections) and k edges from the current time slice (intra-slice connections) consistent with the BFS order induced by the optimal tree-augmented network (tDBN). This approach increases exponentially, in the number of variables, the search space of the state-of-the-art tDBN algorithm. Concerning worst-case time complexity, given a Markov lag m, a set of n random variables ranging over r values, and a set of observations of N individuals over T time steps, the bcDBN algorithm is linear in N, T and m; polynomial in n and r; and exponential in p and k. We assess the bcDBN algorithm on simulated data against tDBN, revealing that it performs well throughout different experiments.

Clonal diversity of extended-spectrum beta-lactamase producing Escherichia coli isolates in fecal samples of wild animals.

The clonal diversity of extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli isolates from nine different species of wild animals from distinct regions of Portugal and Spain and their content in replicon plasmids were analyzed. Among the initial 53 ESBL-producing E. coli isolates that were studied (from previous studies), 28 were selected, corresponding to different animal origins with distinct ESBL types and pulsed-field gel electrophoresis (PFGE) patterns. These 28 isolates produced different ESBLs ascribed to the following families: CTX-M, SHV and TEM. The isolates were classified into three phylogenetic groups: B1 (n = 11), A (n = 10) and D (n = 7). The seven E. coli of phylogroup D were then typed by multilocus sequence typing and ascribed to four distinct sequence types: ST117, ST115, ST2001 and ST69. The clonal diversity and relationship between isolates was studied by PFGE. Lastly, the plasmids were analyzed according to their incompatibility group using the PCR-based-replicon-typing scheme. A great diversity of replicon types was identified, with up to five per isolate. Most of the CTX-M-1 and SHV-12 producing E. coli isolates carried IncI1 or IncN replicons. The diversity of ESBL-producing E. coli isolates in wild animals, which can be disseminated in the environment, emphasizes the environmental and health problems that we face nowadays.

First report on MRSA CC398 recovered from wild boars in the north of Portugal. Are we facing a problem?

The aim of the present study was to evaluate the resistance of Staphylococcus aureus recovered from wild boars, to analyze their genetic lineages, and to investigate the susceptibility to oxacillin. Samples from mouth and nose of 45 wild boars (Sus scrofa) were collected during hunt activity from November 2012 to January 2013 in the North of Portugal. S. aureus isolates were recovered from 30 of these samples (33%); one isolate/sample was further studied. The susceptibility of the isolates was tested by disk-diffusion test against 14 antimicrobial agents and minimal inhibitory concentration was used to test oxacillin according to EUCAST guidelines. The genetic lineages of S. aureus were characterized by agr-typing, spa-typing and MLST. From the 30 isolates, 18 S. aureus were susceptible to all antibiotics tested and 7 presented resistance to one or more of the following antibiotics: penicillin (n=3), oxacillin (n=4), cefoxitin (n=1), clindamycin (n=2), gentamicin (n=1), fusidic acid (n=1), ciprofloxacin (n=2), tetracycline (n=1) and linezolid (n=1). One MRSA CC398 (spa-type t899) isolate was detected (oxacillin MIC=32mg/L and mecA-positive), which presented resistance to penicillin, tetracycline, and ciprofloxacin and contained the genes of immune evasion cluster (IEC) system (type B). The 29 methicillin-susceptible isolates were typed as ST1 (t1533), ST133 (t3583), ST1643 (t10712), ST2328 (t3750) and the new STs (3220, 3222, 3223, 3224) associated to new spa-types t14311 and t14312. The agr-types I, II, III and IV were identified. It is a matter of concern when MRSA and some specific lineages of S. aureus are taken as commensal habitants of the skin and nose of wild animals and are characterized with resistance to various antimicrobial agents in clinical use.

Antimicrobial-resistant Escherichia coli and Enterococcus spp. isolated from Miranda donkey (Equus asinus): an old problem from a new source with a different approach.

PURPOSE: The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals. METHODOLOGY: In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic-resistant and virulent factors was analysed by PCR. RESULTS: A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3')-IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole-resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %). CONCLUSION: To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichiacoli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic-resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

Três médicos e uma mãe / Three doctors and a mother / Tres médicos y una madre

A papilomatose laríngea, doença rara potencialmente fatal, carateriza-se pela proliferação de papilomas no trato respiratório, múltiplos, recorrentes, cuja etiologia é a infeção por papilomavírus humano (HPV). Menina, 21 meses, filha de mãe com serologia positiva para vírus da imunodeficiência humana (VIH) e HPV. Em acompanhamento nas consultas de Pediatria do Desenvolvimento do Hospital, Pediatra Particular e Médico de Família (MF). Aos 18 meses, na consulta de acompanhamento do MF, a mãe preocupada salienta a fala sussurrada e choro rouco da filha com diagnóstico frequente de laringite aguda no Pediatra e MF nos últimos 3 meses, motivando a referenciação à Otorrinolaringologia e posterior diagnóstico de papilomatose laríngea. A abordagem da disfonia na criança evita o uso inapropriado de corticoides, inibidores da bomba de prótons e antibioticoterapia. Neste relato sobressai a desvantagem associada ao seguimento por múltiplos médicos, sendo o MF fundamental para reunir e integrar a informação clínica, permitindo a continuidade de cuidados.

Laryngeal papillomatosis is a rare and potentially fatal disease characterized by the proliferation of recurrent respiratory papillomas, whose etiology is human papillomavirus (HPV) infection. We report a clinical case of a 21-month girl, whose mother is sero-positive to human immunodeficiency virus (HIV) infection and HPV. This girl attended multiple medical consultations: Development Pediatrics (at the hospital), private Pediatrician and General Practitioner (GP). At 18 months, in the GP surveillance consultation, the concerned mother referred whispered talking, hoarse crying and frequent diagnosis of acute laryngitis at the Pediatrician in the last 3 months. She was referenced to otorhinolaryngology with subsequent diagnosis of laryngeal papillomatosis. The approach to childhood dysphonia avoids inappropriate use of corticosteroids, proton pump inhibitors and antibiotics. This report highlights the disadvantage of the surveillance by multiple doctors and the key role of the GP in gathering and integrating clinical information, allowing the continuity of care.

La papilomatosis laríngea, una enfermedad rara y potencialmente mortal, se caracteriza por la proliferación de papilomas respiratorios recurrentes, cuya etiología es la infección por el virus del papiloma humano (VPH). Se relata el caso de una niña de 21 meses, hija de una madre seropositiva al virus de la inmunodeficiencia humana (VIH) y VPH. Vigilada en las consultas de Pediatría de Desarrollo del Hospital, Pediatría Privada y Médico de la Familia (MF). A los 18 meses, en la consulta de vigilancia del MF, la madre preocupada destaca habla susurrada, llanto ronco y diagnóstico frecuente de la laringitis aguda en la pediatra en los últimos 3 meses. Se referenció a otorrinolaringología con posterior diagnóstico de papilomatosis laríngea. El enfoque de la disfonía infantil evita el uso inapropiado de los corticosteroides, inhibidores de la bomba de protones y antibioterapia. En este informe se destaca la desventaja asociada al seguimiento por parte de varios médicos, y el papel clave del MF para reunir e integrar la información clínica, lo que permite la continuidad de la atención.

Genetic Diversity and Antibiotic Resistance Among Coagulase-Negative Staphylococci Recovered from Birds of Prey in Portugal.

Wild animal populations in contact with antimicrobials and antimicrobial resistant bacteria that are daily released into the environment are able to become unintentional hosts of these resistant microorganisms. To clarify this issue, our study evaluated the presence of antibiotic resistance determinants on coagulase-negative staphylococci recovered from birds of prey and studied their genetic relatedness by pulsed-field gel electrophoresis (PFGE). The unusual vga(A) and erm(T) genes, which confer resistance to clindamycin and erythromycin, respectively, were detected in Staphylococcus sciuri or Staphylococcus xylosus strains and the tet(K) gene in Staphylococcus kloosii. The PFGE patterns showed that three S. xylosus (isolated of Strix aluco and Otus scops) and two S. sciuri (recovered from Strix aluco and Milvus migrans) were clonally indistinguishable. These animals could be a source of unusual antimicrobial resistance determinants for highly used antibiotics in veterinary clinical practice.

Biobased monoliths for adenovirus purification.

Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

Acquired antibiotic resistance among wild animals: the case of Iberian Lynx (Lynx pardinus).

The selective pressure generated by the clinical misuse of antibiotics has been the major driving force leading to the emergence of antibiotic resistance among bacteria. Antibiotics or even resistant bacteria are released into the environment and contaminate the surrounding areas. Human and animal populations in contact with these sources are able to become reservoirs of these resistant organisms. Then, due to the convergence between habitats, the contact of wild animals with other animals, humans, or human sources is now more common and this leads to an increase in the exchange of resistance determinants between their microbiota. Indeed, it seems that wildlife populations living in closer proximity to humans have higher levels of antibiotic resistance. Now, the Iberian Lynx (Lynx pardinus) is a part of this issue, being suggested as natural reservoir of acquired resistant bacteria. The emerging public health concern regarding microbial resistance to antibiotics is becoming true: the bacteria are evolving and are now affecting unintentional hosts.

Antimicrobial resistance determinants in Staphylococcus spp. recovered from birds of prey in Portugal.

Antibiotic resistance among wild animals represent an emerging public health concern. The objective of this study was to analyze the staphylococcal nasal microbiota in birds of prey and their content in antimicrobial resistance determinants. Nasal samples from 16 birds of prey were collected, swabs were dipped and incubated into BHI broth [6.5% NaCl] and later seeded on manitol salt agar and oxacillin-resistance screening agar base media. Staphylococcal colonies were isolated from both media and were identified by biochemical and molecular methods. Susceptibility testing to 18 antimicrobial agents was performed by disk-diffusion method. Six of the 16 tested animals carried staphylococci (37.5%) and 7 isolates of the following species were recovered: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus sciuri rodentium, Staphylococcus cohnii urealitycum, and Staphylococcus gallinarum. The S. aureus isolate was penicillin-resistant (with blaZ gene) but methicillin-susceptible and was ascribed to spa-type t012, sequence-type ST30 and agr-type III. The S. epidermidis isolate carried blaZ, mecA, mrs(A/B), mphC, tet(K), drfA, and fusC genes, ica operon, and was typed as ST35. The genes ant6'-Ia, tet(K), tet(L), dfrG, cat221, cat194, and cat223 were detected in S. saprophyticus or S. gallinarum isolates. Birds of prey seem to be a natural reservoir of S. aureus and coagulase-negative staphylococci resistant to multiple antibiotics. Due to the convergence between habitats, the contact between wildlife, other animals and humans is now more common and this involves an increased possibility of interchange of these microorganisms in the different ecosystems.

Clonal diversity of ESBL-producing Escherichia coli in pigs at slaughter level in Portugal.

We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy pigs, and to evaluate their clonality and associated resistance. Forty-nine percent of pigs sampled (n=35/71) in a slaughterhouse in Portugal revealed ESBL-producing E. coli isolates. Most isolates produced CTX-M-1 enzyme (71.4%; n=25/35), followed by CTX-M-9 (11.4%; n=4/35), CTX-M-14 (5.7%; n=2/35), SHV-12 (5.7%; n=2/35), and CTX-M-32 (5.7%; n=2/35). Ninety-four percent of the isolates presented a phenotype of multi-resistance. Most isolates belonged to phylogroups B1 (42.8%; n=15/35) and A (40%; n=14/35). Multilocus sequence typing (MLST) analysis revealed nine sequence types (STs) under six clonal complexes (CCs) and nine singletons, including overrepresentation of CC10 and three new STs (ST2524, ST2525, ST2528). We observed the frequent presence of CTX-M-producing E. coli in pigs at slaughter level, most of them belonging to CC10, commonly recovered from clinical samples.

Gilthead seabream (Sparus aurata) as carriers of SHV-12 and TEM-52 extended-spectrum beta-lactamases-containing Escherichia coli isolates.

In recent years, bacterial resistance to beta-lactam antibiotics has risen dramatically in Escherichia coli isolated from animals that could pass through the food chain to humans. One hundred eighteen fecal samples of Sparus aurata were tested for extended-spectrum beta-lactamase (ESBL)-containing E. coli recovery. Susceptibility to 16 antimicrobial agents was performed by disk diffusion. ESBL-phenotypic detection was carried out by double-disk test, and the presence of genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction and sequencing. The detection of other antimicrobial resistance mechanisms and phylogenetic groups was also performed in recovered isolates as well as their clonal diversity by pulsed-field gel electrophoresis. Five of the 118 fecal samples harbored ESBL-positive E. coli isolates (4.2%), and one isolate per sample was completely characterized. These five ESBL-positive E. coli isolates contained the bla(TEM-52) or bla(SHV-12) genes, as well as a variety of other resistance genes (cmlA, tetA, aadA, sul1, sul2, and sul3). Four isolates harbored class 1 integrons with the following gene cassettes in their variable region: dfrA1 + aadA1 (one isolate) and sat + psp + aadA2 (three isolates). Four unrelated pulsed-field gel electrophoresis patterns were identified among the five ESBL-positive isolates, and they were ascribed to phylogroups A and B1. The intestinal tract of S. aurata might constitute a reservoir of ESBL-producing E. coli isolates.