Margarida Sousa

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.

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.

Genetic characterisation of extended-spectrum β-lactamases in Escherichia coli isolated from retail chicken products including CTX-M-9 containing isolates: a food safety risk factor

1. Bacterial resistance to β-lactam antibiotics has risen dramatically in Escherichia coli from food animals. In a previous study, 29 randomly selected chicken products, collected in Portugal, were analysed for the presence of extended-spectrum β-lactamases (ESBLs)-producing E. coli; and during this study the genetic characterisation of ESBLs genes was investigated. 2. The presence of genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by PCR followed by sequencing. Additionally, other mechanisms of antimicrobial resistance, phylogenetic groups and the presence of virulence determinants were evaluated among the isolates. 3. β-lactamases genes were identified as follows: bla CTX-M-14 (n = 4), bla CTX-M-1 (n = 2), bla CTX-M-9 (n = 4) and bla TEM-52 (n = 13). Mutations at positions −42, −18, −1, and +58 of ampC promoter region were identified in 4 non-ESBL-producing isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates; the aadA gene detected in 8 of 10 streptomycin-resistant isolates; the aac(3)-II gene in all gentamicin-resistant isolates; the cmlA gene in the chloramphenicol-resistant isolate; and sul1 and/or sul2 and/or sul3 genes were found in all trimethoprim-sulfamethoxazole-resistant isolates. The intI1 gene was detected in 8 trimethoprim-sulfamethoxazole-resistant isolates and the intI2 gene in 4 isolates; one gene cassette arrangements were identified among class 1 integrons (dfrA1 + aadA1) and among the class 2 integrons (dfrA1 + sat2 + aadA1). Among cefotaxime-resistant isolates, 16 belonged to A or B1 phylogenetic groups, while 11 isolates were classified into the D or B2 phylogroups. At least one virulence-associated gene (aer, fimA, or papC) was detected in 74·1% of the cefotaxime-resistant isolates. 4. Because ESBLs-producing bacteria are resistant to a broad range of β-lactams, infections caused by these organisms complicate therapy and limit treatment options.

Water Content Determination in Biodiesel: Optimization of Methodology in Coulometric Karl Fischer Titration

Coulometric Karl Fischer (KF) titration is a highly accurate technique employed for moisture determination in non-aqueous solvents. Methodology for coulometric titration in a KF apparatus coupled to an autosampler oven was optimized in order to improve the accuracy of moisture determination in biodiesel derived from soybean. Several parameters were investigated, and three of them were optimized according to a central composite 23 star experimental design. The highest accuracy for moisture determinations corresponded to a relative standard deviation of 1.48 %. It was reached when the KF titrator was operated with electrode without diaphragm, the sample mass was 4 g, and the oven temperature was set to 170°C. Other parameters, like carrier gas flux and extraction time, presented minimal or no effects for improving the accuracy of moisture determination.

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

Abstract The clonal diversity of extended‐spectrum‐&bgr;‐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.

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 Escherichia coli 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.

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. Jub. Iberian Lynx (Lynx pardinus) photo by Carlos M García (https://www.alguazul.com). Brezo and Brisa. Iberian Lynx (Lynx pardinus). From the Ex-situ Conservation Programme (www.lynxexsitu.es).

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.