Background: The availability of comprehensive data on the ecology and molecular epidemiology of Staphylococcus aureus/MRSA in wild animals is necessary to understand their relevance in the “One Health” domain. Objective: In this study, we determined the pooled prevalence of nasal, tracheal and/or oral (NTO) Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) carriage in wild animals, with a special focus on mecA and mecC genes as well as the frequency of MRSA and methicillin susceptible S. aureus (MSSA) of the lineages CC398 and CC130 in wild animals. Methodology: This systematic review was executed on cross-sectional studies that reported S. aureus and MRSA in the NTO cavities of wild animals distributed in four groups: non-human primates (NHP), wild mammals (WM, excluding rodents and NHP), wild birds (WB) and wild rodents (WR). Appropriate and eligible articles published (in English) between 1 January 2011 to 30 August 2021 were searched for from PubMed, Scopus, Google Scholar, SciElo and Web of Science. Results: Of the 33 eligible and analysed studies, the pooled prevalence of NTO S. aureus and MRSA carriage was 18.5% (range: 0–100%) and 2.1% (range: 0.0–63.9%), respectively. The pooled prevalence of S. aureus/MRSA in WM, NHP, WB and WR groups was 15.8/1.6, 32.9/2.0, 10.3/3.4 and 24.2/3.4%, respectively. The prevalence of mecC-MRSA among WM/NHP/WB/WR was 1.64/0.0/2.1/0.59%, respectively, representing 89.9/0.0/59.1/25.0% of total MRSA detected in these groups of animals.The MRSA-CC398 and MRSA-CC130 lineages were most prevalent in wild birds (0.64 and 2.07%, respectively); none of these lineages were reported in NHP studies. The MRSA-CC398 (mainly of spa-type t011, 53%), MRSA-CC130 (mainly of spa types t843 and t1535, 73%), MSSA-CC398 (spa-types t571, t1451, t6606 and t034) and MSSA-CC130 (spa types t843, t1535, t3625 and t3256) lineages were mostly reported. Conclusion: Although the global prevalence of MRSA is low in wild animals, mecC-mediated resistance was particularly prevalent among MRSA isolates, especially among WM and WB. Considering the genetic diversity of MRSA in wild animals, they need to be monitored for effective control of the spread of antimicrobial resistance.

The purpose of this study was to analyse the prevalence and genetic characteristics of ESBL and acquired-AmpC (qAmpC)-producing Escherichia coli isolates from healthy and sick dogs in Portugal. Three hundred and sixty-one faecal samples from sick and healthy dogs were seeded on MacConkey agar supplemented with cefotaxime (2 µg/mL) for cefotaxime-resistant (CTXR) E. coli recovery. Antimicrobial susceptibility testing for 15 antibiotics was performed and the ESBL-phenotype of the E. coli isolates was screened. Detection of antimicrobial resistance and virulence genes, and molecular typing of the isolates (phylogroups, multilocus-sequence-typing, and specific-ST131) were performed by PCR (and sequencing when required). CTXRE. coli isolates were obtained in 51/361 faecal samples analysed (14.1%), originating from 36/234 sick dogs and 15/127 healthy dogs. Forty-seven ESBL-producing E. coli isolates were recovered from 32 sick (13.7%) and 15 healthy animals (11.8%). Different variants of blaCTX-M genes were detected among 45/47 ESBL-producers: blaCTX-M-15 (n = 26), blaCTX-M-1 (n = 10), blaCTX-M-32 (n = 3), blaCTX-M-55 (n = 3), blaCTX-M-14 (n = 2), and blaCTX-M-variant (n = 1); one ESBL-positive isolate co-produced CTX-M-15 and CMY-2 enzymes. Moreover, two additional CTXR ESBL-negative E. coli isolates were CMY-2-producers (qAmpC). Ten different sequence types were identified (ST/phylogenetic-group/β-lactamase): ST131/B2/CTX-M-15, ST617/A/CTX-M-55, ST3078/B1/CTX-M-32, ST542/A/CTX-M-14, ST57/D/CTX-M-1, ST12/B2/CTX-M-15, ST6448/B1/CTX-M-15 + CMY-2, ST5766/A/CTX-M-32, ST115/D/CMY-2 and a new-ST/D/CMY-2. Five variants of CTX-M enzymes (CTX-M-15 and CTX-M-1 predominant) and eight different clonal complexes were detected from canine ESBL-producing E. coli isolates. Although at a lower rate, CMY-2 β-lactamase was also found. Dogs remain frequent carriers of ESBL and/or qAmpC-producing E. coli with a potential zoonotic role.

Background: Enterobacteriaceae are major players in the spread of resistance to β-lactam antibiotics through the action of CTX-M β-lactamases. We aimed to analyze the diversity and genetic characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates from patients in a Northern Portuguese hospital. Methods: A total of 62 cefotaxime/ceftazidime-resistant E. coli (n = 38) and K. pneumoniae (n = 24) clinical isolates were studied. Identification was performed by MALDI-TOF MS. Antimicrobial susceptibility testing against 13 antibiotics was performed. Detection of ESBL-encoding genes and other resistance genes, phylogenetic grouping, and molecular typing (for selected isolates) was carried out by PCR/sequencing. Results: ESBL activity was detected in all 62 E. coli and K. pneumoniae isolates. Most of the ESBL-producing E. coli isolates carried a blaCTX-M gene (37/38 isolates), being blaCTX-M-15 predominant (n = 32), although blaCTX-M-27 (n = 1) and blaCTX-M-1 (n = 1) were also detected. Two E. coli isolates carried the blaKPC2/3 gene. The lineages ST131-B2 and ST410-A were detected among the ESBL-producing blood E. coli isolates. Regarding the 24 ESBL-producing K. pneumoniae isolates, 18 carried a blaCTX-M gene (blaCTX-M-15, 16 isolates; blaCTX-M-55, 2 isolates). All K. pneumoniae isolates carried blaSHV genes, including ESBL-variants (blaSHV-12 and blaSHV-27, 14 isolates) or non-ESBL-variants (blaSHV-11 and blaSHV-28, 10 isolates); ten K. pneumoniae isolates also carried the blaKPC2/3 gene and showed imipenem-resistance. ESBL-positive E. coli isolates were ascribed to the B2 phylogenetic group (82%), mostly associated with ST131 lineage and, at a lower rate, to ST410/A. Regarding K. pneumoniae, the three international lineages ST15, ST147, and ST280 were detected among selected isolates. Conclusions: Different ESBL variants of CTX-M (especially CTX-M-15) and SHV-type (specially SHV-12) were detected among CTX/CAZR E. coli and K. pneumoniae isolates, in occasions associated with carbapenemase genes (blaKPC2/3 gene).

The aim of the study was to analyze the mechanisms of resistance in extended-spectrum beta-lactamase (ESBL)- and acquired AmpC (qAmpC)-producing Escherichia coli isolates from healthy and sick cats in Portugal. A total of 141 rectal swabs recovered from 98 sick and 43 healthy cats were processed for cefotaxime-resistant (CTXR) E. coli recovery (in MacConkey agar supplemented with 2 µg/mL cefotaxime). The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method was used for E. coli identification and antimicrobial susceptibility was performed by a disk diffusion test. The presence of resistance/virulence genes was tested by PCR sequencing. The phylogenetic typing and multilocus sequence typing (MLST) were determined by specific PCR sequencing. CTXRE. coli isolates were detected in seven sick and six healthy cats (7.1% and 13.9%, respectively). Based on the synergy tests, 11 of 13 CTXRE. coli isolates (one/sample) were ESBL-producers (ESBL total rate: 7.8%) carrying the following ESBL genes: blaCTX-M-1 (n = 3), blaCTX-M-15 (n = 3), blaCTX-M-55 (n = 2), blaCTX-M-27 (n = 2) and blaCTX-M-9 (n = 1). Six different sequence types were identified among ESBL-producers (sequence type/associated ESBLs): ST847/CTX-M-9, CTX-M-27, CTX-M-1; ST10/CTX-M-15, CTX-M-27; ST6448/CTX-M-15, CTX-M-55; ST429/CTX-M-15; ST101/CTX-M-1 and ST40/CTX-M-1. Three of the CTXR isolates were CMY-2-producers (qAmpC rate: 2.1%); two of them were ESBL-positive and one ESBL-negative. These isolates were typed as ST429 and ST6448 and were obtained in healthy or sick cats. The phylogenetic groups A/B1/D/clade 1 were detected among ESBL- and qAmpC-producing isolates. Cats are carriers of qAmpC (CMY-2)- and ESBL-producing E. coli isolates (mostly of variants of CTX-M group 1) of diverse clonal lineages, which might represent a public health problem due to the proximity of cats with humans regarding a One Health perspective.

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Antimicrobial resistance is an important One Health challenge that encompasses the human, animal, and environmental fields. A total of 111 Escherichia coli isolates previously recovered from manure (n = 57) and indoor air (n = 54) samples from a broiler farm were analyzed to determine their phenotypes and genotypes of antimicrobial resistance and integron characterization; in addition, plasmid replicon analysis and molecular typing were performed in extended-spectrum-beta-lactamase (ESBL) producer isolates. A multidrug-resistance phenotype was detected in 46.8% of the isolates, and the highest rates of resistance were found for ampicillin, trimethoprim–sulfamethoxazole, and tetracycline (>40%); moreover, 15 isolates (13.5%) showed susceptibility to all tested antibiotics. None of the isolates showed imipenem and/or cefoxitin resistance. Twenty-three of the one hundred and eleven E. coli isolates (20.7%) were ESBL producers and carried the blaSHV-12 gene; one of these isolates was recovered from the air, and the remaining 22 were from manure samples. Most of ESBL-positive isolates carried the cmlA (n = 23), tet(A) (n = 19), and aac(6′)-Ib-cr (n = 11) genes. The following genetic lineages were identified among the ESBL-producing isolates (sequence type-phylogroup-clonotype): ST770-E-CH116–552 (n = 12), ST117-B2-CH45–97 (n = 4), ST68-E-CH26–382/49 (n = 3), ST68-E-CH26–49 (n = 1), and ST10992-A/B1-CH11–23/41/580 (n = 4); the latter two were detected for the first time in the poultry sector. At least two plasmid replicon types were detected in the ESBL-producing E. coli isolates, with IncF, IncF1B, IncK, and IncHI1 being the most frequently found. The following antimicrobial resistance genes were identified among the non-ESBL-producing isolates (number of isolates): blaTEM (58), aac(6′)-Ib-cr (6), qnrS (2), aac(3)-II (2), cmlA (6), tet(A)/tet(B) (22), and sul1/2/3 (51). Four different gene-cassette arrays were detected in the variable region of class 1 (dfrA1-aadA1, dfrA12-aadA2, and dfrA12-orf-aadA2-cmlA) and class 2 integrons (sat2-aadA1-orfX). This work reveals the worrying presence of antimicrobial-resistant E. coli in the broiler farm environment, with ESBL-producing isolates of SHV-12 type being extensively disseminated.

Bacteriocins are antimicrobial peptides with relevance in the modulation of human and animal microbiota that have gained interest in biomedical and biotechnological applications. In this study, the production of bacteriocin-like inhibitory substances (BLIS) was tested among a collection of 890 staphylococci of different origins (humans, animals, food, and the environment) and species, both coagulase-positive (CoPS, 238 isolates of 3 species) and coagulase-negative staphylococci (CoNS, 652 isolates of 26 species). Of the 890 staphylococci, 60 (6.7%) showed antimicrobial activity by the spot-on-lawn method against at least one of the 25 indicator bacteria tested. BLIS-producer (BLIS+) isolates were detected in 8.8% of CoPS and 6.0% of CoNS. The staphylococcal species with the highest percentages of BLIS+ isolates were S. chromogenes (38.5%), S. pseudintermedius (26.7%), and S. warneri (23.1%). The production of BLIS was more frequently detected among isolates of pets, wild animals, and food. Moreover, 13 BLIS+ isolates showed wide antimicrobial activiy spectrum, and 7 of these isolates (of species S. aureus, S. pseudintermedius, S. sciuri, and S. hominis) demonstrated antimicrobial activity against more than 70% of the indicator bacteria tested. The genetic characterization (by PCR and sequencing) of the 60 BLIS+ isolates revealed the detection of (a) 11 CoNS and CoPS isolates carrying putative lantibiotic-like genes; (b) 3 S. pseudintermedius isolates harboring the genes of BacSp222 bacteriocin; and (c) 2 S. chromogenes isolates that presented the gene of a putative cyclic bacteriocin (uberolysin-like), being the first report in this CoNS species. Antimicrobial susceptibility testing was performed in BLIS+ isolates and one-third of the CoNS isolates showed susceptibility to all antibiotics tested, which also lacked the virulence genes studied. These BLIS+ CoNS are good candidates for further characterization studies.

The role of the air as a vehicle of bacteria dissemination in the farming environment has been previously reported, but still scarcely studied. This study investigated the bacteria density/diversity of the inside and outside air and of litter samples at a broiler farm. Samples were collected considering two seasons, three outside air distances (50/100/150 m) and the four cardinal directions. Selective media was used for staphylococci, enterococci, and Enterobacteriaceae recovery. A high number of bacteria was detected in the litter (2.9 × 105–5.8 × 107 cfu/g) and in the inside air (>105 cfu/m3), but a low emission of bacteria was evidenced in the outside air (<6 cfu/m3). Moreover, the bacteria detected in the farm’s outside air decreased the further from the farm the sample was taken. A total of 544 isolates were identified by MALDI-TOF (146 from the litter, 142 from inside air and 256 from outside air). From these, 162 staphylococci (14 species; S. saprophyticus 40.7%), 176 Enterobacteriaceae (4 species; E. coli 66%) and 190 enterococci (4 species; E. hirae 83%) were detected. E. hirae was the predominant species, and identical PFGE clones were detected in inside and outside samples. The detection of identical DNA profiles in E. hirae isolates from inside and outside samples suggests the role of the air in bacterial dissemination from the inside of the broiler farm to the immediate environment.