Longitudinal study of ESBL/AmpC-producing Enterobacterales strains sharing between cohabiting healthy companion animals and humans in Portugal and in the United Kingdom.

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales (ESBL/AmpC-E) are an increasing healthcare problem in both human and veterinary medicine. The aim of this study was to investigate the possible sharing of ESBL/AmpC-E strains between healthy companion animals and humans of the same household in Portugal (PT) and the United Kingdom (UK). In a prospective longitudinal study, between 2018 and 2020, faecal samples were collected from healthy dogs (n=90), cats (n=20) and their cohabiting humans (n=119) belonging to 41 PT and 44 UK households. Samples were screened for the presence of ESBL/AmpC-E and carbapenemase-producing bacteria. Clonal relatedness between animal and human strains was established by using REP-PCR fingerprinting method, followed by whole-genome sequencing (WGS) of selected strains. ESBL/AmpC-E strains were detected in companion animals (PT=12.7%, n=8/63; UK=8.5%, n=4/47) and humans (PT=20.7%, n=12/58; UK=6.6%, n=4/61) in at least one timepoint. REP-PCR identified paired multidrug-resistant ESBL/AmpC-producing Escherichia coli strains from companion animals and owners in two Portuguese households (4.8%) and one UK household (2.3%). WGS analysis of nine E. coli strains from these three households confirmed that interhost sharing occurred only between the two animal-human pairs from Portugal. Three shared strains were identified: one CTX-M-15-producing E. coli strain in a cat-human pair (O15-H33-ST93) and two CTX-M-15- and CTX-M-55/CMY-2-producing E. coli strains, in a dog-human pair (O8:H9-ST410 and O11:H25-ST457, respectively) at different timepoints. These E. coli clonal lineages are human pandemic, highlighting the role of companion animals living in close contact with humans in the dissemination and persistence of antimicrobial resistance in the household environment.

OXA-181-Producing Extraintestinal Pathogenic Escherichia coli Sequence Type 410 Isolated from a Dog in Portugal.

Two multidrug-resistant and carbapenemase-producing Escherichia coli clones of sequence type 410 were isolated from fecal samples of a dog with skin infection on admission to an animal hospital in Portugal and 1 month after discharge. Whole-genome sequencing revealed a 126,409-bp Col156/IncFIA/IncFII multidrug resistance plasmid and a 51,479-bp IncX3 blaOXA-181-containing plasmid. The chromosome and plasmids carried virulence genes characteristic for uropathogenic E. coli, indicating that dogs may carry multidrug-resistant E. coli isolates related to those causing urinary tract infections in humans.

In vitro antimicrobial efficacy of two medical grade honey formulations against common high-risk meticillin-resistant staphylococci and Pseudomonas spp. pathogens.

BACKGROUND: Antimicrobial resistance is a problem in human and animal healthcare. Honey may be used for its wound healing properties and antimicrobial effects. OBJECTIVE: To investigate the antimicrobial activity of two commercially available medical grade honeys (MGHs) against Staphylococcus spp. and Pseudomonas spp. isolates. METHODS AND MATERIALS: Two formulations, MGH1 (40% w/v honey) and MGH2 (80% w/v Manuka honey), were tested in vitro for minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against 11 Staphylococcus and 11 Pseudomonas isolates at low [1.5 × 104  colony forming units (cfu)/well] and high (1.5 × 106  cfu/well) concentrations of inoculum, representing systemic and cutaneous bacterial loads during infection, respectively. RESULTS: MGH2 showed a lower MIC against staphylococci than MGH1, although this was not statistically significant. MGH1 had stronger bactericidal effects against staphylococci than MGH2, although this effect was statistically significant only at the higher bacterial concentration (P < 0.01). For Pseudomonas spp., MGH1 had significantly higher antimicrobial activity (both MIC and MBC) than MGH2 against all isolates tested and at both bacterial concentrations (P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: Both MGHs were effective in vitro against common cutaneous pathogens including meticillin-resistant staphylococci and Pseudomonas species. The higher efficacy of the MGH1 formulation against Pseudomonas and its consistent effects against staphylococci, while containing only half of the amount of honey compared to MGH2, invites further investigation of the mechanisms and clinical applications of MGH1.

Evidence of Sharing of Klebsiella pneumoniae Strains between Healthy Companion Animals and Cohabiting Humans.

This study aimed to characterize the fecal colonization and sharing of Klebsiella pneumoniae strains between companion animals and humans living in close contact. Fecal samples were collected from 50 healthy participants (24 humans, 18 dogs, and 8 cats) belonging to 18 households. Samples were plated onto MacConkey agar (MCK) plates with and without cefotaxime or meropenem supplementation. Up to five K. pneumoniae colonies per participant were compared by pulsed-field gel electrophoresis (PFGE) after XbaI restriction. K. pneumoniae strains with unique pulse types from each participant were characterized for antimicrobial susceptibility, virulence genes, and multilocus sequence type (MLST). Fecal K. pneumoniae pulse types were compared to those of clinical K. pneumoniae strains from animal and human patients with urinary tract infections (n = 104). K. pneumoniae colonization was detected in nonsupplemented MCK in around 38% of dogs (n = 7) and humans (n = 9). K. pneumoniae strains isolated from dogs belonged to sequence type 17 (ST17), ST188, ST252, ST281, ST423, ST1093, ST1241, ST3398, and ST3399. None of the K. pneumoniae strains were multidrug resistant or hypervirulent. Two households included multiple colonized participants. Notably, two colonized dogs within household 15 (H15) shared a strain each (ST252 and ST1241) with one coliving human. One dog from H16 shared one PFGE-undistinguishable K. pneumoniae ST17 strain with two humans from different households; however, the antimicrobial susceptibility phenotypes of these three strains differed. Two main virulence genotypes were detected, namely fimH-1 mrkD ycfM entB kfu and fimH-1 mrkD ycfM entB kpn These results highlight the potential role of dogs as a reservoir of K. pneumoniae to humans and vice versa. Furthermore, to our best knowledge, this is the first report of healthy humans and dogs sharing K. pneumoniae strains that were undistinguishable by PFGE/MLST.

Klebsiella pneumoniae causing urinary tract infections in companion animals and humans: population structure, antimicrobial resistance and virulence genes.

OBJECTIVES: To characterize the population structure, antimicrobial resistance and virulence genes of Klebsiella spp. isolated from dogs, cats and humans with urinary tract infections (UTIs). METHODS: Klebsiella spp. from companion animals (n = 27) and humans (n = 77) with UTI were tested by the disc diffusion method against 29 antimicrobials. Resistant/intermediate isolates were tested by PCR for 16 resistance genes. Seven virulence genes were screened for by PCR. All Klebsiella pneumoniae from companion animals and third-generation cephalosporin (3GC)-resistant isolates from humans were typed by MLST. All Klebsiella spp. were compared after PFGE XbaI macro-restriction using Dice/UPGMA with 1.5% tolerance. RESULTS: bla CTX-M-15 was detected in >80% of 3GC-resistant strains. K. pneumoniae high-risk clonal lineage ST15 predominated in companion animal isolates (60%, n = 15/25). Most companion animal ST15 K. pneumoniae belonged to two PFGE clusters (C4, C5) that also included human strains. Companion animal and human ST15-CTX-M-15 K. pneumoniae shared a fimH-1/mrkD/entB/ycfM/kfu virulence profile, with a few (n = 4) also harbouring the yersiniabactin siderophore-encoding genes. The hospital-adapted ST11 K. pneumoniae clonal lineage was detected in a cat (n = 1) and a human (n = 1); both were MDR, had 81.1% Dice/UPGMA similarity and shared several virulence and resistance genes. Two 3GC-resistant ST348 strains with 86.7% Dice/UPGMA similarity were isolated from a cat and a human. CONCLUSIONS: Companion animals with UTI become infected with high-risk K. pneumoniae clonal lineages harbouring resistance and virulence genes similar to those detected in strains from humans. The ST15-CTX-M-15 K. pneumoniae clonal lineage was disseminated in companion animals with UTI. Caution must be applied by companion animal caretakers to avoid the spread of K. pneumoniae high-risk clonal lineages.

Increase in antimicrobial resistance and emergence of major international high-risk clonal lineages in dogs and cats with urinary tract infection: 16 year retrospective study.

Objectives: To evaluate temporal trends in antimicrobial resistance, over 16 years, in bacteria isolated from dogs and cats with urinary tract infection (UTI) and the clonal lineages of bacteria harbouring critical antimicrobial resistance mechanisms. Methods: Antimicrobial susceptibility testing was conducted for 948 bacteria isolated from dogs and cats with UTI (1999-2014). Resistance mechanisms were detected by PCR, namely ESBL/AmpC in third-generation cephalosporin (3GC)-resistant Escherichia coli and Proteus mirabilis, mecA in methicillin-resistant staphylococci, and aac(6')-Ieaph(2″)-Ia and aph(2″)-1d in high-level gentamicin-resistant (HLGR) enterococci. Resistant bacteria were typed by MLST, and temporal trends in E. coli and Enterobacteriaceae antimicrobial resistance were determined by logistic regression. Results: Enterobacteriaceae had a significant temporal increase in resistance to amoxicillin/clavulanate, 3GCs, trimethoprim/sulfamethoxazole, fluoroquinolones, gentamicin and tetracycline (P < 0.001). An increase in MDR was also detected (P < 0.0001). 3GC resistance was mainly caused by the presence of blaCTX-M-15 and blaCMY-2 in E. coli and the presence of blaCMY-2 in P. mirabilis. Two major 3GC-resistant E. coli clonal lineages were detected: O25b:H4-B2-ST131 and ST648. The mecA gene was detected in 9.2% (n = 11/119) of Staphylococcus spp., including MRSA clonal complex (CC) 5 (n = 2) and methicillin-resistant Staphylococcus epidermidis CC5 (n = 4). A temporal increase in MDR methicillin-resistant Staphylococcus pseudintermedius was detected (P = 0.0069). Some ampicillin-resistant and/or HLGR Enterococcus spp. were found to belong to hospital-adapted CCs, namely Enterococcus faecalis ST6-CC6 (n = 1) and Enterococcus faecium CC17 (n = 8). Conclusions: The temporal increase in antimicrobial resistance and in MDR bacteria causing UTI in dogs and cats creates important therapeutic limitations in veterinary medicine. Furthermore, the detection of MDR high-risk clonal lineages raises public health concerns since companion animals with UTI may contribute to the spread of such bacteria.

Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius Strains Isolated from Small Animals.

Staphylococcus pseudintermedius is often associated with pyoderma, which can turn into a life-threatening disease. The dissemination of highly resistant isolates has occurred in the last 10 years and has challenged antimicrobial treatment of these infections considerably. We have compared the carriage of virulence genes and biofilm formation between methicillin-resistant and methicillin-susceptible S. pseudintermedius (MRSP and MSSP, respectively) isolates and their in vitro gene expression profiles by transcriptome sequencing (RNA-seq). Isolates were relatively unevenly distributed among the four agr groups, and agr type III predominated in MRSP. Five virulence genes were detected in all isolates. Only the spsO gene was significantly associated with MSSP isolates (P = 0.04). All isolates produced biofilm in brain heart infusion broth (BHIB)-4% NaCl. MSSP isolates produced more biofilm on BHIB and BHIB-1% glucose media than MRSP isolates (P = 0.03 and P = 0.02, respectively). Virulence genes encoding surface proteins and toxins (spsA, spsB, spsD, spsK, spsL, spsN, nucC, coa, and luk-I) and also prophage genes (encoding phage capsid protein, phage infection protein, two phage portal proteins, and a phage-like protein) were highly expressed in the MRSP isolate (compared with the MSSP isolate), suggesting they may play a role in the rapid and widespread dissemination of MRSP. This study indicates that MRSP may upregulate surface proteins, which may increase the adherence of MRSP isolates (especially sequence type 71 [ST71]) to corneocytes. MSSP isolates may have an increased ability to form biofilm under acidic circumstances, through upregulation of the entire arc operon. Complete understanding of S. pseudintermedius pathogenesis and host-pathogen signal interaction during infections is critical for the treatment and prevention of S. pseudintermedius infections.

Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period.

OBJECTIVES: The objective of this study was to investigate the evolution of resistance to antimicrobials, corresponding mechanisms and molecular characteristics of Staphylococcus spp., between 1999 and 2014. METHODS: Susceptibility to 38 antimicrobials was determined for 632 clinical staphylococcal isolates obtained from companion animals (dogs, cats, horses and other animals). Twenty antimicrobial resistance genes, including mecA and mecC, were screened by PCR. Methicillin-resistant staphylococci were characterized by spa (Staphylococcus aureus), SCCmec, MLST and PFGE typing. Statistical analyses were performed using SAS v9.3 and differences were considered relevant if P ≤ 0.05. RESULTS: The mecA gene was identified in 74 staphylococcal isolates (11.6%): 11 MRSA (40.7%), 40 methicillin-resistant Staphylococcus pseudintermedius (MRSP; 8.7%) and 23 methicillin-resistant CoNS (26.7%). Resistance to the majority of antimicrobials and the number of mecA-positive isolates increased significantly over time. Eighteen spa types were identified, including two new ones. MRSA isolates were divided into three PFGE clusters that included ST22-IV, ST105-II, ST398-V and ST5-VI. Most methicillin-resistant Staphylococcus epidermidis isolates were of clonal complex (CC) 5, including a new ST, and clustered in eight PFGE clusters. MRSP were grouped into five PFGE clusters and included ST45-NT, ST71-II-III, ST195-III, ST196-V, ST339-NT, ST342-IV and the new ST400-III. Methicillin-resistant Staphylococcus haemolyticus clustered in two PFGE clusters. CONCLUSIONS: The significant increase in antimicrobial-resistant and mecA-positive isolates in recent years is worrying. Furthermore, several isolates are MDR, which complicates antimicrobial treatment and increases the risk of transfer to humans or human isolates. Several clonal lineages of MRSA and methicillin-resistant S. epidermidis circulating in human hospitals and the community were found, suggesting that companion animals can become infected with and contribute to the dissemination of highly successful human clones. Urgent measures, such as determination of clinical breakpoints and guidelines for antimicrobial use, are needed.

European multicenter study on antimicrobial resistance in bacteria isolated from companion animal urinary tract infections.

BACKGROUND: There is a growing concern regarding the increase of antimicrobial resistant bacteria in companion animals. Yet, there are no studies comparing the resistance levels of these organisms in European countries. The aim of this study was to investigate geographical and temporal trends of antimicrobial resistant bacteria causing urinary tract infection (UTI) in companion animals in Europe. The antimicrobial susceptibility of 22 256 bacteria isolated from dogs and cats with UTI was determined. Samples were collected between 2008 and 2013 from 16 laboratories of 14 European countries. The prevalence of antimicrobial resistance of the most common bacteria was determined for each country individually in the years 2012-2013 and temporal trends of bacteria resistance were established by logistic regression. RESULTS: The aetiology of uropathogenic bacteria differed between dogs and cats. For all bacterial species, Southern countries generally presented higher levels of antimicrobial resistance compared to Northern countries. Multidrug-resistant Escherichia coli were found to be more prevalent in Southern countries. During the study period, the level of fluoroquinolone-resistant E. coli isolated in Belgium, Denmark, France and the Netherlands decreased significantly. A temporal increase in resistance to amoxicillin-clavulanate and gentamicin was observed among E. coli isolates from the Netherlands and Switzerland, respectively. Other country-specific temporal increases were observed for fluoroquinolone-resistant Proteus spp. isolated from companion animals from Belgium. CONCLUSIONS: This work brings new insights into the current status of antimicrobial resistance in bacteria isolated from companion animals with UTI in Europe and reinforces the need for strategies aiming to reduce resistance.

Efficacy of medical grade honey in the management of canine otitis externa - a pilot study.

BACKGROUND: The high prevalence of antimicrobial resistance within otic pathogens has created a need for alternative therapies of otitis externa (OE). Evidence suggests that medical grade honey (MGH) may be effective against drug-resistant pathogens. HYPOTHESIS/OBJECTIVES: The efficacy of a commercial MGH compound was assessed in an open clinical trial. We hypothesized that it would be an effective alternative to conventional treatments. ANIMALS: Client-owned dogs (n = 15) with a confirmed diagnosis of infectious OE were enrolled in this pilot study. METHODS: Dogs were prescribed MGH (1 mL daily per ear) until cure was achieved or for a maximum of 21 d. Evaluation was based on weekly clinical scores, cytological progression and owner assessments of pruritus. Swab samples were submitted for culture and susceptibility testing. MGH was tested for biocidal activity against the bacterial isolates. RESULTS: Medical grade honey promoted rapid clinical progress, with 70% of dogs achieving clinical cure between days 7 and 14 and over 90% having resolved by Day 21. There was a decrease in clinical scores throughout the duration of the trial (P < 0.001) and owner-assessed pruritus also decreased significantly (P < 0.05). In vitro assays of the biocidal activity of MGH showed activity against all bacterial isolates, including meticillin-resistant strains of Staphylococcus pseudintermedius (MRSP) and other species of drug-resistant bacteria. CONCLUSION AND CLINICAL IMPORTANCE: Medical grade honey was successful in both clinical and laboratory settings, thus demonstrating its potential of becoming an alternative treatment for canine OE.

Clonal diversity, virulence patterns and antimicrobial and biocide susceptibility among human, animal and environmental MRSA in Portugal.

OBJECTIVES: The objective of this study was to identify the Staphylococcus aureus clonal types currently circulating in animals, humans in contact with animals and the environment in Portugal based on genetic relatedness, virulence potential and antimicrobial/biocide susceptibility. METHODS: Seventy-four S. aureus isolates from pets, livestock, the environment and humans in contact with animals were characterized by SCCmec typing, spa typing, PFGE and CC398-specific PCR, by antimicrobial and biocide susceptibility testing and by detection of resistance genes and genes for efflux pumps. Representative strains were analysed by DNA microarray and MLST. RESULTS: The S. aureus isolates represented 13 spa types and 3 SCCmec types and belonged to three clonal complexes (CC5, CC22 and CC398). Most of the isolates were multiresistant and harboured the resistance genes that explained the resistance phenotype. The qacG and qacJ genes for biocide resistance were detected in 14 isolates (all MRSA CC398), while 4 isolates (3 CC5 and 1 CC22) had insertions in the -10 motif of the norA promoter. Isolates of the clonal lineages associated with pets (CC5 and CC22) harboured specific sets of virulence genes and often a lower number of resistance genes than isolates of the clonal lineage associated with livestock animals (CC398). CONCLUSIONS: We found, for the first time in animals in Portugal, four strains belonging to CC5, including ST105-II, a lineage that has been previously reported as vancomycin-resistant S. aureus in Portugal. Moreover, for the first time the qacG and qacJ genes were detected in MRSA CC398 strains. Active surveillance programmes detecting MRSA not only in livestock animals but also in companion animals are urgently needed.

Influence of Particle Size and Extraction Methods on Phenolic Content and Biological Activities of Pear Pomace.

The main goal of this research was to investigate how particle size influences the characteristics of pear (Pyrus Communis L.) pomace flour and to examine the impact of different pre-treatment methods on the phenolic content and associated bioactivities. Pear pomace flour was fractionated into different particle sizes, namely 1 mm, 710 µm, 180 µm, 75 µm and 53 µm. Then two extraction methods, namely maceration with methanol and two-step extraction with hexane via Soxhlet followed by ultrasound extraction with methanol, were tested. Total phenolic and total flavonoid contents ranged from 375.0 to 512.9 mg gallic acid/100 g DW and from 24.7 to 34.6 mg quercetin/100 g DW, respectively. Two-step extraction provided antioxidant activity up to 418.8 (in FRAP assay) and 340.0 mg Trolox/100 g DW (in DPPH assay). In order to explore various bioactive properties, this study assessed the inhibitory effects of enzymes, specifically α-amylase and ß-glucosidase (associated with antidiabetic effects), as well as angiotensin-converting enzyme (linked to potential antihypertensive benefits). Additionally, the research investigated antibacterial potential against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, revealing significant results (p < 0.05), particularly in the case of the two-step extraction method. This investigation underscores the substantial value of certain food industry wastes, highlighting their potential as bioactive ingredients within the framework of a circular economy.

ESBL/pAmpC-Producing Escherichia coli Causing Urinary Tract Infections in Non-Related Companion Animals and Humans.

Urinary tract infections (UTI) caused by Escherichia coli are frequently diagnosed in humans and companion animals. Extended-spectrum beta-lactamase (ESBL)- and cephalosporinase (pAmpC)-producing Escherichia coli are worldwide-disseminated and frequently multidrug-resistant, hence leading to treatment failure and public health concerns. This study aimed to characterize and compare ESBL/pAmpC-producing E. coli strains causing community-acquired UTI in companion animals and non-related humans. Third-generation cephalosporin (3GC)-resistant E. coli (companion animals n = 35; humans n = 85) isolated from patients with UTI were tested against 14 antimicrobials following CLSI guidelines. PCR-based assays were used to detect the major E. coli phylogenetic groups, pathogenicity associated-islands (PAIs), virulence genes, and ESBLs/pAmpC resistance genes. ESBL/pAmpC-producing E. coli isolates were typed by multi-locus sequence typing (MLST) and PCR. E. coli strains from companion animals and humans shared two MDR high-risk clonal lineages: ST131 and ST648. To the best of our knowledge, this study reports the first description of E. coli ST131 clade C1-M27 and the clonal lineage ST131 clade A in humans with community-acquired UTI in Portugal. Considering that companion animals with UTI are generally treated at home by the owners, measures should be implemented to avoid the spread of multidrug-resistant high-risk clones to humans and their household environment.

Sharing of Clinically Important Antimicrobial Resistance Genes by Companion Animals and Their Human Household Members.

The aims of this study were to implement a rapid easy methodology, to characterize the antimicrobial resistance gene (AMR) gut content associated with Enterobacteriales and staphylococci; and to evaluate statistical association between AMRs present in fecal samples from healthy companion animals and their human household members. Fecal samples were collected from 27 humans and 29 companion animals living in close contact in 20 households. Nineteen healthy humans without daily contact with companion animals were the control group. After DNA extraction, ß-lactamase families and 10 genes of other antimicrobial classes were screened by PCR. Furthermore, third-generation cephalosporin-resistant, carbapenem-resistant, and colistin-resistant Enterobacteriales and methicillin-resistant staphylococci were screened by bacteriological methods. The blaTEM-1B gene with a P3 promotor was the most frequent ß-lactam-resistant gene detected in humans and companion animals from households (33.3%, and 17.2%, respectively). The sul2 was the most frequently shared gene by humans and animals from the same household. In 50% of households at least one AMR was detected simultaneously in companion animal/owner pairs. Healthy humans and companion animals carried several AMRs of clinical importance. To the best our knowledge, this study reports the first detection of the blaSHV-27 gene in fecal samples from healthy humans in Portugal and in Europe.

Epidemiological Study of Pesticide Poisoning in Domestic Animals and Wildlife in Portugal: 2014-2020.

Nowadays the intentional poisoning of domestic and wild animals is a crime in the European Union (EU), but as in the past the poison is still used in rural areas of a number of European countries to kill animals that were considered harmful for human activities. From January 2014 up until October 2020, the Laboratory of Pharmacology and Toxicology of the Faculty of Veterinary Medicine (LFT-FMV) has done the analytical detection of poisoning substances in 503 samples of wildlife and domestic animals and pesticides residues were found in 239 of the samples analyzed. In this retrospective study, toxicology results from domestic species (dog, cat, sheep, cows, and horses), wildlife species (red foxes, birds of prey, lynx, and wild boar), and food baits, are presented. During this period the samples analyzed at the LFT-FMV, were received from all over the country. Analytical detections were performed via solvent extraction followed by thin layer chromatography. Molluscicides (47%, n = 109) and Carbamates (24%, n = 57) were found to be the first category of pesticides involved in intoxications, in both domestic and wild animals, followed by rodenticides (13%, n = 30)-in this group second and third generation, were the most represented; Strychnine is the third (11%, n = 26) even though this pesticide has been banned in Portugal since 1988 and in the European Union since 2006 and finally Organophosphates (5%, n = 11) in the small number. This study allowed to realize that a great number of positive samples involved banned pesticides (i.e., Aldicarb and Strychnine) but, at the same time, many positives cases were due to the exposure to commercially available products (i.e., Methiocarb and Anticoagulant rodenticides). Also, it's possible to identify the areas where domestic species are the most affected (i.e., Setubal and Lisboa) and the areas where the wild animals are the mainly affected species (i.e., Faro, Castelo Branco, and Bragança).

Detection of multidrug resistance and extended-spectrum/plasmid-mediated AmpC beta-lactamase genes in Enterobacteriaceae isolates from diseased cats in Italy.

OBJECTIVES: The aim of this study was to determine the antimicrobial susceptibility of Enterobacteriaceae isolated from cats affected by diseases commonly encountered in practice, and to characterise the third-generation cephalosporin (3GC)-resistance molecular mechanisms involved. METHODS: Clinical samples (n = 100) included 58 rectal swabs from cats with diarrhoea, 31 nasal swabs from cats with clinical signs of upper respiratory tract disease, four ear swabs from cats with otitis, three conjunctival swabs from cats with conjunctivitis, two oral swabs from cats with stomatitis, one swab from a skin abscess and one urine sample from a cat with cystitis. A total of 125 Enterobacteriaceae were isolated from 90 cats. Escherichia coli was the most frequently isolated species (n = 65), followed by Enterobacter species (n = 20), Proteus species (n = 13), Citrobacter species (n = 12) and others (n = 15). Bacterial susceptibility testing was performed with respect to eight antimicrobial classes. Beta (ß)-lactamase genes were identified by PCR and nucleotide sequencing. RESULTS: Overall, the higher frequency of resistance was to amoxicillin-clavulanate (61.3%), trimethoprim/sulfamethoxazole (33.6%) and cefotaxime (32.8%). Thirty-six percent of the isolates (n = 45) were resistant to 3GCs. Of these isolates, 34 were tested by PCR and nucleotide sequencing and 23 were confirmed as encoding ß-lactamase genes. Fourteen 3GC-resistant isolates harboured extended-spectrum ß-lactamases (ESBLs) belonging to groups CTX-M-1 (n = 12, two of which were CTX-M-79), CTX-M-2 (n = 1) and CTX-M-9 (n = 1), as well as SHV-12 (n = 1) and TEM-92 (n = 1). Nine isolates had CMY-2 plasmid-mediated AmpC ß-lactamases (pAmpC). Thirty-one percent (n = 39) of the isolates were multidrug resistant (MDR) and were isolated from 34% (n = 31/90) of the cats. CONCLUSIONS AND RELEVANCE: A high frequency of MDR and ESBL/pAmpC ß-lactamase-producing Enterobacteriaceae were detected among bacteria isolated from a feline population in southern Italy with a variety of common clinical conditions, which poses limitations on therapeutic options for companion animals. We describe the first detection of CTX-M-79 and TEM-92 ESBL genes in isolates from cats.

Clonal relatedness of Proteus mirabilis strains causing urinary tract infections in companion animals and humans.

Proteus mirabilis is a major cause of urinary tract infection (UTI) in humans and companion animals. This study aimed to evaluate the antimicrobial resistance, virulence and clonal relatedness of P. mirabilis isolated from dogs, cats and humans with UTI. P. mirabilis isolated from companion animals (N = 107) and humans (N = 76) with UTI were compared by PFGE analysis after overnight NotI macro-restriction using Dice/UPGMA with a 1.5% tolerance. Strains were characterized for antimicrobial resistance by disk diffusion. Twenty-four resistance genes and four virulence genes were screened by PCR. Thirty-nine clusters (similarity >80%) and 73 single pulse-types were detected. Nine clusters included P. mirabilis isolated from community and hospital patients, including strains with 100% similarity. A high number of clusters (43.6%, n = 17/39) included strains from companion animals and humans. Similarity between some companion animal and human strains varied between 80-100%. One strain from a dog was 100% similar to one human community-acquired P. mirabilis. One P. mirabilis from a cat was found to be 94.7% and 92.4% similar to community and hospital patient strains, respectively. P. mirabilis CMY-2-producers did not cluster all together. Nevertheless, cluster C36 included five P. mirabilis from companion animals (similarity 85.8%-95.7%), of which, four (80%) were multidrug-resistant CMY-2-producers. This study shows that companion animals and humans become infected with closely related P. mirabilis strains. The high number of clusters containing companion animals and human strains points to the zoonotic nature of P. mirabilis. These results underline the potential role of companion animals as reservoirs and in the dissemination of uropathogenic P. mirabilis to humans and vice versa.

Risk Factors for Nasal Colonization by Methicillin-Resistant Staphylococci in Healthy Humans in Professional Daily Contact with Companion Animals in Portugal.

Methicillin-resistant staphylococci (MRS), namely Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP), are opportunistic agents of great importance in human and veterinary medicine. The aims of this study were to investigate the frequency, persistence, and risk factors associated with nasal colonization by MRS in people in daily contact with animals in Portugal. Seventy-nine out of 129 (61.2%) participants were found to be colonized by, at least, one methicillin-resistant (MR) staphylococci species (MR Staphylococcus epidermidis [n = 68], MRSA [n = 19], MR Staphylococcus haemolyticus [n = 7], MRSP [n = 2], and other coagulase-negative staphylococci [n = 4]). Three lineages were identified among the MRSA isolates (n = 7): the major human healthcare clone in Portugal (ST22-t032-IV, n = 3), the livestock-associated MRSA (ST398-t108-V, n = 3), and the New York-/Japan-related clone (ST105-t002-II, n = 1). MRSP isolates belonged to the European clone ST71-II-III. We identified two risk factors for nasal colonization by MRS in healthy humans: (i) being a veterinary professional (veterinarian and veterinary nurse) (p < 0.0001, odds ratio [OR] = 6.369, 95% confidence interval [CI, 2.683-15.122]) and (ii) have contacted with one MRSA- or MRSP-positive animal (p = 0.0361, OR = 2.742, 95% CI [1.067-7.045]). The follow-up study revealed that the majority (85%) remain colonized. This study shows that MRS in veterinary clinical practice is a professional hazard and highlights the need to implement preventive measures to minimize spread.

First report on antimicrobial resistance and molecular characterisation of Salmonella enterica serotype Typhi isolated from human specimens in Luanda, Angola.

OBJECTIVES: Typhoid fever is a common infection in Africa and, despite scarce surveillance reports, its incidence is commonly considered high by the Angolan health system. Drug-resistant Salmonella enterica serotype Typhi has emerged, making antimicrobial susceptibility testing essential to provide clinical guidance. This is the first report analysing the antimicrobial resistance patterns and population structure of the few S. enterica ser. Typhi isolated from patients with typhoid fever in Luanda, Angola. METHODS: Isolates were collected by the Angolan National Institute of Public Health between September 2013 and May 2014. Ten isolates were identified by the API 20E system and serotyping, and the genus was confirmed by PCR. All isolates were tested for antibiotic susceptibility, and the presence of resistance genes [blaCTX-M, blaSHV, blaTEM, blaOXA-1, several plasmid-borne genes encoding AmpC ß-lactamases, sul and qnr genes, dfrIa, dfrA12, aac(6')-Ib, cmlA and floR] were screened by PCR. Isolates were typed by PFGE and MLST. RESULTS: Several isolates were identified with resistance to trimethoprim/sulfamethoxazole (n=6), ß-lactams (n=5) and chloramphenicol (n=1) and reduced susceptibility to ciprofloxacin (n=2). PFGE revealed eight closely related restriction patterns, and MLST grouped these into three sequence types (ST1, ST2 and ST8), with ST2 being predominant. CONCLUSION: This first epidemiological report provides a preliminary description of S. enterica ser. Typhi strains circulating in Luanda and emphasises the need for continuous monitoring of this pathogen to provide information in order to implement better epidemiological strategies for the control of typhoid fever in Angola.