Patrícia Antunes

Incidence of Salmonella from poultry products and their susceptibility to antimicrobial agents.

The incidence of Salmonella in 60 samples of poultry products of national origin available for consumers obtained from two local butcher shops and one canteen of the city of Porto and the susceptibility to antimicrobial agents allowed for human or animal therapy were evaluated. The results show that poultry samples are frequently contaminated with Salmonella (60%), belonging to 10 different serotypes. Salmonella enteritidis and S. hadar were the most prevalent serotypes. In addition, a high number (75%) of the Salmonella isolates was resistant to one or more antimicrobial agents and eight different resistance profiles were recorded. Resistance to nalidixic acid and enrofloxacin was demonstrated for 50% of the isolates and the occurrence of resistant and multiresistant S. enteritidis isolates were less frequent than for S. hadar. This study suggests a high incidence of Salmonella on Portuguese poultry products and shows that they could be a potential vehicle of resistant Salmonella foodborne infections.

Dissemination of Sulfonamide Resistance Genes (sul1, sul2, and sul3) in Portuguese Salmonella enterica Strains and Relation with Integrons

ABSTRACT In 200 sulfonamide-resistant Portuguese Salmonella isolates, 152 sul1, 74 sul2, and 14 sul3 genes were detected. Class 1 integrons were always associated with sul genes, including sul3 alone in some isolates. The sul3 gene has been identified in isolates from different sources and serotypes, which also carried a class 1 integron with aadA and dfrA gene cassettes.

Dissemination of sul3-Containing Elements Linked to Class 1 Integrons with an Unusual 3′ Conserved Sequence Region among Salmonella Isolates

ABSTRACT A sul3 domain (IS440-sul3-orf1-IS26) was found linked to an unusual 3′ conserved sequence region (qacH) of class 1 integrons and detected among nontyphoid Salmonella isolates (n = 47) from different sources. Three types of integrons differing in the gene cassette array (dfrA12-orfF-aadA2-cmlA1-aadA1, dfrA12-orfF-aadA2/1, and estX-psp-aadA2-cmlA1-aadA1) were found associated with this sul3 domain. They were associated with particular clones and specific high-molecular-weight plasmids.

Salmonellosis: the role of poultry meat

Salmonellosis remains one of the most frequent food-borne zoonoses, constituting a worldwide major public health concern. Currently, at a global level, the main sources of infection for humans include meat products, including the consumption of contaminated poultry meat, in spite of the success of Salmonella control measures implemented in food-animal production of industrialized countries. In recent years, a shift in Salmonella serotypes related to poultry and poultry production has been reported in diverse geographical regions, being particularly associated with the spread of certain well-adapted clones. Moreover, antimicrobial resistance in non-typhoidal Salmonella is considered one of the major public health threats related with food-animal production, including the poultry production chain and poultry meat, which is an additional concern in the management of salmonellosis. The circulation of the same multidrug-resistant Salmonella clones and/or identical mobile genetic elements encoding antibiotic resistance genes from poultry to humans highlights this scenario. The purpose of this review was to provide an overview of the role of poultry meat on salmonellosis at a global scale and the main problems that could hinder the success of Salmonella control measures at animal production level. With the increasing globalization of foodstuffs like poultry meat, new problems and challenges might arise regarding salmonellosis control, making new integrated intervention strategies necessary along the food chain.

Spread of multidrug-resistant Enterococcus to animals and humans: an underestimated role for the pig farm environment

OBJECTIVES The aim of this study was to discover the potential role of the pig farm environment in the spread of multidrug-resistant (MDR) Enterococcus strains, including high-risk clones, to animals and humans. METHODS Enterococcus isolates were recovered from a variety of samples (n = 82; swine, feed/medicines/antiseptics and pig farm facilities) from six Portuguese farms, most using antibiotics. Antimicrobial susceptibility/conjugation assays were performed by standard procedures, bacterial identification/screening of antibiotic resistance genes were performed by PCR and clonality was determined using PFGE/multilocus sequence typing. RESULTS Enterococcus isolates resistant to antibiotics (n= 473) were recovered from samples of different origin (swine, feed/antiseptics, animal residues and pig farm facilities), but only the clinically relevant species Enterococcus faecium (n = 171) and Enterococcus faecalis (n = 78) were included for further comprehensive molecular analysis. Isolates resistant to vancomycin, ampicillin, tetracyclines, erythromycin and aminoglycosides were better recovered in Slanetz-Bartley medium with these antibiotics present than in media not supplemented with antibiotics (P < 0.05). E. faecium was more frequently resistant to ampicillin, ciprofloxacin or nitrofurantoin and E. faecalis to tetracyclines, chloramphenicol or aminoglycosides (P < 0.05). Glycopeptide and erythromycin resistance rates were similar in both species. The transfer of resistance to several antibiotics, including vancomycin and ampicillin, was demonstrated. Clones associated with human infections were detected in different samples from the same farm [E. faecium from sequence type (ST) 78 lineage and E. faecalis ST16; manure, waste lagoons, faeces and drinking water] and in geographically distant farms [E. faecium clonal complex (CC) 5; E. faecalis CC21 and ST16]. CONCLUSIONS The pig farm environment has an underestimated potential role in the transmission of MDR Enterococcus to animals and, possibly, to humans. The continuous contact of swine with MDR Enterococcus by different routes (e.g. feed, dust, air and rooms) might decrease the impact of restrictive antibiotic use policies and reinforces the need for different and preliminary interventions at the husbandry management level.

Incidence and susceptibility to antimicrobial agents of Listeria spp. and Listeria monocytogenes isolated from poultry carcasses in Porto, Portugal

The occurrence of Listeria spp. and Listeria monocytogenes in 63 samples of Portuguese poultry carcasses obtained from two local butcher shops and one canteen in the city of Porto, Portugal, and the susceptibility of these bacteria to antimicrobial agents allowed for use in human or animal therapeutics were evaluated. All poultry samples were contaminated with Listeria spp., and L. monocytogenes was isolated from 41% (26 of 63) of the samples. Other Listeria species, including L. innocua, L. welshimeri, and L. seeligeri, were also isolated from poultry samples. A multiplex polymerase chain reaction method was used for the identification of all of the Listeria isolates; this method showed total conformity with the conventional method of biochemical identification and proved to be more reliable, faster, and less arduous. In addition, high percentages of Listeria spp. (84%) and L. monocytogenes (73%) isolates were found to be resistant to one or more antimicrobial agents of different groups, and 12 different resistance profiles were recorded. The frequency of the resistance of L. monocytogenes isolates to enrofloxacin and clindamycin is notable. The results of this study suggest a high incidence of L. monocytogenes on Portuguese poultry products available for consumers and indicate that poultry could be a potential vehicle of foodborne infections due to strains of L. monocytogenes that are resistant to antimicrobial agents.

Salmonella cross-contamination in swine abattoirs in Portugal: Carcasses, meat and meat handlers

In this study the occurrence of Salmonella in swine, pork meat and meat handlers along with their clonal relatedness is evaluated at abattoir level. Samples from the lymph nodes, carcass surface and meat of 100 pigs and 45 meat handlers were collected in eight abattoirs (July 2007-August 2008). Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis (PFGE). From the pigs tested, 42 produced at least one positive sample. A relatively high frequency of Salmonella occurrence was found in the ileoceacal lymph node samples (26.0%), followed by carcass (16.0%) and meat samples (14.0%). However, ileoceacal lymph nodes that test positive for Salmonella are not found to be a predictor of positive test results further on in the process. Besides the slaughterhouse environment, meat handlers were identified as a possible source of subsequent contamination, with 9.3% of the sample testing positive. Diverse Salmonella enterica serotypes were detected, mainly S. Typhimurium and the monophasic variant S. 4,[5],12:i:-, but also S. Derby, S. Rissen, S. Mbandaka, S. London, S. Give, S. Enteritidis and S. Sandiego, in total corresponding to 17 PFGE types. Our results demonstrate that besides a high level of Salmonella swine contamination at pre-harvest level, slaughtering, dressing, cutting and deboning operations are contributing to the occurrence of clinically relevant clones (e.g. S. Typhimurium DT104 and S. 4,[5],12:i:-) in pork products. This study also highlights the possibility of an ongoing Salmonella community being spread by abattoir workers.

Microbiological quality of ready-to-eat salads: an underestimated vehicle of bacteria and clinically relevant antibiotic resistance genes.

The increase demand for fresh vegetables is causing an expansion of the market for minimally processed vegetables along with new recognized food safety problems. To gain further insight on this topic we analyzed the microbiological quality of Portuguese ready-to-eat salads (RTS) and their role in the spread of bacteria carrying acquired antibiotic resistance genes, food products scarcely considered in surveillance studies. A total of 50 RTS (7 brands; split or mixed leaves, carrot, corn) were collected in 5 national supermarket chains in Porto region (2010). They were tested for aerobic mesophilic counts, coliforms and Escherichia coli counts as well as for the presence of Salmonella and Listeria monocytogenes. Samples were also plated in different selective media with/without antibiotics before and after enrichment. The E. coli, other coliforms and Enterococcus recovered were characterized for antibiotic resistance profiles and clonality with phenotypic and genetic approaches. A high number of RTS presented poor microbiological quality (86%--aerobic mesophilic counts, 74%--coliforms, 4%--E. coli), despite the absence of screened pathogens. In addition, a high diversity of bacteria (species and clones) and antibiotic resistance backgrounds (phenotypes and genotypes) were observed, mostly with enrichment and antibiotic selective media. E. coli was detected in 13 samples (n=78; all types and 4 brands; phylogenetic groups A, B1 and D; none STEC) with resistance to tetracycline [72%; tet(A) and/or tet(B)], streptomycin (58%; aadA and/or strA-strB), sulfamethoxazole (50%; sul1 and/or sul2), trimethoprim (50%; dfrA1 or dfrA12), ampicillin (49%; blaTEM), nalidixic acid (36%), ciprofloxacin (5%) or chloramphenicol (3%; catA). E. coli clones, including the widespread group D/ST69, were detected in different samples from the same brand or different brands pointing out to a potential cross-contamination. Other clinically relevant resistance genes were detected in 2 Raoultella terrigena carrying a bla(SHV-2) and 1 Citrobacter freundii isolate with a qnrB9 gene. Among Enterococcus (n=108; 35 samples; Enterococcus casseliflavus--40, Enterococcus faecalis--20, Enterococcus faecium--18, Enterococcus hirae--9, Enterococcus gallinarum--5, and Enterococcus spp.--16) resistance was detected for tetracyclines [6%; tet(M) and/or tet(L)], erythromycin [3%; erm(B)], nitrofurantoin (1%) or ciprofloxacin (1%). The present study places ready-to-eat salads within the spectrum of ecological niches that may be vehicles for antibiotic resistance bacteria/genes with clinical interest (e.g. E. coli-D-ST69; bla(SHV-2)) and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed.

Co-transfer of resistance to high concentrations of copper and first-line antibiotics among Enterococcus from different origins (humans, animals, the environment and foods) and clonal lineages

OBJECTIVES We studied the occurrence of diverse copper (Cu) tolerance genes from Gram-positive bacteria and their co-transfer with antibiotic resistance genes among Enterococcus from diverse sources. METHODS Enterococcus (n = 922) of several species and from human, animal, environment and food samples were included. Antimicrobial and CuSO4 susceptibility and conjugation assays were performed by standard procedures, bacterial screening of Cu and antibiotic resistance genes by PCR, and clonality by PFGE/multilocus sequence typing. RESULTS tcrB and cueO genes occurred in 15% (n = 137/922) and 14% (n = 128/922) of isolates, respectively, with the highest occurrence in piggeries (P < 0.05). They were more frequent among Enterococcus faecium (tcrB: 23% versus 8% in Enterococcus faecalis and 12% in other species; cueO: 25% versus 5% and 9%, respectively; P < 0.05). A correlation between phenotypic and genotypic assays was observed for most E. faecium (CuSO4 MIC50 = 24 mM in tcrB/cueO(+) versus CuSO4 MIC50 = 12 mM in tcrB/cueO(-)), but not for other species. Co-transfer of Cu tolerance (associated with tcrB, cueO or an unknown mechanism) with erythromycin, tetracycline, vancomycin, aminoglycosides or ampicillin resistance was demonstrated. A variety of PFGE types was detected among isolates carrying Cu tolerance mechanisms, some identified in sequence types (STs) often linked to human infections (E. faecium from ST18 and ST78 clonal lineages and E. faecalis clonal complex 2). CONCLUSIONS Cu tolerance might contribute to the selection/maintenance of multidrug-resistant Enterococcus (including resistance to first-line antibiotics used to treat enterococcal infections) due to the use of Cu compounds (e.g. antiseptics/animal feed supplements). The distribution of the multicopper oxidase cueO and the co-transfer of ampicillin resistance along with Cu tolerance genes are described for the first time.

Ready-to-eat street-vended food as a potential vehicle of bacterial pathogens and antimicrobial resistance: An exploratory study in Porto region, Portugal.

The ready-to-eat street vending commerce, as street mobile food vendors, has grown exponentially worldwide, representing in some countries a significant proportion of food consumed by the urban population. However, the microbiological food safety hazards of mobile vending units in industrialized countries are scarcely evaluated. To assess the microbiological quality and safety of this type of food and try to achieve the connection of its contamination with hygienic conditions of food-handlers, we analyzed hotdogs (n = 10), hamburgers (n = 10) and hands (n = 9) from ten street-vending trailers in the Porto region. Food and food-handler samples were tested for Enterobacteriaceae and coliform counts, Escherichia coli and coagulase-positive staphylococci counts/detection and presence of Salmonella. Aerobic mesophilic counts and detection of Listeria monocytogenes (Pulsed Field Gel Electrophoresis-PFGE and serotyping) were also tested in food samples. E. coli isolates were confirmed by MALDI-TOF and characterized for clonality (phylogenetic groups-PhG, PFGE and Multilocus Sequence Typing), antibiotic resistance (disk diffusion, PCR/sequencing) and intestinal pathogenic virulence factors (PCR/sequencing). All food samples presented poor microbiological quality (100% Enterobacteriaceae and coliforms; 20% E. coli (4 hamburgers, 4 trailers) and 20% (2 hamburgers/2 hotdogs, 3 trailers) were positive for L. monocytogenes (2 PFGE-types belonging to serotype 1/2a and 4b). Salmonella and coagulase-positive staphylococci were not detected. Food-handlers carried Enterobacteriaceae and coliforms (100%), E. coli (11%) and/or coagulase-positive staphylococci (44%). E. coli was detected in 12 samples (n = 30-food/food-handlers; phylogenetic groups A0/A1/B1) with 33% resistant to one or more antibiotics. Two multidrug resistant atypical E. coli pathotype strains (astA-ST165(CC165)/food-handler, eaeA-ST327/food) were detected. Three out of eight E. coli clonal lineages [ST409/ST976(CC10)/ST297] and the two L. monocytogenes clones were spread in different samples/trailers, suggesting cross-contamination or a common source of contamination. This exploratory study, in Porto region, showed ready-to-eat street foods from vending trailers as potential vehicles of clinically relevant L. monocytogenes serotypes and/or E. coli carrying clinically relevant virulence/antibiotic resistance features, and food-handlers as a critical risk factor in this expanding food sector.