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Research Project
Veterinary and Animal Science Research Centre
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Listeria monocytogenes from Food Products and Food Associated Environments: Antimicrobial Resistance, Genetic Clustering and Biofilm Insights
Publication . Silva, Adriana; Silva, Vanessa; Gomes, João Paulo; Coelho, Anabela; Batista, Rita; Saraiva, Cristina; Esteves, Alexandra; Martins, Ângela; Contente, Diogo; Diaz-Formoso, Lara; Cintas, Luis M.; Igrejas, Gilberto; Borges, Vítor; Poeta, Patrícia
Listeria monocytogenes, a foodborne pathogen, exhibits high adaptability to adverse environmental conditions and is common in the food industry, especially in ready-to-eat foods. L. monocytogenes strains pose food safety challenges due to their ability to form biofilms, increased resistance to disinfectants, and long-term persistence in the environment. The aim of this study was to evaluate the presence and genetic diversity of L. monocytogenes in food and related environmental products collected from 2014 to 2022 and assess antibiotic susceptibility and biofilm formation abilities. L. monocytogenes was identified in 13 out of the 227 (6%) of samples, 7 from food products (meat preparation, cheeses, and raw milk) and 6 from food-processing environments (slaughterhouse-floor and catering establishments). All isolates exhibited high biofilm-forming capacity and antibiotic susceptibility testing showed resistance to several classes of antibiotics, especially trimethoprim-sulfamethoxazole and erythromycin. Genotyping and core-genome clustering identified eight sequence types and a cluster of three very closely related ST3 isolates (all from food), suggesting a common contamination source. Whole-genome sequencing (WGS) analysis revealed resistance genes conferring resistance to fosfomycin (fosX), lincosamides (lin), fluoroquinolones (norB), and tetracycline (tetM). In addition, the qacJ gene was also detected, conferring resistance to disinfecting agents and antiseptics. Virulence gene profiling revealed the presence of 92 associated genes associated with pathogenicity, adherence, and persistence. These findings underscore the presence of L. monocytogenes strains in food products and food-associated environments, demonstrating a high virulence of these strains associated with resistance genes to antibiotics, but also to disinfectants and antiseptics. Moreover, they emphasize the need for continuous surveillance, effective risk assessment, and rigorous control measures to minimize the public health risks associated to severe infections, particularly listeriosis outbreaks. A better understanding of the complex dynamics of pathogens in food products and their associated environments can help improve overall food safety and develop more effective strategies to prevent severe health consequences and economic losses.
Nocturnal Birds of Prey as Carriers of Staphylococcus aureus and Other Staphylococci: Diversity, Antimicrobial Resistance and Clonal Lineages
Publication . Silva, Vanessa; Lopes, Ana Filipa; Soeiro, Vanessa; Caniça, Manuela; Manageiro, Vera; Pereira, José Eduardo; Maltez, Luís; Capelo, José Luis; Igrejas, Gilberto; Poeta, Patrícia
Owls are nocturnal predators that inhabit urbanized and farmlands. They are in direct contact with other animals, both livestock and small wild rodents that they mostly feed on. Staphylococci can be both commensal and pathogenic bacteria that are widespread across the various ecological niches. We aimed to isolate staphylococci from owls and to characterize their antimicrobial resistance, virulence factors and genetic lineages. Swab samples were collected from the throat and cloaca of 114 owls admitted to two rehabilitation centers in Portugal. The identification of staphylococci species was performed by MALDI-TOF. Staphylococci antimicrobial resistance and virulence genes were investigated by means of the disk diffusion method and PCR. Staphylococcus aureus isolates were characterized by MLST, agr and spa-typing. Of the tested animals, 66 isolates were recovered, including 10 different species of staphylococci, of which 25 were coagulase-positive (CoPS) and 41 were coagulase-negative (CoNS). Twenty-three S. aureus were isolated, of which one mecC-MRSA was identified. The isolates were mainly resistant to penicillin, aminoglycosides, clindamycin and tetracycline. mecC-MRSA belonged to ST1245 and spa-type t843 and the remaining S. aureus were ascribed to 12 STs and 15 spa types. A high diversity of clonal lineages was identified among the S. aureus isolated from wild owls. Owls feed mainly on small rodents often exposed to waste and anthropogenic sources, which may explain the moderate prevalence of S. aureus in these animals.
Staphylococcus aureus and Methicillin-Resistant Coagulase-Negative Staphylococci in Nostrils and Buccal Mucosa of Healthy Camels Used for Recreational Purposes
Publication . Silva, Vanessa; Caniça, Manuela; Manageiro, Vera; Verbisck, Newton; Tejedor-Junco, María Teresa; González-Martin, Margarita; Corbera, Juan Alberto; Poeta, Patrícia; Igrejas, Gilberto
Several different species of animals host staphylococci as normal microbiota. These animals can be a source of staphylococci zoonotic infections. People with routine or occupational exposure to infected/colonized animals are at risk of a potential transmission. Therefore, we aimed to investigate the presence of S. aureus and other staphylococci in camels used for recreational purposes as well as their antimicrobial resistance, virulence factors and genetic lineages. A total of 172 samples were collected from 86 healthy camels (nose and mouth) from different farms located in the Canary Islands, Spain. Antimicrobial susceptibility testing was performed against 14 antimicrobial agents. The presence of virulence genes was studied by PCR. Multilocus sequence typing, spa typing and agr typing were performed in all S. aureus isolates. From the 86 camels tested, 42 staphylococci were isolated, of which there were 11 S. aureus, 13 S. lentus, 12 S. sciuri, 3 S. xylosus, S. epidermidis, S. hominis and S. chromogenes. Staphylococci isolates were resistant to penicillin, ciprofloxacin, clindamycin and fusidic acid. All S. aureus isolates harbored the hla, hlb and hld virulence genes. S. aureus isolates were ascribed to three sequence types (STs) and three spa types. All S. aureus isolates belonged to agr type III. Camels from Gran Canaria used in recreational purposes have a moderate prevalence of S. aureus and other coagulase-negative staphylococci. Nevertheless, S. aureus isolates are susceptible to almost all antibiotics tested.
A One Health Approach Molecular Analysis of Staphylococcus aureus Reveals Distinct Lineages in Isolates from Miranda Donkeys (Equus asinus) and Their Handlers
Publication . Silva, Vanessa; Alfarela, Cláudia; Caniça, Manuela; Manageiro, Vera; Nóvoa, Miguel; Leiva, Belen; Kress, Maria; Capelo, José Luís; Poeta, Patrícia; Igrejas, Gilberto
Donkeys (Equus asinus) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate S. aureus and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of S. aureus strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in S. aureus isolates. From the 49 donkey samples, 4 S. aureus (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of S. aureus and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the mecA, aph (3')-IIIa, ant (4')-Ia, tetM, tetK, lnuA, ermB, ermC, dfrA and dfrG genes. S. aureus isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the blaZ, aph (3')-IIIa, tetL, tetM and tetK genes. All S. aureus isolates from donkeys belonged to ST49 and spa-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 spa-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant S. sciuri. S. aureus transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.
Hygiene Indicators and Salmonellae on Surfaces of Swine Carcasses from Two Slaughterhouses in Northern Portugal
Publication . Moura-Alves, Márcio; Carvalho, Marta; Baggio Ribeiro, Deise Helena; Barbosa, Joana; Silveira, Leonor; Pista, Ângela; Pinto, Helena Patrícia; Saraiva, Cristina; Teixeira, Paula; Esteves, Alexandra
Abstract: The monitoring of carcass surface contamination along the slaughter line enables verification of slaughter operation hygiene and the use of good manufacturing practices. Pork meat is a common source of human nontyphoidal salmonellosis, one of the most frequently reported foodborne illnesses worldwide. This study was conducted to gather data on microbial loads before and after evisceration on the surfaces of swine carcasses in two slaughterhouses. The presence of Salmonella enterica was evaluated only after evisceration on carcass surfaces and in livers and floor drains (environmental samples) because pigs are common carriers of this pathogen. The contamination of carcass surfaces was evaluated by delimitation of surface area with sterilized templates (100 cm2), and surface samples were collected with gauze swabs. Total aerobic mesophilic bacteria, Enterobacteriaceae, and Escherichia coli were enumerated. Significant differences (P < 0.05) in counts of mesophilic bacteria, Enterobacteriaceae, and E. coli were found on the external carcass surfaces, with higher counts after evisceration. The neck and abdominal areas had higher levels of mesophilic bacteria, Enterobacteriaceae, and E. coli and a high prevalence of Salmonella. Salmonella was detected in only one of the studied slaughterhouses; 19 (7.3%) of 259 analyzed carcass samples were positive for Salmonella, and Salmonella was detected in two livers and two floor drains. A total of 52 Salmonella isolates (44 from carcasses, 5 from livers, and 3 from drains) were recovered. Three Salmonella serovars (Typhimurium 4,5:i- , Wernigerone, and Derby) were identified, and 53.8% of the 52 isolates were multidrug resistant. The results reveal the need for continuous improvement of slaughtering operations and implementation of good manufacturing practices to ensure the safety of pork produced in Portugal.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
6817 - DCRRNI ID
Funding Award Number
UIDB/00772/2020