PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Phd thesis

Thesis results summary 

Through the experimental studies conducted in Northern Italy (Piedmont and Lombardy) livestock farms, it was possible to demonstrate the simultaneous presence of two different resistant bacterial groups, extended-spectrum β-lactamase (ESBL)-producing E. coli and methicillin-resistant staphylococci (MRS), that were never detected in combination in Italian poultry and  swine farms. Our results were revelant not only because of the aforementioned novel finding (contemporary presence of ESBL-producing E. coli and MRS), but also because they were carried out in different productive stages, including the farm environment. Taking into account different animal productive phases, it was possible to understand that animals, namely chicks and post-weaning piglets are already coloniesed with MRS since the first days (chicks) or weeks (piglets) of life. This bacterial colonisation can be due to environmental contamination, especially for the staphylococcal species S. sciuri and S. lentus, that were recovered often in the nearby farm environment, or can be linked to piglet to piglet transmission and a maternal colonisation, especially for the swine productive chain. In fact, the highest percentages of MRS were recovered in the youngest animals, weaning and post-weaning piglets, and in sows. Furthermore, some swine MRS possessed a mutated mecA gene, that in some specifical cases (e.g. S. xylosus with G737A nucleotide mutation) was never detected in livestock and human medicine. We recovered other mutations, sometimes in combination, like T675A and G682A, which were sequenced in mecA genes amplified from S. aureus and other staphylococcal species (S. sciuri and S. haemolyticus) from two different productive phases of an intensive swine farm.

Looking to ESBL-producing E. coli in poultry sector, animals were carriers during the first days of life, with an important decrease in the next weeks of the productive cycle. This event can be associated to parental colonisation and in ovo administration of third- generation cephalosporins. The same early carrier status was detected in swine sector, where the highest prevalence of ESBL-producing E. coli was observed during the weaning and post-weaning stages of piglets. For this important resistant bacterial species, we appreciated the potential pathogenicity, using the next generation molecular technique of Whole genome sequencing (WGS). Through WGS it was possible to unravel in samples from healthy animals, the presence of enterotoxins’ associated genes (astA and stb) and various virulence genes linked to other E. coli human and animal pathotypes. Moreover, we found a possible association of ESBL-producing E. coli and MRS prevalence recovered in swine sector, with antibiotic usage at farm level. In fact, we detected higher prevalence for both bacterial groups in animal sectors, such as post-weaning, where antibiotics are more often administered. Although these results were only observed in 4 swine farms, a wider group of farms will be useful to understand if a direct link between antibiotic usage and resistant bacteria can be established. Surprisingly, we recovered oxazolidinone associated resistant genes, cfr and optrA, from different staphylococcal species, in which these genes were never reported in Italy. These results were relevant not only because these genes confer resistance to last resort human antibiotics, such as oxazolidinones, but also because these strains were multidrug resistant, and they were sampled from healthy animals. Further studies are necessary to understand if the aforementioned genes are cromosomally encoded or are located on plasmids, to understand if they can be transmitted among diverse staphylococcal species. Also, it will be important to test farmers not only for these specific resistant staphylococci but also for ESBL-producing E. coli, to comprehend if the same bacterial strains are present in animals and humans at strict contact with them.

Exposure assessment of farm workers to resistant bacteria was also an important part of the thesis. We found which are the most at risk farm practices for MRS and ESBL-producing E. coli exposition (carcasses removal and litter removal)  during daily work in poultry farm; moreover, we unraveled the most dangerous farm practices in swine sector. The study conducted in an intensive farm, revealed that removal of dead pigs >25kg (gilts), fecundation of gilts, and piglets’ tattooing and castration were the most at risk activities for ESBL-producing E. coli exposition; instead for MRS exposition, piglets’ tattooing and castration resulted the riskiest for farmers. All these findings are really important and should be shared with farm workers in order to prevent transmission of above mentioned resistant bacteria and to promote the use of personal protective equipment especially during at risk farm practices.

Considering our experimental studies carried out in poultry and swine productive sectors, our results are extremely important in contributing to fill the gap about AMR at livestock level. This is true especially for MRS, such as S. sciuri or S. xylosus, which are often not considered during AMR surveillance in livestock productive chains, preferring just to monitor MRSA presence. These findings do not represent the end of research in livestock sector, but indicate the need to strenghten AMR surveillance in livestock productive chain, including farm management factors such as antibiotic usage, biosecurity and overall farm hygiene; in fact, all these components can leverage and promote the dissemination of resistant bacteria on farm, as was supported by our studies. Future studies should focus on understanding if horizontal gene transfer can happen easily at farm level among different staphylococcal species (e.g. S. aureus and S. sciuri) that possess mecA or oxazolidinone resistance associated genes. In this way it will be perhaps necessary to include MRS and not only MRSA in the routine sourveillance in livestock sector. Moreover, WGS should be implemented in the official controls not only for the AMR part but also to enlight if important virulence genes are carried by bacterial strains sampled from healthy and asymptomatic animals.

Research activities

Publications, conferences and posters

Bonvegna M, Grego E, Sona B, Stella MC, Nebbia P, Mannelli A, Tomassone L. Research article. Occurrence of Methicillin-Resistant Coagulase-Negative Staphylococci (MRCoNS) and Methicillin-Resistant Staphylococcus aureus (MRSA) from Pigs and Farm Environment in Northwestern Italy. Antibiotics, 2021; 10(6):676. doi: 10.3390/antibiotics10060676.

Bonvegna M, Elena Grego, Laura Tomassone. Abstract publication. Oxazolidinone resistance-associated genes cfr and optrA in MDR CoNS from healthy pigs in Italy. JAC-Antimicrobial Resistance, Volume 4, Issue Supplement_1, February 2022, dlac004.015, https://doi.org/10.1093/jacamr/dlac004.015

Bonvegna M, Dellepiane L, Stella MC, Tomassone L, Nebbia P, Piovano F, Mannelli A. Oral presentation, National Symposium. Detection of methicillin-resistant staphylococci and ESBL- producing Escherichia coli in a conventional broiler farm in North-western Italy. V Simposio SIPA, 12 and 19 November 2020.

Bonvegna M, Sona B, Stella MC, Nebbia P, Mannelli A, Tomassone L. Poster, International Congress. Occurrence of methicillin-resistant coagulase-negative staphylococci and extended-spectrum beta-lactamases producing Escherichia coli in swine farms from North-western Italy. World One Health Congress, 30 October- 3 November 2020.

Bonvegna M, Sona B, Stella MC, Nebbia P, Mannelli A, Tomassone L. Oral presentation, International Conference. Investigation of methicillin-resistant coagulase-negative staphylococci and extended-spectrum beta-lactamases- producing Escherichia coli in swine farms from North-western Italy. 2nd International Conference of European College of Veterinary Microbiology, 25-27 October 2020.

Bonvegna M, Grego E, Tomassone L. Poster, International conference. Oxazolidinone resistance–associated gene cfr in multidrug resistant coagulase negative staphylococci (MDRCoNS) from healthy pigs in Italy. Infection 2021 - British Society for Antimicrobial Chemotherapy (BSAC) Winter Conference, 9-10 December 2021

Bonvegna M, Christensen H, Kristensen T, Kudirkiene E, Elmerdahl Olsen J, Tomassone L. Oral presentation, National conference, Veterinary Department, University of Turin. To be or not to be: ID card of a “non-pathogenic” multi-drug resistant (MDR) Escherichia coli isolated in a piglet. Game of Research, 16 December 2021.

Bonvegna M, Grego E, Tomassone L. Poster, International conference. First detection of the oxazolidinone resistance–associated genes cfr and optrA in methicillin- resistant coagulase negative staphylococci (MRCoNS) from healthy pigs in Italy. 4th International Conference on Animal Health Surveillance (ICAHS4), 3-5 May 2022.

Colombino E, Zoppi S, Alborali GL, Bonvegna M, et al. Research article. Bacterial and Viral Pathogens in Swine of North-West Italy. Journal of Comparative Pathology, 2022; 191. 39. doi: 10.1016/j.jcpa.2021.11.086.