PLANT SPECIES’ DEPENDANT IN VITRO IMMUNE RESPONSE AND CHANGES IN THE PORTED BACTERIOME IN SMALL VERSUS LARGE RUMINANTS

Abstract Low-input farms, where cohabitation of various species is frequent, ensure welfare by providing outdoor free-roaming and also offering the opportunity for expression of physiological behaviour of the animals. The study aimed at investigating the bacteriome and the immune potential of cohabitants (bovine and sheep) from a low input farm. The research was carried out on nasal swabs and blood samples from bovine (n=7), and sheep (n=17) cohabiting on the same low-input farm. The swabs were subjected to clasical bacteriology techniques and bichemical identification by API tests (Bio Merieux France). To monitor the in vitro blast transformation capacity of lymphocytes, aliquots of blood were mixed 1:4 with RPMI1640 (Sigma Aldrich, USA), further divided in 200µl aliquots in duplicate in 96 well-plates and supplemented with a mitogen (PHA), alcohol control and alcoholic extract of Symphytum officinale (1.5 µl/well). The plates were incubated at 37⁰C for 72 h. The glucose residue was quantified by spectrophotometry (SUMAL PE2, Karl Zeiss, Jena) and blastogenic indices (SI%) were calculated. The groups were compared by Student’s t test for statistical significance of the results. P. aeruginosa, A. hydophila/caviae, E. cloacae, Pasteurella pneumotropica/M. haemolytica, Sphingomonas paucimobilis were isolated from bovine with an average MAR index of 0.288, while P. oryzihabitans, P. aeruginosa, P. fluorescens/P. putida, E. cloacae, Shigella spp. and Ewingella americana with a MAR index of 0.37 were isolated from sheep. The spontaneous SI was higher in cows (55.7± 10.3%) while PHA induced SI was higher in bovine (53.2±9.21%) and sheep (37.81±5.08%). The Symphytum officinale extract did not influence the SI in either bovine or sheep. Considering the similar influential factors acting on cohabiting animals on a low-input farm, there was a species-specific immune system controlled MAR resistance of the bacteriome, which the Symphytum officinale extract could not influence. Key words: bovine, sheep, bacteriome, in vitro cellular response, MAR index, Symphytum officinal

MICROFLORA PURTATĂ ȘI RISCUL ANTIBIOTICOREZISTENȚEI LA OVINE ÎN CREŞTEREA SEMI-INTENSIVĂ

Cea mai mare parte a florei bacteriene purtate se regăsește în tractul respirator și digestiv al animalelor sănătoase sau al oamenilor și nu exercită nici un efect patogen în circumstanțe fiziologice. Atunci când factorii favorizanți induc diminuarea imunității, tulpinile aparent inofensive pot deveni extrem de patogene pentru animale și consumatori sau oameni care lucrează în sectorul animal. Evaluarea încărcăturii microbiene de portaj la ovine crescute pe paşune în turme mixte cu caprine Identificarea populaţiilor bacteriene din mediul de viaţă a ovinelor Evaluarea rezistenţei la antibiotice a tulpinilor izolate de la ovine, cât şi a bacteriilor din mediu de viaţă a animalelor. Materiale și metode Scopul studiului a fost acela de a identifica și compara tulpinile bacteriene digestive și respiratorii portate de la oi (624) și capre (76) într-un efectiv mixt. Cercetările au fost efectuate pe 30 de animale sănătoase (15 capre și 15 oi) din același efectiv. Tampoanele nazale și rectale au fost cultivate pe bulion simplu timp de 24 de ore la 37°C și inseminare pe plăci de agar cu glucoză pentru încă 24 de ore la 37°C, inoculate și pe agar MacConkey și Chapman. Izolatele au fost identificate prin utilizarea sistemului de testare API 20 E pentru familia Enterobacteriaceae și API 20 Staph. pentru coloniile bacteriene crescute pe mediu Chapman. Testarea sensibilității la antibiotice s-a făcut prin metoda Kirby Bauer. Pentru interpretarea statistică a diferențelor de prevalență a fost utilizat testul exact Fisher (r pentru Windows). Rezultate și discuții Bacteriile identificate în turma mixta de oi şi capre demonstrează că examenul clinic şi diagnosticul de sănătate clinică nu sunt suficiente pentru eliminarea riscului patologic şi zoonotic, avându-se în vedere procentele relativ ridicate de bacterii condiţionat patogene. Există posibilitatea ca gradul mare de încărcare microbiană în saivanele în care sunt adăpostite oile şi caprele să fie datorat lipsei de igienă corespunzătoare, fiind impuse astfel verificări periodice ale parametrilor igienico-sanitari. O importanţă epidemiologică deosebită o au, indiferent de habitat, bacteriile cu potenţial patogen zoonotic cum sunt: Escherichia coli, izolată din toate arealele studiate, Serratia ficaria şi Serratia fonticola izolate de pe păşune. Unele specii bacteriene (Erwinia spp., Pantoea spp., Serratia ficaria), sunt bacterii fitopatogene, care prin acţiunea lor depreciază calitativ furajul animalelor. Notarea aprecierilor de susceptibil, intermediar, rezistent precum şi a variantelor intermediare a acestora (0,1,2,3 şi 4 ) Rezistenţa relativ ridicată la antibiotice din gama penicilinelor demonstrează expunerea îndelungată la acest antibiotic, a cărui eficienţa terapeutică este de multe ori variabilă Cele mai bune rezultate s-au obţinut la gentamicină şi doxiciclină cu toate acestea se recomandă o atenţie deosebită la alegerea unui antibiotic, indicaţia terapeutică fiind bazată pe rezultatele antibiogramei Rezultatele obţinute la testarea meticilinei pun în evidenţă tulpini de Staphylococcus xylosus şi Staphylococcus lentus emergente care se fac remarcate prin rezistenţă deosebită la acest antibiotic Concluzii Bacterii de origine fecală precum E. coli au fost identificate în sistemul respirator al caprelor, dar nu și al oilor, în timp ce tulpinile de E. cloacae au fost reprezentate în tractul respirator al oilor, dar nu și al caprelor. Deși turma a fost mixtă, au existat diferențe între izolatele bacteriene, ceea ce sugerează o susceptibilitate și un comportament de hrănire diferențiat la aceste specii. Cu toate acestea, izolatele bacteriene purtate de la capre și oi clinic sănătoase ar putea exercita efecte patogenice în circumstanțe stresante, subliniind importanța identificării precoce a agenților patogeni și a managementului sanitar precis al persoanelor auzite

A COMPARATIVE EVALUATION OF PLANT EXTRACTS’ INHIBITING EFFECTES ON PORTED BACTERIA IN PIGS AND SMALL RUMINANTS

Abstract Continuously increasing antimicrobial resistance urges for research in identifying new active compounds agains bacterial pathogens. This research aimed to monitor the potential of traditional medicinal plants in efficient control of antibiotic resistant bacteriome in swine and small ruminants raised on low-input outdoor farms from North Western Romania. Aerobic bacterial strains (n=14) originating from the nasal cavities of extensively raised swine and sheep were subjected to biochemical identification (Vitek®2 Compact System) and further tested for susceptibility to antibiotics (n=12, antibiotic classes=6, Kirby-Bauer method). Simultaneously, the aromatogram technique was applied to alcoholic extracts Calendula officinalis, Saturaja hortensis, Coriandrum sativum, Artemisia absinthium, Cucurbita pepo, Allium sativum and essential oils of Anethum graveolens, Zingiber officinale, Geranium spp. and Lavandula angustifolia. The antibiogram indicated a multiple antibiotic resistance (MAR) index > 0.2 in 86% of the pigs and 33% in sheep (overall MAR=0.34 and 0.13, respectively). In pigs the highest average of inhibition diameters was observed with chloramphenicol (20.75±0.92 mm) and norfloxacin (20.68±1.55 mm), while the lowest was shown by cefotaxime (7.5±0.79 mm); in sheep the amikacin was the most efficient. Out of the plant extracts, C. pepo was the most effective in pigs (12 out of 14 strains) while the in sheep, the highest efficacy was recorded for the essential oil of Geranium spp. (26.87±8.2 mm) and the lowest activity was noted for the Anethum graveolens oil (15.04±2.2 mm). These plants could enhance the welfare of the animals by reducing the potentially pathogenic, antibiotic resistante bacterial load, as an alternative to classical antibiotic therapy. These results open the perspective of using essential oils in the prevention of staphylococcal infections. Key words: low input farms, sheep, pigs, plant extracts, antimicrobial resistanc

TAXONOMY INFLUENCES THE EFFICACY OF PLANT EXTRACTS AGAINST ANTIBIOTIC RESISTANT BACTERIOME

Abstract Antimicrobial resistance of the microbiome poses lately a high risk to human and animal patients and also to environment health. The missuse and lack of control in antimicrobial treatments lead to escalation of the antibiotic resistance phenomenon. The research aimed at investigating to which extent the taxonomy of plants intervenes in conditioning the antimicrobial resistance in both ATCC strains (Escherichia coli ATCC 25922 and Pseudomonas spp. 10145) and clinical isolates. The pathogenic bacteria were obtained from animal patients with diverse pathologies and identified for diagnostic purposes by standard microbiological technniques using rapid biochemical tests: GN 24 (Tody Laboratories, România) for Gram-negative, and GP 24 (Tody Laboratories, România) for Gram-positive strains as Escherichia coli, Pseudomonas putrefaciens¸ Pseudomonas fluorescens, and Staphylococcus sciuri, Staphylococcus aureus. The evaluation of both antibiotic susceptibility and the activity of essential oils against multiresistant bacteria were tested by the Kirby-Bauer disc diffusion method using amoxicillin/clavulanic acid, penicillin, imipenem, gentamycin, streptomycin, florfenicol, cefquinome, erythromycin, tylosin, tulathromycin, oxytetracycline, and doxycycline. The assessment included 10 essential oils derived from plants belonging to families Lauraceae (Cinnamomum zeylanicum), Myrtaceae (Melaleuca alternifolia), Geraniaceae (Pelargonium capitatum), Lamiaceae (Mentha piperita, Thymus vulgaris, Lavandula angustifolia, Ocimum basilicum, Salvia glutinosa). The extracts of Lauraceae family showed a significant antimicrobial activity against all strains, at least comparable or even superior to that of the most efficient antibiotics, regardless of their concentration. These results promote the further therapeutic experimentation of cinnamon essential oil as an efficient alternative to antibiotics in clinical trials. Key words: plant taxonomy, essential oils, clinical isolates, ATCC strains, antibiotic resistanc

INCIDENCE OF CUTANEOUS Staphylococcus species IN EXTENSIVELY RAISED SWINE

The commensal/opportunistic bacteriome in various animal species includes potentially pathogenic S. aureus. Widespread in humans, on the skin or mucosae, it induces a highly variable pathology, depending on its toxins and virulence, on the host and also on the environment. Much less is known about other staphylococci and their involvement in swine pathology. This research envisaged the incidence of Staphylococcus spp., in samples collected from extensively raised clinically healthy pigs, of the same age, during the period 2019-2020. The samples (n=49) were provided from farms of different sizes and in different years. Cotton swabs were used to sample secretions from anterior nares of individual pigs, and from the skin behind the ears of the animals. Classical microbiology methods were used to isolate Staphylococcus spp. and the isolates were identified using biochemical tests (API Staph, BioMerieux). Forty species of Staphylococcus were identified: Staphylococcus xylosus (47,5%), Staphylococcus lentus (30%), and Staphylococcus sciuri (22,5%). These bacterias seemed often present as a commensal animal-associated bacteria, but în some cases they could become pathologic în some diseases like bovine mastitis, and exudative epidermitis. Key words: swine, extensive raising, skin, staphylococci, pathogenicity

ANTIBIOTIC PROFILE OF BACTERIA ISOLATED FROM THE SKIN SURFACE FROM EXTENSIVELY RAISED SWINE

Antibiotic resistance of Staphylococcus is increasing worldwide and is becoming a global concern. The present report exposes the study of the antibiotic susceptibility in isolates of Staphylococcus spp obtained from farm pigs. The samples were collected from healthy pigs, from different farms, in the period between 2019-2020. Forty strains of Staphylococcus were isolated from the anterior nares of individual pigs, and from the skin behind the ears of the animals. These isolates were tested against 12 different antimicrobial agents using the Kirby-Bauer disk-diffusion method. The antibiotic susceptibility profile of different bacteria isolates was studied to detect MAR, MDR. Multiple antibiotic resistance (MAR) indices were determined and it was above 0.2 in 24 of the total of 40 bacteria studied. The highest MAR index was detected in Staphylococcus lentus (0.5) and Staphylococcus xylosus (0.41) and 26 (65%) strains was MDR. In conclusion, the high levels of antibiotic resistance generally correlates with high antibiotic usage and antibiotics used are a risk to humans with occupational contact with livestock antibiotics. Key words: staphylococcus, antibiotic resistance, MDR, MAR