Introduction .The incorrect use of antibiotics in veterinary medicine has contributed to reduce the pathogen susceptibility to antibiotics. Through direct contact or food chain the resistant bacteria can be transferred to humans (2). In dairy cows, differently from other production animals, milk discharge during antibiotic administration and related withdrawal time has a direct economical effect on production. This issue has determined a more responsible use of antibiotics. Moreover, in mastitis field antibiotics are used mainly to treat clinical cases and not for prophylaxis, except when blanket dry-cow therapy is applied. For this reason monitoring susceptibility to antibiotic-resistance in dairy cows could identify different trend respect to other food animals. Gram-negative bacteria are frequently cause of clinical mastitis and, as part of enteric flora, they are subjected to the selective pressure of systemic drug treatments. For this reason they are subjected to specific monitoring program for antibiotic resistance. This paper describes a study about in vitro antimicrobial susceptibility of Gram negative bacteria isolated from bovine clinical mastitis in Northern Italy. Materials and Methods Among Gram negative bacteria isolated from bovine clinical mastitis in Northern Italy during the years 2010/2011, 99 isolates were selected: 53 E.coli, 28 Klebsiella spp, 14 Serratia spp, 2 Enterobacter spp. e 2 Citrobacter spp. Pathogens isolation was carried out in milk samples following the National Mastitis Council guideline (3). Species identification was performed through commercial kits API 20 and API 20 NE (Biomerieux Marcy l’Etoile, France). In vitro antimicrobial susceptibility was evaluated using minimal inhibitory concentrations (MICs) broth dilution test, according to the procedure described in Clinical and Laboratory Standards Institute (CLSI) guideline. CLSI resistance breakpoints were used (1). Sensititre susceptibility plates were used according to supplier instructions (Trek Diagnostics System, East Grinstead, UK). The antibiotics used are listed in table 1. After incubation the plates were manually read recording the last dilution able to inhibit bacterial growth. Results and Discussion The 36% of tested E. coli resulted resistant towards ampicillin, while using penicillins associated to clavulanic acid the percentage of resistant isolates declined to 7,5%. Serratia spp isolates demonstrated a higher resistance level to amoxicillin/clavulanic acid, with susceptibility reduced to 74%. Cephalosporins confirmed a very high efficacy in the regards of E coli, as 94,3% of isolates were susceptible to 1st generation cephalosporins and 100% to 3rd generation (ceftiofur and cefpodoxime). Similar results were obtained for Klebsiella spp, while all Serratia spp tested isolates revealed some resistance to 1st generation cephalosporins, maintaining susceptibility to 3rd generation cephalosporins. Among drugs active specifically towards Gram-negatives bacteria, chloramphenicol and sulfonamide associated with trimethoprim demonstrated high efficacy to all tested species. Average susceptibility was 85% for sulfonamide with trimethoprim, and 95% for chloramphenicol. Finally, only one E. coli isolate resulted resistant to fluoroquinolons with an intermediate MIC for marbofloxacin and enrofloxacin, while all the others 98 Gram-negative isolates tested were susceptible (MIC < 0,5 g/mL). Complete data are shown in table 1. Table 1. In vitro susceptibility and resistance of E. coli and Klebsiella spp., Serratia spp., Enterobacter spp. and Citrobacter spp. (joined in “other Gram-negatives”) towards tested antibiotics. Antibiotics Sensible E. coli (%) Resistant E. coli (%) Sensible other Gram-negatives (%) Resistant other Gram-negatives (%) Ampicillin 64,2% 35,8% 30,4% 69,6% Amoxicillin/clavulanic acid 92,5% 7,5% 73,9% 26,1% Amikacin 100,0% 0,0% 100,0% 0,0% Cefpodoxime (3rd gen. Ceph.) 100,0% 0,0% 100,0% 0,0% Ticarcillin/clavulanic acid 94,3% 5,7% 89,1% 10,9% Sulfonamide/thrimethoprim 86,8% 13,2% 89,1% 10,9% Cefazolin (1°st gen. Ceph.) 94,3% 5,7% 63,0% 37,0% Gentamicin 100,0% 0,0% 100,0% 0,0% Imipenem 100,0% 0,0% 100,0% 0,0% Penicillin 0,0% 100,0% 0,0% 100,0% Doxiciclin 75,5% 24,5% 71,7% 28,3% Ceftiofur (3rd gen. Ceph.) 100,0% 0,0% 100,0% 0,0% Marbofloxacin 98,1% 1,9% 100,0% 0,0% Enrofloxacin 98,1% 1,9% 100,0% 0,0% Chloramphenicol 94,3% 5,7% 97,8% 2,2% In conclusion, this study demonstrates that Gram-negative bacteria commonly isolable in case of mastitis are susceptible to most antibiotics, included fluoroquinolons, sulfonamide and chloramphenicol. Reduced susceptibility to some beta-lactams in E.coli are avoided associating clavulanic acid, an inhibitor of beta-lactamases. Serratia spp. is an exception probably due to specific beta-lactamases produced, which are resistant to inhibitors. This findings confirm the importance of milk culture in mastitis cases to achieve an etiological diagnosis, that allows to choose treatment or culling for chronically infected animals. The aims of treatment procedures in farm animal should be avoiding abuse of antibiotic therapy and reducing selection of resistant strains. Periodical survey and detection of resistance phenomenon are recommended. References 1. CLSI. (2008). Performances standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: approved standard – third edition CLSI document M31-A3. CLSI Wayne, PA, (USA): Clinical and Laboratory Standard Institute. 2. Friedman D.B., Kanwat CP, Headrick ML, Patterson NJ, Neely JC, Smith LU. (2007) Importance of prudent antibiotic use on dairy farms in south carolina: a pilot project on farmers’ knowledge, attitudes and practices. Zoonoses Public Health. 54, 366–375. 3.NATIONAL MASTITIS COUNCIL (1999). Laboratory and field handbook on bovine mastitis. National Mastitis Council, Madison WI.

SUSCEPTIBILITY TO MAIN ANTIBIOTICS OF GRAM-NEGATIVE BACTERIA FROM BOVINE MASTITIS IN ITALIAN HERDS / A. Barberio, C. Locatelli, R. Muliari, S. Bonamico, V. Bronzo, P. Moroni. ((Intervento presentato al 52. convegno National Mastitis Council Annual Meeting (NMC) tenutosi a San Diego nel 2013.

SUSCEPTIBILITY TO MAIN ANTIBIOTICS OF GRAM-NEGATIVE BACTERIA FROM BOVINE MASTITIS IN ITALIAN HERDS

C. Locatelli;V. Bronzo;P. Moroni
2013

Abstract

Introduction .The incorrect use of antibiotics in veterinary medicine has contributed to reduce the pathogen susceptibility to antibiotics. Through direct contact or food chain the resistant bacteria can be transferred to humans (2). In dairy cows, differently from other production animals, milk discharge during antibiotic administration and related withdrawal time has a direct economical effect on production. This issue has determined a more responsible use of antibiotics. Moreover, in mastitis field antibiotics are used mainly to treat clinical cases and not for prophylaxis, except when blanket dry-cow therapy is applied. For this reason monitoring susceptibility to antibiotic-resistance in dairy cows could identify different trend respect to other food animals. Gram-negative bacteria are frequently cause of clinical mastitis and, as part of enteric flora, they are subjected to the selective pressure of systemic drug treatments. For this reason they are subjected to specific monitoring program for antibiotic resistance. This paper describes a study about in vitro antimicrobial susceptibility of Gram negative bacteria isolated from bovine clinical mastitis in Northern Italy. Materials and Methods Among Gram negative bacteria isolated from bovine clinical mastitis in Northern Italy during the years 2010/2011, 99 isolates were selected: 53 E.coli, 28 Klebsiella spp, 14 Serratia spp, 2 Enterobacter spp. e 2 Citrobacter spp. Pathogens isolation was carried out in milk samples following the National Mastitis Council guideline (3). Species identification was performed through commercial kits API 20 and API 20 NE (Biomerieux Marcy l’Etoile, France). In vitro antimicrobial susceptibility was evaluated using minimal inhibitory concentrations (MICs) broth dilution test, according to the procedure described in Clinical and Laboratory Standards Institute (CLSI) guideline. CLSI resistance breakpoints were used (1). Sensititre susceptibility plates were used according to supplier instructions (Trek Diagnostics System, East Grinstead, UK). The antibiotics used are listed in table 1. After incubation the plates were manually read recording the last dilution able to inhibit bacterial growth. Results and Discussion The 36% of tested E. coli resulted resistant towards ampicillin, while using penicillins associated to clavulanic acid the percentage of resistant isolates declined to 7,5%. Serratia spp isolates demonstrated a higher resistance level to amoxicillin/clavulanic acid, with susceptibility reduced to 74%. Cephalosporins confirmed a very high efficacy in the regards of E coli, as 94,3% of isolates were susceptible to 1st generation cephalosporins and 100% to 3rd generation (ceftiofur and cefpodoxime). Similar results were obtained for Klebsiella spp, while all Serratia spp tested isolates revealed some resistance to 1st generation cephalosporins, maintaining susceptibility to 3rd generation cephalosporins. Among drugs active specifically towards Gram-negatives bacteria, chloramphenicol and sulfonamide associated with trimethoprim demonstrated high efficacy to all tested species. Average susceptibility was 85% for sulfonamide with trimethoprim, and 95% for chloramphenicol. Finally, only one E. coli isolate resulted resistant to fluoroquinolons with an intermediate MIC for marbofloxacin and enrofloxacin, while all the others 98 Gram-negative isolates tested were susceptible (MIC < 0,5 g/mL). Complete data are shown in table 1. Table 1. In vitro susceptibility and resistance of E. coli and Klebsiella spp., Serratia spp., Enterobacter spp. and Citrobacter spp. (joined in “other Gram-negatives”) towards tested antibiotics. Antibiotics Sensible E. coli (%) Resistant E. coli (%) Sensible other Gram-negatives (%) Resistant other Gram-negatives (%) Ampicillin 64,2% 35,8% 30,4% 69,6% Amoxicillin/clavulanic acid 92,5% 7,5% 73,9% 26,1% Amikacin 100,0% 0,0% 100,0% 0,0% Cefpodoxime (3rd gen. Ceph.) 100,0% 0,0% 100,0% 0,0% Ticarcillin/clavulanic acid 94,3% 5,7% 89,1% 10,9% Sulfonamide/thrimethoprim 86,8% 13,2% 89,1% 10,9% Cefazolin (1°st gen. Ceph.) 94,3% 5,7% 63,0% 37,0% Gentamicin 100,0% 0,0% 100,0% 0,0% Imipenem 100,0% 0,0% 100,0% 0,0% Penicillin 0,0% 100,0% 0,0% 100,0% Doxiciclin 75,5% 24,5% 71,7% 28,3% Ceftiofur (3rd gen. Ceph.) 100,0% 0,0% 100,0% 0,0% Marbofloxacin 98,1% 1,9% 100,0% 0,0% Enrofloxacin 98,1% 1,9% 100,0% 0,0% Chloramphenicol 94,3% 5,7% 97,8% 2,2% In conclusion, this study demonstrates that Gram-negative bacteria commonly isolable in case of mastitis are susceptible to most antibiotics, included fluoroquinolons, sulfonamide and chloramphenicol. Reduced susceptibility to some beta-lactams in E.coli are avoided associating clavulanic acid, an inhibitor of beta-lactamases. Serratia spp. is an exception probably due to specific beta-lactamases produced, which are resistant to inhibitors. This findings confirm the importance of milk culture in mastitis cases to achieve an etiological diagnosis, that allows to choose treatment or culling for chronically infected animals. The aims of treatment procedures in farm animal should be avoiding abuse of antibiotic therapy and reducing selection of resistant strains. Periodical survey and detection of resistance phenomenon are recommended. References 1. CLSI. (2008). Performances standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: approved standard – third edition CLSI document M31-A3. CLSI Wayne, PA, (USA): Clinical and Laboratory Standard Institute. 2. Friedman D.B., Kanwat CP, Headrick ML, Patterson NJ, Neely JC, Smith LU. (2007) Importance of prudent antibiotic use on dairy farms in south carolina: a pilot project on farmers’ knowledge, attitudes and practices. Zoonoses Public Health. 54, 366–375. 3.NATIONAL MASTITIS COUNCIL (1999). Laboratory and field handbook on bovine mastitis. National Mastitis Council, Madison WI.
mastitis; Gram-negative bacteria; antibiotics; susceptibility; MIC
Settore VET/05 - Malattie Infettive degli Animali Domestici
SUSCEPTIBILITY TO MAIN ANTIBIOTICS OF GRAM-NEGATIVE BACTERIA FROM BOVINE MASTITIS IN ITALIAN HERDS / A. Barberio, C. Locatelli, R. Muliari, S. Bonamico, V. Bronzo, P. Moroni. ((Intervento presentato al 52. convegno National Mastitis Council Annual Meeting (NMC) tenutosi a San Diego nel 2013.
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