Antibacterial susceptibility profiles of subclinical mastitis pathogens isolated from cows in Batna and Setif Governorates ( East of Algeria )

Sub-clinical mastitis is a main pathology of dairy husbandry because it is not clinically recognized by the owners and the veterinarians. For this reason, its economic loss is usually underestimated in milk production. This study has been undertaken in order to evaluate the epidemiologic situation of sub-clinicalmastitis in Batna and Setif governorates (East of Algeria). For this purpose, a detailed bacteriological study of all bacterial strains isolated from sub-clinical mastitis followed by a study of their antibacterial susceptibility profiles has been undertaken. 89 bacterial strains distributed as follows were studied: 27 strains of staphylococci among which 23 were coagulase-negative staphylococci (CNS) that are generally incriminated in sub-clinical mastitis. 39 strains of streptococci among which 10 were Lactococcus lactis ssp lactis strains. 23 strains of enterobacteria represented mainly by Escherichia coli (E.coli). All these bacterial strains were isolated from cow milk of 3 different farms. The antibacterial susceptibility profiles have revealed a susceptibility of the isolated strains to a large number of antibiotics mainly to the Neomycin, the Cephalexin and the Spiramycin. Keys words: Sub-clinical mastitis, antibiotic, susceptibility profiles, milk, cow, bacterial strain. Introduction belong to 3 different herds. Milk samples were taken just before the second milking time (afternoon). 89 Sub-clinical mastitis is still the most frequent bacterial strains were isolated from these milk samples and dangerous pathology of dairy herds. The from which 39 strains of streptococci, 27 strains of identification of the bacterial strains agent of this staphylococci and 23 strains of enterobacteria were mastitis in association with the study of their identified. antibacterial susceptibility profiles to the common antibiotics conduct to make the best choice of the Methods antibiotic therapy of mastitis. However, the suggestion The study of the antibacterial susceptibility that the study of the antibacterial susceptibility profiles has been achieved using the diffusion method profiles may serve to eliminate all the antibiotics on agar medium according to the swab method of towards which bacterial strains are resistant is not Kirby Bauer du Comité de l'Antibiogramme de la always true. It is admitted that strains that are SociétéFrançaise de Microbiologie (CA-SFM) susceptible in vitro may be resistant in vivo (Bouchot (Soussy, 2002) or the disc method. The latter consists et al., 1985), the reverse argument is equally possible: in placing on the surface of a culture medium strains that are resistant in vitro may be susceptible in uniformly inoculated with a bacterialstrain. Each disc vivo (Constable and Morin, 2003). The large use of contains a known amount of a known antibiotic. The antibiotics is the most common strategy of control of antibiotic diffuses around the disc with a decreasing mastitis especially during the dry period by gradient of concentration as far as we go far from the intramammary administration of long acting disc. After incubation, a growth inhibition zone is antibiotics in tubes. formed around the disc. The diameter of the growth inhibition zone is as high as the bacterial strain is Materials susceptible to the antibiotic contained in the disc (Faroult, 1998). 50 lacting cows were sampled. These cows Vet. World, 2011, Vol.4(12):537-541 RESEARCH www.veterinaryworld.org Veterinary World, Vol.4 No.12 December 2011 537 Antibacterial susceptibility profiles of subclinical mastitis pathogens isolated in Batna and Setif Governorates The antibacterial susceptibility profiles have been noted either for pathogenic or the total bacterial been performed according to the standards of the CAstrains isolated from cases of subclinical mastitis SFM (1996). (Table 3) . The results are obtained by measuring the Table-2. Frequency of global susceptibility of diameter of the growth inhibition zone around the bacterial strains to some antibiotics (N= 89) antibiotic disc for each isolated bacterial strain. Antibiotic Break Number % % % points (N) resistant intermediary susceptible Results Penicillin 28 29 63 36.5 0 63.5 Amoxicillin 14 21 52 11.54 13.46 75 In the present study many bacterial strains have Kanamycin 15 17 27 11.11 0 88.89 Neomycin 13 18 50 0 0 100 been isolated with four predominant species such as S. Gentamycin 11 17 50 2 10 88 Chloramphenicol 12 22 50 0 16 84 xylosus and E. coli with a frequency of isolation of Oxytetracyclin 14 19 50 26 2 72 17,97% for each and Lactococcus lactis ssp lactis and Spiramycin 19 24 64 26.56 9.37 64.07 Lincomycin 17 21 27 40.74 11.11 48.15 Streptococcus uberis with a frequency of isolation of Rifampicin 24 29 27 7.41 18.52 74.07 Cephalexin 12 18 38 2.63 0 97.37 11,24% for each. Colistin 10 13 21 14.29 52.38 33.33 It is worth noting the presence at a reasonable Cefoxitin 15 25 49 4.08 32.65 63.27 Trimethoprim + 10 16 75 4 2.67 93.33 frequency of other bacterial species mainly Sulfamids Enterococcus (7,86%), Aerococcus viridans and S. Doxycyclin 14 19 64 21.88 10.94 67.18 Streptomycin 13 15 27 3.70 0 96.30 lentus (5, 62%) (Table1). Ciprofloxacin 22 25 50 6 6 88 Cefazolin 12 18 19 26.32 36.84 36.84 Table-1. Frequency of bacterial strains isolation Erythromycin 13 23 58 0 8.62 91.38 from subclinical Mastitis Table-3. Frequency of global susceptibility of Species Number of strains Frequency (%) pathogenic bacterial strainsto some antibiotics Lactococcus lactisssplactis 10 11,24 (N= 79) Streptococcus uberis 10 11,24 Aerococcus viridans 5 5,62 Antibiotic Break Number % % % Gemella morbillorum 2 2,25 points (N) resistant intermediary susceptible Enterococcus 7 7,86 Penicillin 28 29 53 32.07 0 67.93 Streptococcus bovis II 1 3 3,38 Amoxicillin 14 21 43 13.95 16.28 69.77 Streptococcus sanguinis 1 1,12 Kanamycin 15 17 27 11.11 0 88.89 Streptococcus mitis 1 1,12 Neomycin 13 18 50 0 0 100 Staphylococcus aureus 4 4,50 Gentamycin 11 17 50 2 10 88 S. xylosus 16 17,97 Chloramphenicol 12 22 50 0 16 84 S. lentus 5 5,62 Oxytetracyclin 14 19 50 26 2 72 S. hominis 1 1,12 Spiramycin 19 24 54 29.63 9.26 61.11 S. epidermidis 1 1,12 Lincomycin 17 21 27 40.74 11.11 48.15 E.coli 16 17,97 Rifampicin 24 29 27 7.41 18.52 74.07 Proteus 2 2,25 Cephalexin 12 18 28 0 0 100 Enterobacter 4 4,50 Colistin 10 13 21 14.29 52.38 33.33 Serratia 1 1,12 Cefoxitin 15 25 49 4.08 32.65 63.27 Total 89 100 Trimethoprim 10 16 69 2.90 1.45 95.65 + Sulfamids The study of the global susceptibility of the Doxycyclin 14 19 54 20.37 16.67 62.96 Streptomycin 13 15 27 3.70 0 96.30 bacterial strains (Gram+ and Gram-) including Ciprofloxacin 22 25 50 6 6 88 Cefazolin 12 18 19 26.32 36.84 36.84 Lactococcus lactis ssp lactis to the different tested Erythromycin 13 23 48 0 10.42 89.58 antibiotics has revealed a complete susceptibility to the Neomycin (100%), the Cephalexin (97,37%), the The global susceptibility of Gram+ cocci S t rep tomycin (96 ,30%) , the assoc ia t ion including Lactococcus lactis ssp lactis to the tested TrimethoprimSulfamids (93,33%) and to the antibiotics has revealed a complete (100%) susceptibility to the Neomycin and the Amoxicillin. Erythromycin (91,38%). A certain resistance has been Afterwards, we have found the Cephalexin noted to the Lincomycin, the Colistin and the Cefazolin with a rate of 51,85%, 66,37% and 63,16% (97,37%), the Streptomycin and the Chloramphenicol respectively (R+I) (Table 2). (96,30% each), the association TrimethoprimIn contrast, the global susceptibility of Sulfamids (92,73%), the Cefoxitin (92,59%), the pathogenic bacterial strains has also revealed a Erythromycin (91,38%). On the other hand, some complete susceptibility to the Neomycin and the resistance has been noted against the Lincomycin Cephalexin. A high degree of susceptibility has also (51,85% of resistant Gram+ cocci) (Table 4). been showed to the Streptomycin and to the The global susceptibility of staphylococci to the association Trimethoprim-Sulfamids with a rate of tested antibiotics has revealed a complete (100%) 96,30% and 95,65% respectively. It is worth noting susceptibility to the Neomycin. Next, in a declining that the almost the same level of susceptibility has order we have found the Streptomycin, the Ciprofloxacin, www.veterinaryworld.org Veterinary World, Vol.4 No.12 December 2011 538 Antibacterial susceptibility profiles of subclinical mastitis pathogens isolated in Batna and Setif Governorates Microbiologie (CA-SFM). (1996) Technical recommenare responsible of bovine mastitis and the study of dations for in-vitro susceptibility testing. Clinical their susceptibility profiles to the commonly used Microbiology and Infection ; 2, Suppl 1 : S 11 – S 25. antibiotics permit to the veterinarians to make the most 5. Comité de l'Antibiogramme de la Société Française de judicious choice of the antibiotic that is clinically Microbiologie (CA-SFM1). (2008) Groupe de travail : antibiogramme vétérinaire ducomite de l'antibiogramme de active against the isolated pathogenic bacteria. Indeed, la société française de microbiologie. Nouveautés des the epidemiological study of the most frequently recommandations vétérinaires 2008. http://www.sfm.asso.fr/ incriminated bacterial species facilitates the use 6. Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM2). (2010) Recommandations antibiotics during the dry period of the mammary 2010 .SFM (la Société Française de Microbiologie). gland. Consequently, the quantity and the quality of http://www.sfm.asso.fr/ milk production will be greatly improved. 7. Constable P.D., Morin D.E. (2003) Treatment of clinical The present study has revealed a frequency of mastitis: using antimicrobial susceptibility profiles for treatment decisions. Vet. Clin. Food Anim.,19 : 139-155. susceptibility of 100% to the Neomycin and to a less 8. De Oliveira A.P., Watts J.L., Salmon S.A., Aarestrup F.M. extent to the Cephalexin and the Spiramycin. Some (2000) Antimicrobial s


Introduction
belong to 3 different herds.Milk samples were taken just before the second milking time (afternoon).89 Sub-clinical mastitis is still the most frequent bacterial strains were isolated from these milk samples and dangerous pathology of dairy herds.The from which 39 strains of streptococci, 27 strains of identification of the bacterial strains agent of this staphylococci and 23 strains of enterobacteria were mastitis in association with the study of their identified.antibacterial susceptibility profiles to the common antibiotics conduct to make the best choice of the Methods antibiotic therapy of mastitis.However, the suggestion The study of the antibacterial susceptibility that the study of the antibacterial susceptibility profiles has been achieved using the diffusion method profiles may serve to eliminate all the antibiotics on agar medium according to the swab method of towards which bacterial strains are resistant is not Kirby Bauer du Comité de l'Antibiogramme de la always true.It is admitted that strains that are SociétéFrançaise de Microbiologie (CA-SFM) susceptible in vitro may be resistant in vivo (Bouchot (Soussy, 2002) or the disc method.The latter consists et al., 1985), the reverse argument is equally possible: in placing on the surface of a culture medium strains that are resistant in vitro may be susceptible in uniformly inoculated with a bacterialstrain.Each disc vivo (Constable and Morin, 2003).The large use of contains a known amount of a known antibiotic.The antibiotics is the most common strategy of control of antibiotic diffuses around the disc with a decreasing mastitis especially during the dry period by gradient of concentration as far as we go far from the intramammary administration of long acting disc.After incubation, a growth inhibition zone is antibiotics in tubes.
formed around the disc.The diameter of the growth inhibition zone is as high as the bacterial strain is

Materials
susceptible to the antibiotic contained in the disc (Faroult, 1998).50 lacting cows were sampled.These cows The antibacterial susceptibility profiles have been noted either for pathogenic or the total bacterial been performed according to the standards of the CA-strains isolated from cases of subclinical mastitis SFM (1996).
(Table 3) .The results are obtained by measuring the antibiotics has revealed a complete susceptibility to the Neomycin (100%), the Cephalexin (97,37%), the The global susceptibility of Gram+ cocci S t r e p t o m y c i n ( 9 6 , 3 0 % ) , t h e a s s o c i a t i o n including Lactococcus lactis ssp lactis to the tested Trimethoprim-Sulfamids (93,33%) and to the antibiotics has revealed a complete (100%) Erythromycin (91,38%).A certain resistance has been susceptibility to the Neomycin and the Amoxicillin.noted to the Lincomycin, the Colistin and the Afterwards, we have found the Cephalexin Cefazolin with a rate of 51,85%, 66,37% and 63,16% (97,37%), the Streptomycin and the Chloramphenicol respectively (R+I) (Table 2).
(96,30% each), the association Trimethoprim-In contrast, the global susceptibility of Sulfamids (92,73%), the Cefoxitin (92,59%), the pathogenic bacterial strains has also revealed a Erythromycin (91,38%).On the other hand, some complete susceptibility to the Neomycin and the resistance has been noted against the Lincomycin Cephalexin.A high degree of susceptibility has also (51,85% of resistant Gram+ cocci) (Table 4).been showed to the Streptomycin and to the The global susceptibility of staphylococci to the association Trimethoprim-Sulfamids with a rate of tested antibiotics has revealed a complete (100%) 96,30% and 95,65% respectively.It is worth noting susceptibility to the Neomycin.Next, in a declining that the almost the same level of susceptibility has order we have found the Streptomycin, the Ciprofloxacin, ) and the Gentamycin and the Cefoxitin negative staphylococci (CNS) it was complete (100%) (96,60% each).The some proportion of resistance has for the Neomycin and the Ciprofloxacin.Afterwards, been shown against the Lincomycin (51,85%) (Table 5).
we have found the Chloramphenicol, the Streptomycin and the combination Trimethoprim-Sulfamids revealed a complete susceptibility (100%) of Staphylococcus aureus strains to many antibiotics The global susceptibility of enterobacteria have including the Penicillin, the Kanamycin, the revealed a complete susceptibility (100%) to the Neomycin, the Gentamycin, the Chloramphenicol, the Neomycin and the Ciprofloxacin.Next, in a declining Lincomycin, the Cefoxitin, the combination order we have found the Chloramphenicol (95,65%) Trimethoprim-Sulfamids, the Doxycyclin, the and the association Trimethoprim-Sulfamids (95%).Ciprofloxacin and the Erythromycin.However, 75% Indeed, enterobacteria has shown a susceptibility the and 50% of susceptibility has been shown to both the the Amoxycillin (81,25%) the Colistin (66,67%) and Oxytetracyclin, the Streptomycin and the Rifampicin the Cefazolin (63,16%) (Table 8).respectively (Table 6).
The present study has revealed the Streptococci ones a very low frequency of resistant strains has been noted (Guerin-Faublee and Brun, 1999).However, strains of bovine mastitis origin were more susceptible some cases of resistance have been reported in Greece to most antibiotics compared the staphylococci or the on strains of bovine origin (Fthenakis, 1998).Indeed, enterobacteria.For example, a complete (100%) Erskin et al. (2002), have reported an increase of susceptibility has been noted for the Amoxicillin and susceptibility to the Erythromycin of strains of to the Cephalexin (97,36%).
S.aureus isolated from cases of bovine mastitis in the A degree of resistance (43,24%) has been shown USA.This finding is in complete accordance with the against the Doxycyclin.Lactococcus lactis ssp lactis results of the present study.Perreten et al. (1998) have strains are more susceptible to antibiotics than the revealed that strains of coagulase-negative streptococci strains.A complete susceptibility (100%) staphylococci showed resistance towards the has been noted to the Amoxicillin and the chloramphenicol and the Lincomycin.This finding Erythromycin (Table 9).confirm our results only for the Lincomycin (with a have showed 30% of resistance to the Amoxycillin (Onerba, 2006).

Discussion
C. Streptococci: According to the data published A. Staphylococci: Most of the studied strains have before 2000, concerning Streptococcus uberis, the expressed an important susceptibility to the antibiotic frequency of resistance to the Erythromycin varied used.This observation is in accordance with some from 2 to 25% and from 8 to 13% to the Spiramycin previous studies (De Oliveira et al., 2000; Erskine et (Bouveron, 2000).In France, during 1999France, during -2000France, during , the al., 2002)).
frequency of resistance to the Erythromycin reached In France, according to the data of the Resapath 25 to 26% according to data published by the Afssa (Afssa, 2000), 86% of the bovine strains of coagulase-(Afssa, 2000).In the present study, the frequency of positive staphylococci (CPS) and 91% of coagulase resistance to the Erythromycin and the Spiramycin negative staphylococci (CNS) isolated during 1999was 18,18% and 41,38% respectively.This increase in 2000 (most of which were isolated from mammary the frequency of resistance may be explained by the infections) were susceptible to the Erythromycin, 30% high frequency of isolation of Streptococcus uberis as and 49% to the Spiramycin and 96% and 73% to the it represented 34,48% of the total isolated streptococci.Lincomycin respectively.These results are in accordance Conclusion with our findings except for the Lincomycin that presented a certain level of resistance of 51,85%.
The identification of the pathogenic strains that

Table - 2. Frequency of global susceptibility of diameter
of the growth inhibition zone around the bacterial

strains to some antibiotics (N= 89) antibiotic
disc for each isolated bacterial strain.

Table - 9. Frequency of susceptibility global of
frequency of resistance of 60,87 (R+I)).streptococci isolated to some antibiotic (N= 39) B. Enterobacteria: The proportions of resistance to disc Points resistant intermediary susceptible For example, a clear resistance to the Amoxicillin has * : NCCLS.** : CA-SFM1.