Available at www.veterinaryworld.org/Vol.6/Oct-2013/9.pdf RESEARCH ARTICLE Open Access Isolation and antibiogram of Staphylococcus, Streptococcus and Escherichia coli isolates from clinical and subclinical cases of bovine mastitis

Aim: The present study was aimed to isolate and evaluate the continuous change in the pattern of drug resistance showed by different mastitogenic organisms, isolated from clinical and subclinical cases of mastitis. Materials and Methods: The study was carried out using 150 milk samples received from various clinical and subclinical cases, from which the causative organisms were isolated and subjected to in vitro antibiotic sensitivity test. Results: The bacteriological analysis of the samples indicated the presence of both Gram positive and Gram negative organisms followed by isolation of isolates like Staphylococcus, E. coli, Streptococcus, Bacillus, Corynebacterium, Listeria, Klebsiella. The in vitro sensitivity of Staphylococcus, E. coli and Streptococcus isolates revealed that they were more sensitive towards newer antimicrobials like Levofloxacin and Enrofloxacin. Conclusion: The prevalence of Staphylococcus was found to be maximum followed by Streptococcus and E. coli among the isolated organisms. Levofloxacin and Enrofloxacin were found to be most effective against the targeted isolates.


Introduction
like T.B, brucellosis, diphtheria, scarlet fever and Q fever [4]. Now there has also been increasing evidence Mastitis is a multi-etiological complex disease of bidirectional transmission of Methicillin Resistant which is characterized by physical, chemical and Staphylococcus aureus (MRSA) in humans and bacteriological changes in milk and pathological animals. Once exposed to MRSA, animals become changes in glandular tissue of udder [1]. It is a reservoir of infection for human beings. So the devastating disease haunting the dairy industry increased antimicrobial resistance of the organisms in worldwide and is a matter of great concern for leading animals treated with antibiotics and their zoonotic milk producing country like India because of the losses transmission continues to be a matter of great concern incurred due to high morbidity, discarded milk, globally [5]. The success of bovine mastitis therapy treatment costs and reduced milk production, thus basically depends on the understanding of clinical drawing in more attention towards its treatment and presentation and antimicrobial susceptibility of the control. It has also been found out that mastitis alone etiological agent, among various other factors [6]. One contributes to 70% of the losses during milk production important reason for therapy failure in the management which could have been avoided by means of prompt of mastitis could arise from various factors involving curative and control measures [2]. Particularly the pathological changes in the udder, etiology, lower Indian dairy industry suffers a monetary blow of over efficacy of antimicrobials, and improper veterinary Rs. 6000 crores per year [3]. Apart from the economic services [7]. But above all, antimicrobial susceptibility losses, mastitis can have serious implications on public testing, typically using a disk diffusion assay is often health. Mastitis which is mostly caused by the performed as part of the routine laboratory evaluation interaction of multiple pathogenic agents (primarily of milk samples with an attempt to provide guidelines bacteria), can expose human beings to various morbific for the treatment of mastitis, especially identification organisms through infected milk, thus serving as a of the pathogens causing clinical and subclinical media for transmission of various zoonotic diseases mastitis and thus forming a crucial part in development of mastitis control program. OIE has also recommended monitoring and observation of effectivity of antimicrobials towards pathogenic and commensal bacteria affecting animals [8].
Since the isolation of organism and choice of standard protocol [10,11]. antimicrobial agent is often considered empirical in the The in vitro antibiotic sensitivity pattern was studied management of mastitis, the present study was by Kirby-Bauer disc diffusions method using 13 conducted to observe the sensitivity and resistance number of antibiotic discs (Himedia) such as profile of mastitis causing isolates (particularly Levofloxacin (5 mcg/disc), Enrofloxacin (5 mcg/disc),

Materials and Methods
mcg/disc), Cephalexin (30 mcg/disc), Amoxycillin (10 Milk samples (n=150) from clinical and subclinical mcg/disc), Ciprofloxacin (5 mcg/disc), Ofloxacin (5 cases of mastitis were received by Department of mcg/disc). The inoculum was prepared by transferring Microbiology, College of Veterinary Science and 4-5 colonies from pure culture to BHI broth and Animal Husbandry, Orissa University of Agriculture incubated at 37°C for 6-8 hours until moderate and Technology, from various regions of Bhubaneswar turbidity developed. Then the inoculums turbidity was and areas around, for isolation, identification and matched with standard 0.5 McFarland and diluted with antibiogram of the mastitogenic organisms during the NSS if required. The standardized inoculum was then period from 06.01.2009 to 05.08.2011. All the samples applied onto the MHA agar plate by soaking with were showing positive result for Modified California sterile cotton swab and allowed the inoculums to dry. Mastitis test. Each sample was taken by means of a The discs were then placed aseptically equidistant from sterile inoculating loop and introduced into liquid each other on the surface of the agar plates. The plates media (BHI broth) and incubated for substantial were incubated at 37°C for 24-48 hours for growth of microorganisms. Following growth in broth development of inhibition zone. The diameters of zone a loopful of culture was streaked onto muller hinton of inhibition were then measured in millimeter and agar (MHA), eosin methylene blue (EMB) agar, noted down [12]. The interpretation regarding the mannitol salt agar (MSA), and blood agar and degree of susceptibility (resistant, moderate and highly incubated for 24-48 hour at 37°C. The different sensitive) was made as per Clinical and Laboratory colonies were marked and noted basing on their colony Standards Institute (formerly NCCLS) chart provided characteristics and growth on selective media. The by the antibiotic disc manufacturer. colonies on blood agar were further analyzed basing on

the hemolytic pattern. The morphological and staining
Identification & isolation of organisms: Out of the 150 characteristics of the organisms were determined by samples processed, 6 are found to be sterile as no microscopic examination of Gram stained smears and a growth was observed in liquid and solid media, which tentative analysis regarding the type of isolates was may have been due to prior medication before made. The isolates were identified according to cultural, collection of milk from the udder [13]. Bacteriological morphological and biochemical characteristics screening of the samples revealed 17 samples being complying with methods of Cruickshank [9]. The pure cultures whereas the remaining 127 invariably biochemical test and sugar fermentation properties for showed the presence of mixed cultures, which may be identification of isolates were conducted as per species to be highest in mastitis. coli was made on basis of their motility, staining, Antibiogram of the isolates: It was revealed during the cellular morphology, characteristic growth on selective isolation and identification that Staphylococcus, media (MSA, EMB and blood agar), hemolytic pattern Streptococci and E. coli are the major pathogens on blood agar, biochemical test and sugar fermentation responsible for clinical and sub clinical cases of properties whereas the inference regarding other mastitis and study of their response to various isolates was made as per the methods followed above antibiotics will help in the management and control of except for the growth on selective media. Isolation of these bacteria from milk samples has been previously the condition in day to day animal husbandry practices reported [2,14,15,16].
in an efficient way. On carrying out the antibiogram of Of the 144 samples, Staphylococcus was found in the targeted isolates (Staphylococcus spp., Streptococcus      [29] showed that E. antibiotics like Pencillin-G, Cephalexin, and Amoxycillin.
coli were sensitive to Gentamicin, Ciprofloxacin, Chlora-The sensitivity pattern for Streptomycin, Ceftriaxone, mphenicol, Cephalothin, Amoxycillin and Oxytetracycline. Gentamicin and Oxytetracycline showed an alarming The increased resistance to these therapeutic indication of increasing resistance among the bacterial agents can be attributed to injudicious use of antibiotics organisms to these therapeutic agents . This in the mastitis cases [4,14,17,20,21,26,30] along study was in close agreement with many workers [22, with negligence towards the bacteriological examination 26]. Farooq et al.
[23] during their study period have of the suspected milk. Though species, regional and found that Norfloxacin, Gentamicin and Chloramphenicol were the most effective drugs where as Amoxycillin, managemental variations leads to variation of results in Kanamycin, Sulphamethaxazole, Streptomycin and the antibiogram study but also the preferences of Penicillin-G showed lesser effectiveness against antibiotics used by various workers leads to inaccurate mastitis isolates. antibiotics continues further.

Antibiogram of Staphylococcus isolates: Staphylococcus
Conclusion is the most important and prevalent mastitogenic Development of resistance among bacteria is a organism occurring globally, including India [2]. In matter of great concern in the field of veterinary case of studies relating to Staphylococcus, Unakal et al. medicine especially when the organisms renders an [5] have found that the highest numbers of Staphylococcus animal unproductive, thus posing a direct threat to aureus isolates were susceptible to Ceftriaxone country's economy. So as a well planned therapy is very followed by Cefotaxime, Methicillin, Ciprofloxacin, essential in control and cure of mastitis, conducting an Erythromycin, Amikacin, Gentamicin, Amoxycillin, antibiogram profile of the mastitogenic organisms Ampicillin, whereas the lowest susceptibility was provides a clear insight towards management of shown in Penicillin-G. However the present study mastitis. In the present study, the prevalence of contrastingly showed a decreasing trend of sensitivity Staphylococcus was found to be maximum followed by of Staphylococcus towards Ceftriaxone combination Streptococcus and E. coli among the isolated organisms. of drugs [