Antibiotic resistance trend of Staphylococcus aureus isolated between 2010 and 2012 from mastitis cases in Azawak zebu in Niger

1 Bacteriology, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, SartTilman, B43a, B-4000 Liège, Belgium. 2 Bacteriology, Department of Diagnosis, Epidemiological Investigations and Applied Research, BP 485, Niamey, Niger. 3 Microbiology, Immunology and Infectious Pathology, Interstate school of Veterinary Science and Medicine, BP 5077, Dakar, Senegal. 4 Department of Animal Production, Faculty of Veterinary Medicine, University of Liege, Boulevard de Colonster, 20, building B43, 4000 Liege, Belgium.


INTRODUCTION
Mastitis is the inflammation of the mammary gland most frequently caused by bacterial infection.Bacterial mastitis is a disease causing considerable economic loss in cattle farming.Milk deficits during mastitis vary from 25 to 48% depending on the intensity of inflammation and the stage of lactation (Gebreyohannes et al., 2009).Treatment (use of antibiotics) costs can be expensive and the incurable cows must be reformed, a preventive method truly efficient *Corresponding author.E-mail: karimlebelge@yahoo.fr.Tel: +3243664052, +22796573021.Fax: +3243664261, +22720732147.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0International License but also very expensive (Radostits et al., 2007).At the Sahelian station in Toukounous (Niger) reformed cows are sold to private farmers for fattening before slaughter.
Several bacterial species can cause bovine mastitis (Radostits et al., 2007).In many countries including Niger, Staphylococcus aureus is one of the most frequently isolated species from subclinical and clinical mastitis (Pradeep et al., 2003;Bada et al., 2005;Harouna et al., 2009;Issa et al., 2013).As a consequence of the wide use of antibiotics in dairy cattle farms, several mastitiscausing bacterial species have become resistant to the most frequently used antibiotics (Werckenthin et al., 2001).Nevertheless, antimicrobial therapy still plays an important role in mastitis control by reducing the infection levels and preventing new cases (Rabello et al., 2005).
In Niger, zebu cattle of the Azawak breed offer the best dairy aptitude and since 1975 the Niger government has opted for a policy of development and improvement of the breed.The experimental station in Toukounous was therefore set up with the objective of improving the dairy production by genetic selection and of promoting its distribution to individual farmers across the whole country (MRA, 2002).But, according to the available data in the yearly reports, the extensive use of antibiotics of the βlactam (amoxicillin, ampicillin), tetracyclin (tetracyclin, oxytetracylin), aminoglycoside (streptomycin, gentamicin) and sulfonamide families is noticeable, more especially for the treatment of intestinal and mammary gland infections (SSET, 2009).However, the influence of this wide antibiotic use on the level of resistance of mastitis-causing bacterial species is unknown.It is therefore important to study not only the prevalence of the different antibiotic resistance profiles of the most frequent mastitis-causing bacterial species, but also their evolution in time to foresee the efficiency of the treatments in the future.
Therefore the objective of this survey was to follow the evolution of antibiotic resistance of the S. aureus isolates during a three year period of time (2010-2012) from cases of mastitis in Azawak zebu at the Sahelian experimental station of Toukounous, Niger.

Bacterial isolation and identification
A total of 164 milk samples were collected between 2010 and 2012 from cows of the three Azawak zebu herds (elites, non-elites and primiparous) of the Toukounous station with subclinical mastitis.The bacteriological analysis of milk samples and the identification of S. aureus were performed as described previously (Issa et al., 2013).

Antibiotic susceptibility testing and resistance profiles
The susceptibility of the S. aureus isolates to seven antibiotics was determined by the disc diffusion method on Mueller-Hinton agar plates (Becton Dickinson, Belgium) as described by Bauer et al. (1966).Isolates were tested with discs with tetracyclin (30 UI), penicillin G (10 UI), gentamicin (10 UI), trimethoprim-sulfamethoxazole (1.25 µg / 23.75 µg), enrofloxacin (5 µg), clindamycin (2 UI) and oxacillin (5 µg) (Becton Dickinson, Belgium).The diameter in mm of the inhibition zone of each tested antibiotic was compared with the recommendations of the French Committee Guidelines for susceptibility testing (Comité de l'Antibiogramme vétérinaire de la Société Française de Microbiologie, 2010) to determine the susceptibility/resistance profile of each isolate.The data were processed in a spreadsheet program (Excel for Windows, Microsoft).

PCR characterization
All the isolates were tested for the presence of blaZ gene (coding for β-lactamase) by polymerase chain reaction (PCR) with forward primer 5'-TAA GAG ATT TGC CTA TGC TT-3' and reverse primer 5'-TTA AAG TCT TAC CGA AAG CAG-3' (Olsen et al., 2006).DNA extraction was carried out using the ChargeSwitch DNA Mini Bacteria Kit (Invitrogen, USA) according to the manufacturer's instructions for staphylococci.The PCR reaction mixture contained: 1U of Taq DNA polymerase (New England Biolabs, USA), 2.5 µl of 2 mM deoxynucleoside triphosphates (Eurogentec, Belgium), 2.5 µl of 10X ThermoPol Reaction Buffer, 1 µl of each primer (10 µM) and 1.5 µl of a DNA template in a total volume of 25 µl.The PCR reactions were performed using a Mastercycler® (Eppendorf, France).The PCR-amplified DNA fragment of 519 bp was isolated by electrophoresis in a 1.5% agarose gel and photographed under UV light after ethidium bromide staining.

Statistical analysis
To compare the evolution of the percentages of resistant S. aureus to the different antibiotics over the years (2010, 2011 and 2012), the Fisher's Exact Test was performed (SAS, 2001).Differences were considered significant at p <0.05.
The average resistance rates over the three years differ according to the antibiotic.As many as 20 of the 43 S. aureus isolates (46%) were resistant to penicillin (Figure 1); much less isolates were resistant to oxacillin (12%), gentamicin (9%), trimethoprim-sulfamethoxazole (9%), tetracyclin (7%), clindamycin (5%) and enrofloxacin (2%).The annual resistance rates in 2010 (22 isolates), 2011 (9 isolates) and 2012 (12 isolates) were the following: penicillin (64, 22 and 33%), oxacillin (14, 22 and 0%), gentamicin (4, 0 and 0%), trimethoprim-sulfamethoxazole (14, 11 and 0%) tetracyclin (14, 0 and 0%), clindamycin (4, 11 and 0%) and enrofloxacin (0, 0 and 8%).The Fisher's Exact Test confirmed a significant difference (p<0.05) in the resistance percentages to oxacillin, enrofloxacin and clindamycin over the 3 years but not to the other antibiotics.All but three of the 21 resistant isolates were resistant to penicillin and 11 of them were multidrug resistant (Table 2): six isolates were resistant to two antibiotics, four to three antibiotics and one to 5 antibiotics.These results are probably related to the frequent use of β-lactams in the treatment of any suspected bacterial infection at the station followed by tetracyclins alone or in combination with aminoglycosides in second intention.The average resistance rates in Toukounous are comparable, sometimes lower, to those obtained in previous studies performed in different African countries and in Pakistan.For instance, 64, 56, 54 and 100% of the S. aureus isolates respectively in Tunisia, Pakistan, Senegal and Uganda, are resistant to penicillin (Ben Hassen et al., 2002;Arshad et al., 2006;Kadja et al., 2010;Kateete et al., 2013).The results are also similar for oxacillin and tetracyclin.Oxacillin resistance rate is 7.6, 10 and 15% respectively in Nigeria, Senegal and Morocco (Bendahou et al., 2008;Kadja et al., 2010;Suleiman et al., 2012); whereas tetracyclin resistance rate is 13 and 15% in two previous studies in Niger in 2007 and 2009 (Harouna et al., 2009;Issa et al., 2013).Finally, 25% of the isolates of this study are multiresistant (resistance to at least two antibiotics), a result also comparable to the 23% reported by Zanette et al. (2010), but lower than the 62% reported by Meideiros et al. (2009), both in Brazil.
Thirteen cows could be followed between 2010 and 2012 to assess the individual antibiotic resistance evolution (Table 3).The antibiotic resistance profiles of the S. aureus isolated from the same cow differed from one year to another suggesting that the isolates are not clonal and that reinfection of the udder occurred during the three years.The Pulsed Field Gel Electrophoresis profiles (manuscript in preparation) confirm the hypothesis that isolates from the same cow are indeed not clonal.
Of the 20 isolates of S. aureus resistant to the penicillin by the in vitro disc diffusion test, 18 (90%) tested positive by PCR for the blaZ gene suggesting that the production of βlactamase by most penicillin-resistant S. aureus of this study is encoded by the blaZ gene (Zscheck and Murray, 1993).In conclusion, our study provides recent data on the distribution of antimicrobial resistance at the pilot farm of Toukounous.The massive resort to β-lactam antibiotics to treat any condition is most probably responsible for the high rate of resistance observed at the farm.Besides the resistance problem, it has also been reported that intramammary infection remained significantly more often chronic if it was caused by blaZ-positive than blaZnegative isolates of S. aureus (Taponen et al., 2003).It is therefore an absolute necessity to be more cautious and strict in the application of treatments with antibiotics, particularly in the use of the β-lactams.

Table 1 .
Number of S. aureus isolated at Toukounous according to the heard and to the year.

of sampled animal / No of isolated S. aureus
Figure 1.Antibiotic resistance profiles of the 43 S. aureus isolates in 2010, 2011 and 2012.

Table 2 .
Resistance profiles of the 21 S. aureus isolates resistant to at least one antibiotic.

Table 3 .
Antibiotic resistance profiles of the S. aureus isolates from 13 cows over three years (2010 to 2012).Clin = clindamycin, Gen= gentamicin, S = sensitive to all tested antimicrobials, 0 = no S. aureus isolated from milk sample.