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Temporal profile of antimicrobial resistance exhibited by strains of Staphylococcus spp. isolated from cases of bovine mastitis for 20 years (1992-2011)/Perfil temporal da resistencia antimicrobiana exibido por cepas de Staphylococcus spp. isoladas de casos de mastite bovina durante 20 anos (1992-2011).

INTRODUCTION

Mastitis is the major infectious disease in dairy herds (CARVALHO et al., 2007; GETAHUM et al., 2008) and it is responsible for major economic losses in milk industry. Additionally, it represents the major reason for using antimicrobials in animals worldwide (SARAN & CHAFFER, 2000). Among the etiological agents, Staphylococcus spp. is cited as the most prevalent micro-organism involved in subclinical mastitis (KREWER et al., 2013). Staphylococcus aureus is the most pathogenic species (CARVALHO et al., 2007) and is characterized by high invasiveness and formation of fibrous tissue at the site of infection, leading to chronic cases that are characterised by low cure rates (SANTOS & FONSECA, 2007). Moreover, coagulase-negative Staphylococcus has received attention as an emerging etiological cause of subclinical mastitis, and as S. aureus, it is reported as resistant to multiple antimicrobials commonly used in mastitis treatment (WEESE & DUIJKEREN, 2010).

The emergence of antimicrobial resistance has consequences in both human and animal health. The selective pressure imposed by the constant use of antimicrobials and the presence of resistance genes are the most important features in the occurrence of this phenomenon (PHILLIPS et al., 2004). Several studies with microorganisms isolated from the milk of cows with mastitis reported different patterns of susceptibility to antimicrobial agents (FREITAS et al., 2005; MEDEIROS et al., 2009; KREWER et al., 2013). Nevertheless, there is a lack of information regarding the temporal changes in the susceptibility of these pathogens to antimicrobial agents used in the treatment of this disease (LINDEMAN et al., 2013). The aim of this retrospective study was to determine the antimicrobial resistance profile of Staphylococcus spp. isolated from bovine milk samples with mastitis over a period of 20 years (1992-2011).

MATERIALS AND METHODS

The results of antimicrobial susceptibility tests of 2,430 isolates of Staphylococcus spp. were reviewed and analysed. Data were obtained from records of bovine milk samples analysed between 1992 and 2011 in the diagnostic service of the Bacteriology Laboratory of the Federal University of Santa Maria (LABAC-UFSM).

The samples were isolated in blood agar (Himedia Laboratories[R]) supplemented with 5% of sheep blood, and incubated aerobically at 35 [+ or -] 2[degrees]C for 4h. Bacterial identification was performed using morphological analysis and biochemical tests (QUINN et al., 1994). Since in most of the cases the identification of Staphylococcus spp. species was not realized, the results were organized into the genus Staphylococcus spp.

The antimicrobial susceptibility tests were performed in vitro using the method of agar disk diffusion (BAUER et al., 1966) for the following antimicrobials: 1) beta-lactams: oxacillin (1[micro]g), penicillin (10IU), ampicillin (10[micro]g) and cephalexin (30[micro]g); 2) fluoquinolone: norfloxacin (10[micro]g); 3) tetracycline: tetracycline (30[micro]g); 4) sulphonamide: sulphazotrim (25[micro]g); 5) aminoglycosides: gentamicin (25[micro]g) and neomycin (30[micro]g). The results were analysed in accordance with the Clinical and Laboratory Standards Institute (CLSI, 2002).

Isolates were classified as susceptible, intermediate or resistant to antimicrobials. In order to facilitate the calculation, intermediate results were considered resistant. For quality control of the susceptibility tests, a reference strain from the American Type Culture Collection (ATCC) (S. aureus ATCC 25923) was used. During the study period, the number of isolates varied in relation to antimicrobial agents in the same year, possibly due to unavailability of certain antibiotic discs in the laboratory.

The resistance of Staphylococcus spp. to each antimicrobial was evaluated based on the percentage of resistant isolates compared to the number of samples tested in each year. Comparisons using the Kruskal-Wallis test were performed. The data regarding to the resistance of Staphylococcus spp. between two decades were analyzed, regardless of antimicrobial tested by means of chi-square. A Bonferroni test was applied to compare the means when differences between classes or antimicrobials were identified. The SAS (2001) statistical program was used to perform the analyses. In order to evaluate the temporal trends in antimicrobial susceptibility during the study period, regression analysis was performed with data transformed to arcsine square root, for which the choice of models was based on the significance of linear, quadratic and cubic coefficients using a "t" test at 5% probability (SAS 2001).

RESULTS

The results of the in vitro susceptibility tests showed that 729 (30%) of Staphylococcus spp. were susceptible to all antimicrobials tested. The resistance profiles of the isolates are summarized in table 1. It was possible to conclude that the resistance to the antimicrobials was higher in the first decade (1992-2001: 29.2%) than the second decade (20022011: 17.1%) (P<0.0004).

Among the classes of antimicrobials, beta-lactams (34.3%) and tetracycline (28%) showed the highest resistance mean, however there is no statistical difference between the results of these two classes (P<0.0001). The other classes presented similar resistance means (from 8.5% to 11.6%). Among the antimicrobials from the same classes, different resistance rates were observed (P<0.0001). The highest resistance was observed for beta-lactams, with 46.2% of the isolates resistant to penicillin, 43.9% to ampicillin and 34.7% to oxacillin. Different results were found for cephalexin, since only 6.9% of the isolates were resistant to this beta-lactam antibiotic. 8.5% of the isolates were resistant to norfloxacin, which was similar to the results found for sulphazotrim (9.1% of resistance). Among aminoglycosides, the highest rate of resistance was demonstrated for neomycin (15.7%), while 7.6% of isolates were resistant to gentamicin.

The dynamic behaviour of the resistance of each isolate is presented in figure 1. During the 20 years studied, there was a reduction in the resistance of Staphylococcus spp. to penicillin ([R.sup.2]=50.38; P=0.0005) and ampicillin ([R.sup.2]=53.65; P=0.0002). However, a decrease in the resistance rates to oxacillin was observed during the first decade, followed by an increase during the second decade ([R.sup.2]=82.72; P<0.0001) (Figure 1a). It was not observed by regression analysis any evolution pattern of resistance to cephalexin (Figure 1a), norfloxacin (Figure 1b) and tetracycline (Figure 1c). Nevertheless, it was possible to verify a trend towards decreased resistance of the isolates to sulphazotrim ([R.sup.2]=18.80; P=0.0561) (Figure 1d). The regression analysis for the class of aminoglycosides showed a decline in the resistance to neomycin during the first decade, followed by an increase in resistance in the second decade ([R.sup.2]=30.43; P=0.0372). However, it was not possible to observe any trend for gentamicin (Figure 1e).

DISCUSSION

Antimicrobial resistance has been the subject of several studies, and currently it is considered the main risk to global health (SPELLBERG et al., 2013). Therefore, it is important to know the dynamics of antimicrobial resistance in order to select effective drugs to eradicate causative agents (GUARDABASSI et al., 2010). Similar to the results found in the present study, other authors also reported high resistance rate to penicillin and ampicillin (MACHADO et al., 2008; MEDEIROS et al., 2009). This result was expected since historically beta-lactams are widely used in mastitis therapy, as well as other bacterial diseases in cattle (MEDEIROS et al., 2009).

At the same time, the regression of resistance to penicillin and ampicillin over the 20 years observed, confirms some evidences observed in previous studies. For instance, Gram-positive bacteria isolated from cows with mastitis showed decreased resistance to beta-lactams (ERSKINE et al., 2002; MAKOVEC & RUEGG, 2003). Additionally, other reports showed an increased availability of several classes of antimicrobials for veterinary use. This range of new available antimicrobials in veterinary can partially explain the decreased resistance of S. aureus to penicillin in countries with historically high levels of resistance (AARESTRUP & SCHWARZ, 2006).

The lower resistance of Staphylococcus spp. against cephalexin might be attributed to its relatively recent use in veterinary medicine and due to the fact that cephalosporins are stable to [beta]-lactamases (SPINOSA et al., 2002). Furthermore, other reasons might be the lower availability of veterinary medicine formulations containing this drug (SINDAN, 2013) and the high cost of these products.

The rate of new intramammary infections is approximately four times higher during the dry period compared to the lactation period (SANTOS & FONSECA, 2007). Thus, it is crucial to preserve the susceptibility to beta-lactams and aminoglycosides, since the available formulations for the treatment of mastitis in the dry period consist predominantly of these two antibiotic classes (SINDAN, 2013). Especially in herds where contagious mastitis is well controlled and they are more susceptible to develop environmental mastitis, it is important that these drugs remain effective against the causative microorganisms.

The high level of Staphylococcus spp. resistant to gentamicin has been attributed to the indiscriminate use of this drug (FREITAS et al., 2005); however, in this study, the average of resistance was relatively low. Similar results were reported in other regions of Brazil where isolates from cases of mastitis showed high sensitivity to gentamicin (MEDEIROS et al., 2009). Among aminoglycosides, the highest resistance rate was found for neomycin suggesting that this antimicrobial was used more often in cattle over the study period.

Although the high level of tetracycline resistant isolates was observed, it was not possible to note any trend of increasing or reducing them over the 20 years. In general, antimicrobial tetracycline class has a broad spectrum of action and is widely used in veterinary medicine mainly for mastitis therapy, respiratory diseases and other bovine diseases (MEDEIROS et al., 2009). Although the in vitro sensitivity tests have been requested because of the occurrence of mastitis, it was not possible to determine the form of presentation of the disease or previous antimicrobial treatments for most of the samples. However, the resistance profiles observed in the present study, suggest the occurrence of different degrees of selection pressure from different antimicrobials on strains of Staphylococcus spp. over the years.

The reduction of resistance is a phenomenon occurring in certain microorganisms, which might happen when selection pressure is removed in certain populations, allowing resistant strains to be replaced by susceptible strains (PHILLIPS et al., 2004). Possible reasons for this phenomenon might be the greater prudence of the use of antimicrobials in dairy herds (ERSKINE et al., 2002) and the improvement of the biochemical constitution of the drugs, which has been correlated with an increase in the antimicrobial susceptibility of S. aureus isolated from humans (DUARTE & SA, 2011). Suggested strategies to reduce the use of antimicrobial treatments in dairy cattle include the treatment of subclinical mastitis cases in the dry period only, and selective dry cow therapy, a strategy that restricts the treatment to infected quarters. The use of this measure can help to reduce the probability of selection of resistant strains among bacterial populations (GUARDABASSI et al., 2010).

Although similar results have been reported to those found in previous studies of antimicrobial agents used in bovine mastitis in other countries (ERSKINE et al., 2002; MAKOVEC & RUEGG, 2003; LINDEMAN et al., 2013), one should be cautious when establishing comparisons to patterns of resistance of these studies due to (i) sampling criteria, (ii) regional differences of pathogen population, (iii) and differences between laboratory protocols and guidelines for interpretation (AARESTRUP & SCHWARZ, 2006). Furthermore, knowledge and monitoring of local resistance patterns are fundamental to the construction of effective treatment strategies. Prospective studies in this direction, with the comparison of the change in antimicrobial susceptibility after therapy are also highly desirable (ERSKINE et al., 2002).

CONCLUSION

The results indicated no trend towards increased resistance of Staphylococcus spp. to the most tested antimicrobials. However, it is necessary to monitor the resistance of these antimicrobial agents constantly in order to guarantee a therapeutic reserve.

http://dx.doi.org/10.1590/0103-847Si cr20140928

ACKNOWLEDGEMENTS

The authors acknowledge the Conselho Nacional de Pesquisa e Desenvolvimento Tecnologico, CNPq/Brazil, for the professor scholarship of research productivity.

REFERENCES

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CARVALHO, L.B. et al. Contagem de celulas somaticas e isolamento de agentes causadores de mastite em bufalas (Bubalus bubalis). Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, v.59, p.242-245, 2007. Available from: <http://www.scielo.br/pdf/abmvz/v59n1/39.pdf>. Accessed: Jun. 10, 2013. doi:10.1590/S0102-09352007000100039.

CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI) (National Committee for Clinical Laboratory Standards). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria Isolated from animals. Approved standard M31-A2. Wayne, Pa: CLSI, 2002. 86p.

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GETAHUM, K. et al. Bovine mastitis and antibiotic resistance patterns in Selalle smallholder dairy farms, central Ethiopia. Tropical Animal Health and Production, v.40, p.261-268, 2008. Available from: <http://link.springer.com/article/10.1007/s11250-007-9090-5>. Accessed: Jun. 10, 2013. doi: 10.1007/s11250-007-9090-5.

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KREWER, C.C. et al. Etiology, antimicrobial susceptibility profile of Staphylococcus spp. and risk factors associated with bovine mastitis in the states of Bahia and Pernambuco. Pesquisa Veterinaria Brasileira, v.33, p.601-606, 2013. Available from: <http://www.scielo.br/scielo.php?pid=S0100736X2013000500009&script=sci_arttext>. Accessed: Jun. 10, 2013. doi : 10.1590/S0100-736X2013000500009.

LINDEMAN, C.J. et al. Susceptibility to antimicrobial agents among bovine mastitis pathogens isolated from North American dairy cattle, 2002-2010. Journal of Veterinary Diagnostic Investigation, v.25, p.581-591, 2013. Available from: <http://vdi.sagepub.com/content/25/5/581.long>. Accessed: Jun. 13, 2013. doi: 10.1177/1040638713498085.

MACHADO, T.R.O. et al. Antimicrobial susceptibility of coagulase-negative staphylococci isolated from mastitic cattle in Brazil. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, v.60, p.278-282, 2008. Available from: <http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-09352008000100041>. Accessed: Jun. 10, 2013. doi: 10.1590/S0102-09352008000100041.

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PHILLIPS, I. et al. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. Journal of Antimicrobial Chemotherapy, v.53, p.28-52, 2004. Available from: <http://jac.oxfordjournals.org/content/53/1/28.short>. Accessed: Jun. 20, 2013. doi: 10.1093/jac/dkg483.

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SPINOSA, H.S. et al. Farmacologia aplicada a medicina veterinaria. 3.ed. Rio de Janeiro: Guanabara Koogan, 2002. 752p.

WEESE, J.S.; VAN DUIJKEREN E. Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in veterinary medicine. Veterinary Microbiology, v. 140, p.418-429, 2010. Available from: <http://www.ncbi.nlm.nih.gov/pubmed/19246166>. Accessed: Jun. 20, 2013. doi: 10.1016/j. vetmic.2009.01.039.

Ananda Paula Kowalski (I) Grazieli Maboni (I) Julia Pires Espindola (I) Ariane Foletto (I) Guerino Bandeira Junior (I) Luciana Potter (II) Sonia de Avila Botton (I) Agueda Castagna de Vargas (I) *

(I) Departamento de Medicina Veterinaria Preventiva, Centro de Ciencias Rurais (CCR), Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000, Predio 44, Sala 5137, Campus, Camobi, 97105-900, Santa Maria, RS, Brasil. E-mail: agueda.vargas@gmail.com.

* Corresponding author.

(II) Departamento de Zootecnia, CCR, UFSM, Santa Maria, RS, Brasil.

Received 06.21.14 Approved 10.21.14 Returned by the author 01.29.15 CR-2014-0928.R1

Table 1--Profile of antimicrobial resistance
of Staphylococcus spp. derived from bovine
milk samples (1992-2011) (1).

Year              OXA       PEN       AMP
Antimicrobials

1992              6/6       6/6       43/47
                  100%      100%      91.5%
1993              58/62     40/63     49/61
                  93.5%     63.5%     80.3%
1994              51/82     61/83     58/83
                  62.2%     73.5%     69.9%
1995              76/117    67/127    58/135
                  64.9%     52.7%     42.9%
1996              104/176   81/171    97/180
                  59.1%     47.4%     53.9%
1997              91/192    75/164    84/182
                  47.4%     45.7%     46.1%
1998              78/389    252/419   261/463
                  20%       60.1%     56.4%
1999              42/368    180/364   179/365
                  11.4%     49.4%     49%
2000              36/220    117/222   104/207
                  16.4%     52.7%     50.2%
2001              13/105    41/107    39/107
                  12.4%     38.3%     36.4%
2002              22/73     22/73     22/73
                  30.1%     30.1%     30.1%
2003              11/61     19/61     15/61
                  18%       31.1%     24.6%
2004              4/43      14/43     11/43
                  9.3%      32.6%     25.6%
2005              10/43     17/43     14/43
                  23.2%     39.5%     32.6%
2006              5/54      12/54     10/54
                  9.3%      22.2%     18.5%
2007              12/49     15/49     9/49
                  24.5%     30.6%     18.4%
2008              14/50     14/50     18/50
                  28%       28%       36%
2009              9/32      22/69     18/67
                  28.1%     31.9%     26.9%
2010              5/26      12/20     12/27
                  19.2%     60%       44.4%
2011              5/30      11/31     14/31
                  16.7%     35.5%     45.2%

Year              CEF       NOR       TET
Antimicrobials

1992              NT        NT        17/45
                                      37.8%
1993              NT        NT        8/30
                                      26.7%
1994              NT        NT        NT

1995              NT        7/47      11/28
                            14.9%     39.3%
1996              NT        15/161    27/73
                            9.3%      37%
1997              5/99      8/194     53/192
                  5%        4.1%      27.6%
1998              70/423    41/463    146/469
                  16.5%     8.8%      31.1%
1999              18/360    23/359    76/361
                  5%        6.4%      21%
2000              11/163    26/208    60/221
                  6.7%      12.5%     27.1%
2001              5/107     7/107     25/107
                  4.7%      6.5%      23.4%
2002              2/73      1/73      13/73
                  2.7%      1.4%      17.9%
2003              1/61      5/61      13/61
                  1.6%      8.2%      21.3%
2004              1/43      1/43      9/43
                  2.3%      2.3%      20.9%
2005              3/43      6/43      14/43
                  7%        13.9%     32.6%
2006              1/54      0/54      14/54
                  1.8%      0%        25.9%
2007              2/49      2/49      7/49
                  4.1%      4.1%      14.3%
2008              7/50      10/50     22/50
                  14%       20%       44%
2009              4/69      12/68     17/68
                  5.8%      17.6%     25%
2010              3/35      4/48      12/39
                  8.6%      8.3%      30.8%
2011              0/31      2/31      9/31
                  0%        6.4%      29%

Year              SUT       GEN       NEO
Antimicrobials

1992              5/45      3/47      2/5
                  11.1%     6.4%      40%
1993              12/84     3/63      11/58
                  14.3%     4.7%      19%
1994              23/83     6/81      23/82
                  27.7%     7.4%      28%
1995              14/131    7/135     15/129
                  10.7%     5.2%      11.6%
1996              30/169    9/180     29/173
                  17.7%     5%        16.7%
1997              7/193     17/197    14/188
                  3.6%      8.6%      7.4%
1998              44/415    36/387    86/453
                  10.6%     9.3%      19%
1999              12/357    14/372    23/367
                  3.4%      3.7%      6.3%
2000              21/218    21/154    33/221
                  9.6%      13.6%     14.9%
2001              4/107     3/107     7/106
                  3.7%      2.8%      6.6%
2002              8/73      8/73      20/73
                  11%       11%       27.4%
2003              13/61     4/61      11/61
                  4.9%      6.6%      18%
2004              2/43      2/43      3/43
                  4.6%      4.6%      7%
2005              1/43      2/43      6/43
                  2.3%      4.6%      13.9%
2006              2/54      2/54      1/54
                  3.7%      3.7%      1.8%
2007              6/49      3/49      3/49
                  12.2%     6.1%      6.1%
2008              5/50      7/50      6/50
                  10%       14%       12%
2009              1/69      1/69      3/69
                  1.4%      1.4%      4.3%
2010              4/39      5/35      7/33
                  10.3%     14.3%     21.2%
2011              3/31      6/31      10/31
                  9.7%      19.3%     32.3%

(1) Number of resistant Staphylococcus spp. OXA = oxacillin;
PEN = penicillin; AMP = ampicillin; CEF = cephalexin;
NOR = norfloxacin; TET = tetracycline; SUT = sulphazotrim;
GEN = gentamicin; NEO = neomycin; NA = not analysed.
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Title Annotation:microbiologia; texto en ingles
Author:Kowalski, Ananda Paula; Maboni, Grazieli; Espindola, Julia Pires; Foletto, Ariane; Bandeira, Guerino
Publication:Ciencia Rural
Date:Jun 1, 2015
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