Antimicrobial resistance profiles of Staphylococcus aureus clusters on small dairy farms in southern Brazil

Perfil de resistência a antimicrobianos de grupos clonais de Staphylococcus aureus isolados de pequenas propriedades leiteiras do sul do Brasil

Lilian K. Girardini Daniel S. Paim Thais C. Ausani Graciela V. Lopes Debora C.P. Pellegrini Maria Aparecida V.P. Brito Marisa Cardoso About the authors

ABSTRACT:

In intensive dairy farming, persistent intramammary infection has been associated with specific Staphylococcus (S.) aureus strains, and these strains may be resistant to antimicrobials. The objective of this study was to evaluate the antimicrobial resistance phenotypes of S. aureus isolates and to assess the distribution and the persistence of clonal groups in small dairy herds of southern Brazil. Milk samples were collected from all lactating cows from 21 dairy farms over a two-year period, totaling 1,060 samples. S. aureus isolates were tested for susceptibility to thirteen antimicrobials using the disk diffusion method. The total DNA of the isolates was subjected to SmaI digestion followed by pulsed-field gel electrophoresis (PFGE). Banding patterns differing by ≤4 bands were considered members of a single PFGE cluster. The frequency of S. aureus isolation ranged from 3.45% to 70.59% among the 17 S. aureus-positive herds. Most S. aureus isolates (87.1%) were susceptible to all antimicrobials; resistance to penicillin (18.2%) was the most frequently observed. The 122 isolates subjected to macrorestriction analysis were classified into 30 PFGE-clusters. Among them, only 10 clusters were intermittent or persistent over the two-year period. The majority (93.6%) of isolates belonging to persistent and intermittent clusters were susceptible to all tested antimicrobials. S. aureus intramammary colonization in small dairy farms of southern Brazil is most frequently caused by sporadic PFGE clusters, although some persistent clusters can arise over time. Both sporadic and persistent isolates were highly susceptible to antimicrobials.

INDEX TERMS:
Mastitis; PFGE clusters; Methicillin-resistant Staphylococcus aureus; MRSA.

RESUMO:

A infecção intramamária persistente em bovinos leiteiros tem sido associada com estirpes de Staphylococcus (S.) aureus específicos, os quais podem ser resistentes a antimicrobianos. Os objetivos deste estudo foram avaliar os fenótipos de resistência aos antimicrobianos de isolados de S. aureus e a distribuição e persistência de grupos clonais em pequenos rebanhos leiteiros do sul do Brasil. As amostras de leite foram coletadas de todas as vacas em lactação de 21 propriedades leiteiras, ao longo de um período de dois anos, perfazendo um total de 1.060 amostras. Isolados de S. aureus foram testados quanto à resistência frente a treze antimicrobianos, pelo método de disco-difusão. O DNA total dos isolados foi clivado com a enzima Smal e submetido a eletroforese em gel de campo pulsado (PFGE). Padrões de bandas diferentes por ≤4 bandas foram considerados como pertencentes ao mesmo grupo clonal. A freqüência de S. aureus variou de 3,45% até 70,59%, entre os 17 rebanhos com isolamento positivo de S. aureus. A maioria dos isolados de S. aureus (87,1%) foi suscetível a todos os antimicrobianos; resistência à penicilina (18,2%) foi a mais freqüentemente observada. Os 122 isolados submetidos à análise de macrorestrição foram classificados em 30 grupos clonais de PFGE. Entre eles, apenas dez grupos clonais foram intermitentes ou persistentes ao longo do período de dois anos. A maioria (93,6%) dos isolados pertencentes a grupos clonais persistentes e intermitentes foram suscetíveis a todos os antimicrobianos testados. Concluiu-se que a colonização intramamária em bovinos de pequenas propriedades leiteiras do Sul do Brasil é mais frequentemente causada por grupos clonais esporádicos de S. aureus, embora alguns grupos clonais persistentes possam ocorrer ao longo do tempo. Em ambos os grupos clonais os isolados foram majoritariamente suscetíveis a antimicrobianos.

TERMOS DE INDEXAÇÃO:
Mastite; grupos clonais; PFGE; Staphylococcus aureus resistente à meticilina; MRSA.

Introduction

Staphylococcus (S.) aureus is the main coagulase-positive species of Staphylococci associated with intramammary infections in dairy cattle and can lead to clinical or subclinical mastitis (Young et al. 2001Young B., Platt D., Logue D., Ternent H. & Fitzpatrick J. 2001. Bovine Staphylococcus aureus mastitis: strain recognition and dynamics of infection. J. Dairy Res. 68:377-388.). Although S. aureus can colonize the skin and mucous membranes of cows, the udder is the most important reservoir of these bacteria (Capurro et al. 2010Capurro A., Aspán A., Unnerstad E.H., Waller K.P. & Artursson K. 2010. Identification of potential sources of Staphylococcus aureus in herds with mastitis problems. J. Dairy Sci. 93:180-191.). It was demonstrated that S. aureus intramammary infection can be widespread among cows within the same herd, and genotyping has shown that common clonal groups can be persistent in a herd over time (Mork et al. 2012Mork T., Jorgensen H.J., Sunde M., Kvitle B., Sviland S., Waage S. & Tollersrud T. 2012. Persistence of staphylococcal species and genotypes in the bovine udder. Vet. Microbiol. 159:171-180., Piccinini et al. 2012Piccinini R., Tassi R., Daprà V., Pilla R., Fenner J., Carter B. & Anjum M. 2012. Study of Staphylococcus aureus collected at slaughter from dairy cows with chronic mastitis. J. Dairy Res. 79:249-255.). Among the genotyping techniques, pulsed-field gel electrophoresis has been frequently used to determine whether S.aureus isolates are close related and associated with persistent intramammary infection (Middleton et al. 2002Middleton J., Fox L.K., Gay J.M., Tyler J.W. & Besser T.E. 2002. Use of pulsed-field gel electrophoresis for detecting differences in Staphylococcus aureus strain populations between dairy herds with different cattle importation practices. Epidemiol. Infect. 129:387-395., Haveri et al. 2008Haveri M., Hovinen M., Roslöf A. & Pyörälä S. 2008.Molecular types and genetic profiles of Staphylococcus aureus strains isolated from bovine intramammary infections and extramammary sites. J. Clin. Microbiol. 46:3728-3735., Mork et al. 2012Mork T., Jorgensen H.J., Sunde M., Kvitle B., Sviland S., Waage S. & Tollersrud T. 2012. Persistence of staphylococcal species and genotypes in the bovine udder. Vet. Microbiol. 159:171-180.).

Antimicrobial resistance in S. aureus is a concern for both human and animal health, because multi-resistant strains represent a challenge to effective treatment. Particularly, methicillin-resistant S. aureus (MRSA), which are resistant to almost all types of β-lactam antimicrobials, have been a concern worldwide (Lee 2003Lee J.H. 2003. Methicillin (oxacillin)-resistant Staphylococcus aureus strains isolates from major food animals and their potential transmission to humans. Appl. Environ. Microbiol. 69:6489-6494., Van Duijkeren et al. 2004Van Duijkeren E., Box A.T.A., Heck M.E.O.C., Wannet W.J.B. & Fluit A.C. 2004. Methicillin-resistant staphylococci isolated from animals. Vet. Microbiol. 103:91-97.). The presence of MRSA in dairy cows has been investigated because it was demonstrated that cattle may serve as a source of emergence of new MRSA strains in humans (Juhász-Kaszanyitzky et al. 2007Juhász-Kaszanyitzky E., Jánosi S., Somogyi P., Dán A., Bloois L.G., Van Duijkeren E. & Wagenaar J.A. 2007. MRSA transmission between cows and humans. Emerg. Infect. Dis. 13:630-632., Vanderhaeghen et al. 2010Vanderhaeghen W., Cerpentier T., Adriaensen C., Vicca J., Hermans K. & Butaye P. 2010. Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitits in Belgian cows. Vet. Microbiol. 144:166-171.). Therefore, colonization of dairy cattle with antimicrobial-resistant S. aureus impacts milk production and may additionally represent an infection hazard to people who work in close contact with cows or consume raw milk (Juhász-Kaszanyitzky et al. 2007Juhász-Kaszanyitzky E., Jánosi S., Somogyi P., Dán A., Bloois L.G., Van Duijkeren E. & Wagenaar J.A. 2007. MRSA transmission between cows and humans. Emerg. Infect. Dis. 13:630-632.).

In southern Brazil, small dairy farms based on family labor contribute a large amount of the total milk production. These independent farmers usually deliver the milk production to processing plants; however, some of the milk is also consumed by the family. One major challenge for small dairy farm production is the lack of technical assistance, leading to failures in animal management and control measures to prevent udder infection (Stumpf et al. 2000Stumpf W.J., Bittencourt D. & Gomes J.F. 2000. Sistemas de pecuária de leite: uma visão na região de clima temperado. Embrapa Clima Temperado, Pelotas, RS.). Often, farmers treat animals without any previous knowledge of antimicrobial susceptibility. These practices may influence pathogen transmission and resistance, resulting in an epidemiological scenario quite different from that of dairy farms with large-scale production. Therefore, the aim of this study was to assess the antimicrobial-resistance profiles and pulsotypes of S. aureus isolates causing intramammary infection in small dairy herds.

Materials and Methods

Herds and sampling. The study population consisted of 1,185 small dairy herds located in a region of approximately 4,821 square kilometers of the state of Rio Grande do Sul, Brazil (UTM zone 22S, 6.69.000N, 350.000E, 6.830.000N, 450.000E). First, farms were stratified according to the average number of cows in lactation (≤10, 11-15, 16-20, 21-25 and ≥25). Next, the sample size was calculated, taking into account an expected S. aureus isolation frequency of 20% (Brito et al. 2001Brito M.A.V.P., Brito J.R.F., Silva M.A.S. & Carmo R.A. 2001. Concentração mínima inibitória de dez antimicrobianos para amostras de Staphylococcus aureus isoladas de infecção intramamária bovina. Arq. Bras. Med. Vet. Zootec. 53 (5):10-17.), with a precision of 10% at the 95% confidence level. The number of farms of each stratum to be included in the study was determined such that their percent representation in the total target population was maintained (80% of farms had ≤15 cows in lactation). In each stratum, farms were randomly selected for sampling using Microsoft Excel 2010 software. In the 21 farms included in the study, an average of 13 cows (minimum 3 and maximum 50) was in lactation. All sampled farms used mechanical milking and washed the udder before milking. Most farms applied post-dipping treatment with iodine, although no pre-dipping protocol was used. On all farms, clinical mastitis was treated without prior antimicrobial resistance testing.

Samples were collected every six months over a two-year period on all farms. During each sampling event, milk samples were collected from all lactating cows in the herd. Animals that were receiving antimicrobial treatment were excluded from sampling. The procedures for the collection and transport of milk samples followed the recommendations of the National Mastitis Council (2004)National Mastitis Council 2004. Microbiological procedures for the diagnosis of bovine udder infection and determination of milk quality. 4th ed. NMC, Verona.. Milk samples were collected before the milking process began. The teats were washed and dried individually with disposable paper towels before sampling. The first three streams of milk were discarded, and the teats were then disinfected with 70% alcohol. A composite sample of milk from all teats of each cow was collected in one sterile screw-cap flask. Samples were transported under refrigeration to the laboratory for processing.

Bacterial isolation and identification. Ten microliters of each milk sample was streaked onto 5% sheep blood agar and incubated at 37°C for 24 - 48 hours. After incubation, predominant colonies (at least three colonies present on the agar plate) with similar morphology were isolated and identified as described previously by the National Mastitis Council (2004)National Mastitis Council 2004. Microbiological procedures for the diagnosis of bovine udder infection and determination of milk quality. 4th ed. NMC, Verona. and Markey et al. (2013)Markey B., Leonard F., Archambault M., Cullinane A. & Maguire D. 2013. Clinical Veterinary Microbiology. 2nd ed. Mosby Elsevier, London.. Gram positive cocci with positive catalase and coagulase test results that displayed acetoin production (Voges-Proskauer test) were classified as Staphylococcus aureus.

Antimicrobial susceptibility testing and detection of MRSA. Isolates of S. aureus were tested for antimicrobial susceptibility using the disk diffusion test on Müller-Hinton agar (Oxoid, Thermo Scientific, UK) according to the guidelines of the Clinical and Laboratory Standards Institute (2008)Clinical and Laboratory Standards Institute. 2008. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard. 3rd ed. CLSI, Wayne.. The following antimicrobial disks (Oxoid, Thermo Scientific, UK) were tested: cephalothin (30μg), ceftiofur (30μg), clindamycin (2μg), enrofloxacin (5μg), gentamicin (10μg), oxacillin (10μg), penicillin (10 IU), sulfonamide (300μg), sulfa + trimethoprim (25μg) and tetracycline (30μg). S. aureus ATCC 25923 was used as the quality control. Isolates displaying inhibition zones ≥29mm to penicillin were subjected to the zone edge evaluation of the penicillin disk-diffusion test as recommended (CLSI 2009). Fuzzy zone edges were considered as indicative of no beta-lactamase production.

Isolates that were found to be resistant to oxacillin by the disk diffusion test were subjected to minimum inhibitory concentration (MIC) determination, screened by the Etest® (bioMérieux, Marcy l'Etoile, Lyon, France), and checked for the presence of the mecA gene by PCR, as described by Murakami et al. (1991)Murakami K., Minamide W., Wada K., Nakamura E., Teraoka H. & Watanabe S. 1991. Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J. Clin. Microbiol. 29: 2240-2244.. DNA was extracted after disrupting the bacterial cell wall with Guanidine EDTA-Sarkosyl (Rademaker & De Bruijn 1997Rademaker J.L.W. & De Bruijn F.J. 1997. Characterization and classification of microbes by REP-PCR genomic fingerprinting and computer-assisted pattern analysis, p.151-171. In: Caetano-Anollés G. & Gresshoff P.M. (Eds), DNA Markers: protocols, applications, and overviews. J. Wileyand Sons, New York.). The PCR reactions were performed in a total volume of 50 μl composed of 45μL of PCR MasterMix, 1 μL of each primer (mecA F: AAA TCG ATG GTA AAG GTT GGC; mecA R: AGT TCT GCA GTA CCG GAT TTG C) and 3 μL of template DNA. Amplification was performed for 40 cycles as follows: initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 3 min with a final extension at 72°C for 5 min. Ten microliters of PCR product was analyzed using 2% agarose gel electrophoresis. The reactions were performed in a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). S. aureus ATCC 25923 was used as a negative control for the mecA gene. A mecA-positive strain of S. epidermidis, which was previously confirmed by sequencing of the mecA gene, was used as a positive control (Santos et al. 2015Santos F., Mendonça L., Reis D., Guimarães A., Lange C., Ribeiro J., Machado M. & Brito M. 2015. Presence of mecA-positive multidrug-resistant Staphylococcus epidermidis in bovine milk samples in Brazil. J. Dairy Sci. (In publication)).

Macrorestriction of the total DNA and pulsed-field gel electrophoresis (PFGE). One isolate of S. aureus from each S. aureus-positive cow was submitted to macrorestriction and PFGE analysis, according to a protocol previously proposed by McDougal et al. (2003)McDougal L.K., Steward C.D., Killgore G.E., Chaitram J.M., McAllister S.K. & Tenover F.C. 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J. Clin. Microbiol. 41:5113-5120.. Initially, a single colony of the isolates was inoculated into 5mL of brain heart infusion broth and incubated at 37°C for 24h. The concentrations of the cells were adjusted to an absorbance at 610nm of 0.9 to 1.1 using a spectrophotometer. Two hundred-microliter aliquots of the adjusted cell suspension were centrifuged. The pellet was resuspended in 300μl of Tris-EDTA (TE) buffer (10 mM Tris HCl, 1 mM EDTA [pH 8.0]) and incubated in a water bath at 37°C for 10 min. Three hundred-microliter aliquots of 1.8% of agarose were added to the cell suspension, gently mixed, and dispensed into wells of a plug mold. DNA-soaked agarose plugs were submitted to treatment with 4μL lysostaphin (no. L-7386: Sigma, 1mg / mL). The solidified plugs were transferred to 3 mL of buffer lysis solution (6mM Tris-HCl, 1 M NaCl, 100mM EDTA, 0.5% Brij-58, 0.2% sodium deoxycholate, and 0.5% sodium-lauryl-sarcosine) and incubated at 37°C in a water bath with stirring for 4h or overnight. The plugs were washed four times with 4mL of TE buffer for 10-15 min each and stored at 4°C.

Next, a DNA-plug portion was subjected to digestion with 20 U of the enzyme SmaI (Invitrogen, Carlsbad, CA, USA) for 2-3 hours at 25°C. Electrophoresis was performed with a 1% agarose gel using 0.5X Tris-borate-EDTA buffer with a CHEF DR-II system (BioRad Laboratories, Hercules, CA) at 6 V/cm for 20h at 14oC, with an initial switching time of 5 seconds and a final switching time of 40 seconds. Salmonella Braenderup (ATCC# BAA-664) was included as a size reference. After PFGE, the gel was stained with ethidium bromide (2μg/mL, Sigma, St. Louis, USA) for 20 min in a covered container and destained in distilled water for 45 min. The gels were photographed and documented using a Kodak 2200 system (Rochester, New York, USA).

Banding patterns were compared using the Gel-Compar II software package (Applied Maths, Kortrijk, Belgium). Percent similarities between isolate fingerprints were determined on the basis of the Dice correlation coefficient. A band position tolerance of 1% was used for the analysis of PFGE patterns. Dendrograms were generated by unweighted pairwise grouping with mathematical averaging (UPGMA). Isolates were considered as belonging to a common cluster when the PFGE pattern differed by ≤ 4 bands. A cluster was considered transient when isolated only once over a two-year period in a dairy farm; intermittent when it was isolated in more than one sampling event; and persistent when isolated at two or more subsequent samplings.

Results

From 1,060 milk samples collected, 395 (37.2%) tested negative for bacterial growth. Staphylococcus aureus was isolated from 136 samples (12.8%), and other Staphylococcus spp. were present in 290 samples (27.3%) (Table 1). Other microorganisms (Corynebacterium sp., Streptococcus sp., Enterococcus sp., Nocardia sp., Trueperella pyogenes, Escherichia coli, Klebsiella sp. and yeast) were found in 239 samples (22.5%). On 17 farms, S. aureus was isolated at frequencies ranging from 2.6 to 52.9% of the samples; on 14 farms S. aureus isolates were obtained in two or more samplings; and five farms were positive in all four samplings.

Table 1
Bacteriological test results from milk samples collected on small dairy farms in southern Brazil

Among the 132 isolates of S. aureus tested by the disk diffusion method, the majority (87.1%; 115/132) were susceptible to all antimicrobial agents. Resistance was detected to penicillin (18.2%; 16/132), tetracycline (2.3%; 3/132), and enrofloxacin (0.7%; 1/132). All penicillin susceptible isolates (n=116) displayed a fuzzy edge zone on the disk diffusion test and were considered as no beta-lactamase producers. All strains were susceptible to cephalothin, ceftiofur, clindamycin, gentamicin, sulfonamide, and sulfa + trimethoprim. Regarding methicillin-resistance, three isolates (2.3%) were resistant in the disk diffusion test to oxacillin. The MICs presented by these strains ranged from 0.19 to 0.75μg.mL-1 in the Etest (resistance ≥4μg.mL-1). All three isolates were also negative for mecA detection and, therefore, were considered methicillin-susceptible.

The most prevalent resistance profile included resistance only to penicillin and was observed in 14 isolates (10.6%). Only one isolate was resistant to antimicrobials belonging to three different classes of antimicrobial agents (penicillin, enrofloxacin and tetracycline) and was thus considered multi-resistant.

Among the 122 S. aureus isolates digested by SmaI, 102 different PFGE banding patterns were obtained. The patterns presented between 6 and 16 bands ranging from 33.3 to ~1140 kb. Fourteen patterns were found in more than one isolate (from two to five); the remaining patterns were represented by single isolates. The 102 PFGE banding patterns were grouped into 30 clusters of patterns that differed by ≤4 bands (Fig.1). Four clusters (P5, P8, P9 and P17) encompassed almost the half of the S. aureus isolates. Ten clusters (P4, P5, P6, P8, P9, P14, P17, P19, P26 and P27) were found in more than one sampling event at the same respective farms and were classified as intermittent or persistent (Table 2). These clusters encompassed 63 (51.6%) of the S. aureus isolates submitted to PFGE profiling. On one farm (K), the same cluster (P9) was persistent over the two-year period of testing. On two farms (Q and G), up to three persistent or intermittent clusters were found concomitantly infecting different cows. Among the isolates belonging to persistent and intermittent clusters, the great majority (59/63; 93.6%) were susceptible to all tested antimicrobials.

Fig.1
Dendrogram representing the similarity grouping of Staphylococcus aureus clusters. The total DNA of isolates was digested with SmaI and separated by pulsed-field gel electrophoresis (PFGE).

Table 2
Distribution of persistent and intermittent S. aureus PFGE-pulsotypes among dairy farms sampled in a two-year period in southern Brazil

Discussion

In this study, milk of lactating cows with and without symptoms of mastitis was sampled during a two-year period in order to assess the presence of persistent Staphylococcus aureus strains causing intramammary infection in small dairy herds in southern Brazil. In 62.3% (665/1060) of the samples, at least one bacterial species was isolated, and among them the genus Staphylococci was the most frequent (426/665, 64.1%). The colonization of the udder with coagulase-negative staphylococci is frequently reported in dairy herds, and it may cause mild subclinical mastitis (Mendonça et al. 2012Mendonça E.C.L., Marques V.F., Melo D.A., Alencar T.A., Coelho I.S., Coelho S.M.O. & Souza M.M.S. 2012. Caracterização fenotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite bovina. Pesq. Vet. Bras. 32(9):859-864., Mork et al. 2012Mork T., Jorgensen H.J., Sunde M., Kvitle B., Sviland S., Waage S. & Tollersrud T. 2012. Persistence of staphylococcal species and genotypes in the bovine udder. Vet. Microbiol. 159:171-180.). In our study, the cows were not examined for alterations in the mammary gland or the milk somatic cell counts were assessed; therefore, it is not possible to draw any conclusion about the implications of the coagulase-negative staphylococci isolation for the udder's health of the sampled animals. On the contrary, the isolation of S. aureus from 12.8% (136/1060) of the milk samples from lactating cows, belonging to 17 (17/21, 80.9%) of those farms, demonstrates the relevance of this pathogen in the region. Among the 17 positive herds, in 14 (82.3%) S. aureus was isolated in two or more sampling events indicating that S. aureus strains were circulating among the animals over time. Staphylococcus aureus is a frequent cause of clinical and subclinical mastitis, and chronically infected cows in a herd are an important reservoir of this pathogen (Ericsson et al. 2009Ericsson U.H., Lindberg A., Person W.K., Ekman T., Artursson K., Nilsson-Ost M. & Bengtsson B. 2009. Microbial aetiology of acute clinical mastitis and agent-specific risk factors. Vet. Microbiol. 137:90-97., Mork et al. 2012Mork T., Jorgensen H.J., Sunde M., Kvitle B., Sviland S., Waage S. & Tollersrud T. 2012. Persistence of staphylococcal species and genotypes in the bovine udder. Vet. Microbiol. 159:171-180.). Considering the highly contagious nature of the infection, the presence of infected cows may constitute a serious risk of the perpetuation and spread of S. aureus to susceptible animals and the contamination of milk. Although the inability to eradicate S. aureus from dairy herds may be related to failures in standard milking time hygiene as well as in dry cow mastitis therapy (Hutton et al. 1990Hutton D.T., Fox L.K. & Hancock D.D. 1990. Mastitis control practices: difference between herds with high and low milk somatic cell counts. J. Dairy Sci. 73:1135-1143.), other studies raised the hypothesis that certain strains could be more difficult to eliminate from the udder (Smith et al. 1998Smith T.H., Fox L.K. & Middleton J.R. 1998. Outbreak of mastitis caused by one strain of Staphylococcus aureus in closed dairy herd. J. Am. Vet. Med. Assoc. 212:553-556.).

Early attempts for discrimination of S. aureus strains were based on typing methods, such as ribotyping and phagotyping, and resulted in the identification of common strains in different herds and geographical areas (Mathews et al. 1994Mathews K.R., Kumar S.J., O'Conner S.A., Harmon R.J., Pankey J.W., Fox L.K. & Oliver S.P. 1994. Genomic fingerprints of Staphylococcus aureus of bovine origin by polymerase chain reaction-based DNA fingerprinting. Epidemiol. Infect. 107:177-185., Aarestrup & Jensen 1997Aarestrup F.M. & Jensen N.E. 1997. Prevalence and duration of intramammary infection in Danish heifers during the peripartum period. J. Dairy Sci. 80:307-312.). Later on, PFGE started to be considered the most discriminatory tool to resolve clonal relationship and proved to be superior to other tested techniques (Tenover et al. 1994Tenover F.C., Arbeit R., Archer G., Biddle J., Byrne S., Goering R., Hancock G., Herbert G.A., Hill B., Hollis R., Jarvis W.R., Kreiswirth B., Eisner W., Maslow J., McDougal L.K., Miller J.M., Mulligan M. & Pfaller M.A. 1994. Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus. J. Clin. Microbiol. 32:407-415., Olive & Bean 1999Olive D.M. & Bean P. 1999. Principles and applications of methods for DNA-based typing of microbial organisms. J. Clin. Microb. 37:1661-1669.). Using PFGE, it was demonstrated that S. aureus strains are more likely to be unique to a herd than to be found in multiple herds, and once introduced in a herd these strains may become persistent (Joo et al. 2001Joo Y.S., Fox L.K., Davis W.C., Bohach G.A. & Park Y.H. 2001. Staphylococcus aureus associated with mammary glands of cows: genotyping to distinguish different strains among herds. Vet. Microbiol. 80:131-138., Mork et al. 2012Mork T., Jorgensen H.J., Sunde M., Kvitle B., Sviland S., Waage S. & Tollersrud T. 2012. Persistence of staphylococcal species and genotypes in the bovine udder. Vet. Microbiol. 159:171-180.). In our study, the 122 S. aureus isolates digested by SmaI could be grouped into 30 PFGE clusters, among which three (P5, P8 and P9) encompassed strains originated from four to five different herds. Seven clusters included strains present in two herds and the other 20 clusters were found in only one herd. In this sense, our results are in accordance with the conclusion of Joo et al. (2001)Joo Y.S., Fox L.K., Davis W.C., Bohach G.A. & Park Y.H. 2001. Staphylococcus aureus associated with mammary glands of cows: genotyping to distinguish different strains among herds. Vet. Microbiol. 80:131-138. that S. aureus strains are more likely to be associated with the herd than to be widespread among multiple herds in a region.

Regarding the persistence of clusters, ten were considered persistent or intermittent in the farms over the two-year period. Among them, seven clusters were found at least once in up to five herds and two of them (P5 and P9) proved to be intermittent or persistent in two herds each. Although those clonal groups represent one third of the total of clusters, they are distributed among herds as well as persistent. Other studies also pointed out that some clonal groups can cause persistent intramammary infections over months to years (Aarestrup et al. 1995Aarestrup F.M., Wegener H.C. & Rosdahl V.T. 1995. A comparative study of Staphylococcus aureus strains isolated from bovine subclinical mastitis during 1952-1956 and 1992. Acta Vet. Scand. 36:237-243., Buzzola et al. 2001Buzzola F.R., Quelle L., Gomez M.I., Catala M., Steele-Moore L., Berg D., Gentilini E., Denamiel G. & Sordelli D.O. 2001. Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina. Epidemiol. Infect. 126:445-452.). Since they persist in the udder, they are secreted over time and have a higher potential for spreading. Persistent strains most likely have properties that make them particularly fit to survive in the udder. Genes encoding virulence factors, or those required for biofilm formation, have been reported in persistent strains isolated from the udders of cows (Cucarella et al. 2004Cucarella C., Tormo A.T., Úbeda C., Trotonda M.P., Monzón M., Peris C., Amorena B., Lasa I. & Penadés J.R. 2004. Role of biofilm-associated protein Bap in the pathogenesis of bovine Staphylococcus aureus. Infect. Immun. 72:2177-2185., Haveri et al. 2008Haveri M., Hovinen M., Roslöf A. & Pyörälä S. 2008.Molecular types and genetic profiles of Staphylococcus aureus strains isolated from bovine intramammary infections and extramammary sites. J. Clin. Microbiol. 46:3728-3735., Piccinini et al. 2012Piccinini R., Tassi R., Daprà V., Pilla R., Fenner J., Carter B. & Anjum M. 2012. Study of Staphylococcus aureus collected at slaughter from dairy cows with chronic mastitis. J. Dairy Res. 79:249-255.). Another possible explanation for persistence of clonal groups could be the repeated treatment with antimicrobials, which would lead to the elimination of susceptible strains but allow for the persistence of resistant strains (Rajala-Schultz et al. 2009Rajala-Schultz P.J., Torres A.H., DeGraves F.J., Gebreyes W.A. & Patchanee P. 2009. Antimicrobial resistance and genotypic characterization of coagulase-negative staphylococci over dry period. Vet. Microbiol. 134: 55-64.).

In our study, antimicrobial resistance was not frequent among the S. aureus strains, and the majority (87.1%) of the S. aureus isolates, including the persistent and intermittent isolates, was susceptible to all tested antimicrobials. Even the resistance to penicillin, which is highly prevalent among S. aureus strains in Brazilian herds (Medeiros et al. 2009Medeiros E.S., Mota A.M., Santos M.V., Freitas M.F.L., Pinheiro Júnior J.W. & Teles J.A.A. 2009. Perfil de sensibilidade microbiana in vitro de linhagens de Staphylococcus spp. isoladas de vacas com mastite subclínica. Pesq. Vet. Bras. 29(7):569-574., Silva et al. 2012Silva E.R., Pereira A.M.G., Moraes W.S., Santoro K.R. & Silva T.R.M. 2012. Perfil de sensibilidade antimicrobiana in vitro de Staphylococcus aureus isolado de mastite subclínica bovina. Revta Bras. Saúde Prod. Anim. 13(3):701-711.), was found in only 18.2% of strains. Among the sampled farms, only clinical mastitis cases were treated with antimicrobials and no susceptibility test was performed. In Brazilian herds, a higher chance of S. aureus resistant to penicillin and ampicillin was found in farms that did not perform microbiological cultures and susceptibility tests (Beuron et al. 2014Beuron D.C., Cortinhas C.S., Botaro B.G., Macedo S.N., Gonçalves J.L., Brito M.A.V.P. & Santos M.V. 2014. Risk factors associated with the antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis. Pesq. Vet. Bras. 34(10):947-952.). In the aforementioned study, herds with a higher number of lactating cows (from 10 to >60) were sampled, while in our study 80% of the sampled farms had less than 15 cows in lactation. The size of herds was found to influence the adoption of culture tests, and large herds were found to perform test more often than small herds. In fact, most herds of our study were small and didn't perform culture tests, which may explain the lack of association of antimicrobial resistance and none antimicrobial susceptibility testing. Another study (Pol & Ruegg 2007Pol M. & Ruegg P.L. 2007. Relationship between antimicrobial drug usage and antimicrobial susceptibility of Gram-positive mastitis pathogens. J. Dairy Sci. 90:262-273.) pointed out that the higher resistance to penicillin in S. aureus strains was attributed to the adoption of dry cow treatment and the long-term exposure to antimicrobials. However, in our study the low frequency of resistant strains cannot be related to the dry cow therapy, which, according to the farmers, was seldom adopted in those herds. The costs of laboratory diagnosis and treatment of chronically infected animals are often not affordable in small herds, and antimicrobial treatment is usually performed only in clinically affected animals. On the other hand, the lack of subclinical mastitis diagnosis may contribute to the persistence of udder infection with susceptible isolates, which could have been eliminated by antimicrobial treatment.

Even considering the penicillin susceptible profile of the tested S. aureus strains, we screened MRSA strains by the agar diffusion test using oxacillin discs and submitted the positive strains to mecA detection and E-test. Although three isolates were positive in the screening test, none of them carried mecA and the MIC values were much lower than the CLSI-breakpoint for susceptibility (≤2μg/mL), which may suggest that the strains were in fact not resistant. A variant of mecA has been described (Cuny et al. 2011Cuny C., Layer F., Strommenger B. & Witte W. 2011. Rare occurrence of methicillin-resistant Staphylococcus aureus CC130 with a novel mecA homologue in humans in Germany. PLos ONE 6(9):e24360.) and identified in bovine mastitis and in humans (García-Álvarez et al. 2011García-Álvarez L., Holden M.T., Lindsay H., Webb C.R., Brown D.F., Curran M.D., Walpole E., Brooks K., Pickard D.J., Teale C., Parkhill J., Bentley S.D., Edwards G.F., Girvan E.K., Kearns A.M., Pichon B., Hill R.L., Larsen A.R., Skov R.L., Peacock S.J., Maskell D.J. & Holmes M.A. 2011. Methicillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect. Dis. 11(8):595-603.). However, our isolates presented a different phenotypic resistance profile of strains carrying variant mecA, which were resistant to oxacillin in the screening test and displayed high MIC values.

In summary, although persistent clusters were present, a characteristic typically reported in intensive production systems, the predominance of sporadic PFGE clusters demonstrates the wide variability of S. aureus found on small dairy farms. This scenario might be related to several factors. Most dairy farmers in the region are not aware of the risks of S. aureus colonization to milk production and human health. Furthermore, most small farmers failed to maintain hygiene measures before and after milking, which may facilitate the presence of S. aureus on the skin of the udder and its access to the mammary gland. The challenges of S. aureus body colonization of lactating cows brought on by their immediate environment have already been demonstrated (Capurro et al. 2010Capurro A., Aspán A., Unnerstad E.H., Waller K.P. & Artursson K. 2010. Identification of potential sources of Staphylococcus aureus in herds with mastitis problems. J. Dairy Sci. 93:180-191.), highlighting the importance of hygiene measures and grouping or culling practices of infected animals in control programs. The lack of these measures on the sampled farms may have been responsible for the new S. aureus clusters that were introduced into the mammary gland, as well as for their persistence and continuous circulation among animals or between animals and their environment.

Conclusions

In summary, the results suggest that Staphylococcus aureus intramammary colonization on small dairy farms in southern Brazil is mostly caused by sporadic PFGE-clusters, although some persistent clusters can be present over time.

In both groups, isolates showed to be highly susceptible to antimicrobials.

Acknowledgements

This project was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 578430/ 2008-8). D. Paim and T. Campos received scholarships from CNPq.

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Publication Dates

  • Publication in this collection
    Oct 2016

History

  • Received
    26 Jan 2016
  • Accepted
    24 June 2016
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