Abstract
Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.
Mastitis, which may be clinical (severe) or subclinical (moderate), is an important mammary gland disease that is usually caused by bacterial infection. If untreated, it constitutes a serious problem in dairy herds with considerable economic consequences, mainly due to reduced milk production and discarded milk (27). Although several bacterial pathogens can cause mastitis, Staphylococcus aureus is one of the most important etiologic agents in mastitis of cows, goats, and sheep (20). Moreover, S. aureus is probably the most infectious agent because it causes a chronic and deep infection in the mammary glands that is extremely difficult to cure (19).
The prevalence of subclinical and clinical bovine mastitis caused by S. aureus ranges from 5 to 50% in different countries (17). Similarly, the prevalence of S. aureus intramammary infections in goats ranged from 5.6 to 17% (29) and between 5 and 11% in clinical cases of ovine mastitis (21), whereas lower rates, between 0.22 and 2.06%, have been reported for subclinical mastitis in sheep (33). In contrast to studies of bovine, caprine, and ovine infections, only a few studies of staphylococcal mastitis concerning buffalo intramammary infections have been published, and the importance of subclinical mastitis in this dairy species may not be enough to significantly influence milk production as in other domestic ruminants (22). In Brazil, in the rural areas of Rio de Janeiro State, bovine subclinical mastitis was reported to be higher than in other areas of the country and close to 40% (5, 34), whereas S. aureus was identified in up to 37% of isolates from goats with subclinical mastitis (28).
Knowledge of the distribution of S. aureus in dairy herds might help to formulate strategies for reducing the spread of infection. During the past decade, the epidemiology of S. aureus mastitis in dairy cattle has been studied using various molecular typing methods. Several studies revealed that only a few specialized clones are responsible for most of the cases of mastitis on a single farm and that some of these S. aureus clones might have a broad geographic distribution (1, 9, 14, 15, 30).
Molecular studies of S. aureus isolates from dairy farms in Brazil are still limited and concentrate exclusively on cows (2, 16, 25, 26, 34) and goats (29). The aim of the present study was to characterize isolates from cases of bovine, caprine, ovine, and buffalo S. aureus mastitis by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) and contribute to the comprehension of the S. aureus genetic population in Brazilian dairy herds.
MATERIALS AND METHODS
Origin of the bacterial isolates.
Seventy-nine staphylococcal isolates were obtained from milk samples of cows (n = 12), sheep (n = 16), goats (n = 18), and buffalo (n = 33) with subclinical mastitis, hosted in 18 small diary herds (30 to 100 animals) located in 10 municipalities of the State of Rio de Janeiro, Brazil (Fig. 1). The bovine samples were collected between June 1996 and April 1997 from 10 herds (herds D to H and M to Q), the buffalo samples were collected between March and June 2003 from 5 herds (herds I to L and R), the caprine samples were collected in October 2003 from herds A and B, and the ovine samples were collected in November 2003 from herd C. In addition, anal samples were randomly collected in November 2005 from five ostriches created in an ostrich breeding (breeding X).
FIG. 1.
Map of Rio de Janeiro State showing the locations of the different municipalities, the herds from which the samples were collected, and the geographic clonal distribution. Letters designate the herds or ostrich breeding. The type of animal hosted in each herd is indicated on the left size of the figure. Symbols indicate the clonal type(s) recovered in each herd. No S. aureus isolates were recovered from herds M to R.
Identification of staphylococcal isolates.
A California mastitis test of milk samples was performed using a commercially available test (Fatec, São Paulo, Brazil). The milk samples were collected after cleaning the teats, discarding a few streams of milk, and scrubbing the teat ends with cotton balls moistened with 70% alcohol. California mastitis test-positive samples were transported to the laboratory on ice. Ostriches were sampled by introducing sterile cotton swabs (Venturi Transystem; Copan Innovation, Italy) into the anus. All samples were incubated at 37°C for 18 h and then inoculated on 5% sheep blood agar plates. The bacterial genus was determined on the basis of Gram staining, colony morphology, and catalase testing.
Identification of S. aureus isolates.
The colonies identified as Staphylococcus were subjected to biochemical tests for oxidase (DrySlide oxidase; Difco) and aerobic mannitol fermentation. All isolates that fermented mannitol were subsequently tested with a Staphytech Plus kit (Oxoid, Basingstoke, United Kingdom) and were also tested for acetoin production.
Antimicrobial susceptibility tests.
Susceptibility tests were performed by the disk diffusion method for penicillin, oxacillin, cephalothin, cefoxitin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, gentamicin, and tetracycline according to the guidelines of the Clinical Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (24). Susceptibility to vancomycin was tested according to the European Antimicrobial Resistance Surveillance System (EARSS) protocol (8): 10 μl of the stationary-phase culture grown overnight in brain heart infusion broth was plated onto a Mueller-Hinton agar plate containing 5 μg/ml of the antibiotic and incubated for 48 h at 36°C.
PFGE.
PFGE was performed after SmaI digestion as described by Chung et al. (3). The resulting band patterns were analyzed by visual inspection, followed by analysis with BioNumerics software (version 4.0; Applied Maths, Gent, Belgium) for relatedness evaluation. Dendrograms were generated from similarity matrixes calculated with the Dice coefficient, and patterns were clustered by the unweighted-pair group method with arithmetic averages, using an optimization and a tolerance of 1%. Profiles with more than 80% similarity were considered closely related.
DNA isolation for PCR essays.
Chromosomal DNA was extracted by incubating cells grown overnight in an agar plate with 20 μl of 1× TE (10 mM Tris, 1 mM EDTA, pH 8) and lysostaphin at 0.5 mg/ml for the lysis step for 30 min, followed by a denaturation step for 15 min at 95°C. The mixture was harvested at 13,000 rpm for 5 min, and 2 μl of the supernatant was used as a DNA template in the PCRs.
Detection of the mecA, PVL, and ETD genes.
The presence of the mecA gene was determined by PCR as described by Murakami et al. (23). The genes encoding the Panton-Valentine leukocidin (PVL) and exfoliative toxin D (ETD), i.e., lukS-PV lukF-PV and etd, respectively, were amplified by PCR as described previously (12, 18).
spa typing and MLST.
spa typing was performed as previously described (11). The spa types were assigned through the Ridom web server (http://www.ridom.de/spaserver/). spa types with similar repeat profiles were grouped together as part of the same lineage (spa lineage) identified in the present study by the same capital letter.
MLST was carried out as described by Enright et al. (6), with the exception that primer arcCF2 (5′-CCT TTA TTT GAT TCA CCA GCG-3′) (4) was used. spa typing and MLST PCR products were purified with a High Pure PCR product purification kit (Roche, Basel, Switzerland) and used as templates for sequencing of both strands at Macrogen, Seoul, South Korea. MLST alleles and sequence types (STs) were identified using the MLST database (http://www.mlst.net), which was also employed to perform searches for earlier reports on the STs identified during the present study. Trace files of putative novel alleles and the allelic profiles of novel STs were sent to the database curator for allele and/or ST number assignment and entry into the database.
RESULTS
Species identification.
Among the 87 staphylococcal isolates, 58 isolates fermented mannitol. Out of these, 30 isolates produced agglutination with the Staphytech plus kit, showed acetoin production, and were classified as S. aureus.
Antimicrobial susceptibility tests.
The isolates showed full susceptibility to oxacillin, cephalothin, cefoxitin, trimethoprim-sulfamethoxazole, gentamicin, tetracycline, and vancomycin. Among the 30 isolates, 10 (33%) were resistant to penicillin, 2 (6.5%) showed intermediate resistance to clindamycin, and 2 (6.5%) were intermediately resistant to erythromycin. Twenty isolates (67%) were susceptible to all antimicrobial agents tested. None of the isolates amplified the mecA gene, confirming methicillin susceptibility.
Molecular typing.
Digestion of the chromosomal DNA of the isolates with the endonuclease SmaI and separation of the fragments by PFGE revealed five types (A, B, C, D, and F) (Table 1). PFGE type A represented over half of the isolates (n = 19 [63%]) and comprised six subtypes (A1 to A6). A total of 12 isolates representative of each PFGE subtype were studied by spa typing, generating eight spa types that could be grouped in three spa lineages. Although PFGE types B and F showed congruence between PFGE and spa type clustering, being grouped in two distinct clones by both methodologies, PFGE types A, C, and D were associated with the same spa lineage (Table 1). MLST performed on a representative of each PFGE pattern identified five STs (out of which three were novel STs), confirming the existence of five distinct clones among the 30 MSSA isolates. Table 1 shows the molecular characterization of strains representing the five clonal types defined by PFGE, spa typing, and MLST. Representatives of all PFGE subtypes were tested for the presence of the PVL and ETD genes, and no isolate harbored any of these genes.
TABLE 1.
Molecular characteristics of ostrich colonization and bovine, caprine, ovine, and buffalo milk S. aureus isolates from Rio de Janeiro State, Brazil
| Strain | PFGE type | No. of isolates | No. of isolates for indicated animal species
|
spa typing data
|
MLST data
|
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Bovine | Caprine | Ovine | Buffalo | Ostrich | Repeat succession | Type | Lineage | Allelic profile | ST | |||
| VET-BZ1 | A1 | 7 | 1 | 4 | 2 | r07r23r12r21r17r34r34r33r34 | t359 | A | 3-1-1-1-1-5-96a | 747a | ||
| VET-BZ3 | A2 | 1 | 1 | r07r23r12r21r17r34r34r33r34 | t359 | A | ||||||
| VET-BZ12 | A3 | 1 | 1 | r07r23r12r21r17r34r3434r34r34r34r33r34 | t1182 | A | ||||||
| VET-BZ15 | A4 | 6 | 1 | 2 | 2 | 1 | r07r23r12r21r17r34r34 r34r33r34 | t267 | A | |||
| VET-BZ47 | A5 | 3 | 2 | 1 | r07r23r12r21r17r34r34r33r34 | t359 | A | |||||
| VET-BZ46 | A6 | 1 | 1 | r07r23r12r21r17r34r34 r34r33r34 | t267 | A | ||||||
| VET-BZ29 | B1 | 3 | 3 | r03r16r12r21r17r16r17r17r17r23r24 | t1180 | B | 89a-66-46-2-7-50-18 | 750a | ||||
| VET-BZ55B | B2 | 2 | 2 | r03r16r21r17r23r13r17r17r17r17r17r23r24 | t1181 | B | ||||||
| VET-BZ45 | C | 1 | 1 | r07r23 | t605 | A | 3-68-1-4-1-5-40 | 126 | ||||
| VET-BZ70 | D1 | 2 | 2 | r07r23r21r16r34r33r13 | t127 | A | 1-1-1-73a-1-1-1 | 751a | ||||
| VET-BZ71 | D2 | 1 | 1 | r07r23r21r16r34r33r13 | t127 | A | ||||||
| VET-BZ66 | F | 2 | 2 | r26r23r17r34r17r20r17r12r17r16 | t002 | C | 1-4-1-4-12-1-10 | 5 | ||||
Allele or ST newly registered at www.mlst.net.
Clonal distribution among the different animals.
The three isolates colonizing ostriches and the three isolates from cases of caprine mastitis, as well as five of the six isolates recovered from cows, were classified as PFGE type A. The remaining bovine isolate belonged to clone C. The 13 buffalo isolates were distributed over all clonal types, with the exception of clone C, while the ovine isolates belonged to clone A or B. Table 1 displays the clonal distribution among the bovine, ovine, caprine, buffalo, and ostrich isolates.
Geographic clonal distribution.
Clonal type A, represented by 19 isolates, was distributed over six municipalities of Rio de Janeiro State (Fig. 1). The five isolates of type B and the three isolates of type D were found in two farther apart municipalities. Clones C and F, represented by single isolates, were found in different municipalities in coexistence with other clones.
DISCUSSION
Studies of the molecular epidemiology of S. aureus collected from cows and goats in Brazil are still very scarce, and there is no published study of S. aureus from sheep or buffalo. In the present study, we characterized S. aureus isolates from cases of bovine, ovine, caprine, and buffalo subclinical mastitis collected in different herds and different municipalities of Rio de Janeiro State, Brazil, in order to contribute to a better knowledge of the distribution of S. aureus in Brazilian dairy herds.
The antimicrobial susceptibility test revealed that 20 strains (67%) were susceptible to all antimicrobial agents tested, a susceptibility rate slightly higher than that found by Rabello et al. (25), who reported a susceptibility rate of 45% for all antimicrobial agents among bovine S. aureus isolates from farms in Rio de Janeiro State. In addition, we observed no tetracycline resistance and a lower percentage of penicillin resistance (33% versus 55%) (16, 25). The discrepancies observed might be related to different antimicrobial policies adopted for the herds enrolled in the two studies.
The genetic diversity among S. aureus isolates recovered from cases of bovine, ovine, caprine, and buffalo mastitis as well as among a few isolates colonizing ostriches was studied by PFGE, spa typing, and MLST, which showed that the 30 isolates were distributed over five clonal types, out of which 63% belonged to a single clone (PFGE A, spa type t359, ST747). This clone was found in several herds located in different regions of Rio de Janeiro State, substantiating the existence of a limited number of clones of S. aureus which are responsible for at least some of the cases of ruminant mastitis in this Brazilian state. This is in agreement with the findings of others that demonstrated that only a few specialized clones were responsible for most of the cases of bovine mastitis and that these S. aureus clones have a broad geographic distribution (1, 9, 30). Moreover, Cabral et al. (2) reported that only a limited number of clones were responsible for the cases of S. aureus bovine mastitis on various farms in six different municipal districts of two regions of São Paulo State, Brazil. It is important to note that the isolates recovered from cows were collected 7 to 9 years prior to the other isolates, which could be relevant for the interpretation of some of the data. However, although S. aureus clonal replacements over time have been frequently reported in several countries, in Brazil a single nosocomial methicillin-resistant S. aureus (MRSA) clone, the so called pandemic Brazilian MRSA clone (ST239, SCCmecIII), has been responsible for the overwhelming majority of nosocomial infections for the last 20 years (31, 35). An investigation of more S. aureus strains from the state of Rio de Janeiro and from other states of Brazil, including more contemporary isolates from cows, would give more information about the geographic distribution and temporal evolution of these mastitis-causing clones.
None of the S. aureus isolates harbored the PVL or ETD gene. In contrast, Zecconi et al. (37) identified the PVL gene in more than 50% of the bovine isolates tested, a value that is close to the ones observed in human isolates (36) but not in animal strains (32).
Three out of the five STs found in the present study, including the major ST747 clone, were newly registered and integrated one novel allele each (Table 1). It is not unusual for animal isolates to possess novel alleles in comparison to human strains (13), which may be simply due to the investigation of a new population, as most analyses so far have focused on human clinical isolates. Greater sampling and analyses of other S. aureus populations would likely yield novel alleles. The absence, at the time of this writing, of strains from other locations in the S. aureus MLST database (http://www.mlst.net) with the novel alleles yqiI-96, arcC-89, and gmk-73 may indicate localized selective pressure and a consequently confined diversification.
A single isolate of ST747, the major ST found in the present study, was found in a milk isolate from a cow with bovine mastitis from the region of Rio de Janeiro and later reported (http://www.mlst.net). Single-locus variants (SLV) or double-locus variants (DLV) of ST747 have been detected among isolates from cases of bovine mastitis collected in The Netherlands (STs 70 and 71), Brazil (STs 742 and 746) (http://www.mlst.net), the United States (STs 97, 115, 124, and 347), and Chile (STs 97, 355, 357, and 358) (30). In addition, Smith et al. (30) showed that ST97 (SLV of ST747) and its derivatives are adapted not only to the mammary gland but also to the bovine environment and have achieved a worldwide distribution. Similarly, SLV or DLV of ST750, the second most frequent ST in this study, have been found among isolates from cases of bovine mastitis collected in Norway (ST131, 132, and 133), Portugal (ST133), and Brazil (ST745) and among caprine isolates from Norway (ST481) and France (ST712) but never among human isolates (http://www.mlst.net).
Although one isolate recovered from buffalo milk belonged to ST5, a genetic background that is mainly associated with cases of human disease or colonization (7), all other STs found in the present study were exclusively detected among animal strains. These results showed that human isolates are different from ruminant and ostrich isolates, supporting the concept of host specificity among S. aureus strains, as suggested by others (10, 13, 14). Gilot and van Leeuwen (10) showed that the transfer of strains between humans and cows is a possible but infrequent event. Therefore, the detection of ST5 in a single buffalo milk sample could be due to human contamination.
Clonal type A was found among all the animal species in the present study, suggesting that S. aureus isolates from cows, sheep, goats, buffalo, and ostriches do not represent separate genetic populations. These findings agree with those of Mørk et al. (20), who genotyped Norwegian S. aureus isolates from cases of bovine, caprine, and ovine mastitis by PFGE and found that a small number of closely related genotypes are responsible for a great proportion of S. aureus mastitis cases in cows, ewes, and goats in Norway and that these genotypes exhibit little or no host preference among these species. Similarly, Jorgensen et al. (13) showed that certain S. aureus genotypes, with a wide geographic distribution, are frequently found in both Norwegian bovine and caprine bulk milk. Moreover, van Leeuwen et al. (32) showed that mastitis-related strains isolated from diverse host species (sheep, goat, and cow) were genetically clustered, signifying tissue specificity and that the combination of virulence factors plays an important role in host or even tissue specificity in S. aureus infections.
In summary, the phenotypic and genotypic characterization of S. aureus isolates from bovine, ovine, caprine, and buffalo milk samples as well as a few colonization ostrich samples showed that a major clonal type is responsible for subclinical mastitis in different regions of the state of Rio de Janeiro, Brazil. In addition, this clone does not demonstrate host specificity among the different animal species and seems to be capable of both infection and colonization.
Acknowledgments
This work was supported by Project FCG 61052 from Fundação Calouste Gulbenkian and Project POCTI/SAU-ESP/57841/2004 from Fundação para a Ciência e Tecnologia, Portugal, both awarded to H. de Lencastre, and by project E-26/170.509/2004 from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), awarded to O. Vieira-da-Motta.
We are grateful to Teresa Conceição for technical support.
Footnotes
Published ahead of print on 20 April 2007.
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