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Coagulase-negative staphylococci (CNS; n = 417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.
Coagulase-negative staphylococci are the microorganisms most commonly isolated from bovine milk in many countries, and they are an important cause of mastitis (
). The CNS are opportunistic pathogens that are usually diagnosed as a group without species identification. They cause subclinical IMI that result in an increase in SCC and reduced milk quality, leading to economic losses (
). Because simple subclinical CNS infections can be self-limiting, they are usually not treated with antibiotics. However, CNS often appear with other major pathogens such as Staphylococcus aureus, Streptococcus spp., or coliform bacteria. In these cases and in persistent CNS infections, the cows undergo antimicrobial treatment. Currently, β-lactam antimicrobials (including penicillin and cephalosporins), aminoglycosides (gentamicin and neomycin), and macrolides (spiramycin) are commonly used to treat mastitis in Switzerland (
Büttner, S., O. Flechtner, C. Müntener, and G. Overesch. 2011. Bericht über den Vertrieb von Antibiotika in der Veterinärmedizin und das Antibiotikaresistenzmonitoring bei Nutztieren in der Schweiz (ARCH-VET 2010). Federal Veterinary Office and Swissmedic, Bern, Switzerland. www.swissmedic.ch/archvet-d.asp.
). The CNS may also harbor antimicrobial resistance elements and pathogenicity islands, such as the staphylococcal cassette chromosome (SCCmec) element (
Methicillin-resistant coagulase-negative staphylococci in the community: high homology of SCCmec IVa between Staphylococcus epidermidis and major clones of methicillin-resistant Staphylococcus aureus.
The arginine catabolic mobile element and staphylococcal chromosomal cassette mec linkage: Convergence of virulence and resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus.
) that can be transferred to Staph. aureus. Arginine catabolic mobile elements are genomic islands in Staph. epidermidis that are associated with host colonization, fitness, and pathogenicity. Mobility of ACME is associated with recombinase genes present on the SCCmec elements (
Epidemiologic distribution of the arginine catabolic mobile element among selected methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates.
The arginine catabolic mobile element and staphylococcal chromosomal cassette mec linkage: Convergence of virulence and resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus.
). The SCCmec elements contain the mec genes—mecA or mecC (mecALGA251)—which encode alternative penicillin-binding proteins (PBP 2a) and confer resistance to all β-lactam antimicrobials (
Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus.
). Most Staph. sciuri isolates are susceptible to β-lactam antimicrobials. However, alterations in the promoter regions of mecA1 upregulate mecA1 expression and confer methicillin resistance (
Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus.
Expression of high-level methicillin resistance in Staphylococcus aureus from the Staphylococcus sciuri mecA homologue: Role of mutation(s) in the genetic background and in the coding region of mecA.
Development of methicillin resistance in clinical isolates of Staphylococcus sciuri by transcriptional activation of the mecA homologue native to the species.
Antimicrobial resistance of coagulase-negative staphylococci from bovine subclinical mastitis with particular reference to macrolide-lincosamide resistance phenotypes and genotypes.
) or the genetic background of multidrug-resistant CNS strains in bovine milk.
We identified different CNS species in milk from cows with clinical and subclinical bovine mastitis, characterized their antimicrobial resistance mechanisms, and determined whether specific methicillin-resistant and multidrug-resistant CNS clones are common in dairy cows.
Materials and Methods
Origin of Milk Samples
Coagulase-negative staphylococci (n = 417) were isolated from milk (n = 370) obtained from cows diagnosed with clinical (n = 115) and subclinical (n = 255) mastitis and control samples (n = 47) in Switzerland. Control samples were collected from cows that had suffered from mastitis previously and had been treated; the control milk samples contained <150,000 cells/mL. The 417 isolates came from 363 different cows and from 2 different mammary quarters of 7 cows. The 363 cows originated from 195 different farms (nf) in the cantons of Berne (nf = 91), Jura (nf = 56), Fribourg (nf = 26), Vaud (nf = 8), Lucerne (nf = 5), Valais (nf = 4), Solothurn (nf = 3), Aargau (nf = 1), and Thurgau (nf = 1). In 47 cases, 2 different CNS strains were found in the same milk sample.
Isolation and Identification of CNS
Milk samples were centrifuged at 590 × g for 10 min at room temperature. The milk pellets were cultivated on tryptone soy agar containing 5% defibrinated sheep blood (Becton, Dickinson and Co., Franklin Lakes, NJ) and incubated at 37°C for 18 to 24 h. Staphylococci were selected based on colony morphology, gram-positive staining of cocci, and catalase production and were subcultured on tryptone soy agar containing 5% defibrinated sheep blood.
The isolates were identified by matrix-assistedlaserdesorption/ionization time-of-flightmassspectrometry (MALDI-TOF MS) analysis using the ethanol–formic acid extraction method for better resolution (Microflex LT, Bruker Daltonics GmbH, Bremen, Germany; Application Note MT-80, Bruker Daltonics GmbH). Species identification was considered valid when the matching score with reference spectra of the MALDI Biotyper v3.0 database (Bruker Daltonics GmbH) was ≥2, according to the criteria proposed by the manufacturer. Isolates whose measured spectra had score <2.0 were further identified by DNA sequencing of the 16S rDNA (
). The CNS strains were stored at −80°C in trypticase soy medium containing 30% glycerin (Becton, Dickinson and Co.).
DNA Extraction and Amplification
To obtain total DNA, cells were incubated in 100 µL of Tris-EDTA buffer containing 0.1 mg/mL lysostaphin for 15 min at 37°C; then, 450 µL of lysis buffer (0.1 M Tris-HCl, pH 8.5, 0.05% Tween 20, 0.24 mg/mL proteinase K) was added and incubated at 60°C for 45 min. The DNA was then denatured at 95°C for 15 min. The PCR was performed with HOT FIREPol DNA Polymerase (Solis BioDyne, Tartu, Estonia) using the primers and conditions listed in Table 1.
Table 1Primers and primer sequences used in the study
Target gene
Primer name (F = forward; R = reverse) and sequence
Clinical and Laboratory Standards Institute. 2009. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Vol. 29, no. 2. 8th ed. Approved standard M07-A8. Clinical and Laboratory Standards Institute, Wayne, PA.
) using Sensititre susceptibility plates (NLEUST plates; Trek Diagnostics Systems, East Grinstead, UK) that contained the following 19 antimicrobials: chloramphenicol, ciprofloxacin, clindamycin, dalfopristin-quinupristin, erythromycin, fusidic acid, gentamicin, kanamycin, linezolid, mupirocin, oxacillin, penicillin, rifampicin, streptomycin, sulfamethoxazole, tetracycline, tiamulin, trimethoprim, and vancomycin. The resistance breakpoints were those proposed for CNS in the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, www.eucast.org; Table 2), except for streptomycin and kanamycin, for which breakpoints came from the French Society for Microbiology (www.sfm-microbiologie.org). The production of β-lactamase was tested on nitrocefin dry slides (Becton, Dickinson and Co.) using colonies grown on Mueller Hinton agar for 18 h at 37°C with 0.05 µg/mL penicillin to induce β-lactamase production (
Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission.
Denotes a significant difference (P=0.03) in the number of oxacillin-resistant isolates from clinical and subclinical mastitis cases, as determined by Fisher’s exact test.
Denotes a significant difference (P=0.03) in the number of oxacillin-resistant isolates from clinical and subclinical mastitis cases, as determined by Fisher’s exact test.
Denotes a significant difference (P=0.02) in the number of sulfamethoxazole-resistant isolates from clinical mastitis cases and control milk, as determined by Fisher’s exact test.
Denotes a significant difference (P=0.02) in the number of sulfamethoxazole-resistant isolates from clinical mastitis cases and control milk, as determined by Fisher’s exact test.
0.0
Trimethoprim
R >4.0
18
4.3
6
5.2
11
4.3
1
2.1
Clindamycin
R >0.5
14
3.4
3
2.6
10
3.9
1
2.1
Chloramphenicol
R >8.0
13
3.1
4
3.5
8
3.1
1
2.1
Gentamicin
R >1.0
10
2.4
5
4.4
5
2.0
0
0.0
Kanamycin
R >16.0
10
2.4
5
4.4
5
2.0
0
0.0
Quinupristin-dalfopristin
R >2.0
0
0.0
0
0.0
0
0.0
0
0.0
Rifampicin
R >0.5
0
0.0
0
0.0
0
0.0
0
0.0
Ciprofloxacin
R >1.0
0
0.0
0
0.0
0
0.0
0
0.0
Mupirocin
R >256
0
0.0
0
0.0
0
0.0
0
0.0
a Denotes a significant difference (P = 0.03) in the number of oxacillin-resistant isolates from clinical and subclinical mastitis cases, as determined by Fisher’s exact test.
b Denotes a significant difference (P = 0.02) in the number of sulfamethoxazole-resistant isolates from clinical mastitis cases and control milk, as determined by Fisher’s exact test.
Characterization of the mec Genes and SCCmec Elements
All isolates displaying a MIC for oxacillin above the resistance breakpoint (MIC >0.25 µg/mL), which suggests the presence of an alternative penicillin-binding protein (based on Clinical and Laboratory Standards Institute and EUCAST), were additionally tested by PCR for the mecA, mecA1, and mecC genes (
) using the primers listed in Table 1. The complete nucleotide sequences of the mecA genes were obtained by PCR amplification with the mecA-F7 and mecA-R7 primers (Table 1). Sequencing was performed on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA). The SCCmec types were determined by the Kondo method (
Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: Rapid identification system for mec, ccr, and major differences in junkyard regions.
Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus.
) in the promoter region by restriction analysis of PCR products amplified with primers mecAscK1-F and mecAsc-R (Table 1). The 335-bp PCR product was tested for PsiI cleavage using the manufacturer’s suggested conditions (New England BioLabs, Beverly, MA); PsiI recognizes the mutated promoter sequence TATAAT but not the wild-type sequence TATATT.
Genotyping of Methicillin-Resistant CNS
Methicillin-resistant, mecA-positive CNS isolates and multidrug-resistant, mecA-negative Staph. epidermidis isolates were genotyped by pulsed-field gel electrophoresis (PFGE). Analysis of SmaI-digested chromosomal DNA was performed as described previously (
Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission.
). Digested DNA was separated by gel electrophoresis in a contour-clamped homogeneous electric field DRIII device (Bio-Rad Laboratories Inc., Richmond, CA) with a ramped pulse time of 5 to 40 s at 6 V/cm for 21 h at 12°C. The lambda ladder PFG marker (New England BioLabs) was used as a size reference. The digital PFGE pattern images were analyzed with the BioNumerics software (Applied Maths, Kortrijk, Belgium), and the PFGE profiles were defined by the DNA banding patterns and criteria of
All Staph. epidermidis isolates (n = 15) were examined by multilocus sequence typing (MLST), which is based on the sequencing of internal fragments of 7 housekeeping genes (
Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis.
) and ACME. The presence and type of ACME was determined using the primer pairs AIPS.27 and AIPS.28 for arcA and AIPS.45 and AIPS.46 for opp3 gene clusters (
The arginine catabolic mobile element and staphylococcal chromosomal cassette mec linkage: Convergence of virulence and resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus.
Antimicrobial resistance phenotypes (Table 2) were compared using the Fisher exact test. This test is useful when the sample size is small (zero in some cells), and the test evaluates the hypothesis that the 2 column percentages in a 2 × 2 table are equal. Statistical analysis was performed with the statistical software NCSS 2007 (www.ncss.com). The overall level of statistical significance was set to P < 0.05.
Results
Prevalence and Identification of CNS
In total, 97.8% of the CNS isolates (n = 408) were clearly identified at the species level by MALDI-TOF MS analysis. The most frequent CNS species were Staph. xylosus, Staph. chromogenes, Staph. sciuri, and Staph. haemolyticus (Table 3). The remaining 2.2% were identified by 16S rDNA analysis as Staph. chromogenes (n = 1), Staph. saprophyticus (n = 1), or novel Staphylococcus species (n = 7; Table 3). Neither clinical nor subclinical mastitis could be correlated with the presence of individual bacterial species. Similar species were identified in control milk samples with low SCC. Of the 417 CNS, 268 isolates were the only species present in the milk from which they originated, and 149 isolates were present together with at least one other species. Staphylococcus xylosus (n = 92), Staph. chromogenes (n = 56), Staph. haemolyticus (n = 22), and Staph. sciuri (n = 21) were the predominant species among the 268 isolates that had only a single CNS species present (Table 4). Among the mixed cultures, 18 CNS isolates were co-purified with Staph. aureus (12.1%), 83 isolates were present with Streptococcus spp. (55.7%), and 48 isolates (32.2%) were coincident in milk with at least one other bacterium (e.g., Trueperella pyogenes, Escherichia coli, Corynebacterium bovis, or a mix of more than 3 different bacteria; Table 4). In the milk samples, none of the CNS was found more frequently as a single agent than together with other bacteria, with the exception of Staph. chromogenes, which was not often found together with Staph. aureus or streptococci (Table 4).
Table 3Prevalence of CNS and distribution of different CNS strains in clinical and subclinical mastitis milk and control milk
Indicates at least one species other than Staphylococcus aureus and Streptococcus spp. (e.g., Trueperella pyogenes, Escherichia coli, Corynebacterium bovis) or a mix of more than 3 different bacteria.
(n = 48)
No.
No.
%
No.
%
No.
%
No.
%
Staphylococcus xylosus
150
92
34.3
7
38.9
34
41.0
17
35.4
Staphylococcus chromogenes
70
56
20.9
1
5.6
5
6.0
8
16.7
Staphylococcus sciuri
37
21
7.8
1
5.6
10
12.0
5
10.4
Staphylococcus haemolyticus
35
22
8.2
0
0.0
9
10.8
4
8.3
Staphylococcus devriesei
18
13
4.9
0
0.0
3
3.6
2
4.2
Staphylococcus warneri
17
11
4.1
1
5.6
3
3.6
2
4.2
Staphylococcus simulans
16
14
5.2
0
0.0
2
2.4
0
0.0
Staphylococcus epidermidis
15
10
3.7
2
11.1
2
2.4
1
2.1
Staphylococcus fleurettii
12
4
1.5
1
5.6
4
4.8
3
6.3
Staphylococcus succinus
9
5
1.9
2
11.1
2
2.4
0
0.0
Staphylococcus vitulinus
9
2
0.7
3
16.7
0
0.0
4
8.3
Staphylococcus hyicus
6
2
0.7
0
0.0
3
3.6
1
2.1
Staphylococcus equorum
6
5
1.9
0
0.0
1
1.2
0
0.0
Staphylococcus saprophyticus
2
0
0.0
0
0.0
2
2.4
0
0.0
Staphylococcus auricularis
2
1
0.4
0
0.0
1
1.2
0
0.0
Staphylococcus capitis
2
2
0.7
0
0.0
0
0.0
0
0.0
Staphylococcus cohnii
2
2
0.7
0
0.0
0
0.0
0
0.0
Staphylococcus hominis
1
0
0.0
0
0.0
1
1.2
0
0.0
Staphylococcus lentus
1
1
0.4
0
0.0
0
0.0
0
0.0
Staphylococcus spp.
7
5
1.9
0
0.0
1
1.2
1
2.1
1 Indicates at least one species other than Staphylococcus aureus and Streptococcus spp. (e.g., Trueperella pyogenes, Escherichia coli, Corynebacterium bovis) or a mix of more than 3 different bacteria.
Analysis of Antimicrobial Resistance Phenotypes and Genotypes
Oxacillin resistance, which is the indicator of mec gene-mediated methicillin resistance, was the most frequent resistance phenotype (47.0% of isolates), followed by resistance to fusidic acid (34.1%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), and erythromycin (7.0%; Table 2). Resistance to 2 or more antibiotics was observed in 15.1% of the CNS isolates. Multidrug-resistant isolates were found in milk from clinical (n = 21) and subclinical (n = 34) mastitis cases and in control milk (n = 8). Oxacillin resistance was significantly more frequent in clinical mastitis isolates (56.5%) than in subclinical mastitis isolates (43.9%), whereas sulfamethoxazole resistance was significantly more frequent in clinical mastitis isolates (11.3%) than in control milk. No significant difference in resistance between isolates was observed for the other antimicrobials tested (Table 2).
Oxacillin resistance was attributed to the mecA gene present in 9.7% (n = 19) of the oxacillin-resistant isolates (n = 196). The mecA gene was detected in Staph. fleurettii (11/12), Staph. epidermidis (6/15), Staph. haemolyticus (1/37), and Staph. xylosus (1/155) isolates. The mecA or mecC gene was not detected in the other 177 oxacillin-resistant isolates (90.3%; Table 5). These isolates exhibited an oxacillin MIC of 0.5 or 1.0 µg/mL, which is just above the clinical resistance breakpoint. Twenty of these 177 oxacillin-resistant mecA- and mecC-negative isolates contained a blaZ gene expressing a β-lactamase. The remaining isolates were also negative for blaZ (Table 5). Among them, all Staph. sciuri isolates (n = 37) contained the mecA1 gene, and exhibited low-level resistance to oxacillin (MIC between 0.5 and 1.0 µg/mL). The low oxacillin resistance was due to the absence of the T→A mutation in the −10 promoter sequence (
Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus.
Resistance to other antimicrobials correlated with the presence of the associated resistance genes (Table 5): the β-lactamase gene blaZ; the tetracycline efflux genes tet(L) and tet(K); the streptomycin adenyltransferase and nucleotidyltransferase genes ant(6)-Ia and str; the chloramphenicol acetyltransferase genes catpC221 and catpC223; the gentamicin acetyltransferase gene aac(6')-Ie; the kanamycin-neomycin phosphotransferase genes aph(2')-Ia and aph(3′)-III; the macrolide and lincosamide 23S rRNA methylase genes erm(B) and erm(C); the macrolide efflux gene msr; the lincosamide nucleotidyltransferase gene lnu(A); and the trimethoprim-resistant dihydrofolate reductase genes dfr(A), dfr(D), dfr(G), and dfr(K). In a few strains, resistance to erythromycin, clindamycin, streptomycin, gentamicin, chloramphenicol, and trimethoprim could not be explained by the presence of any of the tested genes, suggesting new antimicrobial resistance mechanisms in CNS (Table 5).
Resistance to fusidic acid was not due to the known fusidic acid resistance genes fus(B), and fus(C), suggesting the appearance of new resistance genes or mutations in the elongation factor G fus(A) (
). Similarly, no known tiamulin resistance genes (vga or lsa) were detected in the tiamulin-resistant strains. Resistance to sulfonamides was not further characterized.
Multiple combinations of these genes were found in 4.8% of the CNS isolates. The most frequent resistance genes detected in combination were those conferring resistance to oxacillin, tetracycline, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, and clindamycin (Table 6). The presence of several genes in one isolate was linked to the presence of mecA in Staph. epidermidis (n = 6), Staph. sciuri (n = 3), Staph. haemolyticus (n = 1), and Staph. fleurettii (n = 1). The other mecA positive isolates [Staph. fleurettii (n = 10) and Staph. xylosus (n = 1)] contained only the mecA gene. The CNS isolates lacking mecA, but containing several other resistance genes were classified as Staph. chromogenes (n = 3), Staph. epidermidis (n = 3), Staph. haemolyticus (n = 1), and Staph. warneri (n = 2; Table 6). All CNS isolates containing several resistance genes were found in milk samples from cows presenting with subclinical or clinical mastitis (Table 6). Although one Staph. xylosus isolate contained a mecA gene, multidrug resistance was never observed in Staph. xylosus, the most frequently detected CNS in our study.
Table 6Occurrence of multiple antimicrobial resistance genes in CNS isolated from the milk of 20 different cows suffering from bovine mastitis
The CNS strains containing the mecA gene (n = 19) were further analyzed for mecA sequences, clonality, and SCCmec and ACME types. Two different mecA genes with slight sequence differences were detected in Staph. epidermidis isolates (Figure 1). Four mecA genes that slightly differed from each other were found in Staph. fleurettii (Figure 1). The mecA genes detected in Staph. haemolyticus and Staph. xylosus also differed slightly from each other and from those in Staph. epidermidis and Staph. fleurettii (Figure 1).
Figure 1Genetic background and properties of methicillin-resistant CNS from bovine mastitis milk from Switzerland. The phylogenetic tree was constructed from pulsed-field gel electrophoresis (PFGE) patterns of 19 methicillin-resistant CNS isolates. Cluster analysis was generated by Bionumerics 6.6 (Applied Maths, Kortijk, Belgium). The dotted line indicates the cut-off value of ≥90% determining clonality between the isolates, according to the criteria of
). (a) mecA of Staphylococcus haemolyticus M1570/10 (EMBL accession no. HE978799); (b) mecA identical to mecA of Staphylococcus epidermidis RP26A (EMBL accession no. CP000029); (c) mecA of Staph. epidermidis M8/10 (EMBL accession no. HE978797); (d) mecA of Staph. epidermidis M703/10 (EMBL accession no. HE978798); (e) mecA identical to mecA of Staphylococcus fleurettii M143/10 (EMBL accession no. HE978795); (f) mecA of Staph. fleurettii M3783/09 (EMBL accession no. HE978796); (g) mecA of Staph. fleurettii M205/10 (EMBL accession no. HE978794); (h) mecA of Staph. fleurettii M4460/09 (EMBL accession no. HE861945); (i) mecA of Staphylococcus xylosus M1545/10 (EMBL accession no. HE978800); (j) mecA of Staph. fleurettii has been reported to be chromosomally encoded and only contains part of the class A mec gene complex; it is not associated with a staphylococcal cassette chromosome (SCCmec) element (
Analysis of methicillin-resistant Staph. epidermidis (n = 6) and Staph. fleurettii (n = 11) by PFGE showed that strains of the same species were not clonally related, except for 4 Staph. fleurettii from 4 different farms that showed 2 similar PFGE patterns (Figure 1). Methicillin-resistant Staph. epidermidis belonged to ST59 (n = 2), ST55, ST89, and the new strains ST452 and ST454, whereas Staph. epidermidis strains lacking the mecA gene (methicillin-sensitive Staph. epidermidis, MSSE) and displaying a multidrug-resistance profile belonged to ST111 (n = 4), ST184 (n = 1), ST293 (n = 1), and to the new strain ST453 (n = 1); MSSE also showed a different PFGE profile (data not shown).
Several different SCCmec elements were detected among the methicillin-resistant CNS isolates (Figure 1). Staphylococcus epidermidis strains contained SCCmec IV (n = 4), SCCmec V (n = 1), and one nontypeable SCCmec related to types IV and VI. Staphylococcus haemolyticus (n = 1) and Staph. xylosus (n = 1) both contained a nontypeable SCCmec. The mecA gene of Staph. fleurettii strains (n = 11) were associated with the class A mec gene complex and could not be assigned to a known SCCmec element (Figure 1). Two methicillin-resistant Staph. epidermidis strains, ST59-SCCmec IV and ST454-SCCmec V, contained a type 2 and type 3 ACME, respectively; ACME type 1 was detected in 4 MSSE ST111 and in 1 MSSE ST456. None of the Staph. epidermidis isolates carried the biofilm-formation operon ica (Figure 1).
Discussion
Many diverse CNS species have been identified in bovine milk, and MALDI-TOF MS is a reliable and rapid method to identify CNS species (
). We observed that a short ethanol-formic acid extraction is necessary for accurate identification. The CNS in milk were frequently detected as single bacterial species, suggesting that these species were the infectious agents. However, the presence of these CNS species was not correlated with a clinical mastitis diagnosis (Table 3). The most frequently occurring species in this study were Staph. xylosus, Staph. chromogenes, Staph. sciuri, and Staph. haemolyticus, as reported in other studies (
). Although Staph. xylosus is not known to cause mastitis, it was detected in 35.9% of the milk samples in our study and as a single species in 22.8% of those samples, emphasizing previous conclusions that Staph. xylosus is an underestimated pathogenic CNS in bovine mastitis (
). Additionally, two-thirds of the Staph. xylosus isolates were resistant to oxacillin but lacked a known mec gene. The absence of a mecA gene was also observed in other oxacillin-resistant CNS isolates (Table 5). The presence of a blaZ overexpressing β-lactamase may explain decreased susceptibility to oxacillin in some of the strains, as has been described in borderline oxacillin-resistant Staph. aureus (
). For the other oxacillin-resistant isolates lacking mec and blaZ genes, independent mechanisms (which may not be related to an acquired resistance gene) explain the decreased susceptibility to oxacillin with MIC in the range of 0.5 to 2.0 µg/mL. Oxacillin resistance in Staph. sciuri may also depend on mecA1 gene overexpression. Alterations to the promoter region of mecA1 are necessary for high-level mecA1 expression and oxacillin resistance in Staph. sciuri (
Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus.
Expression of high-level methicillin resistance in Staphylococcus aureus from the Staphylococcus sciuri mecA homologue: Role of mutation(s) in the genetic background and in the coding region of mecA.
Development of methicillin resistance in clinical isolates of Staphylococcus sciuri by transcriptional activation of the mecA homologue native to the species.
Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus.
). However, the MIC for these isolates were between 0.5 and 1.0 µg/mL, values that are above the CLSI and EUCAST resistance breakpoints. The clinical and therapeutic relevance of decreased susceptibility to oxacillin remains to be clarified. The oxacillin breakpoint may be set low to properly gauge resistance in CNS from bovine mastitis cases (
), and detection of acquired mec genes may be necessary for correct interpretation of the antibiogram.
The mecA gene was detected in Staph. epidermidis, Staph. fleurettii, Staph. haemolyticus, and Staph. xylosus. Three mecA genes that differed from each other in only a few base pairs were found in the methicillin-resistant Staph. fleurettii and Staph. epidermidis isolates, suggesting the independent acquisition of the mecA gene in these species. This conclusion is supported by the observation that the different SCCmec elements were detected in individual Staph. epidermidis isolates, and that all but one Staph. fleurettii isolate contained a mecA gene associated with the class A mec gene complex. It has been reported that the mecA-containing region in Staph. fleurettii is not associated with a SCCmec element but is encoded chromosomally within a part of the class A mec gene complex (
Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: Rapid identification system for mec, ccr, and major differences in junkyard regions.
) detects the chromosomal class A mec gene complex in Staph. fleurettii. Genetic diversity was confirmed by PFGE, which showed that, except for 2 pairs of Staph. fleurettii with similar PFGE profiles, all methicillin-resistant CNS isolates had different PFGE profiles. Despite different PFGE profiles, Staph. epidermidis isolates belonging to the ST111 and ST59 groups were predominant in bovine mastitis cases, suggesting that a specific clonal lineage of Staph. epidermidis has adapted to the udder environment (
). The ACME were mainly observed in the Staph. epidermidis ST59 or ST111 groups, suggesting that it may also play a role in host adaptation in cows. Additionally, Staph. epidermidis was the predominant CNS species among those that contained multiple antimicrobial resistance genes. Multiple resistance genes were also found in Staph. sciuri, Staph. chromogenes, Staph. haemolyticus, and Staph. fleurettii, and these genes were frequently associated with the presence of the mecA gene. Genes conferring resistance to clinically relevant antimicrobials such as the penicillins, macrolides, lincosamides, and aminoglycosides were also detected. In total, 15.1% of the isolates studied were resistant to more than 2 antimicrobials, and some strains were virtually resistant to all antimicrobials authorized for the treatment of mastitis. The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment.
Our study demonstrated that CNS species in milk from cows experiencing mastitis are generally susceptible to the antimicrobials commonly used for treatment. However, CNS have the potential to acquire resistance genes, leading to therapeutic failures. Some multidrug-resistant isolates, especially Staph. epidermidis, Staph. chromogenes, and Staph. haemolyticus, are present in bovine mastitis milk and may resist antimicrobial treatment. An antibiogram is therefore recommended for targeted therapy, and chronically infected cows should be culled from the herd.
Acknowledgments
This study was supported by Grant No.1.11.21 to V. P. from the Swiss Federal Veterinary Office (BVET); J. P. R. received a scholarship from the Federal Commission for Scholarships for Foreign Students. We thank Alexandra Collaud, Alexandra Rossano, Juliette Wipf, Stefanie Ackermann, and Susanne Rickli from the Institute of Veterinary Bacteriology (University of Bern, Switzerland) for technical assistance. We also thank Michèle Bodmer from the Clinic for Ruminants (University of Bern, Switzerland) and Marcus Doherr and Martin Reist from the Veterinary Public Health Institute (University of Bern, Switzerland) for advice.
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