Molecular identification of coagulase-negative staphylococci isolated from bovine mastitis and detection of β-lactam resistance

Introduction: Bovine mastitis is a frequent cause of economic loss in dairy herds. Coagulase-negative staphylococci (CoNS) are increasing in importance as cause of bovine intramammary infection throughout the world in recent years. CoNS have been isolated from milk samples collected from cows with clinical and subclinical mastitis in several countries. Identification of mastitis pathogens is important when selecting appropriate antimicrobial therapy. Methodology: A total of 93 strains of Staphylococcus spp isolated from bovine mastitis in Argentina between 2010 and 2013 were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) using the gap gene and were tested for the presence of bla Z and mec A genes by PCR and for the susceptibility to penicillin and cefoxitin by disk diffusion. Results: The most common CoNS species was S. chromogenes 46.2% (43/93), followed by S. devriesei 11.8% (11/93) and S. haemolyticus 9.7% (9/93). The bla Z gene was detected in 19 (20.4%), but only 16 (17.2%) isolates were resistant to penicillin; the mec A was detected in 6 (6.5%) isolates but only 4 (4.3) were resistant to cefoxitin. The 6 mec A-positive isolates showed oxacillin MICs ≥ 0.5 μg/ml. Discussion: CoNS are important minor mastitis pathogens and can be the cause of substantial economic losses. The presence of methicillin resistant isolates emphasizes the importance of identification of CoNS when an intramammary infection is present because of the potential risk of lateral transfer of resistant genes among staphylococcal species.


Introduction
Bovine mastitis is a disease that affects the health of dairy cows worldwide resulting in decreased milk production and quality [1].Staphylococci are causing the majority of mastitis cases in different dairy regions around the world.There are differences in pathogenicity between the coagulase-positive and coagulase-negative staphylococcal (CoNS) species.As a group, CoNS have traditionally been considered to be of minor pathogenicity, although in recent years they are increasing in importance as causes of bovine mastitis resulting in tissue damage, decrease in milk production and persistent bovine intramammary infections [2], and have been isolated from milk samples collected from cows with clinical and subclinical mastitis in several countries [3][4][5] .
Different commercial kits based on specific biochemical reactions often fail in the identification of different species of CoNS [6].Several genomic targets have been used for the molecular identification of CoNS, including genes for 16S rRNA, gap, hsp60, sodA, tuf and rpoB [7].Identification of mastitis pathogens is important to select appropriate antimicrobial therapy.-lactam antibiotics are frequently used in intramammary therapy.Bacterial resistance to -lactam mechanisms include production of -lactamases, encoded by the blaZ gene, and production of a low affinity penicillin-binding protein (PBP2a), encoded by the mecA gene.The latter, designated as methicillin-resistance (MR) precludes therapy with any of the currently available -lactam antibiotics [8].CoNS are reservoirs of resistance genes, potentially threatening public health [9].MR-CoNS in agricultural animals may act as important reservoirs for the transfer of antimicrobial resistance genes [10].It has been suggested that Staphylococcus epidermidis, after horizontal transfer, facilitates the potential of S. aureus to resist antibiotic treatment, manifesting in methicillin-resistant S. aureus (MRSA).
The aim of this study was to identify CoNS species isolated from mastitic milk of cows and examine genotypic antimicrobial resistance profiles and to compare results obtained by phenotypic and genotypic profiling of resistance to penicillin (PEN) and cefoxitin (FOX), -lactams antibiotics.

CoNS strains
A total number of 93

DNA isolation
CoNS were routinely cultured on brain-heart infusion broth and incubated for 24 hours at 37°C.The whole-cell DNA was isolated using Wizard Genomic DNA purification kit (Promega, Madison, USA).

Species identification by the gap PCR-RFLP
Species identification was achieved by using PCRrestriction fragment length polymorphism (PCR-RFLP).The gap gene encoding for glyceraldehydes-3phospate dehydrogenase, was selected as a target for PCR amplification.PCR amplification tests were performed as in Yugueros et al. [19] with some modifications.Briefly, the PCR mixture (50 µl) was composed of 10 µl of 10X PCR amplification buffer, 0.2 mM of dNTPs (Promega, Madison, USA), 0.1 mM of each primer, 1.5 mM Cl 2 Mg, 0.5U of Taq DNA polymerase (Promega) and 6 µl of DNA extract.The PCR mixtures were subjected to denaturation for 2 minutes at 94°C, followed by 40 cycles of 20 seconds at 94°C, 30 seconds at 60°C, and 40 seconds at 72°C, and a final 5 minutes at 72°C using a termocycler (Eppendorf, Hamburg, Germany).Partial gap gene sequencing (Macrogen, Seoul, Korea) was performed for CoNS species as quality control.The amplification products (~931 bp) were digested with 1U of AluI (Fermentas, Waltham, USA) overnight at 37ºC.The resulting DNA fragments were analyzed by 2.5% agarose (Amresco Inc.Irvine, Canada) gel electrophoresis during 2 hours at 70V.To differentiate S. chromogenes from S. intermedius, PCR products were digested with 1U of RsaI (Fermentas, USA) overnight at 37ºC.For an unambiguous species identification of S. caprae, S. saprophyticus and S. xylosus, PCR products were digested with 1U of TaqI (Fermentas, USA) overnight at 65ºC.The same enzyme was used to differentiate S. sciuri subsp.sciuri and S. sciuri subsp.carnaticus.To determine an unambiguous species identification of S. cohnii subsp.urealyticus and S. devriesei, the gap gene amplicons were discriminated by digestion with 1U of ClaI (Fermentas, USA) overnight at 37ºC.

RFLP in silico
RFLP patterns for the endonucleases AluI, ClaI, RsaI and TaqI were determined using Restriction Mapper software and the GenBank database for CoNS partial gap sequences.

Susceptibility testing
Antimicrobial susceptibility for PEN and FOX were determined in vitro using the agar disc diffusion method [21] with 10U PEN disc (BBL) and 30 µg FOX disc (BBL) on Mueller-Hinton agar (Britania, Argentine).Plates were incubated for 24 hours at 35°C before reading diameters.Isolates were categorized as sensitive (S), intermediate (I) or resistant (R) according to CLSI interpretative criteria [21].The mecA positive isolates were tested for oxacillin (OXA) MIC by E-test strips (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions.

CoNS Identification by the gap PCR-RFLP
The gap gene allowed to differentiate 14 species.Table 2 shows the species identified in this study and their PCR-RFLP patterns.All amplicons were digested with AluI.For some species it was necessary to use another endoenzyme due to a difficult differentiation by the gap PCR-RFLP when employing only AluI.Table 3 shows the species identified by PCR-RFLP in this study.The most common CoNS species was S. chromogenes 46.2% (43/93), followed by S. devriesei 11.8% (11/93) and S. haemolyticus 9.7% (9/93).

Antimicrobial resistance
According to PCR results (Table 3), 19 CoNS were β-lactam resistant carrying the blaZ gene and 6 were MR carrying the mecA gene.In three isolates (1 S. hyicus and 2 S. warneri) resistance to PEN was not observed unless the blaZ gene was detected.All MR species had OXA MICs ≥ 0.5 μg/ml, however the cefoxithin zone diameter > 25 mm identified these isolates as OXA susceptible.Correlation between cefoxithin zone diameters and oxacillin MICs for the 6 MR-CoNS was not found.Among S. chromogenes strains, the most common CoNS species, β-lactam resistance accounted for 16.3% (7/43), less than in other species.Six of these strains were positive to blaZ gene and mecA gene was detected in only one strain.Among S. devriesei strains, the second most common CoNS species, no β-lactam resistance was observed.For S. haemolyticus, the third most common species, β-lactam resistance was more common than in other species reaching 55.6% (5/9).Among the S. hyicus strains, blaZ gene was detected in only one strain corresponding to 20% (1/5) but no expression to PEN was observed by disk diffusion test.For S. warneri, a 100% (4/4) of strains were β-lactam resistant carrying the blaZ gene but only 2 strains were resistant to PEN.Among the 4 S. epidermidis strains, resistance was observed in 3, one carrying the blaZ gene and the other ones carrying the mecA gene or both genes.βlactam resistance was observed in the only S. capitis strain.Finally, in one S. saprophyticus isolate PEN resistance was observed but no blaZ gene was detected.

CoNS Identification by the gap RFLP-PCR
A total of 14 CoNS species were identified; the most frequently isolated species was S. chromogenes (46.2%) followed by S. devriesei (11.8%) and S. haemolyticus (9.7%).Comparing to other countries, for what concerns the first most common species our results agree with those reported in Belgium, USA, Netherlands, Switzerland and Germany [11][12][13][14][15].However, in Sweden [16] the most common species identified was S. epidermidis.The second most common species did not agree with other studies.In USA and Netherlands [12,13] S. epidermidis was reported as the second most common species, while S. xylosus was the second in Belgium and Switzerland [11,14], and S. simulans in Germany and Sweden [15,16].With respect to the third species most frequently identified in our study, our percentages agree with those reported in Switzerland [14], S. haemolyticus was reported as the most frequently isolated species in Turkey [17].Among the least common species identified, they were the same as those reported in other countries [9,11,16,18].The S. devriesei species was identified using the ClaI restriction enzyme; we could not identify this species before, staying only within a group.In agreement with Yugueros et al. [19], genotypic identification based on gap genes is a useful diagnostic tool for the suitable identification of CoNS isolated from animal origin.

Antimicrobial resistance
In this study, β-lactam resistance is low and coincides with Sawant et al. [13] in USA but in other countries as Netherlands or Korea the resistance is higher [9,22].For S. chromogenes, the most common CoNS species, resistance to PEN was 13.6%, coinciding with studies in USA and Netherlands [13,9].For S. haemolyticus, the third most common CoNS species, resistance to PEN exhibited 55% not coinciding with other authors; it might be due to the low number of S. haemolyticus isolates in this study.
One blaZ negative isolate was resistant to PEN, which may be explained by mutations in primer binding sites of resistance genes [23] or by βlactamase hyper-producing strains [22].
Resistance to PEN was not detected in all isolates carrying the blaZ gene, this may be due to the lack of gene expression without induction, as described for S. aureus [20].From the 6 isolates carrying the mecA gene, the resistance to FOX was exhibited in 4. Two isolates were categorized as susceptible to FOX based on the disk diffusion method, but were categorized as resistant to OXA because they showed MICs ≥ 0.5μg/ml.Constitutive presence of mecA or its homologues in the absence of phenotypic resistance to oxacillin has been described for S. sciuri [24].Correlation between phenotypic and genotypic resistance sometimes is not found.CLSI guidelines indicate that checking for the presence of mecA gene by PCR is the most reliable method for detection of MR [21].In the current work this profile was detected in 3 species, the most important fact being that the mecA gene was found in S. epidermidis, a putative zoonotic pathogen [9].
Comparing our results, MR prevalence among CoNS was similar to that found in USA and Switzerland [13,14].However, CoNS-MR isolated from bovine mastitis is higher in Netherlands while in Korea presents a lower incidence [9,22].The presence of mecA has been detected in various species of staphylococci [13].S. epidermidis and S. haemolyticus represents the most frequently observed species among MR-CoNS [15].However, MR was obtained in S. epidermidis, S.cohnii subsp.urealyticus and S. chromogenes.
Multidrug resistance of CoNS can be associated to the presence of β-lactam resistance genes [8].In this study multidrug resistance (unpublished observations) was not common in mecA positive CoNS.
CoNS are a heterogeneous group in which each species had variable antimicrobial resistant rate.Thus, CoNS of mastitis origins should be identified at the species rather than at genus level for effective antimicrobial agent selection.Carriage of antimicrobial resistant genes by CoNS species in cattle may be relevant because it potentially poses a human health hazard.

Table 1 .
Primers for identification and resistance profile of 93 CoNS from bovine milk.
1The partial gap gene sequencing (Macrogen, Korea) was performed for some CoNS species as gold standard test.Sequences were compared with sequences available in GenBank using Basic Local Alignment Search Tool (BLAST) program (data not shown).

Table 3 .
Genotypic and phenotypic resistance profile of CoNS species identified by PCR-RFLP of the gap gene.