Coagulase-Positive Staphylococcus: Prevalence and Antimicrobial Resistance
Staphylococcus pseudintermedius is the most prevalent coagulase-positive Staphylococcus inhabitant of the skin and mucosa of dogs and cats, causing skin and soft tissue infections in these animals. In this study, coagulase-positive Staphylococcus species were isolated from companion animals, veterinary professionals, and objects from a clinical veterinary environment by using two particular culture media, Baird-Parker RPF agar and CHROMagar Staph aureus. Different morphology features of colonies on the media allowed the identification of the species, which was confirmed by performing a multiplex polymerase chain reaction (PCR). Among 23 animals, 15 (65.2%) harbored coagulase-positive Staphylococcus, being 12 Staphylococcus pseudintermedius carriers. Four out of 12 were methicillin-resistant S. pseudintermedius (MRSP). All veterinary professionals had coagulase-positive Staphylococcus (CoPS) species on their hands and two out of nine objects sampled harbored MRSP. The antimicrobial-resistance pattern was achieved for all isolates, revealing the presence of many multidrug-resistant CoPS, particularly S. pseudintermedius. The combined analysis of the antimicrobial-resistance patterns shown by the isolates led to the hypothesis that there is a possible crosscontamination and dissemination of S. aureus and S. pseudintermedius species between the three types of carriers sampled in this study that could facilitate the spread of the methicillin-resistance phenotype.
Introduction
Coagulase-positive Staphylococcus (CoPS) species are commensal bacteria present in skin and nasal flora; however, they can cause opportunistic infections in animals and humans.1,2 Among CoPS, Staphylococcus pseudintermedius has particular importance in the veterinary setting, mainly in small animals, being associated with dermatological problems such as pyoderma, postoperative wound infection, and otitis.2–4 Although the zoonotic potential of S. pseudintermedius is not well defined yet, it has been isolated from human infections and humans in contact with animals.5–7 In addition, since the phenotypic differentiation of CoPS species is difficult, it is probable that S. pseudintermedius has been misidentified in routine laboratory diagnostics with other CoPS, especially Staphylococcus intermedius and Staphylococcus aureus, and, thus, its prevalence may have been underestimated.4
Several methods to isolate and identify S. pseudintermedius have been documented. A combination of biochemical tests (D-mannitol test, arginine dihydrolase test, and β-gentibiose test) are particularly used to phenotypically differentiate other Staphylococcus species from S. pseudintermedius.8 Molecular methods such as Pulsed-Field Gel Electrophoresis, multiplex polymerase chain reaction (PCR), and PCR-Restriction Fragment Length Polymorphism are the most effective for S. pseudintermedius identification.2,6,9
Similarly to S. aureus, S. pseudintermedius can acquire the mecA gene, which is located on staphylococcal cassette chromosome mec (SCCmec) elements and confers resistance to β-lactam antibiotics by encoding an altered penicillin-binding protein.10 The number of cases reporting methicillin-resistant S. pseudintermedius (MRSP) has been increasing and, usually, these MRSP are multidrug-resistant.4–12
The phenotypic identification of MRSP species can be made by antimicrobial susceptibility testing using a minimum inhibitory concentration (MIC) breakpoint for oxacillin of ≥0.5 mg/L in broth dilution methods or by measuring an inhibition halo with a diameter ≤17 mm when using 1μg of oxacillin/disc in the agar diffusion methods, following the interpretative criteria of Clinical and Laboratory Standards Institute (formerly NCCLS) of 2004.13–15 Both tests can be highly consistent to detect MRSP species; however, the detection of mecA gene by PCR is still the most reliable and, also, confirmatory method to identify methicillin-resistance Staphylococcus species.10,16,17
The main objective of this study was to find a new isolation method that could differentiate S. pseudintermedius from other CoPS using culture media. Two agar media were used: the Baird-Parker RPF agarc, which has been mostly used in Food Microbiology for the direct detection and enumeration of coagulase-positive Staphylococci; and CHROMagare Staph aureus, which is a selective medium for the isolation, enumeration, and identification of S. aureus from clinical and food sources. Subsequently, the prevalence of two CoPS species, S. pseudintermedius and S. aureus, isolated from domestic animals, veterinary professionals, and the environment of a veterinary hospital was achieved. The antimicrobial resistance profile of CoPS isolates was also determined. Finally, there was an attempt to establish a possible correlation between all collected samples.
Materials and Methods
Sampling
Companion Animals
Between February and May 2013, a total of 23 animals (21 dogs and two cats) were enrolled at the Veterinary Hospital of the University of Porto after the positive consent of the owners in order to collect samples from the skin and from oral and nasal mucosae. Two samples were collected from each body site, shortly after the animal observation by a veterinarian, using a pre-moistened sterile swab inoculated in 5 mL of brain heart infusion brotha (BHI) supplemented with 0.1% polysorbate 80b (T80)−BHI+T80. During the sample collection procedure, an inquiry was made to the owners in order to obtain some information about potential risk factors for the presence of S. aureus or S. pseudintermedius, such as animal age, sex, residential area, and animal health status.
Veterinary Professionals
A total of nine veterinary professionals, including veterinarians, technicians, and veterinary nurses, affiliated with the Veterinary Hospital of the University of Porto were recruited and consented the samples collection, at the same day, for the isolation of S. aureus and S. pseudintermedius, from hands and nasal mucosa. The hand sample was collected with moistened sterile gauze and the nasal sample with two sterile swabs. Gauze and swabs were then placed in 50 mL and 5 mL of BHI+T80, respectively.
Clinical Environment
On a single day, samples were collected from nine different objects and surfaces (e.g., floors, top parts of medical examination stands, computer keyboard, cages) of the veterinary hospital environment. There was no information about the disinfection status of the objects/surfaces. Sample collection was done with sterile gauze that was placed in 50 mL of BHI+T80. Afterwards, 1 mL was taken to perform a 1:10 dilution in 9 mL of BHI+T80.
All samples were kept in the broth medium no longer than 1 hr until processing in the laboratory.
Bacterial Isolates and Antimicrobial Susceptibility Testing
The following procedure was similarly performed for all samples (animals, veterinary professionals, and clinical environment samples).
At the laboratory, each sample was incubated at 37°C. After 6 hr of incubation, an aliquot of 30 μL was inoculated onto Baird-Parker RPFc. After completing 18 hr of incubation, an aliquot of 30 μL was streaked onto Baird-Parker RPFc and 60 μL were spread on the same culture media supplemented with oxacillind (2 μg/mL). All plates were incubated at 37°C for further evaluation of coagulase-positive activity at 24, 28, 32, and 48 hr.
During the observation period, every Baird-Parker RPFc plate presenting typical coagulase-positive colonies (white halo surrounding a well-delimited round-shape colony, whose color vary from grey to black) was subcultured by streaking onto CHROMagare Staph aureus. Two to five colonies isolated from each sampling site were selected for subculture. CHROMagare Staph aureus plates were incubated at 37°C for 24 hr. After that time, a maximum of four colonies exhibiting typical S. aureus morphology (mauve-colored colonies) or S. pseudintermedius (purple and blue colonies) were selected for antimicrobial susceptibility testing and storage. Antimicrobial susceptibility testing was performed by the agar disc diffusion method on Mueller-Hinton agar, following the Clinical and Laboratory Standards Institute guidelines and interpretative criteria (formerly NCCLS) of 2004,13 according to a previous study,14 for a panel of 23 antimicrobial agentsf: fucsidic acid (FD, 10 μg), amoxicilin (AMC, 10 μg), ampicillin (AMP, 10 μg), kanamycin (K, 30 μg), cefoxitin (FOX, 30 μg), ciprofloxacin (CIP, 5 μg), clindamycin (DA, 2 μg), chloramphenicol (C, 30 μg), erythromycin (E, 15 μg), streptomycin (S, 10 μg), gentamicin (CN, 10 μg), imipenem (IPM, 10 μg), lomefloxacin (LOM, 10 μg), neomicin (N, 10 μg), nitrofurantoin (F, 300 μg), oxacillin (OX, 1 μg), penicillin (P, 10 μg), quinupristin-dalfopristin (QD, 15 μg), rifampicin (RD, 5 μg), teicoplanin (TEC, 30 μg), tetracycline (TE, 30μg), trimethoprim/sulfamethoxazole (SXT, 25 μg), and vancomycin (VA, 30 μg). Staphylococcus aureus ATCC 25293 was used as a quality control strain.
Species Identification by PCR
The DNA was extracted from isolated colonies that presented coagulase-positive activity in Baird-Parker RPFc and the mauve, dark mauve, purple, and blue colors in CHROMagare Staph aureus. A total of 41 DNA extractions were performed using lysostaphind (100 μg/ml) and proteinase Kg (100 μg/ml). Then, a multiplex PCR for the species-specific detection of nuc gene was performed by using the primers as previously described for the identification of three species of coagulase-positive staphylococci: S. pseudintermedius, S. aureus, and S. intermedius.2 The reaction mixture for the PCR, with a total volume of 50 μL, consisted of 33 μL of distilled water, 5 μL reaction buffer (×10),g 1 μL of dNTPs 10 mM,h 2,5 μL of each primer, 1μL of Taq DNA polymerase 500 Ug, and 5 μL of DNA. The reaction mixture was performed in a thermocycleri at 95°C for 1 min, followed by 30 cycles at 95°C for 1 min, 53°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 7 min. Samples (5 μL) of PCR products were analyzed by electrophoresis in a 1.5% (w/v) agarose gel at 100 V for 60 min. Gels were stained with ethidium bromide and observed in a UV transilluminator.i
Results
Overall, 138, 18, and 9 samples were collected from companion animals (dogs and cats), veterinary professionals, and objects/surfaces of the clinical environment, respectively. Staphylococcus aureus and S. pseudintermedius were phenotypically detected in the media Baird-Parker RPFc and CHROMagare Staph aureus and genotypically confirmed by multiplex PCR. Staphylococcus pseudintermedius presented a white color in Baird-Parker RPFc, with creamy consistence. Its coagulase halo was not as exuberant as the one presented by S. aureus colonies, which showed black to grey color with pasty consistence. In CHROMagare Staph aureus, the main observation was the different color presented by S. pseudintermedius and S. aureus colonies. The first ones presented a color between purple and blue with aqueous consistency, while S. aureus colonies presented mauve to dark mauve color with mucous consistency (see Supplementary Figure I). The use of CHROMagare Staph aureus was particularly useful in one oral sample to allow the differentiation of colonies that appeared to be one single Staphylococcus species in Baird-Parker RPFc. For every sample analyzed, each purple and blue colony in CHROMagare was identified by PCR (data not shown) as being S. pseudintermedius and the mauve and dark mauve colonies were identified as S. aureus species. The multiplex PCR confirmed not only the species identification but also the “purity” of the colonies with different colors. For each colony tested, the presence of one Staphylococcus species excluded the presence of the other.
During the coagulase activity observation on Baird-Parker RPFc, it was found that S. pseudintermedius presented a coagulase-positive activity only after 28 hr of incubation at 37°C, instead of the 24 hr needed for S. aureus isolates to show the positive activity.
Prevalence of CoPS Isolates in Companion Animals
Among the 23 animals, 15 (65.2%) had CoPS species and the remaining eight were non-CoPS carriers; 14 (93.3%) out of the 15 were dogs, only one was a cat (6.7%) The distribution of the two CoPS isolated from the two kinds of animals and among different body sites of the animals is shown in Table 1. A detailed analysis of these 15 CoPS-animals allowed us to observe that eight (53.3%) were S. pseudintermedius exclusive carriers, three (20.0%) were S. aureus exclusive carriers, and four (26.7%) of them carried both S. aureus and S. pseudintermedius. The oral mucosa was the site where S. aureus was most isolated. Two out of the seven S. aureus were methicillin-resistant S. aureus (MRSA). Regarding S. pseudintermedius, it was similarly present in the three body sites sampled, skin being the site that provided the major number of S. pseudintermedius isolates (Table 1). Moreover, it must be highlighted that from the 12 S. pseudintermedius carriers, four harbored MRSP. The antimicrobial resistance exhibited by the two Staphylococcus species isolated from the animals is presented on Table 2, showing multidrug-resistance particularly by S. pseudintermedius isolates. Diverse antimicrobial-resistance patterns were shown by S. pseudintermedius and S. aureus isolated from different body sites of the animals (Table 3).
Prevalence of CoPS in Veterinary Professionals
The analysis of the samples collected from the veterinary professionals showed that all of them had CoPS species on their hands. However, only two presented CoPS in the nasal mucosa and were identified as being S. aureus. Eight (88.8%) S. aureus and five (55.6%) S. pseudintermedius were isolated from nine hand samples. All the S. aureus were methicillin-sensitive (MSSA) and only one of the S. pseudintermedius was MRSP. The resistance pattern of the isolates is shown in Table 4.
Prevalence of CoPS in Objects and Surfaces of the Veterinary Hospital
Only three (33.3%) out of the nine samples collected from the veterinary objects harbored CoPS. Two were S. pseudintermedius and both were methicilin-resistant. Staphylococcus aureus was isolated in only one object and was a MSSA. In particular, one MRSP isolate showed resistance to a high number of antimicrobials (Table 5).
Discussion
Taking into account the present results, it may be appropriate to draw attention to the potential use of CHROMagare Staph aureus as a very selective medium to isolate not only S. aureus but also S. pseudintermedius. This medium can overcome other culture media such as Mannitol-Salt agar and Blood Agar due to its selectivity for CoPS species and by hampering the proliferation of contaminant bacteria.18 The origin of color differentiation between S. aureus and S. pseudintermedius colonies on CHROMagare Staph aureus remains uncertain; however, it is probably related to the chromogenic mixture mentioned by the manufacturer or to the pH indicator present in this culture medium. Though this medium appears to be reliable for S. aureus and S. pseudintermedius identification, molecular methods, such as PCR or Pulsed-Field Gel Electrophoresis, should always be recommended as confirmatory methods.
Regarding the prevalence of CoPS in the companion animals, it was observed that S. pseudintermedius isolates prevailed over S. aureus, which is not surprising.3,7,19 Staphylococcus aureus was mostly isolated from oral mucosa, whereas S. pseudintermedius was equally present in the three body sites sampled. In fact, this finding is not in agreement with other reports, which stated that nasal and anal regions were the body sites more commonly colonized by S. pseudintermedius.4,20 It is also important to refer that S. pseudintermedius with different resistance profiles were isolated from the same animal and from the same sampled site.
All S. pseudintermedius were multidrug-resistant, showing resistance toward at least two antimicrobial agents, which is in accordance with previous observations.10,11,17,21,22 Moreover, four out of 12 S. pseudintermedius isolated from the animals were MRSP. The high number of MRSP found in the companion animals may be related to a regular use of antimicrobial agents to treat these animals. Available data indicates that in Portugal, the use of antimicrobial agents in animals, including the use of drugs that are critically important to human medicine, is one of the highest amongst 19 European countries; unfortunately, there is no detailed information regarding the use of antimicrobials in companion animals.23 The growing number of household pets and their increasing health care standards has led to an augmented number of geriatric animals, which have an extensive medical history, including antimicrobial drug administration, and longer contact with owners, increasing both the risk of antimicrobial-resistance emergence and inter-species clonal spread.
In our work, the percentage of MRSP doubles the one of MRSA, supporting previous results and represents an additional concern to the European efforts that are already trying to combat the spread of MRSA.24,25 The close contact of small animals with people and also with other animals can promote the spread of resistant clones, namely methicillin-resistant clones, and may explain the increasing of MRSP species in small animals, even in healthy ones.17,26 A recent study that screened healthy dogs in Portugal for the presence of nasal MRSA concluded that those dogs may be a reservoir of MRSA that could be transmitted to humans, either by direct contact (skin and mouth) or indirectly (via the household environment).27 Thus, the high number of MRSA isolated from healthy dogs may also contribute to the disquieting scenario of MRSA in Portugal (according to the European Centre for Disease Prevention and Control, the proportion of MRSA amongst S. aureus clinical isolates in Portugal in the year 2011 was higher than 50%).28
All the veterinary professionals sampled in this study harbored CoPS species. These results substantiate that these professionals are very likely to be colonized by CoPS species, as are the pet owners.5,19,26,29 However, the potential risk to the health of veterinary professionals is still to be investigated.
Regarding the clinical environment, only a small percentage of objects harbored CoPS. However, a larger number of collected samples would certainly provide more information about these two Staphylococcus species present in the clinical environment. The presence of a MRSP with such a high antibiotic resistance pattern in the clinical environment can be worrisome in terms of public health and underlines the need for an exhaustive disinfection of clinical surfaces, as well as good hand hygiene, on the part of all veterinary professionals.
The combined analysis of isolates from small animals, veterinary professionals, and clinical environment led us to conclude that there was a MSSA phenotype common to eight veterinary professionals, one clinical object (computer keyboard), and three animals (AMPR PR). A MSSP phenotype was common to one veterinary professional and four dogs (AMPR PR) and two MRSP isolates (one from a dog and one from the cage floor) showed the same resistance pattern, comprising of simultaneous resistance against ampicillin, lomefloxacin, oxacillin, clindamycin, ciprofloxacin, amoxicillin/clavulanic acid, erythromycin, neomicin, sulfamethoxazole/trimethoprim, kanamycin, streptomycin, and penicillin. These findings may be an indication of possible cross-contamination and dissemination of S. aureus and S. pseudintermedius clones among the three types of carriers analyzed in this study. Although the colonization mechanism remains unknown, a longitudinal study could provide additional information on how these contaminations might occur.
Conclusion
In this study, a phenotypic identification method, using CHROMagare Staph aureus, turned out to be very reliable in the identification of S. aureus and S. pseudintermedius isolated from animal, human, and abiotic sources and, thus, can be very helpful in veterinarian clinical diagnostic practices. CoPS isolated herein showed diverse antimicrobial-resistance patterns and several methicillin-resistant Staphylococcus species were found in the different sources sampled, underlining that dissemination of resistance clones is very likely to happen in the veterinary environment. Therefore, our results highlight the necessity of taking precautions in order to avoid the spread of multidrug-resistant strains, and, in particular, methicillin-resistant Staphylococcus, among animals and humans (owners and veterinary professionals).
Contributor Notes
The online version of this article (available at www.jaaha.org) contains supplementary data in the form of one figure.
