Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay

Bacillus cereus causes two types of gastrointestinal diseases: emesis and diarrhea. The emetic type of the disease is attributed to the heat-stable depsipeptide cereulide and symptoms resemble Staphylococcus aureus intoxication, but there is no rapid method available to detect B. cereus strains causing this type of disease. In this study, a polymerase chain reaction (PCR) fragment of unknown function was identified, which was shown to be specific for emetic toxin producing strains of B. cereus. The sequence of this amplicon was determined and a PCR assay was developed on this basis. One hundred B. cereus isolates obtained from different food poisoning outbreaks and diverse food sources from various geographical locations and 29 strains from other species belonging to the B. cereus group were tested by this assay. In addition, 49 non-B. cereus group strains, with special emphasis on food pathogens, were used to show that the assay is specific for emetic toxin producing B. cereus strains. The presented PCR assay is the first molecular tool for the rapid detection of emetic toxin producing B. cereus strains.     

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5%) were found to be slime producers by Christensen's test tube method whereas 58 strains (31%) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1%) and erythromycin (47.1%) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2%) and 36 (62.1%) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4%) of 129 S. aureus strains were positive for beta-lactamase enzyme. However, 78 (81.25%) of 96 beta-lactamase positive S. aureus strains were beta-lactamase positive ORSA isolates, but none of them had vancomycin resistance.     

High throughput multiple locus variable number of tandem repeat analysis (MLVA) of Staphylococcus aureus from human, animal and food sources

Staphylococcus aureus is a major human pathogen, a relevant pathogen in veterinary medicine, and a major cause of food poisoning. Epidemiological investigation tools are needed to establish surveillance of S. aureus strains in humans, animals and food. In this study, we investigated 145 S. aureus isolates recovered from various animal species, disease conditions, food products and food poisoning events. Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis (MLVA), known to be highly efficient for the genotyping of human S. aureus isolates, was used and shown to be equally well suited for the typing of animal S. aureus isolates. MLVA was improved by using sixteen VNTR loci amplified in two multiplex PCRs and analyzed by capillary electrophoresis ensuring a high throughput and high discriminatory power. The isolates were assigned to twelve known clonal complexes (CCs) and--a few singletons. Half of the test collection belonged to four CCs (CC9, CC97, CC133, CC398) previously described as mostly associated with animals. The remaining eight CCs (CC1, CC5, CC8, CC15, CC25, CC30, CC45, CC51), representing 46% of the animal isolates, are common in humans. Interestingly, isolates responsible for food poisoning show a CC distribution signature typical of human isolates and strikingly different from animal isolates, suggesting a predominantly human origin.     

Enterotoxigenic potential of Staphylococcus intermedius.

Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In contrast to Staphylococcus aureus, a systematic screening for the enterotoxins has not yet been performed on the genomic level for the coagulase-positive species S. intermedius. Therefore, the enterotoxigenic potential of 281 different veterinary (canine, n = 247; equine, n = 23; feline, n = 9; other, n = 2) and 11 human isolates of S. intermedius was tested by using a multiplex PCR DNA-enzyme immunoassay system targeting the staphylococcal enterotoxin genes sea, seb, sec, sed, and see. Molecular results were compared by in vitro testing of enterotoxin production by two immunoassays. A total of 33 (11.3%) S. intermedius isolates, including 31 (12.6%) canine isolates, 1 equine isolate, and 1 human isolate, tested positive for the sec gene. In vitro production of the respective enterotoxins was detected in 30 (90.9%) of these isolates by using immunological tests. In contrast, none of 65 veterinary specimen-derived isolates additionally tested and comprising 13 (sub)species of coagulase-negative staphylococci were found to be enterotoxigenic. This study shows on both molecular and immunological levels that a substantial number of S. intermedius isolates harbor the potential for enterotoxin production. Since evidence for noninvasive zoonotic transmission of S. intermedius from animal hosts to humans has been documented, an enterotoxigenic role of this microorganism in SFP via contamination of food products may be assumed.     

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.     

Antibiotic resistance in Staphylococci isolated from the vaginas of captive female Leontopithecus (Callitrichidae-Primates)

The purpose of this study was to provide current data on Staphylococcus species from the vaginas of clinically normal captive lion tamarins and to determine the antimicrobial susceptibility of these isolates. Samples were collected from 25 adult lion tamarins, processed to isolate Staphylococcus species, and tested for susceptibility to penicillin G, gentamicin, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, streptomycin, ampicillin, and rifampicin. Isolates with the typical characteristics of the genus Staphylococcus were recovered from all 25 samples. Coagulase-negative species were the most common (68% of the isolates), and the most frequently isolated species (10 samples) was S. simulans. Other coagulase-negative species, including S. saprophyticus (n=5), S. epidermidis (n=1), and S. arlettae (n=1), were also recovered. Coagulase-positive Staphylococci were obtained from eight animals (six of from the S. aureus species and two from S. intermedius). Resistance to antibiotics was frequently observed, and 88% of the isolates (23 samples) showed resistance to at least one drug. Resistance to penicillin G was a common finding, and the most active antimicrobial agents were chloramphenicol and gentamicin. Coagulase-positive strains were more frequently resistant to antibiotics (79.7%, average=6.4 drugs) than coagulase-negative strains (38.2%, average=3.0 drugs). The high frequency of resistance observed in those isolates is surprising and very alarming. A detailed history of the use of antimicrobial drugs in these subjects did not reveal any previous exposure to any of the tested antibiotics that could justify the observed resistance rate.     

Identification of Micrococcaceae in Clinical Bacteriology

The cellular morphology, identifying physiological characteristics, and a key to the human genera of Micrococcaceae are presented with flow charts for identification of aerobic and anaerobic isolates. These flow charts can be amended as desired, depending upon the degree of accuracy desired. Micrococcaceae isolates in a 350-bed private general hospital during a 15-week period are tabulated to show relative numbers of the different genera and species, with their probable relationship to infection or contamination. Only 11 of the 220 Micrococcaceae isolates were not Staphylococcus; no Sarcina or Peptococcus were isolated. Of the Staphylococcus isolates, 61% were S. epidermidis. Almost 18% of the S. aureus isolates were coagulase-negative. Of the S. aureus isolates, 80% of the coagulase-positive isolates were infecting agents, as were 67% of the coagulase-negative S. aureus isolates, compared to only 48% of S. epidermidis isolates. Two of four Gaffkya isolates but only one of seven Micrococcus isolates were infecting agents. If coagulase production is used as the sole criterion for speciation of staphylococci, and Micrococcus is not differentiated from Staphylococcus, the term "coagulase-negative staphylococci" does not differentiate three distinct levels of pathogenicity. Coagulase-negative S. aureus is more virulent than S. epidermidis or Gaffkya, which are more virulent than Micrococcus or Sarcina.     

Cytidine deamination assay to differentiate Staphylococcus aureus from other staphylococci

AIMS: Adoption of the property of cytidine (cytosine-beta-d-riboside) deamination in staphylococci to distinguish Staphylococcus aureus from other staphylococci. METHODS AND RESULTS: A total of 560 staphylococcal strains were examined. The test demonstrated a sensitivity of 97.1% and a specificity of 98.8%. Of the 249 S. aureus strains (115 oxacillin-resistant) 58 strains were coagulase-negative S. aureus and another 16 strains were clumping factor-negative S. aureus. The 74 deficient S. aureus strains were identified by 16S rRNA gene sequencing and further investigated by spa typing and 13 spa types were found. CONCLUSIONS: The cytidine deaminase test (CDT) is useful especially for distinguishing coagulase- and clumping factor-negative S. aureus from other staphylococci and the results correlated well with 16S rRNA sequencing and the polymerase chain reaction (PCR) amplification of the nuc gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Cytidine deamination assay differentiates S. aureus from other staphylococci. This method is fast (6 h) and reliable in distinguishing between non-S. aureus and the defective (coagulase-negative, clumping factor-negative) S. aureus isolates which could have major consequences for therapy.     

A Comparison of Methods and the Validity of Deoxyribonuclease Tests for the Characterization of Staphylococci Isolated from Animals

Strains isolated from pigeons belonging to the coagulase-positive species Staphylococcus intermedius , coagulase-negative Staph. hyicus subsp. chromogenes strains from cattle and pigs, and Staph. aureus strains from poultry, gave weakly positive reactions in DNase plate culture tests and heat-resistant DNase tests. Staph. aureus and Staph. intermedius strains from other sources and coagulase-negative and coagulase-positive Staph. hyicus subsp. hyicus strains reacted strongly in these tests. A standardized plate culture test procedure is proposed and the use of DNase tests in the identification of staphylococci isolated from animals is discussed.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

金黄色葡萄球菌拮抗菌株的筛选鉴定及其抗菌物质分析

【背景】植物内生菌的次生代谢产物是新型天然活性物质的重要来源。【目的】从芍药内生细菌中筛选对金黄色葡萄球菌有抑菌活性的菌株和次生代谢产物。【方法】采用平板对峙法筛选拮抗菌株,根据形态学特征和分子生物学的方法鉴定菌株,PCR扩增检测合成脂肽类物质的功能基因;运用牛津杯法依次测定内生细菌发酵液和脂肽类粗提物的抑菌活性,利用Sephadex LH-20凝胶层析分离脂肽类物质,利用基质辅助激光解吸电离飞行时间质谱分析具有抑菌作用的分离组分。【结果】共筛选出13株对金黄色葡萄球菌具有不同程度抑制作用的内生菌株,其中菌株SY11的抑菌作用最为显著,其发酵液和脂肽类粗提物均具有较强的抑制作用。结合形态学鉴定以及16S r RNA基因序列分析,鉴定其为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。PCR扩增检测表明菌株SY11含有3个合成脂肽类物质的功能基因fenA、ituD和srfkn,推测该菌株可能具有合成脂肽类物质的能力。根据具有抑菌活性分离组分的质谱分析结果,推测其有效物质的主要成分为Bacillomycin D。【结论】解淀粉芽孢杆菌SY11对金黄色葡萄球菌有良好抑制效果,其脂肽类粗提物也具有较强的体外抑菌活性。本研究为芍药内生细菌的开发应用奠定了基础。     

The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci

Superoxide dismutase (SOD) profiles of clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All S. aureus clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of S. aureus: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of S. aureus. Southern analysis of eight CoNS species identified only a single sod gene in each case. A full-length sod gene was cloned from Staphylococcus epidermidis and determined to be more similar to sodA than to sodM of S. aureus. Therefore, this gene was designated sodA. The deduced amino acid sequence of the S. epidermidis sodA was 92 and 76% identical to that of the SodA and SodM proteins of S. aureus, respectively. The S. epidermidis sodA gene expressed from a plasmid complemented a sodA mutation in S. aureus, and the protein formed a hybrid with SodM of S. aureus. Both hybrid SOD forms as well as the SodM and SodA proteins of S. aureus and the S. epidermidis SodA protein exist as dimers. These data indicate that sodM is found only in S. aureus and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in S. aureus.     

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.     

Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci

Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have been constructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative species Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15, contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcus species. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called region X. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance of region X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus is strongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressing plasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureus strains Cowan I and A676.     

Comparative Characterization and Biotyping of Staphylococcus aureus Isolates from Human and Bovine Sources

One Hundred and ten alpha and/or delta-haemolytic isolates (collection 1), 50 beta haemolytic isolates (collection 2) from bovine mastitis, and 100 previously phage-typed alpha- and delta-haemolytic isolates (human collection) og Staphylococcus aureus (S. aureus) were tested and biotyped according to the scheme of Hajek & Marsalek (1971). Among collection 1 isolates, 85 (77.3 %) belonged to the human biotype A (human source). Twenty two (20 %) designated as non-allotted strains, possessed characteristics of both animal and human sources. The remaining 3 isolates (2.7 %) in this collection belonged to biotype C (animal source). All collection 2 isolates which were used as control strains for animal sources, belonged to biotype C. The human collection that contained 100 phage-typed haemolytic isolates (representing all human phage groups) were used as a control for the human source. Irrespective of their phage group, these strains predominantly produced alpha and/or delta haemolysins and belonged to the human biotype A. This study also recommended the use of a combined plasma crystal violet agar medium for the presumptive identification of S. aureus biotypes.     

Antimicrobial resistance and species identification of staphylococci isolated from the meat of wild rabbits (<Emphasis Type="Italic">Oryctolagus cuniculus</Emphasis>) in Slovakia

In 2009, a total of 113 strains of staphylococci were isolated from the thigh muscles of ten hunted and 20 farmed wild rabbits (Oryctolagus cuniculus) in the Slovak Republic. Only two isolates (1.8%) possessed coagulase activity, the rest of 111 staphylococcal isolates were coagulase-negative. Among them, six isolates (5.4%) showed the production of DNase. In each isolate, resistance to eight antibiotics by means of agar dilution test was tested. Based on these results, 110 isolates were found to be resistant to at least one antibiotic. Only one isolate was susceptible to all eight antibiotics tested. Another two isolates were susceptible, however, they showed intermediate susceptibility to cefoxitin. Resistance to ampicillin (78.8%), erythromycin (58.4%), penicillin (51.3%) and oxacillin (46.0%) was found most frequently. Twenty-six isolates (23.0%) were resistant to novobiocin. On the other hand, resistance to cefoxitin (8.0%) and gentamicin (1.8%) were quite rare. Fifteen percent of isolates were resistant to one antibiotic, simultaneous resistance to two, three, four and five antibiotics was confirmed in 22.1%, 23.9%, 21.2% and 13.3% of isolates, respectively. Except for two coagulase-positive Staphylococcus aureus isolates (1.8%), seven species of coagulase-negative staphylococci were identified using the MALDI BioTyper (TM) sytem as follows: Staphylococcus warneri (45.1%), Staphylococcus epidermidis (21.2%), Staphylococcus pasteuri (13.3%), Staphylococcus xylosus (8.0%), Staphylococcus capitis (7.1%), Staphylococcus haemolyticus (1.8%) and Staphylococcus cohnii ssp cohnii (1.8%).     

Antibiotic sensitivity of bacterial isolates from cases of canine dermatitis

Among 97 bacterial isolates, 74 strains of Staphylococcus spp developed from 95 swabs taken from skin lesions in dogs. Twenty-eight staphylococcal strains resistant to methicillin and/or oxacillin were identified and mecA expression was confirmed for 14 of these strains. S. aureus and S. intermedius group (SIG) strains were particularly relevant in our cases due to their antibiotic resistance leading to an increased veterinary and public health risk. We suggest a diagnostic protocol based on cytological examination, bacterial identification to species level, and antibiotic sensitivity testing prior to prescribing antibiotic treatment for canine skin diseases.