The Brief Case: Staphylococcus intermedius Group—Look What the Dog Dragged In

DISCUSSION

Members of the Staphylococcus intermedius group (SIG) include S. intermedius, S. pseudintermedius, and S. delphini. SIG members are common colonizers of animal mucosal surfaces, especially those of dogs (2, 3). Though it can be a component of the normal flora of the mouth, nose, and skin, the SIG, particularly S. pseudintermedius, has been implicated as a common cause of pyoderma in dogs (4, 5). In studies assessing carriage rates among dogs and cats, S. pseudintermedius was found to colonize up to 68% of dogs and up to 22% of cats, whereas S. aureus was a colonizer of up to 14% of dogs and 20% of cats (2). Other species of animal-adapted staphylococci include S. schleiferi (canine), S. felis (feline), S. lutrae, S. hyicus (porcine, coagulase variable), and S. agnetis (bovine, coagulase variable).

Members of SIG were recently separated into individual species, with the most recent taxonomic change occurring in 2005 with the creation of the S. pseudintermedius species (4). Staphylococcus delphini, originally isolated from a dolphin specimen, is associated with many types of animals, including birds, cats, horses, and other mammals (4). Despite taxonomic differences, the members of this group are difficult to differentiate phenotypically, suggesting that these species may be interchangeably misidentified (4).

In humans, SIG infections are increasingly recognized. Prior to the advent of MALDI-TOF MS for the identification of bacterial isolates from clinical specimens, SIG members were thought to uncommonly infect humans and were generally associated with dog bites (3–5), foreign-body-related infections, food poisoning, and invasive infections in immunocompromised patients (4). In one study of 39 patients with SIG infections, only 4 had a history of contact with a dog, 2 of which had a SIG member isolated from a dog bite (3). Additionally, SIG isolates were recovered from specimens, including wounds, ears, diabetic ulcers, and cutaneous ulcers (3). In other reports, the SIG was implicated in various other infections, including infective endocarditis and hardware/device infections (6). Differentiating infection from colonization when treating patients with a SIG member can be difficult. For example, the primary-specimen Gram stain of this case showed no polymorphonuclear leukocytes but abundant Gram-positive cocci, suggesting colonization with a SIG member since no inflammatory cells were present. However, this patient was treated because, clinically, this looked like an infection and the swab specimen from this open ulcer grew abundant SIG bacteria.

SIG species are very similar to S. aureus in both clinical presentation and microbiologic characteristics, including coagulase positivity, and it is likely that SIG isolates from human clinical specimens were identified as S. aureus prior to MALDI-TOF MS (6). Historically, coagulase-positive staphylococci isolated from veterinary specimens were identified as SIG, whereas these isolates were identified as S. aureus in human specimens (5). Very few systematic studies have been conducted to evaluate the ability of MALDI-TOF MS instruments to identify SIG isolates. In one study using the Vitek MS (bioMérieux, Durham, NC), four SIG isolates were tested, resulting in three isolates of S. intermedius identified to the genus level and one isolate of S. pseudintermedius identified to the species level (no MALDI-TOF database was specified in the publication). No S. delphini isolates were tested (7). In contrast to that of the Vitek MS, the performance of the Biotyper MALDI-TOF MS in identifying members of the SIG has been more rigorously evaluated. In a study focused on identification of human SIG isolates with the Bruker Biotyper MALDI-TOF MS (database version 3.0), all SIG isolates achieved a score of ≥1.7 (genus-level identification), and 78.9% (15/19) of the S. delphini isolates, 76.5% (13/17) of the S. intermedius isolates, and 89.5% (145/162) of the S. pseudintermedius isolates achieved a score of ≥2.0 (species-level identification) (8).

Like S. aureus, SIG species are catalase positive, coagulase positive, Gram-positive cocci in clusters (Table 1). Like S. aureus, SIG isolates may grow on mannitol salt agar, which is often used in S. aureus colonization screening cultures, and many will ferment d-mannitol (Table 1), producing a yellow colony that might be suggestive of S. aureus (Fig. 1F) (4). However, colonies of SIG isolates can be whiter than S. aureus on SBA plates (Fig. 1A and C). Additionally, SIG isolates are beta-hemolytic, with a double zone of hemolysis (see the clearing and darkening of the agar in Fig. 1D), compared to S. aureus, which has a single zone of beta-hemolysis (Fig. 1B). Despite both being coagulase positive (4), SIG isolates exhibit a slightly different result from S. aureus isolates in latex agglutination testing (i.e., see the more chunky and stringy clumping in Fig. 2A and B), and instances of coagulase-positive S. pseudintermedius misidentification due to negative latex agglutination tests have been documented (4). This misidentification can happen because current Staphylococcus latex agglutination tests employ antibodies to clumping factor and staphylococcal protein A (product of the spa gene) (4). Whereas all SIG species are coagulase positive, only S. intermedius has been shown to have clumping factor (Table 1). Regardless of similar morphologies and characteristics, SIG members and S. aureus can be efficiently differentiated using a variety of biochemical tests (Table 1), including the PYR test (S. aureus negative, SIG positive) (Fig. 2C) (4).

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FIG 1

Colony morphologies of Staphylococcus intermedius group members and Staphylococcus aureus at 24 h of growth. (A) Colony morphology of S. aureus on a sheep blood agar plate. (B) Beta-hemolysis caused by S. aureus. The photo was taken using backlighting to highlight the hemolysis pattern. (C) Colony morphology of S. intermedius group on a sheep blood agar plate. (D) Double zone of beta-hemolysis caused by an S. intermedius group member. The photo was taken using backlighting to highlight the hemolysis pattern. (E) Growth of an S. intermedius group member on a Spectra MRSA chromogenic agar plate (Remel Microbiology Products, Lenexa, KS). (F) Growth of an S. intermedius group member on mannitol salt agar (Remel Microbiology Products).

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FIG 2

Biochemical characteristics of Staphylococcus intermedius group members compared to those of Staphylococcus aureus, and susceptibility testing for S. intermedius group members. (A) Latex agglutination test (coagulase) result for S. aureus; (B) latex agglutination test (coagulase) result for S. intermedius group members; (C) PYR testing results for S. aureus (left, negative) and S. intermedius group members (right, positive); (D) discrepant cefoxitin (FOX 30; 26-mm diameter) and oxacillin (OX 1; 15-mm diameter) results for an S. intermedius group isolate.

Until the early 2000s, very little methicillin resistance (∼5% of isolates) was observed in veterinary SIG isolates, but methicillin resistance in SIG members is on the rise (9). As in S. aureus, methicillin resistance in SIG species is conferred by alternative penicillin binding proteins (PBPs) through the acquisition of the mecA gene, which encodes PBP2a (10). Unlike in S. aureus and the coagulase-negative staphylococci, cefoxitin has proven to be a poor surrogate indicator of methicillin resistance in SIG isolates; compared to the presence of mecA as determined by PCR, the use of cefoxitin as a surrogate indicator of methicillin resistance resulted in both major and very major errors (5). Rather, oxacillin is the appropriate surrogate for methicillin resistance in SIG isolates (Fig. 2D) (5, 10). Oxacillin susceptibility breakpoints for SIG members have been added to CLSI supplement M100 for this purpose (1). It is recognized that this testing can be problematic for some clinical laboratories, since automated identification and susceptibility systems (such as the BD Phoenix and bioMérieux Vitek 2 systems) may not routinely report an oxacillin MIC result (5) and since some clinical laboratories do not use oxacillin for antimicrobial susceptibility testing. For this reason, immunochromatographic testing for the presence of PBP2a has been studied in SIG isolates and was shown to have 100% sensitivity and 100% specificity for the detection of methicillin resistance in a comparison with mecA PCR (10), particularly when induced PBP2a testing with cefoxitin was used (5). Isolates of the SIG can also grow on chromogenic media for the detection of methicillin-resistant S. aureus (MRSA), such as Spectra MRSA agar (Remel Microbiology Products, Lenexa, KS). While SIG isolates do not produce the characteristic “denim blue” colony color of MRSA on these plates (Fig. 1E), the colonies may be considered suspicious for MRSA.

Members of the SIG are human pathogens that may be misidentified when we rely on biochemical tests such as coagulase and latex agglutination for identification of catalase-positive, Gram-positive cocci. Accurate identification has important implications for the interpretation of disk diffusion susceptibility testing, including the determination of methicillin resistance. However, identification using MALDI-TOF MS is reliable for identifying these isolates as members of the SIG.