Evaluation of the Methicillin-Resistant Staphylococcus aureus (MRSA)-Screen Latex Agglutination Test for Detection of MRSA of Animal Origin

Abstract

Methicillin (oxacillin)-resistant staphylococci (MRS) have emerged as major clinical and epidemiological pathogens, and there have been frequent reports of MRS infections in the veterinary field. The MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) was compared with an oxacillin agar screen test, MIC determination, and mecA PCR assay, the “gold standard.” In an analysis of 15 mecA-positive and 48 mecA-negative S. aureus animal isolates, as well as 9 mecA-positive and 147 mecA-negative, coagulase-negative staphylococcal animal isolates, the latex agglutination test surpassed the widely used oxacillin agar screen method and MIC determination, with a sensitivity and a specificity of 100%. The MRSA-Screen test is a reliable and rapid method of detecting MRS in the veterinary field.

Staphylococcus spp. cause severe diseases such as suppurative disease, mastitis, arthritis, and urinary tract infection by introducing numerous virulence factors such as extracellular toxins and enzymes into animal species (20). For humans, these organisms are important causes of food poisoning, pneumonia, wound infections, and nosocomial bacteremia (6). Staphylococcal isolates are frequently resistant to penicillinase-resistant penicillins. Organisms exhibiting this type of resistance are referred to as methicillin (oxacillin)-resistant staphylococci (MRS). These bacteria are also frequently resistant to most of the commonly used antimicrobial agents, including the aminoglycosides, macrolides, chloramphenicol, tetracycline, and fluoroquinolones (10). In addition, MRS strains should be considered to be resistant to all cephalosporins, cephems, and other β-lactams, such as ampicillin-sulbactam, amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, piperacillin-tazobactam, and the carbapenems, regardless of the in vitro test results obtained with those agents (15).

There are many reports on MRS infections in dairy herds with mastitis, as well as in companion animals and horses (2, 4, 8, 9, 17, 18, 21). These reports indicate that MRS infections are problematic in the veterinary field, and these types of strains can be transmitted to humans (9). Therefore, infections by multidrug-resistant pathogens such as MRS require rapid and accurate diagnosis for elimination at an early stage because these strains can cause severe damage to infected sites and may be widespread in the environment. In most routine microbiological settings, detection of methicillin resistance among staphylococcal isolates is based on phenotypic assays such as a disk diffusion test and MIC determination. Genetic confirmation of positive findings based on detection of the mecA gene has also been reported (13, 19). However, these techniques are not yet generally available outside of reference laboratories. The methicillin resistance of Staphylococcus spp. is mediated by the mecA gene, which encodes penicillin-binding protein 2a (PBP2a) (3). A simple latex agglutination assay (MRSA-Screen; Denka Seiken Co., Ltd., Tokyo, Japan) was developed to detect methicillin-resistant Staphylococcus aureus (MRSA), which makes use of a specific monoclonal antibody directed toward the PBP2a antigen (14). This assay has the potential to detect MRSA in a routine microbiology setting because it combines high speed and excellent specificity and sensitivity. This assay has not been tested on isolates of animal origin. In this study, the MRSA-Screen latex agglutination test, which is a simple and rapid assay for detection of various MRS of animal origin, was evaluated to determine the sensitivity of the assay. This test was compared with an oxacillin agar screen test, MIC determination, and the mecA PCR assay, the “gold standard.”

Samples of feces, milk, feed material, and the joints, tracheas, uteri, and meat of cattle, pigs, and chickens were collected at slaughterhouses, meat processing facilities, and farms located throughout Korea, including Chungcheong, Gyeongsang, and Jeolra Provinces, between May 2001 and April 2003. The collected samples were inoculated into either Staphylococcus broth or tryptic soy broth with 70 mg of NaCl per ml and incubated at 35°C for 20 h with constant shaking. The inoculum was subcultured on Baird-Parker medium for 24 to 48 h at 35°C. The typical suspected colonies were tested for Staphylococcus spp. by conventional methods that included Gram staining, tests for colonial morphology, and tests for coagulase using rabbit plasma tubes (Sigma, St. Louis, Mo.). These were also tested by the API STAPH IDENT system (Biomerieux, Lyon, France). The agar screen test for the oxacillin resistance of S. aureus was performed in accordance with NCCLS recommendations (16) with Mueller-Hinton agar containing 4% NaCl and 6 μg of oxacillin per ml. The MIC of oxacillin was determined by an agar dilution method in accordance with NCCLS recommendations (16) with an inoculum of 10⁴ CFU/spot on Mueller-Hinton agar containing 4% NaCl and oxacillin at concentrations ranging from 0.5 to 128 μg/ml for S. aureus and 0.25 to 8 μg/ml for the coagulase-negative staphylococci. The resistance breakpoints of the oxacillin MICs for the S. aureus and coagulase-negative staphylococci were greater than or equal to 4 and 0.5 μg/ml, respectively. A previously described method was used for whole-cell DNA extraction (12). Two microliters of supernatant was used as the template. The presence of the mecA gene was demonstrated by PCR. Amplification of the mecA gene was performed with the primers mecA1 and mecA2 (13). The control organisms included S. aureus ATCC 43300, S. aureus ATCC 25923, and S. epidermidis ATCC 12228. The MRSA-Screen latex agglutination assay was performed in accordance with the manufacturer's protocol. Agglutination was assessed visually within 3 min.

Staphylococcus species were isolated from various samples associated with animals and identified. An agar screen test with oxacillin was performed to determine the phenotypic methicillin (oxacillin) resistance of S. aureus. S. aureus isolates from 28 samples from animal were resistant to oxacillin (Table 1). Thirty-five methicillin-susceptible S. aureus isolates were also collected. A total of 156 coagulase-negative staphylococcal isolates consisting of 45 S. epidermidis, 37 S. saprophyticus, 29 S. simulans, 18 S. xylosus, 17 S. hominis, and 10 S. lentus isolates were tested (Table 2). Of the 28 MRSA animal isolates, 15 were found to be mecA positive and 13 were found to be mecA negative by PCR (Table 1). All of the 35 methicillin-susceptible S. aureus isolates were mecA negative. All of the 15 mecA-positive MRSA isolates were also positive by the MRSA-Screen latex agglutination test. The reactions were determined within 3 min. All of the mecA-negative MRSA and methicillin-susceptible S. aureus isolates were negative by the MRSA-Screen test, with the exception of two mecA-negative isolates that yielded weak latex agglutination after the 10- to 15-min reactions. These two isolates had highly viscous characteristics than the other types of S. aureus. Of the 156 coagulase-negative staphylococci, 9 contained the mecA gene while 147 did not (Table 2). Regardless of the different species, all 9 mecA-positive, coagulase-negative isolates were positive by the MRSA-Screen test and all of the 147 mecA-negative isolates were negative. Therefore, the results of the MRSA-Screen latex agglutination test for PBP2a agreed with those of the mecA PCR. With the PCR as the gold standard method, the MRSA-Screen latex agglutination test demonstrated 100% (24 of 24) sensitivity and 100% (195 of 195) specificity. The oxacillin agar screen test for S. aureus identified all of the 15 mecA-positive isolates, for a sensitivity of 100%. However, it yielded 13 false-positive results for the 48 mecA-negative S. aureus isolates tested in this study, for a specificity of 72.9% (Table 1). The oxacillin MICs for the mecA-positive MRSA isolates determined by the oxacillin agar screen test ranged from 4 μg/ml to greater than 128 μg/ml, and the MICs for the mecA-negative MRSA determined by the oxacillin agar screen test were within relatively lower, ranging from 1 to 16 μg/ml (Table 1). The MICs for the mecA-negative and methicillin-susceptible S. aureus isolates determined by the oxacillin agar screen test were all less than 2 μg/ml. MIC determination for S. aureus showed a sensitivity and a specificity of 100% (15 of 15) and 81.3% (39 of 48), respectively. The MICs for the 9 mecA-positive, coagulase-negative staphylococci were all greater than 0.5 μg/ml, and the MICs for 12 of the 147 mecA-negative, coagulase-negative staphylococci were also greater than 0.5 μg/ml (Table 2). The MIC for the coagulase-negative staphylococci showed a sensitivity and a specificity of 100% (9 of 9) and 91.8% (135 of 147), respectively.

TABLE 1.

Diagnostic test results obtained for MRSA isolates from animals^a

Isolate	Origin	mecA PCR	MRSA-Screenb	Oxacillin-Screen	MIC (μg/ml)
SA006	Chickens	+	+	+	16
SA023	Cattle	−	−c	+	4
SA030	Pigs	−	−	+	8
SA031	Pigs	−	−	+	4
SA032	Pigs	−	−c	+	4
SA033	Pigs	−	−	+	4
SA037	Pigs	−	−	+	2
SA046	Cattle	+	+	+	32
SA049	Chickens	−	−	+	1
SA055	Chickens	−	−	+	2
SA058	Chickens	−	−	+	4
SA095	Chickens	+	+	+	32
SA097	Chickens	+	+	+	32
SA106	Cattle	+	+	+	32
SA107	Cattle	−	−	+	4
SA108	Cattle	−	−	+	4
SA109	Cattle	+	+	+	16
SA120	Cattle	+	+	+	32
SA122	Cattle	+	+	+	32
SA123	Cattle	+	+	+	>128
SA124	Cattle	+	+	+	64
SA125	Cattle	+	+	+	8
SA126	Cattle	+	+	+	16
SA129	Cattle	−	−	+	8
SA130	Cattle	+	+	+	128
SA236	Cattle	+	+	+	64
SA237	Cattle	+	+	+	4
SA240	Cattle	−	−	+	2

^aSymbols: +, positive; −, negative.

^bAll 35 mecA-negative, methicillin-susceptible S. aureus isolates were negative by the MRSA-Screen test, and the oxacillin MICs were less than 2 μg/ml.

^cSlight agglutination of this strain became visible after 10- to 15-min reactions, while other positive reactions were visible within 3 min.

TABLE 2.

Results obtained using the diagnostic tests on coagulase-negative MRS isolates from animals^a

Species (no. of isolates)	No. of isolates		MRSA-Screenb	MIC (μg/ml)
	mecA negative	mecA positive
S. epidermidis (45)	41		−	≤0.25
	2		−	≥4
		2	+	≥4
S. saprophyticus (37)	33		−	≤0.25
	1		−	≥4
		3	+	≥4
S. simulans (29)	24		−	≤0.25
	2		−	≥4
		3	+	≥4
S. xylosus (18)	14		−	≤0.25
	4		−	≥4
S. hominis (17)	15		−	≤0.25
	1		−	2
		1	+	≥4
S. lentus (10)	8		−	<0.25
	1		−	1
	1		−	≥4

^aAll of these coagulase-negative staphylococcal isolates were originated from cattle.

^bSymbols: +, positive; −, negative.

Staphylococcal strains may vary in different hosts and epidemiological potential. This study included S. aureus isolates, as well as coagulase-negative staphylococcal isolates, in order to evaluate animal staphylococcal strains with various genetic backgrounds. However, regardless of the different species, the latex agglutination test detected the PBP2a antigen in the genetically diverse MRS strains and approached the accuracy of the PCR assay for mecA with 100% sensitivity and specificity. Therefore, the latex agglutination test can be applicable to animal MRS strains. The mechanism of methicillin resistance in staphylococcal strains is poorly understood but is believed to involve an interaction of PBP2a and various gene products such as those encoded by the fem (factor essential for methicillin resistance) genes, which are involved in cell wall peptidoglycan synthesis (1, 3, 5).

Despite the standardized recommendations for the susceptibility testing of MRS given by the NCCLS (16), many of the isolates in this study that did not carry mecA were phenotypically resistant to methicillin according to the oxacillin agar screen test and MIC determination. The phenotypic expression of resistance can vary depending on the growth conditions (e.g., the temperature or osmolarity of the medium), making susceptibility testing of MRS by standard microbiological methods potentially difficult (3). Therefore, predicting the presence of mecA may be problematic with these susceptibility tests for methicillin resistance in S. aureus.

mecA-positive, methicillin-susceptible MRS strains become resistant after being incubated with methicillin (3). Induction by β-lactams appears to increase PBP2a, particularly in coagulase-negative staphylococci (7). Other studies have shown that the exposure of several mecA-positive, phenotypically methicillin-susceptible S. aureus isolates to β-lactams results in an increase in the MIC of oxacillin well above the resistance level. This is despite the fact that the initial susceptibility tests revealed that these isolates were vulnerable (11). This suggests that an induction step with β-lactams in conventional phenotypic susceptibility assays for detecting MRS strains is needed.

The PCR assay is considered to be the gold standard for the detection of MRSA. However, this method is too time-consuming and expensive to be practical in a clinical microbiology laboratory. The latex agglutination test has the advantage of a rapid turnaround time from the isolation of an organism to the determination of susceptibility. The test provided the results within 20 min and easily managed the processing of large numbers of samples simultaneously.