Methicillin resistance in both Staphylococcus aureus and coagulase-negative staphylococci (CNS) has been extensively studied over the past three decades. Methicillin-resistant S. aureus (MRSA) was first recognized as a condition of poor clinical significance before observing a sudden increase in European countries by 1968. In the United States, the first hospital outbreak of MRSA was reported in 1968, and an epidemic rate was not observed until 1981. Later, many outbreaks were reported from Africa, Australia, and Asia in 1974, 1987, and 1988, respectively. Once MRSA has been introduced into a hospital, it may cause more than 50% of the institution’s hospital-acquired staphylococcal infections, usually difficult to eradicate.

Methicillin resistance in staphylococci is due to the presence of penicillin-binding protein 2a that exhibits low affinity for ?-lactam antibiotics. This protein is encoded by the mecA gene, located on the chromosome of all MRSA. While this gene encodes for the production of penicillin-binding protein 2a, auxiliary genes such as femA and femB encode proteins that influence the level of methicillin resistance, defining this phenotype as either heterogeneous, which is more prevalent, or as homogeneous. This highly epidemic organism also shows a multi-drug resistance profile, in which an association between methicillin and aminoglycoside resistance is common.

CNS has been less extensively studied than MRSA, but it has become a significant nosocomial pathogen, principally among infections related to medical devices. In clinical practice, S. epidermidis and S. hemolyticus have become the most significant species of CNS among methicillin-resistant CNS. In contrast to MRSA, only 81.5% of S. epidermidis and 58.3% of other CNS carry mecA. However, they do not have femA or femB genes, although S. epidermidis is the more common species and S. hemolyticus has become a meaningful organism because of its ability to develop vancomycin resistance, causing severe nosocomial infections. A possible pathogenic role of other biological properties, such as synergistic hemolysis and slime production, has been studied in CNS, but the association of such properties to methicillin resistance has not been thoroughly explored. Therefore, we performed a laboratory-based case-control study to determine the rate of infections caused by either MRSA or MRCNS. Additionally, we studied the relationship of methicillin resistance with synergistic hemolysis and the slime production of such microorganisms.

A laboratory-based survey was done at a 180-bed tertiary-care center in Mexico City. The surveillance method included a daily visit to the Clinical Microbiology Laboratory to review all inpatient records compiling staphylococci isolates. Whenever this occurred, an investigator interviewed the patient and checked the patient’s ward chart to register the following data: age; sex; underlying disease; antimicrobials used during the previous 4 weeks; length of hospital stay, and clinical manifestations of infection. All consecutive inpatients with a staphylococcal infection were included from May 1991 to October 1992. A non-matched case-control study was performed selecting two controls [patients with hospital-acquired methicillin-susceptible staphylococcal (MSS) infections] per case [patient with methicillin-resistant staphylococcal (MRS) infection].

All infections clinically apparent or in the incubation period at hospital admission were termed community-acquired infections. Nosocomial infection was acquired after a 72-h hospitalization or related to an intervention during the hospital stay. Staphylococcal infections were defined as follows: urinary-tract infection by midstream sample showing >10⁴ CFU/mL or ?10² CFU/mL in urine obtained by needle aspiration of a vesical catheter; intravenous catheter-related infection by detection of > 15 CFU isolated from a catheter segment (2 inches); wound infection by isolation of staphylococcus as the predominant organism in wound secretion; bacteremia by evidence of CNS in at least two blood cultures obtained from different anatomic sites, and for S. aureus, at least one positive blood culture. An episode of infection was defined as symptomatic or asymptomatic by the presence or absence of either clinical symptoms or physical signs.

S. aureus was identified by coagulase production. Species of CNS were defined by means of a commercial micromethod (UniScept 20GP, Analytab-Products, Plainview, NY, USA).

Screening of methicillin resistance was done by inoculating a 10-?L aliquot of a bacterial suspension with 3 × 10⁶ CFU/mL (McFarland turbidity standard no. 1) on Müeller-Hinton agar supplemented with Ca (50 mg/L), Mg (25 mg/L), NaCl (4%), and oxacillin (4 ?g/mL). All growth of at least one well-defined colony on these plates incubated at 30°C for 48 h was suggestive of MRS.

The methicillin-resistance trait was confirmed by microdilution in Müeller-Hinton broth supplemented with cations and NaCl, using a final inoculum of 7.5 × 10⁴ CFU. MRS were microorganisms with a minimal inhibitory concentration (MIC) of oxacillin ?8 ?g/mL, and those with a MIC ?1 ?g/mL were considered MSS. An MIC of oxacillin between ?1 ?g/mL and <8 ?g/mL defined a methicillin-intermediate staphylococcus (MIS). MICs for vancomycin, gentamicin, erythromycin, penicillin, tetracycline, clindamycin, amikacin, ciprofloxacin, and trimethoprim-sulfamethoxazole were carried out according to the recommendations of the National Committee for Clinical Laboratory Standards, using S. aureus ATCC 29213 as control.

Heterogeneous or homogeneous phenotype of MRS were defined according to the efficiency of plating (EOP) defined by the index: % = number of CFU on Müeller-Hinton agar supplemented with 50 ?g/mL of oxacillin divided by the number of CFU on regular Müeller-Hinton agar after incubation at 35°C for 96 h. An EOP index <1% or ?1% determined a phenotype as heterogeneous or homogeneous, respectively.

All clinical isolates were tested for synergistic hemolysis on trypticase soy agar plates containing 5% defibrinated sheep, ox, or human blood cells. S. aureus ATCC 25923, a well-known ?-lysine producer, was streaked perpendicularly to, but not touching, the testing organisms on each of the three plates and incubated at 35°C for 48 h. A positive test was defined as the presence of complete hemolysis observed in the area of partial hemolysis caused by S. aureus ATCC 25923 in either of the three plates.

All CNS were tested for slime production by a qualitative method, inoculating the organisms in a glass tube with trypticase soy broth. After incubation at 35°C for 48 h, the broth was poured off, and the presence of a film on the walls identified an isolate as a slime producer.

Forty-three cases and 86 controls were calculated as the size needed based on: (a) exposure rate to prolonged hospital stay (? days) of 0.20; (b) value of risk associated to exposure equal to or greater than 3; (c) -value of 0.05 (one-sided); and (d) ?-value of 0.20 (one-sided). An association between the epidemiologic characteristics of the population and MRS infection (dependent-variable) was explored by multiple logistic regression analysis [Epidemiological Graphics Estimation and Testing (EGRET) Cytel Software Corporation; Cambridge, MA, USA] of the variables that showed a p value of less than 0.10 in the univariate evaluation. Either the ?² test with Yates’ correction or Fisher’s exact test was used to compare proportions as required by using True Epistat Software (Epistat Services, Richardson, TX, USA). The relationship among biological traits was estimated by the Kappa index, in which one was considered as perfect association while zero (0) suggested correlation by chance. A p value of less than 0.05 was interpreted as significant.