Detection of Staphylococcus aureus Clinical Isolates Harboring the ica Gene Cluster Needed for Biofilm Establishment

The incidence of chronic nosocomial infections by gram-positive bacteria has drastically increased during the last years in association with the more frequent use of in-dwelling medical devices (1, 2). Infections derived from the use of invasive methods, e.g., catheters, are mainly due to staphylococci, especially those strains which produce an extracellular slime and constitute a biofilm, making clinical treatment extremely difficult (4, 5, 7, 9-12). The biofilm development process requires polysaccharidic intercellular adhesin, which is synthesized by the enzymes encoded by the intercellular adhesion cluster (ica) (6, 13, 14).

A great variety of Staphylococcus aureus and Staphylococcus epidermidis strains carry the ica cluster, and some of them constitute biofilm. Loss of the ica locus results in an incapacity to produce polysaccharidic intercellular adhesin and to develop biofilms (1). Staphylococcal infections produced by ica carriers can be more problematic due to the presence of methicillin and mupirocin resistance genes (5, 9, 12). The rapid detection of the ica locus in hospital staphylococcal isolates, together with the simultaneous detection of antibiotic resistance genes, will allow the development of prevention methods to reduce the bacterial capacity to invade the in-dwelling medical devices.

We have analyzed 65 clinical isolates, 7 from catheter samples and 58 clinical isolates randomly selected. The catheter isolates were recovered from the Oncology Medical Service, which follows a specific protocol to avoid catheter colonization, which explains the small number of isolates. The isolates, including S. aureus (60, 2 of them from catheter samples) and S. epidermidis (5, all from catheter samples), recovered during a one-year period, were analyzed by PCR to determine the presence or absence of the genes that confer constitutive methicillin resistance (mecA) and high mupirocin resistance (ileS-2) and a fragment of a gene that identifies S. aureus at the species level (femB) and to detect the presence of the intercellular adhesion gene cluster (ica). Detection of femB, mecA, and ileS-2 genes was performed by applying a triplex PCR method that has been previously described (8). PCR detection of the ica cluster was performed by amplification of a DNA region partially covering the icaA and icaB genes. For S. epidermidis, we used the previously described primers icaAB-F and icaAB-R (3), which yielded a 546-bp fragment, while for S. aureus we designed a pair of primers from the sequence available from the National Center for Biotechnology Information gene bank (locus AF086783): icaA-S (5′ AAA CTT GGT GCG GTT ACA GG 3′) and icaA-E (5′ TCT GGG CTT GAC GTT G 3′) (Roche Diagnostics, Mannheim, Germany). Amplification with this pair of primers generated a 750-bp fragment.

From the 58 clinical isolates randomly selected, 5 (8.6%) methicillin-resistant S. aureus isolates (MRSA) and 1 (1.7%) methicillin-resistant and highly mupirocin-resistant S. aureus isolate (MMRSA) were negative for icaAB, while the other 52 (89.7%) isolates were positive for icaAB (Table 1). Fifty-six of these 58 isolates showed mecA (96.5%), 52 presented icaAB (89.7%), and 40 isolates (61.5%) were characterized by harboring both icaAB and mecA. Eleven of the 58 isolates (16.9%) carry both resistance genes mecA and ileS-2 in addition to the ica cluster, and only 2 of 58 (3.5%) lacked the femB marker. Regarding the seven catheter isolates, two S. aureus isolates showed the icaAB marker but were negative for both resistance markers, i.e., mecA and ileS-2, while only one S. epidermidis isolate presented the icaAB cluster (Table 1).

TABLE 1.

Frequency of detection of femB, mecA, ileS-2, and icaAB loci

Group of Staphylococcus isolates	Total no. of isolates	No. of isolatesa carrying:
		femB	mecA	ileS-2	icaAB	mecA, and icaAB	mecA, and ileS-2	mecA, ileS-2, and icaAB	mecA−, ileS-2−, and icaAB
Randomly selected	58b	56 (96.5), 91.7-100	56 (96.5), 91.7-100	12 (20.7), 9.7-31.4	52 (89.6), 81.5-97.7	40 (69), 56.7-81.2	1 (1.7), <6	11 (19), 8.6-29.4	2 (3.4), <9
Recovered from catheters	7c	2 (28.6), 10.1-47	2 (28.6), 10.1-47	2 (28.6), 10.1-47	3 (42.9), 22.7-63.1		2 (28.6), 10.1-47		3 (42.9), 22.7-63.1

^aValues in parentheses are percentages. For each entry, the second value given is the 95% confidence interval.

^bAll isolates were S. aureus.

^cFive isolates were S. epidermidis, and two isolates were S. aureus.

Our results showed a very high percentage of the ica cluster in nosocomial MRSA and MMRSA isolates. The proportion of ica carriers was slightly lower (42.9%) in the case of catheter isolates, but it reached 89.6% in the case of isolates recovered from mucous membranes and skin.

Frebourg et al. (3) have reported that a high proportion of clinical isolates harboring the ica locus also carry the mecA gene. Here, we observed that 68.9% of the MRSA isolates harbor both loci, ica and mecA. Of these isolates, 18.6% showed a higher virulence potential, since they also presented high mupirocin resistance encoded by the ileS-2 gene.

Therefore, we suggest simultaneous PCR detection of the ica locus and antibiotic resistance genes as a rapid and effective method to be used for discrimination between potentially virulent and nonvirulent isolates, which would be especially relevant for detection of isolates with high capacity to invade in-dwelling medical devices.