Comparison of the Rodac Imprint Method to Selective Enrichment Broth for Recovery of Vancomycin-Resistant Enterococci and Drug-Resistant Enterobacteriaceae from Environmental Surfaces

Abstract

We compared the Rodac imprint technique to selective enrichment broth for detecting vancomycin-resistant enterococci (VRE) and multidrug-resistant Enterobacteriaceae (MDRE) on surfaces. Rodac plates contained tryptic soy agar with 5% sheep blood, vancomycin (6 μg/ml), ceftazidime (2 μg/ml), amphotericin B (2 μg/ml), and clindamycin (1 μg/ml). Two types of broth were used: brain heart infusion (BHI) and BHI plus vancomycin (6 μg/ml) and ceftazidime (2 μg/ml) (BHIVC). Of the 46 surfaces cultured for VRE, 12 (26%) were positive. Of the 12 VRE-positive surfaces, 11 (92%) grew from Rodac, 8 (67%) grew from BHIVC, and 7 (58%) grew from BHI. A larger study is needed for MDRE, as only 4 of 43 surfaces were MDRE positive. The Rodac imprint technique successfully recovered VRE from environmental surfaces.

As the prevalence of patient infection with vancomycin-resistant enterococci (VRE) and other antimicrobial agent-resistant organisms increases in health care settings there have been increased efforts to minimize nosocomial transmission. Some organisms are capable of prolonged survival in the environment, including Clostridium difficile and VRE, and contamination of surfaces has been implicated in patient-to-patient VRE transmission (7, 9). Commonly, three collection methods are used to detect environmental surface contaminants: (i) premoistened swab inoculated onto a selective agar plate, (ii) premoistened swab inoculated into selective enrichment broth, and (iii) direct inoculation by surface to agar contact using the Rodac imprint technique. Studies have demonstrated that recovery of VRE from patient samples obtained with swabs is better when the swabs are placed in enrichment broth compared to direct inoculation onto agar (4–6). Previous studies also have demonstrated that the yield of C. difficile from environmental surfaces is greater when obtained by direct contact with the Rodac imprint technique compared to swab samples placed in enrichment broth or plated on agar (1, 3). However, no studies have been performed to compare the Rodac imprint technique to enrichment broth for recovery of VRE from surfaces and we found no reports evaluating the recovery of multidrug-resistant Enterobacteriaciae (MDRE) from environmental surfaces. Because the Rodac method is less labor-intensive than either swab method, we compared the Rodac imprint technique to the broth enrichment method for the recovery of VRE and MDRE from environmental surfaces.

(This material was presented in part at the 100th Annual Meeting of the American Society for Microbiology, Los Angeles, Calif., 21 to 25 May 2000 [abstr. C-127].)

A total of 49 samples were obtained, including 26 microbiology laboratory surfaces, 14 molecular typing laboratory surfaces, and 9 preinoculated control surfaces. Surfaces cultured were those commonly contacted by the technologists during a routine working day, including bench tops, phones, keyboards, door handles, chairs, pipettors, light switches, and the floor. The surfaces chosen for this study were from areas of the laboratory where clinical cultures positive for VRE and MDRE are handled. The nine control surfaces (six VRE and three MDRE) were preinoculated with various densities of vancomycin-resistant Enterococcus faecalis and multidrug-resistant Escherichia coli (resistant to aztreonam and ceftazidime). The control isolates were obtained from patient samples and are part of the laboratory's quality control collection. To avoid contaminating actual workspace with VRE or MDRE, the control strains were swabbed onto the inside surface of empty, 150-mm-diameter, sterile plastic petri dishes. Cultures were obtained by sampling the petri dish surface. Each control strain was inoculated onto a separate petri dish.

The Rodac contact plate contained medium consisting of tryptic soy agar plus 5% sheep blood, vancomycin (6 μg/ml), amphotericin (2 μg/ml), ceftazidime (2 μg/ml), and clindamycin (1 μg/ml) (VACC). Broth media consisted of brain heart infusion broth without antibiotics (BHI) and brain heart infusion broth with vancomycin (6 μg/ml) and ceftazidime (2 μg/ml) (BHIVC). All media were prepared in the Northwestern Memorial Hospital clinical microbiology laboratory. The Rodac plates also were filled with medium formulated and prepared in our laboratory (VACC). Samples were obtained during routine working hours. Adjacent 10-cm² areas on each surface were sampled with a Rodac imprint plate or a double-headed premoistened culture swab (ampoule crushed to moisten swab). Each sampled area was touched approximately three to five times with a Rodac plate to insure that the entire 10-cm² area was sampled, and then the adjacent area was swabbed. Each of the paired swabs was placed into one of the two broth tubes. The broth tubes and Rodac plates were incubated for 48 h at 35°C prior to workup. All broth tubes were incubated in ambient air, and all plated medium was incubated at 5 to 10% CO₂. At 48 h, turbid BHIVC broths were plated on blood agar and turbid BHI broths were plated on VACC medium. The broth subculture plates were examined at 24 and 48 h for visible growth. All catalase-negative, gram-positive cocci found growing on plates or in broth were identified to the species level by traditional manual biochemical methods, and an agar dilution susceptibility test was performed following NCCLS guidelines (8). The susceptibility test was performed with Mueller-Hinton agar and a final inoculum density of 10⁴ CFU. The plates were incubated at 35°C in ambient air for 16 to 18 h (oxacillin and vancomycin plates were incubated for 24 h). Isolates were tested against vancomycin at concentrations of 2, 6, 16, and 64 μg/ml. Organisms that were identified as an enterococcus and resistant to vancomycin (MIC > 6 μg/ml) were considered VRE. Likewise, all gram-negative bacilli found growing on plates or in broth that were oxidase-negative, glucose-fermenting organisms were identified to the species level by traditional manual biochemical methods, and an agar dilution susceptibility test was performed. Organisms that were identified as a member of the family Enterobacteriaceae and that were resistant to aztreonam (MIC > 16 μg/ml) and ceftazidime (MIC > 16 μg/ml) were considered MDRE. For statistical analysis, results from each broth inoculation method were compared to results from the Rodac imprint technique using the McNemar test for paired samples, and kappa correlation coefficients were determined using SAS software for personal computers (SAS Institute Inc., Cary, N.C.).

Of the 46 surfaces cultured for VRE, 12 were positive (Table 1). When we compared the Rodac technique to broth enrichment, there were no significant differences: 11 of 46 surfaces (24%) were positive by the Rodac technique versus 8 of 46 (17%) by the BHIVC method (P = 0.18) and 7 of 46 (15%) by the BHI method (P = 0.10). The correlation coefficient (κ) for Rodac versus BHIVC was 0.67, and that for Rodac versus BHI was 0.59. VRE was detected on the following surfaces with any of the three methods: control surfaces, the floor of the main bacteriology and molecular epidemiology laboratories, the bench surfaces of all three areas, the cover of the patient result binder in the molecular epidemiology laboratory, and the pipettors in the molecular epidemiology laboratory.

TABLE 1.

Comparison of Rodac imprint with VACC media to broth enrichment for growth of VRE

Method	Controls (n = 6) with VRE at density (CFU/ml) of:						Total no. (%) positive controls	No. (%) positive specimens from surfaces (n = 40)			Total no. (%) positive surfaces	Grand total no. (%)
	Set 1			Set 2				Main bacteriology laboratory (n = 15)	Molecular epidemiology laboratory (n = 14)	Susceptibility testing laboratory (n = 11)
	108	106	105	108	106	105
Rodac imprint	Positive	Positive	Positive	Positive	Positive	Negative	5 (83)	1 (7)	5 (36)	0	6 (15)	11 (24)
Swab in BHIVC	Positive	Positive	Negative	Positive	Positive	Negative	4 (67)	0	3 (21)	1 (9)	4 (10)	8 (17)
Swab in BHI	Positive	Positive	Negative	Positive	Positive	Negative	4 (67)	0	2 (14)	1 (9)	3 (7.5)	7 (15)

For MDRE, only 4 of the 43 surfaces cultured were positive. The 10⁸-CFU/ml density of the E. coli control grew from the Rodac plate and both broth cultures. The 10⁶-CFU/ml density of the E. coli control grew only from the BHI broth. A multidrug-resistant isolate of Enterobacter cloacae was detected on a bench top in the main bacteriology laboratory by the BHI broth alone and on the floor of that area by the Rodac method alone. Due to the low number of positive results, statistical analyses were not performed. Thus, we were unable to adequately compare the three sampling techniques for MDRE due to the small sample size.

In 1995, the Centers for Disease Control and Prevention published recommendations to prevent the spread of VRE in U.S. hospitals (2). These recommendations included that in hospitals where VRE has become endemic, environmental cultures be taken before and after cleaning patient rooms to help determine the effectiveness of cleaning techniques used in rooms housing patients with VRE. A technique to obtain these cultures that is both sensitive and easy to perform would facilitate implementation of these recommendations. Several studies have demonstrated that broth enrichment enhances the recovery of VRE from patient samples compared to the traditional method of plating swabs onto selective agar (4–6). Reisner et al. demonstrated that broth enrichment also increased the recovery of VRE from environmental surface samples compared to swab samples plated on agar (B. S. Reisner, S. Shaw, M. E. Huber, C. E. Woodmansee, S. F. Costa, P. S. Falk, and C. G. Mayhall, Abstr. 4th. Decenn. Int. Conf. Nosocom. Healthcare-Assoc. Infect., abstr. P-T2-50, 2000). However, broth enrichment is more labor-intensive than the traditional method of plating swabs on agar.

Another technique used to obtain samples from environmental surfaces is the Rodac imprint method, where the agar surface of the Rodac plate is pressed against the surface to be cultured. This direct plating method requires less time and materials for specimen inoculation; however, no study has evaluated it for the recovery of VRE. The Rodac method has been used to culture the environment in search of other antimicrobial agent-resistant organisms. Rutala and coworkers reported their experience with the Rodac method to culture environmental surfaces for MRSA during an outbreak in a burn unit and found MRSA in occupied patient rooms, nonoccupied patient rooms, and the contiguous support areas (10). Skoutelis and colleagues used the Rodac imprint technique to culture patient rooms for C. difficile in a nonoutbreak setting and found 16.8% of carpeted rooms and 6.7% of noncarpeted rooms to be positive for this organism (11). Studies have compared the Rodac method to broth enrichment and found that the Rodac method yielded more positive tests than broth enrichment for the detection of C. difficile in the environment (1, 3). Results of our study indicate similar findings for VRE. Although not statistically significant at the P = 0.05 level, our results suggest that the Rodac method recovered more VRE than enrichment broth.

A recent presentation by Lyle and colleagues also demonstrated that Rodac plate cultures were as sensitive as broth enrichment for surfaces that were amenable to imprint cultures (E. A. Lyle, D. W. Blom, G. A. Dewalt, R. A. Weinstein, and M. K. Hayden, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1601, p. 104, 2000), confirming our findings. These workers used Enterococcosel agar with vancomycin (6 μg/ml) in their study. In a previous comparison between VACC and Enterococcosel agar (BBL/Becton Dickinson and Company, Cockeysville, Md.) with vancomycin (6 μg/ml) added, we did not find increased sensitivity in the recovery of VRE with the Enterococcosel agar. Of the 40 VRE samples tested, 37 grew on Enterococcosel agar and 38 grew on VACC at 48 h (unpublished data).

Recovery of MDRE from the environment was uncommon. Only 4 of 43 surfaces were positive for MDRE. At our institution, among patients the prevalence of MDRE colonization or infection is less frequent than that of VRE, resulting in fewer opportunities for environmental contamination in the laboratory.

In our experience the Rodac imprint technique recovered a higher percentage of VRE than the selective enrichment broth method. More frequent environmental surface contamination would be necessary to compare the Rodac method to enrichment broth for detection of MDRE. Enhanced recovery of VRE and the technically simpler methodology make the Rodac method an excellent choice for environmental culturing to detect VRE.