the Detection of Methicillin-Resistant Staphylococcus aureus from Stool Specimens.

JCM Accepts, published online ahead of print on 14 April 2010 J. Clin. Microbiol. doi:10.1128/jcm.02376-09 Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 1 2 Evaluation of a New Selective Medium (BD BBL TM CHROMagar TM MRSA II) for the Detection of Methicillin-Resistant Staphylococcus aureus from Stool Specimens. 3 4 5 6 7 8 9 10 11 12 13 Nancy L. Havill 1* and John M. Boyce 1,2 Hospital of Saint Raphael 1 and Yale University School of Medicine 2, New Haven, CT. Running title: Detection of MRSA is stool specimens. Corresponding author: Nancy L. Havill Hospital of Saint Raphael 1450 Chapel St. New Haven, CT 06511 Phone:203-789-5115 Fax: 203-789-4247 Email: Nhavill@srhs.org 14 15 16 17 18 19 20 21 22 23

2 24 25 26 27 We compared the recovery of MRSA on a new selective chromogenic agar, BD BBL TM CHROMagar TM MRSA II (CMRSAII), to traditional culture media in 293 stool specimens. The recovery of MRSA was greater on the CMRSAII agar. Screening stool samples can identify patients who were previously unknown carriers of MRSA.

3 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Active surveillance cultures to identify patients colonized with methicillinresistant Staphylococcus aureus (MRSA) are recommended as part of an intensive program to control the spread of the organism in healthcare settings 10,14. Although the anterior nares are currently the most common site to be cultured for MRSA colonization 4,7,13,15, other sites including the intestinal tract, can be potential sources of unrecognized colonization for this organism 1,2,6. Patients with S. aureus (SA)(including MRSA) colonization of the intestinal tract are associated with greater contamination of healthcare workers hands and the environment 3. Patients with intestinal colonization with MRSA and concurrent diarrhea contaminate their environment to a significantly greater extent than patients colonized at other body sites 5. Screening stool samples for MRSA can detect patients who are previously unknown carriers and who may not otherwise be cared for using contact precautions, which could potentially lead to increased healthcare associated transmission and subsequent MRSA infections 1,2,6. It has also been suggested that undetected intestinal colonization of MRSA could be the cause of persistent colonization or re-colonization in patients after successful decontamination 8,11. The use of chromogenic agars has been shown to increase sensitivity and decrease time to detection of MRSA when compared with traditional culture methods. 9 However, to date, all currently available chromogenic media used for screening patients for MRSA are recommended only for use with anterior nares specimens. We evaluated a new selective and differential chromogenic medium, BD BBL TM CHROMagar TM MRSA II (CMRSAII)(BD Diagnostics, Sparks, MD), for its ability to detect MRSA in

4 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 specimens from multiple body sites, including stool specimens, as part of a large multicenter trial. (Wendt et al, presented in part at the 48 th Interscience Conference on Antimicrobial Agents and Chemotherapy/46 th Annual Meeting of the Infectious Diseases Society of America, Washington, DC, October 25-28 2008). We report the evaluation of this medium to detect MRSA from stool samples collected at our institution. The study was conducted during the period from December 2007 to February 2008 at a 500-bed university-affiliated hospital. A total of 293 stool specimens collected from all wards in the hospital and submitted to the laboratory for Clostridium difficile toxin assay (CDT) were included in the study. Stools were not rejected on the basis of specimen consistency. The specimens were inoculated onto colistin-nalidixic acid (CNA) agar and immediately following onto CMRSAII. The plates were incubated in ambient air at 36 o C and examined at 24 hours (range 18-28 h) and if negative they were reincubated and examined at 48 hours (range 36-52 h). CMRSAII was incubated in the dark to avoid exposure to light. Colonies morphologically consistent with SA recovered on CNA plates were confirmed as SA with a positive coagulase test ( Staphaureux, Remel, Lenexa, KS) and confirmed as MRSA by cefoxitin disk diffusion. Mauve-colored colonies present on CMRSAII at 24 and 48 hours were presumed to be MRSA and were confirmed as SA with a positive coagulase test. A cefoxitin disk diffusion test was performed if MRSA was not recovered from the CNA plate. Differences in proportions were analyzed using McNemar s test. MRSA was recovered from 62 (21.2%) of 293 stool specimens. CMRSAII recovered 60/62 (96.8%) and CNA media recovered 47/62 (75.8%) (P = 0.004) (Table 1). Of the 60 MRSA isolates recovered on CMRSAII, 44/60 (73.3%) of MRSA were

5 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 identified at 24 hours and 14/60 (26.7%) at 48 hours, whereas the traditional culture method took between 48-96 hours to confirm an isolate as MRSA. When CMRSAII was compared with cefoxitin disk diffusion the overall agreement was 97.3% (285/293) at 24 hours and 99.3% (291/293) at 48 hours. Compared to cefoxitin disk diffusion, CMRSAII had a sensitivity of 84.6% (44/52) at 24 hours and 96.8% (60/62) at 48 hours, and specificity of 100% at both 24 and 48 hours. Traditional culture had a sensitivity of 90.4% (47/52) at 24 hours and 75.8% (47/62) at 48 hours, and a specificity of 100% at both 24 and 48 hours, when compared to cefoxitin disk diffusion. (Table 2) The sensitivity of the traditional culture method at 48 hours was lower than at 24 hours because additional stools were positive on CMRSAII at 48 hours and were confirmed by cefoxitin disk diffusion, but none of these were positive at 48 hours on traditional media. No false positive results were identified on CMRSAII after 48 hours of incubation. For 17 (33.3%) of the 51 unique patients positive for MRSA, the stool isolate was the first MRSA recovered from the patient as determined by electronic chart review. Only four (23.5%) of these patients had a positive CDT and were already placed on contact precautions. Six (35.3%) of the 17 patients had negative nasal MRSA surveillance cultures as part of our active surveillance program. Patients who are at risk for Clostridium difficile diarrhea by virtue of prior hospitalization or antibiotic therapy are also at increased risk of colonization with other multidrug-resistant organisms, including MRSA 12. Screening stool samples that have been sent to the laboratory for CDT assay would increase the likelihood of recovery of MRSA as compared to screening all stool samples. MRSA positive stools were reported and phoned to the nurse to ensure prompt implementation of contact precautions.

6 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 Our study has several limitations. 0ur study represents data collected from a single hospital and may not be representative of all patient populations. This study was conducted within a relatively small time period of three months and may have shown different results if conducted over a longer time period, although these results are consistent with previous data collected at our institution over a period of one year 6. A broth enrichment method was not included as part of the reference method which may have increased the yield of MRSA 9-25% 13, although this method delays the reporting of MRSA for an additional 24 hours. Screening stool samples is only one part of our intensive surveillance program and we have limited resources available to perform such tasks. Although CMRSAII may have less sensitivity than an agar based method with broth enrichment, it is easy to use, has a faster turn around time and requires minimal tech time. In conclusion, we found CMRSAII to be a reliable screening medium to detect MRSA in stool specimens. Recovery of MRSA was greater on CMRSAII than on traditional CNA media. Additionally, CMRSAII has the ability to identity MRSA 24 to 48 hours earlier than the CNA agar screening method. Our study confirms previous studies demonstrating that screening stool samples for MRSA can detect patients who are not previously known to be carriers of MRSA 1,2,6. These findings further substantiate the rationale for including intestinal screening as part of an infection control surveillance strategy. It is possible the increased cost of the chromogenic agar may be offset by the cost savings in reduced transmission of MRSA, but further studies are necessary. Rapid identification of MRSA along with prompt institution of contact precautions may prevent further transmission of this organism in the healthcare setting.

7 120 Acknowledgment: This study was funded in part by BD Diagnostic Systems.

8 121 122 References: 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 1. Acton, D. S., M. J. Plat-Sinnige, W. van Wamel, N. De Groot, and A. Van Belkum. 2009. Intestinal carriage of Staphylococcus aureus: how does its frequency compare with that of nasal carriage and what is its clinical impact? Eur. J. Clin. Microbiol. Infect Dis. 28:115-127. 2. Batra, R., A. C. Eziefula, D. Wyncoll, and J. Edgeworth. 2008. Throat and rectal swabs may have an important role in in MRSA screening of critically ill patients. Intensive Care Med. 34:1703-1706. 3. Bhalla, A., D. C. Aron, and C. J. Donskey. 2007. Staphylcoccus aureus intestinal colonization is associated with increased frequency of S. aureus on skin of hospitalized patients. BMC Infect. Dis. 7:105. 4. Boyce, J. M., and N. L. Havill. 2008. Comparison of BD GeneOhm methicillinresistant Staphylococcus aureus (MRSA) PCR versus CHROMagar MRSA for screening patients for the presence of MRSA strains. J. Clin. Microbiol. 46:350-351. 5. Boyce, J. M., N. L. Havill, J. A. Otter, and N. M. Adams. 2007. Widespread environmental contamination associated with patients with diarrhea and methicillinresistant Staphylococcus aureus colonization of the intestinal tract. Infect. Control Hosp. Epidemiol. 28:1142-1147. 6. Boyce, J. M., N. L. Havill, and B. Maria. 2005. Frequency and possible infection control implications of gastrointestinal colonization with methicllin-resistant Staphyloccus aureus. J. Clin. Microbiol. 43:5992-5995.

9 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 7. Flayhart, D., J. F. Hindler, D. A. Bruckner, G. Hall, R. K. Shrestha, S. A. Vogel, S. S. Richter, W. Howrad, R. Walther, and K. C. Carroll. 2005. Multicenter evaluation of BBL CHROMagar MRSA medium for direct detection of methicillinresistant Staphylococcus aureus from surveillance cultures of the anterior nares. J. Clin. Microbiol. 43:5536-5540. 8. Klotz, M., S. Zimmerman, S. Opper, K. Heeg, and R. Mutters. 2005. Possible risk for re-colonization with methicillin-resistant Staphylococcus aureus (MRSA) by faecal transmission. Int J. Hyg. Environ.-Health. 208:401-405. 9. Lagace-Wiens P. R., M. J. Alfa, K. Manickam and G. K. Harding. 2008. Reductions in workload and reporting time by use of methicillin-resistant Staphylococcus aureus screening with MRSASelect medium compared to mannitolsalt medium supplemented with oxacillin. J. Clin. Microbiol.46:1174-1177. 10. Muto, C.A., J. A. Jernigan, B. E. Ostrowsky, H. M. Richet, W. R. Jarvis, J. M. Boyce, and B. M. Farr. 2003. SHEA guideline for preventing nosocomial transmission of multidrug-resistant strains of Staphylococcus aureus and enterococcus. Infect. Control Hosp. Epidemiol. 24:362-386. 11. Rimland, D., and B. Roberson. 1985. Gastrointestinal carriage of methicillinresistant Staphylococcus aureus. J. Clin. Microbiol. 24:137-138. 12. Safdar, N., and D. G. Maki. 2002. The commonality of risk factors for nosocomial colonization and infection with antimicrobial-resistant Staphylococcus aureus, enterococcus, gram negative bacilli, Clostridium difficile, and candida. Ann Intern Med. 136:834-844.

10 166 167 168 169 170 171 172 173 174 175 176 177 178 13. Safdar, N., L. Narans, B. Gordon, and D. G. Maki. 2003. Comparison of culture screening methods for detection of nasal carriage of methicillin-resistant Staphlylococcus aureus: a prospective study comparing 32 methods. J. Clin. Microbiol. 41:3163-3166. 14. Siegel J., E. Rinehart, M. Jackson, and L. Chiarello. Healthcare infection control practices advisory committee. Manegement of multidrug-resistant organisms in healthcare setting. 2006. Atlanta GA:Centers for disease control and prevention, 2006. 15. Wolk, D. M.., E. Picton, D. Johnson, T. Davis, P. Pancholi, C. C. Ginocchio, S. Finegold, D. F. Welch, M. de Boer, D. Fuller, M. C. Solomon, B. Rogers, M. S. Mehta, and L. R. Peterson. 2009. Multicenter evauation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares. J. Clin. Microbiol. 47:758-764.

11 179 180 181 182 183 184 TABLE 1. Performance of BD BBL TM CHROMagar TM MRSA II compared to Colistin-Nalidixic agar. 185 CMRSAII Positive CMRSAII Negative 186 187 188 CNA agar Positive 45 2 CNA agar Negative 15 231 189 190 191

12 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 TABLE 2. Comparison of BD BBL TM CHROMagar TM MRSA II and Colistin-Nalidixic agar to cefoxitin disk diffusion for detection of MRSA in stool samples. Sensitivity Specificity %, (95 CI) %, (95 CI) 24 hrs 48 hrs 24 hrs 48 hrs CMRSAII 84.6 96.8 100 100 (71.9-93.1) (98.8-99.6) (98.5-100) (98.4-100). CNA agar 90.4 75.8 100 100 (79.0-96.8) (63.3-85.5) (98.5-100) (98.4-100)