Evaluation of CHROMagar Acinetobacter for the Detection of Enteric Carriage 1

of Multi-drug Resistant Acinetobacter baumannii in Critically Ill Patients 2

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N C Gordon1, D W Wareham1,2* 4

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1Division of Infection, Barts & The London NHS Trust, London, UK 6

2Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts & The 7

London School of Medicine and Dentistry, Queen Mary University of London, 8

London, UK 9

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*Corresponding author 12

Dr D Wareham 13

Centre for Infectious Disease, 14

Institute of Cell and Molecular Science 15

4 Newark Street 16

Whitechapel 17

London E1 2AT 18

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Tel: +44(0)20 7882 2317 Fax: +44(0)20 7882 2181 20

Email: d.w.wareham@qmul.ac.uk 21

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Running title: CHROMagar Acinetobacter 23

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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00634-09 JCM Accepts, published online ahead of print on 13 May 2009

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Abstract 26

CHROMagar Acinetobacter was used to screen stool and perineal swabs for enteric 27

carriage of multi-drug resistant A. baumannii in critically ill patients. Results were 28

compared with a molecular assay resulting in a sensitivity and specificity of culture 29

compared to PCR of 91.7% and 89.6% respectively. 30

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Acinetobacter baumannii has emerged as an important nosocomial pathogen, 50

impacting considerably on patient care (6, 9). In many parts of the world, strains 51

resistant to almost all available antimicrobial classes (multidrug-resistant 52

Acinetobacter baumannii, MDRAB) have been reported and implicated in numerous 53

hospital outbreaks, usually in intensive therapy units (ITU) (2). Despite the 54

implementation of infection control measures involving isolation of patients, rigorous 55

hand hygiene and aggressive environmental decontamination, many centers have 56

continued to have ongoing problems, with rapid re-emergence of the organism 57

unless high standards of cleaning are maintained (4, 10). 58

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Although environmental cleaning is of paramount importance, it is likely that patients 60

themselves also represent a significant reservoir of the organism. For many Gram-61

negative bacteria colonization of the digestive tract is an important pre-requisite for 62

the development of nosocomial infections such as ventilator-associated pneumonia 63

(3, 7). Rapid identification of individuals with enteric carriage of MDRAB could 64

therefore be an important component in strategies aimed at limiting the transmission 65

and dissemination of the organism. 66

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CHROMagar Acinetobacter (CHROMagar, Paris, France) is a recently developed 68

selective agar for the rapid identification of MDRAB. It contains agents which inhibit 69

the growth of most Gram-positive organisms as well as carbapenem susceptible 70

Gram-negative bacilli. It incorporates substrates enabling color based preliminary 71

identification of colonies recovered within 18-24 hours of inoculation. In this study we 72

evaluated the ability of CHROMagar Acinetobacter to detect the carriage of MDRAB 73

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in enteric samples from critically ill patients in a unit experiencing problems with 74

MDRAB, compared with a molecular method. 75

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CHROMagar Acinetobacter was prepared from dehydrated powder according to the 77

manufacturer’s instructions. Each batch of media was quality controlled using a 78

representative isolate of the MDRAB OXA-23 clone-1, obtained from the Health 79

Protection Agency, Colindale, UK, previously characterized as resistant to 80

carbapenems (2). Stool samples and / or perineal swabs collected from ITU patients 81

as part of routine screening for Clostridium difficile associated diarrhea or meticillin 82

resistant Staphylococcus aureus (MRSA) carriage were used to inoculate 83

CHROMagar Acinetobacter plates and then 3 ml peptone water broths. Plates were 84

incubated in air at 37 °C and examined after 24 hours for aqua blue colonies 85

indicative of MDRAB (figure 1). The peptone broths were also incubated for 24 hours 86

and used to generate templates for a MDRAB specific molecular assay. 100 µl of 87

turbid broth was heated at 96 °C for 5 min and then centrifuged at 13, 000 rpm to 88

pellet lysed bacterial cells. 2 µl of supernatant was used in a multiplex PCR for the A. 89

baumannii species specific OXA-51-like gene and the OXA-23, -24 and -58-like 90

genes, which mediate carbapenem resistance, using the primers and conditions 91

described by Woodford et al (11). Isolates presumed to be A. baumannii based on 92

their growth and appearance on CHROMagar Acinetobacter were confirmed as such 93

by Gram staining, oxidase testing and multiplex PCR assay. The clonal lineage of 94

each isolate was also determined using another multiplex PCR assay, based on 95

sequence variations in the csuE, ompA and blaOXA-51 alleles. This enables strains to 96

be assigned to clonal complexes with a reliability equivalent to that obtained using 97

pulsed field gel electrophoresis (8). 98

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Sixty-six stool samples and 37 perineal swabs obtained from a total of 70 patients 99

were ultimately included in the analysis. Thirty-three of these were positive by both 100

culture and PCR on peptone broths (table 1). Seven were culture positive but PCR 101

negative whilst 3 were found to be culture negative yet positive by PCR. The gene 102

profiles detected in the PCR assay were identical in every case: OXA-51 +ve, OXA-103

23 +ve, OXA-24 –ve, OXA-58 –ve. 104

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Biochemical and molecular testing of colonies identified as MDRAB using 106

CHROMagar Acinetobacter confirmed that all of the organisms were A. baumannii 107

resistant to β-lactams (including carbapenems), quinolones and aminoglycosides. 108

Multiplex PCR typing placed them as members of European clone II. The only other 109

organisms recovered from samples inoculated onto CHROMagar Acinetobacter were 110

Enterococcus spp which were easily distinguishable by their dark blue color and 111

small colonial morphology. 112

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Of the 40 patients with positive MDRAB cultures, 33 were known to be colonized at 114

other sites. In 7 patients MDRAB was isolated from stool alone and not from any 115

other clinical specimens. The sensitivity and specificity of culture compared with the 116

PCR assay were calculated as 91.7% and 89.6% respectively. 117

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For the 7 samples found to be culture positive using CHROMagar Acinetobacter, but 119

negative in PCRs performed on peptone broths, follow up testing of the colonies 120

confirmed that these were all MDRAB. In 6 of these the patients had already had 121

MDRAB isolated from other clinical specimens. As the broths were inoculated after 122

the CHROMagar plates it is possible that either insufficient material remained on the 123

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swabs or, in the case of the stool samples, there may have been significant inhibition 124

of the PCR reaction. 125

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Our findings support the use of CHROMagar Acinetobacter as a means of identifying 127

enteric carriage of MDRAB, but are limited by the relatively small numbers and the 128

involvement of a single MDRAB clone. Also, as the stool samples were those sent 129

for C. difficile testing, only semi-formed or liquid stools were obtained. The use of the 130

media with formed stools would therefore require further evaluation. A comparison 131

between stool and perineal swab cultures would also help to determine whether 132

perineal swabs can be used as a proxy marker of gut carriage. These are easier to 133

process and are already routinely obtained to screen for other nosocomial pathogens 134

in many clinical settings. 135

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The study also highlights a role for the gut as a significant reservoir for MRAB in 137

addition to environmental contamination. It raises the question of whether selective 138

decontamination of the digestive tract (SDD) using non-absorbable antibiotics such 139

as polymyxin B, to which MDRAB remain susceptible, could be a suitable method of 140

control. The SDD approach has been shown to be effective in reducing the carriage 141

and incidence of nosocomial infections caused by Gram-negative bacteria but has 142

not been widely adopted (1, 5). The availability of a selective culture media such as 143

CHROMagar Acinetobacter, enabling those who may benefit from such a regimen to 144

be rapidly identified, is clearly an important step towards addressing this issue. 145

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Acknowledgments 149

This study was presented in part at the 19th European Congress of Clinical 150

Microbiology and Infectious Diseases, Helsinki, Finland. May, 2009 151

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Figure 1. Appearance on MDRAB on CHROMagar Acinetobacter plates 245

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Table 1: Comparison of culture and molecular tests for the identification of MDRAB 259

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Culture PCR No. of specimens Known Colonized

Negative Negative 60 20

Positive Positive 33 28

Positive Negative 7 6

Negative Positive 3 1

Total 103 55

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