Dio-sensimedia: a novel culture medium for rapid detection of extended spectrum β-lactamases

Background Resistance to contemporary broad-spectrum β-lactams, mediated by extended-spectrum β-lactamases (ESBL), is an increasing problem worldwide. Many of the emerging antimicrobial resistance problems of this decade have been characterized by difficulty in the recognition of resistance in the laboratory, particularly by rapid susceptibility test methods. The plasmid-encoded ESBL represent such a resistance phenomenon that is difficult to recognize. We compared Dio-Sensimedia-ES (DSM-ES; Diomed, Istanbul, Turkey) and Mueller-Hinton (MH) agar in the double-disk synergy test (DDST) as a novel rapid system for detecting ESBL directly from bacterial culture. Methods Sixty ESBL-producing Klebsiella pneumoniae isolates cultured from blood (30), endotracheal aspirates (20), urine (5) and pus (5), as well as 40 Escherichia coli isolates cultured from endotracheal aspirates (15), urine (10), blood (8) and pus (7) were studied. Isolates positive for ESBL by the combined disk tests were tested with the DDST using MH and DSM-ES agar to detect ESBL-mediated resistance in K. pneumoniae and E. coli. DSM-ES agar was also used to determine the susceptibility of Enterobacteriaceae and staphylococci. Results Among 60 ESBL-producing K. pneumoniae isolates, 59 (98.3%) were identified as ESBL-positive by the DDST using MH, and 58 (96.6%), using DSM-ES agar. Of 40 ESBL-producing E. coli isolates, 38 (95%) were ESBL-positive by the DDST on MH agar, and 37 (92.5%), on DSM-ES agar. The average incubation period required for ESBL detection by the DDST on DSM-ES agar was 4 hours. Conclusions Since the DDST results were available within 4 hours when DSM-ES agar was used, the use of this media may significantly lower the length of hospital stay, the total cost for patient care and even the mortality rate by fascilitating early treatment against ESBL-producing organisms.


Background
Microbial resistance through extended-spectrum β-lactamases (ESBL) was first reported in Europe, and subsequently in the United States. Today this resistance mechanism has been recognized globally [1][2][3][4][5][6]. Many clinical microbiology laboratories have problems to detect ESBL-mediated resistance. Controversy exists regarding the clinical importance of such resistance, the choice of optimal laboratory methods to detect it, and surveillance of ESBL-producing organisms. Failure to detect ESBL-mediated resistance has contributed to uncontrolled spread of ESBL-producing organisms and related treatment failures.
The ESBL-producing Gram-negative bacilli possess genes encoding more than one type of the ESBL and enzymes that are responsible for resistance to other antibiotics such as aminoglycosides and fluoroquinolones that are active against Gram-negative bacilli [14,15]. The emergence of multidrug resistance in these virulent pathogens has significantly hampered the efforts to devise effective empiric or directed antibiotic treatment regimens [16].
In our study, we report a novel rapid system for detecting the presence of ESBL directly from microbiological cultures. DSM (Diomed, Istanbul, Turkey) enables observation of the inhibition zones within 4 hours by changing its color in response to the metabolic activity of growing bacteria, even before the bacterial layer has had an opportunity to grow. DSM is an antibacterial susceptibility testing agar media, which is poured in Petri dishes and used to determine susceptibility to antibacterials by the disk diffusion method. The use of this medium in disk diffusion technique is analogous to an improved, chromogenic version of the Kirby-Bauer disk diffusion method.

Bacterial strains and culture media
Sixty ESBL-producing K. pneumoniae isolates cultured from blood (30), endotracheal aspirates (20), urine (5), and pus (5) as well as 40 E. coli isolates cultured from endotracheal aspirates (15), urine (10), blood (8), and pus (7) were studied. The isolates were non-repetitive. All isolates were cultured from patients hospitalized in an intensive care unit at Istanbul Faculty of Medicine, Istan- Modified criteria, i.e., a diameter of <22 mm for ceftazidime; <25 mm, for ceftriaxone; and <27 mm, for cefotaxime or aztreonam were used for interpretation of Kirby-Bauer disk zone diameters. In this study, a combination of ceftazidime (30 µg) and clavulanic acid (10 µg) was used. Zones around K. pneumoniae or E. coli colonies, which were larger than that around the disk containing ceftazidime-clavulanic acid, were deemed to indicate ESBL-positivity.
DMS-ES agar was also used to determine susceptibility of Enterobacteriaceae and staphylococci. This media changes its color from red to yellow as bacteria grow and red circular inhibition zones forms around disks containing the antibacterial agent. All isolates that were positive for ESBL by the combined disk tests were tested with the DDST on Mueller-Hinton (MH) and DSM-ES agar to detect ESBLmediated resistance. E. coli ATCC 25922 and K. pneumoniae ATCC 13883 were used as negative controls.

Double-disk synergy test
The detection of ESBL-mediated resistance by the DDST was performed according to a published protocol [11,12]. Bacterial suspensions were prepared from overnight cultures of clinical isolates to produce a turbidity of a 0.5 McFarland standard. These suspensions were then spread on the surface of MH and DSM-ES agar plates as in the Kirby-Bauer disk diffusion technique. Antibiotic susceptibility disks containing amoxicillin (20 µg) plus clavulanate (10 µg) were placed on the centre of Petri dishes containing the two different media. Ceftazidime (30 µg), ceftriaxone (30 µg), cefpodoxime (10 µg), aztreonam (30 µg), cefotaxime (30 µg) disks were placed 25-30 mm apart circularly around the co-amoxiclav disk. The MH agar plates were incubated for 24 hours at 35°C. DSM-ES agar that is originally red, changed its color to yellow as bacterial culture grew (Fig. 1). DSM-ES agar plates were incubated at 35°C until red inhibition zones became apparent. Red inhibition zones around disks containing antibacterials were visually observed and measured. When the disk containing co-amoxiclav extended to any of the other antibiotic disk inhibition zones, ESBL production was inferred [11,12] (Fig. 2).

Evaluation of the susceptibilities by two methods
Determination of ESBL production using was done by using both DSM-ES and Mueller-Hinton agar. The results of the DDST using the two media were compared.
All the test results were read and interpreted by one of the authors [AAC], who was blinded to the results of microbiological typing.

Results
Among 60 ESBL-producing K. pneumoniae isolates, 59 (98.3%) were identified as ESBL-positive by the DDST on MH agar; and 58 (96.6%), on DSM-ES agar ( Table 1). The average incubation period required for detecting ESBLmediated resistance when using DSM-ES agar was as short as 4 hours.

Discussion
ESBL are encoded on conjugative plasmids, tranposons or integrons. These mobile genetic elements are readily spread under selective antibiotic pressure [21]. Increased clinical use of antibiotics, access to antibiotics without a doctor's prescription in many countries, international travel, and uncontrolled use of antibiotics in the environment and by meat-producers have led to an increase in antibiotic resistance in many bacterial species [22]. Early determination of ESBL-mediated resistance is clinically crucial in cases like meningitis, bacteremia and sepsis in order to start appropriate therapies as early as possible. Even in less emergent situations, early determination of antibacterial susceptibility is important to select the appropriate treatment regimens and increase the success rate of the therapy, lower the rate of side-effects of antibiotics, and decrease health-care costs. Although the DDST detected K. pneumoniae more frequently than E. coli, this test was equally efficent to detect ESBL-mediated resistance in both species. The average incubation period required to detect ESBL-mediated resistance in both species was approximately 4 hours. In this study, the DDST on DSM-ES agar proved to be a reliable, rapid and cost-effective test for detecting of ESBL in K. pneumoniae and E. coli. Since the DDST results can be obtained within 4 to 6 hours when DSM-ES agar is used, the use of this media may significantly lower the length of hospital stay, the total cost for patient care and even the mortality rate by fascilitating early treatment.