JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: , Volume 2, Issue 8, September 2014

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1 EVALUATION OF CHROMAGAR-CTX FOR THE DETECTION OF CTX-M-ESBL- PRODUCING GRAM NEGATIVE BACTERIAL ISOLATES RASHA H. ELSHERIF* LAMIAA A. MADKOUR** REHAM A. DWEDAR*** *Clinical Pathology Department, Faculty of Medicine, Cairo University, Egypt **Microbiology and Immunology Department, Faculty of Medicine, Cairo University, Egypt ***Microbiology and Immunology Department, Faculty of Medicine, Cairo University, Egypt ABSTRACT The present study was performed to determine the sensitivity and specificity of CHROMagar- CTX in the detection of CTX-M producing Gram negative isolates, as well as to determine the prevalence of individual β-lactamase genes in Kasralainy Hospitals. Gram-negative isolates identified as potential ESBL producers by the double disc synergy test were inoculated onto CHROMagar-CTX. In addition, PCR was performed to identify various β- lactamase genes found in these isolates. CHROMagar-CTX revealed a sensitivity of 44% and a specificity of 48% compared to PCR. SHV-1 was the most predominant β-lactamase gene, while TEM-1 + SHV-1 was the most prevalent gene combination. The study highlights the importance of rapid cost-effective screening methods for ESBL detection. CHROMagar-CTX requires further evaluation on larger panels of isolates. KEYWORDS: Chromagar, Enterobacteriaceae, β-lactamase, ESBL INTRODUCTION With an alarming increase in multi-drug resistant pathogens, the β-lactamase-harboring organisms have posed a major threat to public health as well as to infection control practices 9. Resistance to the β-lactam antibiotics is mediated by β-lactamases and - to less extent - by alterations of drug efflux pumps. Functionally, the β-lactamases are divided into 4 groups; penicillinases, extended spectrum β-lactamases (ESBLs), carbapenemases and Amp-C cephalosporinases 9. Of these, ESBLs - which are encoded by mobile genes - confer resistance to penicillins, cephalosporins, monobactams and related oxyimino β-lactams 8. Most ESBLs belong to the CTX-M, SHV (Sulfhydryl variable) and TEM (Temoniera) families 15. The rising incidence of ESBL-harboring microorganisms has led to a worrisome increase in the use of carbapenems, which may, eventually; result in pan-resistant organisms Consequently, several tests have been developed to detect ESBL-producers; including the double disc synergy test where ESBL producers, in the presence of a β-lactamase inhibitor

2 (e.g. clavulanic acid); show enhanced susceptibility to a cepholsporin or monobactam 9. The ESBL E-tests have also been proposed; but being costly, they are impractical in routine laboratories. Although these tests are undoubtedly useful; yet, the presence of KPCs and Amp-C cephalosporinases may result in false-positive and negative results respectively. In addition, automated methods may be sensitive in ESBL detection; yet, their use is limited due to the high cost and the need for specialized equipment and expertise 9. Thus, the rising diversity of ESBLs, together with the overlapping of non-esbl resistance mechanisms has made the identification of ESBL producers a pressing challenge 7. On the other hand, chromogenic culture media are considered rapid methods that allow both ESBL detection and presumptive organism identification 9. Hence, the current study was performed to evaluate CHROMagar-CTX, for detection and presumptive identification of Gram negative microorganisms harboring CTX-M-ESBL, as well as to identify the prevalent genes coding for β-lactamases at Kasralainy Hospitals in Egypt. Subjects and Methods Clinical bacterial isolates A total of 170 Gram-negative bacterial isolates, identified as ESBL by both the disc diffusion method and the confirmatory double disc synergy test (DDST), were incorporated in this study. The specimens were collected from the ICU ward in Kasralainy Hospitals, Cairo University, in the period from May 2013 to September The clinical isolates were isolated from the following samples: 59 isolates from urine samples, 39 from respiratory samples, 31 from wound swabs, 24 from blood cultures, 16 from pus samples and one isolate from ascitic fluid aspirate. The isolates were identified on the basis of conventional microbiological procedures; including colony morphology on blood agar and MacConkey's agar (Mast Diagnostics, Merseyside, UK), Gram's staining and biochemical reactions. Antimicrobial susceptibility was determined by the Kirby-Bauer disc diffusion method as per the CLSI recommendations 5. ESBL CLSI screening test Isolates showing an inhibition zone of 22 mm with ceftazidime (30 μg), 25 mm with cefriaxone (30 μg) and 27 mm with cefpodoxime (30 μg) were identified as potential ESBL producers 5. All discs were obtained from Mast Diagnostics, Merseyside, UK. None of the isolates was resistant to cefoxitin disc (30 μg). 152

3 E. coli ATCC and P. aeruginosa ATCC were used as quality control strains. Interpretative criteria for each tested antimicrobial were those recommended by the CLSI. Double disc synergy test for ESBL confirmation As described by Jarlier et al. 12, synergy was determined between a disc of amoxicillinclavulanate (20 μg/10 μg) (augmentin) and a 30-μg disc of third-generation cephalosporin (Mast Diagnostics, Merseyside, UK). The discs were placed at a distance of 20 mm from center to center on a Mueller-Hinton Agar (MHA) plate swabbed with the test isolate. An extension of the edge of the inhibition zone of cephalosporin toward the augmentin disc was interpreted as positive for ESBL production 4,12. CHROMagar-CTX The 170 potential ESBL producers were tested with CHROMagar. CHROMagar ECC was used as the basal medium to which the supplement CHROMagar CTX (CHROMagar, France) was added 13. Different suspected ESBL strains were streaked onto CHROMagar- CTX and incubated at 37 C for hours. Interpretation of the outcome was as follows; E. coli CTX-M appear as blue colonies, other Enterobacteriacae CTX-M appear as mauve colonies. On the other hand, Gram positive strains and non-ctx-m ESBL Gram negative strains are inhibited. Some Acinetobacter spp. and Pseudomonas spp., globally-known to be frequently multi-drug resistant, could grow on the medium as colorless colonies. Genotyping of the clinical isolates Genotypic identification of β-lactamases with PCR (Thermo Scientific GeneJet Mini Kit, Sigma Aldrich) was done using primers (Table 1) targeting the blactx-m, blatem-1, blashv-1 and blaoxa genes 14. Statistical methodology: Data were statistically described in terms of percentages when appropriate. Accuracy was represented using the terms sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy. All statistical calculations were done using computer programs SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 17 for Microsoft Windows. 153

4 Results: The present study included 170 Gram-negative bacterial isolates that were presented to the Microbiology Laboratory in the period from May 2013 to September Among the 170 suspected ESBL-producers, Klebsiella spp. was the most common pathogen isolated from tested samples, constituting 61/170 (35.8%) of the total isolates, followed by Pseudomonas aeruginosa 37/170 (21.7%), Escherichia coli 36/100 (21%) and Acinetobacter baumanii 28/170 (16.4%). The prevalence of various β-lactamase genes among the different bacterial isolates is shown in Figure 1 and table 2. It can be noted that SHV-1 was the predominant gene, followed by TEM-1. The most common combination of β-lactamase genes was TEM-1 + SHV-1 followed by CTX-M + SHV-1. No strain was positive for all the four tested genes. Using PCR, the different clinical samples exhibited variable distribution of β-lactamase genes; with urine samples showing the highest prevalence followed by the respiratory specimens (Table 3). Polymerase chain reaction was used to confirm the presence of β-lactamase genes (CTX- M, OXA, TEM-1 and SHV-1). PCR results are compared to those of DDST in Table 4. On CHROMagar-CTX, E. coli yielded blue colonies, while other Enterobacteriaceae yielded mauve colonies (Figure 2). On the other hand, Acinetobacter and Pseudomonas yielded colorless colonies (Figure 3). Remarkably, the results of CHROMagar-CTX revealed great discrepancy with those of PCR, as shown in tables 5, 6 and 7 as well as Figure 4. Discussion: The prevalence of β-lactamase-producers among Gram-negative bacteria has been rising worldwide. In the current study, β-lactamase genotypes showed high prevalence of SHV-1 gene. However, a study from Turkey 2 and a study from Iran 16 identified TEM-1 as the most prevalent gene. On the other hand, a study from Saudi Arabia revealed that CTX-M was the most prevalent gene 11. This proves that the distribution of β-lactamase genes can vary according to countries and institutions, although the prevalence of β-lactamase-producers is a global problem 6. As previously reported 10,11, our study showed that the majority of ESBL β-lactamase producers were recovered from urine samples. 154

5 Moreover; the occurrence of more than one ESBL β-lactamase gene in a single isolate has been increasingly detected 1,3. In our study, the most common ESBL β-lactamase gene combination was TEM-1 + SHV-1 followed by CTX-M + SHV-1. In a study by Hassan & Abdalhamid 11, the most prevalent combination was CTX-M + SHV. However, in a study by Harada et al. 10 the coexistence of TEM and CTX-M-9 was the most common combination. Considering the alarming rise of ESBLs and the specific importance of CTX-M-ESBL, the development of screening media for rapid ESBL detection has become the need of the time. Such media could provide simultaneous ESBL detection as well as identification of the causative organism 13. A chromogenic medium; CHROMagar CTX was evaluated in the present study. Strikingly, it showed a sensitivity of 44% and a specificity of 48%. However; in a study by Randall et al. 13, CHROMagar-CTX showed a sensitivity of 100% and a specificity of 64.2%. A chromogenic ESBL screening medium would be of great value for epidemiological studies, and more importantly, for optimizing therapeutic strategies. When ideally the screening medium should be 100% sensitive and 100% specific, this remains an unrealistic goal. The CHROMagar CTX requires further evaluation on larger panels of isolates. Author disclosure statement: No competing interests exist References: 1. Al-Agamy MH, Shibl AM, Tawfik AF Prevalence and molecular characterization of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in Riyadh, Saudi Arabia. Ann Saudi Med; 29: Bali EB, Acik L & Sultan N Phenotypic and molecular characterization of SHV, TEM, CTX-M and extendedspectrum beta lactamase produced by Escherichia coli, Acinobacter baumannii and Klebsiella isolates in a Turkish hospital. Afr J Microbiol Res; 4(8), Bindayna K, Khanfar HS, Senok AC, et al Predominance of CTX-M genotype among extended spectrum beta lactamase isolates in a tertiary hospital in Saudi Arabia. Saudi Med J; 31: Bradford PA Extended-spectrum beta-lactamases in the 21 st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev; 14: Clinical and Laboratory Standards Institute (CLSI) Performance standards for antimicrobial susceptibility testing; Tenth Edition, Wayne PA. 6. Coque TM, Baquero F, Canton R Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill; Drieux L, Brossier F, Sougakoff W, et al Phenotypic detection of extended-spectrum beta-lactamase production in Enterobacteriaceae: review and bench guide. Clin Microbiol Infect; 14(Suppl. 1): Emery CL, Weymouth LA Detection and clinical significance of the extendedspectrum β-lactamases in a tertiary care medical center. J Clin Microbiol; 35:

6 9. Gazin M, Paasch F, Goossens H, et al Current trends in culture-based and molecular detection of extended-spectrum-β-lactamase-harboring and carbapenem-resistant Enterobacteriaceae. J Clin Microbiol; 50(4), Harada Y, Morinaga Y, Yamada K, et al Clinical and molecular epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli in a Japanese tertiary hospital. J Med Microb Diagn; 2(127), Hassan H, Abdalhamid B Molecular characterization of extended-spectrum betalactamase producing Enterobacteriaceae in a Saudi Arabian tertiary hospital. JIDC; 8 (3). 12. Jarlier V, Nicolas MH, Fournier G, et al Extended broad-spectrum-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis; 10: Randall LP, Kirchner M, Teale CJ, et al Evaluation of CHROMagar CTX, a novel medium for isolating CTX-M-ESBL-positive Enterobacteriaceae while inhibiting AmpCproducing strains. J Antimicrob Chemother; 63: Sana T, Rami K, Racha B, et al Detection of genes TEM, OXA, SHV and CTX-M in 73 clinical isolates of Escherichia coli producers of extended spectrum Beta-lactamases and determination of their susceptibility to antibiotics. IAJAA;1(1). 15. Thomson KS Extended-spectrum-β-lactamase, AmpC, and carbapenemase issues. J Clin Microbiol; 48(4), Zaniani FR, Meshkat Z, Nasab MN et al The prevalence of TEM and SHV genes among extended-spectrum beta-lactamases producing Escherichia coli and Klebsiella pneumoniae. IJBMS; 15(1), 654. Tables Table 1: Primer sequences and PCR cycles for the tested genes 14 : Primer Primer sequences PCR cycles CTX-M1-A2 CTX-M1-B2 CTT CCA GAA TAA GGA ATC CCG TTT CCG CTA TTA CAA 1 cycle of 10 minutes (min) at 94 o C, 30 cycles of [1min at 94 o C, 1 min at 48 o C, 1min at 72 o C], 1 cycle of 7 min at 72 o C. TEM-1/F TEM-1/R ATG AGT ATT CAA CAT TTC CG CTG ACA GTT ACC AAT GCT TA 1 cycle of 5 min at 96 o C; 35 cycles of [1 min at 96 o C, 1 min at 58 o C,1 min at 72 o C]; 1 cycle of 10 min at 72 o C. SHV-1/F SHV-1/R GGT TAT GCG TTA TAT TCG CC TTA GCG TTG CCA GTG CTC 1 cycle of 5 min at 96 o C; 35 cycles of [1min at 96 o C, 1min at 60 o C, 1 min at 72 o C; 1 cycle of 10 min at 72 o C]. OXA-1/F OXA-1/R ACA CAA TAC ATA TCA ACT TCG C AGT GTG TTT AGA ATG GTG ATC 1 cycle 5min at 96 o C; 35 cycles of [1min at 96 o C, 1min at 60 o C, 2min at 72 o C]; 1 cycle of 10 min at 72 o C. 156

7 Table 2: Prevalence of β-lactamase genes among different isolates as detected by PCR. Isolate Klebsiella Klebsiella Pseud. E. Acinetobacter Enterob Proteus Proteus Total pneumonia oxytoca aeruginosa coli baumanii acter mirabilis vulgaris No. of strains CTX-M No ve % OXA No ve % TEM-1 No ve % SHV-1 No ve % CTX-M No & SHV +ve % OXA &TEM- 1 +ve OXA & SHV-1 +ve TEM-1 & SHV- 1 +ve No % No % No % All -ve No % No. = Number Table 3: Prevalence of β-lactamase genes among different clinical specimens Specimen CTX-M +ve OXA +ve TEM-1 +ve SHV-1 +ve Number Percent (%) Number Percent (%) Number Percent (%) Number Percent (%) Urine Respiratory specimens Pus and wound swabs Blood Ascitic fluid Total Table 4: Comparison between DDST and PCR in the detection of β-lactamase genes Methods PCR Total - + DDST Total

Table 5: Comparison between CHROMagar-CTX and PCR in the detection of CTX-M harboring isolates Methods PCR for CTX-M Total - + Growth on CHROMagar- - 74 10 84 CTX + 78 8 86 Total 152 18 170 Table 6:

8 Table 5: Comparison between CHROMagar-CTX and PCR in the detection of CTX-M harboring isolates Methods PCR for CTX-M Total - + Growth on CHROMagar CTX Total Table 6: Comparison between CHROMagar-CTX and PCR in the detection of non-ctx-m β- lactamase -producers Methods PCR for other β-lactamase genes Total - + Growth on CHROMagar CTX Total Table 7: Parameters of CHROMagar-CTX compared to PCR Parameter Value Percent (%) Sensitivity Specificity Positive predictive value Negative predictive value Accuracy Prevalence Figures: Fig. 1: Ethidium bromide-stained agarose gel showing amplicons obtained when DNA was amplified with SHV-1, TEM-1, CTX-M and OXA primers using PCR 158

9 A B Fig. 2: Blue colonies of E. coli (A) and mauve colonies of Klebsiella (B) on CHROMagar-CTX Fig. 3: Colorless colonies of Acinetobacter on CHROMagar-CTX Fig. 4: Parameters of CHROMagar-CTX 159

SUMMARY. Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M,

SUMMARY. Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M, SUMMARY Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M, The doctoral thesis entitled Prevalence of Enterobacteriaceae producing extendedspectrum beta-lactamases (ESBL) isolated from broilers