![Salimian Rizi K et al.
Avicenna J Clin Microb Infec. 2015;2(3):e296914
ment of bacteriology, TMU, and medical laboratory de-
partment of hospitals in Tehran, Iran.
Authors’ Contributions
Kobra Salimian Rizi performed the laboratory work.
Shahin Najar Peerayeh designed the work study, and Bita
Bakhshi advised the work study.
Funding/Support
This study was supported by a grant from Tarbiat
Modares University, Faculty of Medical Sciences, Tehran,
IR Iran.
References
1. Mezzatesta ML, Gona F, Stefani S. Enterobacter cloacae complex:
clinical impact and emerging antibiotic resistance. Future Micro-
biol. 2012;7(7):887–902.
2. Sanders WEJ, Sanders CC. Enterobacter spp.: pathogens
poised to flourish at the turn of the century. Clin Microbiol Rev.
1997;10(2):220–41.
3. Levison ME, Mailapur YV, Pradhan SK, Jacoby GA, Adams P, Em-
ery CL, et al. Regional occurrence of plasmid-mediated SHV-7, an
extended-spectrum beta-lactamase, in Enterobacter cloacae in
Philadelphia Teaching Hospitals. Clin Infect Dis. 2002;35(12):1551–4.
4. Maynard C, Bekal S, Sanschagrin F, Levesque RC, Brousseau R,
Masson L, et al. Heterogeneity among virulence and antimi-
crobial resistance gene profiles of extraintestinal Escherichia
coli isolates of animal and human origin. J Clin Microbiol.
2004;42(12):5444–52.
5. Livermore DM, Canton R, Gniadkowski M, Nordmann P, Rosso-
lini GM, Arlet G, et al. CTX-M: changing the face of ESBLs in Eu-
rope. J Antimicrob Chemother. 2007;59(2):165–74.
6. Boyd DA, Tyler S, Christianson S, McGeer A, Muller MP, Willey
BM, et al. Complete nucleotide sequence of a 92-kilobase plas-
mid harboring the CTX-M-15 extended-spectrum beta-lactamase
involved in an outbreak in long-term-care facilities in Toronto,
Canada. Antimicrob Agents Chemother. 2004;48(10):3758–64.
7. Coque TM, Novais A, Carattoli A, Poirel L, Pitout J, Peixe L, et al.
Dissemination of clonally related Escherichia coli strains ex-
pressing extended-spectrum beta-lactamase CTX-M-15. Emerg
Infect Dis. 2008;14(2):195–200.
8. Roopa TJ, Sudha SS. Antimicrobial susceptibility of extended
spectrum beta –lactamase (ESBL) producing uropathogens iso-
lated from ICU patients. Int J Biological Technology. 2010;1:23–31.
9. Szabo D, Bonomo RA, Silveira F, Pasculle AW, Baxter C, Linden PK,
et al. SHV-type extended-spectrum beta-lactamase production is
associated with Reduced cefepime susceptibility in Enterobacter
cloacae. J Clin Microbiol. 2005;43(10):5058–64.
10. Aibinu IE, Ohaegbulam VC, Adenipekun EA, Ogunsola FT, Odug-
bemi TO, Mee BJ. Extended-spectrum beta-lactamase enzymes
in clinical isolates of Enterobacter species from Lagos, Nigeria.
J Clin Microbiol. 2003;41(5):2197–200.
11. CLSI.. Performance standards for antimicrobial susceptibility testing,
20th information supplement (M100-S20).: Clinical and Laboratory
Standards Institute; 2012.
12. Mendonca N, Louro D, Castro AP, Diogo J, Canica M. CTX-M-15,
OXA-30 and TEM-1-producing Escherichia coli in two Portuguese
regions. J Antimicrob Chemother. 2006;57(5):1014–6.
13. Harajly M, Khairallah MT, Corkill JE, Araj GF, Matar GM. Frequen-
cy of conjugative transfer of plasmid-encoded ISEcp1 - blaCTX-
M-15 and aac(6')-lb-cr genes in Enterobacteriaceae at a tertiary
care center in Lebanon - role of transferases. Ann Clin Microbiol
Antimicrob. 2010;9:19.
14. Hausner M, Wuertz S. High rates of conjugation in bacterial bio-
films as determined by quantitative in situ analysis. Appl Environ
Microbiol. 1999;65(8):3710–3.
15. Phornphisutthimas S, Thamchaipenet A, Panijpan B. Conjuga-
tion in Escherichia coli: A laboratory exercise. Biochem Mol Biol
Educ. 2007;35(6):440–5.
16. Iabadene H, Messai Y, Ammari H, Ramdani-Bouguessa N, Lounes
S, Bakour R, et al. Dissemination of ESBL and Qnr determinants
in Enterobacter cloacae in Algeria. J Antimicrob Chemother.
2008;62(1):133–6.
17. Mirsalehian A, Akbari NF, Peymani A, Kazemi B, Jabal A, Mirafshar
SM. Prevalence of extended spectrum beta-lactamase-producing
Enterobacteriaceae by phenotypic and genotypic methods in in-
tensive care units in Tehran, Iran. Daru. 2008;16(3):169–73.
18. Kasap M, Fashae K, Torol S, Kolayli F, Budak F, Vahaboglu H. Char-
acterization of ESBL (SHV-12) producing clinical isolate of Entero-
bacter aerogenes from a tertiary care hospital in Nigeria. Ann
Clin Microbiol Antimicrob. 2010;9:1.
19. Morand PC, Billoet A, Rottman M, Sivadon-Tardy V, Eyrolle L,
Jeanne L, et al. Specific distribution within the Enterobacter
cloacae complex of strains isolated from infected orthopedic
implants. J Clin Microbiol. 2009;47(8):2489–95.
20. Huang ZM, Shan H, Mi ZH, Yang HY, Wu L, Chu QJ, et al. [Analysis
on 168 rRNA methylase genes and aminoglycoside modifying
enzymes genes in Enterobacter cloacae in China]. Zhonghua Liu
Xing Bing Xue Za Zhi. 2008;29(4):369–73.
21. Jabeen K, Zafar A, Hasan R. Frequency and sensitivity pattern
of Extended Spectrum beta Lactamase producing isolates in
a tertiary care hospital laboratory of Pakistan. J Pak Med Assoc.
2005;55(10):436–9.
22. Amin H, Zafar A, Ejaz H, Jameel NU. Phenotypic characterization
of ESBL producing Enterobacter cloacae among children. Pak J
Med Sci. 2013;29(1):144–7.](https://image.slidesharecdn.com/d63ff183-5954-4c51-b7a3-3af856566733-150925104103-lva1-app6892/85/ajcmi-02-03-29691-edited-by-SC-4-320.jpg)
ajcmi-02-03-29691 edited by SC
- 1. Avicenna J Clin Microb Infec. 2015 August; 2(3): e29691. DOI: 10.17795/ajcmi-29691 Published online 2015 August 23. Research Article Emergence of blaCTX-M-15 Gene and Its Transferability in Enterobacter spp. IsolatedFromtheHospitalsof Tehran,Iran Kobra Salimian Rizi 1,* ; Shahin Najar Peerayeh 1 ; Bita Bakhshi 1 ; Mohammad Rahbar 2 1 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran 2 Department of Microbiology, Iranian Reference Health Laboratory, Deputy of Health, Ministry of Health and Medical Education, Tehran, IR Iran *Corresponding author: Kobra Salimian Rizi, Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran. Tel: +98-9382657837, Fax: +98-1288006544, E-mail: alimian.k@gmail.com Received: May 11, 2015; Accepted: June 30, 2015 Background: Enterobacter spp. is increasingly recognized as an important nosocomial pathogen and implicated in many episodes of hospitalacquiredinfections. Objectives: The current study aimed to describe distribution and transferability of blaCTX-M-15 gene, and the antibiotic susceptibility pattern in theclinical isolates of Enterobacterspp. Materials and Methods: A total of 110 Enterobacter isolates were collected from five hospitals in Tehran, Iran from May 2012 to April 2013. EnterobacterspecieswereidentifiedbyAPI20Esystem.Antibacterialsusceptibilitywasdeterminedbydiskdiffusionmethod,andextended spectrum beta lactamase (ESBL) production was confirmed by combined disk method. The blaCTX-M-15 gene was identified by PCR with specific primers.Thetransferabilityof theblaCTX-M-15was testedbyconjugationwithbrothmattingmethod. Results: The prevalence of Enterobacter species was E. cloacae (78.2 %), E. aerogenes (6.13 %) and E. sakazakii (8.2%). They were from different clinical sources. Maximal resistance in Enterobacter isolates was noticed against Augmentin®, trimethoprim - sulfamethoxazole and cefoxitin 75.5%, 64.5%, and 59.1%, respectively. Fourteen isolates, showed ESBL phenotype. The blaCTX-M-15 gene frequency in Enterobacter isolates was 11.8%.Threeconjugativeplasmids containingblaCTX-M-15werefoundinoneEnterobacterisolate. Conclusions:ItwasthefirstreportontheblaCTX-M-15geneemergenceinclinicalEnterobacterspp.inIran.Thecurrentstudydemonstrated thepredominantpresenceof thegeneencodingCTX-M-15amongESBLproducingEnterobacterspp.commonlywithalargeplasmid,inthe setting. Keywords: Conjugation; Drug Resistance; ESBLs; Enterobacter Copyright © 2015, Hamadan University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non- Commercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages,providedtheoriginalworkisproperlycited. 1. Background The genus Enterobacter includes the facultative anaero- bic Gram-negative bacteria belonging to Enterobacte- riaceae family and widely found in the environment. Recently, the Enterobacter spp. has taken on clinical signif- icance and has emerged as nosocomial pathogens, espe- cially, from intensive care units (1, 2). Enterobacter species are significant causes of nosocomial infections and are intrinsically resistant to aminopenicillins, cefazolin and cefoxitin due to the production of constitutive chromo- somal AmpC β-lactamases (3). ESBLs (Extended-Spectrum Beta-Lactamases) are typi- cally inhibitor-susceptible β-lactamases that hydrolyze penicillins, cephalosporins and aztreonam and are mostly encoded by mobile genes. The most frequently encountered ESBLs belong to the CTX-M, SHV, and TEM families (4). In clinical strains, CTX-M-encoding genes are commonly located on plasmids which vary in size from 7 - 200 kb (5). Many of these plasmids are conjugative with transfer frequencies ranging from 10-2 - 10-7 (6). To date, more than 60 types CTX-M ESBLs belonging to five evo- lutionary groups are described. In most clinical isolates CTX-M-15 is the most frequent CTX-M type, and is reported in Enterobacteriaceae isolates from many regions of the world (7-9). It is necessary to know the frequency of ESBL positive strains in hospitals to formulate a policy of em- pirical therapy in high risk units where infections due to resistant organisms are much higher (8-10). 2. Objectives The current study aimed to describe the antibiotic sus- ceptibility pattern of the clinical isolates and blaCTX-M-15 gene distribution in clinical isolates of Enterobacter spp., and the transferability of the blaCTX-M-15 by conjugation. 3. Materials and Methods 3.1. Bacterial Isolates and Identification A total of 110 Enterobacter isolates were collected from hospitals in Tehran from May 2012 to April 2013. Isolates were identified by conventional methods or the API 20E system (bioMerieux, Inc., Hazelwood, MO).
- 2. Salimian Rizi K et al. Avicenna J Clin Microb Infec. 2015;2(3):e296912 3.2. Antibiotic Susceptibility Testing The antibiotic susceptibility of integron positive iso- lates, was determined by disk diffusion method on Muel- ler-Hinton agar plates (Merck, Darmstadt, Germany) as recommended by the Clinical Laboratory Standards Institute (CLSI) (11). The disks contained the following antibiotics (Mast, UK): Augmentin® (30 μg), imipenem (10 μg), co-trimoxazole (25 μg), tetracycline (30 μg), chlor- amphenicol (30 μg), ciprofloxacin (5 μg), aztreonam (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefoxitin (30 μg), tobramycin (10 μg), amikacin (30 μg), gentamicin (10 μg), and cefepime (50 µg). They were obtained from mast Pharmaceutical Inc. U.K. E.coli ATCC 25922 was used as control for antimicrobial susceptibility test. 3.3. ESBL Confirmation by Combination Disk Method The isolates showing reduced susceptibility to ceftazi- dime or cefotaxime were tested for ESBLs production by the combination disk method according to CLSI guide- lines (CLSI). Combination disk method was performed using four disks: cefotaxime (CTX) (30 μg), cefotaxime (30 μg) + clavulanic acid (10 μg), ceftazidime (CAZ) (30 μg), and ceftazidime (30 μg) + clavulanic acid (10 μg). A 5 mm increase in a zone diameter for the tested antimicrobial agent (CAZ or CTX) in combination with clavulanic acid versus its zone when tested alone was considered as ES- BLs positive. Quality control for ESBL production was per- formed using E. coli ATCC 25922 as negative control. Mini- mumInhibitoryConcentrations(MICs)of ceftazidimeand cefotaximeweredeterminedforESBLsisolatesbytheE-test (AB Biodisk, Solna, Sweden) according to CLSI guidelines. 3.4. Polymerase Chain Reaction Analysis The DNA of ESBL-producing isolates were extracted by boiling method and used as template in PCR assay. Amplification reactions were performed in a total vol- ume of 25 μL of reaction mixture containing 5 μL of 10 × PCR buffer, 2.5 mM Mgcl2, 200 mM dNTP, and 1.25 units of Taq polymerase, 10 pmol of each primer and 1 μL of the sample DNA. The following specific primers with 850 bp length were designed for the PCR reactions (F): 5’-AGAATAAGGAATCCCATGGTT and (R): 5’-GCAAGACCT- CAACCTTTTCC (12). Cycling conditions were as follows: Initial denaturation at 94°C for 5 minutes; 35 cycles of 94°C for 1minute, 55°C for 45 seconds, and 72°C for 1 min- ute followed by a final extension at 72°C for 7 minutes. Klebsiella pneumoniae TMU4 was used as positive control. 3.5. Conjugation Experiments The isolates with blaCTX-M-15 gene were used as donor strains in conjugation experiments. Conjugation transfer assay was performed in broth culture with E. coli 15ARr (cefotaxime sensitive and rifampicin resistant) as the re- cipient. Before conjugation transfer assay, donor strains were tested for sensitivity to rifampicin and resistance to cefotaxime on nutrient agar containing rifampicin (50 mg/mL) and cefotaxime (100 mg/mL). Donor and recipi- ent cells were mixed at a ratio of 1:10. The trans-conjugants were selected on nutrient agar containing cefotaxime (100 mg/mL) supplemented with rifampicin (50 mg/mL) (13). 3.6. Conjugation Frequency Conjugation frequency was also expressed as the per- centage of transconjugants per added donor cell in 1 mL (13-15). Colony forming unit per mL (cfu/mL) was used in- stead of the number of cells. The CFU of donors and trans- conjugants from the dilution plates were measured with selective antibiotics (cefotaxime and rifampicin) (13-15). Donor number was determined by plating 10-4, 10-5 and 10-6 dilutions. For transconjugants all dilutions (from 1 - 10-6) were plated. 3.7. PCR Analysis and Determination of MIC of Transconjugants DNA of trans-conjugants were obtained by the plasmid extraction kit (BIONEER®) and screened for blaCTX-M-15 gene. MICs of ceftazidime and cefotaxime were deter- mined for trans-conjugants by the E-test (AB Biodisk, Solna, Sweden) according to CLSI guidelines (CLSI 2012). 4. Results It was observed that 74.55% of the isolates were from the urine culture, 13.64% from the chip throat, 6.36% from wound, 3.64% from the blood culture and 0.91 % from the eye infection. Enterobacter cloacae (78.2%), E. aerogenes (6.13%) and E. sakazakii (8.2%) were determined. The fre- quency of clinical Enterobacter spp. isolates was higher in females (60%) than males (40%). Analysis of the anti- microbial susceptibility profile of the isolates showed that they were all susceptible to imipenem. Of the 110 isolates, 95.5% were resistant to Augmentin®, 64.5% resis- tant to trimethoprim-sulfamethoxazole, 22.7 % resistant to ceftazidime, and 23.6% resistant to cefotaxime (Table 1). Of the 110 Enterobacter isolates, 1 (0.9%) was susceptible to all of the tested antimicrobials and 85 (77.3%) were multidrug-resistant and showed resistance to more than two antimicrobial families. Combined disc test was performed on 29 isolates. Fourteen Enterobacter isolates showed ESBL phenotype. All of the fourteen Enterobacter isolates had the blaCTX-M-15 gene (Figure 1). The transferability of the blaCTX-M-15 was tested by con- jugation. A conjugative plasmid containing blaCTX-M-15 was found in three Enterobacter isolates. Antibiotic sus- ceptibility profile of transconjugant strains and donor strains are showed in Table 2. Conjugation frequency was calculated by the number of transconjugants in 1 mL per the number of donor cells in 1 mL which was 0.9 × 10-5. These results were confirmed by PCR. The MIC of paren- tal isolates and transconjugants were similar and includ- ed cefotaxime 256 and for ceftazidime.
- 3. Salimian Rizi K et al. 3Avicenna J Clin Microb Infec. 2015;2(3):e29691 Table 1. Percentage of Isolates Susceptible, Moderately Suscep- tible or Resistance to Each Antibiotic a,b Antibiotic Susceptible Intermediate Resistant AUG 2 (1.8) (2.7) 3 (95.5) 105 SXT 36 (32.7) (2.7) 3 71 (64.5) CHL 89 (81) 7 (6.4) 14 (12.8) CIP 97 (88.2) 6 (5.5) 7 (6.4) IPM 110 (100) 0 (0) 0 (0) T 52 (47.3) 9 (8.2) 49 (44.5) AN (91) 100 0 (0) 10 (9.1) TOB 89 (81) 1 (0.9) 20 (18.2) GM 91 (77.8) 3 (2.7) 16 (14.5) CTX 81 (73.6) 3 (2.7) (23.6) 26 CAZ 83 (75.5) 2 (1.8) 25 (22.7) ATM 87 (79.1) 1 (0.9) 22 (20) CPM 93 (84.5) 5 (4.5) 12 (11) FOX 50 (45.5) 0 (0) 60 (54.5) a The valves are present as No. (%). b Abbreviations:AN,amikacin;ATM,aztreonam;AUG,Augmentin®;CAZ, ceftazidime;CHL,chloramphenicol;CIP,ciprofloxacin;CTX,cefotaxime; CPM, cefepime; FOX, cefoxitin; GM, gentamicin; T, tetracycline; TOB; tobramycin; and SXT, trimethoprim-sulfamethoxazole. Figure 1. Detection of CTX-M-15 Enzyme by Amplification of blaCTX-M Gene Lane M, 100 bp plus blue DNA ladder (Gene ON); lane 1: control. positive; lane 2: control negative; lanes 3 - 5: clinical isolates. Table 2. Antibiotic Susceptibility Profile of the Donor and Transconjugant Strains a,b Code Antibiotic Susceptibility Profile (Donor Strains) Antibiotic Susceptibility Profile (Transconjugant Strains) 1 SXTR, AUGR, CHLR, CIPR, IPMS, TR, ANS, TNR, GMR, CTXR, CAZR, ATMR, CPMR, FOXR SXTR, AUGR, CHLR, CIPI, IPMS, TR, ANS, TNR, GMR, CTXR, CAZR, ATMR, CPMR, FOXR 2 SXTR, AUGR, CHLS, CIPS, IPMS, TR, ANR, TNR, GMR, CTXR, CAZR, ATMR, CPMR, FOXR SXTR, AUGR, CHLR, CIPR, IPMS, TR, ANS, TNR, GMR, CTXR, CAZR, ATMR, CPMR, FOXR 3 SXTR, AUGI, CHLS, CIPS, IPMS, TS, ANS, TNR, GMR, CTXR, CAZR, ATMR, CPMR, FOXR SXTR, AUGI, CHLS, CIPS, IPMS, TS, ANS, TNR, GMR, CTXR, CAZR, ATMR, CPMS, FOXR a Abbreviations: AN; amikacin; ATM, aztreonam; AUG, Augmentin®; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CPM, cefepime; CTX ,cefotaxime; FOX, cefoxitin; GM, gentamicin; IMP; imepenem; SXT, trimethoprim-sulfamethoxazole; T, tetracycline; TN, tobramycin;. b The letters I, R, and S refer to intermediate, resistant and susceptible strains, respectively. 5. Discussion Enterobacter spp. is increasingly identified as a cause of serious nosocomial infections. In recent years, ESBL pro- duction among these isolates is becoming a major clini- cal concern because of its ability to develop resistance to several classes of antimicrobial agents and has high po- tential for transmission of resistance to other bacterial species (16). In the current study, the most prevalent clini- cal Enterobacter species was E. cloacae (78.2 %) that showed the important role of this species in human infections, which was in agreement with other reports (17-20). In the current study, the frequency of ESBL-producing clinical Enterobacter spp. was 12.73% which was different from the other results in Pakistan (50%, 14.93%) (21, 22), Nigeria (37.5%) (10) and Pennsylvania, the USA (33.33%) (9). Three of the fourteen isolates with blaCTX-M-15 gene (21.4%), had a conjugative large plasmid. The low frequen- cy is perhaps due to the type of conjugation method em- ployed in in the current study. Based on these findings, larger multi-center studies are needed to determine the molecular epidemiology of En- terobacter isolates, the distribution of CTX-M ESBL as well as the presence of conjugative plasmids among Entero- bacteriaceae in the hospital populations. The current study results showed that the MIC of the transconjugants and parental strains to CAZ and CTX were similar and the resistance determinants to CAZ and CTX were transferred on a conjugative large plasmid. As a result, third-genera- tion of cephalosporin, fluoroquinolones and imipenem are suggested to be used as frontline remedial antibiotics to treat Enterobacter infections. Careful monitoring and employing appropriate infection control policy are nec- essary to prevent further emergence and spread of resis- tant organisms in the hospitals. Acknowledgements Authors sincerely thank the staff members of depart-
- 4. Salimian Rizi K et al. Avicenna J Clin Microb Infec. 2015;2(3):e296914 ment of bacteriology, TMU, and medical laboratory de- partment of hospitals in Tehran, Iran. Authors’ Contributions Kobra Salimian Rizi performed the laboratory work. Shahin Najar Peerayeh designed the work study, and Bita Bakhshi advised the work study. Funding/Support This study was supported by a grant from Tarbiat Modares University, Faculty of Medical Sciences, Tehran, IR Iran. References 1. Mezzatesta ML, Gona F, Stefani S. Enterobacter cloacae complex: clinical impact and emerging antibiotic resistance. Future Micro- biol. 2012;7(7):887–902. 2. Sanders WEJ, Sanders CC. Enterobacter spp.: pathogens poised to flourish at the turn of the century. Clin Microbiol Rev. 1997;10(2):220–41. 3. Levison ME, Mailapur YV, Pradhan SK, Jacoby GA, Adams P, Em- ery CL, et al. Regional occurrence of plasmid-mediated SHV-7, an extended-spectrum beta-lactamase, in Enterobacter cloacae in Philadelphia Teaching Hospitals. Clin Infect Dis. 2002;35(12):1551–4. 4. Maynard C, Bekal S, Sanschagrin F, Levesque RC, Brousseau R, Masson L, et al. Heterogeneity among virulence and antimi- crobial resistance gene profiles of extraintestinal Escherichia coli isolates of animal and human origin. J Clin Microbiol. 2004;42(12):5444–52. 5. Livermore DM, Canton R, Gniadkowski M, Nordmann P, Rosso- lini GM, Arlet G, et al. CTX-M: changing the face of ESBLs in Eu- rope. J Antimicrob Chemother. 2007;59(2):165–74. 6. Boyd DA, Tyler S, Christianson S, McGeer A, Muller MP, Willey BM, et al. Complete nucleotide sequence of a 92-kilobase plas- mid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother. 2004;48(10):3758–64. 7. Coque TM, Novais A, Carattoli A, Poirel L, Pitout J, Peixe L, et al. Dissemination of clonally related Escherichia coli strains ex- pressing extended-spectrum beta-lactamase CTX-M-15. Emerg Infect Dis. 2008;14(2):195–200. 8. Roopa TJ, Sudha SS. Antimicrobial susceptibility of extended spectrum beta –lactamase (ESBL) producing uropathogens iso- lated from ICU patients. Int J Biological Technology. 2010;1:23–31. 9. Szabo D, Bonomo RA, Silveira F, Pasculle AW, Baxter C, Linden PK, et al. SHV-type extended-spectrum beta-lactamase production is associated with Reduced cefepime susceptibility in Enterobacter cloacae. J Clin Microbiol. 2005;43(10):5058–64. 10. Aibinu IE, Ohaegbulam VC, Adenipekun EA, Ogunsola FT, Odug- bemi TO, Mee BJ. Extended-spectrum beta-lactamase enzymes in clinical isolates of Enterobacter species from Lagos, Nigeria. J Clin Microbiol. 2003;41(5):2197–200. 11. CLSI.. Performance standards for antimicrobial susceptibility testing, 20th information supplement (M100-S20).: Clinical and Laboratory Standards Institute; 2012. 12. Mendonca N, Louro D, Castro AP, Diogo J, Canica M. CTX-M-15, OXA-30 and TEM-1-producing Escherichia coli in two Portuguese regions. J Antimicrob Chemother. 2006;57(5):1014–6. 13. Harajly M, Khairallah MT, Corkill JE, Araj GF, Matar GM. Frequen- cy of conjugative transfer of plasmid-encoded ISEcp1 - blaCTX- M-15 and aac(6')-lb-cr genes in Enterobacteriaceae at a tertiary care center in Lebanon - role of transferases. Ann Clin Microbiol Antimicrob. 2010;9:19. 14. Hausner M, Wuertz S. High rates of conjugation in bacterial bio- films as determined by quantitative in situ analysis. Appl Environ Microbiol. 1999;65(8):3710–3. 15. Phornphisutthimas S, Thamchaipenet A, Panijpan B. Conjuga- tion in Escherichia coli: A laboratory exercise. Biochem Mol Biol Educ. 2007;35(6):440–5. 16. Iabadene H, Messai Y, Ammari H, Ramdani-Bouguessa N, Lounes S, Bakour R, et al. Dissemination of ESBL and Qnr determinants in Enterobacter cloacae in Algeria. J Antimicrob Chemother. 2008;62(1):133–6. 17. Mirsalehian A, Akbari NF, Peymani A, Kazemi B, Jabal A, Mirafshar SM. Prevalence of extended spectrum beta-lactamase-producing Enterobacteriaceae by phenotypic and genotypic methods in in- tensive care units in Tehran, Iran. Daru. 2008;16(3):169–73. 18. Kasap M, Fashae K, Torol S, Kolayli F, Budak F, Vahaboglu H. Char- acterization of ESBL (SHV-12) producing clinical isolate of Entero- bacter aerogenes from a tertiary care hospital in Nigeria. Ann Clin Microbiol Antimicrob. 2010;9:1. 19. Morand PC, Billoet A, Rottman M, Sivadon-Tardy V, Eyrolle L, Jeanne L, et al. Specific distribution within the Enterobacter cloacae complex of strains isolated from infected orthopedic implants. J Clin Microbiol. 2009;47(8):2489–95. 20. Huang ZM, Shan H, Mi ZH, Yang HY, Wu L, Chu QJ, et al. [Analysis on 168 rRNA methylase genes and aminoglycoside modifying enzymes genes in Enterobacter cloacae in China]. Zhonghua Liu Xing Bing Xue Za Zhi. 2008;29(4):369–73. 21. Jabeen K, Zafar A, Hasan R. Frequency and sensitivity pattern of Extended Spectrum beta Lactamase producing isolates in a tertiary care hospital laboratory of Pakistan. J Pak Med Assoc. 2005;55(10):436–9. 22. Amin H, Zafar A, Ejaz H, Jameel NU. Phenotypic characterization of ESBL producing Enterobacter cloacae among children. Pak J Med Sci. 2013;29(1):144–7.