The strain and the susceptibility tests
A strain of Enterobacter aerogenes was isolated and identified by standard methods from the blood of a two-year-old male patient, admitted (8th November 1999) to a tertiary care hospital in a Southwestern city of Nigeria, with clinical diagnosis of febrile convulsion. The case note is, however, not available for the history and outcome of the patient. The strain was identified by API 20E (bioMérieux Marcy l'Etoile, France) according to the instructions provided by the manufacturer.
Disk diffusion test was performed on Mueller-Hinton agar (disks and agar media were from Oxoid, Basingstoke UK) for the phenotypic identification of ESBLs as described elsewhere . Briefly, the cefotaxime (CTX; 30-μg) disk was placed 20 mm away from the amoxicillin (20-μg)-clavulanate (10-μg) (AMC) disk, the ceftazidime (CAZ; 30-μg) disk was placed at 30 mm distance, and the cefepime (FEP; 30-μg) disk was placed at 30 mm distance. For the phenotypic detection of the AmpC enzyme, a cefoxitin (FOX; 30-μg) disk was placed on the agar, as well.
The MICs of key antibiotics were determined by the broth micro-dilution test using Mueller-Hinton Broth (Oxoid, Basingstoke UK) as recommended by CLSI. End-points were interpreted after 18 h of incubation at 37°C. E. coli ATCC 25922 and E. coli DH10B were included as control strains. Powder forms of antibiotics were obtained from local companies: ampicillin (Mustafa Nevzat), piperacillin & tazobactam (Wyeth), clavulanate (DEPA), cefepime (Bristol-Myers Squibb), cefotaxime (Toprak), ceftazidime (Glaxo-SmithKline), imipenem (Merck), meropenem (Astra-Zeneca), ciprofloxacin (Bayer), gentamicin (Bilim) and tobramycin (Nobel). The final concentration of clavulanate was 4 mg L-1.
Plasmids were isolated by the alkaline lysis or the Kado Liu methods , run on 0.8% agarose gels and visualized under UV light. Transconjugation and transformation experiments were performed with E. coli J53-2 (RifR) and electro-competent E. coli DH10B strains as recipients, respectively [11, 12].
DNA & RNA isolation, PCR and RT-PCR
DNA templates for PCR experiments were prepared by simply boiling a dense bacterial suspension in ddH2O and a 10 min of centrifugation at 16.000 × g. DNA-free RNAs were isolated with RNeasy Mini Kit (Qiagen), and run on denaturating gel conditions to check the integrity of RNAs and the lack of visible DNA contamination. cDNAs were immediately synthesized by random hexamer primers with Revert Aid first strand cDNA synthesis kit (Fermentas, Lithuania).
PCR reactions were set in 50 μl final volumes made up of 1× buffer, 1.5 U Taq polymerase (Fermentas, Lithuania), 1.5 mM MgCl2, 0.8 mM dNTPs, 50 pmol primers each. RT-PCR was set up with the same master mixture except, 1× SYBR Green I (Sigma) was added. Reactions were prepared on ice and run on Quantica (Techne) RT-PCR Thermal cycler as described elsewhere . Specificity of the product was assessed by the dissociation curve analysis made by the Quantica software and the relative mobility of the PCR products on the agarose gels.
Primers, ShvF1-5'-ATTACCATgAgCgATAACAg-3' and ShvR1-5'-CATTCAgTTCCgTTTCCC-3' were used (55°C annealing temperature) in RealTime-PCR to amplify a 133 bp fragment internal to blaSHV-12 gene. The primer ShvF1 and the primer DeoRR1-5'-CCAggTggTCACCAATgATT-3' were used (50°C annealing temperature) to amplify a 927 bp fragment which extends from the 3' end of the blaSHV-12 gene to the 402 bp of the transcriptional regulator gene. To amplify the entire sequence (861 bp) of blaTEM gene, the primers TemA-5'-ATgAgTATTCAACAT TTCCgTg-3' and TEMD-5'-TTACCAATgCTTAATCAgTgAg-3' (annealing temp. 52°C) were used.
Crude cell extracts were prepared by sonication. Analytical isoelectric focusing (IEF) was performed on a 5% polyacrylamide gel containing ampholytes (pH range, 3-10; Bio-Rad Laboratories, USA) with a Model 111 Mini IEF Cell (Bio-Rad). SHV-1 (pI 7.6), OXA-14 (pI 6.2) and TEM-1 (pI 5.4) were included as references. Enzymes were focused at a constant 1 W for 45 min and detected by overlaying the gel with 1 mM nitrocefin solution.
Cloning and sequencing
High molecular weight (HMW) genomic DNA was isolated using the procedure described by Chen and Kuo . For plasmid DNA isolation, alkaline lysis method was used (Sambrook and Russell, 2001). Twenty micrograms each of HMW genomic DNA and plasmid DNA were digested with 0.1 units of Bsp 143I (Fermentas) for 20 min at 37°C and 0.5 microgram of the cloning vector, pZero (Invitrogen), was digested with 5 units of Bam HI (Fermentas) for one hour at 37°C. Digested DNA and the vector were phenol/chloroform extracted and compatible arms were ligated overnight at 16°C. Two microliters of ligation mix was then transformed to electrocompetent E. coli DH10B and the recombinant clones were selected on agar plates supplemented with ampicillin (100 mcg/mL) plus zeocin (50 mcg/mL).
Dye terminator cycle sequencing with the ABI Prism BigDye Terminator kit (Applied Biosystems, Foster City, Calif.) were used to obtain the sequences. The assay was carried out according to the standardprotocol. Data was collected on an ABI 377 automated fluorescence sequencer.