Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria

Background We studied the beta-lactamases of an E. aerogenes isolate recovered from the blood of a two-year-old patient. The isolate demonstrated a disk-diffusion phenotype typical for an AmpC-ESBL co-producer. Methods Microbiology studies were performed according to standard protocols. The resistance gene was identified by transconjugation and cloning experiments. Results By transconjugation only a narrow spectrum beta-lactamase (TEM-1) encoded on a small plasmid was transmitted. The ESBL was cloned and expressed in an E. coli host. Sequence analysis of the recombinant plasmid revealed blaSHV-12 associated to the insertion sequence, IS26. Conclusion This is the first study demonstrated the occurrence of SHV-12 in Nigeria.


Background
Enterobacter species, in particular, E. cloacae and E. aerogenes are able to compromise antibacterial treatment by over expressing the chromosomal AmpC betalactamase [1,2]. Emergence and spread of Class A extended-spectrum beta-lactamases (ESBLs) among these species are further complications [3].
ESBLs confer resistance to expanded-spectrum betalactam antibiotics. The majority of these enzymes are derived by amino acid substitutions from the narrow spectrum precursors, TEM-1, 2 and SHV-1. TEM-type ESBLs generally disseminate on transposons, Tn1, Tn2 and Tn3 [4]. SHV-type ESBLs, on the other hand, are typically associated to IS26 and disseminate through IS26 dependent mobilization events from the K. pneumoniae chromosome [5,6]. Eventually, the extended-spectrum derivates of TEM and SHV enzymes are now ubiquitous.
We detected an ESBL producing E. aerogenes clinical isolate from Nigeria. Since, the data in the literature regarding the occurrence and the dissemination of ESBLs in Nigeria is limited [7,8], we characterized the beta-lactamases of this isolate by microbiological and molecular means

Methods
The strain and the susceptibility tests A strain of Enterobacter aerogenes was isolated and identified by standard methods from the blood of a twoyear-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.

Plasmid studies
Plasmids were isolated by the alkaline lysis or the Kado Liu methods [10], run on 0.8% agarose gels and visualized under UV light. Transconjugation and transformation experiments were performed with E. coli J53-2 (Rif R ) 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 ddH 2 O 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 MgCl 2 , 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 [13]. 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.

Cloning and sequencing
High molecular weight (HMW) genomic DNA was isolated using the procedure described by Chen and Kuo [14]. 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 Bsp143I (Fermentas) for 20 min at 37°C and 0.5 microgram of the cloning vector, pZero (Invitrogen), was digested with 5 units of BamHI (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.

Results
The isolate, identified as E. aerogenes (EaN146), exhibited a phenotype by the disk diffusion test that is typical for an AmpC-ESBL co-producer [9]. Briefly, there was no zone around the FOX disk which was specifically caused by the over-expressed AmpC enzyme whereas the zone around the FEB disk was enlarged on the AMC side, indicating the existence of a Class A ESBL.
By transformation we were able to transfer a narrow spectrum enzyme encoded on a <7 kb cryptic plasmid (data not shown) to DH10B recipient (TN146) which was identified as TEM-1 by sequencing on both sides of the PCR product.
By shotgun cloning, on the other hand, we recovered a positive clone (CN146) possessing an ESBL. Sequencing of the recombinant plasmid (pCEaN146) from this clone revealed a 5656-bp insert (Figure 1; GenBank accession # EU534207) on which we determined an open reading frame (ORF) encoding the ESBL, SHV-12 and an insertion sequence, IS26, upstream (73-bp) of the bla gene with perfect 14 bp terminal inverted repeats [15]. With the aid of ORF finding software (MB DNA Analysis v6.84) and Blast comparison against the sequences submitted to GenBank we identified three more intact ORFs and one another ORF disrupted by IS26. Interestingly, we detected a 9-bp direct repeat sequence (GTGCTGCTG) flanking on the left and right boundaries of a 3002-bp which might be an incomplete transposable unit.
MICs of the strains are shown in Table 1. E. aerogenes is highly resistant to beta-lactam antibiotics except carbapenems. In this strain clavulanate apparently potentiated the activity of cefepime which is relatively stable against the AmpC type enzyme and hydrolyzed only by the ESBL, but not enhanced the activity of ceftazidime which is hydrolyzed by the clavulanate resistant AmpC enzyme [9].
The IEF experiment is presented in Figure 2. Only two enzymes (pI 5.4 & pI 9) were visible in the lane of E. aerogenes, one representing the AmpC (pI 9) and the other representing the TEM-1 (pI 5.4). At pI 8.2, after several minutes of incubation a faint band, possibly corresponding to SHV12, appeared while the other bands diffused preventing us to record a good image (data not shown). To confirm the expression of SHV-12 in EaN146, we performed a RT-PCR experiment with primers ShvF1 and ShvR1. Template DNAs were transcribed from the mRNAs of EaN146, CN146 and TN146. This experiment demonstrated that, although low in amount, EaN146 is expressing SHV-12.

Discussion
This study characterized the beta-lactamases from a clinical isolate of E. aerogenes and demonstrated the occurrence of IS26 associated bla SHV-12 in Nigeria. SHV-12 was first identified in 1997 in Switzerland and later reported from various continents including Africa [16][17][18][19][20]. Reports of SHV-12 producing E. cloacae and Klebsiella blood isolates from Tanzania and Cameroon and now E. aerogenes from Nigeria indicate a high endemicity of SHV-12 possessing Enterobacteriaceae in the Western coast of Africa and therefore attract interest.
SHV-1 is supposed to be a species specific enzyme encoded mainly on the chromosome of K. pneumoniae [5,21]. Evolutionary analysis of submitted sequences indicated that extended-spectrum variants evolved on two branches from bla SHV-1 , both mediated by IS26 depended mobilization events from the chromosome of K. pneumoniae [6]. These analyses revealed that bla SHV-  this study has evolved from bla SHV-2a as supposed by the evolutionary approach [6]. IS26 is an 820-bp long insertion sequence that typically generates 8 bp target duplication upon transposition [15]. It is demonstrated for IS1 that the length of target site duplication sequences may vary according to the sequence of the integrated site [22]. This phenomenon has not been studied for IS26. In this study we identified a 9 bp direct repeat on the boundaries of a 3002 bp region bearing bla SHV-12 and the DeoR type regulator gene. This 3002 bp region may be a replicating unit.
Data for ESBLs from Nigeria are rare in the literature [7,8,23]. In a study by Soge et al., CTX-M-15 producing K. pneumoniae clinical isolates were characterized [7]. In another study, the authors characterized ESBLs by phenotypic means among E. aerogenes [8].

Conclusions
This is the first study reporting the occurrence and the genetic support of ESBL bla SHV-12 gene in Nigeria.