Prevalence of bla SHV genes in clinical isolates of Klebsiella pneumoniae at Saint Camille medical Center in Ouagadougou. Isolation of bla SHV11 -like gene

Five bacterial strains (4 Klebsiella pneumoniae and 1 Escherichia coli ) representative of pathogenic species and resistant to bbbb -lactam antibiotics are investigated to isolate the genes responsible of bbbb lactamase activity. The use of engineering techniques enables us to show the widespread of bla SHV genes particularly in clinical isolates of K. pneumoniae . Our results highlighted an atypical bla SHV-11 gene.


INTRODUCTION
The major mechanism of bacterial resistance to β-lactam antibiotics is the production of β-lactamases. These enzymes are able to inactivate the β-lactam antibiotic by hydrolysing the β-lactam ring (Abraham and Chain, 1940;Frère et al, 1991;Reid et al, 1987). It is reported that up 90% ampicillin resistance is due to the production of TEM-1 β-lactamase in Escheridia coli (Livermore, 1995) and SHV-1 β-lactamase in Klebsiella pneumoniae (Tzouvelekis and Bonomo, 1999).
Many bacteria commonly isolated from human biological specimens like E. coli, klebsiella pneumoniae Shigella spp., and Salmonella spp. responsible for infection of gastroenteritis, urinary disease and the inflammation of the ear (children) were collected at the Saint Camille medical center of Ouagadougou and these strains exhibit *Corresponding author. E-mail: b_zeba@yahoo.fr. Fax: (226) 33 06 08. Tel: (226)29 08 33. an important β-lactamase activity against the usual βlactam-antibiotics (Zeba et al., 2003). The present work is an attempt to establish what types of β-lactamase genes are occurring in our area may be related to these activities. Therefore, one E. coli and 4 K. pneumoniae strains were screened. This is the first epidemiological study at this level, carried in Ouagadougou and Burkina Faso.

Culture conditions
These clinical wild strains were grown on trypticase soy agar (Difco) plates (solid medium) or in Luria Bertani (LB Difco).  The E. coli competent DH5-α cells used in transformation experiments were grown in SOC medium which allow quick recuperation of cells and ensure maximum transformation efficiency.

Shotgun cloning
Genomic DNAs of the strains were extracted and purified by using Wizard kits of genomic DNA purification (Wizard genomic DNA purification kit, Promega corporation, Madison, WI, USA). Different types of Fragments from genomic DNAs were obtained by using specific different restriction enzymes (Sau3AI, BamHI, BglII, EcoRI etc.) These fragments were ligated into cloning vector PK18, carrying kanamycin resistance gene and transformed into E. coli competent DH5-α cells. The PK18 recombinant clones were selected on plates containing 50 µg of kanamycin per ml + 60 mM IPTG + 40 µg of Xgal per ml (KIX plates).

PCR
The PCR is both carried out to detect the β-lactamase genes or to amplify and isolate them (Mabilat and Goussard (1993), Brinas et al. (2002). PCR amplifications of the blaTEM, blaSHV, and blaampC genes were carried out using specific primers pairs (for example SHVF and SHVR, TEMF and TEMR, ampCF and ampCR) for amplification of correspondent genes (Sutcliffe, 1978;Mercier and Levesque, 1990) when the template is genomic DNA or shotgun clones. Theses primers were obtained from Eurogentec Bel. S.A. The PCR products were ligated into pGEM-T Easy Vector carrying ampicillin resistance gene and as previously, transformed into E. coli competent DH5-α cells. The correspondents clones were always selected on plates containing 50µg of ampicillin per ml + 60 mM IPTG and 40 µg per ml X-gal (AIX plates).

RESULTS AND DISCUSSIONS
Four recombinants plasmids carrying ampicillin-resistant genes were recovered from genomic DNAs of strains No. 291, 312, 392 and 1201. The attempt to obtain recombinant plasmids from strain 1004 DNA with this approach failed.
The PCR with primers specific for the bla TEM , bla SHV , and bla AMPC genes were performed both directly on genomic DNAs and on recombinant plasmids. Positive PCR results were obtained from recombinant plasmids and from genomic DNA 291, 312, 392 and 1201 with primer of SHV (Figure 1). Positive PCR result was obtained with genomic DNA of strain N o . 1004 with primer TEM (result not reported). PCR with primer AmpC was negative on all the strains investigated.
These results clearly point out that the K. pneumoniae strain N° 291, 312, 392 and 1201 harbour bla SHV genes and the E. coli N°1004 have bla Tem gene. Among the 4 identified bla SHV genes, 2 were isolated by cloning PCR products into pGEM-T Easy Vector (   (coming from fragment 5 of Figure 1)]. An attempt to sequence fragment N°3 of Figure 2 (coming from fragment 6 of Figure 1) failed.
Substrate profiles of β-lactamase from eleven K. pneumoniae genes and those currently studied (Zeba et al, 2003) indicate a close behavioural similarity in their spectral activity. These phenotype similarities can be related to those of the genotypes. Thus the bla SHV gene may be common among K. coli species. Our results also indicate the existence of parental gene of bla SHV (bla SHV-1 ) on clinical wild strains of K. pneumoniae in Ouagadougou (Burkina Faso) and highlight an atypical bla SHV11 gene . This gene of 861 bp (clone 2, Figure 3) was isolated from K. pneumoniae N°392. It is not identical with bla SHV-11 genes available in gene Bank (NCBI-DB). The nucleotide, alignment of bla  gene and that of clone 5 reveals substitutions at position 324 (cytosine replace thymine), 357 (thymine replace cytosine), 762 (cytosine replace thymine) and 795 (cytosine replace thymine). It appears that the change always occurs between cytosine and thymine. Therefore, on the basis of nucleotide sequences, the two genes (classical bla SHV-11 and clone 5) are slightly different, but the translation of the two genes yields the same variant SHV-11 β-lactamase. Our investigations are continuing to establish clearly the different types of bla SHV genes encountered in Burkina Faso. The PCR analysis of the single E. coli detects bla TEM type gene. We hope to further investigate these and similar genes in subsequent studies.