Phenotypic and molecular detection of TEM and SHV beta-lactamases produced by organisms of the family enterobacteriaceae; study on blood culture clinical isolates from intensive care unit

ABSTRACT

The increasing emergence of clinical isolates of the family Enterobacteriaceae with Extended-spectrum beta-lactamases [ESBLs] phenotypes remains a major reason for antimicrobial resistance problems, especially in intensive care units [ICUs]. ESBLs are enzymes produced by some species of Gram negative bacilli and have the ability to inactivate a wide range of beta-lactam antibiotics. The distinguishing feature of these enzymes is that they have extended substrate profiles that confer resistance to the expanded-spectrum cephalosporins [cefotaxime, ceftriaxone, ceftazidime, cefepime and others] and aztreonam. More than 150 different ESBLs have been characterized, and most of these are derived from point mutations affecting the TEM-1 and SHV-1 enzymes. The aim of this study was to evaluate the reliability of double disc diffusion method for the detection of ESBLs in several Enterobacteriaceae blood culture clinical isolates and to detect of point mutation in blaTEM and blaSHV genes in ESBLs producing strains. Eighty six blood culture specimens collected from septicaemic ICU patients admitted to the Internal Medicine Department of Alexandria Main University Hospital, during the period between February through November 2006. Out of 86 specimens, 37 gave positive results for different members of the family Enterobacteriaceae. Screening test for ESBLs was done to determine the sensitivity of the isolates to third generation cephalosporins [3GC] ceftazidime, cefotaxime, and ceftriaxone each 30 micro g/disc. Double Disc Synergy test [DDST] was also done to determine synergy between a disc of augmentin [20 micro g amoxycillin and 10 micro g clavulanic acid] and 30 micro g disc of each 3GC antibiotics [cefotaxime and ceftazidime]. Fifteen isolates [40.5%] out of the 37 were suspected to be ESBLs producers by the screening test E.coli 10/37 [27%], Klebsiella spp 4/37 [10.8%], Enterobacter spp. 1/37 [2.7%] and none of the Acinitobacter spp. 0/37 [0%]. By doing the DDST [confirmatory], 7/37 [18.9%] E.coli strains 3/37 [8.1%] Klebsiella spp. and 1/37 [2.7%] Enterobacter spp. gave positive results. Among the isolates positive by screening test; 2/10 [20%] E.coli strains gave positive PCR results for bla[TEM-1] gene and 2/10 [20%] gave positive result for bla[SHV-1] gene. One of these 2 strains showed positive results for both genes. Among the 4 Klebsiella spp., positive also by screening test, 1/4 [25%] strain gave positive result for bla[TEM-1] gene and 2/4 [50%] gave positive result for bla[SHV-1] gene. The Enterobacter spp. gave positive result for blaSHV-1 gene. The DNA sequence analysis of the bla[TEM-1] and bla[SHV-1] genes amplification products was performed. The sequencing of TEM gene of strain No.4 revealed that it encoded TEM-1 beta-lactamase. The sequencing of SHV gene of strain No.14 revealed the G to A nucleotide change that gives the glycine to serine substitution at position 238, denoting that it encoded SHV-2. ESBL producers are associated with increased morbidity and mortality, especially amongst patients on intensive care and high-dependency units. Accurate laboratory detection is important to avoid clinical failure due to inappropriate antimicrobial therapy