Study of Resistance to 82 Clinical Cases Enterobacteriaceae to Beta-lactam Antibiotics

Knowledge of antimicrobial resistance patterns in E. coli, the predominant pathogen associated with urinary tract infections (UTI) is important as a guide in selecting empirical antimicrobial therapy. To describe the antimicrobial susceptibility of E. coli associated with UTI in a major university hospital in Tehran (Iran), seventy-six clinical isolates of E. coli were studied for susceptibility to β-lactam antibiotics by the disc diffusion method and Minimal Inhibitory Concentrations determination. All isolates were resistant to ampicillin, amoxicillin and oxacillin. Resistance to the other tested antibiotics was shown to be 93.4% to cefradine, 76.3% to carbenicillin, 47.3% to cefazoline, 50% to cefalexin and 32.8% to cephalothin while 1.3% expressed resistance to cefoxitime, and 2.6% were resistant to ceftizoxime and ceftriaxone. Substrate hydrolysis by ultra violet spectroscopy showed that 87.4% harbored penicillinases, 9% produced cephlosporinases and 3.6% degraded both substrates. Clavulanic acid inhibited enzyme activity in 82.9%, of which 78.95% was penicillinases (group IIa) and 3.95% was cephalosporinases (group IIb) of the Bush classification system. These results indicate that E. coli can posses a variety of β-lactamases that are responsible for β-lactam resistance. Members of the family Enterobacteriaceae, particularly Escherichia coli is the most common causes of urinary tract infections in hospitals and societies. Betalactam antibiotics, particularly the third and fourth generation of cephalosporins are effective in treating these infections.


Introduction
Urinary Tract Infections (UTI) are the second most common infections present in community practice (Gonzalez & Schaeffer, 1999).Members of Enterobacteriaceae, specifically, E. coli are the main causes of urinary infections (Gupta, 2003).Extensive use of β-lactams in veterinary medicine and human practice is believed to be associated with selection of resistance in both pathogenic and nonpathogenic isolates of E. coli (Livermore, 1995).More than two hundred βlactamase enzymes are recognized which are classified into 4 main groups and 8 subgroups (Bush & Jacoby, 1996;Bush, 1989).The resistance of Enterobacter spp. to β-lactam antibiotics is most frequently mediated by production of TEM, SHV and AmpC β-lactamase (Barnaud et al., 2001).In the last decade, production of plasmidmediated ESBL which hydrolyzes a wide range of the most recently developed cephalosporins, has been recognized as an additional important emerging mechanism of resistance among members of the family Enterobacteriaceae including clinical isolates of E. coli (Bradford, 2001;Pitout et al., 1998).The first plasmid mediated βlactamases (TEM-1) was described in E. coli in 1960 and within a few years, it was found in many different genera of Gram-negative bacteria (Bradford, 2001).The AmpC family of β-lactamases occurs both chromosomally and plasmid-mediated in E. coli and plasmid encoded AmpC β-lactamases are found to be responsible for global outbreaks (Cudron, Moland, & Sanders, 1997;Eftekhar, 2005).We studied 76 urinary isolates of E. coli for their susceptibility to 12 β-lactam antibiotics.Preferred substrate hydrolysis was performed to determine the class of β-lactamases.DNA amplification of βlactamase types TEM, SHV and AmpC genes was carried out by PCR using type specific primers of blaTEM, ampC and SHV genes for all of the isolates.

Detection and Identification of Bacteria
Identifying bacteria by biochemical version and was confirmed by API 20E test.
The results showed that among 82 cases of Escherichia coli cases, 76 cases were Citrobacter freundii, a case was Enterobacter, a case was Klebsiella pneumoniae, a case was Klebsiella oxytoca, and a case was Hafnia.

A) by disk diffusion method:
All tested bacteria were resistant to antibiotics like amoxicillin, ampicillin and Oxacillin and only 9% of cases were resistant to coamoxiclave.As it is shown in Figure 1 and Figure 2, resistance to Cefradine and carbenicillin was 97.5% and 79.20%; and resistance to other antibiotics was 20.7% Cephalothin, 26.8% cefalexin and 31.7%cefazoline, 1.2% to Cefoxitime, 1.2% Ceftizoxime 1.2%, and Ceftiaxime.

B) The method MIC:
The results of the MIC are shown in Figure 3. MIC test showed susceptibility to the samples.As it is seen in Figure 4, colonies of bacteria with beta-lactamase enzyme have a white halo.As it is shown in Figure 5, depending on the rate of hydrolysis of benzyl penicillin and Cefaloridine to each other (spectrophotometry) 87.4% organisms contain enzyme penicillinase, 9% organisms contain enzymes Cephalosprinase and 3.6 percent contain both enzymes equally.
Figure 5.The results of hydrolysis of the substrate by Spectrophotometer

Lactamase Classification Results
None of the beta-lactamase enzymes tested showed no inhibition with EDTA.This does not mean that Group III was among the beta-lactamase.However, 91% enzymes were inhibited by clavulanic acid, and taking into account the results of hydrolysis of the substrate (Figure 5) showed that 87.4% organisms were penicillinase that were resistant to clavulanic acid, and this class of beta-lactamase belonged to Group 2a, 3.6% organisms equally showed penicillinase and cephalosporinase and can be inhibited by clavulanic acid and belonged to the Group 2b, and 9% were penicillinase that cannot be inhibited by clavulanic acid which belonged to Group 4. Among the studied organisms, Group I, i.e. cephalosporinases were not resistant to clavulanic acid (Figure 6).

Figure 1 .
Figure 1.Results of Penicillin antibiotics The results of the colony by Iodometer.A) Bacteria containing beta-lactamase B) bacteria lacking the enzyme beta-lactamase 3.1.3The Results of Substrate Hydrolysis

Figure 6 .Figure 7 .
Figure 6.The results of the classification of beta-lactamases