TLA-1: a New Plasmid-Mediated Extended-Spectrum β-Lactamase from Escherichia coli

Abstract

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two ␤-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single ␤-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 ␤-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5␣. Sequencing of the bla TLA-1 gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A ␤-lactamases:70 SXXK, 130 SDN, and 234 KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A ␤-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A ␤-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 ␤-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum ␤-lactamase of Ambler class A.The main mechanism of resistance to ␤-lactam antibiotics in members of the family Enterobacteriaceae is the production of ␤-lactamases (21, 35). Expanded-spectrum cephalosporins (cefotaxime, ceftazidime) have been specifically designed to resist degradation by the older broad-spectrum ␤-lactamases such as TEM-1, TEM-2, and SHV-1. With the use of these antibiotics in vivo, extended-spectrum ␤-lactamases (ESBLs) have been selected; these ESBLs most often are mutants of these older enzymes and carry a limited number of amino acid substitutions (G. Jacoby and K. Bush, http://www.lahey.org/studies /webt.htm). There is also a small but growing family of plasmid-mediated ESBLs that are not related to TEM or SHV ␤-lactamases, such as 10,14,15) and Toho (17,23), that preferentially hydrolyze cefotaxime and that belong to Ambler class A. In addition, there has been a worldwide emergence of novel ␤-lactamases, mainly among members of the family Enterobacteriaceae, that hydrolyze expanded-spectrum ␤-lactams. While they maintain the main properties of the class A ␤-lactamases, they are not closely related to the TEM, SHV, or CTX-M families of ␤-lactamases. Most of these ESBLs are plasmid mediated and include the PER-1, PER-2, VEB-1, CblA, and CepA enzymes. These ␤-lactamases are not species specific, since they have also been isolated from clinically significant gram-negative species that are not mem...

Antimicrobial resistance of bacterial strains isolated from orange juice products

“…The results of the antibiotic sensitivity test were interpreted and are presented as the resistance of bacterial isolates to the antibiotics (Table 1), and the antibiotic resistance pattern among the bacterial isolates (Table 2). In several earlier works on similar studies carried out on foodstuffs including orange juices, researches have been focused mainly on the strains of E. coli and S. aureus (Umoh et al, 1990;Grewal and Tiwari, 1990;Abbar and Kaddar, 1991;Singh et al, 1995;Desai and Kamat, 1998;Leegar et al, 1999;Silva et al, 2000;Rahman Khan and Malik, 2001). However, in this work, a broad overview of the study was conducted and this is why the resistance pattern and the mechanisms in selected strains of all the bacteria isolated from the orange juices were studied.…”

“…Several workers have reported the production of β-lactamase by bacteria with high resistance to β-lactam antibiotics (Nandivada and Amyes, 1990;Esperson, 1998;Rahman Khan and Malik, 2001). There have also been reports of the spread of β-lactamase genes into species that previously were not known to possess them (Mascaretti et al, 1999;Kawakami et al, 2000;Silva et al, 2000).…”

“…In several studies that have been conducted on the resistance of food-borne pathogens, emphasis had been on E. coli and S. aureus (Umoh et al, 1990;Grewal and Tiwari, 1990;Abbar and Kaddar, 1991;Singh et al, 1995;Desai and Kamat, 1998;Leegar et al, 1999;Silva et al, 2000;Rahman Khan and Malik, 2001). However, in the present study, an attempt was made to evaluate the resistance of selected strains of all the bacterial strains that were isolated from the orange fruit products.…”

The High Resolution Crystal Structure for Class A β-Lactamase PER-1 Reveals the Bases for Its Increase in Breadth of Activity

“…from Salmonella typhimurium (10), VEB-1 from E. coli (57), CME-1 from Chryseobacterium (flavobacterium) meningosepticum (58), TLA-1 from E. coli (59), CBLA from Bacteroides uniformis (15), CEPA from B. fragilis (13) and CFXA from B. vulgatus (14). The phylogenetic tree shown in Figure 6 indicates that these proteins constitute a distinct cluster of enzymes, and the X-ray structure of PER-1 suggests that this partition reflects major structural differences In retrospect and in view of the major structural differences between the PER-1 and the TEM enzymes, it is not surprising that site directed mutagenesis of PER-1 (34,35) could not relate the cephalosporinase activity of PER-1 to the residue type at positions 164, 179, 238 and 240.…”

Clonal and Horizontal Dissemination of Klebsiella pneumoniae Expressing SHV-5 Extended-Spectrum β-Lactamase in a Mexican Pediatric Hospital

“…There is limited information in Mexico concerning molecular studies on the type of ESBL selected in vivo in multidrugresistant enterobacteria (13,24,25,26). In this work we used a molecular approach to determine the epidemiology of an outbreak produced by an endemic multidrug-resistant K. pneumoniae strain.…”