Detection of extended-spectrum plasmid-mediated beta-lactamases by disk diffusion.

Abstract

The most common plasmid-mediated β-lactamases in the Enterobacteriaceae are TEM-1, TEM-2 and SHV-1, which have a weak activity against first-generation cephalosporins. Neither oximino β-lactams, in particular cefotaxime and ceftazidime, nor monobactams and carbapenems were hydrolyzed by TEM-1 or −2 or SHV β-lactamases until enzyme mutants emerged in the mid-1980s. Because of their greatly extended substrate range, these mutant enzymes were called extended-spectrum β-lactamases (ESBLs) [1Philippon A Labia R Jacoby G Extended-spectrum β-lactamases.Antimicrob Agents Chemother. 1989; 33: 1131-1136Crossref PubMed Scopus (499) Google Scholar]. ESBLs were first recognized in Europe, in Germany [2Knothe A Shah P Kremery V Antal M Mitsuhashi S Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens.Infection. 1983; 11: 315-317Crossref PubMed Scopus (732) Google Scholar] and in France [3Brun-Buisson C Legrand P Philippon A Montravers F Ansquer M Duval J Transferable enzymatic resistance to third-generation cephalosporins during nosocomial outbreak of multiresistant Klebsiella pneumoniae.Lancet. 1987; ii: 302-306Abstract Scopus (339) Google Scholar,4Sirot D Sirot J Labia R Morand A Courvalin P Darfeuille-Michaud A Transferable resistance to third-generation cephalosporins in clinical isolates of Klebsiella pneumoniae: identification of CTX-1, a novel β-lactamase.Antimicrob Agents Chemother. 1987; 20: 323-333Crossref Scopus (293) Google Scholar], and have, since then, been reported around the world [5Jacoby GA Medeiros AM More extended-spectrum β-lactamases.Antimicrob Agents Chemother. 1991; 35: 1697-1704Crossref PubMed Scopus (654) Google Scholar]. There has been a rapid increase in the number and variety of ESBLs and in the mechanisms that regulate their production and spread. Klebsiella pneumoniae is by far the most common species in which ESBLs have been recognized (75% of ESBL-producing strains). ESBLs have been detected in nearly all other species of Enterobacteriaceae, but occur muchless frequently. Recent surveys of hospital isolates of K. pneumoniae in Europe indicate that 14% to 16% produce ESBLs, whereas alow frequency (<0.1%) is observed in Escherichia coli [6Sirot D Goldstein FW Soussy CJ et al.Resistance to cefotaxime and seven other β-lactams in members of the family Enterobacteriaceae: a 3-year survey in France.Antimicrob Agents Chemother. 1992; 36: 1677-1681Crossref PubMed Scopus (131) Google Scholar,7Goldstein FW Péan Y Rosato A et al.Characterisation of ceftriaxone-resistant Enterobacteriaceae: a multicentre study in 26 French hospitals.J Antimicrob Chemother. 1993; 32: 595-603Crossref PubMed Scopus (57) Google Scholar]. Reports of ESBLs in Proteus mirabilis have been relatively rare, perhaps because of thelow frequency of plasmid acquisition, or of failure to detect enzyme production in this species [8Nordmann P Nass T Labia R Les nouvelles β-lactamases des bacilles à gram négatif.La lettre de l'infectiologue. 1994; 5: 151-155Google Scholar]. ESBLs differ from the parent enzyme, either TEM or SHV penicillinases, by one to four amino acid changes which alter the substrate specificity and are predominantlylocated at positions 104, 164, 205, 237, 238 and 240 (according to Ambler numbering). In recent years, there have been numerous reports of ESBLs from different parts of the world, with the TEM-type enzymes forming thelargest group (more than 20 types), while the variety of ESBLs reported in the SHV family islimited (n=6). ESBLs derived from TEM or SHV vary considerably according to thelevel of resistance they confer to cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (ATM). With the exception of CTX-1/TEM-3 and TEM-4, which confer resistance to approximately 32 mg/L of cefotaxime, TEM derivatives are responsible forlow or moderate MIC values (0.06 to 4 mg/L). MICs of ceftazidime are higher (4 to 512 mg/L) than those of cefotaxime. The MICs of aztreonam (0.1 to 128 mg/L) are never higher than those of ceftazidime, except for TEM-22, which confers a higher MIC of aztreonam (128 mg/L) than that of ceftazidime (4 mg/L). SHV-2 to SHV-5 derivatives are more potent ESBLs, since they confer MICs >32 mg/L of cefotaxime, ceftazidime and aztreonam [5Jacoby GA Medeiros AM More extended-spectrum β-lactamases.Antimicrob Agents Chemother. 1991; 35: 1697-1704Crossref PubMed Scopus (654) Google Scholar]. These enzymes have no detectable activity against cephamycins and carbapenems. All the SHV and TEM extended-spectrum derivatives are sensitive to the β-lactamase inhibitors clavulanate, sulbactam and tazobactam. Of the three inhibitors, clavulanate is the most effective, whereas sulbactam is only of marginal benefit, especially against strains producing enzymes of the SHV family. The double disk synergy (DDS) test described by Jarlier et al. [9Jarlier V Nicolas MH Fournier G Philippon A Extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns.Rev Infect Dis. 1988; 10: 867-878Crossref PubMed Scopus (1333) Google Scholar], based on synergy between β-lactamase inhibitors and cefotaxime, ceftazidime or aztreonam, enables the detection of ESBLs in strains misclassified as susceptible on the basis of standard interpretative criteria for the disk diffusion method. Disks of CTX, CAZ or ATM are placed around a disk of amoxicillin + clavulanate (AMC) 30 mm apart (center to center). The DDS test is always positive with K. pneumoniae and E. coli strains producing an ESBL-enzyme (Figure 1). Moreover, the presence of an ESBL is strongly suggested by highlevels of resistance to penicillins and to first- and second-generation cephalosporins, except cephamycins, and by frequently associated resistance to aminoglycosides (AAC-6′). In ESBL-producing P. mirabilis, inhibition diameters of CTX, CAZ and ATM are >28 mm. Theselarge diameters are often responsible for false-negative double disk synergy tests when the disks are placed as described (Figure 2A). A positive synergy test is observed (Figure 2B) when disks are further apart (40 mm center to center) or when the disks of CTX, CAZ and ATM are quartered in order to reduce their potency. In species such as Enterobacter cloacae hyperproducing a cephalosporinase in addition to an ESBL, the highlevels of resistance (MICs >32 mg/L) to CTX, CAZ and ATM are responsible for a false-negative synergy test (because of reduced zone diameters). Synergy can be detected by reducing the distance between the disks to 20 mm, or by a synergy test with a disk of cefepime or cefpirome which areless hydrolyzed than CTX, CAZ and ATM by the cephalosporinase (Figure 3). Certain species of Enterobacteriaceae produce a chromosomal β-lactamase which hydrolyzes third-generation cephalosporins and aztreonam when hyper-produced and which is inhibited by clavulanic acid. The DDS test may be positive in Klebsiella oxytoca, Citrobacter diversus, Proteus vulgaris, Proteus penneri, Kluyvera ascorbata and Serratia fonticola, even if the strain does not produce an ESBL. In some species, mainly P. mirabilis but also E. coli or Klebsiella producing certain TEM-derived ESBLs, low or moderate MIC values (MIC <lower breakpoint) are frequently observed with cefotaxime, ceftriaxone aztreonam and the most recent cephalosporins cefepime or cefpirome. All these β-lactams are hydrolyzed by ESBL to various extents and, even if thelevel of resistance conferred islow, the strains must be classified as ‘Intermediate’ to all third-generation cephalosporins, since it is not known if such isolates are clinically susceptible to these drugs. Morevoer, therapy with third generation cephalosporins could be responsible for the emergence of a resistant strain producing an ESBL enzyme with an additional mutation increasing the hydrolytic activity of the parent enzyme. In urinary tract infections, a therapeutic option is the Use of a combination penicillin + β-lactamase inhibitor. Combination of a third-generation cephalosporin, such as cefotaxime, with sulbactam could also be of interest. In severe infections, the most potent β-lactams are the carbapenems that are highly resistant to the hydrolytic activity of all the TEM- and SHV-related β-lactamases. The 7-α-methoxy cephalosporins, such as cefoxitin, cefotetan andlatamoxef, are often effective. Of these, cefotetan andlatamoxef are the most active. However, cephamycins should beused with caution to treat infections caused by ESBL-producing K. pneumoniae because of the relative ease with which clinical strains decrease the expression of outer membrane proteins.