ß-Lactamases involved in resistance to broad-spectrum cephalosporins in Escherichia coli and Klebsiella spp. clinical isolates collected between 1994 and 1996, in Barcelona (Spain)

Montserrat Sabaté, Elisenda Miró*, Ferran Navarro, Clara Vergés, Roxana Aliaga, Beatriz Mirelis and Guillermo Prats

Departament de Microbiologia, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma, Av. Sant Antoni MªClaret, 167, 08025 Barcelona, Spain

Received 29 March 2001; returned 2 August 2001; revised 21 December 2001; accepted 14 March 2002.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The aim of this study was to evaluate the incidence of decreased susceptibility to broad-spectrum cephalosporins in Enterobacteriaceae that lack inducible chromosomal bla genes, and to determine the enzymes responsible for resistance. From all clinically relevant Enterobacteriaceae strains isolated between 1994 and 1996, 88 of 7054 Escherichia coli, seven of 581 Klebsiella pneumoniae and 23 of 166 Klebsiella oxytoca strains were studied because of their decreased susceptibilities to broad-spectrum cephalosporins (as reflected in intermediate susceptibilities and/or positive synergy tests and/or irregular crenellated inhibition zones). The most frequent mechanism implicated in decreased susceptibility to broad-spectrum cephalosporins displayed by E. coli and K. oxytoca was hyperproduction of chromosomal ß-lactamase, followed by plasmid-mediated SHV-1 hyperproduction in E. coli. In our hospital, the incidence of plasmid-mediated extended-spectrum ß-lactamases (ESBLs) between 1994 and 1996 was low. ESBLs were found in only 10 (0.14%) E. coli strains (six CTX-M-9, two TEM-12 and two SHV-2), in one (0.17%) K. pneumoniae strain (SHV-2) and in no K. oxytoca strains. The relatively wide variety of ß-lactamases that were detected among these common bacteria isolated from a single medical centre, including non-TEM- and non-SHV-derived ESBLs, appears epidemiologically remarkable.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Resistance to ß-lactam antibiotics in Gram-negative bacteria can be due to three mechanisms: decreased accumulation of the drugs by the cell, hydrolysis of the antibiotics by ß-lactamases or alterations to the penicillin-binding proteins that reduce their affinities for the drugs. The most common mechanism of resistance in Gram-negative bacteria causing clinically significant infection remains the production of ß-lactamases, including chromosome- and plasmid-encoded enzymes. The widespread occurrence of plasmid-mediated TEM-1, TEM-2, SHV-1 and OXA-1 ß-lactamases was therapeutically resolved by the introduction of cephamycins, broad-spectrum cephalosporins, such as cefotaxime and ceftazidime, and monobactams. However, Gram-negative bacteria quickly acquired resistance to these drugs by the following means: (i) derepression of chromosomal class C ß-lactamase synthesis in species with inducible enzyme production; (ii) hyperproduction of a ß-lactamase; (iii) acquisition of plasmid-encoded extended-spectrum ß-lactamases (ESBLs); or (iv) production of plasmid-mediated cephamycinases.1

In recent years, several outbreaks caused by ESBL production in Enterobacteriaceae have been reported. The enzymes found most commonly are TEM derivatives and, to a lesser extent, SHV and OXA derivatives.2,3 However, in the past 12 years a small, growing family, the CTX-M enzymes, has been detected in a variety of Enterobacteriaceae species, from widely separated geographical regions.417 Additionally, different subtypes of cephamycinases, encoded by plasmids derived from the ampC genes of Citrobacter freundii (CMY-2, CMY2b, LAT-1, LAT-2 and BIL-1),1823 Enterobacter cloacae (MIR-1 and ACT-1),24,25 Morganella morganii (DHA-1)26 and others of unknown phylogenetic origin (CMY-1, MOX-1 and FOX-1–3),2731 are spreading widely.

The aims of this study were to evaluate the incidence of decreased susceptibility to broad-spectrum cephalosporins in species of Enterobacteriaceae without inducible chromosomally encoded class C ß-lactamases over a significant period, and to determine the enzymes responsible for resistance.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

Among all clinically relevant Enterobacteriaceae strains without inducible chromosomally encoded class C ß-lactamases isolated in our laboratory between 1994 and 1996, a total of 88 of 7054 Escherichia coli, seven of 581 Klebsiella pneumoniae and 23 of 166 Klebsiella oxytoca strains were studied. They were selected because of their decreased susceptibilities to broad-spectrum cephalosporins, as indicated by an inhibitory zone diameter smaller than that indicative of intermediate breakpoint values and/or a positive synergy test between clavulanic acid, cefotaxime, ceftazidime or aztreonam, and/or an irregular crenellated zone consisting of ‘scatter colonies’, with small or normal-sized zones of inhibition around the discs of cephalosporins. Subsequently, the disc diffusion synergy tests were repeated using different disc separation distances, and the MICs of the ß-lactams were determined.

One strain per patient was selected for further study. A total of 247 strains were isolated from urine, six from blood, seven from wounds and one from sputum.

Antibiotic susceptibility testing

Initially, antibiotic susceptibility was determined by disc diffusion assay according to NCCLS recommendations.32 The antibiotics tested were ampicillin, ticarcillin, piperacillin, co-amoxiclav, cefazolin, cefoxitin, cefotaxime, ceftazidime and aztreonam. MICs were determined using two-fold antibiotic dilution series in agar according to NCCLS recommendations.32 E. coli ATCC 25922 and E. coli ATCC 35218 were used as control strains for antibiotic susceptibility studies.

Transfer of ß-lactam resistance by conjugation

Bacterial matings were performed on solid medium as described previously,33 using E. coli K-12 C600 (NalR) and E. coli HB101 (NalR KmR) as recipients. Ampicillin (100 mg/L) and kanamycin (50 mg/L) or nalidixic acid (50 mg/L) were used for selection of transconjugants.

Analytical isoelectric focusing of ß-lactamase

Crude cell extracts containing ß-lactamase were prepared from 250 mL Luria–Bertani broth cultures (Oxoid, Basingstoke, UK). Cells were pelleted by centrifugation and washed in double-distilled water and repelleted. Cells were resuspended in water (5 mL) and treated with an ultrasonicator (Labsonic2000; Biotech International, Leicester, UK) for three cycles of 15 s at 4°C. Cell debris was removed by centrifugation and the supernatants of the sonic extracts were frozen at –20°C until tested.

ß-Lactamases were characterized initially by isoelectric focusing in polyacrylamide gels with a pH gradient from 4 to 11 (SERVALYT 4-9 T, 9-11 T; Serva, Heidelberg, Germany) as described previously.33,34 ß-Lactamase activities in the gel were detected iodometrically using penicillin and ceftriaxone as substrates.34 The ß-lactamases TEM-1, TEM-2, OXA-1, SHV-2, SHV-4, SHV-5, CTX-M-4, CTX-M-9 and AmpC from E. coli K12 were used as standards.

ß-Lactamase assays

Hydrolysis of ß-lactam antibiotics was monitored spectrophotometrically using a Biochrom 4060 spectrophotometer (Pharmacia, Uppsala, Sweden) as described previously.33 Briefly, one unit of enzymic activity is defined as the amount of enzyme that hydrolyses 1 mmol of substrate in 1 min at 25°C in 0.1 M phosphate buffer (pH 7). The molar extinction coefficients used were as follows: penicillin (233 nm), 2.34/mM/cm; cefaloridine (260 nm), 13.19/mM/cm; cefotaxime (264 nm), 14.21/mM/cm; ceftazidime (260 nm), 20/mM/cm. Hyperproduction of chromosomal ß-lactamase in E. coli was determined according to Martínez-Martínez et al.35 Protein concentration was measured by the Bradford method.

PCR

PCR amplification was performed as described previously by various authors. For blaTEM, we used primers TEM-P3 and TEM-P4;36 for blaSHV genes, primers were SHVA and SHVB;36,37 for blaCTX-M-9 genes, primers were CTX-M-9IATG and CTX-M-9ISTOP;16,38 and for the blaOXY-1 and blaOXY-2 alleles of K. oxytoca, primer pairs were OXY-1A and OXY-1B, and OXY-2A and OXY-2B,39 respectively (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1.  Nucleotide sequences of the oligonucleotides used for PCR amplification and for DNA sequencing
 
To amplify the chromosomal ampC of E. cloacae and C. freundii and related genes encoding the plasmid-mediated cephamycinases CMY-2–7 and LAT-1–4, we used the degenerate primers ampC A1 and ampC A2 (Table 1) designed by Koeck et al.22 These primers did not amplify ampC of E. coli.

The promoter region of blaSHV was amplified using primers FOR2 and REV2 (Table 1). Amplification conditions were as follows: denaturation at 94°C for 5 min; 27 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2.5 min; and a final extension period of 72°C for 7 min.

The blaOXA-1 gene was amplified using primers OXA1/4A and OXA1/4B (Table 1). The amplification conditions were: denaturation at 94°C for 5 min; 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; and a final extension period of 72°C for 7 min.

Strains producing the ß-lactamases used as controls in isoelectric focusing studies were used as controls in these experiments.

DNA sequencing

Direct DNA sequencing of PCR products was carried out using the dideoxy method16 with fluorescent primers and the Automatic Laser Fluorescent DNA Sequencer (ALF; Pharmacia). Primers used were those listed in Table 1.

Nucleotide sequences and deduced amino acid sequences were analysed using software available via the Internet at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) and the Pedro’s BioMolecular Research Tools website (www.ub.es/dbqm/inicial/rt_all.htm).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Eighty-eight of 7054 E. coli strains (1.2%), seven of 581 K. pneumoniae strains (1.2%) and 23 of 166 K. oxytoca strains (13.8%) were selected, between 1994 and 1996, because of their decreased susceptibility or resistance to broad-spectrum cephalosporins.

Hyperproduction of AmpC chromosomal ß-lactamase present in E. coli (Table 2)

Among the 88 E. coli strains, 48 (54.5%) were resistant to co-amoxiclav (MIC 16–>128 mg/L; MIC90 128 mg/L) and cefoxitin (MIC 32–>128 mg/L; MIC90 > 128 mg/L), and showed decreased susceptibilities or resistance to cefotaxime (MIC <= 0.5–32 mg/L; MIC90 8 mg/L), ceftazidime (MIC 1–>64 mg/L; MIC90 32 mg/L) and aztreonam (MIC <= 4–64 mg/L; MIC90 8 mg/L). Isoelectric points (pIs) of the ß-lactamases in crude cell extracts of all 48 isolates were >=9. Twenty-one of these isolates contained a second ß-lactamase, all with the same pI of 5.4, which showed no activity against ceftriaxone in the isoelectric focusing gel, indicating production of a TEM-1 ß-lactamase. The resistance and enzyme patterns are indicative of the hyperproduction of the chromosomal class C ß-lactamase or the production of a plasmid-encoded cephamycinase.3,35 No amplicons were obtained for any of the strains using the degenerate primers (ampC A1 and ampC A2) that amplify the chromosomal ampC genes of E. cloacae and C. freundii and the genes of the more common plasmid-encoded cephamycinases CMY-2–7 LAT-1–4, BIL-1, MIR-1 and ACT-1.1825 The results indicate that the strains do not produce a plasmid-mediated cephamycinase belonging to this group. Moreover, we were able to disregard the possibility of the production of a plasmid-encoded cephamycinase belonging to the group that includes CMY-1, CMY-8, MOX-1, FOX-1, FOX-2 and FOX-3,2731 and the DHA-126 cephamycinase that belongs to another group, because each of these ß-lactamases has a pI <= 8.25. Bacterial mating experiments with three of the strains as donors failed to demonstrate transfer of the broad-spectrum resistance phenotype. Kinetic studies of ß-lactamase activity from six strains showed that cloxacillin (250 µM) inhibited 52.4–91.1% of the ß-lactamase activity, whereas clavulanic acid (2 µM) inhibited 9.1–48.1%, and ß-lactamase activities ranged from 18.7 to 132 mmol/min/g (Table 3). These data support the hypothesis that AmpC ß-lactamase hyperproduction is the main mechanism of this resistance phenotype. Nevertheless, the MICs for these strains could be influenced by other mechanisms, such as a change in outer membrane permeability.35


View this table:
[in this window]
[in a new window]
 
Table 2.  ß-Lactamases detected in E. coli and Klebsiella strains resistant to broad-spectrum cephalosporins
 

View this table:
[in this window]
[in a new window]
 
Table 3.  ß-Lactamase activity against cefaloridine with or without ß-lactamase inhibitors
 
E. coli contains a chromosomal ampC gene, with a weak promoter and a loop attenuator control element that accounts, at least in part, for the very low enzyme production usually seen.4042 Strains with the wild-type gene and standard promoter are inherently susceptible to ampicillin. Occasionally, the enzyme is hyperproduced, and these strains are resistant to ampicillin, cefazolin, cefoxitin and have reduced susceptibilities to co-amoxiclav and the oxyiminocephalosporins.3,42 Such hyperproduction can be caused by gene amplification on the bacterial chromosome,43 by mutations that create a stronger promoter44 or by attenuator mutations that abolish the transcription termination complex, increasing ß-lactamase production.41

Hyperproduction of SHV-1 (Table 2)

Twenty-eight of 88 (31.8%) E. coli strains and six of seven (85.7%) K. pneumoniae strains gave an irregular crenellated inhibition zone, consisting of ‘scatter colonies’ with small or normal-sized zones, around discs containing co-amoxiclav or ceftazidime. All strains were found to be resistant to penicillins; most are also resistant to narrow-spectrum cephalosporins and have reduced susceptibilities to co-amoxiclav (MIC 8–32 mg/L; MIC90 32 mg/L) and ceftazidime (MIC 1–32 mg/L; MIC90 32 mg/L), but are susceptible to cefoxitin (MIC 2–4 mg/L; MIC90 4 mg/L), cefotaxime (MIC <= 0.5 mg/L) and aztreonam (MIC <= 2 mg/L). Co-amoxiclav and ceftazidime showed synergy against all strains in disc tests, but with a small and atypical area of growth inhibition enhancement. These characteristics are compatible with production of ESBLs. Transconjugants were obtained from matings with five E. coli strains and two K. pneumoniae isolates. Analytical isoelectrofocusing revealed that the E. coli transconjugants and the parental donor strains each had a single band of ß-lactamase activity focusing at pH 7.6 and showed the same levels of resistance to co-amoxiclav (MIC 8–32 mg/L) and ceftazidime (MIC 4–16 mg/L) as the parental strains. PCR products lacking a NheI restriction site were obtained with primers specific for blaSHV, indicating production of SHV-1 ß-lactamase.37 K. pneumoniae transconjugants displayed low level resistance to ceftazidime (MIC <= 0.75 mg/L) and co-amoxiclav (MIC <= 4 mg/L), consistent with production of SHV-1.

Hyperproduction of SHV-1 by E. coli and K. pneumoniae clinical strains, resulting in decreased susceptibility to broad-spectrum cephalosporins, has been described previously.32,4547

With respect to the E. coli isolates investigated, we reported previously33 that the mechanism of decreased susceptibility to ceftazidime is due to an approximate five-fold hyperproduction of SHV-1. To determine whether SHV-1 hyperproduction is due to a mutation(s) in the promoter region, we obtained PCR amplicons of 500 bp corresponding to the region from two of the E. coli strains. The sequences of the DNA fragments were identical to that of a control strain, in which SHV-1 production is normal. Therefore, we concluded that the increased production of SHV-1 in these particular E. coli isolates is not due to a mutated promoter region. Further studies are needed to investigate whether hyperproduction of SHV-1 in the clinical E. coli strains reflects multiple copies of the blaSHV-1 gene carried on a plasmid or a multicopy plasmid carring the blaSHV-1 gene.

It has been reported that K. pneumoniae expresses chromosomally encoded ß-lactamases such as SHV-1 or related ß-lactamases (LEN-1)3,48 that confer relatively low degrees of resistance to compounds such as ampicillin and carbenicillin.49 The resistance mechanism in the six K. pneumoniae isolates may therefore be hyperproduction of chromosomally encoded SHV-1 ß-lactamase or the additive effect of chromosomal- and plasmid-encoded SHV-1.

SHV-2-like production (Table 2)

Two of 88 (2.3%) E. coli strains and one of seven (14.3%) K. pneumoniae strains exhibited a clear synergy between broad-spectrum cephalosporins and co-amoxiclav, with MICs of cefotaxime of 64 mg/L, ceftazidime 32 mg/L and aztreonam 4 mg/L, indicating production of ESBLs. All three strains produced ß-lactamases with pIs of 7.6 and yielded blaSHV PCR products containing a NheI site.37 Substrate profiles showed high activity of cefotaxime (380.3, 43.4 and 25.3 mmol/min/g). These results indicate the expression of a SHV-2-like enzyme.

Production of TEM-12 in E. coli (Table 2)

Two of 88 strains of E. coli, with MICs of 0.25 mg/L of cefotaxime, 16 and 32 mg/L of ceftazidime and 1 and 2 mg/L of aztreonam, each produced a ß-lactamase with a pI of 5.2; both yielded blaTEM PCR amplicons, the sequences of which revealed carriage of blaTEM-12.36 However, we were unable to detect ß-lactamase activity in cell extracts with broad-spectrum cephalosporins.

CTX-M-9 like production (Table 2)

Six of 88 strains of E. coli produced two ß-lactamases with pIs of 5.4 and c. 8. Each of these strains, with MICs of 16 or 32 mg/L of cefotaxime, 1 or 2 mg/L of ceftazidime and 1–8 mg/L of aztreonam, yielded a PCR product using primers specific for blaTEM, consistent with production of a TEM-1 ß-lactamase (equating with the ß-lactamase with pI 5.4, which showed no activity on isoelectric focusing with ceftriaxone as a substrate). PCR assay for blaCTX-M-9 was positive, consistent with production of a ß-lactamase with pI 8. One of these strains has been described previously.16

OXA-30 production

For two of 88 (2.3%) E. coli strains there was distinct but weak synergy between cefotaxime and co-amoxiclav, which suggests production of ESBLs. MIC determinations showed that both strains were resistant to penicillins and to narrow-spectrum cephalosporins, with reduced susceptibility or resistance to co-amoxiclav (MIC > 16 mg/L), cefotaxime (MIC 4 mg/L) and ceftazidime (MIC 2–4 mg/L), but were susceptible to aztreonam (MIC <= 0.25 mg/L). Isoelectric focusing studies showed production of a ß-lactamase with a pI of c. 7.4. PCR assays with OXA1/4 primers were positive, and the enzyme substrate profiles showed low activity of cefotaxime (2 and 4.8 mmol/min/g), which indicates the production of an extended-spectrum oxacillinase. The sequences of the amplicons from these two strains were identical to that of blaOXA-30,50 which differs from blaOXA-1 at codon 131 [Arg (AGA) to Gly (GGA)].

Hyperproduction of blaoxy chromosomal ß-lactamases present in K. oxytoca (Table 2)

Twenty-three of 166 (13.8%) K. oxytoca strains showed decreased susceptibilities to broad-spectrum cephalosporins and resistance to aztreonam (MIC 8–>64 mg/L; MIC90 > 64 mg/L), but were susceptible or showed decreased susceptibilities to cefotaxime and ceftazidime (MICs <= 2 mg/L). The pIs obtained for ß-lactamases from these isolates were c. 8 (one strain), 7.8 (seven strains), c. 6.8 (13 strains), c. 6.4 (one strain) and 5.2 (one strain). Two strains that produced ß-lactamases with pI 7.8 each expressed a second ß-lactamase with pI 5.4.

The eight strains with ß-lactamases of pI 7.8 or 8 were PCR positive with primers specific for OXY-1, representing a 4.8% carriage of blaOXY-1, and the 15 strains with ß-lactamases of pI 5.2, 6.4 or 6.8 were PCR positive with primers specific for OXY-2, representing a 9% carriage rate for blaOXY-2. These results suggested to us that resistance to broad-spectrum cephalosporins in all 23 strains is mediated by the hyperproduction of chromosomal K1 ß-lactamase. K. oxytoca, like K. pneumoniae, carries a chromosomally encoded class A ß-lactamase. The ß-lactamase genes of K. oxytoca have been assigned to two main subgroups: blaOXY-1 and blaOXY-2.51 In this study, OXY-1-type ß-lactamase is represented by two different forms, with pIs 7.8 and 8, with most isolates producing the pI 7.8 form (seven of eight strains). OXY-2 ß-lactamases had pIs 5.2, 6.4 and 6.8, with most producing the pI 6.8 form (13 of 15 strains). The results obtained for OXY-1 are similar to previous findings.52 The OXY-2 results are different to those previously reported, where the most frequent form was the ß-lactamase with a pI of 5.2, representing 59% of all OXY-2 enzymes. The susceptibility patterns for ß-lactam hydrolysis revealed that OXY-2 enzymes hydrolyse some ß-lactams [ceftazidime (MIC <= 0.12–4 mg/L; MIC90 4 mg/L), cefotaxime (MIC 0.5–4 mg/L; MIC90 4 mg/L) and aztreonam (MIC 8–>64 mg/L; MIC90 > 64 mg/L)] better than OXY-1 enzymes [ceftazidime (MIC <= 0.12–0.5 mg/L; MIC90 0.5 mg/L), cefotaxime (MIC <= 0.12–0.5 mg/L; MIC90 0.5 mg/L) and aztreonam (MIC 8–16 mg/L; MIC90 16 mg/L)]. We found that the isolates with the OXY-1 variant were less resistant to ß-lactams than those with OXY-2, in agreement with previous reports.39

Plasmid-determined ß-lactamases are reported to be rare in K. oxytoca. Reig et al.53 found that only 13 of 131 strains produced such enzymes (usually TEM-1). Leung et al.54 did not find any plasmid-encoded ß-lactamases in K. oxytoca. Our results showed that two (8.7%) of the 23 K. oxytoca strains with decreased susceptibilities to broad-spectrum cephalosporins produced ß-lactamases with pIs of 5.4. PCR and isoelectric focusing analysis indicated the presence of blaTEM-1 in these isolates.

Taken together these results show that the most frequent mechanism implicated in decreased susceptibility to broad-spectrum cephalosporins of E. coli, K. pneumoniae and K. oxytoca is hyperproduction of a chromosomal ß-lactamase, followed by plasmid-mediated SHV-1 hyperproduction in E. coli. Resistance to broad-spectrum cephalosporins is still rare in Spain.36,55,56 In our hospital, the incidence of ESBLs was very low and similar for E. coli (0.14%) and K. pneumoniae (0.17%). The ESBLs found in the study were SHV-2 and TEM-12 and the recently described CTX-M-9,16 which was more common than the other two (six of 10 ESBLs detected).

The incidence of ESBLs has been followed in our hospital from 1997 to 1999. In this period, 37 of 7705 (0.5%) E. coli, eight of 491 (1.6%) K. pneumoniae and one of 913 (0.1%) Salmonella enterica38 produced an ESBL. The CTX-M-9 enzyme accounted for >70% of the total of ESBLs detected in our laboratory among E. coli.

Like the CTX-M family enzymes, cephamycinases encoded by plasmids are another growing family that is spreading widely. During the two periods of time evaluated, we did not detect plasmid-encoded class C ß-lactamases. However, in January 2000 we identified the first isolate from our hospital that produces CMY-2, a Salmonella enterica serovar Mikawasima. Two additional strains producing a CMY-2 enzyme (one K. pneumoniae and one Proteus mirabilis strain) were later isolated.57

Of note is the relatively wide variety of ß-lactamases that were detected among these common bacteria isolated in a single medical centre, especially since few were TEM- and SHV-based ESBLs.


    Acknowledgements
 
We are grateful to D. Sirot for his helpful advice and comments and to C. Roig for excellent technical assistance. We thank the Fundación Mª Francisca de Roviralta for financial support. This work was supported partially by governmental grants FIS 97/0623, FIS 98/1522 and FIS 98/1293.


    Footnotes
 
* Corresponding author. Tel: +34-93-2919071; Fax: +34-93-2919070; E-mail: emiro{at}hsp.santpau.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Piddock, L. J. V., Walters, R. N., Jin, Y. F., Turner, H. L., Gascoyne-Binzi, D. M. & Hawkey, P. M. (1997). Prevalence and mechanism of resistance to ‘third-generation’ cephalosporins in clinically relevant isolates of Enterobacteriaceae from 43 hospitals in the UK, 1990–1991. Journal of Antimicrobial Chemotherapy 39, 177–87.[Abstract]

2 . Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 1211–33.[Free Full Text]

3 . Livermore, D. M. (1995). ß-Lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]

4 . Barthélémy, M., Peduzzi, J., Bernard, H., Tancrède, C. & Labia, R. (1992). Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum ß-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca. Biochimica et Biophysica Acta 1122, 15–22.[ISI][Medline]

5 . Bauernfeind, A., Casellas, J. M., Goldberg, M., Holley, M., Jungwirth, R., Mangold, P. et al. (1992). A new plasmidic cefotaximase from patients infected with Salmonella typhimurium. Infection 20, 158–63.[ISI][Medline]

6 . Bauernfeind, A., Grimm, H. & Schweighart, S. (1990). A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection 18, 294–8.[ISI][Medline]

7 . Bauernfeind, A., Stemplinger, I., Junwirth, R., Ernst, S. & Casellas, J. M. (1996). Sequences of ß-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their sequences with those of other ß-lactamases. Antimicrobial Agents and Chemotherapy 40, 509–13.[Abstract]

8 . Bernard, H., Tancrede, C., Livrelli, V., Morand, A., Barthélémy, M. & Labia, R. (1992). A novel plasmid-mediated extended-spectrum ß-lactamase not derived from TEM- or SHV-type enzymes. Journal of Antimicrobial Chemotherapy 29, 590–2.[ISI][Medline]

9 . Bradford, P. A., Yang, Y., Sahm, D., Grope, I., Gardovska, D. & Storch, G. (1998). CTX-M-5, a novel cefotaxime-hydrolyzing ß-lactamase from an outbreak of Salmonella typhimurium in Latvia. Antimicrobial Agents and Chemotherapy 42, 1980–4.[Abstract/Free Full Text]

10 . Gazouli, M., Sidorenko, S. V., Tzelepi E., Kozlova, N. S., Gladin, D. P. & Tzouvelekis, L. S. (1998). A plasmid-mediated ß-lactamase conferring resistance to cefotaxime in a Salmonella typhimurium clone found in St Petersburg, Russia. Journal of Antimicrobial Chemotherapy 41, 119–21.[Abstract]

11 . Gazouli, M., Tzelepi, E., Markogiannakis, A., Legakis, N. J. & Tzouvelekis, L. S. (1998). Two novel plasmid-mediated cefotaxime-hydrolyzing ß-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium. FEMS Microbiology Letters 165, 289–93.[ISI][Medline]

12 . Gazouli, M., Tzelepi, E., Sidorenko, S. V. & Tzouvelekis, L. S. (1998). Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A ß-lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis. Antimicrobial Agents and Chemotherapy 42, 1259–62.[Abstract/Free Full Text]

13 . Gniadkowski, M., Schneider, I., Palucha, A., Jungwirth, R., Mikiewicz, B. & Bauernfeind, A. (1998). Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing ß-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrobial Agents and Chemotherapy 42, 827–32.[Abstract/Free Full Text]

14 . Ishii, Y., Ohno, A., Taguchi, H., Imajo, S., Ishiguro, M. & Matsuzawa, H. (1995). Cloning and sequence of the gene encoding a cefotaxime-hydrolysing class A ß-lactamase isolated from Escherichia coli. Antimicrobial Agents and Chemotherapy 39, 2269–75.[Abstract]

15 . Ma, L., Ishii, Y., Ishiguro, M., Matuzawa, H. & Yamaguchi, K. (1998). Cloning and sequencing of the gene encoding TOHO-2, a class A ß-lactamase preferentially inhibited by tazobactam. Antimicrobial Agents and Chemotherapy 42, 1181–6.[Abstract/Free Full Text]

16 . Sabaté, M., Tarragó, R., Navarro, F., Miró, E., Vergés, C., Barbé, J. et al. (2000). Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing ß-lactamase (CTX-M-9) from Escherichia coli in Spain. Antimicrobial Agents and Chemotherapy 44, 1970–3.[Abstract/Free Full Text]

17 . Tzouvelekis, L. S., Gazouli, M., Markogiannakis, A., Paraskaki, E., Legakis, N. J. & Tzelepi, E. (1998). Emergence of resistance to third-generation cephalosporins amongst Salmonella typhimurium isolates in Greece: report of the first three cases. Journal of Antimicrobial Chemotherapy 42, 273–5.[Free Full Text]

18 . Bauernfeind, A., Hohl, P., Schneider, I., Jungwirth, R. & Frei, R. (1998). Escherichia coli producing a cephamycinase (CMY-2) from a patient from the Libyan–Tunisian border region. Clinical Microbiology and Infection 4, 168–70.[Medline]

19 . Bauernfeind, A., Stemplinger, I., Jungwirth, R. & Giamarellou, H. (1996). Characterization of the plasmidic ß-lactamase CMY-2, which is responsible for cephamycin resistance. Antimicrobial Agents and Chemotherapy 40, 221–4.[Abstract]

20 . Fosberry, A. P., Payne, D. J., Lawlor, E. & Hodgson, J. E. (1994). Cloning and sequence analysis of blaBIL-1, a plasmid-mediated class C ß-lactamase gene in Escherichia coli BS. Antimicrobial Agents and Chemotherapy 38, 1182–5.[Abstract]

21 . Gazouli, M., Tzouvelekis, L. S., Prinarakis, E., Miriagou, V. & Tzelepi, E. (1996). Transferable cefoxitin resistance in enterobacteria from Greek hospitals and characterization of a plasmid-mediated group 1 ß-lactamase (LAT-2). Antimicrobial Agents and Chemotherapy 40, 1736–40.[Abstract]

22 . Koeck, J. L., Arlet, G., Phillipon, A., Basmaciogullari, S., Thien, H. V., Buisson, Y. et al. (1997). A plasmid-mediated CMY-2 ß-lactamase from an Algerian clinical isolate of Salmonella senftenberg. FEMS Microbiology Letters 152, 255–60.[ISI][Medline]

23 . Tzouvelekis, L. S., Tzelepi, E. & Mentis, A. F. (1994). Nucleotide sequence of a plasmid-mediated cephalosporinase gene (blaLAT-1) found in Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 38, 2207–9.[Abstract]

24 . Bradford, P. A., Urban, C., Mariano, N., Projan, S. J., Rahal, J. J. & Bush, K. (1997). Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrobial Agents and Chemotherapy 41, 563–9.[Abstract]

25 . Papanicolaou, G. A., Medeiros, A. A. & Jacoby, G. A. (1990). Novel plasmid-mediated ß-lactamase (MIR-1) conferring resistance to oxyimino- and {alpha}-methoxy ß-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 34, 2200–9.[ISI][Medline]

26 . Barnaud, G., Arlet, G., Verdet, C., Gaillot, O., Lagrange, P. H. & Phillippon, A. (1998). Salmonella enteritidis: AmpC plasmid-mediated inducible ß-lactamase (DHA-1) with an ampR gene from Morganella morganii. Antimicrobial Agents and Chemotherapy 42, 2352–8.[Abstract/Free Full Text]

27 . Bauernfeind, A., Stemplinger, I., Jungwirth, R., Wilhelm, R. & Chong, Y. (1996). Comparative characterization of the cephamycinase blaCMY-1 gene and its relationship with other ß-lactamase genes. Antimicrobial Agents and Chemotherapy 40, 1926–30.[Abstract]

28 . Bauernfeind, A., Wagner, S., Jungwirth, R., Schneider, I. & Meyer, D. (1997). A novel class C ß-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins. Antimicrobial Agents and Chemotherapy 41, 2041–6.[Abstract]

29 . González, M., Perez-Diaz, J., Ayala, J., Casellas, J. M., Martínez-Beltran, J., Bush, K. et al. (1994). Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated ß-lactamase with two molecular variants. Antimicrobial Agents and Chemotherapy 38, 2150–7.[Abstract]

30 . Horii, T., Arakawa, Y., Ohta, M., Ichiyama, S., Wacharotayankun, R. & Kato, N. (1993). Plasmid-mediated AmpC-type ß-lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum ß-lactams, including moxalactam. Antimicrobial Agents and Chemotherapy 37, 984–90.[Abstract]

31 . Marchese, A., Arlet, G., Shito, G. C., Lagrange, P. H. & Philippon, A. (1998). Characterization of FOX-3, an AmpC-type plasmid-mediated ß-lactamase from an Italian isolate of Klebsiella oxytoca. Antimicrobial Agents and Chemotherapy 42, 464–7.[Abstract/Free Full Text]

32 . National Committee for Clinical Laboratory Standards. (1995). Performance Standards for Antimicrobial Susceptibility Testing—Sixth Informational Supplement: Approved Standard M100-S6. NCCLS, Villanova, PA.

33 . Miró, E., Del Cuerpo, M., Navarro, F., Sabaté, M., Mirelis, B. & Prats, G. (1998). Emergence of clinical Escherichia coli isolates with decreased susceptibility to ceftazidime and synergic effect with co-amoxiclav due to SHV-1 hyperproduction. Journal of Antimicrobial Chemotherapy 42, 535–8.[Abstract]

34 . Barthélémy, M., Guionie, M. & Labia, R. (1979). ß-Lactamases: determination of their isoelectric points. Antimicrobial Agents and Chemotherapy 13, 696–8.

35 . Martínez-Martínez, L., Conejo, M. C., Pascual, A., Hernández-Allés, S., Ballesta, S., Ramírez de Arellano-Ramos, E. et al. (2000). Activities of imipenem and cephalosporins against clonally related strains of Escherichia coli hyperproducing chromosomal ß-lactamase and showing altered porin profiles. Antimicrobial Agents and Chemotherapy 44, 2534–6.[Abstract/Free Full Text]

36 . Sabaté, M., Vergés, C., Miró, E., Mirelis, B., Navarro, F., Del Rio, E. et al. (1999). Incidencia de betalactamasas de espectro ampliado en Escherichia coli en un hospital universitario durante 1994–96. Enfermedades Infecciosas y Microbiologia Clínica 17, 401–4.

37 . Nüesch-Inderbinen, M. T., Hächler, H. & Kayser, F. H. (1996). Detection of genes coding for extended-spectrum SHV betalactamases in clinical isolates by a molecular method, and comparison with the E test. European Journal of Clinical Microbiology and Infectious Diseases 15, 398–402.[ISI][Medline]

38 . Simarro, E., Navarro, F., Ruiz, J., Miró, E., Gómez, J. & Mirelis, B. (2000). Salmonella enterica serovar Virchow with CTX-M like ß-lactamase around Spain. Journal of Clinical Microbiology 38, 4676–8.[Abstract/Free Full Text]

39 . Gheorghiu, R., Yuan, M., Hall L. M. C. & Livermore, D. M. (1997). Bases of variation in resistance to ß-lactams in Klebsiella oxytoca isolates hyperproducing K1 ß-lactamase. Journal of Antimicrobial Chemotherapy 40, 533–41.[Abstract]

40 . Jaurin, B. & Grundstrom, T. (1981). ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of ß-lactamase of the penicillinase type. Proceedings of the National Academy of Sciences, USA 78, 4897–901.[Abstract]

41 . Jaurin, B., Grundstrom, T., Edlund, T. & Normark, S. (1981). The E. coli beta-lactamase attenuator mediates growth rate-dependent regulation. Nature 290, 221–5.[ISI][Medline]

42 . Nelson, E. C. & Elisha, B. G. (1999). Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 43, 957–9.[Abstract/Free Full Text]

43 . Edlund, T., Grundström, T. & Normark, S. (1979). Isolation and characterization of DNA repetitions carrying the chromosomal ß-lactamase gene of Escherichia coli K12. Molecular and General Genetics 173, 115–25.[Medline]

44 . Caroff, N., Espaze, E., Berard, I., Richet, H. & Reynaud, A. (1999). Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum ß-lactamase production. FEMS Microbiology Letters 173, 459–65.[ISI][Medline]

45 . Petit, A., Ben Yaghlane-Bouslama, H., Sofer, L. & Labia, R. (1992). Does high level production of SHV-type penicillinases confer resistance to ceftazidime in Enterobacteriaceae? FEMS Microbiology Letters 92, 89–94.[ISI]

46 . Rice, L. B., Carias, L. C., Hujer, A. M., Bonafede, M., Hutton, R., Hoyen, C. et al. (2000). High-level expression of chromosomally encoded SHV-1 ß-lactamase and an outer membrane protein change confer resistance to ceftazidime and piperacillin–tazobactam in a clinical isolate of K. pneumoniae. Antimicrobial Agents and Chemotherapy 44, 363–7.

47 . Sanders, C. C., Iaconis, J. P., Bodey, G. P. & Samonis, G. (1988). Resistance to ticarcillin–potassium clavulanate among clinical isolates of the family Enterobacteriaceae: role of PSE-1 ß-lactamase and high levels of TEM-1 and SHV-1 and problems with false susceptibility in disc-diffusion tests. Antimicrobial Agents and Chemotherapy 32, 1365–9.[ISI][Medline]

48 . Babini, G. S. & Livermore, D. M. (2000). Are SHV ß-lactamases universal in Klebsiella pneumoniae? Antimicrobial Agents and Chemotherapy 44, 2230.[Free Full Text]

49 . Labia, R., Fabre, C., Masson, J. M., Barthélémy, M., Heitz, M. & Pitton, J. S. (1979). Klebsiella pneumoniae strains moderately resistant to ampicillin and carbenicillin: characterization of a new ß-lactamase. Journal of Antimicrobial Chemotherapy 5, 375–82.[ISI][Medline]

50 . Siu, L. K., Lo, J. Y. C, Yuen, K. Y., Chau, P. Y., Ng, M. H. & Ho, P. L. (2000). ß-Lactamases in Shigella flexneri isolates from Hong Kong and Shanghai and a novel OXA-1-like ß-lactamase, OXA-30. Antimicrobial Agents and Chemotherapy 44, 2034–8.[Abstract/Free Full Text]

51 . Fournier, B., Roy, P. H., Lagrange, P. H. & Phillippon, A. (1996). Chromosomal ß-lactamase genes of Klebsiella oxytoca are divided into two main groups: blaoxy-1 and blaoxy-2. Antimicrobial Agents and Chemotherapy 40, 454–9.[Abstract]

52 . Fournier, B. & Roy, P. H. (1997). Variability of chromosomally encoded ß-lactamases from Klebsiella oxytoca. Antimicrobial Agents and Chemotherapy 41, 1641–8.[Abstract]

53 . Reig, R., Roy, C., Hermida, M., Teruel, D. & Coira, A. (1993). A survey of ß-lactamases from 618 isolates of Klebsiella spp. Journal of Antimicrobial Chemotherapy 31, 29–35.[Abstract]

54 . Leung, M., Shannon, K. & French, G. (1997). Rarity of transferable ß-lactamase production by Klebsiella species. Journal of Antimicrobial Chemotherapy 39, 737–45.[Abstract]

55 . Fernández-Aránguiz, A., Alonso, R., Colom, K., Gallego, L., Morla, A., Garaizar, J. et al. (1991). Estudio multicéntrico de la resistencia a la cefotaxima durante el año 1991: Detección y caracterización de nuevas ß-lactamasas de espectro ampliado. Revista Española de Quimioterapia 4, 137–44.

56 . Fernández-Rodríguez, A., Reguera, J. A., Pèrez-Díaz, J. C., Picazo, J. J. & Baquero, F. (1992). Primera epidemia española de resistencia plasmídica a cefalosporinas de tercera generación: implicación de SHV-2. Enfermedades Infecciosas y Microbiología Clínica 10, 456–61.

57 . Navarro, F., Perez-Trallero, E., Marimón, J. M., Aliaga, R., Gomariz, M. & Mirelis, B. (2001). CMY-2-producing Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Escherichia coli strains isolated in Spain (October 1999–December 2000). Journal of Antimicrobial Chemotherapy 48, 383–9.