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.
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Abstract |
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Introduction |
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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-13),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.
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Materials and methods |
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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 LuriaBertani 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).
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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 Pedros BioMolecular Research Tools website (www.ub.es/dbqm/inicial/rt_all.htm).
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Results and discussion |
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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 0.532 mg/L; MIC90 8 mg/L), ceftazidime (MIC 1>64 mg/L; MIC90 32 mg/L) and aztreonam (MIC
464 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-27 LAT-14, 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 52.491.1% of the ß-lactamase activity, whereas clavulanic acid (2 µM) inhibited 9.148.1%, 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
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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 832 mg/L; MIC90 32 mg/L) and ceftazidime (MIC 132 mg/L; MIC90 32 mg/L), but are susceptible to cefoxitin (MIC 24 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 832 mg/L) and ceftazidime (MIC 416 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 18 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 24 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 0.124 mg/L; MIC90 4 mg/L), cefotaxime (MIC 0.54 mg/L; MIC90 4 mg/L) and aztreonam (MIC 8>64 mg/L; MIC90 > 64 mg/L)] better than OXY-1 enzymes [ceftazidime (MIC
0.120.5 mg/L; MIC90 0.5 mg/L), cefotaxime (MIC
0.120.5 mg/L; MIC90 0.5 mg/L) and aztreonam (MIC 816 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.
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Acknowledgements |
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Footnotes |
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References |
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