a Departamento de Microbiologia, Faculdade de Farmácia, Universidade de Lisboa, Av. das Forças Armadas; b Laboratório de Microbiologia Clínica do Hospital de Santa Maria, Av. Prof. Egas Moniz, 1600 Lisbon; c Departamento de Microbiologia, Faculdade de Farmácia, Universidade do Porto, R. Anibal Cunha 164, 4050 Porto; d Departamento de Química (Centro de Química Fina e BiotecnologiaCQFB), Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Monte da Caparica, Portugal
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Abstract |
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Introduction |
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Materials and methods |
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A survey of K. pneumoniae resistant to ceftazidime from the Hospital de Santa Maria in Lisbon was carried out between April 1995 and March 1996. A total of 130 isolates was isolated from the intensive care unit, as well as from the internal medicine, surgery and paediatric wards. These isolates were from urine (72), blood cultures (25), respiratory tract (15) and other clinical specimens (18) including wounds, drainage fluids, catheter and faeces. In addition, eight isolates from sporadic cases (collected between 1991 and 1993 and during 1997) were also obtained from the neonatal intensive care unit (NICU) of the same hospital (three from respiratory tract, two from blood cultures, one from urine and two from environmental analysis).
All isolates were identified with the API 20E System (API System, bioMérieux, Lisbon, Portugal). Antibiotic susceptibility testing and genotypic analysis were performed on all isolates. Seventy-nine isolates, representative of different pulsed-field gel electrophoresis (PFGE) patterns (four isolates from between 1991 and 1993, 74 isolates from the 130 isolated in 1995 and 1996, and one isolate from 1997), were selected for conjugation studies, isoelectric focusing (IEF), ß-lactamase assays, plasmid characterization, DNA amplification and nucleotide sequence determination.
The nalidixic acid-resistant mutant of Escherichia coli K12 C6005 was used as the recipient for mating experiments. E. coli C600 (pCFF14) expressing CAZ-1/TEM-5, E. coli C600 (pCFF74) expressing CAZ-6/TEM-24, E. coli C600 (pCFF84) expressing CAZ-7/TEM-16 (all provided by Dr C. Chanal), E. coli J53 (pMG223) expressing TEM-10 and E. coli J53 (pMG225) expressing TEM-26 (both provided by Dr G. Jacoby) were used as references for ß-lactamases studies. E. coli TG1 and E. coli TOP 10F' (Alfagene, Lisbon, Portugal) were used in the transformation reactions.
Antibiotic susceptibility testing
Susceptibility to antimicrobial agents was determined by the standard disc diffusion method on MuellerHinton agar as described by the NCCLS.6 The following agents were tested: amoxycillin, amoxycillin plus clavulanic acid, cefoxitin, cefuroxime, ceftazidime, cefotaxime, aztreonam, imipenem, gentamicin, amikacin, netilmicin, norfloxacin, pefloxacin and nalidixic acid. The double-disc synergy test between clavulanic acid and extended-spectrum cephalosporins was used to detect production of ESBLs according to the method described by Ben Redjeb et al.7 The MICs of ceftazidime, aztreonam, cefepime, cefotaxime, cefuroxime and ceftazidime plus clavulanic acid were determined using Etest strips (AB Biodisk, Solna, Sweden) on Mueller Hinton agar.
Genotypic analysis by PFGE
Chromosomal DNA from each isolate was prepared and digested with XbaI (Biolabs) as described by Maslow et al.8 The restriction fragments generated were separated on a 1% agarose gel (SeaKem; B. Brown, Lisbon, Portugal) in 0.5x TBE buffer, pH 8.0. A Bio-Rad CHEF DRII apparatus (Paci, Lisbon, Portugal) was used at 200 V and 15°C for 22 h, with pulse times of 140 s, ramped linearly. A ladder of bacteriophage lambda concatemers (IZASA, Lisbon, Portugal) was used to determine the size of XbaI restriction fragments. Gels were interpreted as recommended by Tenover et al.9
Conjugal transfer of ceftazidime resistance
Conjugation was performed using the plate-mating method.10 Transconjugants were selected on agar containing ceftazidime and nalidixic acid at concentrations of 30 and 50 mg/L, respectively.
Isoelectric focusing
Crude preparations of ß-lactamases from K. pneumoniae and from their transconjugants were obtained by sonicating the cells in phosphate buffer, pH 7.0. IEF was performed by the method of Matthew et al.11 using an LKB Multiphor apparatus with PAG plates (pH 3.08.0; Pharmacia LKB, Lisbon, Portugal). ß-Lactamase activity was detected by the chromogenic nitrocefin test. Standard enzymes (including CAZ-1/TEM-5, CAZ-6/TEM-24, CAZ-7/TEM-16, TEM-10 and TEM-26) were used as pI markers.
ß-Lactamase assays
Enzymes were extracted from the transconjugants as described above. Hydrolysis of ß-lactam antibiotics (ceftazidime, cefotaxime and aztreonam) was monitored spectrophotometrically at 25°C in 0.1 M phosphate buffer, pH 7.0, using a Shimadzu UV-160A spectrophotometer, with each measurement taken at least in duplicate. The computer program Madonna12 was used to calculate kinetic parameters (Km and Vmax) by four methods of calculation (direct linear plot, LineweaverBurk, HanesWoolf and Eadie Hofstee).
Plasmid DNA
Plasmid DNA was isolated from K. pneumoniae and transconjugants by alkaline lysis.13 DNA samples were electrophoresed in 0.7% agarose gels at 12 V/cm for 2 h. Plasmid size was estimated using plasmid molecular weight standards. Restriction enzyme digestion was performed as described by Sambrook et al.14 with KpnI, PstI, BamHI and EcoRI (IZASA).
DNA amplification by PCR
A 1006 bp fragment of the TEM ß-lactamase gene carried by the isolates was amplified by PCR from the uncut plasmid DNA of the K. pneumoniae and respective transconjugants. TEM primers A and B15 (Table I) were used. Amplification conditions were the following: 32 cycles of 94°C for 1 min, 60°C for 1 min and 70°C for 1 min, with a final elongation step of 10 min.
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Determination of the nucleotide sequence of the ß-lactamase gene was performed using the dideoxynucleotide chain termination method.17 Two additional primers, C and D (Table I), were chosen using the program PCRPLAN (PCGene; IntelliGenetics, Geneva, Switzerland), to obtain the complete sequence of both strands of the 1006 bp fragment.
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Results |
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All 138 isolates of K. pneumoniae were resistant to amoxycillin, ceftazidime, aztreonam, gentamicin and netilmicin, but they were susceptible to imipenem, norfloxacin, pefloxacin and nalidixic acid. All had reduced susceptibility to amikacin, cefoxitin, cefuroxime and cefotaxime. All isolates exhibited a degree of resistance to ceftazidime or aztreonam much higher than that to cefotaxime. The presence of clavulanic acid enhanced the antibacterial activities of ceftazidime and aztreonam.
All the isolates had MICs > 32 mg/L for ceftazidime. However, these isolates had various degrees of susceptibility to aztreonam (MIC ranging from 16 to >256 mg/L), cefotaxime (MIC range 0.1253.0 mg/L), cefepime (MIC range 0.383.0 mg/L) and cefuroxime (MIC range 1.512 mg/L). The MICs of ceftazidime plus clavulanic acid ranged from <0.125 to 0.75 mg/L, and showed that the ß-lactamase inhibitor clavulanic acid restored susceptibility to ceftazidime. The MICs for the E. coli transconjugants were identical to those for the parent isolates.
Genomic macrorestriction analysis by PFGE
PFGE of genomic DNA after digestion with XbaI produced an average of 21 fragments ranging in size from less than 36 kb to approximately 700 kb. Among the 138 isolates analysed, 26 different patterns or PFGE types were revealed. These were classified as: profiles IVIII (Figure 1 and Table II
), and profile X, which included all other isolates (18) with unique banding patterns. Among isolates demonstrating profiles I and II, one or two variants showed differences of one band. Among the 130 survey isolates there were 18 distinct genotypes. Profile I was identified in 63 isolates and profile II was found in 37 isolates. Profiles IIIVIII were identified in 20 isolates. A high level of genetic heterogeneity was found among the remaining 10 isolates from the survey. This same heterogeneity was observed among the isolates obtained from the sporadic cases at the NICU.
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IEF showed that the isolates had strongly reactive ß-lactamases of pI 5.6. In addition, these organisms had ß-lactamases that focused almost identically to the TEM-1 and SHV-1 (pI 5.4 and 7.6, respectively). Some isolates produced one band of pI 6.8. The transconjugants produced only one band of enzyme activity with a pI of 5.6.
Kinetic parameters
The ß-lactamase with a pI of 5.6 hydrolysed ceftazidime (Vmax 13.8 x 101 µM/min/mg total protein; Km 110.1 µM) faster than cefotaxime (Vmax 1.38 x 101 µM/min/mg total protein; Km 13.9 µM) or aztreonam (Vmax 0.47 x 101 µM/min/mg total protein; Km 42.9 µM). Identical values were found with reference enzymes TEM-10 and TEM-26.
Characterization of plasmids encoding ß-lactamases
Different plasmid profiles were observed among the K. pneumoniae isolates. In all isolates analysed, the presence of a single large plasmid of about 50 kb was detected. In addition to this large plasmid, plasmids of lower molecular weights were detected only in some isolates (Figure 2). In the E. coli transconjugants, acquisition of resistance to ß-lactam and aminoglycoside antibiotics was correlated with the 50 kb plasmid. Restriction patterns of these plasmids showed small differences for the various enzymes tested (data not shown).
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The TEM ß-lactamase genes from the transconjugants were amplified and sequenced. As they produced ESBLs with a pI of 5.6, which aligned with TEM-10 and TEM-26, they were amplified with TEM primers (Table I). For all isolates a 1006 bp PCR product was obtained. The deduced amino acid sequence was identical for each isolate analysed, and included a serine at position 164 and a lysine at position 240 (numbering of Ambler et al.18), indicating the presence of TEM-10.
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Discussion |
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PFGE has been proposed as a method for epidemiological studies, being considered useful in the investigation of the source, transmission and spread of nosocomial infections caused by the methicillin-resistant Staphylococcus aureus,19 group B streptococci,20 K. pneumoniae2124 and Salmonella.25 Plasmid profile analysis has also been proved to be useful for the study of outbreaks of infection with K. pneumoniae.21,2628 When combined with PFGE it is an effective tool for investigating the epidemiology of ESBL-producing K. pneumoniae isolates.28 In our study identical plasmids were detected in different isolates, suggesting that propagation of the outbreak prominently involved plasmid spread, although clonal spread of isolates was seen in all individual wards and predominated in the paediatric and surgical wards. The epidemiology of K. pneumoniae is complex. Gouby et al.22 reported an outbreak in a geriatric department in which the origin of the isolates involved in the outbreak was clonal. The dissemination of SHV-4 ß- lactamases in French hospitals, reported by Arlet et al.,23 was due to the propagation of a single bacterial isolate. In contrast, Bingen et al.21 reported an outbreak in which a surprisingly high number of genetically unrelated isolates was involved.
The present study determined the incidence of ESBL production amongst clinical isolates from a hospital in Lisbon. This is the first report of ESBL TEM-10 identified in K. pneumoniae isolates in a Portuguese hospital. Ferreira et al.29 carried out a survey in the Hospital de São João, in the north of Portugal, where they identified, by IEF, 14.3% of K. pneumoniae isolates producing ESBLs with pI > 7.6. The prevalence of TEM-10 in the Hospital de Santa Maria may be related to the increased usage of extendedspectrum cephalosporins in recent years. However, it is difficult to assess whether or not this resistance developed as a direct result of cephalosporin therapy. According to Hibbert-Rogers et al.,30 even after discontinuation of ceftazidime and other cephalosporins, continued colonization of patients by ESBL-producing Klebsiella isolates has been observed.
ESBLs are usually encoded by genes within plasmids that are easily transmitted among different members of the Enterobacteriaceae. The accumulation of resistance genes results in isolates that contain multi-resistance plasmids. The emergence of these multiply resistant Klebsiella isolates at the Hospital de Santa Maria has been accompanied by a relatively high stability of plasmids containing the blaTEM-10 gene. This does not seem to be associated with a significant increase in the use of extended-spectrum cephalosporins. The observation of a plasmid-mediated ESBL present in two isolates from the environment shows that these genes may be maintained in the absence of hospital pressures and that ESBL-producing organisms are no longer confined to nosocomial infections.31
In summary, this study shows that the ESBL TEM-10 present among K. pneumoniae isolates is widely disseminated in different wards, and its persistence could be explained by dissemination of both the multi-resistance plasmids harbouring the bla gene and the isolates themselves.
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Acknowledgments |
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Notes |
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References |
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Received 22 February 1999; returned 6 August 1999; revised 15 September 1999; accepted 3 January 2000