Extended-Spectrum--Lactamase-Producing Enterobacteriaceae in Yaounde, Cameroon
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微生物临床杂志 2005年第7期
Laboratory of General Biology, Faculty of Science, University of Yaounde I, P.O. Box 812 Yaounde, Cameroon
A Stampor "School of Public Health" Medical School, University of Zagreb, Croatia
Faculty of Medicine and Biomedical Sciences, University of Yaounde I, P.O. Box 1364 Yaounde, Cameroon
Laboratory of Bacteriology, Yaounde Central Hospital, Yaounde, Cameroon
Institute of Medical Microbiology, University of Zürich, P.O. Box, CH-8028 Zürich, Switzerland
NENT/NANT, Institute of Veterinary Bacteriology, University of Berne, Lnggassstrasse 122, P.O. Box, CH-3001 Berne, Switzerland
ABSTRACT
Organisms producing extended-spectrum -lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Cameroon. A total of 259 Enterobacteriaceae strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these Enterobacteriaceae strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strains—i.e., 6 Klebsiella pneumoniae, 4 Escherichia coli, 1 Citrobacter freundii, and 1 Enterobacter cloacae strain—was transferred to E. coli HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprim-sulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of -lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow- to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible E. coli strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The -lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.
INTRODUCTION
Many extended-spectrum -lactamases (ESBLs) are plasmid-mediated derivatives from TEM- and SHV-type enzymes and cause resistance to expanded-spectrum cephalosporins. They belong to Bush group 2be (6). Since their initial description in Germany in 1983 (13), ESBLs have diversified and spread worldwide. Several ESBLs appear to be particularly widely disseminated, being found in many countries, whereas others seem to occur more commonly in one or few countries (4). The various national patterns of antibiotic consumption in hospitals probably account for the differences in distribution of these enzymes. In an attempt to detect and study the dissemination of ESBLs in a central African country (Cameroon), we collected and characterized producers of such enzymes among clinical isolates of Enterobacteriaceae at Yaounde Central Hospital between 1995 and 1998. The ESBL SHV-12 was found in several species of Enterobacteriaceae for the first time in Cameroon.
MATERIALS AND METHODS
Bacterial strains. A total of 259 isolates, members of the Enterobacteriaceae family were collected from patients in Yaounde Central Hospital (Table 1). Isolates were collected over 3-year period (April 1995 to March 1998) from urine, pus, and blood. The isolates were identified by conventional techniques (9) and were confirmed by the API 20 E (bioMerieux, France). There were no replicate strains isolated from any patient in the present study. Escherichia coli HK 225 (12), which is resistant to rifampin and streptomycin, was used as a recipient strain for transfer experiments by conjugation. E. coli ATCC 25922 was used as a control strain for antimicrobial susceptibility testing and as a negative control for PCR experiments. E. coli K12R111 (provided by Danielle Sirot) encoding TEM-1 and plasmid pMPA encoding SHV-2A were used as positive controls for blaTEM and blaSHV genes, respectively.
Antimicrobial susceptibility testing and detection of ESBL producers. Antimicrobial susceptibility was determined by disk diffusion tests according to the methods of the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) (16) by using disks from BBL Microbiology Systems (Cockeysville, Md.). The tested antibiotics were amoxicillin, amoxicillin-clavulanate, piperacillin, cefazolin, cefoxitin, cefotaxime, ceftazidime, aztreonam, imipenem, gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole.
The double-disk (DD) synergy test (11) was used for detection of ESBLs in clinical and transconjugant strains. A central disk of amoxicillin-clavulanate was surrounded by disks with cefotaxime, ceftriaxone, ceftazidime, and aztreonam at a distance of ca. 19 mm (center to center) on a Mueller-Hinton agar plate (Difco Laboratories, Detroit, MI) inoculated according to the standard procedures (16). Distortion of the peripheral inhibition zones of surrounding antibiotics toward the central disk with clavulanate was indicative for an ESBL. The tests were repeated with a disk spacing of 15 mm (center to center).
MICs for positive DD synergy test strains were determined by using the E-test (AB Biodisk, Solna, Sweden) according to the instructions of the manufacturer.
Conjugation experiments. Transfer of resistance phenotypes was performed by a liquid mating method (19) in brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.). Culture mixtures were incubated overnight at 37°C with clinical isolates as donors and E. coli HK 225 as the recipient. After conjugation, bacterial suspensions were plated onto agar containing 50 μg of ampicillin, 10 μg of ceftazidime, 2 μg of cefotaxime, 100 μg of rifampin, and 2,000 μg of streptomycin per ml. The resulting transconjugants were purified and identified with API 20 E strips. The frequencies of transfer were determined per input donor cell.
Characterization of -lactamases. Transconjugant strains were grown at 37°C with shaking in brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.) containing ceftazidime (10 μg/ml), cefotaxime (2 μg/liter), and ampicillin (50 μg/liter). The cells were harvested during late log phase (8) by centrifugation at 3,000 x g at 4°C, washed twice in 0.1 M phosphate buffer (pH 7.0), and resuspended in 1/50 of the original volume. After cell rupture by sonication on ice, the cellular debris was removed by two centrifugations at 5,000 x g, each for 30 min at 4°C. Both times, the supernatant was carefully removed without solid particles. The crude extracts were stored at –20°C.
The extracts' ability to hydrolyze -lactam antibiotics (amoxicillin, amoxicillin-clavulanate, piperacillin, cefazolin, cefoxitin, ceftriaxone, cefotaxin, ceftazidime, aztreonam, and imipenem) was evaluated by a microbiological method (15). Disk diffusion test were performed according to the methods of the CLSI (16), and antimicrobial disks were immediately impregnated with 10 μl of crude extract after their application. The inhibition zones produced against E. coli HK 225 were compared to those observed by untreated disks. A reduction of the inhibition zone diameter as a consequence of the applied extract was considered evidence for -lactamase activity.
Molecular analysis techniques. DNA of transconjugants strains was extracted with phenol and chloroform, precipitated with isopropanol, and resuspended in TE buffer (3). bla genes were amplified by PCR with the following oligonucleotide primers: TEM OT-1-F (5'-TTGGGTGCACGAGTGGGTTA-3') and OT-2-R (5'-TAATTGTTGCCGGGAAGCTA-3'), which amplified a 465-bp fragment of the blaTEM gene (2), and SHV-F (5'-CGC CGG GTT ATT CTT ATT TGT CGC-3') and SHV-R (5'-TCT TTC CGA TGC CGC CGC CAG TCA-3'), which amplified a 1,017-bp fragment of the blaSHV gene (17). The cycle conditions were as follows: 30 cycles, with 1 cycle consisting of denaturation at 95°C for 30 s, annealing at 52°C (TEM) or 68°C (SHV) for 30 s, and extension at 72°C for 1 min. Each PCR program was preceded by a denaturation step of 94°C for 5 min and followed by a final denaturation at 72°C for 10 min. The SHV PCR products were digested with the restriction enzyme NheI (17).
Sequencing of the PCR products was performed by using the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) according to the recommendations of the manufacturer in an ABI Prism 310 genetic analyzer. Sequence data were processed and analyzed with the version 9.0 GCG Sequence Analysis Software Package (Genetics Computer Group, Madison, WI).
Sequence accession number. The nucleotide sequence of blaSHV-12 from C. freundii 09 is filed in the GenBank database under the accession number AY940490.
RESULTS
Detection of ESBL-producing isolates. A total of 259 isolates (124 urine isolates, 120 isolates from pus, and 15 isolates from blood) belonging to the family Enterobacteriaceae were studied, among which 31 (12%) potential ESBL producers were identified by the DD synergy test. Characteristic clavulanate-induced distortions of inhibition zones that were indicative for ESBL production were found in 86, 90, 62, and 97% of the strains around the disks containing cefotaxime, ceftriaxone, ceftazidime, and aztreonam, respectively. The ESBL producer's isolates were investigated, and the ESBL-producing phenotype was found most frequently among Klebsiella species (18.8%; K. pneumoniae [n = 11] and K. oxytoca [n = 1]), followed by Citrobacter species (17.6%; C. freundii [n = 3]) and E. coli (14.3%) (Table 1). Of the ESBL-producing strains, 15 were from urine, 11 were from pus, and 5 were from blood; isolates were mainly from patients in the intensive care unit (ICU) (38.7%) and the surgical (45.1%) ward (Table 2).
MICs of extended-spectrum cephalosporins exhibited a notable variability among different isolates, but MICs of CAZ were always 2 μg/ml (Table 2). All of the isolates were resistant to amoxicillin, piperacillin, and cephalothin, and most of them were also resistant to amoxicillin-clavulanate, gentamicin, and trimethoprim-sulfamethoxazole. All of the isolates were susceptible to imipenem, and most of them were also susceptible to cefoxitin, ciprofloxacin, and amikacin (Table 2).
Transferability of ESBL genes. Among potential ESBL producers, 12 (38.7%) were transferred oxyimino-cephalosporin resistance to E. coli HK 225. The transfer frequencies were between 9 x 10–8 and 6.7 x 10–4 per input donor. The isolates included six of K. pneumoniae, four of E. coli, one of E. cloacae, and one of C. freundii. Resistance determinants against the following non--lactam antibiotics were cotransferred to E. coli HK 225: gentamicin (six cases), gentamicin and trimethoprim-sulfamethoxazole (two cases), and trimethoprim-sulfamethoxazole (one case). In contrast, resistance to cefoxitin or to other antibiotic classes was not transferred (Table 3).
-Lactamase type. Supernatants containing crude -lactamase extracts from transconjugants did not affect the inhibition zones of E. coli HK 225 around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The supernatants did, however, significantly reduce the inhibition zones around disks containing amoxicillin, piperacillin, cefazolin, cefotaxime, ceftriaxone, ceftazidime, and aztreonam (data not shown). These results were consistent with the presence of the detected ESBLs.
All 12 transconjugants were subjected to molecular detection and characterization procedures aimed at blaTEM and blaSHV genes. From all 12 strains, no PCR product was obtained with TEM specific primers, whereas a characteristic 1,017-bp amplimer was synthesized, which proved to be degradable by the restriction enzyme NheI into two fragments of 770 and 247 bp in length. This positive PCR/NheI test indicated that an SHV-ESBL was produced by all transconjugants and hence by the 12 corresponding donor clinical isolates (Table 3).
Nucleotide sequencing revealed that all 12 amplimers were 100% homologous to each other along their entire length. This also confirmed the PCR/NheI results and showed that the deduced amino acid sequences differed in the following positions (Ambler numbering [1]) from the SHV-1 standard (4): leucine 35glutamine, glycine 238serine, and glutamic acid 240lysine. Thus, SHV-12 (http://www.lahey.org/studies/webt.htm) was identified in all of the 12 strains (Table 3). Moreover, two silent mutations at codons 138 and 268 were the same as those described in the original sequence of blaSHV-12 (EMBL accession no. X98105 [18]), namely, CTG and ACG, as opposed to CTA and ACC, respectively, in the standard blaSHV-1 sequence. However, one additional G-to-C transversion immediately after the TAA stop codon was detected (position 935 in X98105).
The blaSHV-12 sequence and flanking regions of C. freundii 09 were deposited in GenBank (accession no. AY940490) as the representative for the 12 isolates listed in Table 4.
The 12 SHV-12-producing isolates exhibited a relatively narrow clinical distribution. They were mainly obtained from the surgical ward between 1996 and 1998 (Table 4).
DISCUSSION
The aim of this study was to determine the prevalence and characterize ESBL producers among members of the family Enterobacteriaceae in Yaounde Central Hospital. We found isolates that expressed an ESBL producer phenotype among every genus that was tested. ESBLs were present in 31 (12%) among the 259 Enterobacteriaceae isolates evaluated here. The prevalence among genera is varied, with rates of 18.8% for Klebsiella spp. and 1.8 to 17.6% for all other genera. Of 31 ESBL producer isolates, 12 (38.7%) transferred the ESBL gene to E. coli HK 225 and were also found to produce SHV-12 ESBL. These data represent the first report of the prevalence of ESBL producers among Enterobacteriaceae in Cameroon.
In the present study, most ESBL producers were collected from patients in the surgical ward and the ICU. In these wards, isolates are exposed to great antibiotic pressure. Furthermore, many of these patients are particularly vulnerable to infection because they are immunocompromised or have an easy avenue of access for bacteria (23).
The susceptibility test data showed that the ESBL producers which were resistant to most -lactams and non--lactams such as gentamicin and trimethoprim-sulfamethoxazole were multidrug-resistant strains. The ESBL producers usually carry a multiresistant plasmid, the genes conferring resistance to -lactam and non--lactam antibiotics (10, 23). All of the ESBL producers were susceptible to imipenem, and most were also susceptible to cefoxitin, amikacin, and ciprofloxacin. If the patients are infected by ESBL producers, carbapenem may be used (5, 14).
The results of the present study are evidence of an ongoing outbreak during the sampling period from 1996 to 1998 at Yaounde Central Hospital of ESBL-producing organisms attributable to at least four species of Enterobacteriaceae: K. pneumoniae, E. coli, C. freundii, and E. cloacae. Although the blaSHV-12 gene was cotransferable along with other resistance determinants upon conjugation and was found in several species, it seemed possible that the determinant is located on a large low-copy broad-host-range plasmid, as is usually the case (10, 21). In the present study, we focused on determinants that were easily transferable by conjugation in vitro (more than a third of the ESBL isolates from Yaounde Central Hospital) because these are the most clinically relevant factors with regard to the speed of dissemination.
Within the collection of 31 strains that yielded positive DD results, ceftazidime and aztreonam showed the lowest (62%) and highest (96%) rates of detection, respectively. These results indicate that at maximum sensitivity, when looking for ESBLs, several oyximino-cephalosporins and aztreonam should be used simultaneously for DD testing. This is in agreement with the recommendations by Coudron et al. (7). Moreover, this finding underlines the fact that synergy tests and other physiological tests in general are of limited sensitivity in detecting ESBLs, a fact that has been stated already in 1995 (14) and confirmed by careful studies involving site-directed mutagenesis, as well as different copy number cloning vectors in isogenic systems (20).
ESBLs are now a problem for hospitalized patients worldwide. The rates of ESBL producers among Klebsiella sp. and E. coli at our center are 18.8 and 14.3%, respectively. This prevalence is lower for Klebsiella sp. and higher for E. coli than that reported by the SENTRY worldwide surveillance program, in which the ESBL prevalences in K. pneumoniae and E. coli were 45 and 8.5% (Latin America), 25 and 7.9% (Western Pacific), or 23 and 5.3% (Europe), respectively (22).
In conclusion, the present study emphasizes the importance of screening for ESBLs even in countries where such enzymes have not been reported previously. The plasmid-mediated ESBLs have been already disseminated to four different species of Enterobacteriaceae and escalated into a multiclinic outbreak at Yaounde Central Hospital by the time they were discovered.
ACKNOWLEDGMENTS
We thank the Yaounde Central Hospital administration and the Head of the Biotechnology Centre for allowing us to carry out part of this study at their facilities. We thank the technicians of the laboratory of Bacteriology of the Yaounde Central Hospital Julienne Mvondo, Madeleine Tchonko, Solange Ntoual, Eudosie Dongmo, Marguerite Wodo, Marie Abiazen, and Marie Claire Epape for help in this investigation. We also thank Marie Christine Fonkoua (Centre Pasteur du Cameroun) for generously contributing to the strain collection and Gilbert Kuepouo and Gideon Ajeagah for reading the manuscript.
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A Stampor "School of Public Health" Medical School, University of Zagreb, Croatia
Faculty of Medicine and Biomedical Sciences, University of Yaounde I, P.O. Box 1364 Yaounde, Cameroon
Laboratory of Bacteriology, Yaounde Central Hospital, Yaounde, Cameroon
Institute of Medical Microbiology, University of Zürich, P.O. Box, CH-8028 Zürich, Switzerland
NENT/NANT, Institute of Veterinary Bacteriology, University of Berne, Lnggassstrasse 122, P.O. Box, CH-3001 Berne, Switzerland
ABSTRACT
Organisms producing extended-spectrum -lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Cameroon. A total of 259 Enterobacteriaceae strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these Enterobacteriaceae strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strains—i.e., 6 Klebsiella pneumoniae, 4 Escherichia coli, 1 Citrobacter freundii, and 1 Enterobacter cloacae strain—was transferred to E. coli HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprim-sulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of -lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow- to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible E. coli strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The -lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.
INTRODUCTION
Many extended-spectrum -lactamases (ESBLs) are plasmid-mediated derivatives from TEM- and SHV-type enzymes and cause resistance to expanded-spectrum cephalosporins. They belong to Bush group 2be (6). Since their initial description in Germany in 1983 (13), ESBLs have diversified and spread worldwide. Several ESBLs appear to be particularly widely disseminated, being found in many countries, whereas others seem to occur more commonly in one or few countries (4). The various national patterns of antibiotic consumption in hospitals probably account for the differences in distribution of these enzymes. In an attempt to detect and study the dissemination of ESBLs in a central African country (Cameroon), we collected and characterized producers of such enzymes among clinical isolates of Enterobacteriaceae at Yaounde Central Hospital between 1995 and 1998. The ESBL SHV-12 was found in several species of Enterobacteriaceae for the first time in Cameroon.
MATERIALS AND METHODS
Bacterial strains. A total of 259 isolates, members of the Enterobacteriaceae family were collected from patients in Yaounde Central Hospital (Table 1). Isolates were collected over 3-year period (April 1995 to March 1998) from urine, pus, and blood. The isolates were identified by conventional techniques (9) and were confirmed by the API 20 E (bioMerieux, France). There were no replicate strains isolated from any patient in the present study. Escherichia coli HK 225 (12), which is resistant to rifampin and streptomycin, was used as a recipient strain for transfer experiments by conjugation. E. coli ATCC 25922 was used as a control strain for antimicrobial susceptibility testing and as a negative control for PCR experiments. E. coli K12R111 (provided by Danielle Sirot) encoding TEM-1 and plasmid pMPA encoding SHV-2A were used as positive controls for blaTEM and blaSHV genes, respectively.
Antimicrobial susceptibility testing and detection of ESBL producers. Antimicrobial susceptibility was determined by disk diffusion tests according to the methods of the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) (16) by using disks from BBL Microbiology Systems (Cockeysville, Md.). The tested antibiotics were amoxicillin, amoxicillin-clavulanate, piperacillin, cefazolin, cefoxitin, cefotaxime, ceftazidime, aztreonam, imipenem, gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole.
The double-disk (DD) synergy test (11) was used for detection of ESBLs in clinical and transconjugant strains. A central disk of amoxicillin-clavulanate was surrounded by disks with cefotaxime, ceftriaxone, ceftazidime, and aztreonam at a distance of ca. 19 mm (center to center) on a Mueller-Hinton agar plate (Difco Laboratories, Detroit, MI) inoculated according to the standard procedures (16). Distortion of the peripheral inhibition zones of surrounding antibiotics toward the central disk with clavulanate was indicative for an ESBL. The tests were repeated with a disk spacing of 15 mm (center to center).
MICs for positive DD synergy test strains were determined by using the E-test (AB Biodisk, Solna, Sweden) according to the instructions of the manufacturer.
Conjugation experiments. Transfer of resistance phenotypes was performed by a liquid mating method (19) in brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.). Culture mixtures were incubated overnight at 37°C with clinical isolates as donors and E. coli HK 225 as the recipient. After conjugation, bacterial suspensions were plated onto agar containing 50 μg of ampicillin, 10 μg of ceftazidime, 2 μg of cefotaxime, 100 μg of rifampin, and 2,000 μg of streptomycin per ml. The resulting transconjugants were purified and identified with API 20 E strips. The frequencies of transfer were determined per input donor cell.
Characterization of -lactamases. Transconjugant strains were grown at 37°C with shaking in brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.) containing ceftazidime (10 μg/ml), cefotaxime (2 μg/liter), and ampicillin (50 μg/liter). The cells were harvested during late log phase (8) by centrifugation at 3,000 x g at 4°C, washed twice in 0.1 M phosphate buffer (pH 7.0), and resuspended in 1/50 of the original volume. After cell rupture by sonication on ice, the cellular debris was removed by two centrifugations at 5,000 x g, each for 30 min at 4°C. Both times, the supernatant was carefully removed without solid particles. The crude extracts were stored at –20°C.
The extracts' ability to hydrolyze -lactam antibiotics (amoxicillin, amoxicillin-clavulanate, piperacillin, cefazolin, cefoxitin, ceftriaxone, cefotaxin, ceftazidime, aztreonam, and imipenem) was evaluated by a microbiological method (15). Disk diffusion test were performed according to the methods of the CLSI (16), and antimicrobial disks were immediately impregnated with 10 μl of crude extract after their application. The inhibition zones produced against E. coli HK 225 were compared to those observed by untreated disks. A reduction of the inhibition zone diameter as a consequence of the applied extract was considered evidence for -lactamase activity.
Molecular analysis techniques. DNA of transconjugants strains was extracted with phenol and chloroform, precipitated with isopropanol, and resuspended in TE buffer (3). bla genes were amplified by PCR with the following oligonucleotide primers: TEM OT-1-F (5'-TTGGGTGCACGAGTGGGTTA-3') and OT-2-R (5'-TAATTGTTGCCGGGAAGCTA-3'), which amplified a 465-bp fragment of the blaTEM gene (2), and SHV-F (5'-CGC CGG GTT ATT CTT ATT TGT CGC-3') and SHV-R (5'-TCT TTC CGA TGC CGC CGC CAG TCA-3'), which amplified a 1,017-bp fragment of the blaSHV gene (17). The cycle conditions were as follows: 30 cycles, with 1 cycle consisting of denaturation at 95°C for 30 s, annealing at 52°C (TEM) or 68°C (SHV) for 30 s, and extension at 72°C for 1 min. Each PCR program was preceded by a denaturation step of 94°C for 5 min and followed by a final denaturation at 72°C for 10 min. The SHV PCR products were digested with the restriction enzyme NheI (17).
Sequencing of the PCR products was performed by using the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) according to the recommendations of the manufacturer in an ABI Prism 310 genetic analyzer. Sequence data were processed and analyzed with the version 9.0 GCG Sequence Analysis Software Package (Genetics Computer Group, Madison, WI).
Sequence accession number. The nucleotide sequence of blaSHV-12 from C. freundii 09 is filed in the GenBank database under the accession number AY940490.
RESULTS
Detection of ESBL-producing isolates. A total of 259 isolates (124 urine isolates, 120 isolates from pus, and 15 isolates from blood) belonging to the family Enterobacteriaceae were studied, among which 31 (12%) potential ESBL producers were identified by the DD synergy test. Characteristic clavulanate-induced distortions of inhibition zones that were indicative for ESBL production were found in 86, 90, 62, and 97% of the strains around the disks containing cefotaxime, ceftriaxone, ceftazidime, and aztreonam, respectively. The ESBL producer's isolates were investigated, and the ESBL-producing phenotype was found most frequently among Klebsiella species (18.8%; K. pneumoniae [n = 11] and K. oxytoca [n = 1]), followed by Citrobacter species (17.6%; C. freundii [n = 3]) and E. coli (14.3%) (Table 1). Of the ESBL-producing strains, 15 were from urine, 11 were from pus, and 5 were from blood; isolates were mainly from patients in the intensive care unit (ICU) (38.7%) and the surgical (45.1%) ward (Table 2).
MICs of extended-spectrum cephalosporins exhibited a notable variability among different isolates, but MICs of CAZ were always 2 μg/ml (Table 2). All of the isolates were resistant to amoxicillin, piperacillin, and cephalothin, and most of them were also resistant to amoxicillin-clavulanate, gentamicin, and trimethoprim-sulfamethoxazole. All of the isolates were susceptible to imipenem, and most of them were also susceptible to cefoxitin, ciprofloxacin, and amikacin (Table 2).
Transferability of ESBL genes. Among potential ESBL producers, 12 (38.7%) were transferred oxyimino-cephalosporin resistance to E. coli HK 225. The transfer frequencies were between 9 x 10–8 and 6.7 x 10–4 per input donor. The isolates included six of K. pneumoniae, four of E. coli, one of E. cloacae, and one of C. freundii. Resistance determinants against the following non--lactam antibiotics were cotransferred to E. coli HK 225: gentamicin (six cases), gentamicin and trimethoprim-sulfamethoxazole (two cases), and trimethoprim-sulfamethoxazole (one case). In contrast, resistance to cefoxitin or to other antibiotic classes was not transferred (Table 3).
-Lactamase type. Supernatants containing crude -lactamase extracts from transconjugants did not affect the inhibition zones of E. coli HK 225 around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The supernatants did, however, significantly reduce the inhibition zones around disks containing amoxicillin, piperacillin, cefazolin, cefotaxime, ceftriaxone, ceftazidime, and aztreonam (data not shown). These results were consistent with the presence of the detected ESBLs.
All 12 transconjugants were subjected to molecular detection and characterization procedures aimed at blaTEM and blaSHV genes. From all 12 strains, no PCR product was obtained with TEM specific primers, whereas a characteristic 1,017-bp amplimer was synthesized, which proved to be degradable by the restriction enzyme NheI into two fragments of 770 and 247 bp in length. This positive PCR/NheI test indicated that an SHV-ESBL was produced by all transconjugants and hence by the 12 corresponding donor clinical isolates (Table 3).
Nucleotide sequencing revealed that all 12 amplimers were 100% homologous to each other along their entire length. This also confirmed the PCR/NheI results and showed that the deduced amino acid sequences differed in the following positions (Ambler numbering [1]) from the SHV-1 standard (4): leucine 35glutamine, glycine 238serine, and glutamic acid 240lysine. Thus, SHV-12 (http://www.lahey.org/studies/webt.htm) was identified in all of the 12 strains (Table 3). Moreover, two silent mutations at codons 138 and 268 were the same as those described in the original sequence of blaSHV-12 (EMBL accession no. X98105 [18]), namely, CTG and ACG, as opposed to CTA and ACC, respectively, in the standard blaSHV-1 sequence. However, one additional G-to-C transversion immediately after the TAA stop codon was detected (position 935 in X98105).
The blaSHV-12 sequence and flanking regions of C. freundii 09 were deposited in GenBank (accession no. AY940490) as the representative for the 12 isolates listed in Table 4.
The 12 SHV-12-producing isolates exhibited a relatively narrow clinical distribution. They were mainly obtained from the surgical ward between 1996 and 1998 (Table 4).
DISCUSSION
The aim of this study was to determine the prevalence and characterize ESBL producers among members of the family Enterobacteriaceae in Yaounde Central Hospital. We found isolates that expressed an ESBL producer phenotype among every genus that was tested. ESBLs were present in 31 (12%) among the 259 Enterobacteriaceae isolates evaluated here. The prevalence among genera is varied, with rates of 18.8% for Klebsiella spp. and 1.8 to 17.6% for all other genera. Of 31 ESBL producer isolates, 12 (38.7%) transferred the ESBL gene to E. coli HK 225 and were also found to produce SHV-12 ESBL. These data represent the first report of the prevalence of ESBL producers among Enterobacteriaceae in Cameroon.
In the present study, most ESBL producers were collected from patients in the surgical ward and the ICU. In these wards, isolates are exposed to great antibiotic pressure. Furthermore, many of these patients are particularly vulnerable to infection because they are immunocompromised or have an easy avenue of access for bacteria (23).
The susceptibility test data showed that the ESBL producers which were resistant to most -lactams and non--lactams such as gentamicin and trimethoprim-sulfamethoxazole were multidrug-resistant strains. The ESBL producers usually carry a multiresistant plasmid, the genes conferring resistance to -lactam and non--lactam antibiotics (10, 23). All of the ESBL producers were susceptible to imipenem, and most were also susceptible to cefoxitin, amikacin, and ciprofloxacin. If the patients are infected by ESBL producers, carbapenem may be used (5, 14).
The results of the present study are evidence of an ongoing outbreak during the sampling period from 1996 to 1998 at Yaounde Central Hospital of ESBL-producing organisms attributable to at least four species of Enterobacteriaceae: K. pneumoniae, E. coli, C. freundii, and E. cloacae. Although the blaSHV-12 gene was cotransferable along with other resistance determinants upon conjugation and was found in several species, it seemed possible that the determinant is located on a large low-copy broad-host-range plasmid, as is usually the case (10, 21). In the present study, we focused on determinants that were easily transferable by conjugation in vitro (more than a third of the ESBL isolates from Yaounde Central Hospital) because these are the most clinically relevant factors with regard to the speed of dissemination.
Within the collection of 31 strains that yielded positive DD results, ceftazidime and aztreonam showed the lowest (62%) and highest (96%) rates of detection, respectively. These results indicate that at maximum sensitivity, when looking for ESBLs, several oyximino-cephalosporins and aztreonam should be used simultaneously for DD testing. This is in agreement with the recommendations by Coudron et al. (7). Moreover, this finding underlines the fact that synergy tests and other physiological tests in general are of limited sensitivity in detecting ESBLs, a fact that has been stated already in 1995 (14) and confirmed by careful studies involving site-directed mutagenesis, as well as different copy number cloning vectors in isogenic systems (20).
ESBLs are now a problem for hospitalized patients worldwide. The rates of ESBL producers among Klebsiella sp. and E. coli at our center are 18.8 and 14.3%, respectively. This prevalence is lower for Klebsiella sp. and higher for E. coli than that reported by the SENTRY worldwide surveillance program, in which the ESBL prevalences in K. pneumoniae and E. coli were 45 and 8.5% (Latin America), 25 and 7.9% (Western Pacific), or 23 and 5.3% (Europe), respectively (22).
In conclusion, the present study emphasizes the importance of screening for ESBLs even in countries where such enzymes have not been reported previously. The plasmid-mediated ESBLs have been already disseminated to four different species of Enterobacteriaceae and escalated into a multiclinic outbreak at Yaounde Central Hospital by the time they were discovered.
ACKNOWLEDGMENTS
We thank the Yaounde Central Hospital administration and the Head of the Biotechnology Centre for allowing us to carry out part of this study at their facilities. We thank the technicians of the laboratory of Bacteriology of the Yaounde Central Hospital Julienne Mvondo, Madeleine Tchonko, Solange Ntoual, Eudosie Dongmo, Marguerite Wodo, Marie Abiazen, and Marie Claire Epape for help in this investigation. We also thank Marie Christine Fonkoua (Centre Pasteur du Cameroun) for generously contributing to the strain collection and Gilbert Kuepouo and Gideon Ajeagah for reading the manuscript.
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