Countrywide Spread of Community- and Hospital-Acquired Extended-Spectrum -Lactamase (CTX-M-15)-Producing Enterobacteriaceae in Lebanon
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微生物临床杂志 2005年第7期
Laboratoire de Microbiologie, Faculte des Sciences Pharmaceutiques et Biologiques, Universite Paris 5-Rene Descartes, Paris, France
Saint George Hospital, and University of Balamand, Beirut, Lebanon
Lebanese University, Beirut, and Nini Hospital, Tripoli, Lebanon
Arz Hospital, Al Metn, Lebanon
Rizk Hospital, Beirut, Lebanon
Saint Joseph Hospital, Beirut, Lebanon
Htel Dieu de France Hospital, Beirut, Lebanon
C. H. de Versailles, Le Chesnay, France
ABSTRACT
A prospective study was carried out to assess the extent of carriage of extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected -lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of Escherichia coli (n = 56), Klebsiella pneumoniae (n = 9), Enterobacter cloacae (n = 6), and Citrobacter freundii (n = 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing E. coli presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing E. coli in Lebanon, it represents the first report of an SHV-5a-producing C. freundii.
INTRODUCTION
-Lactam antibiotics are widely used in the treatment of bacterial infections. However, the production of extended-spectrum -lactamases (ESBLs), one of the resistance mechanisms encountered in Enterobacteriaceae, has been associated with several treatment failures. Indeed, ESBLs are capable of efficiently hydrolyzing extended-spectrum cephalosporins (cefotaxime and ceftazidime) and are highly susceptible to inhibition by clavulanic acid and tazobactam (17). In addition to the classical TEM and SHV enzymes, several ESBLs of predominantly Ambler's molecular class A have recently been reported, including the CTX-M enzymes (2). Since the second half of the 1980s, ESBLs disseminate dangerously into wide geographic regions (4). This spread may be the consequence of plasmid transmission among nonrelated Enterobacteriaceae, as observed in French hospitals (27). It may also be mediated by the clonal dissemination of the enzyme-producing microorganisms (10). Sometimes, transmission of ESBL-producing strains between hospitals, followed by their clonal expansion and/or the horizontal transfer of plasmids carrying the ESBL gene, may even occur (1).
Although ESBL-producing strains have been reported by several countries, very few resistance data representing the Middle East have been published. Moreover, the majority of the studies represent hospital-based patients, whereas the carriage rates of ESBL from fecal flora of patients or community-based subjects remains unknown. Infections caused by ESBL-producing strains are increasing in frequency in Lebanon, a Mediterranean country in the Middle East with a dynamic and ethnically diverse population of 3.5 million. A study done in Lebanon at Saint George Hospital revealed that, among the 4,299 isolates of Escherichia coli and 1,248 isolates of Klebsiella pneumoniae tested over the 5 years between 1997 and 2001, ESBL production was observed with 2% of the E. coli and 20% of the K. pneumoniae strains (7). However, no epidemiological survey of ESBL-producing strains has been performed until now in this country. The present prospective multicentric study was done in order to report the extent of carriage of ESBL-producing Enterobacteriaceae and to characterize the types of ESBLs produced at both hospital and community levels in Lebanon.
(This study was presented in part at the ECC & RICAI 2004 Congress, Paris, France, on 1 to 3 December 2004 [abstr. 340/770].)
MATERIALS AND METHODS
Study design. This study was a prospective laboratory-based surveillance study in which fecal samples collected from hospitalized patients, health care personnel, and healthy subjects were screened for the presence of possible ESBL-producing strains. A total of 1,442 fecal samples (1,440 rectal swabs and 2 feces samples) were collected between 1 January 2003 and 31 March 2003 from 378 patients admitted to the intensive care units (including the neonatal intensive care unit) of five Lebanese tertiary care general hospitals located in different areas of Lebanon: Rizk Hospital, Beirut (RH); Saint George Hospital, Beirut (SGH); Arz Hospital, Al Metn (AH); Nini Hospital, Tripoli (North Lebanon) (NN); and Saint Joseph Hospital, Beirut (SJH). Patients were checked for carriage of ESBL-producing strains upon admission, 72 h later, and then at weekly intervals over a 3-month period. The 58 health care workers of the same intensive care units were checked (rectal swabs) for ESBL-producing strains once at the beginning of the study during the same period. A total of 382 fecal samples (rectal swabs) were collected between 1 April 2003 and 31 June 2003 from 382 young healthy students (HS) from the Lebanese University-Faculty of Public Health section 3 (in Tripoli, North Lebanon) and section 4 (in Bekaa, East Lebanon).
Bacterial strains. All samples were suspended at participating sites in normal saline in order to form a 10-μg/ml suspension. From these, 100 μl was streaked on Trypticase soy-5% sheep blood agar containing 6 μg of vancomycin/ml, 2 μg of amphotericin B/ml, 2 μg of ceftazidime/ml, and 1 μg of clindamycin/ml (VACC medium) (11). After 24 and 48 h of incubation at 35°C, colonies appearing on the plates were sent to Saint George Hospital for strain identification by using the API 20E system (bioMerieux) and for resistance phenotype analysis. To these strains were added six ESBL-producing strains isolated from stools of patients admitted to the intensive care unit of the Htel Dieu de France Hospital, Beirut (HDF), between 1 January 2003 and 31 March 2003.
Antibiotic susceptibility testing and ESBL production detection. ESBL production was detected by the double-disk synergy test according to the method of Jarlier et al. (12). Antibiotic susceptibility of ESBL-producing strains was determined by the disk diffusion method according to the recommendations of the French Society of Microbiology (32). The following commercial disks were tested: amoxicillin-clavulanic acid (20 μg-10 μg), cefoxitin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), gentamicin (10 μg), trimethoprim-sulfamethoxazole (1.25 μg-23.75 μg), amikacin (10 μg), and imipenem (10 μg). The MICs of ceftazidime and cefotaxime with or without clavulanic acid (at a fixed concentration of 2 μg/ml) were determined by using serial twofold dilutions of antibiotics in Mueller-Hinton agar and inocula of 105 CFU per spot. For each subject, if several ESBL-producing isolates were detected and all were from the same species and presented similar antibiograms, only one was selected for further study, and the others were considered to be repeat isolations of the same strain.
Analytical IEF of -lactamases and bioassays for penicillin G, ceftazidime, and cefotaxime hydrolyzing activities. Isoelectric focusing (IEF) was performed according to the method of Labia and Barthelemy (15) on all selected isolates. Crude cell-free preparations of -lactamases were obtained by three sonications, each for 10 s, at an amplitude of 12 μm, with intermittent cooling on ice. Supernatants (15 μl) of each preparation were subjected to IEF on prepared polyacrylamide gels and separated with 350 V for 18 h at 4°C with the Multiphor II system (Pharmacia-Biotech, Orsay, France). After IEF, -lactamase activity was visualized by the iodometric method using gels containing penicillin G, ceftazidime, or cefotaxime. -Lactamases were identified by comparison to reference enzymes run in tracks adjacent to the test samples.
PCR detection and sequencing of blaTEM, blaSHV, blaCTX-M, and blaOXA-1 genes. PCRs and sequence analysis were used in order to identify the gene responsible for the ESBL phenotype in all strains. It should be noted that not all genes were sequenced in strains where multiple -lactamases genes existed. The isoelectric points (pIs) of the -lactamases were used to predict the most likely ESBL candidate, and this gene was sequenced. For example, if a strain contained blaTEM and the IEF results showed a -lactamase with a pI of 5.4, an assumption was made that this -lactamase most likely represented the non-ESBL TEM-1 and the other gene was sequenced.
All strains were examined for the presence of blaTEM, blaSHV, blaCTX-M, and blaOXA-1 using PCR conditions as previously prescribed (5, 21, 26). Primers MAb/F (5'-GGGGAGCTCATAAAATTCTTGAAGAC) and MAb/R (5'-GGGGGATCCTTACCAATGCTTAATCA) were used for amplifications of the entire blaTEM genes. Primers SHV/F (5'-GCCCGGGTTATTCTTATTTGTCGC) and SHV/R (5'-TCTTTCCGATGCCGCCGCCAGTCA) were used for amplifications of the entire blaSHV genes. CTX-M-type genes were detected by using universal primers CTX-M-U1 (5'-ATGTGCAGYACCAGTAARGTKATGGC) and CTX-M-U2 (5'-TGGGTRAARTARGTSACCAGAAYCAGCGG) (where R is purine, Y is pyrimidine, and S is G or C). Primers OXA-1/F (5'-ATGAAAAACACAATACATATC) and OXA-1/R (5'-AATTTAGTGTGTTTAGAATGG) were used for partial amplification of blaOXA-1 genes. Both DNA strands of PCR products were sequenced by using the above-cited primers for blaTEM and blaSHV genes. Entire CTX-M-type genes were amplified for sequencing purposes by using primer forward CTX-M-1-F (5'-GGTTAAAAAATCACTGCGTC-3'; located at positions 3 to 22) and primer reverse CTX-M-15-R (5'--TTACAAACCGTCGGTGACGA-3'; located at positions 876 to 857) as described previously (34). The sequence of the blaTEM, blaSHV, and blaCTX-M ORF PCR products were determined by using a Big Dye PCR (Perkin-Elmer/Cetus) and analyzed on an ABI Prism 310 DNA Genetic Analyzer (Perkin-Elmer Applied Biosystems).
Pulsed-field gel electrophoresis typing. The GenePath Group VI reagent kit and Genepath instruments (Bio-Rad Laboratories, Hercules, CA) were used to analyze the genome of strains representing the predominant species of ESBL-producing Enterobacteriaceae in Lebanon. Bacteria inserted in agarose plugs were lysed with lysozyme and treated with proteinase K. Genomic DNA was digested with XbaI, and the fragments were separated in agarose gels by electrophoresis based on a contour-clamped homogeneous electric field method according to the manufacturer's recommendations. After being stained with ethidium bromide, the gels were placed on a UV transilluminator, and the images were digitized with the Gel Doc 1000 system (Bio-Rad). To compare the profiles among different gels, a reference strain was routinely deposited in duplicate on each gel, thereby improving the assessment of strain relatedness. DNA relatedness was determined with Molecular Analyst RFLP software (Bio-Rad). Macrorestriction analysis was limited to DNA fragments of at least 50 kb. A similarity index was determined for each pair of strains by using the Dice coefficient, with a 0.80% band tolerance. Clustering correlation coefficients were calculated by using the unweighted pair-group method of arithmetic averages (UPGMA). Isolates were considered to be within a genotype (Gt) if the similarity coefficient was 80%. The lambda ladder PFG marker (Biolabs; Ozyme, Saint Quentin en Yvelines, France) was used as the DNA range size marker.
RESULTS
Isolate selection and antibiotic susceptibility. A total of 118 strains isolated from 72 subjects were identified as ESBL producers. For the subjects at the origin of several ESBL-producing isolates, it appeared that for each one of them the strains isolated were from the same species and presented similar antibiograms. Therefore, only one representative strain by subject was chosen for advanced characterization. A total of 72 ESBL producers were therefore selected, including 56 E. coli, 9 K. pneumoniae, 6 Enterobacter cloacae, and 1 Citrobacter freundii. They were detected in all participating hospitals, as well as in the community. The 72 ESBL carriers represented 61 patients out of 378 (16%), 2 health care workers out of 58 (3.4%), and 9 healthy subjects out of 382 (2.4%). The occurrence of ESBL-producing strains in the studied population of patients was mainly detected upon admission (32 patients out of 61). For patients who were not contaminated upon admission, a hospital stay longer than 1 month increased the risk of carriage of ESBL-producing strains. Antimicrobial susceptibility tests by the disk diffusion method indicated strong synergy between amoxicillin-clavulanic acid and either cefotaxime or ceftazidime. All ESBL-producing strains were resistant to extended-spectrum cephalosporins ceftazidime and cefotaxime (90% of the MICs were >32 mg/liter) but remained susceptible to imipenem. In the presence of 2 μg of clavulanic acid per ml, the susceptibility of these strains to ceftazidime and cefotaxime was restored. E. coli and K. pneumoniae isolates were susceptible to cefoxitin. A total of 43% of the strains were resistant to gentamicin, 17% were resistant to amikacin, and 79% were resistant to trimethoprim-sulfamethoxazole.
-Lactamase contents of isolates. The -lactamase content of 72 ESBL producers was analyzed, and the results are shown in Table 1. The majority of the isolates (n = 60) were found to produce -lactamases with a pI of 8.6. Other -lactamases of various pI values were also identified in some of the isolates, and these included enzymes with pIs of 7.3 and 8.2. The most frequent were pI 7.3 -lactamases observed in 25 strains isolated from healthy subjects and from all hospitals except HDF. -Lactamases with pI 8.2 were detected in 13 stains isolated from five hospitals (AH, HDF, NN, SJH, and SGH), including C. freundii HDF6, and in two strains isolated from healthy students (HS23 and HS79). In addition to enzymes with these pIs, all strains had another band of -lactamase activity with a pI of 5.4, and Klebsiella strains harbored a -lactamase with a pI of 7.6.
Identification of -lactamases with oxyiminocephalosporin-hydrolyzing activities. The bioassay experiments were carried out with protein extracts obtained from the isolates. The results are presented in Table 1. In all isolates, the pI 7.3, 8.2, and 8.6 -lactamases demonstrated cefotaxime- and ceftazidime-hydrolyzing activity under the conditions used in the assay.
Some other -lactamases that were observed as separate IEF bands in protein extracts of studied isolates, such as those with pIs of 5.4 (probably TEM-1) and 7.6 (probably species-specific K. pneumoniae enzymes) did not show cefotaxime- or ceftazidime hydrolyzing activity in the bioassay under the conditions used.
Detection and distribution of bla genes. Universal primers were used in PCR experiments to detect the blaTEM, blaSHV, blaCTX-M, and blaOXA-1 genes. All isolates producing -lactamases with pI 8.6 revealed PCR products coding for the CTX-M family of ESBLs. Detection of blaSHV genes was observed in isolates that produced the pI 7.6 and the pI 8.2 -lactamases. Isolates producing -lactamases with pI 7.3 were associated with the blaOXA-1 gene. Amplicons corresponding to blaTEM gene were detected in all of the strains, representing most likely the pI 5.4 -lactamase.
The complete nucleotide sequences of the blaSHV and blaCTX-M genes were determined for the 72 analyzed strains. The results of sequence analysis are shown in Table 1. All strains having amplicons with CTX-M type PCR were found to contain blaCTX-M-15 gene. Strains having amplicons with SHV type PCR revealed the presence of the blaSHV-5a.
A total of 60 strains were therefore found to harbor the blaCTX-M-15 gene and 13 strains were found to harbor the SHV-5a gene. The only C. freundii strain included in that study carried the SHV-5a ESBL gene. A total of 25 strains, all belonging to the E. coli species carried OXA-1-type gene. One E. coli strain isolated from a healthy subject (HS61) carried both CTX-M-15 and SHV-5a genes.
DNA macrorestriction. The application of this technique allowed the comparison of the genome of 47 strains of E. coli from different origins (patients, n = 36; health care workers, n = 2; healthy subjects, n = 9). Digestion with the restriction enzyme XbaI yielded 7 to 17 fragments over 50 kb in size (Fig. 1). Thirty-eight Gts were defined, linked by a similarity coefficient of >22%. Thirty-one Gts had only one strain, five had two subtypes, and three had three subtypes. The three strains of the Gt 10 did not seem to have an epidemiological link, since they were isolated from patients in three geographically separated units. It was the same for both strains of Gt 30. On the other hand, the two strains of Gt7 may be linked epidemiologically since they were isolated from patients of the same ward after a short lapse of time. It was the same for Gt 31. The two strains of Gt 13 could be linked, both of them being isolated from healthy subjects. The two strains of Gt 14 were genetically related, one isolated from a health care worker and the other from a patient of the same ward. Furthermore, two strains of Gt 28 were linked to the third strain isolated from healthy subject.
DISCUSSION
Although several studies addressed the issue of the emergence of ESBL-producing Enterobacteriaceae worldwide, no epidemiological survey has been carried out in Lebanon until now. It has only been reported that resistance to -lactam antibiotics is on the rise among clinical isolates of different Lebanese hospitals, expressing the need for an exhaustive research. The present study corresponds to the first national surveillance on ESBL-producing Enterobacteriaceae isolated from six Lebanese health care facilities and from the community.
A total of 72 nonduplicate ESBL-producing strains were collected from patients, health care workers, and healthy subjects from different regions of Lebanon. The percentage of ESBL-producing E. coli strains was higher than the one of ESBL-producing K. pneumoniae, a result that agrees with the Canadian epidemiological study (20) but is opposed to the one observed in previous studies done in Korea (13) and in the United Kingdom (22).
Molecular experiments revealed that CTX-M-15 was the prevalent ESBL produced. Unlike most CTX-Ms that preferentially hydrolyze cefotaxime, CTX-M-15, an Asp-240-Gly variant of CTX-M-3, increased the catalytic efficiency against ceftazidime (28). The same Asp-240-Gly substitution has also been reported in CTX-M-16 (3). Another substitution known to increase the hydrolyzing activity of ceftazidime is due to the Pro-167 Ser substitution in CTX-M-19 (29). CTX-M-15 was produced by 83% of the characterized strains and was detected in E. coli, K. pneumoniae, and E. cloacae species. Due to the large number of samples analyzed (n = 1,882), only one selecting medium, including ceftazidime (not cefotaxime), was chosen according to previous phenotypic results revealing a higher percentage of resistance to ceftazidime than cefotaxime among ESBL-producing strains isolated in a Lebanese hospital (7). Moreover, ceftazidime was reported as the best single test antibiotic for detecting extended-spectrum -lactamase production (14). Nevertheless, even though our findings show the predominance of CTX-M-15 enzyme, the medium used cannot detect classical CTX-Ms, and our results may thus underestimate the already extensive spread of CTX-M -lactamases in this country. The rapid emergence of the CTX-Ms as the predominant ESBL type is not an isolated Lebanese phenomenon. A recent report on ESBL types in Enterobacteriaceae in Argentinean public hospitals found that CTX-Ms accounted for roughly 70% of all ESBLs found (31), with similar findings in studies conducted in Japan (35), China (23), the United Kingdom (22), and Spain (33). CTX-M-15 has already been identified in several countries, including Taiwan (36), Turkey (16), France (8), and United Kingdom (24). Moreover, the recent detection in Lebanon of a Salmonella enterica serotype Typhimurium carrying the blaCTX-M-15 gene falls within the same track and supports our findings (18).
In the remaining isolates, SHV-5a ESBL was detected. Among the SHV-type -lactamases, SHV-5 and related enzymes seem to be the most prevalent ESBLs all over the world and were responsible for outbreaks of nosocomial infections in several countries (9, 19, 30). In the present study, SHV-5a was found to be produced by different species of the family Enterobacteriaceae, including C. freundii species. Although a variety of ESBL-producing C. freundii strains have been reported (6, 25), we believe this to be the first reported case of a SHV-5a-producing strain of C. freundii. One of the E. coli strains isolated from a healthy student carried both blaSHV-5a and blaCTX-M-15 genes. This cocarriage of ESBL genes is not a novel finding but is uncommon. TEM-derived ESBL enzymes were not detected in the present study.
The majority of epidemiological studies of ESBL-producing Enterobacteriaceae have only looked at hospital isolates; therefore, the search for these enzymes in community isolates in Lebanon is of interest. The isolation of ESBL-producing Enterobacteriaceae in six hospitals, as well as in the stools of healthy subjects, is indicative of the spread of these organisms in the whole country. The high degree of diversity of pulsed-field gel electrophoresis phenotypes of E. coli isolates producing either CTX-M-15 or SHV-5a enzymes suggested that the clonal dissemination of ESBL-producing strains, even if existing in certain hospitals (Gt 10 and Gt 30), did not play the predominant role in the overall spread. Plasmids may have been able to transfer within bacterial populations of separated hospitals by means of patient transfer, which is a common practice in Lebanon, or through acquisition from the community. The transmission of certain CTX-M-15 producing strains may have preceded the dissemination of plasmids carrying bla genes in Lebanon.
In summary, the data presented here illustrate the complexity and extent of the spread of ESBL-producing Enterobacteriaceae strains in Lebanon. Our results point out mainly the emergence and the dramatic dissemination of CTX-M-15-producing E. coli in this country. The fecal carriage of ESBL-producing isolates during no outbreak situations in hospitalized patients and the establishment of these isolates in the community represent an opportunity for these isolates to become endemic.
ACKNOWLEDGMENTS
We are very grateful to G. Paul and L. Gilly from the bacteriology laboratory of the CHU Cochin for helpful assistance in the IEF experiments.
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Saint George Hospital, and University of Balamand, Beirut, Lebanon
Lebanese University, Beirut, and Nini Hospital, Tripoli, Lebanon
Arz Hospital, Al Metn, Lebanon
Rizk Hospital, Beirut, Lebanon
Saint Joseph Hospital, Beirut, Lebanon
Htel Dieu de France Hospital, Beirut, Lebanon
C. H. de Versailles, Le Chesnay, France
ABSTRACT
A prospective study was carried out to assess the extent of carriage of extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected -lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of Escherichia coli (n = 56), Klebsiella pneumoniae (n = 9), Enterobacter cloacae (n = 6), and Citrobacter freundii (n = 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing E. coli presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing E. coli in Lebanon, it represents the first report of an SHV-5a-producing C. freundii.
INTRODUCTION
-Lactam antibiotics are widely used in the treatment of bacterial infections. However, the production of extended-spectrum -lactamases (ESBLs), one of the resistance mechanisms encountered in Enterobacteriaceae, has been associated with several treatment failures. Indeed, ESBLs are capable of efficiently hydrolyzing extended-spectrum cephalosporins (cefotaxime and ceftazidime) and are highly susceptible to inhibition by clavulanic acid and tazobactam (17). In addition to the classical TEM and SHV enzymes, several ESBLs of predominantly Ambler's molecular class A have recently been reported, including the CTX-M enzymes (2). Since the second half of the 1980s, ESBLs disseminate dangerously into wide geographic regions (4). This spread may be the consequence of plasmid transmission among nonrelated Enterobacteriaceae, as observed in French hospitals (27). It may also be mediated by the clonal dissemination of the enzyme-producing microorganisms (10). Sometimes, transmission of ESBL-producing strains between hospitals, followed by their clonal expansion and/or the horizontal transfer of plasmids carrying the ESBL gene, may even occur (1).
Although ESBL-producing strains have been reported by several countries, very few resistance data representing the Middle East have been published. Moreover, the majority of the studies represent hospital-based patients, whereas the carriage rates of ESBL from fecal flora of patients or community-based subjects remains unknown. Infections caused by ESBL-producing strains are increasing in frequency in Lebanon, a Mediterranean country in the Middle East with a dynamic and ethnically diverse population of 3.5 million. A study done in Lebanon at Saint George Hospital revealed that, among the 4,299 isolates of Escherichia coli and 1,248 isolates of Klebsiella pneumoniae tested over the 5 years between 1997 and 2001, ESBL production was observed with 2% of the E. coli and 20% of the K. pneumoniae strains (7). However, no epidemiological survey of ESBL-producing strains has been performed until now in this country. The present prospective multicentric study was done in order to report the extent of carriage of ESBL-producing Enterobacteriaceae and to characterize the types of ESBLs produced at both hospital and community levels in Lebanon.
(This study was presented in part at the ECC & RICAI 2004 Congress, Paris, France, on 1 to 3 December 2004 [abstr. 340/770].)
MATERIALS AND METHODS
Study design. This study was a prospective laboratory-based surveillance study in which fecal samples collected from hospitalized patients, health care personnel, and healthy subjects were screened for the presence of possible ESBL-producing strains. A total of 1,442 fecal samples (1,440 rectal swabs and 2 feces samples) were collected between 1 January 2003 and 31 March 2003 from 378 patients admitted to the intensive care units (including the neonatal intensive care unit) of five Lebanese tertiary care general hospitals located in different areas of Lebanon: Rizk Hospital, Beirut (RH); Saint George Hospital, Beirut (SGH); Arz Hospital, Al Metn (AH); Nini Hospital, Tripoli (North Lebanon) (NN); and Saint Joseph Hospital, Beirut (SJH). Patients were checked for carriage of ESBL-producing strains upon admission, 72 h later, and then at weekly intervals over a 3-month period. The 58 health care workers of the same intensive care units were checked (rectal swabs) for ESBL-producing strains once at the beginning of the study during the same period. A total of 382 fecal samples (rectal swabs) were collected between 1 April 2003 and 31 June 2003 from 382 young healthy students (HS) from the Lebanese University-Faculty of Public Health section 3 (in Tripoli, North Lebanon) and section 4 (in Bekaa, East Lebanon).
Bacterial strains. All samples were suspended at participating sites in normal saline in order to form a 10-μg/ml suspension. From these, 100 μl was streaked on Trypticase soy-5% sheep blood agar containing 6 μg of vancomycin/ml, 2 μg of amphotericin B/ml, 2 μg of ceftazidime/ml, and 1 μg of clindamycin/ml (VACC medium) (11). After 24 and 48 h of incubation at 35°C, colonies appearing on the plates were sent to Saint George Hospital for strain identification by using the API 20E system (bioMerieux) and for resistance phenotype analysis. To these strains were added six ESBL-producing strains isolated from stools of patients admitted to the intensive care unit of the Htel Dieu de France Hospital, Beirut (HDF), between 1 January 2003 and 31 March 2003.
Antibiotic susceptibility testing and ESBL production detection. ESBL production was detected by the double-disk synergy test according to the method of Jarlier et al. (12). Antibiotic susceptibility of ESBL-producing strains was determined by the disk diffusion method according to the recommendations of the French Society of Microbiology (32). The following commercial disks were tested: amoxicillin-clavulanic acid (20 μg-10 μg), cefoxitin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), gentamicin (10 μg), trimethoprim-sulfamethoxazole (1.25 μg-23.75 μg), amikacin (10 μg), and imipenem (10 μg). The MICs of ceftazidime and cefotaxime with or without clavulanic acid (at a fixed concentration of 2 μg/ml) were determined by using serial twofold dilutions of antibiotics in Mueller-Hinton agar and inocula of 105 CFU per spot. For each subject, if several ESBL-producing isolates were detected and all were from the same species and presented similar antibiograms, only one was selected for further study, and the others were considered to be repeat isolations of the same strain.
Analytical IEF of -lactamases and bioassays for penicillin G, ceftazidime, and cefotaxime hydrolyzing activities. Isoelectric focusing (IEF) was performed according to the method of Labia and Barthelemy (15) on all selected isolates. Crude cell-free preparations of -lactamases were obtained by three sonications, each for 10 s, at an amplitude of 12 μm, with intermittent cooling on ice. Supernatants (15 μl) of each preparation were subjected to IEF on prepared polyacrylamide gels and separated with 350 V for 18 h at 4°C with the Multiphor II system (Pharmacia-Biotech, Orsay, France). After IEF, -lactamase activity was visualized by the iodometric method using gels containing penicillin G, ceftazidime, or cefotaxime. -Lactamases were identified by comparison to reference enzymes run in tracks adjacent to the test samples.
PCR detection and sequencing of blaTEM, blaSHV, blaCTX-M, and blaOXA-1 genes. PCRs and sequence analysis were used in order to identify the gene responsible for the ESBL phenotype in all strains. It should be noted that not all genes were sequenced in strains where multiple -lactamases genes existed. The isoelectric points (pIs) of the -lactamases were used to predict the most likely ESBL candidate, and this gene was sequenced. For example, if a strain contained blaTEM and the IEF results showed a -lactamase with a pI of 5.4, an assumption was made that this -lactamase most likely represented the non-ESBL TEM-1 and the other gene was sequenced.
All strains were examined for the presence of blaTEM, blaSHV, blaCTX-M, and blaOXA-1 using PCR conditions as previously prescribed (5, 21, 26). Primers MAb/F (5'-GGGGAGCTCATAAAATTCTTGAAGAC) and MAb/R (5'-GGGGGATCCTTACCAATGCTTAATCA) were used for amplifications of the entire blaTEM genes. Primers SHV/F (5'-GCCCGGGTTATTCTTATTTGTCGC) and SHV/R (5'-TCTTTCCGATGCCGCCGCCAGTCA) were used for amplifications of the entire blaSHV genes. CTX-M-type genes were detected by using universal primers CTX-M-U1 (5'-ATGTGCAGYACCAGTAARGTKATGGC) and CTX-M-U2 (5'-TGGGTRAARTARGTSACCAGAAYCAGCGG) (where R is purine, Y is pyrimidine, and S is G or C). Primers OXA-1/F (5'-ATGAAAAACACAATACATATC) and OXA-1/R (5'-AATTTAGTGTGTTTAGAATGG) were used for partial amplification of blaOXA-1 genes. Both DNA strands of PCR products were sequenced by using the above-cited primers for blaTEM and blaSHV genes. Entire CTX-M-type genes were amplified for sequencing purposes by using primer forward CTX-M-1-F (5'-GGTTAAAAAATCACTGCGTC-3'; located at positions 3 to 22) and primer reverse CTX-M-15-R (5'--TTACAAACCGTCGGTGACGA-3'; located at positions 876 to 857) as described previously (34). The sequence of the blaTEM, blaSHV, and blaCTX-M ORF PCR products were determined by using a Big Dye PCR (Perkin-Elmer/Cetus) and analyzed on an ABI Prism 310 DNA Genetic Analyzer (Perkin-Elmer Applied Biosystems).
Pulsed-field gel electrophoresis typing. The GenePath Group VI reagent kit and Genepath instruments (Bio-Rad Laboratories, Hercules, CA) were used to analyze the genome of strains representing the predominant species of ESBL-producing Enterobacteriaceae in Lebanon. Bacteria inserted in agarose plugs were lysed with lysozyme and treated with proteinase K. Genomic DNA was digested with XbaI, and the fragments were separated in agarose gels by electrophoresis based on a contour-clamped homogeneous electric field method according to the manufacturer's recommendations. After being stained with ethidium bromide, the gels were placed on a UV transilluminator, and the images were digitized with the Gel Doc 1000 system (Bio-Rad). To compare the profiles among different gels, a reference strain was routinely deposited in duplicate on each gel, thereby improving the assessment of strain relatedness. DNA relatedness was determined with Molecular Analyst RFLP software (Bio-Rad). Macrorestriction analysis was limited to DNA fragments of at least 50 kb. A similarity index was determined for each pair of strains by using the Dice coefficient, with a 0.80% band tolerance. Clustering correlation coefficients were calculated by using the unweighted pair-group method of arithmetic averages (UPGMA). Isolates were considered to be within a genotype (Gt) if the similarity coefficient was 80%. The lambda ladder PFG marker (Biolabs; Ozyme, Saint Quentin en Yvelines, France) was used as the DNA range size marker.
RESULTS
Isolate selection and antibiotic susceptibility. A total of 118 strains isolated from 72 subjects were identified as ESBL producers. For the subjects at the origin of several ESBL-producing isolates, it appeared that for each one of them the strains isolated were from the same species and presented similar antibiograms. Therefore, only one representative strain by subject was chosen for advanced characterization. A total of 72 ESBL producers were therefore selected, including 56 E. coli, 9 K. pneumoniae, 6 Enterobacter cloacae, and 1 Citrobacter freundii. They were detected in all participating hospitals, as well as in the community. The 72 ESBL carriers represented 61 patients out of 378 (16%), 2 health care workers out of 58 (3.4%), and 9 healthy subjects out of 382 (2.4%). The occurrence of ESBL-producing strains in the studied population of patients was mainly detected upon admission (32 patients out of 61). For patients who were not contaminated upon admission, a hospital stay longer than 1 month increased the risk of carriage of ESBL-producing strains. Antimicrobial susceptibility tests by the disk diffusion method indicated strong synergy between amoxicillin-clavulanic acid and either cefotaxime or ceftazidime. All ESBL-producing strains were resistant to extended-spectrum cephalosporins ceftazidime and cefotaxime (90% of the MICs were >32 mg/liter) but remained susceptible to imipenem. In the presence of 2 μg of clavulanic acid per ml, the susceptibility of these strains to ceftazidime and cefotaxime was restored. E. coli and K. pneumoniae isolates were susceptible to cefoxitin. A total of 43% of the strains were resistant to gentamicin, 17% were resistant to amikacin, and 79% were resistant to trimethoprim-sulfamethoxazole.
-Lactamase contents of isolates. The -lactamase content of 72 ESBL producers was analyzed, and the results are shown in Table 1. The majority of the isolates (n = 60) were found to produce -lactamases with a pI of 8.6. Other -lactamases of various pI values were also identified in some of the isolates, and these included enzymes with pIs of 7.3 and 8.2. The most frequent were pI 7.3 -lactamases observed in 25 strains isolated from healthy subjects and from all hospitals except HDF. -Lactamases with pI 8.2 were detected in 13 stains isolated from five hospitals (AH, HDF, NN, SJH, and SGH), including C. freundii HDF6, and in two strains isolated from healthy students (HS23 and HS79). In addition to enzymes with these pIs, all strains had another band of -lactamase activity with a pI of 5.4, and Klebsiella strains harbored a -lactamase with a pI of 7.6.
Identification of -lactamases with oxyiminocephalosporin-hydrolyzing activities. The bioassay experiments were carried out with protein extracts obtained from the isolates. The results are presented in Table 1. In all isolates, the pI 7.3, 8.2, and 8.6 -lactamases demonstrated cefotaxime- and ceftazidime-hydrolyzing activity under the conditions used in the assay.
Some other -lactamases that were observed as separate IEF bands in protein extracts of studied isolates, such as those with pIs of 5.4 (probably TEM-1) and 7.6 (probably species-specific K. pneumoniae enzymes) did not show cefotaxime- or ceftazidime hydrolyzing activity in the bioassay under the conditions used.
Detection and distribution of bla genes. Universal primers were used in PCR experiments to detect the blaTEM, blaSHV, blaCTX-M, and blaOXA-1 genes. All isolates producing -lactamases with pI 8.6 revealed PCR products coding for the CTX-M family of ESBLs. Detection of blaSHV genes was observed in isolates that produced the pI 7.6 and the pI 8.2 -lactamases. Isolates producing -lactamases with pI 7.3 were associated with the blaOXA-1 gene. Amplicons corresponding to blaTEM gene were detected in all of the strains, representing most likely the pI 5.4 -lactamase.
The complete nucleotide sequences of the blaSHV and blaCTX-M genes were determined for the 72 analyzed strains. The results of sequence analysis are shown in Table 1. All strains having amplicons with CTX-M type PCR were found to contain blaCTX-M-15 gene. Strains having amplicons with SHV type PCR revealed the presence of the blaSHV-5a.
A total of 60 strains were therefore found to harbor the blaCTX-M-15 gene and 13 strains were found to harbor the SHV-5a gene. The only C. freundii strain included in that study carried the SHV-5a ESBL gene. A total of 25 strains, all belonging to the E. coli species carried OXA-1-type gene. One E. coli strain isolated from a healthy subject (HS61) carried both CTX-M-15 and SHV-5a genes.
DNA macrorestriction. The application of this technique allowed the comparison of the genome of 47 strains of E. coli from different origins (patients, n = 36; health care workers, n = 2; healthy subjects, n = 9). Digestion with the restriction enzyme XbaI yielded 7 to 17 fragments over 50 kb in size (Fig. 1). Thirty-eight Gts were defined, linked by a similarity coefficient of >22%. Thirty-one Gts had only one strain, five had two subtypes, and three had three subtypes. The three strains of the Gt 10 did not seem to have an epidemiological link, since they were isolated from patients in three geographically separated units. It was the same for both strains of Gt 30. On the other hand, the two strains of Gt7 may be linked epidemiologically since they were isolated from patients of the same ward after a short lapse of time. It was the same for Gt 31. The two strains of Gt 13 could be linked, both of them being isolated from healthy subjects. The two strains of Gt 14 were genetically related, one isolated from a health care worker and the other from a patient of the same ward. Furthermore, two strains of Gt 28 were linked to the third strain isolated from healthy subject.
DISCUSSION
Although several studies addressed the issue of the emergence of ESBL-producing Enterobacteriaceae worldwide, no epidemiological survey has been carried out in Lebanon until now. It has only been reported that resistance to -lactam antibiotics is on the rise among clinical isolates of different Lebanese hospitals, expressing the need for an exhaustive research. The present study corresponds to the first national surveillance on ESBL-producing Enterobacteriaceae isolated from six Lebanese health care facilities and from the community.
A total of 72 nonduplicate ESBL-producing strains were collected from patients, health care workers, and healthy subjects from different regions of Lebanon. The percentage of ESBL-producing E. coli strains was higher than the one of ESBL-producing K. pneumoniae, a result that agrees with the Canadian epidemiological study (20) but is opposed to the one observed in previous studies done in Korea (13) and in the United Kingdom (22).
Molecular experiments revealed that CTX-M-15 was the prevalent ESBL produced. Unlike most CTX-Ms that preferentially hydrolyze cefotaxime, CTX-M-15, an Asp-240-Gly variant of CTX-M-3, increased the catalytic efficiency against ceftazidime (28). The same Asp-240-Gly substitution has also been reported in CTX-M-16 (3). Another substitution known to increase the hydrolyzing activity of ceftazidime is due to the Pro-167 Ser substitution in CTX-M-19 (29). CTX-M-15 was produced by 83% of the characterized strains and was detected in E. coli, K. pneumoniae, and E. cloacae species. Due to the large number of samples analyzed (n = 1,882), only one selecting medium, including ceftazidime (not cefotaxime), was chosen according to previous phenotypic results revealing a higher percentage of resistance to ceftazidime than cefotaxime among ESBL-producing strains isolated in a Lebanese hospital (7). Moreover, ceftazidime was reported as the best single test antibiotic for detecting extended-spectrum -lactamase production (14). Nevertheless, even though our findings show the predominance of CTX-M-15 enzyme, the medium used cannot detect classical CTX-Ms, and our results may thus underestimate the already extensive spread of CTX-M -lactamases in this country. The rapid emergence of the CTX-Ms as the predominant ESBL type is not an isolated Lebanese phenomenon. A recent report on ESBL types in Enterobacteriaceae in Argentinean public hospitals found that CTX-Ms accounted for roughly 70% of all ESBLs found (31), with similar findings in studies conducted in Japan (35), China (23), the United Kingdom (22), and Spain (33). CTX-M-15 has already been identified in several countries, including Taiwan (36), Turkey (16), France (8), and United Kingdom (24). Moreover, the recent detection in Lebanon of a Salmonella enterica serotype Typhimurium carrying the blaCTX-M-15 gene falls within the same track and supports our findings (18).
In the remaining isolates, SHV-5a ESBL was detected. Among the SHV-type -lactamases, SHV-5 and related enzymes seem to be the most prevalent ESBLs all over the world and were responsible for outbreaks of nosocomial infections in several countries (9, 19, 30). In the present study, SHV-5a was found to be produced by different species of the family Enterobacteriaceae, including C. freundii species. Although a variety of ESBL-producing C. freundii strains have been reported (6, 25), we believe this to be the first reported case of a SHV-5a-producing strain of C. freundii. One of the E. coli strains isolated from a healthy student carried both blaSHV-5a and blaCTX-M-15 genes. This cocarriage of ESBL genes is not a novel finding but is uncommon. TEM-derived ESBL enzymes were not detected in the present study.
The majority of epidemiological studies of ESBL-producing Enterobacteriaceae have only looked at hospital isolates; therefore, the search for these enzymes in community isolates in Lebanon is of interest. The isolation of ESBL-producing Enterobacteriaceae in six hospitals, as well as in the stools of healthy subjects, is indicative of the spread of these organisms in the whole country. The high degree of diversity of pulsed-field gel electrophoresis phenotypes of E. coli isolates producing either CTX-M-15 or SHV-5a enzymes suggested that the clonal dissemination of ESBL-producing strains, even if existing in certain hospitals (Gt 10 and Gt 30), did not play the predominant role in the overall spread. Plasmids may have been able to transfer within bacterial populations of separated hospitals by means of patient transfer, which is a common practice in Lebanon, or through acquisition from the community. The transmission of certain CTX-M-15 producing strains may have preceded the dissemination of plasmids carrying bla genes in Lebanon.
In summary, the data presented here illustrate the complexity and extent of the spread of ESBL-producing Enterobacteriaceae strains in Lebanon. Our results point out mainly the emergence and the dramatic dissemination of CTX-M-15-producing E. coli in this country. The fecal carriage of ESBL-producing isolates during no outbreak situations in hospitalized patients and the establishment of these isolates in the community represent an opportunity for these isolates to become endemic.
ACKNOWLEDGMENTS
We are very grateful to G. Paul and L. Gilly from the bacteriology laboratory of the CHU Cochin for helpful assistance in the IEF experiments.
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