JAC Advance Access originally published online on October 29, 2007
Journal of Antimicrobial Chemotherapy 2008 61(1):64-72; doi:10.1093/jac/dkm403
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Original research |
Complex molecular epidemiology of extended-spectrum β-lactamases in Klebsiella pneumoniae: a long-term perspective from a single institution in Madrid
1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal and CIBER-ESP, Madrid, Spain 2 Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y Cajal, Madrid, Spain
* Corresponding author. Tel: +34-91-3368330; Fax: +34-91-3368809; E-mail: rcanton.hrc{at}salud.madrid.org
Received 6 July 2007; returned 15 August 2007; revised 17 August 2007; accepted 25 September 2007
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Abstract |
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Objectives: To analyse all extended-spectrum-β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates recovered from 2001 to 2004 in our institution and to compare this period with that of 1989 to 2000.
Methods: All K. pneumoniae isolates recovered during the studied period were screened for ESBL production. One isolate per patient was selected for ESBL characterization and for population structure, including phylogenetic groups, and plasmid analysis.
Results: Ninety-three (3.2% mean) ESBL-producing K. pneumoniae isolates recovered from 61 patients (26%, medical wards; 18%, surgical wards; 25%, ICU; and 31%, outpatients) were identified. Outpatients significantly increased (P < 0.01) when compared with 1989–2000 (7%). The number of different ESBLs increased with persistence of previously identified enzymes (TEM-4, SHV-2, CTX-M-9 and CTX-M-10) and emergence of new ESBLs (TEM-110, SHV-11, SHV-12, CTX-M-14 and CTX-M-15). A polyclonal structure, including epidemic clones with specific ESBLs (TEM-4, SHV-12 and CTX-M-15), was observed. Phylogenetic analysis showed that most isolates (74.6%) belonged to KpI-type with a clear relationship between KpIII-type and CTX-M-10 producers. Persistence of specific plasmids associated with specific ESBLs (TEM-4, SHV-12, CTX-M-10 and CTX-M-15) was observed. Co-resistance analysis revealed an increment in resistance to trimethoprim (41.5% versus 10.3%), sulphonamide (54.7% versus 29.3%) and nalidixic acid (34.0% versus 6.9%) when compared with 1989–2000.
Conclusions: K. pneumoniae is still an important ESBL producer in our institution with a complex epidemiology. The main features were a few outbreaks with persistence of specific plasmids, emergence of new enzymes and an increment in community isolates. These results should be taken into account for the implementation of epidemiological containment measures.
Keywords: ESBLs , K. pneumoniae , long-term studies
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Introduction |
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Extended-spectrum β-lactamases (ESBLs) were initially described in Germany and France during the 1980s and they are now reported worldwide.1 The ESBLs first identified derived from TEM-1, TEM-2 and SHV-1 enzymes, however the CTX-M ESBLs, which were identified at a later date, are now the largest group within ESBLs.1,2 At the time of the emergence of ESBLs, they were consistently associated with nosocomial outbreaks of Klebsiella pneumoniae isolates, mainly in intensive care units (ICUs). This situation changed when ESBLs emerged in the community and Escherichia coli became the main pathogen harbouring these enzymes.3,4 Nevertheless, K. pneumoniae is still an important ESBL producer not only in the nosocomial setting but also in the community.5–11
Previous studies have demonstrated large outbreaks due to specific multiresistant K. pneumoniae strains expressing ESBLs10,12,13 and have shown a complex epidemiological situation involving different clones, different ESBLs and different genetic elements carrying the blaESBL genes in some institutions.14–16 An increase in the isolation of SHV- and CTX-M-producing K. pneumoniae has also been demonstrated1,7,17,18 in different multicentre studies and as in E. coli, the dissemination of ESBLs has been produced due to clonal expansion and/or plasmid transfer.2,14,19 Recently, the relationship between particular bacterial hosts and ESBLs has been investigated and different studies have addressed an association of ESBL types with specific E. coli phylogenetic groups.20–23 The studies of K. pneumoniae phylogenetic groups remain limited and have been mainly focused on the relationship between these groups and the chromosomal β-lactamases.24 Although all three different phylogenetic groups described so far in K. pneumoniae (KpI, KpII and KpIII) are significantly associated with specific chromosomal SHV-type bla genes, coding for SHV, OKP and LEN enzymes, respectively, as far as we know there is no evidence of the potential association between each phylogenetic group and the production of ESBLs.
In the present study, all ESBL-producing K. pneumoniae isolates recovered from 2001 to 2004 in our institution were studied, updating the complex situation previously described in our hospital.14 In addition, the population structure, including phylogenetic groups, of all ESBL-producing K. pneumoniae isolates recovered since its emergence in 1989 in our hospital was characterized addressing their potential relationship with different ESBL types. The continuous and long-term follow-up of these isolates in our institution demonstrates the changing epidemiology of ESBLs in K. pneumoniae over this period.
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Materials and methods |
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Bacterial isolates
Ninety-three K. pneumoniae clinical isolates from 61 patients (31% ambulatory) obtained from January 2001 to December 2004 at our Microbiology Department of Ramón y Cajal University Hospital (a 1200 bed institution providing paediatric and adult medical and surgical care to the North-East region of Madrid, Spain and covering specialized healthcare to 
550 000 inhabitants) and displaying a phenotype compatible with the presence of an ESBL were studied. All these isolates were included in the study based on their resistance phenotypes and the positive results with the double disc synergy test (DDST).25 DDST was performed with conventional amoxicillin/clavulanate, cefotaxime, ceftazidime and cefepime discs that were applied 20 and 30 mm apart. One isolate per patient was selected for characterization of ESBL and further genetic studies. Bacterial identification and initial antibiotic susceptibility testing were performed using the WIDER system (Fco. Soria Melguizo, Madrid, Spain). For comparative purposes, 62 ESBL-producing K. pneumoniae isolates previously reported14 and recovered in our institution since its time of emergence in 1989 until 2000 were also included to analyse trends of different ESBLs, plasmid persistence and population structure, including phylogenetic groups (see below).
Standard CLSI (2006)26 microdilution was performed for definitive susceptibility pattern. Standard disc diffusion (CLSI) was used to investigate the ESBL-associated resistance profiles to non-β-lactam antibiotics. Discs were purchased from Oxoid (Basingstoke, UK).
Bacteria exponentially growing at 37ºC in Luria–Bertani medium were harvested, and cell-free lysates were prepared by sonication. Isoelectric focusing was performed by applying the crude sonic extract to Phast gels (pH 3–9) in a Phastsystem apparatus (Pharmacia AB, Uppsala, Sweden).27 β-Lactamases with known pIs (5.9, 5.4, 7.6 and 8.1) were used in parallel as controls. Gels were stained with 500 mg of nitrocefin (Oxoid) per litre to identify β-lactamase bands.
Amplification of blaESBL genes was performed using genomic DNA as template from wild-type isolates and specific primers and cycling conditions for the TEM, SHV and the CTX-M groups.14,28 PCR products were purified with a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced using ABI Prism 377 automatic sequencer (PE, Norwalk, CT, USA).
Plasmid analysis and conjugation experiments
The content and size of plasmid was determined in the E. coli transconjugants by Barton's method.29 For fingerprinting analysis, plasmid DNA was obtained by using a QIAGEN Plasmid Midi Kit (Qiagen), digested with EcoRI, PstI and HpaI and electrophoresed in 1% agarose at 100 V for 3 h.
Transfer of bla genes was carried out by broth or filter mating using a rifampicin-resistant E. coli BM21 (nalidixic acid and rifampicin-resistant, lactose fermentation-positive and plasmid-free) as recipient. Luria–Bertani agar plates containing ceftazidime or cefotaxime (2 mg/L) and rifampicin (100 mg/L) were used to select transconjugants.
Phylogenetic group and chromosomal β-lactamase gene family determination
Identification of K. pneumoniae phylogenetic groups (KpI, KpII and KpIII) and determination of different chromosomal β-lactamase gene families (blaSHV, blaOKP and blaLEN) were performed by PCR-based methods previously described.24,30
Pulsed-field gel electrophoresis
Bacterial DNA was prepared as described previously31 using XbaI (Takara Bio, Shiga, Japan) as the restriction enzyme. Digested DNA was separated by electrophoresis in 1.2% agarose gel (Bio-Rad) and 0.5% Tris–borate–EDTA buffer by using a CHEF-DRIII system (Bio-Rad, La Jolla, CA, USA). Conditions were as follows: 14ºC, 6 V/cm, 10–40 s, 22 h. The relationship among isolates was interpreted according to the criteria established by Tenover et al.32
Statistical associations were analysed using 
2 for categorical values and Mantel Haenszel tests for continuous variables. Statistical significance was considered when two-tailed P value was lower than 0.05.
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Results |
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Epidemiological background
Table 1 shows secular trends of all ESBL-producing K. pneumoniae isolates (n = 252) in our institution since the first detection in 1989. Overall, an increment in the number of isolates harbouring ESBLs was observed: 159 isolates were obtained from 1989 to 2000 (13.2/year) and 93 isolates from 2001 to 2004 (23.2/year), but this increment was parallel to that observed in the total number of K. pneumoniae isolates recovered in our laboratory (P = 0.298). It is of note that the increase in isolation of K. pneumoniae isolates since 2001 was mainly due to our departmental process of centralization of all microbiology studies performed in our geographical area. The proportion of ESBL K. pneumoniae isolates ranged from 2.5% to 4.8% during 2001–04 (3.2% mean) and from 0.4% to 18.2% (4.8% mean) during 1989–2000 period,14 the highest figure corresponding to an outbreak situation previously described in the cardiopaediatric ICU during 1997–1998.5,14
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The number of patients per year with ESBL-producing K. pneumoniae isolates during 2001–04 (61 patients, yearly mean 15.2) increased when compared with those from 1989 to 2000 (58 patients, yearly mean 5.3) (Table 1). The origin of patients during 2001–04 was as follows: 16 (26%) were in medical wards, 11 (18%) were in surgical wards, 15 (25%) were in ICUs and 19 (31%) were outpatients. Although not statistically significant (P = 0.03), a decrease in ICU patients with ESBL-positive K. pneumoniae isolates through 2001–04 was observed when compared with those from 1989 to 2000 (41%). In contrast, the proportion of outpatients with ESBL-producing K. pneumoniae isolates significantly (P < 0.01) increased since 2001 (31% versus 7%) (Figure 1).
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The sources of the 61 isolates recovered during 2001–04 were as follows: urine (n = 30; 49.2%), respiratory tract (n = 13; 21.3%), blood (n = 5; 8.2%), wound (n = 8; 13.1%), catheter (n = 2; 3.3%), rectal swab (n = 2; 3.3%) and bile (n = 1; 1.6%). A significant (P = 0.002) increase in urine ESBL-producing K. pneumoniae isolates was detected (30 out of 61 isolates versus 14 out of 62 isolates), as was a non-significant decrease of these isolates in respiratory tract specimens (13 out of 61 isolates versus 22 out of 62 isolates) when compared with those published in our previous series.14
Figure 2 shows the distribution of ESBL enzymes in our institution (one ESBL-producing K. pneumoniae isolate per patient) and secular trend of yearly clone incidence since 1989 until 2004. The increased diversity of ESBLs during 2001–04 was due to the emergence of new enzymes in our geographical area (TEM-110, SHV-11, SHV-12, CTX-M-14 and CTXM-15) with persistence of previously identified enzymes (TEM-4, SHV-2, CTX-M-9 and CTX-M-10). Some types detected in 1989–2000, such as SHV-2a, were not present in the later period (Figure 2). Distribution of the different ESBL groups during 2001–04 was as follows: SHV-type, 44% (n = 27); TEM-type, 26% (n = 16); CTX-M-1 cluster, 25% (n = 15); and CTX-M-9 cluster, 5% (n = 3) (Table 2). The number of isolates expressing TEM-type ESBLs significantly (P 
0.001) increased since 1989 as to a lesser extent did SHV types (P = 0.032). Secular trend was not significant when all CTX-M enzymes were considered, probably due to the tenacity of CTX-M-10 along this period. The emergence of CTX-M-15-producing isolates in 2002 and the increase during the last studied year are of note. Moreover, very few isolates expressed CTX-M-9 or CTX-M-14 enzymes.
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Population structure and phylogenetic groups
Population structure analysis (one ESBL-positive isolate per patient) revealed the persistence of the previously described complex situation,14 characterized by the maintenance of a polyclonal structure: 32 different clones were detected during 2001–04 (mean per year 9.7) including epidemic clones associated with TEM-4, SHV-12 and CTX-M-15 enzymes (Table 2). Most of the TEM-4 isolates (12/13) presented the same PFGE-type (Kp41T). Most of the isolates belonging to this clone were recovered from outpatients (8/12, 66.7%) and to a lesser extent from ICU (2/12, 16.7%) and medical (2/12, 16.7%) patients. An opposite scenario was observed in our institution during 1989–2000, when TEM-4 was mainly identified in ICU patients. Eleven out of 16 SHV-12 isolates from 2001 to 2004 showed the same PFGE pattern (Kp49S), but were isolated at different hospital locations (medical, 54.5%; ICU, 36.4%; and surgical, 9.1%) and none of them were outpatients. Most of the CTX-M-15-producing isolates (6 out of 8) belonged to the same PFGE-type (Kp47C), all of them isolated since 2002. In this case, with the exception of one isolate identified in an outpatient, they were detected in hospitalized patients at different locations (Table 2).
Analysis of phylogenetic groups of representative isolates of each clone (1989–2004) showed that most of them belonged to KpI-type (74.6%), less to the KpIII-type (20.9%), and only 3% belonged to KpII-type. Interestingly, there was a clear relationship between the KpIII group and production of CTX-M-10 enzymes (44.5% of CTX-M isolates). All but one KpIII-type isolate was positive for LEN chromosomal β-lactamase gene (Table 3).
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Plasmid analysis
The presence of specific plasmids among different isolates was studied in the transconjugants (one each per clone). For widely distributed ESBLs such as TEM-4, SHV-12 and CTX-M-15, more than one isolate per clone was analysed to ascertain plasmid diversity and stability. Two different plasmids (pRYCE11 and pRYCE26) were identified in TEM-4-producing isolates: (i) pRYCE11 was isolated in different clones (Kp41T and Kp70T) from 2002 to 2004 (Table 2) and showed an identical EcoRI, PstI and HpaI restriction pattern to that of plasmid associated with the outbreak at the cardiopaediatric ICU in our institution during 4 years (1995–99);14 and (ii) pRYCE26 was only found in Kp41T clones.
Four plasmid types carrying blaSHV-12 were observed (pRYCE28, pRYCE32, pRYCE33 and pRYCE38). The pRYCE28 (60 kb), which was described in the transconjugants obtained from Kp49S clone, persisted from 2001 to 2003 in different wards (36.4% ICU, 54.5% medical and 9.1% surgical). It is noteworthy that plasmid pRYCE38 (clone Kp63S) was also present in two different clones (Kp58S and Kp59S) containing SHV-2.
In the case of CTX-M-10-producing isolates, three different plasmid RFLP types (pRYCE29, pRYCE30 and pRYCE31) were described. Plasmid pRYCE30 was present in three CTX-M-10-producing clones (Kp52C, Kp53C and Kp54C), which were isolated from 2001 to 2003 in different hospital areas (Table 2). All CTX-M-15 (Kp47C and Kp66C) studied clones harboured the same plasmid (pRYCE34) of 180 kb isolated from 2002 to 2004.
Susceptibility patterns of non-β-lactam antibiotics revealed an increment of trimethoprim (41.5% versus 10.3%), sulphonamide (54.7% versus 29.3%), and nalidixic acid (34.0% versus 6.9%) resistances during 2001–04 when compared with the corresponding figures during 1989–2000.14 In contrast, gentamicin (37.7% versus 63.8%) and tobramycin (43.4% versus 63.7%) resistances decreased during 2001–04. These results may be related to different co-resistances associated with different ESBLs. Isolates with TEM-type ESBLs showed high tobramycin resistance rates (93.3%), and all of them were resistant to gentamicin. This resistance was transferred in most cases (71.4%). In the case of SHV-types, 92% of isolates were resistant to sulphonamides but this resistance phenotype was not transferred, although trimethoprim resistance was achieved in most transconjugants. Six out of seven isolates harbouring CTX-M-10 enzymes were susceptible to all the non-β-lactam antibiotics tested. All isolates harbouring blaCTX-M-15 were resistant to nalidixic acid and 75% were resistant to kanamycin.
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Discussion |
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During recent years, attention has been focused on the increment of ESBL-producing E. coli, whereas a decrease in K. pneumoniae isolates has been observed in some studies.10,18 This fact is also due to the contribution of E. coli in the dissemination of ESBLs, particularly in the community setting.3,4,33 Nevertheless, as we demonstrate in the present study, the complex epidemiology of ESBL-producing K. pneumoniae is still a major concern. We have confirmed: (i) the maintenance of ESBL-producing K. pneumoniae isolates from 1989 to 2004 in our institution; (ii) the increased prevalence in the community during recent years; (iii) the emergence and spread of new ESBL variants, as described for E. coli;2 and (iv) the current dominance of CTX-M enzymes over other ESBLs. Moreover, population structure analysis has revealed the presence of long-term hospital epidemic K. pneumoniae clones associated with specific plasmids harbouring specific blaESBL. Conversely, persistent plasmids associated with specific blaESBL genes have been able to disperse within different clones.
Most of the studies on ESBL in K. pneumoniae have been performed during outbreaks5,9,12 or at a certain time-point 15,17,18,34 and, with few exceptions,10,14 none of them studied a long-term follow-up in the same institution. In a previous publication, we described our experience with all K. pneumoniae isolates expressing ESBL recovered in our hospital since its emergence, in 1989, until 2000.14 The present study represents an update and continuous follow-up of our previous description, revealing the local natural history of such isolates. The frequency of ESBL-producing K. pneumoniae isolates during 2001–04 was lower than that found in our previous study (3.2% versus 4.8%).14 This fact could be explained due to an outbreak situation that occurred during 1997 and 1998 in which the frequency of these isolates was 12.1% and 18.1%, respectively.5,14 Our current frequency of isolates is lower than that of a recent multicentre study performed in our country during 2006 (8%) (A. Pascual, Spain, personal communication). Similar figures have been observed in surveillance studies performed worldwide.35–37 In our study, the overall proportion of ESBL-producing K. pneumoniae isolates did not increase over time. Despite this finding, it is of note that the only compartment in which these isolates have steadily grown is that of community patients (mostly in urinary tract infections) (Table 1).
The continuous follow-up of ESBL-producing K. pneumoniae isolates in our institution showed different ESBL types over time and a great variety of these enzymes during the last period. This situation is similar to that noted in Spanish and Italian surveys performed with Enterobacteriaceae isolates expressing these enzymes.7,18 TEM-4, SHV-2 and CTX-M-10 enzymes have been maintained since the first period studied, whereas SHV-12 and CTX-M-15 emerged and successfully spread during the second period. It is notable that the CTX-M-15 enzyme, which has now been detected worldwide, was recognized in K. pneumoniae only one year after its first description.2,38,39 Moreover, other prevalent ESBLs in E. coli in our country such as CTX-M-9 and CTX-M-14 were rarely found in our K. pneumoniae isolates.4,22
Population structure and plasmid characterization showed that persistence of some enzymes was mainly due to plasmid maintenance among different clones such as TEM-4 and CTX-M-10 enzymes. It is noteworthy that the plasmid pRYCE11 harbouring blaTEM-4 was described over the two different periods associated with different clones and recovered from different hospital wards and also from outpatients. This demonstrates the long-term persistence of a specific plasmid, able to move among different strains in different environments. In the case of CTX-M-10-producing isolates, the same plasmid (pRYCE30) was detected in different clones and years mainly in the nosocomial setting.
This complex allodemic situation is unlike that of SHV-12, one of most prevalent enzymes within the SHV family.7,19,33 In our study, the persistence of an SHV-12 K. pneumoniae clone (Kp49S) harbouring the same plasmid (pRYCE28) was documented in a ‘hidden-outbreak’ during a 2 year period in different wards. Eventually strains within a single clone might harbour the same plasmid (as pRYCE38) with different but highly related β-lactamases (SHV-2 and SHV-12), probably reflecting evolution of ESBLs by mutation as previously described.19,40 In the case of CTX-M-15-producing isolates, the same plasmid (pRYCE34) was detected both in an epidemic and in a non-epidemic clone. Moreover, as occurs with SHV-12, these clones were mainly from hospitalized patients.
To our knowledge, this is the first time that phylogenetic groups have been determined in ESBL-producing K. pneumoniae isolates. Most of our ESBL-producing K. pneumoniae isolates belonged to KpI. Although KpIII was found in a minority of our isolates, it is of interest to note the clear relationship with CTX-M-10-producing isolates, most of them from the nosocomial setting. Nevertheless, this KpIII group has been associated with an environmental origin and linked with the production of the chromosomal LEN β-lactamase,24 a fact that was also observed in our collection. We cannot rule out the possibility of the association of KpIII and CTX-M-10 β-lactamase in the community and later dispersion in the nosocomial setting or association within this compartment. However, potential influx of CTX-M from the community to the nosocomial setting has been demonstrated.41,42
The remarkable stability in our series of the proportion of ESBL-producing K. pneumoniae isolates during the last 15 years contrasts with the progression in prevalence of these enzymes in E. coli, in which an exponential increase of ESBL producers has been noted.2,10,35 Antibiotic-driven selection should be similar for both species, and the differences might be attributed to the different population sizes and clonal variability among human isolates. Both factors might have influenced the exponential increase of ESBLs in E. coli. Indeed the community invasion probably results from a scarce number of different plasmids spreading in a large variety of clones.2,43 Our population analysis suggests that ESBL-producing K. pneumoniae isolates might also increase in frequency in the community due to the increasing accessibility of a high variety of E. coli donors of ESBL plasmids.
In summary, our study demonstrates that K. pneumoniae still plays an important role in the epidemiology of ESBLs, despite the increase of E. coli as a producer of these enzymes. Clonal diversity with few outbreaks, persistence and emergence of ESBLs over time, plasmid transmission, and the increase of community isolates were the main features in our scenario involving K. pneumoniae with ESBLs. This complex epidemiology should be taken into account in the design of studies about the ecology of resistance and the implementation of epidemiological containment measures.
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None to declare.
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Acknowledgements |
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We thank Dr S. Brisse for kindly providing KpI, KpII and KpIII control strains.
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References |
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