Antimicrobial resistance amongst Klebsiella spp. collected from intensive care units in Southern and Western Europe in 1997–1998

Gioia S. Babini and David M. Livermore*

Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion: comparison of 1994...
 References
 
A 1994 survey of 35 intensive care units (ICUs) in Western and Southern Europe found extended-spectrum ß-lactamases (ESBLs) in 220/966 (23%) klebsiellae. A follow-up survey from May 1997 to October 1998 collected klebsiellae from 24 ICUs, including 23 that participated in 1994. Twenty-one ICUs sent 433 eligible isolates, of which 110 (25%) had ESBLs. The prevalence of ESBLs had not changed significantly from 1994 but the proportion of ESBL-producers resistant to piperacillin/tazobactam had risen from 31% to 63% (P < 0.001), and most of this resistance was high level (MICs >= 128 + 4 mg/L). The proportion of Klebsiella oxytoca isolates hyperproducing K1 ß-lactamase rose from 8% in 1994 to 21% in 1997–1998 (P < 0.001). Most klebsiellae (99%) were very susceptible to meropenem (mode MIC 0.03 mg/L) but three had decreased susceptibility (MICs 2–4 mg/L). These could not hydrolyse carbapenems. Aminoglycoside resistance was not significantly changed in prevalence from 1994; ciprofloxacin resistance occurred in 31% of ESBL-producers in both years, but had increased among non-producers (2% in 1994 versus 7% in 1997–1998, P < 0.001).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion: comparison of 1994...
 References
 
Klebsiellae are opportunistic pathogens which frequently cause infections in immunocompromised patients.1 Since the 1980s, they have become the major hosts for extended-spectrum ß-lactamases (ESBLs), most of which are mutants of TEM- and SHV-type ß-lactamases.2 Klebsiellae are also occasional hosts for plasmid-borne AmpC ß-lactamases, and some Klebsiella oxytoca isolates owe cephalosporin and aztreonam resistance to hyperproduction of their chromosomal K1 (KOXY) ß-lactamase.3

A survey of 35 intensive care units (ICUs) in Western and Southern Europe in 19944 found ESBLs in 23% of 966 klebsiellae, AmpC enzymes in 1% and hyperproduction of K1 enzyme in 8% of K. oxytoca. ESBL producers were recovered at 23/35 intensive care units (ICUs), including 20/27 that sent more than ten isolates. A new survey was conducted from May 1997 until October 1998, aiming to monitor changes since 1994. We enrolled 24 centres, including 23 that participated in 1994 (Table IGo).


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Table I. Participating centres and isolates submitted in the 1994 and 1997–1998 surveys
 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion: comparison of 1994...
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Bacteria

Participating centres were asked to submit up to 30 consecutive non-replicate klebsiellae, irrespective of antibiogram, from clinically significant ICU infections. In addition, the centres submitted 20 other Enterobacteriaceae with ESBLs from the same ICUs. They were provided with Etest ESBL detection strips (AB Biodisk, Solna, Sweden) to identify the latter organisms. Collection was from May 1997 to October 1998. On receipt by the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL), isolates were sub-cultured on MacConkey agar (Oxoid, Basingstoke, UK) and were identified with API 20E strips (bioMérieux, Lyons, France).

Antimicrobial agents and susceptibility testing

Ciprofloxacin was from Bayer, Newbury, UK; gentamicin, amikacin, cloxacillin and cefuroxime from Sigma, Poole, UK; ceftazidime from Glaxo Wellcome, Uxbridge, UK; ceftriaxone from Roche, Welwyn Garden City, UK; Ro 48-1256 (an inhibitor of AmpC enzymes),5 from Roche, Basel, Switzerland; piperacillin and tazobactam from Wyeth, Taplow, UK; aztreonam, cefepime, cefotetan and meropenem from Zeneca, Macclesfield, UK; imipenem and cefoxitin from Merck, Hoddesdon, UK; and clavulanate from SmithKline Beecham, Harlow, UK. MICs were determined, as previously,4 on IsoSensitest agar (Oxoid) with inocula of 104 cfu/spot. ß-Lactamase inhibitors were routinely used at 4 mg/L, but cloxacillin 100 mg/L was used to inhibit AmpC enzymes.

Isolates with decreased susceptibility to meropenem

Crude extracts of these isolates were prepared as described by Livermore & Williams6 and assayed against 0.1 mM imipenem and meropenem by spectrophotometry at 297 nm.6

Statistical analyses

Data from the 1994 and 1997–1998 surveys were compared by {chi}2 tests.7


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion: comparison of 1994...
 References
 
Five hundred and six isolates from 21 hospitals were received as klebsiellae and 484 were confirmed as Klebsiella spp.: 342 as Klebsiella pneumoniae (including one K. pneumonia ozaenae), 129 as K. oxytoca and three as Klebsiella ornithinolytica. Two centres collected only ceftazidime-resistant klebsiellae; once these were excluded, the number of klebsiellae analysable for ESBL prevalence fell to 433, comprising 304 K. pneumoniae (including one K. pneumoniae ozaenae), 126 K. oxytoca and three K. ornithinolytica. Only six hospitals sent more than five non-Klebsiella spp. isolates inferred to have ESBLs and, for some of these, the inference was on methods other than the Etests provided. Because of these problems, data for the non-Klebsiella spp. isolates will be analysed separately.

Categorization of isolates by antibiogram

ESBL-positive klebsiellae were putatively identified based on >=16-fold synergy between ceftazidime and ceftazidime + clavulanate,4 and made up 110 of the 433 isolates (25%). Of these, 94 were K. pneumoniae and 16 were K. oxytoca. Table IGo gives their source details: ESBL-producers were recovered from 15/19 centres, including 15/16 that sent ten or more klebsiellae. The proportions of ESBL-producers from individual hospitals ranged from 0 to 83%. The 314 isolates with ceftazidime:ceftazidime + clavulanate MIC ratios of four or less were considered to lack ESBLs. Nine isolates with ceftazidime:ceftazidime + clavulanate MIC ratios of eight were viewed as a borderline group and were excluded from further analysis (see Discussion). Twenty-seven ESBL-negative K. oxytoca were identified as putative hyperproducers of K1 enzyme, based on susceptibility to ceftazidime 1 mg/L but resistance to at least two of cefuroxime 8 mg/L, ceftriaxone 0.5 mg/L and aztreonam 0.5 mg/L. Most were highly resistant to cefuroxime (MICs >= 256 mg/L) and aztreonam (MICs >= 16 mg/L). They represented 21% of the 127 K. oxytoca collected, and were from ten hospitals.

Susceptibility of ESBL-positive and -negative isolates

Cephalosporins, aztreonam and meropenem
All the putative ESBL-producers were resistant to ceftazidime 2 mg/L, whereas all except two non-producers (one K. pneumoniae and one K1 ß-lactamase-hyperproducing K. oxytoca) were susceptible. MIC distributions for ceftriaxone and aztreonam were also well stratified, with 88–94% of ESBL-producers resistant at 1 mg/L. Only five isolates inferred to have neither ESBLs nor hyperproduction of K1 enzyme were resistant to aztreonam 1 mg/L, and six were resistant to ceftriaxone 1 mg/L. Greater MIC overlaps for ESBL-producers and non-producers were seen for cefuroxime and cefoxitin; nevertheless, the modal MIC of cefuroxime for ESBL-producers (64 mg/L) greatly exceeded that for non-producers (2 mg/L). The modal MIC of cefoxitin was 4 mg/L for both ESBL-producers and non-producers; that of cefotetan was 0.5–1 mg/L for ESBL-producers, but 0.06–0.12 mg/L for non-producers.

Regardless of inferred ß-lactamase production, virtually all the klebsiellae were susceptible to meropenem 0.25 mg/L; none the less, MICs 2–4 mg/L were recorded for three isolates: one from Italy and two from a hospital in The Netherlands. These isolates were resistant to all the other ß-lactams tested. Clavulanate 4 mg/L reduced their ceftazidime MIC from 1024 mg/L to 8–64 mg/L, implying ESBL production. Synergy was not seen between ceftazidime and Ro 48-1256 4 mg/L nor cloxacillin 100 mg/L, and was not increased when cloxacillin 100 mg/L was added to ceftazidime–clavulanate. Cell-free extracts of these isolates did not hydrolyse carbapenems.

Piperacillin and piperacillin–tazobactam
All the putative ESBL-producers and hyperproducers of K1 enzyme were resistant to piperacillin 16 mg/L, as were 37/288 isolates with neither mode of resistance. Tazobactam 4 mg/L reduced the modal piperacillin MIC for ESBL non-producers from 8 to 4 mg/L, and 41/110 were susceptible to piperacillin/tazobactam at 16 + 4 mg/L; however, 69/110 ESBL-producers (63%) were resistant to piperacillin/ tazobactam at this concentration and 40% were resistant at 512 + 4 mg/L. Every centre except one, that sent ESBL producers included isolates that were resistant to piperacillin/tazobactam 16 + 4 mg/L. All the hyperproducers of K1 enzyme were highly resistant to piperacillin and piperacillin/tazobactam (MICs >= 128 mg/L). Of the 37 piperacillin-resistant klebsiellae (MIC > 16 mg/L) with neither ESBLs nor hyperproduction of K1 enzyme, 16 were resistant to piperacillin/tazobactam 16 + 4 mg/L.

Non ß-lactams
Aminoglycoside resistance was more frequent (P < 0.001) amongst ESBL-producers than non-producers: thus 61% and 72% of ESBL-producers were resistant to amikacin 4 mg/L and gentamicin 1 mg/L, respectively, compared with 4% and 9.5% of non-producers. Ciprofloxacin resistance (MIC > 1 mg/L) was also more frequent among ESBL-producers than non-producers (31% versus 7%, P < 0.001); nevertheless, resistance was scattered in both groups; thus ciprofloxacin-resistant ESBL-producers were found at seven hospitals and resistant non-producers were found at ten.

Isolates excluded from analysis

Nine isolates for which the ceftazidime:ceftazidime + clavulanate MIC ratio was eight were excluded from the MIC comparisons (above). One was a K. oxytoca inferred to hyperproduce K1 enzyme. Ceftazidime MICs for three of the other eight isolates were 4 mg/L. These were also resistant to aztreonam and ceftriaxone, with MICs >= 4 mg/L, and probably had ESBLs. Ceftazidime MICs for the other five isolates were 0.12–2 mg/L and those of ceftriaxone and aztreonam were <=0.25 mg/L. ESBL production seems unlikely in such organisms.

Laboratory reporting of resistance to cephalosporins for putative ESBL-producers

Each isolate was sent to ARMRL with a case record form indicating, inter alia, the source hospital's susceptibility data. Between 10 and 38% of the putative ESBL-producers had been reported as susceptible to a cephalosporin or to aztreonam, and between 3 and 23.5% as intermediate (Table IIGo). Most of those reported susceptible (74%) had low-level resistance to the compound in question (MICs 4–16 mg/L). In contrast, between 0.3% and 2% of susceptible isolates lacking ESBLs or hyperproduction of K1 enzyme had been reported resistant to one or more cephalosporins. Five K. oxytoca isolates that hyperproduced K1 enzyme had been reported susceptible to ceftriaxone, despite MICs of 8–32 mg/L.


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Table II. Reporting of susceptibility for putative ESBL-producers at the laboratories where they were isolated, in comparison with findings at the ARMRL
 

    Discussion: comparison of 1994 and 1997–1998 surveys
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion: comparison of 1994...
 References
 
This study followed a similar survey in 1994, and aimed to assess whether ESBLs had increased in prevalence among ICU klebsiellae, and whether other Enterobacteriaceae had acquired identical ESBLs to klebsiellae in the same units. In the event, we received few non-klebsiellae, and, for some of these, ESBL production had been inferred outside the protocol. Because of these problems the present paper considers only the klebsiellae.

As in 1994, putative ESBL-producers were identified on the basis of ceftazidime:ceftazidime + clavulanate MIC ratios >=16. The proportion of ESBL-producers was not significantly changed (P > 0.1), regardless of whether all participating hospitals were considered or only those that contributed in both years. Not all the centres that sent ESBL-producers in the 1994 study sent ESBL-producers in the 1997–1998 survey (Table IGo), and vice versa, but this difference may have reflected the occurrence of outbreaks and/or the fact that some centres sent few isolates. More critically, and underscoring their widening distribution, ESBL-producers were found at 15/16 hospitals that sent ten or more klebsiellae in 1997–1998, compared with 20/27 in 1994 (P < 0.05).

The resistance of ESBL-producers to aminothiazolyl cephalosporins needs no discussion, but the status of piperacillin/tazobactam deserves comment. The proportion of isolates resistant to this combination at 16 + 4 mg/L rose from 30% in 1994 to 63% in 1997–1998 (P < 0.001, Table IIIGo), and most of this increase reflected isolates with piperacillin–tazobactam MICs > 512 + 4 mg/L (FigureGo). Piperacillin/tazobactam-resistant ESBL-producers (MIC > 16 + 4 mg/L) were found in 17/23 ICUs that sent ESBL-producers in 1994, but only three of the nine centres that sent more than ten ESBL-producers had >50% of piperacillin/tazobactam resistance (Table IIIGo). In 1997– 1998, ESBL-producers resistant to piperacillin/tazobactam were found in 14 of 15 hospitals that sent ESBL-producers and, of the six centres that sent more than ten ESBLproducers, all but one had >50% piperacillin/tazobactam resistance. Such resistance can reflect hyperproduction of ESBLs, production of multiple ESBLs, or combinations of ß-lactamase and impermeability,8 but the relative importance of these mechanisms is unclear.


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Table III. Summary: comparison of the findings of the 1994 and 1997–1998 surveys
 


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Figure. MIC distribution of piperacillin/tazobactam for klebsiellae with ESBLs in the 1994 ({square}) and 1997–1998 ({blacksquare}) surveys.

 
Another change from 1994 was a significantly increased proportion of K. oxytoca inferred to hyperproduce the K1 chromosomal ß-lactamase, up from 8% to 21% (P < 0.001). Almost all the hyperproducers collected in 1994 were unique isolates,9 and it remains to be determined whether the present increase was due to single-isolate epidemics or to multiple separate isolates.

Cephamycins deserve mention, too. The modal MIC of cefoxitin for ESBL-producers exceeded that for nonproducers in 1994 (16 mg/L versus 2 mg/L) whereas the modal MIC for both groups was 4 mg/L in 1997–1998. Nevertheless, 17% of ESBL-producers collected in 1997–1998 were resistant to cefoxitin 16 mg/L compared with 4% of non-producers (P < 0.001). The modal MICs of cefotetan in 1997–1998 were 0.5–1 mg/L for ESBL-producers compared with 0.06–0.12 mg/L for non-producers. The reduced susceptibility of the ESBL-producers to cephamycins conflicts with the fact that ESBLs do not protect transconjugants against these compounds.10

Aminoglycoside resistance was more common among putative ESBL-producers than non-producers in both years (Table IIIGo) and its prevalence had not significantly changed between the two studies; likewise, there was no significant change in the prevalence of ciprofloxacin resistance among ESBL-producers (31% in both years, P > 0.1, Table IIIGo). On the other hand, ciprofloxacin resistance had increased amongst the ESBL non-producers (2% in 1994 versus 7% in 1997–1998, P < 0.001, Table IIIGo). Moreover, most of the ciprofloxacin resistance among ESBL-producers in 1994 reflected multiple inclusion of a serotype K25 strain, and it may be that resistance is now more widespread among ESBL-different strains.

Three ESBL producers (one from Italy and two from The Netherlands) showed decreased meropenem susceptibility, with MICs of 2–4 mg/L. They were resistant to all other ß-lactams, but lacked carbapenemase activity. Decreased susceptibility to carbapenems in K. pneumoniae has been linked to the simultaneous presence of an acquired AmpC enzyme with the loss of a 42 kDa outer membrane porin11 or to porin loss in the presence of a hyperproduced SHV-type enzyme.12 No synergy was seen between ceftazidime and Ro 48-1256 or cloxacillin in the present cases, contra-indicating AmpC production.

As in 1994, up to 40% of the ESBL-producers had been reported as susceptible to cefotaxime and/or ceftriaxone, and a further 22–23% as intermediate. The continued frequency of reporting ESBL-producers as susceptible to these aminothiazolyl cephalosporins is disturbing when we allow for the wide publicity13,14 given to the clinical inactivity of the compounds against ESBL-producers. Misreporting was rarer for ceftazidime and aztreonam, doubtless reflecting the fact that resistance to these drugs is generally more obvious than that to cefotaxime and ceftriaxone.

In summary, the prevalence of ESBL production amongst klebsiellae from European ICUs was unchanged from 1994, but resistance to piperacillin/tazobactam had increased among ESBL-producers and K. oxytoca isolates hyperproducing K1 enzyme were more frequent. The increased resistance to piperacillin/tazobactam casts a doubt on its role in infections caused by ESBL-producers.


    Acknowledgments
 
We are very grateful to the following for collecting isolates: C. Bebear, Hopital Pellegrin, Université de Bordeaux; G. Bonfiglio, Istituto di Microbiologia, Universita di Catania; F. Baquero, Hospital Ramon y Cajal, Madrid; R. Cisterna, Hospial de Basurto, Bilbao; F. Crokaert, Institut Jules Bordet, Brussels; T. Fosse, Hôpital Saint-Roch, Nice; J. A. Garcia-Rodriguez, Hospital Universitario de Salamanca; J. J. A. Hoogkamp-Korstanje, St Radboud, Academisch Ziekenhuis, Nijmegen; Jacobs, Universitätsklinikum Carl Gustav Carus, Dresden; J. G. M. Koelman, Academisch Ziekenhuis, Vrije Universiteit, Amsterdam; V. Korten, Marmara University Hospital, Istanbul; S. Lauwers, Academisch Ziekenhuis, Vrije Universiteit Brussels; W. L. Manson, Academisch Ziekenhuis, Groningen; Nunes da Costa, Hospital General de Santo Antonio, Oporto; W. Opferkuch, Medizinische Klinik, St Josef Hospital, Bochum; B. Panzig, Ernst-Moritz-Ardnt-Universität, Greifswald; J. D. Perry, Freeman Hospital, Newcastle upon Tyne; M. J. Salgado, Hospital de St Maria, Lisbon; M. Salvado, Laboratoria de Referencia di Catalunya, Barcelona; M. Segovia, Hospital Universitario de Murcia; G. Schito, Istituto di Microbiologia, Universita di Genoa; S. Unal, Hacettepe University School of Medicine, Ankara; G. Verschraegen, Universiteit Gent. We are also very grateful to Zeneca Pharmaceuticals, Alderley Park, UK, for supporting this study financially, and to their affiliates in Belgium, France, Germany, Italy, The Netherlands, Portugal, Spain and Turkey for liaising with the survey participants.


    Notes
 
* Corresponding author. Tel: +44-181-200-4400; Fax: +44-181-200-7449; E-mail: DLivermore{at}phls.nhs.uk

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    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion: comparison of 1994...
 References
 
1 . Podschun, R. & Ullmann, U. (1998). Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clinical Microbiology Reviews 11, 589–603.[Abstract/Free Full Text]

2 . Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 1211–33.[Free Full Text]

3 . Fournier, B., Arlet, G., Lagrange, P. H. & Philippon, A. (1994). Klebsiella oxytoca: resistance to aztreonam by overproduction of the chromosomally encoded ß-lactamase. FEMS Microbiology Letters 116, 31–6.[ISI][Medline]

4 . Livermore, D. M. & Yuan, M. (1996). Antibiotic resistance and production of extended-spectrum ß-lactamases amongst Klebsiella spp. from intensive care units in Europe. Journal of Antimicrobial Chemotherapy 38, 409–24.[Abstract]

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6 . Livermore, D. M. & Williams, J. D. (1996). ß-Lactams: mode of action and mechanisms of antibacterial resistance. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 502–77. Williams & Wilkins, Baltimore, MD.

7 . Swinscow, T. D. (1976). Statistics at square one. XV—The chi-squared tests (continued). British Medical Journal 2, 513–4.[ISI][Medline]

8 . Livermore, D. M. (1993). Determinants of the activity of ß-lactamase inhibitor combinations. Journal of Antimicrobial Chemotherapy 31, Suppl. A, 9–21.[ISI][Medline]

9 . Gheorghiu, R., Yuan, M., Hall, L. M. & Livermore, D. M. (1997). Bases of variation in resistance to ß-lactams in Klebsiella oxytoca isolates hyperproducing K1 ß-lactamase. Journal of Antimicrobial Chemotherapy 40, 533–41.[Abstract]

10 . Jacoby, G. A. & Carreras, I. (1990). Activities of ß-lactam antibiotics against Escherichia coli strains producing extended-spectrum ß-lactamases. Antimicrobial Agents and Chemotherapy 34, 858–62.[ISI][Medline]

11 . Bradford, P. A., Urban, C., Mariano, N., Projan, S. J., Rahal, J. J. & Bush, K. (1997). Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrobial Agents and Chemotherapy 41, 563–9.[Abstract]

12 . MacKenzie, F. M., Forbes, K. J., Dorai-John, T., Amyes, S. G. & Gould, I. M. (1997). Emergence of a carbapenem-resistant Klebsiella pneumoniae. Lancet 350, 783.[ISI][Medline]

13 . Livermore, D. M. (1995). ß-Lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews, 8, 557–84.[Abstract]

14 . Bush, K. (1996). Is it important to identify extended-spectrum ß-lactamase-producing isolates? European Journal of Clinical Microbiology and Infectious Diseases 15, 361–4.[ISI][Medline]

Received 21 June 1999; returned 23 August 1999; revised 22 September 1999; accepted 11 October 1999