New species-related MIC breakpoints for early detection of development of resistance among Gram-negative bacteria in Swedish intensive care units

H. Hanbergera,*, L. E. Nilssonb, B. Claessonc, A. Kärnelld, P. Larssone, M. Rylanderf, E. Svenssonb, M. Sörbergf and L. Söréng

a Division of Infectious Diseases b Clinical Microbiology; University Hospital, S-581 85 Linköping, Sweden; Divisions of Clinical Microbiology c S-541 85 Skövde; d S-141 86 Hudding e Östra S-416 85 Gothenburg; f Karolinska S-171 76 Stockholm; g S-551 85 Jönköping, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The frequency of decreased antibiotic susceptibility among 534 Gram-negative aerobic bacilli from patients admitted to intensive care units at eight hospitals in Sweden during 1997 was evaluated. MICs of cefepime, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, imipenem and piperacillin–tazobactam were determined using Etest. Reduced susceptibility (resistant and intermediate/indeterminate susceptible strains) was defined according to the MIC breakpoints of the British Society for Antimicrobial Chemotherapy (BSAC), the National Committee for Clinical Laboratory Standards (NCCLS) and the new species-related breakpoints of the Swedish Reference Group for Antibiotics (SRGA). The BSAC/NCCLS/SRGA breakpoints for susceptible category (mg/L) of Enterobacteriaceae are: cefepime, not available (NA)/8/0.5; ceftazidime, 2/8/2; ceftriaxone, NA/8/0.5; ciprofloxacin, 1/1/0.12; gentamicin, 1/4/2; imipenem, 4/4/1; and piperacillin–tazobactam, NA/16/16. The most frequently isolated organisms were Escherichia coli (n = 160; 30%), Klebsiella spp. (n = 84; 16%), Enterobacter spp. (n = 77; 14%), Pseudomonas aeruginosa (n = 64; 12%) andProteus spp. (n = 28; 5%). Decreased susceptibility among E. coliusing the BSAC/NCCLS/SRGA respective breakpoints (%) were: cefepime, NA/0/2; ceftazidime, 2/2/2; ceftriaxone, NA/1/2; ciprofloxacin, 2/2/8; gentamicin, 21/0/3; imipenem, 0/0/2; and piperacillin-tazobactam, NA/4/4. Corresponding levels of decreased susceptibility (%) among Klebsiellaspp. were: cefepime, NA/0/5; ceftazidime, 2/1/2; ceftriaxone, NA/1/10; ciprofloxacin, 4/4/19; gentamicin, 25/2/5; imipenem, 0/0/0; and piperacillin-tazobactam, NA/10/10; and among Enterobacter spp. were: cefepime, NA/1/19; ceftazidime, 30/29/30; ceftriaxone, NA/30/36; ciprofloxacin, 3/3/15; gentamicin,18/0/0; imipenem, 0/0/5; and piperacilllin-tazobactam, NA/27/27. In conclusion, the species-related SRGA breakpoints detected Gram-negative isolates with decreased susceptibility in comparison with the native population with higher frequency than did the NCCLS breakpoints. The BSAC breakpoints for susceptible organisms were similar to NCCLS for ciprofloxacin and imipenem, and similar to SRGA for ceftazidime but lower than both NCCLS and SRGA for gentamicin, causing a much higher frequency of decreased susceptibility to gentamicin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since intensive care units (ICUs) usually house patients receiving broad-spectrum antibiotics, they provide an ideal environment for the selection and spread of antibiotic resistant bacteria, as shown in numerous reports on outbreaks of nosocomial infections caused by multi-resistant bacteria.1,2,3,4,5 Surveillance of antibiotic resistance is especially important in ICUs, since infection rates are much higher than in other hospital wards.6,7,8 Jarlier et al.9 reported a high prevalence of decreased susceptibility among Gram-negative bacilli in French ICUs during 1991. In a study of ICUs in Belgium, France, Portugal, Spain and Sweden during 1994–1995, we found an unexpectedly high incidence of decreased antibiotic susceptibility among Gram-negative bacilli in all ICUs, with the exception of Sweden, indicating a threat to the efficacy of currently available empirical drugs.10 However, due to the use of National Committee for Clinical Laboratory Standards (NCCLS) breakpoints,11 which are based on clinical outcome, pharmacology and MIC distributions and are not specifically adapted for early detection of emerging resistance, in these studies, they may have underestimated the in-vitro incidence of isolates with elevated MICs compared with the ‘native’ populations.12 By assessing MIC distribution diagrams, the native population can be viewed relative to strains with decreased susceptibility.

This study investigated the incidence of antibiotic resistance among aerobic Gram-negative bacilli in Swedish ICUs, determining MIC by Etest. The aim was also to evaluate whether and how the new Swedish species-related (SRGA) breakpoints may influence the detection of decreased antibiotic susceptibility among Gram-negative bacteria in Swedish ICUs, compared with the use of NCCLS and the British Society for Antimicrobial Chemotherapy (BSAC) breakpoints.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design and culture collection

A Swedish multicentre susceptibility testing study was performed using aerobic Gram-negative bacilli isolated from patients admitted to ICUs between January and October 1997. ICUs at eight hospitals were included; four were tertiary university hospitals and four were primary county hospitals. Consecutive specimens collected on clinical indications were cultured and tested. A total of 534 Gram-negative strains were isolated from 411 patients admitted to the ICUs over 10 months. Repeat isolates (of the same species from the same patients) were excluded. The data analysis on antibiotic susceptibility was done for pathogens collected from all sites.

Susceptibility testing

MICs of cefepime, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, imipenem and piperacillin–tazobactam were determined using Etest (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions.13,14 All laboratories performed quality control using the reference strains Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853. Ceftazidime was selected as the ß-lactam marker for detection of resistance due to stably derepressed constitutive chromosomal class I ß-lactamases, which hydrolyse most ß-lactam antibiotics except carbapenems,15 and for plasmid-mediated, extended-spectrum ß-lactamases (ESBL).14,15,16

Decreased susceptibility was defined with the MIC breakpoints recommended by the BSAC (www.bsac.org.uk) and NCCLS11 (resistant and intermediate categories), and the new Swedish species-related breakpoints of the SRGA (resistant and indeterminate categories) (www.ltkronoberg.se/ext/raf/raf.htm).

The intermediate category as defined by the NCCLS11 includes isolates with antimicrobial agent MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than for fully susceptible isolates. The intermediate category implies clinical applicability in body sites where the drugs are physiologically concentrated (e.g. quinolones and ß-lactams in urine) or when high dosage of a drug can be used (e.g. ß-lactams). The intermediate also includes a ‘buffer zone’, which should prevent small, uncontrolled technical fac-tors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.11

The indeterminate category of the SRGA implies that the clinical effect with this drug is uncertain. The bacteria has acquired low-level resistance or has a natural decreased susceptibility to the drug. Of the investigated bacteria–drug combinations, the BSAC breakpoints have intermediate categories only for Pseudomonas spp. and gentamicin and ciprofloxacin. The BSAC, NCCLS and SRGA breakpoints for susceptible and resistant categories (mg/L) of Enterobacteriacae and P. aeruginosa are shown in Table I.


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Table I. MIC breakpoints (mg/L) for susceptible and resistant organisms among Enterobacteriaceae and P. aeruginosa according to the BSAC, the SRGA and NCCLS16
 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organisms

The most frequently isolated organisms were E. coli (n = 160; 30%) followed by Klebsiella spp. (n = 84; 16%), Enterobacter spp. (n = 77; 14%), P. aeruginosa (n = 64; 12%) Proteus spp. (n = 28; 5%), Haemophilus spp. (n = 23; 4%), Stenotrophomonas maltophilia (n = 18; 3%), Citrobacter spp. (n = 17; 3%), Acinetobacter spp. (n = 13; 2%), Serratia spp. (n = 12; 2%) and Morganella morganii (n = 11; 2%). These 507 isolates constituted 95% of all Gram-negative isolates collected.

Susceptibility profiles

MIC distributions of cefepime, ceftazidime, ciprofloxacin and imipenem for Enterobacter spp., E. coli and Klebsiella spp. isolates are shown in Figure 1.




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Fig. 1. MIC distributions of Enterobacter spp. (a–e), E. coli (f–j), Klebsiella spp. (k–o) and P. aeruginosa (p) isolates for cefepime, ceftazidime, ciprofloxacin, gentamicin and imipenem. The MIC for the native populations (www.ltkronoberg.se/ext/raf/raf.htm) are shown below the x-axis.)

 
Decreased susceptibility as classified by BSAC, NCCLS and SRGA breakpoints

The decreased antibiotic susceptibility rates among E. coli, Klebsiella spp.,Enterobacter spp., P. aeruginosa and Proteus spp. to the investigated drugs are shown in Figure 2and Table II.



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Fig. 2. Decreased antibiotic susceptibility, as classified by BSAC ({square}), NCCLS () and SRGA () breakpoints, among Enterobacter spp. (a), E.coli (b), Klebsiella spp. (c) and P. aeruginosa (d).

 

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Table II. Susceptible and resistant rates (%) among Gram-negative isolates
 
Cefepime. Decreased susceptibility to cefepime among Klebsiella and Enterobacter spp. was more frequently detected using SRGA breakpoints compared with NCCLS breakpoints, whereas no or only minor differences were seen with the other species investigated (Figures 1 and 2; Table II).

Ceftazidime. No or only minor differences were seen for the species investigated (Figures 1 and 2, Table II).

Ceftriaxone. Decreased susceptibility to ceftriaxone among Klebsiella and Enterobacter spp. was more frequently detected using SRGA breakpoints compared with NCCLS breakpoints, whereas no or only minor differences were seen with the other species investigated (Figures 1 and 2; Table II).

Ciprofloxacin. Decreased susceptibility to ciprofloxacin among E. coli, Klebsiella spp., Enterobacter spp. and Proteus spp. was more frequently detected using SRGA breakpoints compared with BSAC and NCCLS breakpoints, whereas no differences were seen with P. aeruginosa (Figures 1 and 2; Table II).

Gentamicin. Decreased susceptibility to gentamicin among E. coli, Klebsiella spp., Enterobacter spp., Proteus spp. and P. aeruginosa was more frequently detected using BSAC breakpoints compared with NCCLS and SRGA breakpoints (Figures 1 and 2; Table II).

Imipenem. Decreased susceptibility to imipenem among Enterobacter spp. and Proteus spp. was more frequently detected using SRGA breakpoints compared with BSAC and NCCLS breakpoints, whereas no or only minor differences were seen with the other species investigated (Figures 1 and 2; Table II).

Piperacillin–tazobactam. Decreased susceptibility among P. aeruginosa spp. to piperacillin–tazobactam was more frequently detected using BSAC and SRGA breakpoints compared with NCCLS breakpoints, whereas no differences were seen with the other species investigated (Figures 1 and 2; Table II).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is important to have a system for testing antibiotic susceptibility for early detection of emerging resistance. Surveillance studies must be based on precise and accurate quantitative methods that use sufficiently broad ranges of MIC to detect small decreases in susceptibility levels. In this study, the species-related SRGA breakpoints detected Gram-negative isolates with decreased susceptibility in comparison with the native population in higher frequencies than did the NCCLS breakpoints. The BSAC breakpoints for susceptible isolates were similar to NCCLS for ciprofloxacin and imipenem, and similar to SRGA for ceftazidime but lower than both NCCLS and SRGA for gentamicin causing a much higher frequency of decreased susceptibility to gentamicin.

The low-level resistance detected by the SRGA breakpoints, and BSAC breakpoints for gentamicin, is probably due to the presence of a mechanism of resistance. The species-related breakpoint concept (SRGA) may thus be a useful epidemiological tool in hospital infection control to signal changes of susceptibility levels at an early stage. For the prescribing physician it is also crucial to have the most recent information on antibiotic resistance patterns within the hospital or even within specific units to guide empirical therapy. However, by setting the breakpoint for susceptibility closer to the native population, isolates with a minor increase in MIC will be more frequently classified as non-susceptible (Table II; Figures 1 and 2). This microbiological definition of non-susceptible may not necessarily be predictive of clinical failure. Since the Swedish species-related breakpoints are based on MICs for the native population (Figure 1; www.ltkronoberg.se/ext/raf/raf.htm), there is a continuous revision of the breakpoints based on studies on clinical outcome of infections caused by organisms with reduced susceptibility (indeterminate group). It is important not to overpredict decreased susceptibility and exclude the use of well proven drugs since some mechanisms of low-level resistance may be sufficiently weak to be overcome by the high levels of antibiotics achieved in the clinical situation. However, some mechanisms of resistance that are more likely to be selected and expressed, especially when causing infections in the immunocompromised patients might lead to therapeutic failure.17,18 The clinical outcome when treating infections caused by non-susceptible bacteria depends on numerous factors other than the in-vitro result and these should be considered when mak-ing clinical judgements. The site of infection, patient status, pharmacokinetics, pharmacodynamics and previous clinical experience are important considerations.17,18 The SRGA breakpoint for the resistant category is unchanged (www.ltkronoberg.se/ext/raf/raf.htm), while the breakpoint for susceptible is lowered, giving a wider span of MICs for the indeterminate group. It is important to inform the clinician that an isolate in the indeterminate group probably has acquired low-level resistance or has a natural decreased susceptibility to the drug, which should normally not be used to avoid selection of isolates with decreased susceptibility. Recently, four types of category have been suggested by Baquero, namely: S (fully susceptible), absence of mechanisms of resistance; Su (surveillance) or borderline susceptible, presence of mechanisms of resistance (should be genotypically confirmed) which should be viewed as a warning; the presence of the mechanism may not have a clinical impact but such strains cannot be microbiologically considered completely susceptible; I (intermediate), presence of a mechanism of resistance that is predictive of reduced clinical success; R (resistant), presence of a mechanism of resistance that is highly predictive of clinical treatment failure.17,18 However, we believe that the SRGA species-related breakpoints fulfil the requirements as a clinically useful tool provided that isolates classified as indeterminate are evaluated with caution. The prevalence of decreased susceptibility shown in this study is in agreement with those found in an earlier study performed during 1994–1995 at 10 Swedish hospitals.19 However, in a comparative ICU study performed during 1994–1995, the prevalence of decreased susceptibility among Gram-negative bacteria was lower in Sweden compared with Belgium, France, Portugal and Spain when using the NCCLS breakpoints.10 Decreased antibiotic susceptibility across all species and drugs was highest in Portuguese ICUs followed by French, Spanish, Belgian and Swedish ICUs.10

The BSAC breakpoints were similar to NCCLS for ciprofloxacin and imipenem, and similar to SRGA for ceftazidime but lower than both NCCLS and SRGA for gentamicin, causing a much higher frequency of decreased susceptibility to gentamicin. With the exception of gentamicin, the BSAC guidelines are a compromise between the SRGA species-related breakpoints and the NCCLS clinically-oriented susceptibility guidelines 20.

In this study, our data represent all consecutive Gram-negative isolates initially recovered from ICU patients. They include pathogens isolated from patients with community- as well as hospital-acquired infections. No analysis was done to determine whether the isolates tested caused infection or only colonized the critically ill patient. The isolates from specimens other than blood may reflect colonization.However, since colonization is often a prerequisite for infection,21 we found in the previously mentioned study equally high levels of decreased susceptibility among blood isolates compared with isolates from all sites.10 The distribution of Gram-negative bacterial species in this study corresponded well with those reported in the previously mentioned Swedish study,19 but P. aeruginosa was less frequent compared with other European countries.8,9,10

The use of ceftazidime for treatment of Gram-negative ICU infections is favoured by its enhanced activity against P. aeruginosa and low toxicity compared with aminoglycosides. However, the high prevalence of decreased susceptibility to ceftazidime among Enterobacter spp. (Figures 1 and 2; Table II) and other inducible ß-lactamase producers among Enterobacteriacae (data not shown) limits the usefulness of ceftazidime and related drugs as monotherapy. Resistance selection among Enterobacter spp. is probably caused by production of stably derepressed, constitutive, chromosomal, class I ß-lactamases, which hydrolyse most ß-lactam antibiotics except carbapenems.15 Ceftazidime resistance is a serious emerging problem and approximately 40% of Enterobacter cloacae were reported to be resistant to ceftazidime according to ICU studies in the USA during 1987–199122 and 1994–1995.23 Between 26 and 48% of Enterobacter spp. had decreased susceptibility to ceftazidime in ICUs in Belgium, France, Portugal, Spain and Sweden between 1994 and 1995.10 Previous use of third-generation cephalosporins is more likely to cause the selection of resistance to ß-lactams in blood isolates of Enterobacter spp., which are associated with higher mortality.24 The spread of multidrug-resistant Enterobacter aerogenes has also been reported in ICUs in France and Belgium.1,4 Decreased susceptibility among Enterobacter spp. to cefepime, ceftriaxone, ciprofloxacin and imipenem was more frequent based on SRGA breakpoints compared with NCCLS breakpoints, whereas no or only minor differences were seen with the other antibiotics tested (Table II; Figures 1 and 2).

A low level of decreased susceptibility to ceftazidime among E. coli and Klebsiella spp. was found using the BSAC, NCCLS and SRGA breakpoints (Figure 2; Table II), which suggests low prevalence of ESBLs. However, the NCCLS11 have also recommended suspicion of ESBLs among E. coli and Klebsiella spp. with increased MICs (2 mg/L) of ceftazidime since ESBLs may also be present at this level.3 Lower breakpoints are useful for screening ESBLs among E. coli and Klebsiella spp.but require confirmation with specific phenotypic tests such as the Etest ESBL strip used in this study.14

In conclusion, the species-related SRGA breakpoints detected Gram-negative isolates with decreased susceptibility in comparison with the native population in higher frequencies than did the NCCLS breakpoints. The BSAC breakpoints for susceptible were similar to NCCLS for ciprofloxacin and imipenem and similar to SRGA for ceftazidime but lower than both NCCLS and SRGA for gentamicin, causing much higher frequency of decreased susceptibility to gentamicin. The species-related breakpoint concept may be a useful epidemiological tool for detecting changes of susceptibility level at an early stage. The resistance mechanisms involved should be further characterized both genotypically and phenotypically to assess their clinical significance more accurately. For the clinicians, a practical interpretation of the test results should be made available since the microbiological definition of non-susceptible may not be useful for predicting clinical failure. Furthermore, each hospital should have an active programme for ‘on-line’ antibiotic resistance surveillance of key drugs used in the different ICUs, using quantitatively accurate MIC methods to validate the efficacy of their empirical formulary constantly. Armed with these data, we can detect early signals of susceptibility shifts better and implement various strategies to minimize emergence and spread of antibiotic resistance within hospitals and ICUs.12,25


    Acknowledgments
 
We would like to thank Merck Co for their financial support.


    Notes
 
* Corresponding author. Fax: +46-13-159-441; E-mail: hakan.hanberger{at}inf.liu.se Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 22 December 1998; returned 26 March 1999; revised 19 May 1999; accepted 29 June 1999