Antimicrobial susceptibilities among clinical isolates of extended-spectrum cephalosporin-resistant Gram-negative bacteria in a Taiwanese University Hospital

Shio-Shin Jeana, Lee-Jene Tengb,c, Po-Ren Hsueha,b,*, Shen-Wu Hob,c and Kwen-Tay Luha,b

a Section of Infectious Diseases, Department of Internal Medicine, and b Department of Laboratory Medicine, National Taiwan University Hospital c School of Medical Technology, National Taiwan University College of Medicine, 7 Chun-Shan South Road, Taipei, Taiwan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infections caused by Gram-negative bacteria with resistance to extended-spectrum cephalosporins require the identification of effective alternative antimicrobial therapy. To determine the role of other pre-existing or currently available antimicrobial agents in treating infections caused by these multidrug-resistant pathogens, we evaluated the in vitro susceptibilities of these agents in 411 non-duplicate isolates of extended-spectrum cephalosporin-resistant Gram-negative bacteria recovered between January 1999 and December 1999 in a major teaching hospital in Taipei, Taiwan. These isolates included cefotaxime-resistant (MICs 2 mg/L) Escherichia coli (66 isolates) and Klebsiella pneumoniae (77 isolates); cefotaxime-resistant (MICs 64 mg/L) Enterobacter cloacae (59 isolates), Serratia marcescens (52 isolates) and Citrobacter freundii (52 isolates); and ceftazidime-resistant (MICs 64 mg/L) Pseudomonas aeruginosa (50 isolates) and Acinetobacter baumannii (55 isolates). Overall, carbapenems (imipenem and meropenem) had good activity against the cefotaxime-resistant Enterobacteriaceae tested (>90% of isolates were susceptible). However, carbapenems had limited activity against the ceftazidime-resistant P. aeruginosa (only 4% of isolates were susceptible) and A. baumannii (51–56% of isolates were susceptible). Among the E. coli and K. pneumoniae isolates tested, 33.3% and 58.4%, respectively, exhibited extended-spectrum ß-lactamase phenotype, determined by the double disc method. Over 80% of cefotaxime-resistant E. cloacae and C. freundii were susceptible to cefepime, but this agent had limited activity against other bacteria tested. Susceptibilities of these isolates to ciprofloxacin varied, ranging from 25% for A. baumannii to 92% for E. cloacae. Newer fluoroquinolones (moxifloxacin and trovafloxacin) had equal or less activity against these organisms, except for A. baumannii for which their MIC90s (8–16 mg/L) were four- to 16-fold less than that of ciprofloxacin (MIC90 128 mg/L).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Lactam antibiotics are among the most frequently prescribed antimicrobial agents worldwide.1–5 The emergence of resistance to these agents in the past two decades because of increasingly widespread use has resulted in a major clinical crisis.1–4 Gram-negative bacteria resistant to agents such as extended-spectrum cephalosporins, monobactams, carbapenems and ß-lactam–ß-lactamase inhibitor combinations have emerged through the production of a variety of ß-lactamases, alterations in the penicillin-binding proteins and outer membrane permeability, and combinations of multiple mechanisms of resistance.1,2

Resistance to ß-lactam antimicrobial agents, especially extended-spectrum cephalosporins and other antimicrobial agents among clinical isolates of Gram-negative bacteria, is on the rise worldwide.6–12 These antimicrobialresistant pathogens include extended-spectrum cephalosporin- or fluoroquinolone-resistant Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens and Citrobacter freundii, and carbapenem- or ciprofloxacin- resistant Pseudomonas aeruginosa and Acinetobacter baumannii.14 Recent studies in Taiwan have demonstrated a high prevalence of these antimicrobial-resistant bacteria and a trend of increasing resistance under continued antibiotic selective pressure.13–15

The purpose of this study was to determine the in vitro susceptibility of currently available antimicrobial agents against 411 unrelated clinical isolates of extended-spectrum cephalosporin (cefotaxime or ceftazidime)-resistant Gram-negative bacteria and to assess the potential of these agents in the treatment of infections caused by these resistant bacteria.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

Between January 1999 and December 1999, a total of 15 378 isolates of Gram-negative bacteria, including E. coli (3624 isolates), K. pneumoniae (2886 isolates), E. cloacae (1775 isolates), S. marcescens (864 isolates), C. freundii (292 isolates), P. aeruginosa (4467 isolates) and A. baumannii (1470 isolates), were recovered from various clinical specimens of patients treated at the National Taiwan University Hospital (NTUH), a 1800-bed university hospital in Taipei. The standard disc diffusion method, as described by the NCCLS,16 was used routinely to determine the in vitro susceptibility of these isolates to cefazolin, cefotiam and cefotaxime (E. coli, K. pneumoniae, E. cloacae, S. marcescens and C. freundii) or ceftazidime (P. aeruginosa and A. baumannii) and ciprofloxacin. E. coli, K. pneumoniae, E. cloacae, S. marcescens and C. freundii isolates exhibiting resistance to cefazolin and cefotiam and P. aeruginosa and A. baumannii resistant to ceftazidime were preserved for further study. The isolates were stored at -70°C in trypticase soy broth (Difco Laboratories, Detroit, MI, USA) supplemented with 15% glycerol until testing.

Antimicrobial susceptibility testing

MICs of cefotaxime for E. coli, K. pneumoniae, E. cloacae, S. marcescens and C. freundii and those of ceftazidime for P. aeruginosa and A. baumannii were determined using the agar dilution method according to NCCLS guidelines.17 Agar dilution susceptibility testing was performed on isolates of E. coli and K. pneumoniae with cefotaxime MICs >= 2 mg/L (cefotaxime resistant), and E. cloacae, S. marcescens and C. freundii isolates with cefotaxime MICs >= 64 mg/L (cefotaxime resistant), and P. aeruginosa and A. baumannii isolates with ceftazidime MICs >= 64 mg/L (ceftazidime resistant) with various agents (Table 1Go). These antimicrobial agents were provided by their manufacturers and included cefepime and amikacin (Bristol-Myers Squibb, Princeton, NJ, USA); flomoxef (Shionogi & Co., Ltd, Osaka, Japan); ceftazidime (Glaxo, Greenford, UK); cefotaxime and cefpirome (Marion Merrell Dow, Cincinnati, OH, USA); cefoxitin and imipenem (Merck, Sharp & Dohme, Rahway, NJ, USA); meropenem (Sumitomo Pharmaceuticals, Osaka, Japan); ampicillin–sulbactam and trovafloxacin (Pfizer Inc., New York, NY, USA); and ciprofloxacin and moxifloxacin (Bayer Co., West Haven, CT, USA). Sulbactam was added to ampicillin at a 1:2 concentration ratio.


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Table 1. Sources of clinical isolates of extended-spectrum cephalosporin-resistant Gram-negative bacteria recovered from patients treated between January 1999 and December 1999 at the NTUH
 
The isolates were grown overnight on trypticase soy agar plates supplemented with 5% sheep blood (BBL Microbiology Systems, Cockeysville, MD, USA) at 37°C. Bacterial inocula were prepared by suspending the freshly grown bacteria in sterile normal saline and were adjusted to a 0.5 McFarland standard. Using a Steers replicator, an organism density of 104 cfu/spot was inoculated on to the appropriate plate of unsupplemented Mueller–Hinton agar (BBL Microbiology Systems) with various concentrations of antimicrobial agents and incubated at 35°C in ambient air for 16–20 h.

For determining the extended-spectrum ß-lactamase (ESBL) phenotype among cefotaxime-resistant E. coli and cefotaxime-resistant K. pneumoniae isolates, a double disc confirmatory test using cefotaxime, cefotaxime–clavulanate, ceftazidime and ceftazidime–clavulanate antimicrobial discs, was performed and the results were read as recommended by the NCCLS.16 The control strains were E. coli ATCC 25922, K. pneumoniae ATCC 700603 and P. aeruginosa ATCC 27853. Isolates were categorized as susceptible, intermediate or resistant according to the NCCLS guidelines.18 Isolates intermediate or resistant to antimicrobial agents were categorized as non-susceptible to the agents.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The FigureGo shows the percentage of resistance to cefotaxime, ceftazidime and ciprofloxacin of seven Gramnegative bacterial isolates recovered from patients treated at NTUH in 1999 as determined by the standard disc diffusion method. About half of E. cloacae, S. marcescens and C. freundii isolates were non-susceptible to cefotaxime. More than 80% of E. coli, K. pneumoniae, E. cloacae and P. aeruginosa isolates were susceptible to ciprofloxacin. Both ceftazidime and ciprofloxacin had poor activity against A. baumannii.



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Figure. Percentage of seven different species of Gram-negative bacteria that were susceptible to cefotaxime (E. coli, K. pneumoniae, E. cloacae, S. marcescens and C. freundii) or ceftazidime (P. aeruginosa and A. baumannii) (a), and to ciprofloxacin (b) recovered from patients treated at the NTUH between January 1999 and December 1999. Susceptibilities of these isolates were determined using the disc diffusion method as described by the NCCLS.

 
As shown in Table 1Go, a total of 411 isolates with resistance to cefotaxime or ceftazidime were evaluated. The majority (50%) of these isolates were recovered from respiratory tract secretions, followed by blood cultures (34%) and wound pus (9%).

The MICs for E. coli ATCC 25922, K. pneumoniae ATCC 700603 and P. aeruginosa ATCC 27853 were all within the NCCLS control ranges. The MIC ranges, MIC50, MIC90 and the percentages of the 403 clinical isolates that were susceptible, intermediate and resistant to various antimicrobial agents are summarized in Table 2Go.


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Table 2. In vitro susceptibility of clinical isolates of extended-spectrum cephalosporin-resistant Gram-negative bacteria recovered from patients treated between January 1999 and December 1999 at the NTUH
 
Overall, carbapenems (imipenem and meropenem) had good activity against the cefotaxime-resistant Enterobacteriaceae isolates tested (>90% of isolates were susceptible). However, carbapenems had limited activity against ceftazidime-resistant P. aeruginosa (only 4% of isolates were susceptible) and ceftazidime-resistant A. baumannii (56% of isolates were susceptible to imipenem and 51% were susceptible to meropenem). Over 80% of cefotaxime-resistant E. cloacae and C. freundii were susceptible to cefepime, but this agent had limited activity against other bacteria tested. Piperacillin–tazobactam also had poor activity against these isolates, particularly against cefotaxime-resistant K. pneumoniae (susceptibility, 31%), cefotaxime-resistant E. colacae (29%) and ceftazidime-resistant A. baumannii (24%).

Susceptibilities of the 411 isolates to ciprofloxacin varied, ranging from 25% for ceftazidime-resistant A. baumannii to 92% for cefotaxime-resistant E. cloacae isolates. Newer fluoroquinolones (moxifloxacin and trovafloxacin) had equal or less activity against these organisms, except against ceftazidime-resistant A. baumannii for which the MIC90 (8–16 mg/L) of these two agents was four- to 16-fold less than that (MIC90 128 mg/L) of ciprofloxacin.

Susceptibility of the 411 isolates to amikacin varied. Only 15% of ceftazidime-resistant A. baumannii were susceptible to amikacin whereas over four-fifths of the cefotaxime-resistant isolates of E. coli (85%), E. cloacae (92%) and C. freundii (88%) were susceptible to this agent. The rate of resistance of A. baumannii to ampicillin– sulbactam was 66% for ceftazidime-resistant isolates and 100% (MICs 64–128 mg/L) for imipenem-resistant isolates.

Table 3Go shows the susceptibility data for cefotaxime-resistant E. coli and cefotaxime-resistant K. pneumoniae according to their resistance phenotypes. The ESBL phenotype was exhibited by 26.2% of cefotaxime-resistant E. coli isolates and 56.8% of K. pneumoniae isolates tested. Nearly all isolates (95–100%) of cefotaxime-resistant E. coli and cefotaxime-resistant K. pneumoniae exhibiting the ESBL phenotype were susceptible to cefoxitin and flomoxef. All cefotaxime-resistant E. coli isolates of the ESBL phenotype were susceptible to piperacillin–tazobactam except ESBL phenotype cefotaxime-resistant K. pneumoniae, which had a susceptibility rate of 51%. All fluoroquinolones were less active against non-ESBL phenotype isolates of cefotaxime-resistant E. coli and cefotaxime-resistant K. pneumoniae than those against ESBL phenotype isolates. However, the susceptibility rates to amikacin were similar between ESBL and non-ESBL phenotypes of cefotaxime-resistant isolates of E. coli and K. pneumoniae.


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Table 3. In vitro susceptibilities of cefotaxime-resistant (MICs >= 2 mg/L) E. coli and K. pneumoniae isolates recovered from patients treated between January 1999 and December 1999 at the NTUH according to their resistance phenotypes
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial resistance among Gram-negative bacteria to extended-spectrum cephalosporins has complicated the treatment of infections due to these organisms.1,2,10 In recent years there has been a rapid worldwide emergence of these multidrug-resistant pathogens, particularly those causing nosocomial infections. Treatment with a fourth-generation cephalosporin, a carbapenem or a fluoroquinolone with or without an aminoglycoside has become the treatment of choice for the management of these infections.1,10,11,14 With continued antibiotic selective pressure in clinical settings, particularly in hospitals, pathogens resistant to these agents have emerged and pose further problems beyond the lack of available antimicrobial therapy.11,15,19–21

Compared with previous studies from Europe and the Americas,4,6,8,9,11 our isolates of E. cloacae, S. marcescens and C. freundii had higher rates of resistance to cefotaxime and P. aeruginosa and A. baumannii had higher resistance rates to ceftazidime. In the present study of in vitro susceptibilities of recent clinical isolates of cefotaxime- or ceftazidime-resistant Gram-negative bacteria in Taiwan, six important points were demonstrated clearly. First, all isolates of E. coli and K. pneumoniae with ESBL phenotypes were susceptible to flomoxef. These results are in accordance with our previous findings.14 However, this agent is not recommended as the drug of choice for treating infections with these organisms in North America and Europe because of lack of availability in these regions.22

Secondly, compared with previous studies,2,8,9 cefepime had poorer activity against our cefotaxime-resistant K. pneumoniae and ceftazidime-resistant P. aeruginosa isolates. Nevertheless, this agent continued to have good activity against cefotaxime-resistant E. cloacae and C. freundii isolates. Thirdly, our rates of resistance of ceftazidime-resistant isolates of P. aeruginosa and A. baumannii to imipenem and meropenem were considerably higher than those reported by previous Western studies.8,9,23,24 Fourthly, the resistance rates of cefotaxime-resistant isolates of E. coli and S. marcescens and ceftazidime-resistant isolates of P. aeruginosa and A. baumannii to ciprofloxacin were also high.8,9,25 Fifthly, the emergence of carbapenem resistance in clinical isolates of Enterobacteriacae was noted. Finally, the activity of ampicillin–sulbactam against ceftazidime-resistant or imipenem-resistant A. baumannii was poor.

The susceptibility of our ceftazidime-resistant A. baumannii isolates to commonly used antimicrobial agents was low. Loss of specific porin, OprD and overproduction of some ß-lactamases render A. baumannii less susceptible to carbapenems.19,24 For treating infections caused by multidrug-resistant (resistant to extended-spectrum cephalosporins, carbapenems, ciprofloxacin and amikacin) A. bau-mannii, imipenem in combination with amikacin, sulbactam alone (only for non-life threatening infections), or high-dose ampicillin–sulbactam (particularly for treating meningitis due to this organism) has been recommended.19,20,24–26 Unfortunately, all of our isolates of imipenem-resistant A. baumannii were also highly resistant to ampicillin– sulbactam.

The emergence of carbapenem resistance among Gram-negative bacteria other than A. baumanni and particularly in ceftazidime-resistant P. aeruginosa isolates, is also impressive. Overproduction of special ß-lactamases and the emergence of non-metallo-ß-lactamase (IMP-1) and/or loss of the outer membrane proteins have contributed to this resistance.10,21,23 For ceftazidime-resistant P. aeruginosa isolates, although previous reports found that meropenem had better activity than imipenem against P. aeruginosa isolates, our results showed that imipenem and meropenem had similar in vitro activity.13

The high resistance rates of all our clinical isolates of E. coli to ciprofloxacin appears to be a warning sign that agents of this class need to be used with greater caution and with more wisdom, especially in urinary tract or intra-abdominal infections.1,4,27

In conclusion, resistance to all kinds of antimicrobial agents among Gram-negative bacilli is clearly on the increase. This increase has paralleled the introduction, administration and overuse of newer agents of certain classes. In addition to exploring the mechanisms of resistance, all physicians should be obligated to prescribe antimicrobial agents more deliberately. Bacterial strains resistant to most classes of antibiotics will continue to arise unless the inappropriate use of these drugs is curtailed.


    Notes
 
* Corresponding author. Tel: +886-2-2312-3456, ext. 5363; Fax: +886-2-2322-4263; E-mail: hsporen{at}ha.mc.ntu.edu.tw Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 18 April 2001; returned 27 July 2001; accepted 24 September 2001





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