A large outbreak of multiresistant Pseudomonas aeruginosa strains in north-eastern Germany

Brigitte Panziga,*, Gudrun Schröde ra, F.-A. Pittenb and M. Gründlingc

a Institute of Medical Microbiology b Institute of Hygiene and Environmental Medicine c Department for Anaesthesiology and Intensive Medicine, University of Greifswald, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multiply-resistant Pseudomonas aeruginosa were first detected in north-eastern Germany at the end of 1996; since then they have been isolated predominantly from patients in intensive care units. Colonization/infection, especially of the respiratory tract, has been demonstrated in 80 patients, with strains resistant to ß-lactams, carbapenems, aminoglycosides and quinolones. Amikacin showed in-vitro synergy with cefepime, ceftazidime or piperacillin/tazobactam. Horizontal transfer of strains was followed by PFGE and identical strains were detected in the environment, but the source of infection was not established. Rigorous infection control and restricted clinical use of carbapenems limited further dissemination of this outbreak.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is one of the commonest nosocomial pathogens in intensive care units (ICUs). It is intrinsically resistant to a number of structurally unrelated antimicrobial agents, and its resistance to other agents is increasing. During the course of 15 months we followed the circulation of multiply- and highly-resistant P. aeruginosa in different ICUs within the University hospital in Greifswald. Clinical isolates were investigated by phenotyping and genotyping and compared with strains isolated from the environment.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and specimens

Between December 1996 and May 1998, multiply-resistant strains of P. aeruginosa were isolated from routine clinical specimens (tracheal secretions, blood, venous catheter and urine) from 80 ICU patients.

Strains and antimicrobial testing

Primary isolation was performed on Columbia agar containing 5% sheep blood. Antibiotic susceptibility was determined using Mueller-Hinton agar by a disc diffusion method with the following discs (Becton Dickinson GmbH, Heidelberg, Germany): ceftazidime (30 µg), cefsulodine (30 µg), cefepime (30 µg), azlocillin (30 µg), piperacillin/ tazobactam (30 µg/10 µg), imipenem (10 µg), meropenem (10 µg), gentamicin (10 µg), tobramycin (10 µg), amikacin (30 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), aztreonam (30 µg) and polymyxin B (300 µg).

Susceptibility, resistance and moderate susceptibility were defined by the criteria of Deutsches Institut für Normung e.V. (DIN). 1 For selected strains (isolates from the beginning of the outbreak and 15 months later) MIC values were determined by the Etest (AB Biodisk, Solna, Sweden).

Screening tests for synergy were performed by a simplified disc method. When positive results were obtained, further tests were performed using a combination of agar dilution and agar diffusion test. 2

Serotyping

The O serotype was determined using a slide agglutination test with a set of four pools (OMA, OMC, OME, OMF) and 16 monovalent antisera numbered O1 to O16 (Sanofi Diagnostics Pasteur GmbH, Freiburg, Germany).

Genotyping

The genetic relationship of strains with identical multiresistance patterns was investigated by pulsed-field gel electrophoresis (PFGE) using a Gene Path Group 3 Kit (Bio-Rad Laboratories GmbH, Munich, Germany) in a CHEF DR III System (Bio-Rad).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colonization/infection with multiresistant P. aeruginosa strains was observed in 80 patients. The specimens from which multiresistant P. aeruginosa strains were isolated most frequently were tracheal secretions, blood, urine and surgical wound swabs.

All strains were resistant to ß-lactams (ceftazidime, cefepime, cefsulodine, azlocillin and piperacillin/tazobactam), including carbapenems (imipenem, meropenem) and aztreonam, and to aminoglycosides (amikacin, tobramycin, gentamicin) and quinolones (ciprofloxacin, ofloxacin). Only polymyxin B demonstrated in-vitro activity. Agar diffusion tests were confirmed by Etest. Multiple resistance was usually associated with high-level resistance (Table I).


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Table I. Examples of MIC values (mg/L) determined by Etest
 
In-vitro synergy was detected between amikacin and the following antibiotics: cefepime, ceftazidime and piperacillin/tazobactam; there was no synergy between amikacin and imipenem, nor between gentamicin or tobramycin in combination with ß-lactams. The combinations that showed synergy in the primary screening were tested further using the agar dilution- agar diffusion test. The best results were obtained with the combination of amikacin and cefepime, independent of which agents were used in the agar dilution (Table II).


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Table II. In-vitro synergy between cefepime (agar dilution) and amikacin (disc diffusion)
 
Multiply-resistant strains of P. aeruginosa were isolated from the environment (basins and showers) and in the rinsing water of a manually prepared bronchoscope. Swabs collected from medical staff (hands, nose and throat) were consistently negative.

The results of serotyping were ambigious. Weak agglutination was obtained with the serum pool OME. After several passages over cetrimide agar this agglutination became stronger, but examination of the monovalent anti-O sera contained in this pool (O2, O5, O15 and O16) did not confirm a serotype. PFGE showed that isolates from patients and those from the environment were genetically identical or closely related (Figure).



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Figure. CHEF pattern of P. aeruginosa strains following digestion with SpeI.Lane 1: unrelated P. aeruginosa control. Lanes 2- 15: patient and environmental isolates.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance of P. aeruginosa to multiple antimicrobial agents is often associated with the production of extended spectrum mutants of the class D ß-lactamases, OXA-2 and OXA-10, 3,4,5 as OXA-11, -14, -15, -16, -17 and -18.6 Alterations of outer membrane permeability and changes in the active efflux systems can also influence sensitivity to antibiotics.7 Moderate resistance of P. aeruginosa to imipenem usually results from modification of OprD protein expression or loss of D2 porin. 8,9,1010 MICs of about 8 mg/L are typical for this mechanism of resistance. The underlying mechanism of imipenem resistance in our strains cannot be explained simply by alteration of this outer membrane protein, because the MICs were higher (>32 mg/L). It may be that several factors gave rise to resistance to all the ß-lactams, including carbapenems, but the expression of a metallo-ß-lactamase gene of the IMP1 type, first detected in P. aeruginosa strains in Japan, 10 must be assumed. Trials to investigate whether this hydrolytic activity was present in our strains are under way.

Our results concerning the synergic in-vitro interactions of ceftazidime and amikacin correspond to the studies of Giamarellos-Bourboulis et al. 7

The detection of a new group of ß-lactamases that hydrolyse carbapenems and other broad-spectrum ß-lactams has led to a new therapeutic strategy in our hospital. Restricted clinical use of broad- spectrum ß-lactams, including carbapenems, and a rigorous hygienic regime are the most important measures to prevent the spread of the metallo-ß-lactamase gene in P. aeruginosa and other Gram-negative bacilli.


    Acknowledgments
 
We thank Mrs. Helga Schalimow for excellent technical assistance.


    Notes
 
* Corresponding author. Tel: +49-3834-865556; Fax: +49-3834-865561 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Deutsches Institut für Normung e.V. (1995). DIN 58940-4.

2 . Ravizzola, G., Cabibbo, E., Peroni, L., Longo, M., Pollara, P. C., Corulli, M. et al. (1997). In-vitro study of the synergy between ß-lactam antibiotics and glycopeptides against enterococci. Journal of Antimicrobial Chemotherapy 39, 461-70.[Abstract]

3 . Jacoby, G. A. (1994). Genetics of extended-spectrum ß-lactamases. European Journal of Clinical Microbiology and Infectious Diseases 13,Suppl . 1, 2-11.[ISI]

4 . Danel, F., Hall, L. M. C., Gur, D. & Livermore, D. M. (1995). OXA-14, another extended-spectrum variant of OXA-10 (PSE-2) ß -lactamase from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 1881-4.[Abstract]

5 . Mugnier, P., Dubrous, P., Casin, I., Arlet, G. & Collatz, E. (1996). A TEM-derived extended-spectrum ß -lactamase in Pseudomonas. Antimicrobial Agents and Chemotherapy 40, 2488-93.[Abstract]

6 . Livermore, D. M. (1998). New ß-lactamase enzymes of Gram-negative bacteria reported. Current Anti-Infective Therapy 18, 6.

7 . Giamarellos-Bourboulis, E. J., Grecka, P. & Giamarellou, H. (1997). Comparative in vitro interactions of ceftazidime, meropenem, and imipenem with amikacin on multiresistant Pseudomonas aeruginosa. Diagnostic Microbiology and Infectious Diseases 29,81 -6.[ISI][Medline]

8 . Minami, S., Akama, M., Araki, H., Watanabe, Y., Narita, H., Iyobe, S. et al. (1996). Imipenem and cephem resistant Pseudomonas aeruginosa carrying plasmids coding for class B ß-lactamases. Journal of Antimicrobial Chemotherapy 37, 433-44.[Abstract]

9 . Senda, K., Arakawa, Y., Nakashima, K., Ito, H., Ichiyama, S., Shimokata, K. et al. (1996). Multifocal outbreaks of metallo-ß-lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum ß-lactams, including carbapenems. Antimicrobial Agents and Chemotherapy 40, 349-53.

10 . Troillet, K., Samore, M. H. & Carmeli, Y. (1997). Imipenem-resistant Pseudomona aeruginosa: risk factors and antibiotic susceptibility patterns. Clinical Infectious Diseases 25,1094 -8.[ISI][Medline]

Received 23 June 1998; returned 13 July 1998; revised 22 July 1998; accepted 8 October 1998