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Antimicrobial Agents and Chemotherapy, August 2002, p. 2671-2675, Vol. 46, No. 8
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.8.2671-2675.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Incidence, Epidemiology, and Characteristics of Quinolone- Nonsusceptible Streptococcus pneumoniae in Croatia

Glenn A. Pankuch,1 Bülent Bozdogan,1 Kensuke Nagai,1 Arjana Tambic-Andrasevic,2 Slavko Schoenwald,2 Tera Tambic,3 Smilja Kalenic,4 Sanja Plesko,2 Nastja K. Tepes,5 Zdenka Kotarski,6 Marina Payerl-Pal,7 and Peter C. Appelbaum1*

Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania 17033,1 University Hospital of Infectious Diseases "Dr. F. Mihaljevic,",2 Croatian Academy of Medical Sciences,3 Zagreb Clinical Hospital Center,4 Sveti Duh General Hospital,5 University Hospital for Lung Diseases, Zagreb,6 Public Health Laboratory, Cakovec, Croatia7

Received 31 January 2002/ Returned for modification 30 March 2002/ Accepted 23 April 2002


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ABSTRACT
 
Among 585 Streptococcus pneumoniae strains isolated in 22 Croatian hospitals 21 strains (3.6%) were quinolone nonsusceptible. MICs of all quinolones were high for seven strains tested with the same serotype (23F) and mutations in gyrA, parC, and parE. The remaining 14 strains were more heterogeneous and had mutations only in parC and/or parE, and the MICs of quinolones were lower for these strains.


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TEXT
 
The incidence of pneumococci resistant to penicillin G and other ß-lactam and non-ß-lactam compounds has increased worldwide at an alarming rate, including in the United States (1, 2, 3, 8, 13). In the United States in a recent survey, 50.4% of 1,476 clinically significant pneumococcal isolates were not susceptible to penicillin and 33% were resistant to macrolides (10). The problem of drug-resistant pneumococci is compounded by the ability of resistant clones to spread from country to country and from continent to continent (14).

Several recent reports from Hong Kong (8), Canada (5), and Spain (19) have described the increasing incidence of quinolone-nonsusceptible pneumococci. A recent study from our laboratory has documented a high incidence of quinolone nonsusceptibility (8%) among pneumococcal cultures isolated from blood, tracheobronchial fluid, and sputum of adult patients in Zagreb, Croatia (15). The present study includes strains isolated from adult patients from the preceding study (15) and expands on this finding by examining the phenotype and genotype of 585 pneumococci isolated from 22 hospitals in 15 Croatian cities in 2000 and 2001.

Five hundred eighty-five Croatian pneumococcus strains were studied for their susceptibility to penicillin G, amoxicillin-clavulanic acid, erythromycin, ciprofloxacin, levofloxacin, gemifloxacin, gatifloxacin, and moxifloxacin. These clinical strains were isolated from adults in 22 hospitals from 15 Croatian cities (Fig. 1) between November 2000 and April 2001. Strains were frozen at -70 in 2% skim milk and transported by courier to Hershey Medical Center on dry ice within 2 months of isolation. All strains were from individual patients. Patient charts were examined by at least one of the Croatian authors.



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  		FIG. 1. Distribution of the 585 pneumococci isolated from 22 hospitals in 15 Croatian cities in 2000 and 2001.

The patient demographics are shown in Fig. 2. Five hundred eighty-five isolates were from 22 hospitals in 15 cities throughout Croatia. Patient ages varied between 13 and 92 years (mean, 49 years). There was a slightly higher number of isolates from patients older than 50 (53%) than from younger patients. This increase was more important for quinolone-nonsusceptible strains (59%). Most of the isolates (65%) were from male patients, and 86% of the quinolone-nonsusceptible strains were isolated from male patients. Fifty-seven percent of all isolates and 82% of quinolone-resistant strains were nosocomial. Older age and male sex are two important risk factors for pneumococcal pneumonia (12). The number of pneumococcal isolates increased with age in Croatia. This increase was more important for the infections caused by quinolone-nonsusceptible pneumococci. In Finland pneumococcal infections have been found to be more frequent among men than women (11). In Croatia very high proportions of the quinolone-nonsusceptible strains (86%) were isolated from male patients. Among patients with pneumococcal infections, older age, male sex, and hospitalization were found to be the risk factors for isolation of quinolone-nonsusceptible strains.



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  		FIG. 2. Demographics of S. pneumoniae strains isolated in Croatia. Distribution of total strains and quinolone-resistant strains by age (A), sex (B), hospitalization (C), and isolation site (D) is shown. Total strains (585 strains) are indicated by crosshatched bars, and quinolone-nonsusceptible strains (21 strains) are shown by shaded bars, NPH, nasopharyngeal swab; TBA, tracheobronchial aspirates; BC, blood culture; CSF, cerebrospinal fluid.

The main types of infection were upper respiratory tract infection (29%), community-acquired pneumonia (25%), and acute exacerbation of chronic bronchitis (16.4%) for all isolates and upper respiratory tract infection (28.6%), acute exacerbation of chronic bronchitis (14.3%), and pneumonia (14.3%) for quinolone-nonsusceptible strains. Overall isolates were from sputum (36.8%), nasopharynx (34.4%), tracheobronchial aspirates (16%), and blood (7%). Quinolone-nonsusceptible strains were from tracheobronchial aspirates (28.6%), nasopharynx (28.6%), sputum (19%), and blood (9%).

All antimicrobials were obtained from their respective manufacturers. Agar dilution was performed with Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% sheep blood (6).

The MICs for 585 S. pneumoniae isolates are shown in Table 1. Twenty-one strains were quinolone nonsusceptible, defined as a ciprofloxacin MIC of >=4 µg/ml. Of 585 strains, 64% were susceptible to penicillin, 22% were intermediate, and 14% were resistant. Eighteen percent of strains were resistant to macrolides. Two strains (0.3%) were resistant to penicillin, erythromycin, and ciprofloxacin; 21 strains (3.6%) were resistant to penicillin and erythromycin; and 12 strains (2.1%) were resistant to penicillin and ciprofloxacin. Three hundred thirty-three strains (56.4%) were susceptible to all three antibiotics. Amoxicillin-clavulanic acid was active against 99% of total isolates and 95% of quinolone-nonsusceptible strains at a MIC of <=2 µg/ml, the present NCCLS breakpoint (17). Twenty strains (3.4%) were levofloxacin nonsusceptible: 11 of 20 were intermediate resistant (MIC = 4 µg/ml) and 9 of 20 were resistant (MIC > 4 µg/ml) to levofloxacin.


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  		TABLE 1. MICs (micrograms per milliliter) for 585 pneumococcal strainsa

Among quinolones tested gemifloxacin had the lowest MICs against both quinolone-susceptible and quinolone-resistant strains (MIC at which 90% of the isolates tested were inhibited [MIC90] = 0.06 µg/ml), followed by moxifloxacin (MIC90 = 0.25 µg/ml), gatifloxacin (MIC90 = 0.5 µg/ml), levofloxacin (MIC90 = 2 µg/ml), and ciprofloxacin (MIC90 = 2 µg/ml) (Table 1).

To determine quinolone resistance mechanisms, quinolone resistance determinant regions in parC, parE, gyrA, and gyrB genes were amplified by PCR as described by Pan et al. (18), purified with a QIAquick PCR purification kit (Qiagen, Valencia, Calif.), and sequenced in the forward direction unless a new mutation was observed, in which case sequencing was done in both directions directly with an Applied Biosystems Model 373A DNA sequencer.

Twenty-one quinolone-nonsusceptible strains were studied further for their resistance mechanisms (Table 2), for their serotype by the capsule Quellung method (9) with commercial antisera (Statens Seruminstitut, Copenhagen, Denmark) as recommended by the manufacturer, and for their clonality by pulsed-field gel electrophoresis (16, 20). The analysis of the results indicates that quinolone-nonsusceptible strains could be separated into two groups; the first group comprised seven strains for which MICs of all the quinolones tested were relatively high and which were resistant to penicillin. All seven strains were serotype 23F and had mutations in gyrA, parC, and parE that led to the S81Y substitution in GyrA, the S79F substitution in ParC, and the I460V substitution in ParE, respectively. Six of these strains had the same pulsed-field gel electrophoresis type after digestion by SmaI. Five of these strains were isolated in the University Hospital for Lung Diseases in Zagreb (three from bronchial aspirates, one from pleural fluid, and one from blood), and one was isolated from blood in the Public Health Institute in Varazdin (Fig. 1). The strain with a different pulsed-field gel electrophoresis type was from Cakovec.


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  		TABLE 2. Demographics of quinolone-nonsusceptible pneumococcal strains, their susceptibilities, detected mutations, and effluxa

The second group was more heterogeneous. All strains were resistant to ciprofloxacin (MICs >= 4 µg/ml), but the MICs of the newer quinolones for these strains were lower. Of these 14 strains, 5 were serotype 11, 3 were serotype 9, 2 were serotype 14, and 3 were nontypeable. In this group eight strains were genetically related. These strains were isolated in Zagreb (4), Split (3), and Cakovec (1). This group did not have a mutation in gyrA or gyrB, and overall the MICs of quinolones were lower for these strains. They had mutations in parC and/or parE.

The prevalence of pneumococci with reduced fluoroquinolone susceptibility has been shown to be increased in Canada, Hong Kong, and Spain. Quinolone nonsusceptibility was 1.7% in Canada (5), 13.3% in Hong Kong (8), and 7.1% in Spain (19). In Hong Kong quinolone nonsusceptibility rates were higher among penicillin-resistant strains (27.3%) (8). Results of our study document an overall 3.6% incidence of ciprofloxacin-nonsusceptible pneumococci in the areas of Croatia studied. Similar to the Canadian experience (20), many strains, belonging to one clone, clustered in a hospital for chronic lung diseases in Zagreb, treating patients for infections such as acute exacerbations of chronic bronchitis. However, several other serotypes and clones were found, both inside and outside Zagreb. In a recent paper from Hungary (7), the rate of highly levofloxacin resistant pneumococcal strains (4.1%) among penicillin-nonsusceptible strains isolated from sputum in a pulmonary department in Budapest was similar to what we found in Croatia among penicillin-susceptible, -intermediate, and -resistant strains. Resistance mechanisms of these strains were not determined.

Analysis of our data showed the presence of two groups of quinolone-nonsusceptible strains. The main difference among these groups was their level of susceptibility to the newer quinolones gatifloxacin, moxifloxacin, and gemifloxacin. The first group was homogeneous in genotype, phenotype, and serotype; however, strains in the second group were heterogeneous, with different types of mutations, serotypes, and pulsed-field gel electrophoresis type. Finally, gemifloxacin had the lowest MICs of all quinolones tested against both quinolone-susceptible and quinolone-nonsusceptible pneumococcal strains.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from GlaxoSmithKline Laboratories, Collegeville, Pa.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, Hershey Medical Center, P.O. Box 850, Hershey, PA 17033. Phone: (717) 531-5113. Fax: (717) 531-7953. E-mail: pappelbaum{at}psu.edu. Back


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Antimicrobial Agents and Chemotherapy, August 2002, p. 2671-2675, Vol. 46, No. 8
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.8.2671-2675.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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