Antibiotic susceptibility of Kingella kingae isolates from respiratory carriers and patients with invasive infections

Pablo Yagupsky,*, Orna Katz and Nechama Peled

Clinical Microbiology Laboratory, Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva 84101, Israel


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antimicrobial drug susceptibilities of 145 isolates of Kingella kingae to eight antibiotics were determined by the disc diffusion method. In addition, penicillin MICs were determined by the Etest. Study isolates included 37 from blood, 34 from the skeletal system and 74 from respiratory carriers. All isolates were ß-lactamase negative and susceptible to erythromycin, gentamicin, chloramphenicol, tetracycline and ciprofloxacin. A single isolate exhibited resistance to trimethoprim–sulphamethoxazole, and 56 (38.6%) were resistant to clindamycin. The penicillin MIC50 was 0.023 mg/L and the MIC90 was 0.047 mg/L. The distribution of MIC values did not differ according to the site of isolation.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For most of the three decades that have elapsed since the first characterization of Kingella kingae, this Gram-negative cocco-bacillus has been considered a rare cause of human infections.1 In recent years, increasing familiarity of microbiology laboratories with the identification of the organism and improved isolation techniques have resulted in the emergence of K. kingae as a common cause of bacteraemia and skeletal infections in young children.24 With improved culture methods, the annual incidence of invasive K. kingae infections detected among children younger than 24 months living in southern Israel was 27.4 per 100000 and represented one-quarter of that of invasive Haemophilus influenzae type b found in the same population before the introduction of the vaccine.4 In a second study, K. kingae constituted the most common cause of septic arthritis in children younger than 2 years and was isolated in 48% of all culture-proven cases.4

Despite the increasing recognition of K. kingae as an important cause of invasive infections, information on the antibiotic susceptibility profiles of the organism remains limited.1,5,6 This study was conducted to examine the prevalence of antimicrobial drug resistance in a large collection of K. kingae isolates.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study included 142 K. kingae strains isolated in different areas of Israel since the late 1980s, and three American Type Culture Collection (ATCC) strains. The source of isolates was blood (n = 37, including ATCC strains 23331 and 23332), skeletal system (n = 34) and respiratory tract of healthy carriers (n = 74, including ATCC strain 23330). Organisms were isolated in the late 1980s (n = 3), 1991 (n = 6), 1992 (n = 7), 1993 (n = 8), 1994 (n = 57), 1995 (n = 6), 1996 (n = 25), 1997 (n = 13), 1998 (n = 8) and 1999 (n = 9). Isolates were kept frozen at –70°C, and susceptibility tests were performed in batches.

Identification of the organism was based on the typical morphological and physiological characteristics of the species: Gram-negative bacteria that appeared as pairs or short chains of small bacilli with tapered ends; growth and production of ß-haemolysis on trypticase soy agar with added 5% sheep haemoglobin (blood-agar medium) and failure to grow on MacConkey agar; positive oxidase and negative catalase; urease production; motility; and indole reactions and production of acid from glucose and maltose, but not from other sugars.

Isolates were thawed and subcultured twice on blood-agar plates. Antibiotic susceptibilities to trimethoprim– sulphamethoxazole, erythromycin, clindamycin, tetracycline, chloramphenicol, gentamicin and ciprofloxacin were determined by the disc diffusion method of Kirby and Bauer on Mueller–Hinton plates with 5% added sheep blood (Hy Laboratories, Rehovot, Israel). Antibiotic content of the discs (manufactured by Oxoid, Hampshire, UK) was as follows: trimethoprim–sulphamethoxazole 1.25 and 23.75 µg, respectively, erythromycin 15 µg, clindamycin 2 µg, tetracycline 30 µg, chloramphenicol 30 µg, gentamicin 10 µg and ciprofloxacin 5 µg. As there are no standardized criteria for determining antibiotic susceptibility of K. kingae, disc diffusion results were interpreted according to the NCCLS guidelines for Staphylococcus aureus.7

Presence of ß-lactamase was determined by the nitrocefin method. Because different penicillins and cephalosporins are widely used for the treatment of systemic infections, penicillin was selected as the ß-lactam group representative. The MIC of penicillin G was determined by the Etest (AB Biodisk, Solna, Sweden) on the same medium.

In addition, the influence of prolonged incubation of plates and atmosphere on susceptibility testing results was also studied. A random subset of 10 isolates was incubated with and without added 5% CO2, and zones of inhibition around discs and Etest strips were read after 24 and 48 h of incubation. Three capnophilic isolates (including ATCC 23332) were tested in the CO2-enriched atmosphere only.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibition zone diameters around the trimethoprim– sulphamethoxazole, erythromycin, clindamycin, tetracycline, chloramphenicol, gentamicin and ciprofloxacin discs are summarized in the TableGo. A single isolate exhibited resistance to trimethoprim–sulphamethoxazole (no inhibition around the disc) and 56 (38.6%) appeared to be resistant to clindamycin (inhibition zone diameter <=14 mm). All isolates were ß-lactamase negative. The distribution of penicillin MICs is shown in the FigureGo. The MIC values ranged between <=0.002 and 0.064 mg/L. The MIC50 was 0.023 mg/L and the MIC90 was 0.047 mg/L. The distribution of MIC values did not differ between blood, skeletal system and respiratory tract isolates, or according to the date of isolation (data not shown). The MICs of penicillin for ATCC strains 23330, 23331 and 23332 were <=0.002, 0.006 and 0.012 mg/L, respectively.


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Table. Results of susceptibility testing of 145 K. kingae isolates by the disc diffusion method
 


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Figure. Distribution of penicillin MICs for 145 K. kingae isolates.

 
For the 10 random isolates in which the influence of different susceptibility test incubation conditions was investigated, neither incubation of plates for an additional 24 h nor addition of 5% CO2 were found to modify the results.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most of the literature on invasive K. kingae infection consists of single case reports or short series of patients with joint or bone infections or endocarditis. Information on the susceptibility of the organism to antimicrobial drugs is, therefore, fragmentary and the laboratory techniques used for testing are not described in detail in these reports.2,3 In a recently published study, Kugler et al.8 examined the susceptibility of Gram-negative organisms of the HACEK group to a wide array of ß-lactam drugs, fluoroquinolones, rifampicin and trimethoprim–sulphamethoxazole using the Etest. Unfortunately only three K. kingae isolates were included in the study and results of susceptibility testing of these organisms were combined with those obtained with Kingella denitrificans. Only three studies have systematically examined the antibiotic susceptibilities of K. kingae.1,5,6 In the 1980s, Claesson et al.1 tested 13 isolates from Sweden, Norway and Australia by the disc diffusion method, and Prère et al.5 studied three isolates of K. kingae from French children with septic arthritis using the agar-dilution method. More recently, Jensen et al.6 studied 46 clinical isolates from Scandinavian countries by disc diffusion and a macrodilution MIC method. Sixteen isolates were derived from bone or joint exudates, 15 from blood, 11 from respiratory cultures and the remaining isolates were from cerebrospinal fluid, a corneal ulcer, peritoneal fluid and an unknown source.6 In these studies, all K. kingae isolates were found to be susceptible to penicillin and to a wide array of antimicrobial drugs, and resistant to trimethoprim.1,5,6

Other reports of K. kingae isolates from patients with invasive infections, however, suggest that susceptibility of the organism to antibiotics is not uniform.3,9,10 Production of ß-lactamase has been detected in an isolate from an adult AIDS patient with bacteraemia, and in three of five K. kingae isolates from children with bacteraemia or skeletal infections in Iceland.3,9 In addition, sporadic resistance to the trimethoprim–sulphamethoxazole combination and to ciprofloxacin has also been reported.3,10

The results of the present study, which comprises a large number of organisms isolated from individuals living in different geographical areas of Israel and collected over a 12 year period, show that antimicrobial susceptibility patterns of K. kingae are quite uniform and predictable. All test organisms were ß-lactamase negative, exhibited low penicillin MICs and were susceptible to erythromycin, gentamicin, chloramphenicol, tetracycline and ciprofloxacin, and all but one were susceptible to trimethoprim–sulphamethoxazole. This finding is consistent with the clinical observation that invasive K. kingae infections respond promptly to antimicrobial therapy and especially to drugs such as ß-lactams, macrolides or trimethoprim–sulphamethoxazole, which are often administered empirically to young children.14 On the other hand, a large fraction of isolates were found to be resistant to clindamycin. This observation is consistent with the resistance of K. kingae isolates to lincomycin, a related antimicrobial drug, reported in other studies.1,5

Susceptibility of isolates to glycopeptides was not tested because this class of antimicrobial drugs is ineffective against Gram-negative organisms due to the large size of the molecule, which cannot pass through the outer membrane to reach the peptidoglycan target site. In fact, in a previous study we have used a vancomycin-containing selective medium to inhibit growth of Gram-positive flora and facilitate the isolation of K. kingae from pharyngeal cultures.4

In recent years, important paediatric pathogens such as Streptococcus pneumoniae, H. influenzae and Moraxella catarrhalis have developed resistance to ß-lactams, macrolides or trimethoprim–sulphamethoxazole. These organisms are commonly carried in the respiratory tract of young children and are therefore frequently exposed to selective antibiotic pressure. A few years ago, it was demonstrated that K. kingae is also a component of the normal respiratory flora of young children.4 In a prospective study conducted among young attendees at a day-care centre in southern Israel, 109 of 624 (27.5%) throat cultures yielded K. kingae and 35 of 48 (72.9%) children yielded the organism at least once over an 11 month period.4 The results of the present study show that despite the frequent respiratory carriage of the organism, K. kingae remains susceptible to antimicrobial drugs that are commonly prescribed to young children with bacteraemia or skeletal infections.


    Notes
 
* Corresponding author. Tel: +972-764-00507; Fax: +972-764-03541; E-mail: yagupsky{at}bgumail.ac.il Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Claesson, B., Falsen, E. & Kjellman, B. (1985). Kingella kingae infections: a review and a presentation of data from 10 Swedish cases. Scandinavian Journal of Infectious Diseases 17, 233–43.[ISI][Medline]

2 . Goutzmanis, J. J., Gonis, G. & Gilbert, G. L. (1991). Kingella kingae infection in children: ten cases and a review of the literature. Pediatric Infectious Disease Journal 10, 677–83.[ISI][Medline]

3 . Birgisson, H., Steingrimsson, O. & Gudnason, T. (1997). Kingella kingae infections in paediatric patients: 5 cases of septic arthritis, osteomyelitis and bacteraemia. Scandinavian Journal of Infectious Diseases 29, 495–8.[ISI][Medline]

4 . Yagupsky, P. & Dagan, R. (1997). Kingella kingae: an emerging cause of invasive infections in young children. Clinical Infectious Diseases 24, 860–6.[ISI][Medline]

5 . Prère, M. F., Seguy, M., Vezard, Y. & Lareng, M. B. (1986). Sensibilitè aux antibiotiques de Kingella kingae. Pathologie Biologie 34, 604–7.[ISI][Medline]

6 . Jensen, K. T., Schonheyder, H. & Thomsen, V. F. (1994). In-vitro activity of ß-lactam and other antimicrobial agents against Kingella kingae. Journal of Antimicrobial Chemotherapy 33, 635–40.[ISI][Medline]

7 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing: Ninth Informational Approved Standard Supplement M100-S9. NCCLS, Wayne, PA.

8 . Kugler, K. C., Biedenbach, D. J. & Jones, R. N. (1999). Determination of the antimicrobial activity of 29 clinically important compounds tested against fastidious HACEK group organisms. Diagnostic Microbiology and Infectious Diseases 34, 73–6.[ISI][Medline]

9 . Sordillo, E. M., Rendel, M., Sood, R., Belinfanti, J., Murray, O. & Brook, D. (1993). Septicemia due to beta-lactamase-positive Kingella kingae. Clinical Infectious Diseases 17, 818–9.[ISI][Medline]

10 . Giamarellou, H. & Galanakis, N. (1987). Use of intravenous ciprofloxacin in difficult-to-treat infections. American Journal of Medicine 82, 346–51.[ISI][Medline]

Received 28 February 2000; returned 21 June 2000; revised 11 September 2000; accepted 9 October 2000