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The frequency of BRO β-lactamase and its relationship to antimicrobial susceptibility and serum resistance in Moraxella catarrhalis
J Infect Chemother 20 (2014) 6e8
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
The frequency of BRO b-lactamase and its relationship to antimicrobial susceptibility and serum resistance in Moraxella catarrhalis Ryoichi Saito a, *, Shotaro Nonaka a, Yuji Fujinami a, Shiho Matsuoka a, Shinichi Nakajima a, Hiroyuki Nishiyama b, Noboru Okamura a a
Department of Microbiology and Immunology, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan Clinical Laboratory Department, Surugadai Nihon University Hospital, 1-8-13 Kandasurugadai, Chiyoda-Ku, Tokyo 101-8309, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2013 Received in revised form 18 June 2013 Accepted 19 June 2013
We investigated the frequency of BRO b-lactamase and its relationship to antibiotic susceptibility profiles and serum susceptibility. Moraxella catarrhalis clinical isolates (n ¼ 197) were collected from patients with respiratory tract infections in Tokyo between November 2004 and April 2005. Phenotypic and genotypic detection of b-lactamases was performed. The MICs of 6 antibiotics were determined by Etest, and the serum bactericidal assay was conducted by using the culture-and-spot test. Nearly all (192; 97%) of the clinical isolates were b-lactamase producers; of these, 182 (95%) were bro-1 and 10 (5%) were bro-2 positive. MIC50, MIC90, and geometric mean MICs of penicillin, amoxicillin, cefixime, and clarithromycin for BRO-1 isolates were significantly higher than for BRO-2 isolates. The frequency of intermediate and full serum resistance was significantly higher in BRO-1 isolates than in BRO-2 isolates (P ¼ 0.0056), but not BRO-negative isolates (P ¼ 0.1333). We provide the first evidence that the presence of BRO-1 in M. catarrhalis is associated with reduced susceptibility to clarithromycin and b-lactam antibiotics, as well as serum non-sensitive (intermediate and resistant). Ó 2013, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Moraxella catarrhalis b-Lactamase Serum resistance b-Lactam Macrolide
1. Introduction Moraxella catarrhalis, a gram-negative aerobic diplococcus, causes respiratory tract infections such as otitis media, sinusitis, acute bronchitis, and pneumonia [1]. Although M. catarrhalis is generally susceptible to the majority of antibiotics used to treat respiratory tract infections, >90% of clinical isolates worldwide are resistant to penicillin because of the production b-lactamases BRO1 and BRO-2, first described in the 1970s [1e3]. Previous studies have demonstrated that b-lactam MICs for BRO-1 producers were higher than for BRO-2 producers [4,5]. Resistance to serum complement for escape from host defence is an important virulence factor in gram-negative bacteria including M. catarrhalis and has been associated with outer membrane proteins such as UspA2, OMPCD, and CopB [6,7]. The rise and spread of serum-resistant lineages with a high homologous recombination rate and high mutation rate of housekeeping genes has been reported in M. catarrhalis [6,8]. Thus, it has been suggested that it is * Corresponding author. Tel.: þ81 3 5803 5368; fax: þ81 3 5803 5384. E-mail address: [email protected] (R. Saito).
easier for serum-resistant lineages to acquire antibiotic resistanceassociated genes such as b-lactamase. However, our knowledge of the rapid increase of BRO b-lactamase producers, and the relationship between antibiotic resistance and pathogenesis, including serum resistance, is far from complete. Therefore, we investigated the relationship between BRO b-lactamase type and antibiotic or serum susceptibility. 2. Methods 2.1. Bacterial strains and culture conditions A total of 197 M. catarrhalis clinical isolates obtained from nasopharyngeal swab and sputum samples were collected from patients with respiratory tract infections between November 2004 and April 2005 in Tokyo. M. catarrhalis ATCC49143 () and ATCC25238 (þ) were used as controls for a serum bactericidal assay. The M. catarrhalis strains were cultured at 37 C on brain heart infusion (BHI) agar (Oxoid, Tokyo, Japan) and tryptone soya agar (TSA) plates containing 5% sheep blood (Nippon Becton Dickinson, Tokyo, Japan) in an atmosphere comprising 95% air and 5% CO2.
1341-321X/$ e see front matter Ó 2013, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jiac.2013.06.003
R. Saito et al. / J Infect Chemother 20 (2014) 6e8
2.2. Screening of b-lactamase production and detection of bro Nitrocefin-based methods (cefinase disks; Nippon Becton Dickinson, Tokyo, Japan) were used to investigate b-lactamase production. To detect bro, bacterial genomic DNA was prepared by boiling lysis of M. catarrhalis strains. PCR amplification of bro was performed as described in Ref. [2]. bro-1 (165 bp) and bro-2 (144 bp) types were confirmed by size discrimination by electrophoresis in 3.0% agarose and visualization with ethidium bromide. PCRamplified bro-2 was purified and sequenced in the forward and reverse directions. 2.3. Antimicrobial susceptibility testing The minimal inhibitory concentrations (MICs) of penicillin, amoxicillin, amoxicillin-clavulanic acid, cefixime, clarithromycin, and levofloxacin, were determined by Etest (AB Biodisk, Solna, Sweden). 2.4. Serum bactericidal assay The serum bactericidal assay was performed with 50% pooled normal human serum in a culture-and-spot test [9]. The results were classified by colony counts in the droplet area as sensitive strains (0e50 colonies), resistant strains (normal growth), and intermediate strains (intermediate growth) [9]. At least 3 independent assays were performed. 2.5. Statistical analysis
Table 1 Antibiotic MICs (mg/L) and b-lactamase pattern for M. catarrhalis isolates. Antimicrobial agent and parameter
3. Results 3.1. BRO b-lactamase and antimicrobial susceptibility Most of the clinical isolates (192, 97%) were b-lactamase producers by the nitrocefin-based method. Of these, 182 (95%) isolates were bro-1 and 10 (5%) were bro-2 positive. PCR amplicons of bro-2 were 144 bp long and no mutations were found in the 82 bp encoding BRO-2. The MIC ranges, MIC50, MIC90, and geometric MIC means for each isolate are shown in Table 1. The geometric MIC means of penicillin, amoxicillin, amoxicillin-clavulanic acid, cefixime, and clarithromycin for BRO-1-positive isolates were >100, 25, 3, 4, and 2-fold higher than for BRO-negative isolates (P < 0.05), while only those of penicillin, amoxicillin, and cefixime were significantly higher for BRO-2-positive isolates (>10, 5, and 3-fold). Furthermore, the geometric mean MICs of penicillin, amoxicillin, amoxicillin-clavulanic acid, cefixime, and clarithromycin for BRO1-positive isolates were significantly higher than for BRO-2 positive isolates. In contrast, the geometric mean MIC of levofloxacin did not significantly differ between BRO-positive and -negative isolates. 3.2. Serum resistance Of 197 clinical isolates, 19 (9.6%), 37 (18.8%), and 141 (71.6%) were sensitive, intermediate, and resistant in the serum bactericidal assay. Among 182 BRO-1 and 10 BRO-2-positive isolates, 13 (7.1%), 32 (17.6%), and 137 (75.3%) of BRO-1-positive and 4 (40%), 5 (50%), and 1 (10%) BRO-2-positive isolates were sensitive, intermediate, and resistant, respectively. Of 5 BRO-negative isolates, 2
BRO-1
BRO-2
BRO-negative
(n ¼ 182)
(n ¼ 10)
(n ¼ 5)
0.25e2 1 2 0.933d,e
0.063e0.25 0.063 0.063 0.083
0.063e16 0.25 1 0.354d,e
0.063e0.063 0.063 0.063 0.063
0.063e0.25 0.063 0.125 0.092f
0.063e0.063 0.063 0.063 0.063
0.063e0.25 0.063 0.25 0.170d,f
0.063e0.063 0.063 0.063 0.063
0.063e0.125 0.125 0.125 0.095f
0.063e0.125 0.063 0.125 0.083
0.063e0.063 0.063 0.063 0.063
0.063e0.063 0.063 0.063 0.063
Penicillin MIC range 0.5e32 8 MIC50 MIC90 16 a Geometric mean 8.699b Amoxicillin MIC range 0.125e16 MIC50 2 MIC90 4 Geometric mean 1.550b Amoxicillin-clavulanic acid MIC range 0.063e0.5 0.25 MIC50 MIC90 0.25 Geometric mean 0.177b Cefixime MIC range 0.063e1 MIC50 0.25 MIC90 0.5 Geometric mean 0.260b Clarithromycin MIC range 0.063e1 MIC50 0.125 MIC90 0.25 Geometric mean 0.153c Levofloxacin MIC range 0.063e0.25 MIC50 0.063 MIC90 0.063 Geometric mean 0.064 a b
The geometric mean MICs were compared by the Manne Whitney U-test. Categorical variables were evaluated by the Chisquare test. In all analyses, P < 0.05 was considered significant.
7
c d e f
In analysis of geometric mean, 0.063 was calculated with 0.063. P < 0.001 vs. BRO-negative isolates. P < 0.05 vs. BRO-negative isolates. P < 0.01 vs. BRO-negative isolates. P < 0.001 vs. BRO-1 positive isolates. P < 0.01 vs. BRO-1 positive isolates.
(40%) were sensitive and 3 (60%) were resistant. Serumintermediate and -resistant BRO-positive and BRO-1-positive isolates tended to be more frequent than BRO-negative isolates (P ¼ 0.2367 and P ¼ 0.1333, respectively) (Table 2). In contrast, serum-intermediate and -resistant BRO-1-positive isolates were significantly more frequent than BRO-2 positive isolates (P ¼ 0.0056). 4. Discussion More than 90% of global M. catarrhalis clinical isolates are BRO blactamase producers, of which bro-1-positive isolates command a large majority [1,2,4]. Previous studies have demonstrated that BRO-1-positive isolates exhibit high MICs for various b-lactam antibiotics in comparison to BRO-2-positive isolates [2,4]. In Japan, Inoue et al. reported that the frequency of b-lactamase producers was high (>95%) [10], and Ikeda et al. demonstrated that MIC50s of cefixime, cefteram, cefaclor and amoxicillin were 2e8-fold higher for BRO-1 producers than BRO-2 producers [3]. In this study, blactamase producers occupied the majority (97%), and the frequency of bro-1 (95%) and bro-2 (5%) positive isolates was similar to previous findings. Moreover, our results showed that the MIC50, MIC90, and geometric MIC means of b-lactam antibiotics (penicillin, amoxicillin, and cefixime) for BRO-1-positive isolates were significantly higher than for BRO-2-positive isolates, similar to previous findings [2e4]. Although all isolates were susceptible to clarithromycin under CLSI criteria [11], the geometric mean MIC of clarithromycin for BRO-1-positive isolates was significantly higher than for BRO-negative (P < 0.05) and BRO-2-positive (P < 0.01)
8
R. Saito et al. / J Infect Chemother 20 (2014) 6e8
Table 2 Distribution of serum resistance and BRO b-lactamases in M. catarrhalis isolates. Bactericidal assay
BRO Negative
I or R
P Positive
(n ¼ 5)
(n ¼ 192)
3 (60%)
175 (91.1%)
BRO Negative
0.2367
P BRO-1
(n ¼ 5)
(n ¼ 182)
3 (60%)
169 (92.9%)
bro-positive BRO-1
0.1333
P BRO-2
(n ¼ 182)
(n ¼ 10)
169 (92.9%)
6 (60%)
0.0056
I, intermediate; R, resistant.
isolates. The difference in clarithromycin MIC is small, and we could not identify the reason for this difference here; however, we are the first to demonstrate reduced susceptibility to macrolides among BRO-1-positive versus BRO-negative and BRO-2-positive isolates. Serum resistance is a well-known virulence factor for M. catarrhalis. In previous studies, the frequency of serum-resistant isolates is higher in patients with illnesses such as pulmonary infections than in healthy individuals [8,12]. Although Bootsma et al. [13] examined the frequency of serum non-sensitive (intermediate and resistant) isolates and the distribution of BRO b-lactamase types, their relationship has been unclear due to a lack of data. In this study, our observations have demonstrated that serum nonsensitive isolates were mostly BRO-1-positive (P ¼ 0.0056). However, there was no significant difference in the frequency of serum non-sensitive isolates among BRO-positive versus -negative isolates (P ¼ 0.2367) and BRO-1-positive versus BRO-negative isolates (P ¼ 0.1333). A significant difference may have been found if a larger number of isolates had been examined. In summary, our results provide the first evidence that the presence of BRO-1 in M. catarrhalis is associated with reduced susceptibility to clarithromycin and b-lactam antibiotics, as well as serum non-sensitive (intermediate and resistant) in comparison to BRO-negative or BRO-2-positive isolates. In the future, since BRO-1 positive isolates with serum resistance may confer resistance to various antibiotics besides b-lactams and macrolides, we plan to continue our study of the relationship between antibiotic susceptibility, BRO b-lactamases, and virulence factors. Acknowledgements We thank Dr. H. Qiu and Dr. T. Chida for excellent technical assistance. The study did not receive financial support from third parties. All authors declare that they have no conflicts of interest.
References [1] Murphy TF, Parameswaran GI. Moraxella catarrhalis, a human respiratory tract pathogen. Clin Infect Dis 2009;49:124e31. [2] Khan MA, Northwood JB, Levy F, Verhaegh SJC, Farrell DJ, Van Belkum A, et al. bro b-lactamase and antibiotic resistances in a global cross-sectional study of Moraxella catarrhalis from children and adults. J Antimicrob Chemother 2010;65:91e7. [3] Ikeda F, Yokota Y, Mine Y, Yamada T. Characterization of BRO enzymes and blactamase transfer of Moraxella (Branhamella) catarrhalis isolated in Japan. Chemotherapy 1993;39:88e95. [4] Fung CP, Yeo SF, Livermore DM. Susceptibility of Moraxella catarrhalis isolates to b-lactam antibiotics in relation to b-lactamase pattern. J Antimicrob Chemother 1994;33:215e22. [5] Esel D, Ay-Altintop Y, Yagmur G, Gokahmetoglu S, Sumerkan B. Evaluation of susceptibility patterns and BRO b-lactamase types among clinical isolates of Moraxella catarrhalis. Clin Microbiol Infect 2007;13:1023e5. [6] de Vries SPW, Bootsma HJ, Hays JP, Hermans PWM. Molecular aspects of Moraxella catarrhalis pathogenesis. Microbiol Mol Biol Rev 2009;73:389e 406. [7] Hol C, Verduin CM, Vandijke E, Verhoef J, Vandijk H. Complement resistance in Branhamella (Moraxella) catarrhalis. Lancet 1993;341:1281. [8] Wirth T, Morelli G, Kusecek B, van Belkum A, van der Schee C, Meyer A, et al. The rise and spread of a new pathogen: seroresistant Moraxella catarrhalis. Genome Res 2007;17:1647e56. [9] Verduin CM, Hol C, Vandijke E, Faber JAJ, Jansze M, Verhoef J, et al. Assessment of complement-mediated killing of Moraxella (Branhamella) catarrhalis isolates by a simple method. Clin Diagn Lab Immunol 1995;2:365e8. [10] Inoue M, Kaneko K, Akizawa K, Fujita S, Kaku M, Igari J, et al. Antimicrobial susceptibility of respiratory tract pathogens in Japan during PROTEKT years 1e3 (1999e2002). J Infect Chemother 2006;12:9e21. [11] CLSI. Methods for antimicrobial dilution and disk susceptibility testing for infrequently isolated or fastidious bacteria; approved guideline. 2nd ed.; 2010. M45eA2. [12] Hol C, Verduin CM, Vandijke EEA, Verhoef J, Fleer A, Vandijk H. Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis. FEMS Immunol Med Microbiol 1995;11:207e11. [13] Bootsma HJ, van der Heide HGJ, van de Pas S, Schouls LM, Mooi FR. Analysis of Moraxella catarrhalis by DNA typing: evidence for a distinct subpopulation associated with virulence traits. J Infect Dis 2000;181:1376e87.
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
The frequency of BRO b-lactamase and its relationship to antimicrobial susceptibility and serum resistance in Moraxella catarrhalis Ryoichi Saito a, *, Shotaro Nonaka a, Yuji Fujinami a, Shiho Matsuoka a, Shinichi Nakajima a, Hiroyuki Nishiyama b, Noboru Okamura a a
Department of Microbiology and Immunology, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan Clinical Laboratory Department, Surugadai Nihon University Hospital, 1-8-13 Kandasurugadai, Chiyoda-Ku, Tokyo 101-8309, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2013 Received in revised form 18 June 2013 Accepted 19 June 2013
We investigated the frequency of BRO b-lactamase and its relationship to antibiotic susceptibility profiles and serum susceptibility. Moraxella catarrhalis clinical isolates (n ¼ 197) were collected from patients with respiratory tract infections in Tokyo between November 2004 and April 2005. Phenotypic and genotypic detection of b-lactamases was performed. The MICs of 6 antibiotics were determined by Etest, and the serum bactericidal assay was conducted by using the culture-and-spot test. Nearly all (192; 97%) of the clinical isolates were b-lactamase producers; of these, 182 (95%) were bro-1 and 10 (5%) were bro-2 positive. MIC50, MIC90, and geometric mean MICs of penicillin, amoxicillin, cefixime, and clarithromycin for BRO-1 isolates were significantly higher than for BRO-2 isolates. The frequency of intermediate and full serum resistance was significantly higher in BRO-1 isolates than in BRO-2 isolates (P ¼ 0.0056), but not BRO-negative isolates (P ¼ 0.1333). We provide the first evidence that the presence of BRO-1 in M. catarrhalis is associated with reduced susceptibility to clarithromycin and b-lactam antibiotics, as well as serum non-sensitive (intermediate and resistant). Ó 2013, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Moraxella catarrhalis b-Lactamase Serum resistance b-Lactam Macrolide
1. Introduction Moraxella catarrhalis, a gram-negative aerobic diplococcus, causes respiratory tract infections such as otitis media, sinusitis, acute bronchitis, and pneumonia [1]. Although M. catarrhalis is generally susceptible to the majority of antibiotics used to treat respiratory tract infections, >90% of clinical isolates worldwide are resistant to penicillin because of the production b-lactamases BRO1 and BRO-2, first described in the 1970s [1e3]. Previous studies have demonstrated that b-lactam MICs for BRO-1 producers were higher than for BRO-2 producers [4,5]. Resistance to serum complement for escape from host defence is an important virulence factor in gram-negative bacteria including M. catarrhalis and has been associated with outer membrane proteins such as UspA2, OMPCD, and CopB [6,7]. The rise and spread of serum-resistant lineages with a high homologous recombination rate and high mutation rate of housekeeping genes has been reported in M. catarrhalis [6,8]. Thus, it has been suggested that it is * Corresponding author. Tel.: þ81 3 5803 5368; fax: þ81 3 5803 5384. E-mail address: [email protected] (R. Saito).
easier for serum-resistant lineages to acquire antibiotic resistanceassociated genes such as b-lactamase. However, our knowledge of the rapid increase of BRO b-lactamase producers, and the relationship between antibiotic resistance and pathogenesis, including serum resistance, is far from complete. Therefore, we investigated the relationship between BRO b-lactamase type and antibiotic or serum susceptibility. 2. Methods 2.1. Bacterial strains and culture conditions A total of 197 M. catarrhalis clinical isolates obtained from nasopharyngeal swab and sputum samples were collected from patients with respiratory tract infections between November 2004 and April 2005 in Tokyo. M. catarrhalis ATCC49143 () and ATCC25238 (þ) were used as controls for a serum bactericidal assay. The M. catarrhalis strains were cultured at 37 C on brain heart infusion (BHI) agar (Oxoid, Tokyo, Japan) and tryptone soya agar (TSA) plates containing 5% sheep blood (Nippon Becton Dickinson, Tokyo, Japan) in an atmosphere comprising 95% air and 5% CO2.
1341-321X/$ e see front matter Ó 2013, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jiac.2013.06.003
R. Saito et al. / J Infect Chemother 20 (2014) 6e8
2.2. Screening of b-lactamase production and detection of bro Nitrocefin-based methods (cefinase disks; Nippon Becton Dickinson, Tokyo, Japan) were used to investigate b-lactamase production. To detect bro, bacterial genomic DNA was prepared by boiling lysis of M. catarrhalis strains. PCR amplification of bro was performed as described in Ref. [2]. bro-1 (165 bp) and bro-2 (144 bp) types were confirmed by size discrimination by electrophoresis in 3.0% agarose and visualization with ethidium bromide. PCRamplified bro-2 was purified and sequenced in the forward and reverse directions. 2.3. Antimicrobial susceptibility testing The minimal inhibitory concentrations (MICs) of penicillin, amoxicillin, amoxicillin-clavulanic acid, cefixime, clarithromycin, and levofloxacin, were determined by Etest (AB Biodisk, Solna, Sweden). 2.4. Serum bactericidal assay The serum bactericidal assay was performed with 50% pooled normal human serum in a culture-and-spot test [9]. The results were classified by colony counts in the droplet area as sensitive strains (0e50 colonies), resistant strains (normal growth), and intermediate strains (intermediate growth) [9]. At least 3 independent assays were performed. 2.5. Statistical analysis
Table 1 Antibiotic MICs (mg/L) and b-lactamase pattern for M. catarrhalis isolates. Antimicrobial agent and parameter
3. Results 3.1. BRO b-lactamase and antimicrobial susceptibility Most of the clinical isolates (192, 97%) were b-lactamase producers by the nitrocefin-based method. Of these, 182 (95%) isolates were bro-1 and 10 (5%) were bro-2 positive. PCR amplicons of bro-2 were 144 bp long and no mutations were found in the 82 bp encoding BRO-2. The MIC ranges, MIC50, MIC90, and geometric MIC means for each isolate are shown in Table 1. The geometric MIC means of penicillin, amoxicillin, amoxicillin-clavulanic acid, cefixime, and clarithromycin for BRO-1-positive isolates were >100, 25, 3, 4, and 2-fold higher than for BRO-negative isolates (P < 0.05), while only those of penicillin, amoxicillin, and cefixime were significantly higher for BRO-2-positive isolates (>10, 5, and 3-fold). Furthermore, the geometric mean MICs of penicillin, amoxicillin, amoxicillin-clavulanic acid, cefixime, and clarithromycin for BRO1-positive isolates were significantly higher than for BRO-2 positive isolates. In contrast, the geometric mean MIC of levofloxacin did not significantly differ between BRO-positive and -negative isolates. 3.2. Serum resistance Of 197 clinical isolates, 19 (9.6%), 37 (18.8%), and 141 (71.6%) were sensitive, intermediate, and resistant in the serum bactericidal assay. Among 182 BRO-1 and 10 BRO-2-positive isolates, 13 (7.1%), 32 (17.6%), and 137 (75.3%) of BRO-1-positive and 4 (40%), 5 (50%), and 1 (10%) BRO-2-positive isolates were sensitive, intermediate, and resistant, respectively. Of 5 BRO-negative isolates, 2
BRO-1
BRO-2
BRO-negative
(n ¼ 182)
(n ¼ 10)
(n ¼ 5)
0.25e2 1 2 0.933d,e
0.063e0.25 0.063 0.063 0.083
0.063e16 0.25 1 0.354d,e
0.063e0.063 0.063 0.063 0.063
0.063e0.25 0.063 0.125 0.092f
0.063e0.063 0.063 0.063 0.063
0.063e0.25 0.063 0.25 0.170d,f
0.063e0.063 0.063 0.063 0.063
0.063e0.125 0.125 0.125 0.095f
0.063e0.125 0.063 0.125 0.083
0.063e0.063 0.063 0.063 0.063
0.063e0.063 0.063 0.063 0.063
Penicillin MIC range 0.5e32 8 MIC50 MIC90 16 a Geometric mean 8.699b Amoxicillin MIC range 0.125e16 MIC50 2 MIC90 4 Geometric mean 1.550b Amoxicillin-clavulanic acid MIC range 0.063e0.5 0.25 MIC50 MIC90 0.25 Geometric mean 0.177b Cefixime MIC range 0.063e1 MIC50 0.25 MIC90 0.5 Geometric mean 0.260b Clarithromycin MIC range 0.063e1 MIC50 0.125 MIC90 0.25 Geometric mean 0.153c Levofloxacin MIC range 0.063e0.25 MIC50 0.063 MIC90 0.063 Geometric mean 0.064 a b
The geometric mean MICs were compared by the Manne Whitney U-test. Categorical variables were evaluated by the Chisquare test. In all analyses, P < 0.05 was considered significant.
7
c d e f
In analysis of geometric mean, 0.063 was calculated with 0.063. P < 0.001 vs. BRO-negative isolates. P < 0.05 vs. BRO-negative isolates. P < 0.01 vs. BRO-negative isolates. P < 0.001 vs. BRO-1 positive isolates. P < 0.01 vs. BRO-1 positive isolates.
(40%) were sensitive and 3 (60%) were resistant. Serumintermediate and -resistant BRO-positive and BRO-1-positive isolates tended to be more frequent than BRO-negative isolates (P ¼ 0.2367 and P ¼ 0.1333, respectively) (Table 2). In contrast, serum-intermediate and -resistant BRO-1-positive isolates were significantly more frequent than BRO-2 positive isolates (P ¼ 0.0056). 4. Discussion More than 90% of global M. catarrhalis clinical isolates are BRO blactamase producers, of which bro-1-positive isolates command a large majority [1,2,4]. Previous studies have demonstrated that BRO-1-positive isolates exhibit high MICs for various b-lactam antibiotics in comparison to BRO-2-positive isolates [2,4]. In Japan, Inoue et al. reported that the frequency of b-lactamase producers was high (>95%) [10], and Ikeda et al. demonstrated that MIC50s of cefixime, cefteram, cefaclor and amoxicillin were 2e8-fold higher for BRO-1 producers than BRO-2 producers [3]. In this study, blactamase producers occupied the majority (97%), and the frequency of bro-1 (95%) and bro-2 (5%) positive isolates was similar to previous findings. Moreover, our results showed that the MIC50, MIC90, and geometric MIC means of b-lactam antibiotics (penicillin, amoxicillin, and cefixime) for BRO-1-positive isolates were significantly higher than for BRO-2-positive isolates, similar to previous findings [2e4]. Although all isolates were susceptible to clarithromycin under CLSI criteria [11], the geometric mean MIC of clarithromycin for BRO-1-positive isolates was significantly higher than for BRO-negative (P < 0.05) and BRO-2-positive (P < 0.01)
8
R. Saito et al. / J Infect Chemother 20 (2014) 6e8
Table 2 Distribution of serum resistance and BRO b-lactamases in M. catarrhalis isolates. Bactericidal assay
BRO Negative
I or R
P Positive
(n ¼ 5)
(n ¼ 192)
3 (60%)
175 (91.1%)
BRO Negative
0.2367
P BRO-1
(n ¼ 5)
(n ¼ 182)
3 (60%)
169 (92.9%)
bro-positive BRO-1
0.1333
P BRO-2
(n ¼ 182)
(n ¼ 10)
169 (92.9%)
6 (60%)
0.0056
I, intermediate; R, resistant.
isolates. The difference in clarithromycin MIC is small, and we could not identify the reason for this difference here; however, we are the first to demonstrate reduced susceptibility to macrolides among BRO-1-positive versus BRO-negative and BRO-2-positive isolates. Serum resistance is a well-known virulence factor for M. catarrhalis. In previous studies, the frequency of serum-resistant isolates is higher in patients with illnesses such as pulmonary infections than in healthy individuals [8,12]. Although Bootsma et al. [13] examined the frequency of serum non-sensitive (intermediate and resistant) isolates and the distribution of BRO b-lactamase types, their relationship has been unclear due to a lack of data. In this study, our observations have demonstrated that serum nonsensitive isolates were mostly BRO-1-positive (P ¼ 0.0056). However, there was no significant difference in the frequency of serum non-sensitive isolates among BRO-positive versus -negative isolates (P ¼ 0.2367) and BRO-1-positive versus BRO-negative isolates (P ¼ 0.1333). A significant difference may have been found if a larger number of isolates had been examined. In summary, our results provide the first evidence that the presence of BRO-1 in M. catarrhalis is associated with reduced susceptibility to clarithromycin and b-lactam antibiotics, as well as serum non-sensitive (intermediate and resistant) in comparison to BRO-negative or BRO-2-positive isolates. In the future, since BRO-1 positive isolates with serum resistance may confer resistance to various antibiotics besides b-lactams and macrolides, we plan to continue our study of the relationship between antibiotic susceptibility, BRO b-lactamases, and virulence factors. Acknowledgements We thank Dr. H. Qiu and Dr. T. Chida for excellent technical assistance. The study did not receive financial support from third parties. All authors declare that they have no conflicts of interest.
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