Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Antimicrobial resistance and production of beta-lactamases in Bulgarian clinical isolatesMoraxella catarrhalis

Antimicrobial resistance and production of beta-lactamases in Bulgarian clinical... Annals of Microbiology, 59 (1) 169-172 (2009) Antimicrobial resistance and production of beta-lactamases in Bulgarian clinical isolates Moraxella catarrhalis Raina GERGOVA*, Rumyana MARKOVSKA, Ivan MITOV Department of Microbiology, Medical University, Zdrave 2 str., 1431 Sofia, Bulgaria Received 14 July 2008 / Accepted 3 December 2008 Abstract - One hundred and fifteen strains of Moraxella catarrhalis from inpatients and healthy children were collected from 2000 to 2005. MICs to 17 antimicrobial agents from different groups were determined. High resistance rate to penicillin G, aminopenicillins and first generation cephalosporins (98.26%) was found. Susceptibility to cefuroxime, cefotaxime and ceftriaxone was from 86.08 to 97.40%. All M. catarrhalis strains were susceptible to aminopenicillin/inhibitor combinations, ceftazidime, ceftibuten, ciprofloxacin and gentamicin. The susceptibility rates to azithromycin, clarithromycin, tetracycline and trimethoprim/sulfamethoxazole were reduced in range of 6-9%. Presence of bro1 or bro2 genes was revealed in 113 M. catarrhalis strains by PCR. The overall prevalence of the BRO-1 and BRO-2 enzymes was 92.92 and 7.08% respectively. MICs of Penicillin were significantly higher in bro1 positive isolates. Key words: Moraxella catarrhalis; beta-lactamases; bro; PCR. Moraxella catarrhalis is an important causative agent of otorhi- The investigated M. catarrhalis strains (n = 115) were iso- nolaryngological and bronchopulmonary infections in children lated from patients with community-acquired respiratory tract and, particularly, in adults with acute exacerbations of chronic infections and from healthy children between 2000 and 2005. obstructive pulmonary disease (COPD) (Catlin, 1990; Verduin The identification of the isolates was confirmed by Crystal NH et al., 2002; Kais et al., 2006; Taylor et al., 2006; Sethi et al., (BBL). Nitrocefin disk assay (Cefinase disks; BBL) was used to 2007). Many authors have observed the increasing rate of anti- detect beta-lactamase activity. Minimal Inhibitory Concentrations microbial resistance of M. catarrhalis strains, compromising the (MICs) of antimicrobial agents were determined by the E-test empirical choice of the most commonly used antibacterial agents (AB Biodisk, Solna, Sweden) according to CLSI Standards (Brook, 2007). The first M. catarrhalis strain resistant towards Guidelines (National Committee for Clinical Laboratory, 2006). aminopenicillins due to production of beta-lactamases appeared Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC in 1976, and followed by a dramatic increase the resistance of 25922 were used as controls in this procedure. The derived rates M. catarrhalis strains worldwide (Doern et al., 1996; Koseoglu et of resistance were interpreted by using the CLSI 2006 break- al., 2004). Beta-lactamases of M. catarrhalis can be divided into point criteria for Haemophilus influenzae and Neisseria spp., as two closely related types, BRO-1 and BRO-2. Although substrate recommended by Doern et al. (1996) and Zhanel et al. (2003). hydrolysis rates of BRO-1 and BRO-2 are similar, BRO-1 produc- DNA extraction technique was performed according to the manu- ing isolates are more resistant to penicillins than BRO-2 strains facturer’s procedures for Gram-negative bacteria (GFX Genomic (Koseoglu et al., 2004). Blood DNA Purification Kit, Amersham Biosciences, Freiburg, The aim of this study was to investigate the rate of antimi- Germany). The following primers sequences for PCR experi- crobial resistance in 115 M. catarrhalis strains collected within 5 ments were used: left 5’-TTTGGATTGGGGTGAATGAT-3’ and right years in Sofia, Bulgaria, to detect and characterise their beta- 5’-TGGGGCTGGGTGATAAATAG-3’ (Levy and Walker, 2004). Allele lactamases and evaluate the correlation between susceptibility PCR amplification of the bro genes was performed as described patterns and resistance mechanisms. As far as we are aware of previously (Levy and Walker, 2004). DNA from M. catarrhalis this is the first report for the distribution of bro1 and bro2 genes CCUG 353, CIP 103772, CIP 103773, were used as bro-negative, in Bulgaria. bro-1 and bro-2 controls, respectively The determined MICs of 17 antimicrobial agents for 115 nonduplicate M. catarrhalis strains are shown in Table 1. Out of 115 collected M. catarrhalis, 113 were resistant towards * Corresponding Author. Phone: +359 2 9172750; penicillin due to beta-lactamase production (positive nitrocefin Fax: +359 2 9515317; E-mail: renigergova@mail.bg 170 R. GERGOVA et al. TABLE 1 - MICs of 17 antimicrobial agents to 115 strains Moraxella catarrhalis Antimicrobial agents MIC (mg/L) Interpretation Range 50% 90% S (%) I (%) R (%) Penicillin G 0.002 - > 32 16 32 1.74 - 98.26 Ampicillin < 0.016 - 24 4 8 1.74 12.17 86.08 Amoxicillin < 0.016 - 24 4 8 1.74 12.17 86.08 Ampicillin/Sulbactam < 0.016 - 1 0.125 0.125 100 - - Amoxicillin/Clav. acid < 0.016 - 2 0.25 0.25 100 - - Cephalexin 0.016 - 32 8 16 1.74 6.96 91.30 Cefuroxime sodium < 0.016 - 8 2 2 86.08 9.57 4.35 Cefotaxime < 0.016 - 2 0.125 0.25 97.4 2.6 - Ceftriaxone < 0.016 - 2 0.125 0.25 97.4 2.6 - Ceftazidime < 0.006 - 0,5 0.03 0.06 100 - - Ceftibuten < 0.006 - 0,5 0.03 0.125 100 - - Azithromycin < 0.002 - 8 0.03 0.06 93.92 3.48 2.6 Clarithromycin 0.002 - 16 0.06 0.125 93.92 2.6 3.48 Tetracycline 0.125 - 48 0.5 0.5 91.3 1.74 6.96 Ciprofloxacin < 0.016 - 0,5 0.06 0.06 100 - - Gentamicin 0.016 - 0,125 0.06 0.09 100 - - Trimethoprime/Sulfamethoxazole 0.005 - 4 0.25 0.5 90.44 6.96 2.6 CLSI 2006 breakpoint criteria for Haemophilus influenzae and Neisseria spp. disk assay). Beta-lactamases production of M. catarrhalis in our (Melo-Cristino et al., 2006). Many different results for this study (98.26%) is one of the highest in Europe (Schmitz et al., period have been reported (Richter et al., 2000; Jones et al., 2002). The frequency of beta-lactamase-producing Bulgarian 2003). In Estonia 12% resistance to macrolides and 24% to isolates M. catarrhalis is similar like in the United States (Richter trimethoprim/sulfamethoxazole were detected (Altraja et al., et al., 2000), Canada (Zhanel et al., 2003), Estonia (Altraja et 2006). Moraxella catarrhalis has natural resistance to trimetho- al., 2006), Portugal (Melo-Cristino et al., 2006), France and prim and the combination with sulfamethoxazole is bacterio- Greece (Jones et al., 2003) for the same period. The MICs of static and is not recommended for therapy of infections caused beta-lactamase producing M. catarrhalis strains to penicillins and by moraxellae (Catlin, 1990). Tetracycline resistance is usually first generation cephalosporins were four time higher than these coded by Tet B gene, a fragment of nontransferable plasmid of non-producing strains. The isolates susceptible to aminopeni- (Roberts et al., 1991). This may explain the low percentage of cillins and first generation cephalosporins were 1.74%. As it tetracycline resistant strains. was well known the beta-lactamase of M. catarrhalis was 100% In allele PCR 113 beta-lactamase positive strains showed inhibited by sulbactam and clavulanic acid (Kostova et al., 1998; two types bro genes bro1 (92.92%), corresponding with 235 Zhanel et al., 2003). Our results confirmed this fact and showed bp segment and bro2 (7.08%) with 214 bp (Fig. 1). Nitrocefin the possibility of using the combinations of aminopenicillin/beta- disk assay and bro allele PCR showed correspondence in 100%. lactamase inhibitors as empirical therapy of infections due to BRO-1 isolates showed significantly higher penicillin MICs com- moraxellae. The obtained MICs (Table1) showed, that sulbactam pared to BRO-2 isolates. Among patients with otorhinologycal inhibits more effectively in vitro beta-lactamases of M. catarrhalis infections (n = 41) M. catarrhalis strains produced BRO-1 in than clavulanic acid. The MICs of cefuroxime were lower than 92.68%, among these with bronchopulmonary infections (n = these of cephalexin, but some strains demonstrated MIC > 2 52) in 94.23%. In comparison the strains isolated from healthy mg/L in range of low resistance. Our first strain M. catarrhalis children (n = 22), produced BRO-1 in 81.81%. The presence of (from patient with COPD) with decreased sensitivity to third bro2 was associated predominantly with healty children. generation cephalosporins (MICs cefotaxime and ceftriaxone = Beta-lactamase production is chromosomal determined 2 mg/L) was detected in 2004. This strain also produced BRO-1. through bro genes. The 21-bp deletion in the BRO-2 promoter About 20% of the isolates showed MIC 1 mg/L of cefotaxime/ region has notable effect on promoter activity (Levy and Walker, ceftriaxone. For distinction other third generation cephalosporins 2004). This, as well as with mutations or deletions still unknown, such as ceftazidime and ceftibuten showed about tenfold higher might explain the variations in the antibiotic susceptibilities of activity to beta-lactamase positive M. catarrhalis. Their MICs the BRO-1 and BRO-2 producing clinical isolates (Richter et were less than 0.25 mg/L, in most of the cases between 0.032 al., 2000). These chromosomal bro genes could be transferred and 0.064 mg/L (Table1). by non-traditional conjugation from M. catarrhalis, Moraxella Moraxella catarrhalis resistance to other antibiotics still lacunata and Moraxella nonliquefaciens (Wallace et al., 1989), has poor medical significance. Nevertheless isolates with which explains the faster distribution of beta-lactamase positive decreased sensitivity to the other groups of antibiotics have isolates. been determined in the world (Schmitz et al., 2002; Jones et Beta-lactamases produced by moraxellae have indirect al., 2003; Zhanel et al., 2003). We have found resistance to pathogenic role due to their presence in a continuous-culture bio- tetracycline (8.7%) and macrolides (6.08%) higher than the film system with other respiratory pathogens and co-aggregates values in Portugal (1 and 0% respectively) and lower toward with them. It has already been demonstrated that M. catarrhalis trimethoprim/sulfamethoxazole (9.56%), (30% in Portugal) interacts with Streptococcus pneumoniae in this biofilm and that Ann. Microbiol., 59 (1), 169-172 (2009) 171 FIG. 1 - PCR products bro1 (235 bp) and bro2 (214 bp) in Moraxella catarrhalis clinical strains. M. catarrhalis modulates interactions of Streptococcus pyogenes children and those with pharyngo-tonsillitis J. Med. with human epithelial cells and probably increases the intensity Microbiol., 55: 989-992. of mucosal adhesion by respiratory pathogens (Lafontaine et al., Brook I. (2007). Overcoming penicillin failures in the treatment 2004). Despite of the significant etiological role of M. catarrhalis of Group A streptococcal pharyngo-tonsillitis. Int. J. Pediatr. alone in the infection, the strong extracellular beta-lactamase- Otorhi., 71 (10): 1501-8. producing microorganisms may compromise penicillin or cepha- Catlin B.W. (1990). Branhamella catarrhalis: an organism gaining losporin therapy of infection due to S. pneumoniae and S. respect as a pathogen. Clin. Microbiol. Rev., 3: 293-330. pyogenes, isolated in combination with M. catarrhalis (Bootsma, Doern G.V., Brueggemann A.B., Pierce G., Hogan T., Holley H.P., 2000; Lafontaine et al., 2004; Brook and Gober, 2006; Brook Rauch A. (1996). Prevalence of antimicrobial resistance 2007). among 723 outpatient clinical isolates of Moraxella catarrhalis In summary the results showed that nearly all clinical in the United States in 1994 and 1995: results of a 30-center isolates of M. catarrhalis in Bulgaria in the last years were national surveillance study. Antimicrob. Agents Chemother., beta-lactamase positive, predominantly BRO-1 producing. 40: 2884-2886. The data emphasize the importance of M. catarrhalis as an etiological agent spreading beta-lactamases that may inhibit Jones M.E., Blosser-Middleton R.S., Critchley I.A., Karlowsky J.A., some beta-lactams and lead to failure in treatment of mixed Thornsberry C., Sahm D.F. (2003). In vitro susceptibility of infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis: a European multicenter study during 2000-2001. Clin. Microbiol. Infect., 9 (7): 590-599. Acknowledgements Kais M., Spindler C., Kalin M., Ortqvist A., Giske C.G. This work is supported by the Medical University, Sofia, Grant (2006). Quantitative detection of Streptococcus pneumoniae, no. 200432. Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR. Diagn. Microbiol. Infect. Dis., 55 (3): 169-178. REFERENCES Koseoglu O., Ergin A., Hascelik G. (2004). Evalueation of restric- tion endonucleases analysis of BRO beta-lactamases in clini- Altraja A., Naaber P., Tamm E., Meriste S., Kullamaa A., Leesik cal and carrier isolates of Moraxella catarrhalis. Scand. J. Inf. H. (2006). Antimicrobial susceptibility of common pathogens Dis., 36 (4): 431-434. from community-acquired lower respiratory tract infections in Kostova I.P., Changov L.S., Keuleyan E.E., Gergova R.T., Manolov Estonia. J. Chemother., 18: 603-609. I.I. (1998). Synthesis, analysis and in vitro antibacterial Bootsma H.J., van Dijk H., Vauterin P., Verhoef J., Mooi F.R. activity of new metal complexes of sulbactam. Farmaco, 53 (2000). Genesis of BRO ß-lactamase-producing Moraxella (12): 737-740. catarrhalis: evidence for transformation-mediated horizontal Lafontaine E.R., Wall D., Vanlerberg S.L., Donabedian H., transfer. Mol. Microbiol., 36: 93-104. Sledjeski D.D. (2004). Moraxella catarrhalis coaggregates Brook I., Gober A.E. (2006). Increased recovery of Moraxella with Streptococcus pyogenes and modulates interactions of catarrhalis and Haemophilus influenzae in associa- S. pyogenes with human epithelial cells. Infect. Immun., 72 tion with group A ß-haemolytic streptococci in healthy (11): 6689-6693. 172 R. GERGOVA et al. Levy F., Walker E.S. (2004). BRO {beta}-lactamase alleles, anti- of BRO beta-lactamases and resistance to complement in biotic resistance and a test of the BRO-1 selective replace- European Moraxella catarrhalis isolates. J. Clin. Microbiol., 40 ment hypothesis in Moraxella catarrhalis. J. Antimicrob. (4): 1546-1548. Chemother., 53: 371-374. Sethi S., Sethi R., Eschberger K., Lobbins P., Cai X., Grant Melo-Cristino J., Santos L., Ramirez M., Grupo de Estudo B.J., Murphy T.F. (2007). Airway bacterial concentrations Portugues de Bacterias Patogenicas Respiratorias and exacerbations of chronic obstructive pulmonary disease. (2006). Viriato study: update of antimicrobial susceptibility Am. J. Respir. Crit. Care Med., 176 (4): 356-361. data of bacterial pathogens from community-acquired res- Taylor L., Corey M., Matlow A., Sweezey N.B., Ratjen F. piratory tract infections in Portugal in 2003 and 2004. Rev. (2006). Comparison of throat swabs and nasopharyngeal Port. Pneumol., 12 (1): 9-30. suction specimens in non-sputum-producing patients with National Committee for Clinical Laboratory Standards cystic fibrosis. Pediatr. Pulmonol., 41 (9):839-843. (2006). Methods for dilution antimicrobial susceptibility tests Verduin C.M., Hol C., Fleer A., van Dijk H., van Belkum A. (2002). for bacteria that grow aerobically. Approved Standard M7-A4. Moraxella catarrhalis: from emerging to established patho- National Committee for Clinical Laboratory Standards, 2006. gen. Clin. Microbiol. Rev., 15: 125-144. Richter S.S., Winokur P.L., Brueggemann A.B., Huynh H.K., Wallace R.J., Steingrube V.A., Nash D.R., Hollis D., Flanagan C., Rhomberg P.R., Wingert E.M. Doern G.V. (2000). Molecular Brown B.A., Labidi A., Weaver R. (1989). BRO ß-lactamases characterization of the ß-lactamases from clinical isolates of Branhamella catarrhalis and Moraxella subgenus Moraxella, of Moraxella (Branhamella) catarrhalis obtained from 24 including evidence for chromosomal ß-lactamase transfer by U.S. medical centers during 1994-1995 and 1997-1998. Antimicrob. Agents Chemother., 44: 444–446. conjugation in B. catarrhalis, M. nonliquefaciens, and M. lacu- nata. Antimicrob. Agents Chemother., 33: 1845-1854. Roberts M.C., Pang Y.J., Spencer R.C., Winstanley T.G., Brown B.A., Wallace R.J. (1991). Tetracycline resistance in Moraxella Zhanel G.G., Palatnick L., Nichol K.A., Low D.E., Hoban D.J. (Branhamella) catarrhalis: demonstration of two clonal out- (2003). Antimicrobial resistance in Haemophilus influenzae breaks by using pulsed-field gel electrophoresis. Antimicrob. and Moraxella catarrhalis respiratory tract isolates: results Agents Chemother., 35: 2453-2455. of the Canadian respiratory organism susceptibility study, Schmitz F.J., Beeck A., Perdikouli M., Boos S., Mayer S., Scheuring 1997 to 2002. Antimicrob. Agents Chemother., 47: 1875- K., Köhrer K., Verhoef J., Fluit A.C. (2002). Production 1881. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Antimicrobial resistance and production of beta-lactamases in Bulgarian clinical isolatesMoraxella catarrhalis

Download PDF
4 pages

Loading next page...
 
/lp/springer-journals/antimicrobial-resistance-and-production-of-beta-lactamases-in-0p2jN3Ca0W

References (26)

Publisher
Springer Journals
Copyright
Copyright © 2009 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/BF03175616
Publisher site
See Article on Publisher Site

Abstract

Annals of Microbiology, 59 (1) 169-172 (2009) Antimicrobial resistance and production of beta-lactamases in Bulgarian clinical isolates Moraxella catarrhalis Raina GERGOVA*, Rumyana MARKOVSKA, Ivan MITOV Department of Microbiology, Medical University, Zdrave 2 str., 1431 Sofia, Bulgaria Received 14 July 2008 / Accepted 3 December 2008 Abstract - One hundred and fifteen strains of Moraxella catarrhalis from inpatients and healthy children were collected from 2000 to 2005. MICs to 17 antimicrobial agents from different groups were determined. High resistance rate to penicillin G, aminopenicillins and first generation cephalosporins (98.26%) was found. Susceptibility to cefuroxime, cefotaxime and ceftriaxone was from 86.08 to 97.40%. All M. catarrhalis strains were susceptible to aminopenicillin/inhibitor combinations, ceftazidime, ceftibuten, ciprofloxacin and gentamicin. The susceptibility rates to azithromycin, clarithromycin, tetracycline and trimethoprim/sulfamethoxazole were reduced in range of 6-9%. Presence of bro1 or bro2 genes was revealed in 113 M. catarrhalis strains by PCR. The overall prevalence of the BRO-1 and BRO-2 enzymes was 92.92 and 7.08% respectively. MICs of Penicillin were significantly higher in bro1 positive isolates. Key words: Moraxella catarrhalis; beta-lactamases; bro; PCR. Moraxella catarrhalis is an important causative agent of otorhi- The investigated M. catarrhalis strains (n = 115) were iso- nolaryngological and bronchopulmonary infections in children lated from patients with community-acquired respiratory tract and, particularly, in adults with acute exacerbations of chronic infections and from healthy children between 2000 and 2005. obstructive pulmonary disease (COPD) (Catlin, 1990; Verduin The identification of the isolates was confirmed by Crystal NH et al., 2002; Kais et al., 2006; Taylor et al., 2006; Sethi et al., (BBL). Nitrocefin disk assay (Cefinase disks; BBL) was used to 2007). Many authors have observed the increasing rate of anti- detect beta-lactamase activity. Minimal Inhibitory Concentrations microbial resistance of M. catarrhalis strains, compromising the (MICs) of antimicrobial agents were determined by the E-test empirical choice of the most commonly used antibacterial agents (AB Biodisk, Solna, Sweden) according to CLSI Standards (Brook, 2007). The first M. catarrhalis strain resistant towards Guidelines (National Committee for Clinical Laboratory, 2006). aminopenicillins due to production of beta-lactamases appeared Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC in 1976, and followed by a dramatic increase the resistance of 25922 were used as controls in this procedure. The derived rates M. catarrhalis strains worldwide (Doern et al., 1996; Koseoglu et of resistance were interpreted by using the CLSI 2006 break- al., 2004). Beta-lactamases of M. catarrhalis can be divided into point criteria for Haemophilus influenzae and Neisseria spp., as two closely related types, BRO-1 and BRO-2. Although substrate recommended by Doern et al. (1996) and Zhanel et al. (2003). hydrolysis rates of BRO-1 and BRO-2 are similar, BRO-1 produc- DNA extraction technique was performed according to the manu- ing isolates are more resistant to penicillins than BRO-2 strains facturer’s procedures for Gram-negative bacteria (GFX Genomic (Koseoglu et al., 2004). Blood DNA Purification Kit, Amersham Biosciences, Freiburg, The aim of this study was to investigate the rate of antimi- Germany). The following primers sequences for PCR experi- crobial resistance in 115 M. catarrhalis strains collected within 5 ments were used: left 5’-TTTGGATTGGGGTGAATGAT-3’ and right years in Sofia, Bulgaria, to detect and characterise their beta- 5’-TGGGGCTGGGTGATAAATAG-3’ (Levy and Walker, 2004). Allele lactamases and evaluate the correlation between susceptibility PCR amplification of the bro genes was performed as described patterns and resistance mechanisms. As far as we are aware of previously (Levy and Walker, 2004). DNA from M. catarrhalis this is the first report for the distribution of bro1 and bro2 genes CCUG 353, CIP 103772, CIP 103773, were used as bro-negative, in Bulgaria. bro-1 and bro-2 controls, respectively The determined MICs of 17 antimicrobial agents for 115 nonduplicate M. catarrhalis strains are shown in Table 1. Out of 115 collected M. catarrhalis, 113 were resistant towards * Corresponding Author. Phone: +359 2 9172750; penicillin due to beta-lactamase production (positive nitrocefin Fax: +359 2 9515317; E-mail: renigergova@mail.bg 170 R. GERGOVA et al. TABLE 1 - MICs of 17 antimicrobial agents to 115 strains Moraxella catarrhalis Antimicrobial agents MIC (mg/L) Interpretation Range 50% 90% S (%) I (%) R (%) Penicillin G 0.002 - > 32 16 32 1.74 - 98.26 Ampicillin < 0.016 - 24 4 8 1.74 12.17 86.08 Amoxicillin < 0.016 - 24 4 8 1.74 12.17 86.08 Ampicillin/Sulbactam < 0.016 - 1 0.125 0.125 100 - - Amoxicillin/Clav. acid < 0.016 - 2 0.25 0.25 100 - - Cephalexin 0.016 - 32 8 16 1.74 6.96 91.30 Cefuroxime sodium < 0.016 - 8 2 2 86.08 9.57 4.35 Cefotaxime < 0.016 - 2 0.125 0.25 97.4 2.6 - Ceftriaxone < 0.016 - 2 0.125 0.25 97.4 2.6 - Ceftazidime < 0.006 - 0,5 0.03 0.06 100 - - Ceftibuten < 0.006 - 0,5 0.03 0.125 100 - - Azithromycin < 0.002 - 8 0.03 0.06 93.92 3.48 2.6 Clarithromycin 0.002 - 16 0.06 0.125 93.92 2.6 3.48 Tetracycline 0.125 - 48 0.5 0.5 91.3 1.74 6.96 Ciprofloxacin < 0.016 - 0,5 0.06 0.06 100 - - Gentamicin 0.016 - 0,125 0.06 0.09 100 - - Trimethoprime/Sulfamethoxazole 0.005 - 4 0.25 0.5 90.44 6.96 2.6 CLSI 2006 breakpoint criteria for Haemophilus influenzae and Neisseria spp. disk assay). Beta-lactamases production of M. catarrhalis in our (Melo-Cristino et al., 2006). Many different results for this study (98.26%) is one of the highest in Europe (Schmitz et al., period have been reported (Richter et al., 2000; Jones et al., 2002). The frequency of beta-lactamase-producing Bulgarian 2003). In Estonia 12% resistance to macrolides and 24% to isolates M. catarrhalis is similar like in the United States (Richter trimethoprim/sulfamethoxazole were detected (Altraja et al., et al., 2000), Canada (Zhanel et al., 2003), Estonia (Altraja et 2006). Moraxella catarrhalis has natural resistance to trimetho- al., 2006), Portugal (Melo-Cristino et al., 2006), France and prim and the combination with sulfamethoxazole is bacterio- Greece (Jones et al., 2003) for the same period. The MICs of static and is not recommended for therapy of infections caused beta-lactamase producing M. catarrhalis strains to penicillins and by moraxellae (Catlin, 1990). Tetracycline resistance is usually first generation cephalosporins were four time higher than these coded by Tet B gene, a fragment of nontransferable plasmid of non-producing strains. The isolates susceptible to aminopeni- (Roberts et al., 1991). This may explain the low percentage of cillins and first generation cephalosporins were 1.74%. As it tetracycline resistant strains. was well known the beta-lactamase of M. catarrhalis was 100% In allele PCR 113 beta-lactamase positive strains showed inhibited by sulbactam and clavulanic acid (Kostova et al., 1998; two types bro genes bro1 (92.92%), corresponding with 235 Zhanel et al., 2003). Our results confirmed this fact and showed bp segment and bro2 (7.08%) with 214 bp (Fig. 1). Nitrocefin the possibility of using the combinations of aminopenicillin/beta- disk assay and bro allele PCR showed correspondence in 100%. lactamase inhibitors as empirical therapy of infections due to BRO-1 isolates showed significantly higher penicillin MICs com- moraxellae. The obtained MICs (Table1) showed, that sulbactam pared to BRO-2 isolates. Among patients with otorhinologycal inhibits more effectively in vitro beta-lactamases of M. catarrhalis infections (n = 41) M. catarrhalis strains produced BRO-1 in than clavulanic acid. The MICs of cefuroxime were lower than 92.68%, among these with bronchopulmonary infections (n = these of cephalexin, but some strains demonstrated MIC > 2 52) in 94.23%. In comparison the strains isolated from healthy mg/L in range of low resistance. Our first strain M. catarrhalis children (n = 22), produced BRO-1 in 81.81%. The presence of (from patient with COPD) with decreased sensitivity to third bro2 was associated predominantly with healty children. generation cephalosporins (MICs cefotaxime and ceftriaxone = Beta-lactamase production is chromosomal determined 2 mg/L) was detected in 2004. This strain also produced BRO-1. through bro genes. The 21-bp deletion in the BRO-2 promoter About 20% of the isolates showed MIC 1 mg/L of cefotaxime/ region has notable effect on promoter activity (Levy and Walker, ceftriaxone. For distinction other third generation cephalosporins 2004). This, as well as with mutations or deletions still unknown, such as ceftazidime and ceftibuten showed about tenfold higher might explain the variations in the antibiotic susceptibilities of activity to beta-lactamase positive M. catarrhalis. Their MICs the BRO-1 and BRO-2 producing clinical isolates (Richter et were less than 0.25 mg/L, in most of the cases between 0.032 al., 2000). These chromosomal bro genes could be transferred and 0.064 mg/L (Table1). by non-traditional conjugation from M. catarrhalis, Moraxella Moraxella catarrhalis resistance to other antibiotics still lacunata and Moraxella nonliquefaciens (Wallace et al., 1989), has poor medical significance. Nevertheless isolates with which explains the faster distribution of beta-lactamase positive decreased sensitivity to the other groups of antibiotics have isolates. been determined in the world (Schmitz et al., 2002; Jones et Beta-lactamases produced by moraxellae have indirect al., 2003; Zhanel et al., 2003). We have found resistance to pathogenic role due to their presence in a continuous-culture bio- tetracycline (8.7%) and macrolides (6.08%) higher than the film system with other respiratory pathogens and co-aggregates values in Portugal (1 and 0% respectively) and lower toward with them. It has already been demonstrated that M. catarrhalis trimethoprim/sulfamethoxazole (9.56%), (30% in Portugal) interacts with Streptococcus pneumoniae in this biofilm and that Ann. Microbiol., 59 (1), 169-172 (2009) 171 FIG. 1 - PCR products bro1 (235 bp) and bro2 (214 bp) in Moraxella catarrhalis clinical strains. M. catarrhalis modulates interactions of Streptococcus pyogenes children and those with pharyngo-tonsillitis J. Med. with human epithelial cells and probably increases the intensity Microbiol., 55: 989-992. of mucosal adhesion by respiratory pathogens (Lafontaine et al., Brook I. (2007). Overcoming penicillin failures in the treatment 2004). Despite of the significant etiological role of M. catarrhalis of Group A streptococcal pharyngo-tonsillitis. Int. J. Pediatr. alone in the infection, the strong extracellular beta-lactamase- Otorhi., 71 (10): 1501-8. producing microorganisms may compromise penicillin or cepha- Catlin B.W. (1990). Branhamella catarrhalis: an organism gaining losporin therapy of infection due to S. pneumoniae and S. respect as a pathogen. Clin. Microbiol. Rev., 3: 293-330. pyogenes, isolated in combination with M. catarrhalis (Bootsma, Doern G.V., Brueggemann A.B., Pierce G., Hogan T., Holley H.P., 2000; Lafontaine et al., 2004; Brook and Gober, 2006; Brook Rauch A. (1996). Prevalence of antimicrobial resistance 2007). among 723 outpatient clinical isolates of Moraxella catarrhalis In summary the results showed that nearly all clinical in the United States in 1994 and 1995: results of a 30-center isolates of M. catarrhalis in Bulgaria in the last years were national surveillance study. Antimicrob. Agents Chemother., beta-lactamase positive, predominantly BRO-1 producing. 40: 2884-2886. The data emphasize the importance of M. catarrhalis as an etiological agent spreading beta-lactamases that may inhibit Jones M.E., Blosser-Middleton R.S., Critchley I.A., Karlowsky J.A., some beta-lactams and lead to failure in treatment of mixed Thornsberry C., Sahm D.F. (2003). In vitro susceptibility of infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis: a European multicenter study during 2000-2001. Clin. Microbiol. Infect., 9 (7): 590-599. Acknowledgements Kais M., Spindler C., Kalin M., Ortqvist A., Giske C.G. This work is supported by the Medical University, Sofia, Grant (2006). Quantitative detection of Streptococcus pneumoniae, no. 200432. Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR. Diagn. Microbiol. Infect. Dis., 55 (3): 169-178. REFERENCES Koseoglu O., Ergin A., Hascelik G. (2004). Evalueation of restric- tion endonucleases analysis of BRO beta-lactamases in clini- Altraja A., Naaber P., Tamm E., Meriste S., Kullamaa A., Leesik cal and carrier isolates of Moraxella catarrhalis. Scand. J. Inf. H. (2006). Antimicrobial susceptibility of common pathogens Dis., 36 (4): 431-434. from community-acquired lower respiratory tract infections in Kostova I.P., Changov L.S., Keuleyan E.E., Gergova R.T., Manolov Estonia. J. Chemother., 18: 603-609. I.I. (1998). Synthesis, analysis and in vitro antibacterial Bootsma H.J., van Dijk H., Vauterin P., Verhoef J., Mooi F.R. activity of new metal complexes of sulbactam. Farmaco, 53 (2000). Genesis of BRO ß-lactamase-producing Moraxella (12): 737-740. catarrhalis: evidence for transformation-mediated horizontal Lafontaine E.R., Wall D., Vanlerberg S.L., Donabedian H., transfer. Mol. Microbiol., 36: 93-104. Sledjeski D.D. (2004). Moraxella catarrhalis coaggregates Brook I., Gober A.E. (2006). Increased recovery of Moraxella with Streptococcus pyogenes and modulates interactions of catarrhalis and Haemophilus influenzae in associa- S. pyogenes with human epithelial cells. Infect. Immun., 72 tion with group A ß-haemolytic streptococci in healthy (11): 6689-6693. 172 R. GERGOVA et al. Levy F., Walker E.S. (2004). BRO {beta}-lactamase alleles, anti- of BRO beta-lactamases and resistance to complement in biotic resistance and a test of the BRO-1 selective replace- European Moraxella catarrhalis isolates. J. Clin. Microbiol., 40 ment hypothesis in Moraxella catarrhalis. J. Antimicrob. (4): 1546-1548. Chemother., 53: 371-374. Sethi S., Sethi R., Eschberger K., Lobbins P., Cai X., Grant Melo-Cristino J., Santos L., Ramirez M., Grupo de Estudo B.J., Murphy T.F. (2007). Airway bacterial concentrations Portugues de Bacterias Patogenicas Respiratorias and exacerbations of chronic obstructive pulmonary disease. (2006). Viriato study: update of antimicrobial susceptibility Am. J. Respir. Crit. Care Med., 176 (4): 356-361. data of bacterial pathogens from community-acquired res- Taylor L., Corey M., Matlow A., Sweezey N.B., Ratjen F. piratory tract infections in Portugal in 2003 and 2004. Rev. (2006). Comparison of throat swabs and nasopharyngeal Port. Pneumol., 12 (1): 9-30. suction specimens in non-sputum-producing patients with National Committee for Clinical Laboratory Standards cystic fibrosis. Pediatr. Pulmonol., 41 (9):839-843. (2006). Methods for dilution antimicrobial susceptibility tests Verduin C.M., Hol C., Fleer A., van Dijk H., van Belkum A. (2002). for bacteria that grow aerobically. Approved Standard M7-A4. Moraxella catarrhalis: from emerging to established patho- National Committee for Clinical Laboratory Standards, 2006. gen. Clin. Microbiol. Rev., 15: 125-144. Richter S.S., Winokur P.L., Brueggemann A.B., Huynh H.K., Wallace R.J., Steingrube V.A., Nash D.R., Hollis D., Flanagan C., Rhomberg P.R., Wingert E.M. Doern G.V. (2000). Molecular Brown B.A., Labidi A., Weaver R. (1989). BRO ß-lactamases characterization of the ß-lactamases from clinical isolates of Branhamella catarrhalis and Moraxella subgenus Moraxella, of Moraxella (Branhamella) catarrhalis obtained from 24 including evidence for chromosomal ß-lactamase transfer by U.S. medical centers during 1994-1995 and 1997-1998. Antimicrob. Agents Chemother., 44: 444–446. conjugation in B. catarrhalis, M. nonliquefaciens, and M. lacu- nata. Antimicrob. Agents Chemother., 33: 1845-1854. Roberts M.C., Pang Y.J., Spencer R.C., Winstanley T.G., Brown B.A., Wallace R.J. (1991). Tetracycline resistance in Moraxella Zhanel G.G., Palatnick L., Nichol K.A., Low D.E., Hoban D.J. (Branhamella) catarrhalis: demonstration of two clonal out- (2003). Antimicrobial resistance in Haemophilus influenzae breaks by using pulsed-field gel electrophoresis. Antimicrob. and Moraxella catarrhalis respiratory tract isolates: results Agents Chemother., 35: 2453-2455. of the Canadian respiratory organism susceptibility study, Schmitz F.J., Beeck A., Perdikouli M., Boos S., Mayer S., Scheuring 1997 to 2002. Antimicrob. Agents Chemother., 47: 1875- K., Köhrer K., Verhoef J., Fluit A.C. (2002). Production 1881.

Journal

Annals of Microbiology – Springer Journals

Published: Nov 24, 2009

There are no references for this article.