Antimicrobial Agents and Chemotherapy, November 2004, p . 4183-4188, Vol . 48, No . 11

Emergence and Persistence of Macrolide Resistance in Oropharyngeal Flora and Elimination of Nasal Carriage of Staphylococcus aureus after Therapy with Slow-Release Clarithromycin: a Randomized, Double-Blind, Placebo-Controlled Study

Hans F . Berg,^1* Jeroen H . T . Tjhie,² Gert-Jan Scheffer,³ Marcel F . Peeters,¹ Peter H . J . van Keulen,⁴ Jan A . J . W . Kluytmans,⁴ and Ellen E . Stobberingh⁵

Department of Clinical Microbiology, St . Elisabeth Hospital, Tilburg,¹ Department of Clinical Microbiology PAMM Laboratory, Veldhoven,² Department of Clinical Microbiology Amphia Hospital, Breda,⁴ Department of Clinical Microbiology University Medical Centre, Maastricht,⁵ Department of Anaesthesiology, University Medical Centre St . Radboud, Nijmegen, The Netherlands³

Received 30 November 2003/ Returned for modification 24 May 2004/ Accepted 25 July 2004

Since the discovery of erythromycin in 1952, macrolides were presented as alternatives to penicillins, especially for the treatment of infections due to gram-positive microorganisms (18) . Currently, they are recommended as a first-line therapy for adults with community-acquired pneumonia (3) . Unfortunately, resistance to macrolides among Streptococcus pneumoniae, the most common cause of respiratory tract infections, is increasing (6, 19) . There is little doubt that this is mainly the result of the increased use of macrolides .

A small number of papers on the effect of macrolides on the oropharyngeal flora have been published (1, 5, 7, 8, 9) . These studies compared the effects of two or more antimicrobial agents on the oral, gastric or intestinal flora . Most were concerned only with the changes in the microflora quantities . Moreover, none of the studies were randomized and placebo controlled to study the extent of these effects .

The present study was performed to quantify the effect of clarithromycin on the development of resistance in oropharyngeal and nasopharyngeal flora in individual patients . This study was part of an ongoing intervention study in cardiothoracic surgery to study the effects of clarithromycin on the presence of Chlamydia pneumoniae in cardiovascular tissue .

An independent pharmacist dispensed either clarithromycin or placebo tablets according to a computer-generated randomization table which stratified in groups of 10 . The researcher responsible for seeing the patients allocated the next available number on entry into the trial, and each patient collected the corresponding tablets directly from the researcher . The code was revealed to the researcher once recruitment, data collection, and laboratory analyses were complete .

From each patient a nose swab and two throat swabs were obtained on inclusion during the preoperative outpatient visit (visit 1), and on the day of surgery just before the operation a second nose swab and two throat swabs were obtained (visit 2) . A third throat swab was taken 8 weeks after surgery (visit 3) . These swabs were stored in transport medium (Transwab or a Venturi Transystem) at 4°C until further processing .

Microbiological analysis. The nasal swabs were cultured on a blood agar plate and incubated at 35°C for 48 h . At 24 and 48 h of incubation, plates were examined for the presence of Staphylococcus aureus . Putative colonies were identified by using standard identification methods (14) . S . aureus strains were deep-frozen in a bead storage system (Protect; Technical Service Consultants Ltd., Cheshire, England) at –80°C until further testing . Throat swabs were cultured on blood agar plates containing 5 µg of gentamicin per ml and on chocolate agar plates supplied with a bacitracin tablet (Neo-sensitabs; Rosco, Taastrup, Denmark) . These plates were incubated at 35°C in the presence of 5% CO₂ for 48 h . At 24 and 48 h, plates were examined for the presence of potential pathogenic microorganisms (e.g., S . pneumoniae, Haemophilus spp., Streptococcus pyogenes, and Moraxella catharralis) by standard identification methods (15) . All identified strains were frozen at –80°C until further testing .

Sensitivity analysis. Phenotypic resistance was measured by determining the MIC of clarithromycin by using the E-test (AB-Biodisk, Solna, Sweden) according to the manufacturer's guidelines . Haemophilus influenzae ATCC 49247 and S . aureus ATCC 29213 were included as controls . MIC determination was performed by reading the drug concentration at the point of complete inhibition of all growth, including microcolonies, hazes, and isolated colonies, as determined with a plate microscope . Breakpoints for sensitive versus resistant were determined according to NCCLS guidelines . (Haemophilus strains are sensitive when the MIC is 8 µg/ml, intermediate sensitive when the MIC is 16 µg/ml, and resistant when the MIC is 32 µg/ml) . The MIC analyses of strains before and after treatment were performed on the same day under the same conditions .

Indigenous flora. Culture and detection of resistance to erythromycin of the total indigenous oropharyngeal flora were performed for all three throat swabs . The swabs were suspended in 1 ml of 0.9% NaCl and inoculated onto blood agar plates containing 0 and 1 mg erythromycin per liter, using a spiral plater (Eddy Jet; IUL Instruments, I.K.S., Leerdam, The Netherlands) . After 18 h of incubation at 37°C, plates were examined for growth and colonies were identified by macroscopic examination, Gram staining, and, if catalase positive, a coagulase slide test . The proportion of macrolide-resistant flora among the total oropharyngeal flora was determined by establishing the number of gram-positive colonies on each plate and dividing the number of colonies on erythromycin-containing plates by the number of colonies on plates without erythromycin (x 100%) . One of each type of macroscopically different colony growing on the erythromycin-containing plates was additionally tested for erythromycin resistance by disk diffusion (Bauer-Kirby test [NCCLS M2-A6, 6th ed., January 1997]) . These strains that were determined to be resistant by disk diffusion were frozen at –80°C until further testing . Later, additional MIC testing was done by agar dilution with erythromycin . To study the mechanism of resistance, strains (viridans streptococci and coagulase-negative staphylococci) resistant to macrolides were studied for the presence of the following genes conferring resistance to macrolides by PCR as described previously by Sutcliffe et al . (24, 25) and Jensen et al . (10): the ermB (target modification by a ribosomal mutation) and mefAE (macrolide efflux pump mechanism) genes for streptococci and the ermA and ermC (target modification by a ribosomal mutation) and msr (macrolide efflux pump mechanism) genes for staphylococci . Some of these genes are known to occur on common mobile genetic elements such as transposons, which facilitate the spread of these resistance genes . The transposon family Tn916/Tn1545 is known to also harbor erm genes (23) and is common among streptococci . The presence of these genes on Tn916/Tn1545 was analyzed by PCR (20) .

This study was approved by the local medical ethics committee .

Statistical analysis. For statistical analysis, all baseline characteristics were analyzed by using the ² test for the distribution of categorical variables and the Student t test to compare continuous variables . To assess differences in MICs between the two treatment groups and between variables with different standard deviations, a comparison was made by using the Mann-Whitney U test . The related samples were compared by using a Wilcoxon signed rank test . To determine the correlation between the duration of therapy and the development of resistance, linear regression analysis was performed . Statistical significance was accepted when the P value was <0.05 .

 
Pathogens. Before treatment, Haemophilus parainfluenzae was the only potential pathogen found consistently in the throat and S . aureus was the only one found consistently in the nose (in approximately 80 and 35% of patients, respectively) . The prevalence of S . pneumoniae, S . pyogenes, and M . catharralis in throat cultures was low . S . pneumoniae was found in five patients before treatment and in five other patients after treatment . M . catharralis was found five times before treatment and in three other patients after treatment . S . pyogenes was found in two patients before treatment but in none after .

In Table 2, the effect of clarithromycin on oropharyngeal carriage of H . parainfluenzae and on nasal carriage of S . aureus is shown . Carriage of H . parainfluenzae was not significantly affected by clarithromycin therapy . Nasal carriage of S . aureus, however, was significantly reduced in the CL group (from 35.3 to 4.3%) compared to the PB group (from 32.4 to 30.3%) (P < 0.0001; relative risk [RR], 7.0; 95% confidence interval [CI], 3.1 to 16.0) .

 
In Table 3, the percentages of patients with clarithromycin-resistant H . parainfluenzae strains before and after therapy are shown for both groups . This shows a significant increase in resistance in the CL group and not in the PB group (P = 0.007; RR, 1.6; 95% CI, 1.1 to 2.3) .

 
Statistical analysis was not performed on MICs for S . aureus due to the small number of patients with S . aureus after treatment in the CL group .

Indigenous flora. Throat swabs were obtained from 219 patients on visits 1 and 2 . A third swab was obtained from 169 of these 219 patients . Clarithromycin did not reduce the number of CFU per milliliter or the total amount of oral flora significantly . The oral flora in the patients was predominantly streptococci and to a lesser extent coagulase-negative staphylococci . Fewer than 3% also had gram-negative rods, which were not consistently isolated in all three swabs .

Figure 2 shows the percentages of patients with macrolide-resistant gram-positive oropharyngeal flora for both the PB and CL groups at the three different time points . In the CL group the percentage of patients with macrolide-resistant flora in the first swab was identical to that in the placebo group (34%) . Directly after treatment in the CL group, there was a significant shift towards more patients with resistant oral flora (P < 0.0001; RR, 1.9; 95% CI, 1.4 to 2.5), which was more or less the same in the third throat swab (P = 0.003; RR, 1.6; 95% CI, 1.2 to 2.1) . The risk of developing resistance in a subgroup of patients without detectable resistance before therapy was started was significantly higher in the CL group than in the PB group (P < 0.0001; RR, 6.6; 95% CI, 3.2 to 13.5) .

 
Linear regression analysis showed that there was no significant relation between the number of tablets taken and the prevalence of resistance in the CL group .

Quantitative culture was performed to determine for each individual patient with resistant flora what percentage of the total oral flora was resistant . In the pretreatment situation (visit 1), the mean percentages of the total flora that were resistant in the CL and PB groups were 3.4 and 2.3%, respectively . Immediately after treatment (visit 2), the mean percentage of the total flora that was resistant rose to 21.1% in the CL group (versus 1.1% in the PB group; P < 0.0001) . This increase was still present 8 weeks later (11.4 versus 2 · 6%; P < 0.0001) .

Table 4 shows that resistance genes present before treatment were not significantly different between the treatment groups . However, after treatment a significant rise in ermB was seen in the streptococci (P < 0.0001; RR, 3.5; 95% CI, 2.0 to 6.1), as was a significant rise in ermC in staphylococci (P = 0.002; RR, 4.5; 95% CI, 1.6 to 12.8) in the CL group compared to the PB group . This difference was still present 8 weeks after treatment . More than 90% of the macrolide-resistant streptococci contained both macrolide resistance genes and Tn916/Tn1545 fragments .

 
This is the first double-blind, placebo-controlled, randomized study that quantifies the effect of an antibiotic on the development of resistance in the oropharyngeal flora . It shows that administration of a daily dose of 500 mg of slow-release clarithromycin for 2 weeks has major effects that are still present 8 weeks later .

Before patients were included in the study, macrolide resistance was already frequently present . About 35% of patients carried H . parainfluenzae strains that were clarithromycin resistant, and about 35% of the patients had some macrolide-resistant indigenous flora . Whether this rate of resistance is due to macrolide therapy in the more distant past (>3 months) or whether it is the normal rate in the oropharyngeal flora in this population is not clear .

Immediately after treatment, the percentage of patients with macrolide-resistant flora in the CL group more or less doubled; this was found for both macrolide-resistant H . parainfluenzae and for macrolide-resistant indigenous oropharyngeal flora . This effect on the indigenous flora was even greater if only the group of patients that did not carry macrolide-resistant flora was taken into consideration . The relative risk of developing macrolide resistance in the indigenous flora directly after treatment in this group was 6.6 (95% confidence interval, 3.2 to 13.5) .

Unexpectedly, this effect was more or less the same 8 weeks later . Other investigators found that emergence of resistance after clarithromycin therapy had disappeared after 6 weeks (12) . In that study, another group of patients was treated with azithromycin . In this group the resistance rate after 6 weeks was even higher than that after 1 week . The authors speculated that this difference is caused by the extremely long elimination half-life of azithromycin compared to clarithromycin, which leads to subinhibitory concentrations for several weeks . In our study clarithromycin was used in a slow-release form, with a prolonged elimination half-life for once-daily dosing (t_1/2 of 5.3 h and maximum concentration in serum of 1.3 µg/ml, compared to a t_1/2 of 2.7 h for the normal formula) . Although this half-life is not as long as that of azithromycin (up to 72 h), it is possible that the prolonged effect of SR clarithromycin on resistance is partly caused by this prolonged half-life . A big difference, however, is that we were studying patients with gram-positive resistant indigenous flora and H . parainfluenzae, whereas Kastner and Guggenbichler (12) were studying specific pathogens that are not considered to be part of the normal oropharyngeal flora, such as Enterobacter spp., Klebsiella spp., and Pseudomonas spp .

Since we anticipated that the effect of clarithromycin would have largely disappeared after 8 weeks, we did not plan a follow-up beyond that point . Considering our findings, a further follow-up would have been useful . Nevertheless, the conclusion that a significant and sustained effect on resistance is produced by SR clarithromycin for at least 8 weeks is justified .

Furthermore, the percentage of resistance in the total flora in each individual patient was quantified, and very low percentages of resistant flora could be detected with the method used . To check for the effect of the duration of treatment, an analysis that compared the duration of treatment with the percentage of resistant flora was performed . No association was found . This finding supports the hypothesis that development of resistance was caused mainly by selecting macrolide-resistant isolates, because they were already detected after a few tablets . This was also suggested by Leach et al., who concluded that the selective effect of macrolides allowed the growth and transmission of preexisting macrolide-resistant strains (17) . The favorable circumstances for macrolide-resistant flora are illustrated by the fact that more patients had high proportions of the total flora being resistant directly after therapy compared to the pretreatment situation . Although these high proportions decreased slightly after 8 weeks, the percentage of patients with detectable resistance was still as high as it was immediately after therapy . This is particularly worrying, as our results indicate that the resistance genes are associated with mobile genetic elements, which facilitate the spread of these genes to more pathogenic flora as well .

Another clear effect of clarithromycin was the effect on the distribution of the different resistance genes in the oropharyngeal streptococci and staphylococci . A significant rise of the ermB gene (which encodes target modification) was detected in streptococci, as was a significant rise of the ermC gene in staphylococci . Target modification due to methylation of 23S rRNA or ribosomal protein was the most frequently found resistance mechanism in the oropharyngeal flora after CL therapy, which was also found by other investigators (10, 24, 25) .

In conclusion, there was substantial development of resistance that was long-lasting . These findings deserve serious consideration when macrolides are used, especially when this is for indications other than infectious diseases . For example, erythromycin is known for the treatment of gastroparesis as a gastrointestinal prokinetic agent administered orally or intravenously (4) . Also, anti-inflammatory and immunomodulatory properties are mentioned in various pulmonary situations such as cystic fibrosis and diffuse panbronchiolitis (16) . Given the major effect on the development of resistance in the indigenous and pathogenic flora, one should reconsider prescribing macrolides and weigh these results against the evidence of the suggested gain .

Finally, nasal carriage of S . aureus was significantly decreased . In general, systemic antibiotic treatment affects the balance between commensal and pathogenic microflora, but very few antibiotics are capable of decreasing the carriage of S . aureus . A study by Yu et al., for example, showed a decrease in nasal carriage after administration of rifampin (26) . Our study showed that SR clarithromycin is capable of decreasing staphylococcal nasal carriage from 35.3 to 4.3%, which is comparable to the activity of mupirocin nasal ointment (11) . Eradication of S . aureus is important, because elimination of nasal carriage has been found to reduce infection rates and the severe consequences of infection in patients at risk (13) . However, because of the development of resistance, the use of macrolides for this indication is questionable .

In conclusion, our study documents that administration of 500 mg of SR clarithromycin results in a significant elimination of S . aureus nasal carriage but, on the other hand, induces a rapid and prolonged increase in macrolide resistance in oropharyngeal flora . The macrolide resistance genes are similar to those usually detected in pathogenic flora . This study provides indisputable evidence that treatment with clarithromycin results in a major effect on the sensitivity of the oropharyngeal flora to macrolides for at least 8 weeks .

Abbott Pharmaceuticals supported this study with an unrestricted educational grant . The sponsor played no role in study design, data collection, data analysis, data interpretation, writing of the report, or the decision to submit the report for publication .

1. Adamsson, I., C . E . Nord, P . Lundquist, S . Sjöstedt, and C . Edlund. 1999 . Comparative effects of omeprazole, amoxycillin plus metronidazole versus omeprazole, clarithromycin plus metronidazole on the oral, gastric and intestinal microflora in Helicobacter pylori infected patients . J . Antimicrob . Chemother . 44:629-640.
2. Armstrong, G . L., L . A . Conn, and R . W . Pinner. 1999 . Trends in infectious disease mortality in the United States during the 20th century . JAMA 281:61-66.
3. Bartlett, J . G., S . F . Dowell, A . Mandell, T . M . File, D . M . Musher, and M . J . Fine. 2000 . Practice guidelines for management of community-acquired pneumonia in adults . Clin . Infect . Dis . 31:347-382.
4. Bradley, C. 2001 . Erythromycin as a gastrointestinal prokinetic agent . Intensive Crit . Care Nurs . 17:117-119.
5. Brismar, B., C . Edlund, and C . E . Nord. 1991 . Comparative effects of clarithromycin and erythromycin on the normal intestinal microflora . Scand . J . Infect . Dis . 23:635-642.
6. Doern, G . V., M . A . Pfaller, K . Kugler, J . Freeman, and R . N . Jones. 1998 . Prevalence of antimicrobial resistance among respiratory isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY Antimicrobial Surveillance Program . Clin . Infect . Dis . 27:764-770.
7. Edlund, C., G . Alvan, L . Barkholt, F . Vacheron, and C . E . Nord. 2000 . Pharmacokinetics and comparative effects of telithromycin (HMR 3647) and clarithromycin on the oropharyngeal and intestinal microflora . J . Antimicrob . Chemother . 46:741-749.
8. Edlund, C., G . Beyer, M . Hiemer-Bau, S . Ziege, H . Lode, and C . E . Nord. 2000 . Comparative effects of moxifloxacin and clarithromycin on the normal intestinal microflora . Scand . J . Infect . Dis . 32:81-85.
9. Heimdahl, A., and C . E . Nord. 1979 . Effects of phenoxymethylpenicillin and clindamycin on the oral, throat and faecal microflora of man . Scand . J . Infect . Dis . 11:233-242.
10. Jensen, L . B., N . Frimodt-Moller, and F . M . Aarestrup. 1999 . Presence of erm gene classes in gram-positive bacteria of animal and human origin in Denmark . FEMS Microbiol . Lett . 170:151-158.
11. Kalmeijer, M . D., H . Coertjens, P . M . van Nieuwland-Bollen, D . Bogaers-Hofman, G . A . de Baere, A . Stuurman, A . van Belkum, and J . A . Kluytmans. 2002 . Surgical site infections in orthopedic surgery: the effect of mupirocin nasal ointment in a double-blind, randomized, placebo-controlled study . Clin . Infect . Dis . 35:353-358.
12. Kastner, U., and J . P . Guggenbichler. 2001 . Influence of macrolide antibiotics on promotion of resistance in the oral flora of children . Infection 29:251-256.
13. Kluytmans, J., A . van Belkum, and H . Verbrugh. 1997 . Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks . Clin . Microbiol . Rev . 10:505-520.
14. Koneman, E . W., S . D . Allen, W . M . Janda, P . C . Schreckenberger, and W . C . Winn, Jr. 1997 . Color atlas and textbook of diagnostic microbiology, 5th ed., p . 551-554 . Lippincott-Raven Publishers, Philadelphia, Pa.
15. Koneman, E . W., S . D . Allen, W . M . Janda, P . C . Schreckenberger, and W . C . Winn, Jr. 1997 . Color atlas and textbook of diagnostic microbiology, 5th ed., p . 378-379 . Lippincott-Raven Publishers, Philadelphia, Pa.
16. Labro, M . T., and H . Abdelghaffar. 2001 . Immunomodulation by macrolide antibiotics . J . Chemother . 13:3-8.
17. Leach, A . J., T . M . Shelby-James, M . Mayo, M . Gratten, A . C . Laming, B . J . Currie, and J . D . Mathews. 1997 . A prospective study of the impact of community-based azithromycin treatment of trachoma on carriage and resistance of Streptococcus pneumoniae . Clin . Infect . Dis . 24:356-362.
18. Leclercq, R. 2002 . Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications . Clin . Infect . Dis . 34:482-492.
19. Marston, B . J., J . F . Plouffe, T . M . File Jr., B . A . Hackman, S . J . Salstrom, H . B . Lipman, M . S . Kolczak, and R . F . Breiman. 1997 . Incidence of community-acquired pneumonia requiring hospitalization: results of a population-based active surveillance study in Ohio . Arch . Intern . Med . 157:1709-1718.
20. McDougal, L . K., F . C . Tenover, L . N . Lee, J . K . Rasheed, J . E . Patterson, J . H . Jorgensen, and D . J . LeBlanc. 1998 . Detection of Tn917-like sequences within a Tn916-like conjugative transposon (Tn3872) in erythromycin-resistant isolates of Streptococcus pneumoniae . Antimicrob . Agents Chemother . 42:2312-2318.
21. Neu, H . C. 1992 . The crisis in antibiotic resistance . Science 257:1064-1073.
22. Opal, S . M., K . H . Mayer, and A . A . Medeiros. 2000 . Mechanisms of bacterial antibiotic resistance: control of antibiotic resistance, p . 248 . In G . L . Mandell, J . E . Bennett, and R . Dolin (ed.), Mandell, Douglas, and Bennett's principles and practice of infectious diseases . 5th ed . Churchill Livingstone, Philadelphia, Pa.
23. Roberts, A . P., G . Cheah, D . Ready, J . Pratten, M . Wilson, and P . Mullany. 2001 . Transfer of Tn916-like elements in microcosm dental plaques . Antimicrob . Agents Chemother . 45:2943-2946.
24. Sutcliffe, J., A . Tait-Kamradt, and L . Wondrack. 1996 . Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system . Antimicrob . Agents Chemother . 40:817-824.
25. Sutcliffe, J., T . Grebe, A . Tait-Kamradt, and L . Wondrack. 1996 . Detection of erythromycin-resistant determinants by PCR . Antimicrob . Agents Chemother . 40:2562-2566.
26. Yu, V . L., A . Goetz, M . Wagener, P . B . Smith, J . D . Rihs, J . Hanchett, and J . J . Zuravleff. 1986 . Staphylococcus aureus nasal carriage and infection in patients on hemodialysis . Efficacy of antibiotic prophylaxis . N . Engl . J . Med . 315:91-96.

Free Online Full-text Article

What Is Bioremediation?, What Is Biotechnology?, What Is Botulism?, What Is MIC?, What Is Bioengineering?, i, Bacteriology, n, Microorganisms, a, Bacteria, s, Microbiology, i, Bacterium, i, S. cerevisiae, c, Pseudomonas aeruginosa, n, Streptomycin, o, Escherichia coli, a, Streptococci, i, Enterobacteriacea, r, S. cerevisiae, r, Streptococci, i, Yeasts, i, Prokaryotes, o, Bacillus subtilis, i, Streptococci, e, Microbial, o, Escherichia coli, n, Alcaligenes, o, Corynebacter, c, Microorganisms, n, Microorganism, n, Escherichia coli, a, Citrobacter, e, Citrobacter




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005