Background: Streptococcus group B (GBS) or Streptococcus agalactiae is typically associated with neonatal disease and infection in pregnant women. Mortality of GBS sepsis in neonates is over 50% and is particularly high in preterm infants. GBS also causes invasive infection in pregnant and non-pregnant women including urinary tract infection (UTI). Penicillin-derived antibiotics remained as choice drugs for treatment of GBS infection; however, Erythromycin and Clindamycin are useful in cases of allergic to Penicillin. The aim of this study was to investigate the resistance to Erythromycin and Clindamycin, especially inducible Clindamycin resistance, in GBS isolated from urinary samples of women who attended medical offices in Tehran, Iran.Materials and Methods: This study was conducted on 5000 urine samples from Jan. 2011 to Oct. 2012 that 104 GBS were isolated. The isolates were identified as GBS using laboratory criteria. Antimicrobial susceptibility test was done by Erythromycin disk 15µg and Clindamycin disk 2µg for observation inducible resistant D-zone test by double-disk diffusion method with Erythromycin and adjacent Clindamycin.Results: Among the 5000 urine samples 104 (2.08%) were Beta hemolytic GBS. Of the 104 isolated GBS, 22 (21.2%) were resistance, 24 (23%) were intermediate, and 58 (55.8%) were susceptible to Erythromycin; however, 24 (23%) were resistance, 5 (4.8%) were intermediate, and 75 (72.2%) were susceptible to Clindamycin. Of the 22 Erythromycin-resistant isolates, 10 (9.5% in total GBS isolated) displayed the D zone; it means they have inducible Erythromycin resistant to Clindamycin.Conclusion: Various studies in other countries report lower rates of inducible Clindamycin resistance; it indicates the use of more macrolides in the treatment of UTI

Ashrafi-Hafez, Asghar

Farahzadi, Hossein

Frouhesh-Tehrani, Homa

Sharifi, Zohreh

English

Background: Streptococcus group B (GBS) or Streptococcus agalactiae is typically associated with neonatal disease and infection in pregnant women. Mortality of GBS sepsis in neonates is over 50% and is particularly high in preterm infants. GBS also causes invasive infection in pregnant and non-pregnant women including urinary tract infection (UTI). Penicillin-derived antibiotics remained as choice drugs for treatment of GBS infection; however, Erythromycin and Clindamycin are useful in cases of allergic to Penicillin. The aim of this study was to investigate the resistance to Erythromycin and Clindamycin, especially inducible Clindamycin resistance, in GBS isolated from urinary samples of women who attended medical offices in Tehran, Iran.Materials and Methods: This study was conducted on 5000 urine samples from Jan. 2011 to Oct. 2012 that 104 GBS were isolated. The isolates were identified as GBS using laboratory criteria. Antimicrobial susceptibility test was done by Erythromycin disk 15µg and Clindamycin disk 2µg for observation inducible resistant D-zone test by double-disk diffusion method with Erythromycin and adjacent Clindamycin.Results: Among the 5000 urine samples 104 (2.08%) were Beta hemolytic GBS. Of the 104 isolated GBS, 22 (21.2%) were resistance, 24 (23%) were intermediate, and 58 (55.8%) were susceptible to Erythromycin; however, 24 (23%) were resistance, 5 (4.8%) were intermediate, and 75 (72.2%) were susceptible to Clindamycin. Of the 22 Erythromycin-resistant isolates, 10 (9.5% in total GBS isolated) displayed the D zone; it means they have inducible Erythromycin resistant to Clindamycin.Conclusion: Various studies in other countries report lower rates of inducible Clindamycin resistance; it indicates the use of more macrolides in the treatment of UTI.</p

Homa Frouhesh-Tehrani

Asghar Ashrafi-Hafez

Zohreh Sharifi

Hossein Farahzadi

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Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance in Streptococcus Group B Isolated from Urinary Samples of Outpatient Women in Tehran

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  Novelty in Biomedicine NBM 79   Novelty in Biomedicine 2015, 2, 79-83 Original Article   Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance in Streptococcus Group B Isolated from Urinary Samples of Outpatient Women in Tehran   Homa Frouhesh-Tehrani1, Asghar Ashrafi-Hafez2, Zohreh Sharifi3, Hossein Farahzadi4*  1 Noor Pathobiology Laboratory, Tehran, Iran 2 Cancer Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3 Kawsar Human Genetic Research Center, Tehran, Iran 4 Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Abstract Background: Streptococcus group B (GBS) or Streptococcus agalactiae is typically associated with neonatal disease and infection in pregnant women. Mortality of GBS sepsis in neonates is over 50% and is particularly high in preterm infants. GBS also causes invasive infection in pregnant and non-pregnant women including urinary tract infection (UTI). Penicillin-derived antibiotics remained as choice drugs for treatment of GBS infection; however, Erythromycin and Clindamycin are useful in cases of allergic to Penicillin. The aim of this study was to investigate the resistance to Erythromycin and Clindamycin, especially inducible Clindamycin resistance, in GBS isolated from urinary samples of women who attended medical offices in Tehran, Iran. Materials and Methods: This study was conducted on 5000 urine samples from Jan. 2011 to Oct. 2012 that 104 GBS were isolated. The isolates were identified as GBS using laboratory criteria. Antimicrobial susceptibility test was done by Erythromycin disk 15µg and Clindamycin disk 2µg for observation inducible resistant D-zone test by double-disk diffusion method with Erythromycin and adjacent Clindamycin. Results: Among the 5000 urine samples 104 (2.08%) were Beta hemolytic GBS. Of the 104 isolated GBS, 22 (21.2%) were resistance, 24 (23%) were intermediate, and 58 (55.8%) were susceptible to Erythromycin; however, 24 (23%) were resistance, 5 (4.8%) were intermediate, and 75 (72.2%) were susceptible to Clindamycin. Of the 22 Erythromycin-resistant isolates, 10 (9.5% in total GBS isolated) displayed the D zone; it means they have inducible Erythromycin resistant to Clindamycin. Conclusion: Various studies in other countries report lower rates of inducible Clindamycin resistance; it indicates the use of more macrolides in the treatment of UTI. Keywords: Streptococcus Group B, Inducible Resistance, Erythromycin, Clindamycin, Resistance, UTI    *Corresponding Author: Hossein Farahzadi, Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Email: farahzadimic@gmail.com  Please cite this article as: Frouhesh-Tehrani H, Ashrafi-Hafez A, Sharifi Z, Farahzadi H. Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance in Streptococcus Group B Isolated from Urinary Samples of Outpatient Women in Tehran. Novel Biomed. 2015;3(2):79-83.    Introduction Group B Streptococcus (GBS) or Streptococcus agalactiae can be cause of infection in neonates, pregnant women, and non-pregnant adults who those are elderly persons with chronic medical illness1,2. Frouhesh-Tehrani et al.    Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance … NBM                                                                            80                                   Novelty in Biomedicine 2015, 2, 79-83 These infections have frequently been seen since 1970s. According to recent studies GBS infections have a high rate in developed countries3,4. Group B β-hemolytic streptococcus infection is an important cause of early-onset neonatal morbidity and mortality5. Mortality of GBS sepsis in neonates is over 50% and is especially high in preterm infants6. GBS commonly colonizes the female genitourinary tract (10–40% of pregnant women), which is the usual source of early-onset neonatal infection5. Also Streptococcus agalactiae is a component of human intestinal and genitourinary microflora7. Transmission from a colonized pregnant woman to her neonate occurs via the ascending route during labor and delivery. Administration of intrapartum antimicrobial prophylaxis (IAP) to colonized women has resulted in a striking decline in cases of early-onset and maternal GBS disease1. GBS are usually responsible for sepsis and meningitis in the first weeks of life. In spite of regional variations, the incidence for GBS-related neonatal meningitis and sepsis is 0.5-3 per 1000 live births3.  GBS is identified as an infectious agent of invasive disease in non-pregnant adults especially those underlying conditions such as diabetes mellitus, malignancy, or liver disease4. The incidence of invasive GBS infection in non-pregnant adults has increased four-fold recently up to 4.1-7.2 per 100.0003. GBS also cause urinary tract infections (UTIs), which encompass asymptomatic bacteriuria, cystitis, pyelonephritis, urethritis, and urosepsis. GBS asymptomatic bacteriuria is particularly common among pregnant women; however, those most at risk for cystitis due to GBS are the elderly and immunocompromised individuals. Predisposing factors for GBS UTI may include diabetes mellitus and chronic renal failure1. Between 5% and 23% of nonpregnant adults with invasive GBS disease present with a urinary tract infection8. Clinical manifestations of GBS infection in adults are numerous and quite varied. Because group B streptococci may colonize skin and mucosal surfaces and may be isolated from infected sites along with other virulent organisms, their role in pathogenesis has often been questioned. However, studies of invasive GBS infection in which the organisms are isolated from normally sterile sites such as blood or CSF provide direct evidence that group B streptococci are the etiologic agents in many clinical syndromes8. Methods The study subjects were women patients who presenting to different medical offices and experienced clinical and microbiological assessments for urinary tract infection (UTI) because of symptoms indicating infection or as part of routine patient screening. In this study 5000 urine samples was examined to obtain Streptococcus agalactiae between January 2011 and October 2012 in microbiology laboratory of paramedical faculty of Shahid Beheshti University of Medical Sciences in Tehran. The isolates were identified as GBS, if they were: 1) Beta hemolytic catalase-negative, 2) gram-positive cocci resistant both to Trimethoprim Sulphamethoxazole (SXT) and Bacitracin disks on Mueller Hinton blood (MHB) agar medium, 3) positive hippurate hydrolysis test, and 4) positive CAMP test. Antimicrobial susceptibility test was done by Erythromycin disk 15µg and Clindamycin disk 2µg for observation inducible resistant D-zone test by double-disk diffusion method with Erythromycin and adjacent Clindamycin. Antimicrobial susceptibility test by Erythromycin disk 15µg and Clindamycin disk 2µg (from Rosco company) performed on Mueller–Hinton agar plates with 5% defibrinated sheep blood and incubated for 24 hours in 35˚C with 5% CO2 for all confirmed GBS isolates (104 samples), then interpreted according to the Clinical and Laboratory Standards Institute (CLSI) in 2011. Erythromycin-resistant, Clindamycin-susceptible isolates were appraised for inducible Clindamycin resistance by the D-zone test. For observation inducible resistant D-zone test were used double-disk diffusion method with Erythromycin and adjacent Clindamycin. According to standards of CLSI to detect inducible Clindamycin resistance isolates by disk diffusion method, the D-zone test was performed with a 12mm edge-to-edge spacing between Erythromycin and Clindamycin discs. Appearance of flattened configuration of the Clindamycin zone of inhibition adjacent to the Erythromycin disk shows a positive test. Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance …    Frouhesh-Tehrani et al. NBM 81 Novelty in Biomedicine 2015, 2, 79-83 Results Among the 5000 urine samples, 104 (2.08%) were detected as Group B β-hemolytic streptococcus. According to the CLSI recommendations about antimicrobial susceptibility and D zone tests, antimicrobial susceptibility test by Clindamycin and Erythromycin disks was performed for the 104 GBS isolated. 22 (21.2%) isolates were resistance, 24 (23%) isolates were intermediate and 58 (55.8%) isolates were susceptible to Erythromycin. 24 (23%) isolates were resistance, 5 (4.8%) isolates were intermediate and 75 (72.2%) isolates were susceptible to Clindamycin (Table 1). The double disk diffusion test for inducible Clindamycin resistance was performed on all isolates resistant to Erythromycin and susceptible to Clindamycin. Inducible Clindamycin resistance by Erythromycin was displayed by presenting the appearance of a “D” around Clindamycin disk on plate in 10 (9.5% in total GBS isolated) isolates, so these cases showed an positive D zone test (iMLSB phenotype ) (Figure 1). Also resistance to both Erythromycin and Clindamycin disks (cMLSB phenotype) (Figure 2) was indicated in 11 (10.5% in total GBS isolated) isolates and just one (1% in total GBS isolated) case showed a Erythromycin-resistant and Clindamycin-susceptible without D zone (M phenotype) (Figure 3) (Table 2). Discussion Penicillin and Ampicillin are the choice drugs for treatment of β-hemolytic streptococcal infections9. In patients with penicillin allergies or a lack of clinical response, alternative therapies, such as macrolides (e.g., Erythromycin), Lincosamides (e.g., Clindamycin) are often considered for treatment of infections. Group B streptococci are susceptible to Ampicillin, Penicillin, and Cefazolin, but may be resistant to Clindamycin and/or Erythromycin9-11. In a research in Tehran in 2010, among the 498 Group B streptococci isolates taken from adult women’s urine cultures, 24.2% and 16.8% isolates were resistance to Erythromycin and Clindamycin, respectively4.  Resistance frequencies currently range from 6% to 21% for Clindamycin and 12% to 29% for Erythromycin in the United States, while other countries report higher rates12. GBS colonization of the urinary tract in women most likely occurs by an ascending route from the vagina, where GBS can persist asymptomatically. Although in treatment of urinary tract infections by GBS is not used Erythromycin and Clindamycin routinely, whereas source of urinary tract infections can be GBS colonization in gastrointestinal or genital tracts, resistance to Erythromycin and Clindamycin will be important13.  Since the clinical significance of inducible Clindamycin resistance among all β-hemolytic streptococci is unclear, it may not be necessary to perform this induction test on all isolates that are Erythromycin resistant and Clindamycin susceptible; however, all isolates from invasive infections should be tested. When a Group B streptococcus is isolated from a pregnant woman with severe Penicillin allergy, Table 1: Result of antimicrobial susceptibility test by Clindamycin and Erythromycin disks.  Number of samples detected as Group B β-hemolytic streptococcus Erythromycin (%) Clindamycin (%) susceptible intermediate resistance susceptible intermediate resistance 104 samples 55.8% 23% 21.2% 72.2% 4.8% 23%  Table 2: Result of The double disk diffusion test for inducible Clindamycin resistance (phenotypes).  Number of samples detected as Group B β-hemolytic streptococcus iMLSB phenotype (%) cMLSB phenotype (%) M phenotype (%) 104 samples 9.5% 10.5% 1%  Frouhesh-Tehrani et al.    Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance … NBM                                                                            82                                   Novelty in Biomedicine 2015, 2, 79-83 Clindamycin and Erythromycin should be tested and reported9. Recognition of Macrolide–Lincosamide–Streptogramin B (MLSB) phenotype was identified by a disc diffusion method14. The constitutive MLSB (cMLSB) phenotype is associated with high resistance to Erythromycin and Clindamycin, in our study it obtained 10.5% in total GBS isolated. The inducible MLSB (iMLSB) phenotype is associated with resistance to Erythromycin and susceptibility to Clindamycin, with antagonism between the Erythromycin and Clindamycin discs (with positive D zone test), in our study it obtained 9.5% in total GBS isolated. The M phenotype is determined by resistance to Erythromycin, susceptibility to Clindamycin and no antagonism between the two discs (without D zone test), in our study it obtained 1% in total GBS isolated. Resistance to Erythromycin and inducible Clindamycin resistance show limitation of Clindamycin usage when there is a resistance to Erythromycin15. Various studies in other countries report different prevalence of inducible Clindamycin resistance, for example, in Turkey in 2003 of 156 isolates S. agalactiae collected, 28 isolates (18%) expressed the iMLS phenotype and 7 isolates (4.5%) expressed the cMLS phenotype10. in Canada in 2004, Among the 338 GBS isolates tested, 55 (17%) and 26 isolates (8%) were resistant to erythromycin and clindamycin, respectively. Of the 55 erythromycin-resistant isolates, 26 isolates (8%) had constitutive MLSB resistance and 22 isolates (6.5%) had an inducible MLSB resistance phenotype 16. between 1997 and 1999 in two Health Authority Areas in Móstoles and Granada, Spain of 221 Streptococcus agalactiae isolated, 185 isolates (83.7%) were susceptible to erythromycin and azithromycin and 191 isolates (86.4%) were susceptible to miocamycin and clindamycin and 23 isolates (10.4%) had a constitutive MLSB phenotype and 7 isolates (3.2%) had an inducible phenotype17. In Massachusetts in the USA between January 2002 and April 2003, of the 200 isolates of GBS, 44 isolates (22%) were resistant to erythromycin. 32 isolates (16%) were resistant to erythromycin but susceptible to clindamycin. Of these isolates, 21 isolates (10.5%) had increased clindamycin resistance upon induction with erythromycin as determined by the D test and There were 11 erythromycin-resistant, clindamycin-susceptible isolates which did not have inducible resistance to clindamycin in this test18. In Poland in 2010, among 169 GBS isolates detected, 27 isolates (16%) were resistant to erythromycin and 17(10%) resistant to clindamycin. 17 isolates (10%) isolates had a cMLSB phenotype and 7 isolates (4%) isolates had an iMLSB phenotype19. In July 2009, in a laboratory in Louisiana, USA, performed routine susceptibility testing of GBS isolates. Between 1 July 2009 and 31 December 2010, 544 GBS isolates were identified,   Figure 1. iMLSB phenotype (D Test Positive)    Figure 2. cMLSB phenotype    Figure 3. M phenotype  Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance …    Frouhesh-Tehrani et al. NBM 83 Novelty in Biomedicine 2015, 2, 79-83 with 283 (52%) and 178 (33%) demonstrating Erythromycin and Clindamycin resistance, respectively. The highest reported rate of inducible Clindamycin resistance was 11%20. In this way, prevalence of inducible Clindamycin resistance in S. agalactiae in other studies were 2% in New Zealand in 2004, 2% in Australasia in 2005, 2.4% in France in 2008 and 10.3% in Ireland in 201110,16-19.  Conclusion Various studies in other countries report lower rates of inducible Clindamycin resistance. Also rate of resistance to Erythromycin and Clindamycin in Group B Streptococcus isolates was relatively high in this research. These results are may be due to use of more macrolides in the treatment of UTI in our country.  References 1. Ulett GC1, Webb RI, Ulett KB, Cui X, Benjamin WH, Crowley M, et al. Group B Streptococcus (GBS) Urinary Tract Infection Involves Binding of GBS to Bladder Uroepithelium and Potent but GBS-Specific Induction of Interleukin 1a. J Infect Dis. 2010;201:866–70 2. Edwards MS, Baker CJ. Group B streptococcal infections in elderly adults. Clin Infect Dis. 2005;41:839–47. 3. Atalay A, Ölçü M, Perçin D. Antibiotic Susceptibilities and Serotyping of Clinical Streptococcus Agalactiae Isolates. Balkan Med J. 2011;28:362-5 4. Rahbar M, Hajia M, Mohammadzadeh M. Urinary Tract Infections Caused By Group B Streptococcus in Adult Women:  Survey of 11800 Urine Culture Results. Iranian Journal of Pathology. 2012;7(1):32–7 5. Clifford V, Heffernan HM, Grimwood K, Garland S. Variation in Erythromycin and Clindamycin resistance patterns between New Zealand and Australian group B streptococcus isolates. Aust N Z J Obstet Gynaecol. 2011;51(4):328-32   6. Heelan JS, Hasenbein ME, McAdam A J.  Resistance of Group B Streptococcus to Selected Antibiotics, Including Erythromycin and Clindamycin. J. Clin. Microbiol. 2004;42(3):1263–4 7. Schuchat A, Wenger JD. Epidemiology of group B streptococcal disease. Risk factors, prevention strategies and vaccine development. Epidemiol Rev. 1994;16:374-402. 8. Farley MM. Group B Streptococcal Disease in Nonpregnant Adults. Clin Infect Dis. 2001;33:556–61 9. Clinical and Laboratory Standards Institute. 2011. Performance standards for antimicrobial susceptibility testing, 21st informational supplement. CLSI document M100-S21. Clinical and Laboratory Standards Institute, Wayne, PA.  10. Acikgoz ZC, Almayanlar E, Gamberzade S, Gocer S. Macrolide Resistance Determinants of Invasive and Noninvasive Group B Streptococci in a Turkish Hospital. AAC. 2004;48(4):1410–2 11. Raney PM, Tenover FC, Carey RB, McGowan JE Jr, Patel JB. Investigation of inducible Clindamycin and telithromycin resistance in isolates of β-hemolytic streptococci. Diagnostic Microbiology and Infectious Disease. 2006;55:213–8 12. Borchardt SM, Foxman B, DeBusscher JH, Tallman PA, Manning SD, Marrs CF, et al. Frequency of antimicrobial resistance among invasive and colonizing Group B Streptococcal isolates. BMC Infectious Diseases. 2006;57(6) 13. Ulett KB, Ulett GC, Benjamin WH Jr, Zhuo F, Xiao M, Kong F, et al. Diversity of Group B Streptococcus Serotypes Causing Urinary Tract Infection in Adults. JCM. 2009;47(7):2055–60 14. Castor ML, Whitney CG, Como-Sabetti K, Facklam RR, Ferrieri P, Bartkus JM, et al. Antibiotic Resistance Patterns in Invasive Group B Streptococcal Isolates. Infec Dis in Obstetrics and Gynecology 2008. 15. Leclercq R. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin Infect Dis 2002; 34: 482– 492. 16. Desjardins D, Delgaty KL, Ramotar K, Seetaram C, Toye B. Prevalence and Mechanisms of Erythromycin Resistance in Group Aand Group B Streptococcus: Implications for Reporting Susceptibility Results. J. Clin. Microbiol. 2004;42(12):5620–3. 17. Aracil B, Alo´s JI, Minambres M, Oteo J, De La Rosa M, Gomez-Garces JL. Susceptibility of strains of Streptococcus agalactiae to macrolides and lincosamides, phenotype patterns and resistance genes. Clin Microbiol Infect. 2002;8:745–8. 18. Heelan JS, Hasenbein ME, McAdam A J. Resistance of Group B Streptococcus to Selected Antibiotics, Including Erythromycin and Clindamycin. J Clin Microbiol. 2004;42(3):1263–4 19. Brzychczy-Włoch M, Gosiewski T, Bodaszewska M, Pabian W, Bulanda M, Kochan P. 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Assessment of Clindamycin and Erythromycin Resistance, and Inducible Clindamycin Resistance in Streptococcus Group B Isolated from Urinary Samples of Outpatient Women in Tehran

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