|ORIGINAL RESEARCH REPORT
|Year : 2016 | Volume
| Issue : 3 | Page : 132-136
Antibiotic susceptibility/resistant gene profiles of Group B streptococci isolates from pregnant women in a tertiary institution in Nigeria
Charles J Elikwu1, Oyinlola O Oduyebo2, Rose I Anorlu3, Brigitte Konig4
1 Department of Medical Microbiology and Parasitology, Ben Carson School of Medicine, Babcock University, Ilisan-Remo, Ogun State; Department of Medical Microbiology and Parasitology, Lagos University Teaching Hospital, Lagos, Nigeria
2 Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, Lagos, Nigeria
3 Department of Obstetrics and Gynecology, College of Medicine, University of Lagos, Idi-Araba, Lagos, Nigeria
4 Institute of Medical Microbiology and Infection Epidemiology, University of Leipzig, Germany
|Date of Web Publication||4-Jul-2016|
Charles J Elikwu
Department of Medical Microbiology and Parasitology, Ben Carson School of Medicine, Babcock University, Ilisan Remo, Ogun State
Source of Support: None, Conflict of Interest: None
Background: Penicillin is recommended as the first-line agent for intrapartum antibiotic prophylaxis (IAP). Although Group B Streptococcus (GBS) strains are generally susceptible to penicillin with only occasional resistance, they show varying resistance to erythromycin, clindamycin, and tetracycline. Therefore, knowledge of the resistance profile of GBS in the local environment will be useful for administration of appropriate intrapartum antibiotics prophylaxis. Methodology: Rectovaginal swabs collected from pregnant women were cultured for GBS using conventional media. Kirby-Bauer disc diffusion method was performed according to the Clinical and Laboratory Standards Institute guidelines on GBS isolates to determine the antibiotic susceptibility patterns of the isolates. Inducible clindamycin resistance was detected by using the D-zone test. Resistance determinants genes were discerned with conventional polymerase chain reaction. Results: Carriage rates of GBS among pregnant women studied were 19.7%. GBS colonizing the pregnant mothers were uniformly susceptible (100%) to penicillin, vancomycin, and ceftriaxone. Only three (6.5%) of the isolates showed inducible clindamycin resistance. ermA gene was present in all three GBS isolates with inducible macrolide-lincosamide-streptogramin resistance. ermB was absent in all the strains tested. mefA/E gene was carried by two of the macrolide-clindamycin resistance isolates. tetM gene was carried by all isolates with tetracycline resistance phenotypes. Conclusion: In this study, all GBS isolates were susceptible to penicillin G, the recommended antibiotic for IAP. The presence of resistance to erythromycin and inducible resistance to clindamycin excludes the use of these agents as alternatives in cases of penicillin allergy. In this case, vancomycin is the drug of choice as recommended in the updated Centers for Disease Control guideline.
Keywords: Group B streptococci, inducible clindamycin resistance, pregnant women
|How to cite this article:|
Elikwu CJ, Oduyebo OO, Anorlu RI, Konig B. Antibiotic susceptibility/resistant gene profiles of Group B streptococci isolates from pregnant women in a tertiary institution in Nigeria. J Clin Sci 2016;13:132-6
|How to cite this URL:|
Elikwu CJ, Oduyebo OO, Anorlu RI, Konig B. Antibiotic susceptibility/resistant gene profiles of Group B streptococci isolates from pregnant women in a tertiary institution in Nigeria. J Clin Sci [serial online] 2016 [cited 2019 May 24];13:132-6. Available from: http://www.jcsjournal.org/text.asp?2016/13/3/132/185250
| Introduction|| |
Maternal intrapartum beta hemolytic Group B Streptococcus (GBS) colonization is the primary risk factor for early-onset disease in infants.  In the absence of any intervention, approximately 2% of infants born to colonized mothers develop early-onset GBS infections. ,,
To reduce the incidence of neonatal disease caused by beta hemolytic GBS, the Centers for Disease Control (CDC) and Prevention recommends the use of intrapartum antibiotic prophylaxis (IAP) in pregnant women who are vagino-rectal carriers of GBS. , Penicillin is recommended as the first-line agent for IAP, while ampicillin is considered as an acceptable alternative. , In penicillin-allergic women, who are not at high risk for anaphylaxis, cefazolin is considered the agent of choice for intrapartum chemoprophylaxis because of its narrow spectrum of activity and ability to achieve high intra-amniotic concentrations.  In pregnant women at high risk for penicillin anaphylaxis, clindamycin is recommended if the organism is susceptible, and vancomycin is recommended if there is clindamycin resistance or if susceptibility is unknown. ,,
Although GBS strains are generally susceptible to penicillin with only occasional resistance, they show varying resistance rates to erythromycin, clindamycin, and tetracycline. , The most commonly encountered macrolide resistance mechanisms among streptococci are ribosomal modification by a methylase encoded by an erm gene  and drug efflux by a membrane-bound protein encoded by a mef gene.  The presence of the erm methylase confers resistance to erythromycin and inducible or constitutive resistance to lincosamides and streptogramin B (macrolide-lincosamide-streptogramin B phenotype), whereas the presence of the mef pump confers resistance only to 14- and 15-membered macrolides (M phenotype). A second efflux mechanism, encoded by the mreA gene, has been described for GBS. 
In view of the aforementioned, a local knowledge of the resistance profile of GBS will be useful for administration of appropriate antibiotics for prophylaxis. This study aimed to evaluate the antibiotic susceptibility profiles of GBS isolates from a tertiary healthcare institution in Lagos, Southwest Nigeria; and characterize antibiotic resistance genes of GBS isolates to advise the local community on GBS empiric therapy. This will inform the development of rational interventions and antibiotic guidelines in GBS infections.
| Methodology|| |
Study area and population
Participants for this study were recruited from the antenatal clinic of the Lagos University Teaching Hospital (LUTH), a tertiary care facility located in South West Nigeria. Molecular studies were performed at the Institute of Medical Microbiology and Infection Epidemiology, University of Leipzig, Germany.
This was a cross-sectional study conducted at the LUTH, Lagos from December 2010 to October 2011. A pair of vagino-rectal swab were collected from all consenting mothers at gestation age 35-37 weeks presenting at the antenatal clinic. GBS was detected in the maternal specimens by culture and confirmed by polymerase chain reaction (PCR). GBS isolates were tested for antibiotic susceptibility using the disk diffusion method. Phenotypical and molecular antibiotic resistance testing were conducted for isolates that showed resistance for erythromycin and clindamycin (as either of these represents a suitable alternative in case of penicillin allergy) and tetracycline for epidemiological reasons.
Pregnant women at gestational age <35 weeks or those who had had received antibiotics within 2 weeks before presenting at the antenatal clinic were excluded from the study. The Health Research and Ethics Committee of the LUTH, Lagos, Nigeria, approved this study and every participant gave written informed consent before enrollment.
Culture of specimens
Vagino-rectal swabs were collected from 300 pregnant women and were inoculated into Todd-Hewitt broth supplemented with 15 μg/ml nalidixic acid and 10 μg/ml colistin  (Biomerieux, Germany). After overnight incubation at 37°C, broths were sub-cultured onto Columbia blood agar plates with 5% sheep blood (Oxoid, United Kingdom) while the solid media were incubated at 37°C in ambient air for 24-48 h.
Identification of isolates
All isolates under investigation were presumptively identified as GBS if they were beta hemolytic, Gram-positive cocci, catalase negative, and showed positive Christie, Atkins and Munch-Peterson test (CAMP). PCR targeting the GBS species-specific gene, cfb was done for definitive identification. Total DNA extraction from overnight GBS colonies performed using the DN easy blood and tissue kits (Qiagen, Germany) according to the manufacturer's instructions. PCR was performed on GBS DNA extracts using GBS-specific primers ((Sag59) 5'-TTCACCAGCTGTATTAGAAGTA-3' and reverse (Sag190): 5'-GTTCCCTGAACATTATCTTTGAT-3') defined as previously described.  Amplicons were analyzed by electrophoresis in 1.5% agarose gel (Sigma, USA) stained with ethidium bromide using 1 × TAE buffer (buffer solution containing a mixture of Tris base, acetic acid and EDTA) (40 mM Tris-acetate, 1 mM ethylenediaminetetraacetic acid (EDTA); pH 8.0) at 70 V for 60 min. The DNA molecular weight marker (100 bp) (Invitrogen, Germany), was run concurrently to serve as reference for defining the PCR products. Gels were visualized under UV trans-illumination (Biometra, Germany) and the results were transmitted to a computer display system (Biometra, Germany) and stored.
Antibiotic susceptibility testing
All GBS isolates were subjected to antibiotic susceptibility using the disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI)  using the modified Kirby-Bauer method. The antimicrobial agents tested includes penicillin G (10U), clindamycin (2 μg), erythromycin (15 μg), vancomycin (30 μg), ciprofloxacin (5 μg), ceftriaxone (30 μg), tetracycline (30 μg) and co-trimoxazole (25 μg) (Oxoid, UK). The diameter of the zone of inhibition was measured in millimeters using a meter rule and interpreted according to CLSI guidelines with reference to tables of interpretative criteria. Streptococcus agalactiae control strains Ia (2008232728), Ib (2008232729), III (2008232582) and V (2008232731) from CDC were used as control strains. The results were described by frequency number and percentage.
Phenotypic and molecular antibiotic resistance testing were conducted for isolates that showed resistance to erythromycin, clindamycin, and tetracycline.
Phenotypic antibiotic resistance testing
Inducible clindamycin resistance phenotype was performed on all GBS isolates with erythromycin resistance using the D-zone disk diffusion method on Mueller-Hinton agar supplemented with 5% defibrinated sheep blood (Oxoid, UK) using erythromycin and clindamycin disks as recommended by the CLSI.  Isolates with blunting of the inhibition zone around the clindamycin disk adjacent to the erythromycin disk (D-zone positive) were considered to have inducible clindamycin resistance , and were presumed to be resistant. Susceptibility to clindamycin with no blunting defined the M phenotype (efflux mechanism).  The CDC S. agalactiae serotype III (2008232582) was used as a control strain.
Genotypic antibiotic resistance testing
Antibiotic resistance genes of isolates resistant to erythromycin, clindamycin, and tetracycline, was determined. PCR was performed on GBS resistant isolates using primers erm A 1/2, erm B 1/2, and mef AE ½ (BioteZ, Berlin, Germany) for the erm (A), erm (B) and mef (A) genes respectively. The sequences of the primer sets and PCR conditions , were as previously described. All isolates that expressed phenotypic resistance to tetracycline were also tested for the presence of the tet (M) and tet (O) tetracycline resistance determinants  using tet M 1/2 and tet O 1/2 primers respectively (BioteZ, Berlin, Germany). Amplicons were analyzed by electrophoresis in 1.5% agarose gel (Sigma, USA) stained with ethidium bromide using 1 × TAE (40 mM Tris-acetate, 1 mM EDTA; pH 8.0) as stated previously.
| Results|| |
Fifty-nine (19.7%) out of 300 pregnant women were enrolled for the study showed GBS-vaginal colonization. All 46 available GBS isolates colonizing the pregnant mothers were uniformly susceptible (100%) to penicillin, vancomycin, and ceftriaxone while 44 (95.7%) and 41 (89.1%) were resistant to tetracycline and trimethoprim-sulfamethoxazole, respectively [Table 1]. Only three (6.5%) of the isolates showed erythromycin resistance while another three were resistant to clindamycin with a lone intermediate susceptibility. All the three isolates resistant to erythromycin were positive for inducible clindamycin resistance. Erythromycin resistance gene ermA was found in the three erythromycin-clindamycin inducible resistance and absent in the only one strain with intermediate susceptibility [Figure 1]. However, erythromycin resistance gene ermB was absent in all the four strains tested. The mefA/E gene was carried by two of the macrolide-clindamycin resistance isolates [Figure 2]. The tetM gene was carried by all isolates that showed tetracycline resistance phenotypes [Figure 3] while tetO gene was completely absent.
|Figure 1: Gel electrophoresis identification of GBS erm (A) resistant gene|
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|Figure 2: Gel electrophoresis identification of GBS mef (A/E) resistant gene|
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| Discussion|| |
GBS is the leading cause of early-onset neonatal sepsis in most countries of the world. Universal screening is recommended for pregnant women at 35-37 weeks' gestation.  Therefore, we conducted antibiotic susceptibility profiles of GBS isolates with the primary objective of evaluating the antibiogram among GBS isolates from Nigerian pregnant women. In this study, we found that all GBS isolates remain susceptible to penicillin G, the recommended antibiotic for IAP.  However, a study from Obafemi Awolowo University Teaching Hospital, Ile-Ife Nigeria in the same geopolitical zone of our country reported uniform resistance of all GBS isolates to penicillin, ampicillin and clindamycin. 
Though this study recorded a low rate of resistance (6.5%) to erythromycin and another 6.5% to clindamycin, a number of literature has showed that the proportions of GBS isolates with in vitro resistance to clindamycin or erythromycin, have increased over the past two decades. ,,, At this juncture, a limitation of this study is highlighted: A total of 46 GBS isolates analyzed was low compared to 801 isolates by Milledge et al.  whose study stretched over a 5 years period. Even at that, an upward antibiotic resistance trend is predicted given the high rates of antibiotic misuse and abuse in Nigeria.  Resistance to erythromycin is associated frequently but not always with resistance to clindamycin.  The CDC guideline on prevention of perinatal GBS disease recommends that erythromycin is no longer acceptable for empiric prophylaxis because of increasing rates of resistance. , In such a case clindamycin (900 mg intravenously every 8 h until delivery) is the drug of choice if the GBS isolate is susceptible to clindamycin and erythromycin, and if there is no inducible clindamycin resistance. Vancomycin (1 g intravenously every 12 h until delivery) is recommended if testing shows resistance or inducible resistance to clindamycin. If erythromycin and clindamycin susceptibility tests were not performed, or if results are not available at the time of labor, vancomycin should be used in women at high risk of anaphylaxis.  The resistance mechanisms of GBS isolates to erythromycin in this study was accounted for by the presence of the genes that expresses erythromycin methylases and macrolide efflux in the resistant isolates.
Our study provides valuable information about current knowledge of antibiogram of GBS isolates among pregnant women in Nigeria. It is however limited by the small sample size or rather a small size of GBS isolates and the conduct of the study in a lone institution. Nevertheless, the antibiotics situation is similar across the country as Nigeria currently has no policy and guidelines regulating antibiotics in place; we believe that the results of our study were a true reflection of the situation GBS antibiogram among pregnant women in Nigeria.
Financial support and sponsorship
A short-term research grant from DAAD (German Academic Exchange Program).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]