Antibiotic resistance and serotypes of invasive streptococcus pneumoniae Isolated from Patients attending Acute care Health facilities in Kaduna state, Nigeria.

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Lusa H.Y
Department of Medical Microbiology School of Medical Laboratory Sciences AhmaduBello University Teaching Hospital, Zaria.
Olonitola O.S, Ameh J.B., Ado S.A
Department of Microbiology, Faculty of Life Sciences, Ahmadu Bello Univesity, Zaria.
Olayinka A.T
Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Zaria.
All Correspondences to: Lusa H.Y Department of Medical Microbiology School of Medical Laboratory Sciences
AhmaduBello University Teaching Hospital, Zaria. harunalusayadock@gmail.com

ABSTRACT
Background: Bacterial infections that are difficult or impossible to treat are becoming increasingly common and are causing a global health crises due to antibiotic resistance ,while reports have indicated that pneumococcal infection is an important public health concern throughout the world with global burden in causing disease and death comparable to that of Human immunodeficiency virus(HIV), Malaria and Tuberculosis(TB). The Penicillin and Macrolides belong to the groups of most commonly used, clinically important antibiotics for treatments of infections caused by Gram-positive bacteria such as S.aureus, S.pneumoniae and S. pyogenes.Some bacterial pathogens have innate resistance against antibiotics and this typically reflects variations in the structure of their cell envelope.Polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. They also showed there is a link between serotype and growth in-vitro.This study was carried out to determine pneumococcal serotypes and resistance to selected antibiotics among patients with symptoms of respiratory tract infections (RTIs) attending Acute-care health facilities in Kaduna state, Nigeria. Methods: Sputa was collected from patients with suspected cases of RTIs. Culture and sensitivity was conducted, and pneumococci were serotyped. Results: A total of 450 Subjects were enrolled for the study. 207(46.0%) yielded pathogens of which 41(9.1%) were Streptococcus pneumoniae.Pneumococcal isolates were commonly resistant to Cefuroxime 76.2%, Ampicillin 64.4%, erythromycin 45.2 % and Azithromycin 40.5%. Four different pneumococcal serotypes were detected in the study area. Conclusion: This study shows that the pattern of pneumococcal resistance against Penicillin and Erythromycin discourages the common practice of doing sensitivity on one antibiotic and another is used for treatment due to availability or cost implication. The Pneumococcal isolates have shown resistance not only to penicillin and erythromycin but also to alternative antibiotics like Azithromycin, cefuroxime and ceftriaxone. The most common serotypes were 3 and 13.

Key words: Resistance, Sensitivity, Minimum inhibitory concentration (MIC), E-test, serotype Acute-Care Health care facility, Erythromycin, Penicillin, Antibiotics, Pneumococci, RTI, HIV, and TB.

INTRODUCTION

Streptococcus pneumoniae is the most common cause of bacterialacute respiratory infections(ARI)and Pneumococcal resistance is the principal cause of concern regarding treatment failures for ARI (Schrang et al.,2001).Pneumococcus is also the most common cause of community acquired pneumonia (CAP), sporadic bacterial meningitis and bacteremia worldwide. In 2005, WHO estimated that 1.6 million people die of pneumococcal disease every year(Bandettini and Melioli,2012). Nigeria is currently one of the highest pneumococcal disease burdened countries and accounts for 5% of the global burden (Iwalokun et al., 2012; Iliyasu et al., 2014).Thoseexposed to or residing in risk areas such as Acute-care health facilities are at higher risk for invasive pneumococcal disease and death than those residing in communities (Kusteret al.,2014).An association has been established between the type of polysaccharide capsule surrounding the bacteria and harmless colonization versus invasive disease.Antibiotic resistant bacteria that are difficult or impossible to treat are becoming increasingly common and are causing a global health crisis (Abla et al., 2014).Drug resistant infections could kill an extra10 million people across the world every year,if by 2050 they are not tackled they could cost the world around $100 trillion in cost output (AMR Review, 2014).Drug resistance trends are not well documented in most developing countries due to limited laboratory capacity and acute respiratory infections (ARI) are often treated empirically with antibiotics (Schrang et al., 2001). The global emergence of in-vitro antimicrobial resistance and dissemination of penicillin and macrolide resistant Streptococcus pneumoniae strains has become a serious clinical concernworld wide (Mohammad et al., 2012; Alba et al., 2014). Pneumococcal infection is an important public health concern throughout the world with global burden in causing disease and death comparable to that of Human immune-deficiency virus (HIV), Malaria and Tuberculosis (TB) (Iwalokun et al., 2012).Ninety-three capsular serotypes have been identified in Streptococcus pneumoniae, each differing in chemical structure and immunogenicity (Calix and Nahm, 2010).

MATERIALS AND METHODS

Study Area: Kaduna state is located at the Centre of Northern Nigeria, in the Northwest geopolitical zone. The state lies between latitude 90 and 1400 N of the equator with a time of 1 hour ahead of the GMT. The state shares boundaries with Niger state to the west, Zamfara, Katsina and Kano states to the North, Bauchi and Plateau to the east and FCT Abuja and Nasarawa state to the south. It has a population of 6.63 million based on the 2006 census projections. The state is divided into three senatorial zones: Northern, Central, and Southern zones.Kaduna state has 739 Primary health facilities, 29 secondary care facilities and five tertiary hospitals (Abubakar et al., 2013).Kaduna state is administratively divided into 23 local government areas (LGAs) with several districts and wards in each LGA.

Ethical Consideration and Confidentiality Ethical approval was obtained from the Ethical committees of Kaduna State Ministry ofHealth and the targeted facility.Confidentiality was maintained throughout the test analysis.

Sample Collection Sputum samples from 450 patients with suspected cases of RTIs were collected in wide mouth leak-proof containers.Samples were then transported to the laboratory as soon as possible.( CDC; Tests online, 2014).Patients consent was also sort before recruiting them for the study. Purulent part of the sputum samples were inoculated on plates of 5% Blood agar and incubated immediately in presence of 5% CO2 overnight at 37oC(Mohammed, 2013;Iliyasu et al.,2015). Bacterial isolates were identified based on morphological characters, Gram reaction, catalase test, bile solubility, susceptibility to Optochin and hemolysis (Iliyasu et al., 2015).Suspected S.pneumoniae colonies were translucent or mucoid, with raised or having draughtsman appearance and 1-2mm in diameter showing alpha-hemolysis.Microscopic examination was carried out on suspected pneumococcal colonies. The smear was Gram stained. All slides were examined under the oil immersion lens. S.pneumoniaeappear gram-positive, elongated (lanceolate) diplococcus, or forms short chains (Bacteriology-test, 2014). Appearance of gas bubble will indicated positive catalase test (Bacteriology-test, 2014).Evidence of clearing of turbidity in the tube marked test compared with the saline control was considered positive while suspension remaining turbid in both tubes was considered negative solubility to bile (WHO/CDC, 2003). A chemical-impregnated disk containing Optochin was aseptically placed in the center of an inoculated Mueller- Hinton agar surface. The inoculated plate was incubated at 37?C for 24 hours Growth of the lawn up to the margin of the disk indicates the bacterium is resistant to ethylhydrocupreine hydrochloride (optochin). An obvious clear zone around the disk (=14mm in diameter) indicates susceptibility (WHO/CDC, 2003).The susceptibility of S. pneumoniae was determined by the disk diffusion (oxoid, Thermo Fisher, Scientific, Australia) following CLSI guidelines (Wayne, 2006). Breaking points for non-susceptibility and resistance was used to indicate susceptibility of the pneumococci to the antibiotics including:Ampicillin (10µg), erythromycin (15µg), penicillin (10U), Cefuroxime (10µg), Azithromycin (10µg), Ceftriaxone (10µg), Chloramphenicol (30µg) and Tetracycline (30µg).MICs of strains displaying resistance to erythromycin and penicillin from the disk diffusion method were determined by E-test methodology (LiofilChem Diagnostics, Italy). The E-test strips were: Erythromycin (0.016-256µg/mL) and Penicillin G (0.016-256µg/mL). Resistance against the antibiotics wereinterpreted according to MIC test Strip Technical Sheet Streptococcus pneumoniae (MIC Interpretation Guide, 2015). E- Strips(Liofilchem) were applied to an inoculated Mueller-Hinton agar plate E- test and incubated at 37?C for 24 hrs in the presence of 5% CO2. Readings on the scale were considered as the MIC (in µg/mL). Serotyping (PCR Strategy)was carried out. Genomic DNA was extracted from pneumococcal cells of a fresh overnight culture.Amplicons from PCR were analyzed with 1.5% agarose gel electrophoresis (Marcus et al., 2012).The amplicons with expected cps band sizes were purified using the PCR purification kit (Qiagen) according to the manufacturer’s instructions.Sequences were analyzed and viewed with Bionumerics soft ware version5 (Marcus et al.,2012).The amplicon nucleotide sequences were used to interrogate the GenBank data base,and if the highest BLAST bit score gave the serotype.Identification was then considered as a ”match”. Where the query results did not correlate or were not consistent with conventional serotyping methods, the strain/serotype was considered as ”misidentified” (Marcus et al., 2012).

Statistical Analysis The data collected were analyzed using tables and Chi-square tests .Results were considered as significant if the ?2 p-value was ? 0.05 and non-significant if p-value was > 0.05.All statistical analysis were done using IBM/SPSS Software (v20.0).

RESULTS
A total of 450 sputum samples were cultured and isolates included: Streptococcus viridians ‘Group (SVG) 56(27.1%), Staphylococcus aureus 52(25.1%), Streptococcus pneumoniae 41(19.8%), Klebsiella pneumoniae 30(14.5%), Streptococcus pyogenes 20(9.7%), Haemophilus influenzae 5(2.4%), Enterobacter aerogenes 2(1.0%) and Moraxella catarrhalis 1(0.5%).Statistical analysis showed a significant association (P?0.05) with respect to frequency of isolates and geographical distribution as in table 1.
Enterobacter aerogenes 2(1.0%) and Moraxella catarrhalis 1(0.5%).Statistical analysis showed a significant association (P?0.05) with respect to frequency of isolates and geographical distribution as in table 1.

DISCUSSION
Streptococcus pneumoniae is both an aggressive pathogen and a normal part of the human microbiome (Blaschke, 2016).Viridian’s Streptococci bacteria are generally disregarded as the etiologic agent of pneumonia even when isolated from cultures of sterile material.This study show that viridians streptococci should not be ruled out as a cause of pneumonia as reported by Freitas et al. (2006).The high pneumococcal prevalence in this study was likely due to predisposing factors such as; Prior exposure to risk areas such as health care settings and immuno compromised patients or patients with other medical comorbidities (Kusteret al.,2014). Penicillin has been the drug of choice worldwide for the treatment of pneumococcal infections. However penicillin resistant strains have emerged resulting in a shift to the use of other drugs e.g. cefotaxime, chloramphenicol and erythromycin (Dankor et al., 2010).The Pneumococcal isolates have shown resistance to penicillin, erythromycin and other alternative antibiotics as reported byIliyasu in a study carried out in Kano (Iliyasuet al.,2014).Investigations from some parts of Nigeria and other countries have shown high prevalence of resistance to Penicillin and other agents. Penicillin resistant pneumococci (PRP) and Erythromycin resistant pneumococci (ERP) had prevalence of 53.7% and 46.7% respectively, in this study .The prevalence of PRP closely agrees with findings by Akpede et al. (1994) but way below findings’ by Habib et al. (2003), and Kadankai et al. (2009) who recorded prevalence of 85.4% and 93.0% respectively, however rates in this work were higher than that reported by some researchers (Emele, 2000; Oduyebo et al., 2006; Abla et al., 2014) likely due to repeated course of therapy with ß-lactams or macrolide antibiotics (Jorget al.,2016).Erythromycin and other macrolides used in this study as alternatives to ß-lactams for treatment of Respiratory tract infections(RTI) had high resistance rates as compared to works of other researchers (Abla et al., 2014; David et al., 2015), but lower than that recorded by Mohammad et al.,2012. Erythromycin resistance in some Countries were found to be higher than those recorded in this study (Song et al., 2004; Reinert, 2005; Carmargos, 2006; Jenkings, 2008).This difference might be related to the shift in use of other antibiotics in these areas as reported by Dankor et al.(2010). Higherprevalence of Penicillin resistant pneumococci (PRP) and Erythromycin resistant pneumococci (ERP) was obtained among the elderly of age (40-60) years which is in agreement with findings by Nicholas et al. (2013), the reason probably, they are immunocompromised. Year group 20-29 had higher prevalence for penicillin resistance, which is similar to findings by Mohammed, (2013) probably due to nature of exposure to more predisposing factors such as drug abuse, HIV and TB that could lower the immunity. This finding agrees with that reported by Nicholas et al. (2013) in a study carried out in Ghana. Pneumococci are normally exquisitely sensitive to penicillin, which is lethal at concentrations <0.1 µg/mL. In this study high-level (MIC =2µg/ml) PRP and ERP has been associated with bacterial pneumonia which is similar to findings by Mellay et al., (2003);Fukushima et al.,(2008) and Abla et al.,(2014).WHO in 2007 recommended the use of pneumococcal conjugate vaccines (PCV) in all countries, urging that the highest priority for introduction be given to countries with high pneumonia and under-5 mortality.Nigeria only joined other African countries to launch pneumococcal conjugate vaccine (PCV10) as part of its Routine immunization schedule on 22nd December, 2014(WHO, 2015).Accurate serotyping is essential for epidemiologic study of S.pneumoniae [CDC, (nd).The serotypes Sequenced and identified in this study were serotypes 3, 13, 20 and 23. Serotype 3 is included in the 10-valent Pneumococcal Conjugate Vaccine (PCV), the 23F serotype is included in the heptavalent conjugate vaccine. The finding is not in complete agreement with findings of Onyemelukwe and Greenwood,1998 who identified serotypes 1,2 3, and 5 in Zaria and Abla et al., 2013 in Algeria, probably because their subjects comprises both children and adults. Serotypes 13 and 20 are non-vaccine types. Serotypes 3 and 23 are also among the dominant serotypes as reported by WHO, 2007 .The findings are also comparable to that obtained by Tarrago et al., 2008; Dankor et al., 2014 but completely differs from that reported by Falade et al., 2009.

CONCLUSION
The highest pneumococcal resistance to penicillin and erythromycin was obtained among patients’ =50 years of age. A prevalence rate of 23.0% pneumococcal infection was established in this study.The study shows that the isolates did not only show resistance to penicillin but also to other alternative antibiotics such as Erythromycin, Azithromycin, Tetracycline, and cefuroxime and should be used carefully in the future. The indiscriminate use of antibiotics at the community level might be probable cause of resistance obtained in this work. The use of penicillin as the best choice for treatment of infections with S. pneumoniae may need reconsideration because of the emerging trends in the antibiotic resistance. It is also pertinent to note that the resistance pattern shown by Penicillin and Erythromycin discourages a common practice in some of our Health care institutions where sensitivity is done on one antibiotic and another is used for treatment due to availability or cost implication. The implication of this study is a high potential for treatment failure with most antibacterial drugs recommended to empirically treat lower respiratory tract infections. Higher prevalence of Streptococcus viridians Group (SVG) recorded in this study than Streptococcus pneumoniae implies that the diagnosis of pneumococci can be problematic and if not chanced occurrence; only the use of specific gene targets may solve the problem of inaccurate identification of pneumococci. Serotype 3 and 13 are the most prevalent serotypes causing disease in this study. Screening for pneumococcal vaccination status should be authorized in patients age 60yrs or older. Events that reduce selective pressures deriving antimicrobial resistance must be encouraged such as: More appropriate use of antimicrobials, Vaccination must be enforced (PCV and PPSV) .

ACKNOWLEDGEMENT
The authors sincerely thank Ahmadu Bello University Teaching Hospital, Zaria and all the staff of department of Medical Microbiology, ABUTH, Zaria for their support.

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