Background: Children with sickle cell disease have been shown to be at greater risk of bacterial infections from Streptococcus pneumoniae, Hemophilus influenzae and Salmonella enteritidis and from complications of septicaemia, pneumonia, respiratory difficulty and bone pain. The study compared the nasopharyngeal bacterial carriage of 87 children with sickle cell disease (SCD) and 160 children without SCD, ranging in age from four months to fifteen years. The study, conducted at the Wesley Guild Hospital, Ilesa in south- western Nigeria between January and November 2014, also looked at the antibiotic resistance profiles of the bacterial isolates and the presence of specific resistance and virulence genes.

Methods: Samples were collected from each study participant with the aid of a cotton-tipped applicator initially dipped into sterile saline and introduced into nasopharynx of subject, applied onto sterile thioglycolate fluid medium and incubated at 37o C for 24 hr. When growth was noticed, a loopful was retrieved and applied onto blood and chocolate agar as well as other selective and differential media. Bacterial colonies that grew on such media were picked and studied initially by Gram reaction, cultural and biochemical methods. Antibiotic susceptibility testing was done for selected bacterial isolatesalong with PCR demonstration of resistance and virulence genes.

Results: The results showed that the 1-5 and the 5-10 year SCD childrenhad the highest frequency of bone pain episode, hospitalization for malaria, bacterial infections and sepsis and blood transfusions for chronic anemia. Corynebacterium spp predominated among the nasopharyngeal isolates from both SCD and non-SCD children. C. Xerosis accounted for 55.38% among the non- SCD isolates and C. Ulcerans represented 35.84%of the SCD isolates.We recorded low carriage rates for H. influenzae, S. Pneumonia and S.aureus in the nasopharynx of the study participants. Widespread antibiotic resistance was observed among SCD and non-SCD isolates, with demonstration of resistance blaZ and tetK resistance genes and sea, and eta virulence genes by PCR.

Conclusion: The predominance in this study of Corynebacterium spp over organisms such as H. influenzae,S. Pneumonia and S. aureus that are common invasive pathogens of the nasopharynxwas both interesting and unexpected. The predominance suggested a shift in the community nasopharyngeal flora that may have resulted from long-term prophylactic use of antibiotics and successful administration of bacterial vaccines in the study population.

Ako-Nai KA, Uzochukwu CC, Ebhodaghe BI, Kuti PB, Adegoke SA (2015) A Comparative Study of Bacterial Isolates Cultured From the Nasopharynx of Children With and Without Sickle Cell Disease at a Tertiary Healthcare Institution in Nigeria. Ann Pediatr Child Health 3(6): 1074.

•    Nasopharyngeal bacterial carriage
•    Sickle cell disease
•    Healthy control
•    MAR and virulence and resistance genes

Studies have shown that infants are more susceptible to infection because of their relatively immature immune system [1,2]. The nasopharynx of neonates is colonized early in life by bacterial organisms and with increasing frequency and diversity [3]. These organisms are initially acquiredat birth perinatally and postnatally from their mothers and other handlers [4]. The resulting nasopharyngeal flora often includes potential pathogens such as Streptococcus pneumoniae, Hemophilusinfluenzae and Neisseria meningitidis which maycause pneumonia and meningitis [3,4]. The threeorganisms are also a major cause of childhood morbidity and mortality in young children.A study by the World Health Organization (WHO) reported that pneumococcal disease is responsible for an average of 454,000 deaths annually among children <5 years of age [5]. But the introduction of viable vaccines such as the PCV 7 and 13 and the Hib conjugate vaccines has significantly reduced the development of early childhood diseases in developed and developing countries [6-8].

Sickle cell disease is a major hereditary disease with a high prevalence rate in sub-Saharan Africawhere more than 230,000 children are born annually with sickle cell disease [9-11]. An estimated 150,000 children are also born with sickle cell trait (HbAS) annually in Nigeria, with one in four Nigerians carrying the trait. On the other hand, only one in 12 African Americans carries the trait. Although over 700 structural haemoglobin (Hb) variants have been identified worldwide, only HbS and HbC predominate in Africa [12]. Sickle cell disease(SCD) predisposes the host to a number of complications that include septicaemia from infections, chronic anemia, leg ulcers, spleen atrophy and chronic bone pain as well as immune dysregulation [13].We therefore designed our study to determine and compare the pattern of nasopharyngeal bacterial carriage between a cohort of SCD children and children without sickle cell disease who attended the Child Health Clinic of the Wesley Guild Hospital at Ilesa in south-western Nigeria between January and November 2014. The study also compared the antibiotic susceptibility profiles of the dominant bacterial isolates from the two cohorts. Information on haemoglobin genotypes and severity of disease among the SCD children was obtained from medical records and questionnaire interviews of patients and their parents. We also characterized the antibiotic resistanceand virulence genes of thenasopharyngeal isolates of Staphylococcus aureus from the two study cohorts. It is expected that the results obtained from this study would clarify differences in the nasopharyngeal floras of SCD children and non-SCD children as well as suggest improvement in clinical management of associated bacterial infections.

Study center and inclusion criteria

The study was conducted at the Wesley Guild Hospital at Ilesa in south western Nigeria. The hospital is a satellite center of the Obafemi Awolowo University Hospitals Complex at IleIfe, Nigeria. Ilesa is a town of about 277,904 inhabitants and is located at approximately 25 miles from Ile-Ife.Approval for the study was obtained from the Human Subject and Ethical Review Committees of the hospital. Two study cohorts comprising of children with SCD and those without SCD were selectedfrom among young patients who attended the Child Health Clinic of the Ilesa hospital. While participation in the study was voluntary, subjects were recruited after careful explanation of the aims of the study and obtaining informed consent from the parents or guardians.

A total of 87 SCD children and 160 non-SCD children were enrolled in the study. They ranged in age from 4 months to 15 years. Seventy eight (92.85%) of the SCD children were genotyped as HbSS and six (7.24%) as HbSC. The SCD group included 45 (54%) males and 39 (46%) females. Thenon-SCD children included 77 (48%) males and 83 (52%) females. Forty two (42) of the non-SCD children were of HbAS genotype. Information relating to severity of disease in SCD children such as the number of pain episodes, hospitalizations and blood transfusions was extracted from patient medical records.

Sample collection and culture isolate identification

Nasopharyngeal culture samples were obtained from the study subjects by an attending physician using a sterile cotton-tipped applicator that was initially dipped in sterile saline. Samples wereinoculated into duplicate sterile thioglycollate fluid mediaand separately incubated at 37o C aerobically and in anaerobic jarsfor 48hrs.For biochemical characteristics and identification, aloopful of all cultures was streaked onto blood agar (BA), chocolate agar (CA), mannitol salt agar (MSA), eosin methylene blue agar (EMB), sulfide indole motility agar (SIM), Simmon citrate agar and triple sugar iron agar (TSI) (Oxoid LTD, Basingstoke, Hampshire, England). The plates were incubated aerobically and anaerobically at 37o C for 48 hr. Colonies from each culture mediumwere Gram stained and processed for biochemical identification using the Analytical Profile Index (API) 20E and API Staph (Biomerieux, France).Coagulase and catalase tests and sensitivity to Taxo A disc and Taxo P disc(BD Diagnostics, Difco Laboratories, Detriot, USA) were also employed for identification. Antibiotic susceptibility tests were carried out by the Kirby Bauer disc diffusion method using the Mueller Hinton agar. The antibiotics included erythromycin (15 µg), gentamicin (10 µg), agumentin (30 µg), streptomycin (10 µg), tetracycline (10 µg), chloramphenicol (10 µg), nalidixic acid (30 µg), ampicillin(10 µg), nitrofurantoin (200 µg), ciprofloxacin (5 µg), ceftriaxone (30 µg), oxacillin (1 µg) and kanamycin (30 µg). S. aureus ATCC 25923 and Enterobacter aerogenes (American Type Culture Collection, Rockville, USA) were used as control organisms.

DNA extraction of S. aureus isolates

The DNA of each S. aureusisolate was extracted by suspending the bacterial the colony in 200µl of sterile distilled water in labelled eppendorf tube and centrifuged at 13,000rpm for 3mins. The suspension was heated at 100?C for 10 minutes in a microwave machine (Haier thermocool), cold shocked in ice for 2 mins and vortexed (Gallenkamp spin-mix, Germany), recentrifuged at 10,000 rpm for 1 min and then stored at -20o C for DNA amplification [14].

PCR detection of S. aureus nuc, bla Z, tet K, sea and eta genes

Multiple antibiotic resistant S. aureusisolates are known to carry nuc, bla Z and tet K resistancegenes as well as the sea and eta virulence genes that code for production of enterotoxin A and exfoliative toxin A respectively[15]. Details of specific primers that were used to amplify sequences of the virulence and resistance genes as well as thepredicted sizes of the amplified products and specific annealing temperatures are given in Table 2.

A 25µl mixture containing 4µl of 10X buffer, 0.5µl MgCl2 , 3µl dNTPs, 0.2µl Taq polymerase, 1µl of the forward primer, 1µl of the reverse primer and 5µl of extracted DNAwas prepared in a PCR vial. The vial was placed in a programmed thermocycler (iCycler; Bio-Rad, Milan, Italy) with an initial denaturation at 94o C for 5 minutes, followed by 30 cycles of denaturation at 94o C for 1 minute, annealing at 55o C for 1 minute and extension at 72o C for 1 minute. A final extension procedure was carried out at 72o C for 10 minutes.

Agarose gel electrophoresis

Amplified PCR products were electrophoresed on 1% agarose gel containing 0.5 µl of ethidium bromide and run through 100 volt for 25 minutes in 1X TBE buffer in the electrophoresis tank. For each run, a 100 base-pair molecular weight DNA standard (size marker) was used to verify the appropriate size of each amplified PCR product. The DNA bands were then captured and visualized with a short wave ultraviolet transilluminator (UV Transilluminator 2000; Bio-Rad, Milan, Italy) and photographed using a Kodak digital camera.

The study included 87 SCD children and 160 non-SCD children, all ranging in age from 4 months to 15 years. Our results show that 29 (34.5%) of the SCD children were 1-5 years old, 35 (41.6) were 5-10 years of age and 18 (21.4%) were 10-15 years old (Table 1). The 1-5 and the 5-10 year old groups had the highest frequencies of bone pain episode, hospitalization for malaria, bacterial infections and sepsis and blood transfusions for anemia. On the other hand, 60 (37.5%) of the 160 non-SCD children were diagnosed with a variety of ailments that included tonsillitis, pharyngitis, whooping cough, diarrhea, otitis media, pneumonia, maculo-papula rash, tineacapitis, sepsis, impetigo, skin lesions , asthma , conjunctivitis, mumps, stomatitis and cerebral palsy.

Table 2 and Figure 1 show the distribution of the bacterial isolates that were cultured from the nasopharynx of SCD, non-SCD and control children. The gram positives represented about 72% of the total341bacterial isolates compared to 28% for gram negatives. Interestingly, the Corynebacterium species represented 64% and 85% of the bacterial isolates from the nasopharynx of SCD and non-SCD childrenrespectively. Streptococcus pneumoniae, though at a low frequency, was only isolated from SCD children, while Staphylococcus aureus and Bacillus subtilis were more predominant in the non-SCD children than in the SCD children.

Of the 119 bacterial isolates that were cultured from theSCD children,53 (44.5%) were Corynebacterium species that includedC. xerosis (35.8%),C. diphtheriae(20.75%) and C. pseudodiphtheriticum (15.1%). Furthermore, 66(55.46%) of the bacterial isolates from SCD children were gram positive rods.S. aureusand S. pneumoniaerepresented 33% and 25% of the 124 gram positive coccal isolates.Also 41(34.45%) bacterial isolates that were recovered from the nasopharynx of SCD children were gram negative rods.Pseudomonas aeruginosa accounted for 7(17.07%)while14.6% and 12.2% were Streptobacillus moniliformisand Haemophilus influenzae respectively. Klebsiella pneumoniae, and Salmonella enteritidisand Moraxella catarrhalis were less than 5% each.Out of the 222 isolates cultured from non-SCD subjects, 145 (65.31%) were gram positive bacilli, 56(25.22%) were gram negative rods (Table 2). Corynebacterium spp constituted the predominant gram positive organisms at 58.55%, with C. ulcerans at 55.38%, C. xerosis (36.92%) and C. diphtheriaeat 7.69%. Of the 56 gram negative bacilli cultured from the nasopharynx of non-SCD children, 33(58.92%) were Haemophilus influenzae, 18(32.14%) wereMoraxella catarrhalis and 5(8.92%) were Klebsiella pneumoniae.

The antibiotic resistance profiles of S. aureus isolates that were cultured from the nasopharynxof SCD, non-SCD and control children were also determined. Of the 18 S. aureus isolates from the non-SCD children, 14were resistant to ampicillin, 11to erythromycin, 9 to tetracycline, 7 to ceftriaxone, 2 to chloramphenicol, 1 to streptomycin, kanamycin and ciprofloxacin each. All isolates were sensitive to augmentin, oxacillin, gentamycin and nitrofurantoin. In contrast, the three S. aureus isolates from the SCD children were resistant to ampicillin, erythromycin and nalidixic acid. PCR was used to detect the presence of nuc, bla Z, tet K, sea and eta genes in eleven methicillin sensitiveS. aureus isolates. The nuc, blaZ and tetKgenes code for antibiotic resistance while the sea and eta genes code for enterotoxin A and exfoliative toxin A respectively. Table 3 shows a list of the primers that were used to amplify sequences of the virulence and resistance genes as well as the predicted sizes of the amplified products and specific annealing temperatures. Amplified products were electrophoresed on 1% agarose gel containing 0.5 µl of ethidium bromide, along with molecular weight DNA standard size markers to verify the appropriate size of each amplified PCR product. The results, presented in Table 3 and Figures 2, show that all eleven isolates carried the nuc gene while nine isolates carried the blaZ gene. Five isolates carried the sea gene, four carried the tetK gene and three isolates carried the eta gene. One isolate (F9ma) carried all five genes, two isolates (A2m2, B2mi) carried four of the 5 genes, and four isolates (Al16B1, D11Cm, 13ma, l18mb) carried three of the genes while three isolates only carried two of the genes.

Table 1: Indices of disease severity in children with sickle cell disease.

Table 2: Distribution of bacterial isolates cultured from the nasopharynx of children with and without sickle cell disease

Studies have shown that bacteria organisms of the nasopharyngeal flora of SCD children may become invasive and establish blood and lung infections. We sought to investigate whether there were differences in the nasopharyngeal flora and carriage rates of children with SCD and without SCD and in the antibiotic resistance profiles of the bacterial isolates. Studies already showed that children with SCD are significantly more susceptible to infections from S. pneumoniae, H. influenzae,S. enteritidis, malaria and other microbial organisms [11]. Our study included 87children with SCD and 160 children without SCD who attended theWesley Guild Hospital at Ilesa in south-western Nigeria and who ranged in age from 4 months to 15 years. Our data show the indices of disease severity in the SCD children as indicated by the frequency of bone pain episodes, hospitalizations and blood transfusions. The results also show a high frequency of bone pain episode, hospitalization for malaria, bacterial infections and sepsis, and blood transfusions for chronic anemia among the 1-5 and the 5-10 year old SCD children. On the other hand, clinical records showed that 60 (37.5%) of the 160nonSCD children had acute presentations of non-fatal illnesses such as tonsillitis, pharyngitis, whooping cough, diarrhea, otitis media, maculo-papula rash and others. The results are readily explained by the immune dysregulationprocesess that characterize the pathogenesis of SCD.

Information on the distribution of bacterial isolates from the nasopharynx of children with and without SCD is presented in Table 2. Overall, 364 bacterial isolates were cultured from 270 subjects that were recruited for the study. A total of 119 isolates were cultured from 87 sickle cell disease children while 222 bacterial isolates were cultured from 160 children without sickle cell disease.Corynebacterium species were the most prevalent bacteria among the three cohort samples.C. xerosis was the dominant species among the SCD children, while C. ulceranswas isolated most frequently amongthe non- SCD and control subjects.

The predominance in this study of Corynebacterium species over organisms as H. influenzae,S. pneumoniaeand S. Aureus that are common invasive pathogens of the nasopharynx was interesting and unexpected. Our results also show that while the prevalence of H. influenzae 33(14.8%) is relatively high among children without SCD in this study, the prevalence was remarkably low in children with sickle cell disease (4.2%) and control subjects (8.6%). Similar findings have been reported by other investigators [16]. The study found that high carriage rate of S. pneumoniae did not correlate with invasive bacteremia. In our study, the prevalence of nasopharyngeal carriage with both H. influenzae and S. pneumoniae was similarly low for SCD children compared to children without SCD (Table 2). While the reason for low incidence is not apparently clear, the observation may be attributed to long-term prophylactic treatment of children with penicillin and other antibiotics and theeffectiveness of the administration of pneumococcal and Hib vaccines to the study cohorts from early age at the hospital clinic. It is notable from the results that while the prevalence of S. pneumoniae and H. influenzae in the nasopharynx of the study population has been significantly reduced by penicillin and vaccine administration, the two organisms have largely been replaced by various species of Corynebacterium, which are opportunist respiratory pathogens that can cause diphtheria and pharyngitis.

Studies reported from Ile-Ife in the 1990s showeda high prevalence of methicillin resistant S. aureus (MRSA) strains in the anterior nares. However, our present observation is at variance with such previous findings [17]. The results from our present study show that all S. aureusisolates that were recovered from the nasopharynx of the children were methicillin sensitive strains (MSSA). This is an interesting shift of community bacterial flora since the past two decades. Other investigators have reported a similar reduction in the prevalence of MRSA isolatesfrom non-SCD children [18]. Our investigation also revealed that eleven S. aureus isolates from non -SCD children carried resistance and virulence genes. Nine of the 11 S.aureus isolates from the nasopharynx of non- SCD children carried the blaZ resistance gene which confers resistance to the beta- lactam antibiotics. Four isolates carried the tetK virulence gene which confers resistance to tetracycline. Five isolates carried the sea virulence gene that codes for enterotoxins while three of the 11isolates carried the eta virulence gene that codes for the staphyloccocal scalded skin syndrome toxin [15]. This is the first report that has documented the presence of those genes in S. aureus isolates in the study area and the results are significant because of the diverse and differential carriage of both antibiotic resistance genes as well as the virulence genes. In patients with inadequate immunity as in SCD children, the dissemination of such resistant and potentially virulent organisms from the nasopharynx could result in invasive bacteremia, intravascular coagulation and septicaemia [13].

In conclusion the bacterial isolates cultured from the nasopharynx of SCD, non-SCD and control subjects were predominantly Corynebacterium spp. that were sensitive to all the antibiotics used in the study. We examined eleven S. aureus isolatesfor their carriage of antibiotic resistance and virulence genes and found that these genes are present in most of the isolates. Our study revealed low incidence of nasopharyngeal colonization with both H. influenzae and Streptococcus pneumoniae in this centre but the widespread antibiotic resistance among S. aureus isolates among sick children portends serious challenges for therapeutic options.The results of the study suggest the need for a concerted educational program in the appropriate use of prophylactic antibiotics and of an expanded immunization program with coverage for all children.

Table 3: Primers used for the Detection of Resistance and Virulence genes.

Table 4: Characterization of the methicillin sensitive S. aureus (MSSA) obtained from the nasopharynx of non -SCD in Ilesa.

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