Nasopharyngeal carriage, spa types and drug susceptibility profiles of Staphylococcus aureus from healthy children under 5 years in eastern Uganda

Background: Staphylococcus aureus carriage is a known risk factor for staphylococcal disease. However, the carriage rates vary by country, demographic group and profession. This study aimed to determine the S. aureus carriage rate in children in Eastern Uganda, and track Staphylococcus strains that can cause infection in Uganda. Methods: Nasopharyngeal samples from 742 healthy children under 5 years residing in the Iganga/Mayuge Health & Demographic Surveillance Site in Eastern Uganda were processed for isolation of S. aureus. Antibiotic resistance based on minimum inhibitory concentrations (MICs) was determined by the BD Phoenix™ automated identification & susceptibility testing system. Genotyping was performed by spa typing. Results: The processed samples yielded 144 S. aureus isolates (one per child) therefore, the S. aureus carriage rate in children was 19.4% (144/742). Thirty one percent (45/144) of the isolates were methicillin resistant (MRSA) yielding a carriage rate of 6.1% (45/742). All MRSA isolates were susceptible to vancomycin, linezolid and clindamycin however, compared to methicillin susceptible S. aureus (MSSA) (68.8%, 99/144), MRSA isolates were more resistant to non-beta-lactam antimicrobials –tetracycline (91.1%, 41/45), trimethoprim/sulfamethoxazole (73.3%, 33/45), erythromycin (75.6%, 34/45), chloramphenicol (60%, 27/45), gentamicin (55.6%, 25/45) and ciprofloxacin (35.6%, 16/45). Furthermore, one MRSA isolate was mupirocin resistant and 42 (93.3%, 42/45) were multidrug resistant (MDR); three (3%, 3/99) MSSA isolates were mupirocin and clindamycin resistant, while 61 (61.6%, 61/99) were MDR. All MSSA/MRSA

aureus (MSSA/MRSA) carriage rate in children in Eastern Uganda is high and comparable to rates for hospitalized patients in Kampala city. The detection of mupirocin resistance is worrying as it could rapidly increase if mupirocin is administered in a low-income setting.
S. aureus strains of spa types t064, t037 (MRSA) and t645, t4353 (MSSA) are prevalent and could be responsible for majority of staphylococcal infections in Uganda.

Background
A causal relationship between Staphylococcus aureus carriage and infection is well documented [1,2]. The anterior nares and nasopharynx are the most important sites for S. aureus colonization in humans [3]. Generally, the prevalence of S. aureus nasal carriage ranges from 20% to 30% however, it varies by country, profession and demographic group [2,4]. Notably, these estimates are from developed countries and their applicability to African settings where infection control practices and surveillance for antimicrobial resistance are inadequate/nonexistent [5], is debatable. Furthermore, information on the prevalence, population structure and molecular epidemiology of S. aureus carriage in healthy African individuals is scarce [6]. Although the prevalence and correlates of S. aureus nasal carriage in hospitalized adult patients and health workers in Uganda has been documented [7-9], carriage rates in communities especially in children who are more vulnerable to staphylococcal infections, is not known. Generally S. aureus studies in Africa have focused on clinical and/or nosocomial isolates, which limits our understanding of the S. aureus population structure on the continent [1].
The Streptococcus pneumoniae, Haemophillus influenzae and Moraxella catarrhalis nasopharyngeal carriage rates among healthy Ugandan children was found to be high at 58.6% (89/152), 15% (23/152) and 11% (16/152), respectively [10]. These bacteria alongside S. aureus, are the main causes of pneumonia [10], the leading cause of death in children under five years [11]. In this follow-up study, we aimed to determine the S.

Study setting and identification of S. aureus
This cross-sectional study was nested in a previous study that investigated the nasopharyngeal carriage of pneumococci in healthy children less than 5 years of age at the IMHDSS [10], located in Iganga and Mayuge districts in rural Eastern Uganda, between February and October 2011. Following specimen processing for isolation of Strep. pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis during the previous study [10], nasopharyngeal samples (swabs) from 742 children were sub-cultured again for isolation of S. aureus, as highlighted below. Figure 1 highlights the procedure we followed to isolate and identify S. aureus. Briefly, presumptive Staphylococcus colonies were re-streaked on blood agar plates or on tryptic soy agar plates (for samples with no growth on blood agar) and incubated at 37 °C for 24 hours in a CO 2 incubator. To identify S. aureus, we subjected Gram-positive and catalasepositive isolates to three different methods commonly used to identify S. aureus: (i) tube coagulase test, (ii) growth on DNase agar, and (iii) growth on Mannitol salt agar [12].
Isolates that tested positive on all the three methods were considered to be S. aureus; otherwise isolates that tested negative with one or two of the methods were subjected to PCR-detection of the nuc gene as previously described [12]; isolates were considered to be S. aureus if a 270 base pair fragment was identified on agarose gel electrophoresis.
Otherwise nuc gene PCR-negative isolates were further evaluated with the BD Phoenix 100 ID/AST expert system for identification as previously described [13][14][15]. Isolates that tested negative with all the confirmatory tests for identification of S. aureus (i.e. tube coagulase test, nuc gene PCR & BD Phoenix 100 ID/AST) were excluded from further analysis.

Drug susceptibility testing
Except mupirocin susceptibility that was determined by the BD Phoenix 100 ID/AST expert system, drug susceptibility testing was determined by the disc diffusion method according

Genotyping and other molecular procedures
To determine the lineages, spa typing was performed as described previously [16]. Briefly, PCR products were purified with QIAquick PCR purification kit (Qiagen) and sequenced at MBN Laboratories (Kampala, Uganda) or ACGT Inc. (Wheeling, IL, USA) using forward and reverse primers used in PCRs. To obtain spa types, spa sequences were submitted to an online spaTyper server (http://spatyper.fortinbras.us/) and confirmed by cross-checking the repeats with the Ridom Spa Server (http://spaserver2.ridom.de/spatypes.shtml). typing in which all the PCR products were sequenced, DNA sequencing of amplified segments of mecA and PVL genes was performed for selected isolates and confirmed by BLAST-searching at NCBI https://blast.ncbi.nlm.nih.gov/Blast.cgi Additionally, we also compared spa types for S. aureus at the IMHDSS in Eastern Uganda to previously described spa types for S. aureus isolates from Mulago National Referral Hospital in

Demographic characteristics
The characteristics of children sampled were described previously [10,20]. Briefly, about half of the children were less than 2 years old and 50% were female with a mean age of 30 months. Majority (82%) of the children lived in rural areas and their primary source of water was spring water (74%) and well water (26%); only 1% of the children had access to piped water. The median number of children per household was 2 while the median number of household members per bedroom (as a measure of crowding) was 4. Based on reports of mothers 90% of the children had been sick in the previous 2 weeks and the most common symptoms were fever, running nose and cough. Of the children who had been sick around 30% were reported to have been given antibiotics, mostly cotrimoxazole and ampicillin.
Mupirocin is a newer drug used as a topical antibiotic to treat impetigo caused by S.
aureus and S. pyogenes. It is also commonly used for nasal decolonization of MSSA and MRSA in patients and health care workers in the developed countries [5, 32,36]. We have reported a high rate (4%) of mupirocin resistance (high-level) and this is consistent with its reported rates in Africa (5%-50%) [40]. The detection of mupirocin resistance in Uganda is worrying as MRSA decolonization is generally not a common practice in Africa.
As mupirocin use in decolonization regimens leads to rapid emergence of resistance [32], its introduction in African settings for eradication of Staphylococcus colonization will likely spike an increase in resistance [40].
Overall, S. aureus strains of spa types t645, t064, t4353, t002, t318, t037, t355, t084, t3772 & t127 are prevalent in Uganda and generally in Africa [35,41,42]. Consistent with this, a systematic review of the global distribution of spa types revealed that t064 and t037 were the most prevalent spa types in Africa with t037 being exclusively associated with MRSA strains [43]. The exception is t4353, which appears to be common in Uganda setting was reported before [12]. As such, we regarded the nuc negative isolates to be S.
aureus because the Phoenix ID/100 system we used as a tie-breaker confirmed them as so.
It is important to note that currently, the Expert Identification Systems such as the

Additional Files
Additional file 1:  Study flow chart illustrating the procedure we followed to identify S. aureus