Antibiotic Resistance Trends of Nasal Staphylococcal Isolates from Namibian School Children

Nasal colonization with Staphylococcus strains puts children at risk of devel-oping difficult-to-treat staphylococcal infections. Antibiotic resistance data is limited in Namibia. Our study thus aimed to provide resistance trends for nasal staphylococci isolated from school children in the Mariental District. This is the first report on antibiotic resistance trends of staphylococci from Namibian school children. By Kirby-Bauer disk diffusion assay, 352 Staphylococcus aureus and 81 coagulase-negative staphylococci (CoNS) isolates from Namibian school children aged 6 - 14 years underwent susceptibility testing against seven antibiotics. Ninety-six percent S. aureus and 66.7% CoNS were resistant to ampicillin. Ampicillin resistance was significantly higher in S. aureus than in CoNS (P < 0.0001). Ciprofloxacin and gentamicin were the most effective against S. aureus. Ciprofloxacin was also the most effective drug against CoNS. Cefoxitin/methicillin resistance was seen in 14.5% S. aureus isolates and 8.6% of CoNS. Thirty-one antibiotic resistance patterns were observed, most frequently ampicillin (A), ampicillin-erythromycin (AP-E), and ampicillin-tetracycline (AP-T). Altogether 12.5% isolates (50 S. aureus and four CoNS) were multi-drug resistant. From the methicillin-resistant S. aureus (MRSA) isolates, 43.1% were


Introduction
Healthy school children under 16 years are potential carriers of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) and multi-drug resistant strains [1]. According to [2], children are asymptomatic reservoirs for community-associated MRSA (CA-MRSA) which enables these bacteria to rapidly spread within communities. Nasal colonization with S. aureus is a major risk factor for staphylococcal infection [3]. Most staphylococcal infections can easily be cleared with antibiotics, but bacteria that develop resistance towards certain antibiotics make treatment options limited, especially if antibiograms for reference purposes are unavailable. Methicillin-resistant S. aureus is resistant to beta-lactam antibiotics, while some strains are multi-drug resistant. Drug resistant strains are often responsible for chronic, persistent and recurrent infections, which is a challenge for healthcare practitioners.
Antibiotic resistance data is limited in Namibia and few reports on staphylococcal drug resistance exist. Our study aimed to make a contribution towards closing this gap in information by providing resistance data for 433 S. aureus and coagulase-negative staphylococci (CoNS) isolates from nasal swabs of children aged 6 -14 years attending schools in the Mariental District. To our knowledge, this is the first report on resistance trends of nasal staphylococcal isolates from Namibian school children.

Study Area, Population and Sample Collection
This was a cross-sectional study in the town of Mariental, located southeast of Namibia's capital city of Windhoek on the B1 national highway. With written consent from their parents/guardians, the population that was screened for nasal staphylococci consisted of randomly chosen healthy learners attending five schools in the Mariental District. The children were divided into two age-groups: 6 -10 years and 11 -14 years, and consisted of 126 boys and 146 girls. Sample collection was done during March, September and October 2016. One nasal specimen was obtained from each child by gently rotating a sterile Amies transport medium swab (Labocare TM , Johannesburg, South Africa) thoroughly around the perimeter of both nostrils. Specimens were kept frozen at −20˚C until transporting them to the University of Namibia's Biomedical Research Laboratory for processing.

Antibiotic Susceptibility Testing
Antibiotic resistance testing on isolates obtained from the nasal specimens was carried out in 2017. The Kirby-Bauer disk diffusion assay was used to determine antibiotic susceptibility/resistance in isolates [4] [5] [6] [7]. Table 1

Statistical Analysis
A chi-square test for comparison of proportions with MedCalc statistical software (MedCalc statistical software version 16.4.3 {MedCalc software bvba, Ostend, Belgium; https://www.medcalc.org; 2016}) [8] was used to compare percentage differences in antibiotic resistance between S. aureus and CoNS. Statistical significant differences were indicated by a P-value of ≤ 0.05.

Antibiotic Susceptibility/Resistance of Isolates
Altogether 433 staphylococcal isolates, as well as two reference strains, underwent antibiotic susceptibility testing against seven antibiotics ( CoNS, 66.7% of isolates were also resistant to ampicillin (Table 3). However, resistance towards ampicillin was significantly higher in S. aureus than in CoNS (P < 0.0001). Resistance towards ampicillin has become very common in staphylococci and can be ascribed to the action of the enzyme beta-lactamase which is under plasmid control. Plasmids containing resistance genes can be transferred from one bacterium to another [9]. Other studies also indicated high ampicillin As indicated in Table 2 and Table 3, respectively, a total of 83 (23.6%) S. aureus isolates and nine (11.1%) CoNS were resistant to erythromycin (P = 0.0064). These resistance rates are not that high and erythromycin is therefore expected to be effective against staphylococci in most instances. In comparison to our study, a study by Mengistu et al., 2013 [11]    Their percentage resistance is however almost the same as the 32.3% from Mengistu et al., 2013 [11].
In the current study, only 17.0% S. aureus and 8.6% CoNS (P = 0.0595) were resistant to tetracycline, indicating its effectiveness against Staphylococcus. According to Mengistu et al., 2013 [11] 29.6% of staphylococci from CSF displayed resistance towards tetracycline. In agreement with our results, Iileka et al., 2016 [12] reported 17.4% tetracycline resistance in clinical S. aureus strains across the period 2012-2014. The resistance for S. aureus in our study towards this antibiotic is higher than the 4.3% found by de Carvalho et al., 2017 [10] in Brazil.
Rifampicin resistance in our study was relatively low at 18.2% and 17.3% (P = 0.8495) for S. aureus and CoNS isolates, respectively. However, this is somewhat higher than the 7.0% indicated in other Namibian resistance data [13]. Rifampicin resistance in other countries may be higher. In India for instance, Bharathi et al., 2014 [14] observed 64.7% resistance in nasal MRSA from school children.

Rifampicin is among the antibiotics commonly used to treat MRSA infections in
India [14]. Staphylococci can quickly develop resistance to rifampicin [9].
According to our results, ciprofloxacin and gentamicin were most effective against S. aureus, with 99.7% and 93.2% of isolates that were susceptible to these drugs, respectively. Ciprofloxacin was also the most effective drug against CoNS, with 100.0% susceptibility. Resistance between S. aureus and CoNS for the two antibiotics was not statistically significant (P = 0.6220; P = 0.1886 In this study, cefoxitin was used to detect methicillin-resistant bacteria. These bacteria are also resistant to all beta-lactam antibiotics. A total of 51/352 (14.5%) S. aureus isolates were resistant to cefoxitin and therefore identified as MRSA,  [12]. Considering these relatively low percentages, MRSA does not seem to be a major problem in Namibia yet. In general, there are not many studies available on methicillin resistance in CoNS. According to [17], MRSA and MRCoNS can be found together in the human nose and have similar antibiotics resistance genes that can be transferred between bacteria. We could not find studies involving nasal antibiotic resistant staphylococci from healthy school children in our neighboring countries (Angola, Zambia, Botswana and South Africa) to compare our results with.

Resistance Patterns and Multi-Drug Resistance
Altogether 31 antibiotic resistance patterns were observed in this study (Table   4). Isolates with similar resistance patterns could be considered the same strain, unless they acquired these resistance genes from other strains, therefore sharing the same resistance pattern. For S. aureus, 27 different resistance patterns were obtained. For CoNS, there were 14 different patterns. Ten patterns (AP, RP,   Fifty-four out of 433 isolates (12.5%) were resistant against three or more classes of antibiotics and classified as multi-drug resistant. Of these, 50 isolates were S. aureus and four were CoNS. The most common multi-drug resistant pattern for methicillin-susceptible isolates was AP-T-E, displayed by eight S. aureus isolates, and one coagulase-negative isolate. Of the 51 MRSA isolates, 22 (43.1%) were multi-drug resistant, with AP-T-RP-FOX as the most encountered resistance pattern among them (See Figure 1). This is of concern, but is 14

Conclusion
Our study showed the presence of antibiotic-resistant strains among healthy school children. Overall, multi-drug resistance was relatively low. However, some of the MRSA isolates were multi-drug resistant, which is of concern.
Learners should be encouraged to frequently wash their hands to prevent spread of antibiotic-resistant bacteria within the Mariental community and educated on the appropriate use of antibiotics. Ciprofloxacin and gentamicin may effectively be used to treat staphylococcal infections in this study population.