Antibiotic resistance pattern of Staphylococcus aureus with reference to MRSA isolates from pediatric patients

Aim: The extent of methicillin-resistant Staphylococcus aureus (MRSA) infection in Nepalese children is largely unknown. Materials & methods: Six hundred and seventy-two clinical samples collected from 232 patients between June and November 2016 were processed in a microbiology laboratory. Results: Out of 300 culture-positive samples, 52 (17.3%) were S. aureus isolates. Among those 52, 39 (75.0%) were found to be MRSA. The infection rate of S. aureus was shown to be higher in inpatients (55.7%) compared with outpatients (44.3%) at p = 0.637, 95% CI. Thirteen types of antibiotics were used in the antibiotic susceptibility test. MRSA isolates showed 100 and 0% resistance to penicillin and vancomycin, respectively. The D-test showed inducible clindamycin-resistant phenotype in 15.4% of MRSA isolates. Conclusion: This demonstrates the utmost need for routine testing for MRSA in Nepalese hospitals.

aureus [12], although it is found in almost all body parts. With little change in overall mortality, the frequency of community-acquired MRSA and hospital-acquired MRSA infections have increased steadily. They contribute to the failure of empirical therapy. Treatment of these infections has become more difficult because of the emergence of multidrug resistance (MDR) [13]. MDR is defined as resistance to three or more antimicrobial classes. MRSA raises greater concern because of its high virulence capacity [14], high ability to cause a diverse array of life-threatening infections and its capacity to adapt to different environmental conditions [13,15]. The emergence of resistant strains is contributing nosocomial infections [16].
The emergence and the spread of multidrug-resistance S. aureus bacteria is a global threat for the therapeutic management of staphylococcal infections. Studies have shown that asymptomatic carrier children are a potential vector for the dissemination of MRSA in the community [17,18]. However, to our knowledge, the exact extent of MRSA in the children of Nepal has not yet been well assessed. Therefore, this study was an initiative to explore the distribution of MRSA in pediatric patients visiting International Children Friendship Hospital (IFCH). The presence of MRSA among a population of children were evaluated and assessed for the antimicrobial susceptibility profile. The infections in inpatients with those of outpatients were compared. The association between the causative organism with different variables such as age, gender and type of samples were assessed. These are important factors in S. aureus-related infections. We believe that this kind of study will aid physicians in administering first-line treatment by selecting the appropriate therapeutic drug against S. aureus.

Materials
All the chemical, kits, antibiotic discs and microbiological media were purchased from HiMedia Pvt Ltd Co., Mumbai, India.

Sample collection
The study was conducted in the microbiology laboratory of IFCH, Kathmandu, during the period of June 2016 to November 2016. A total of 672 various clinical specimens viz. blood, urine, stool, pus swab from an ear, eye, throat, vaginal and body parts where burn or wound occurred were collected from both inpatients and outpatients. Only those patients who were not taking any medication and referred by a clinician were included in the study. Samples were stored in a sterile, leak-proof, screw-capped container with a proper label (Lab Id no., age, gender, date, time).

Laboratory assessment Isolation & identification
The specimens were processed (i.e., gram staining for microscopic observation and microbial culture) in the microbiology laboratory within 2 h of the collection. A sample of blood was collected in the brain heart infusion (BHI) broth in 1:10 v/v ratio immediately, mixed well and then screw tightened the BHI bottle. The BHI bottles were then incubated at 37 • C for up to 72 h followed by subculture on Blood Agar (BA) and MacConkey Agar (MA) plates. Samples such as pus, urine, stool, etc. were directly inoculated into BA and MA, whereas cerebrospinal fluid (CSF) samples were inoculated into MA and Chocolate (CA). Only the growth obtained in BA and CA plates were further cultured into Mannitol Salt Agar (MSA), an enrichment media for isolation of S. aureus. The BA and CA plates were incubated anaerobically (5-10% CO 2 ), while MA and MSA plates were incubated aerobically at 37 • C for 24 h. Colonies formed in MSA were picked and processed for gram staining. Only cocci were processed for the identification of S. aureus. For conformational identification of S. aureus, catalase test, coagulase (slide and tube) test were performed with the known positive and negative control strains (Figures 1 & 2).

Macroscopic examination
Colony characteristics were examined on MSA and BA medium for the identification of all the isolates. β-hemolytic activity was detected on BA medium ( Figure 3).

Statistical analysis
Microsoft Excel 2013 was used to record the laboratory data and statistical analysis software (IMB-SPSS V21.0) was used to calculate a p-value by using Pearson Chi-Square test. p > 0.05 was considered statistically significant at 95% CI.

Demography of S. aureus
Out of 52 S. aureus-positive samples, 35 (67.3%) and 17 (32.7%) were isolated from males and females, respectively ( Table 3). Distribution of S. aureus in the toddler age group was the most (17 [32.7%]) and it was the least in the adolescent age group (2 [3.8%]) for both genders ( Table 1).

Distribution of MRSA in outpatients & inpatients
In addition to MRSA, response to cefoxitin was also stratified as methicillin-intermediate S. aureus (MISA) and methicillin-sensitive S. aureus (MSSA). The cefoxitin disc diffusion test showed that out of 52 S. aureus isolates, 39  (75.0%), seven (13.5%) and six (11.5%) were identified as MRSA, MISA and MSSA, respectively. Moreover, we found that among these 21 (72.4%), five (17.2%) and three (10.3%) of MRSA, MISA and MSSA, respectively, were isolated from inpatients. The analysis further showed that the incidence of MRSA and MSSA isolations (78.3%) and (13.0%), respectively, were slightly higher in outpatient than in inpatient samples. However, the association between the MRSA occurrence and inpatients was found statistically insignificant (p = 0.637 at 95% CI). On the other hand, the incidence of MISA isolation was lower in inpatient samples (six) compared with outpatient samples (2 [8.7%]). These data clearly show that frequency of MRSA in inpatients is higher compared with outpatient ( Table 2).

Distribution of S. aureus in various infection groups
Out of 35 samples from male patients, 23 (44.2%) were from skin and soft tissue infection (SSTI) patients (skin swab), seven (13.5%) were throat and nasal swabs samples taken from respiratory tract-infected (RTI) patients such as trachetis, sinusitis, laryngitis, pharyngitis/tonsillitis, rhinitis, acute otitis media, etc., five (9.6%) were blood samples and none (0%) were urine samples. Likewise, out of 17 samples from female patients, 11 (21.2%) were from SSTI patients, three (5.77%) were from RTI patients, two (3.9%) were blood samples and one (1.9%) was a urine sample. These results demonstrated that in both male and female pediatric patients, S. aureus strains were more prevalent in SSTIs compared with the remaining other types of infections (Table 3).

Antibiotic resistance pattern of S. aureus
The antimicrobial resistance (AMR) patterns of MRSA and MSSA isolates against antimicrobial agents are summarized in Table 4. More than 25% of MRSA isolates were resistant to ampicillin, ciprofloxacin, cotrimoxazole, erythromycin, clindamycin, azithromycin and tetracycline. Only a few of the MRSA isolates were found to be

Discussion
S. aureus infection causes a health burden, particularly in low-and middle-income countries. MRSA is emerging as a serious threat to public health worldwide. MRSA has significantly increased morbidity and mortality rate of patients along with the increased length of hospital stay. S. aureus infections are a significant clinical problem in medical practice as the organism shows resistance to the commonly used first-line drugs. The prevalence of MRSA, however, varies markedly by country. In this study, the occurrence of S. aureus was studied among the pediatric patients visiting IFCH, using various types of sample. All the samples with clinically detected S. aureus may serve as a reservoir of MRSA, which may transmit the infection in a community. Thus, there is a chance of a rapid increase in the development of community-acquired MRSA infection. Though infected persons with S. aureus are a direct vital source, it is also reported to be isolated from fomites [21]. Fomites can be considered as an indirect transmission route for S. aureus as well as other infectious diseases in hospital [22]. So, environmental sources and fomites cannot be ignored.
In the present study, 52 (17.3%) S. aureus isolates were found out of 300 culture-positive samples. The results are similar to those reported by Mukhiya et al., with growth positivity of 17.4% [23]. Here, the result also shows that children are more prone to S. aureus-associated infection. This might be due to high chances of contact with an infectious agent or their low immunity. Our findings show that S. aureus is a major cause of pyogenic infections. The skin and soft tissue (SST) was the most common site for the infection, likely because this bacterium normally inhabits the skin. Pandey et al. reported the higher isolation of S. aureus from pus and wound (78.37%) in comparison to blood (17.11%) and urine (4.50%) out of 111 S. aureus-containing clinical samples [24]. The current study also shows a parallel result, where 34 (65.4%) S. aureus isolates were found in SST samples, which are more than those obtained from RTIs, blood and UTIs.
In our study, when comparing the number and percentages of S. aureus in the Inpatient Department and Outpatient Department, the rate of S. aureus was higher in inpatients (55.7%) compared with outpatients (44.3%). Our result is in accordance with Rajbhandari et al. in Nepal [25]. They also reported the higher prevalence of S. aureus among the inpatient setting, accounting for 86 (62.3%) as compared with 52 (37.7%) in outpatients. Likewise, if we look at the distribution of MRSA isolates in different types of our patients, we found them to be 21 (40.4%) inpatients and 18 (30.6%) outpatients out of a total 39. These findings also indicate the higher prevalence of nosocomial infections. This higher occurrence of MRSA among inpatients could be due to various hospital associated risk factors such as prolonged hospital stay, antibiotic treatment, underlying immune-compromised condition, hospital environment, instrumentation and use of other invasive devices, which predispose patients to MRSA acquisition.
Over the course of time, S. aureus has developed resistance to different, conventionally used antibiotics. All the MRSA isolates were resistant to more antibiotics than MSSA isolates. A significant difference (p = 0.866) was observed in the case of cefoxitin, gentamicin, amikacin, cotrimoxazole and erythromycin. However, the difference observed in a case of tetracycline and ciprofloxacin was statistically insignificant (p > 0.05). Significant MDR cases of MRSA were 28 (71.8%). It also showed high resistance to cotrimoxazole and erythromycin, of 56.4 and 53.9%, respectively. These two antibiotics and mupirocin are usually used at random to cure a generalized and pyogenic infection. On the other hand, only 15.4% MRSA isolates showed ICR. The increasing rate of the Staphylococcal infections among the patients and the changing patterns of AMR has led to a renewed interest in the use of the clindamycin therapy in treating such infections [26]. The overall findings suggest an alarming situation of AMR accompanied by S. aureus. The development of antibiotic-resistant properties is troublesome and has been described as a serious public health concern, particularly in developing countries [27].
The study showed the frequency of MRSA to be 39 (75%  [31,32]. The authors have attributed this to the indiscriminate use of antibiotics and its accessibility. Prompt detection of MRSA may control for severe epidemics and lessen disease effects by allowing application of appropriate and timely treatment measures. Currently, various molecular techniques are used but they are neither economical nor feasible at small scale [33,34]. Availability of new drugs and inadequate establishment of MRSA treatment brings about the use of antibiotic combinations. Manytime new treatments and therapy are not available against MRSA, regardless of location. For MDR S. aureus strains, a combination of different antibiotics seems the only option. Tetracycline/sulbactam/cefoperazone combination has shown better results than the old combination of ampicillin/sulbactam/cefoperazone antibiotic [22]. This is also supported by similar minimum inhibitory concentration (MIC) results of others group's data [35]. Some variations that occur may be due to differences in infection control measures, antibiotics prophylaxis, empirical therapy, treatments used in each ward and the clonal and often epidemic nature of these microorganisms.
The high rate of MRSA isolation and resistance to penicillin, ampicillin, ciprofloxacin, erythromycin, cotrimoxazole, chloramphenicol etc. shows that these antibacterial agents would be unreliable. Vancomycin seems to be the only antimicrobial agent that showed 100% sensitivity against S. aureus in the study. Hence, vancomycin may be used as the drug of choice for treating MDR-MRSA infections. However, frequent monitoring of vancomycin sensitivity and routine testing should be carried out. Use of vancomycin should be limited to preserve its value. It should be administered only in those cases where there is a clear need. Although all isolates were found to be sensitive to vancomycin, the screening test and MIC determinations are recommended for early detection of impending resistance and monitoring the response to therapy. The regular surveillance of nosocomial infections including monitoring antibiogram of MRSA and MSSA and formulation of definite antibiotic policy may be helpful in reducing the incidence of MRSA infection. The study is, therefore, an opening to facilitate epidemiological studies.
However, apart from antibiotic combinations for the treatment of MRSA, new treatment options using other antibiotics are slowly emerging. Nevertheless, these have their own drawbacks such as drug supply, cost and resistance itself. Therefore, this is not successfully being enforced in practice. Furthermore, the use of new antibiotics offers different antibiotic resistance mechanisms and new resistance properties are likely to emerge. Thus, the use of affordable antibiotics in suitable combinations is the only promising alternative method for MRSA-like bacterial infections. However, developed nations are working on making effective MRSA treatment strategies, including novel drugs or vaccines, gene therapy, bacteriophage engineering and so on by utilizing the latest tools and techniques.
The authors of the current study recommend detecting novel resistance mechanisms such as mecC or uncommon phenotypes such as borderline-resistant oxacillin resistance.

Conclusion
The occurrence of MRSA was found to be 39 (75.0%) out of a total 52 S. aureus isolates. The highest numbers of MRSA isolates were found in the toddler age group and male patients. Methicillin susceptibility of S. aureus was found higher in inpatients than outpatients. MRSA was found to be less resistant to gentamicin, cloxacillin and clindamycin, so these antimicrobial agents can be also used like mupirocin for the treatment of SSTIs and decolonization of MRSA carriers, but the use of such drugs should be limited. Alternatively, drugs like trimethoprim-sulfamethoxazole, teicoplanin, linezolid, quinupristin/dalfopristin, rifamycin, vancomycin and so on, or with suitable combination drugs are prescribed for MRSA treatment.
Both cefoxitin disc diffusion and D-test are the easy, reliable and cost-effective methods for detection of methicillin and inducible MLS B resistance, respectively. Cefoxitin test and ICR (D-test) should be made compulsory in all S. aureus isolates in addition to routine susceptibility test methods, because the routine susceptibility test cannot detect MRSA unless cefoxitin disc is used. Likewise, inducible MLS B resistance is not detected unless erythromycin and clindamycin antibiotics are placed 15-26 mm apart. It should be noted that these are time-consuming and labor-intensive methods. The sensitivity and specificity of these methods are also low and less reliable compared with other detection methods like MIC [36] determination, molecular techniques and so on. Though inducible MLS B resistance is not associated with MRSA and widespread use of MLS B has led to an increasing resistance to MLS B antibiotics, they are still used to treat infections associated with S. aureus. This is also because of the safety and easy availability of these drugs compare to newer classes of antibiotics.
The study shows the MRSA occurrence is prevalent in pediatric patients. This corroborates the findings of previous researchers as discussed. Nevertheless, the total number of S. aureus isolates and the 6 months of study duration are low, which might be a possible source of errors and prevent drawing definite conclusions from this research. Newer classes of drugs are found to be more effective than β-lactam drugs to treat S. aureus infection. Moreover, MRSA infection is still one of the most life-threatening infections in hospitals. Therefore, regular surveillance of MRSA should be carried out in all hospital settings. In addition, restriction of the indiscriminate use of such antibiotics may be an effective strategy to control AMR. We should discourage empirical therapy practices, instead considering microbiological test reports. This might be an effective strategy to tackle the AMR problem and improve the infection treatment plan.

Limitations
We were not able to perform a molecular test for genotyping or Staphylococcal cassettee chromosome mec gene (SCCmec) typing and similarly, MIC test for MRSA confirmation was not done due to lack of resources and timebound factors. Neither risk factor analysis of MRSA carrier nor longitudinal data of MRSA trend was generated due to the limitation of current study settings and expertise in our network. Moreover, the study was conducted in a single hospital, so the findings do not represent a broad population and nonpatients.

Future perspective
This study highlights the importance of routine testing, reporting and monitoring of MRSA, like in other drug resistance cases. Health care workers (HCWs) should be more conscious about AMR and nosocomial infections. In underdeveloped countries like Nepal, confirmation of microbial disease diagnosis and MDR should be done by molecular techniques despite them being uneconomical at small scale. The significant increase in constant AMR surveillance and monitoring of hospital associated infections (HAIs) should be facilitated in order to understand the nature of particular bacterial infections. Empirical therapy including combination drug prescriptions should exist, particularly in developing countries. The study shows male pediatric patients have higher MRSA than female pediatric patients, thus further study should be done to examine this relationship. Generally, male children are actively engaged in outdoor activities and thus exposed to the external environment more than female childrenthis could be one of the reasons for the higher incidence of bacterial infection in males.

Summary points
• Inducible clindamycin resistance was found to be higher among methicillin-resistant Staphylococcus aureus (MRSA) compared with methicillin-sensitive S. aureus. • MRSA is emerging as a serious threat to public health worldwide. It adds burden to a patient by prolonging hospital stay and increasing morbidity and mortality rate. • Vancomycin seems to be the only antimicrobial agent that showed 100% sensitivity against S. aureus in the study.
Hence, vancomycin may be used as the drug of choice for treating multidrug resistance-MRSA infections. • Although all isolates were found sensitive to vancomycin, screening test and minimum inhibitory concentration determination are recommended for early detection of impending resistance and monitoring the response to therapy. • Apart from the antibiotic combination for the treatment of MRSA, new treatment options using other antibiotics are slowly emerging. Currently, the only promising option for developing countries is the use of affordable antibiotics in suitable combinations.