*Corresponding author
Marco Antonio Becerril-Flores.
Instituto de Ciencias de la Salud.
Infections by Staphylococcus aureus are a great worldwide public health problem due to easiness of transmission, via inhalation or direct contact in hospitals. Among infected individuals there are asymptomatic carriers that cause propagation and transmission of these infections within the hospital population. In Mexico, studies about prevalence of infections in healthy carriers by this bacterium are scarce. We investigated the prevalence of pharyngeal infections by S. aureus in asymptomatic children (3 to 8 years old) resident in Pachuca, Hidalgo. There were previous old clinic inspections as part of the study wherein 138 healthy children were studied. Samples of exudates from children were obtained and seeded in selective and differential media and they were morphologically identified as S. aureus by Gram staining. Antibiotics resistance was determined for each sample of bacteria. Prevalence of S. aureus infection was 20.29%, 12.69% in girls, and 26.66% in boys. Pre-school children showed a higher rate of infection than those in elementary school but there was not a significant difference between them (Χ2=0.92, p>0.05). More than 40% of S. aureus strains were ß-hemolytic, and half of the bacteria isolated from children in preschool is resistant to any of the antibiotics studied and resistant to at least one of the antibiotics assayed. The greatest resistance was to ciprofloxacin and erythromycin. Finding children infected with S. aureus as healthy carriers may be, in fact an epidemiologic risk for the entire population. Furthermore, since there are resistant strains of this microorganism, physicians must be very sensible of resistant patterns when selecting antibiotics.
Keywords: S. aureus; Prevalence; Antibiogram; Healthy carrier; Bacterial resistance.
Infection by Staphylococcus aureus, transmitted via inhalation and direct contact, is one of the most common bacterial infections in hospital environments1,2. In Mexico, few studies of the transmission of S aureus have been carried out. It has been considered a public health threat. Nosocomial infections with S. aureus occur in 15% of previously studied pediatric populations. Such infections have an estimated mortality rate of 5%. Such infections have also been associated with ventilation tubes in hospital setting.3,4 Other studies have demonstrated methicillin resistant S. aureus strains.5,6 It is of paramount importance to know how commonly S. aureus is found in our environment, as well as its antibiotic resistance patterns. If it is indeed common, it will be most likely to be spread among the population and cause severe problems in people debilitated by different diseases. For example, in 5% of patients with S. aureus infections in upper respiratory tract, the infection can spread to the inferior tract.7,8 Importantly, recurrent infections in pediatric patients can lead to significant morbidity and mortality. Alternatively, they may recover from their illness while remaining colonized with the bacteria and become a healthy-carrier.9-12 Therefore, knowledge of the prevalence of healthy carriers of antibiotic resistant S. aureus infection is crucial to help physicians provide appropriate empiric treatment against infections and avoid creating resistant strains.13-17
The aim of this work was to investigate the prevalence of S. aureus infection in both symptomatic and asymptomatic pre-school and elementary school children, and demonstrate whether S. aureus isolates are commonly resistant to antibiotics.
Study Population
We conducted a prospective, randomized controlled trial in which 138 children aged three to nine resident in Pachuca, Hidalgo were studied, The sample was calculated taking into account the prevalence of people infected with S. aureus and 15% reported in other studies and considering the 5% error and 95% confidence.3-5 The distances between the residences of each child varied for about 15 km. The level of schooling of children was preschool and elementary education. The study was conducted over a maximum period of 15 days during the month of May 2005. Children were tested for previous infections. We recorded whether they had taken any antibiotics and resided in Pachuca, Hidalgo in the previous year. They also went through medical examination to see if they were asymptomatic. Children were enrolled voluntarily with the permission of their parents by authorization with an informed consent letter.
The protocol was reviewed and approved by the Ethics Committee of the Academic Area of the Institute of Medicine and Health Sciences UAEH. The work was performed according to the Regulations of the General Law on Health Research for Health, which classifies this study as minimal risk.
A sample of pharyngeal exudate from each of the volunteers was taken using a sterile swab, which was transported in a sterile 12X100 mm hermetically closed glass tube. Each sample was seeded in a culture medium of nutrient agar in petri dish incubating at 37 °C for 24 hours.
Each isolated colony was Gram-stained, and those colonies of Gram-positive cocci observed under optical microscope were considered for re-isolation. Once this principle was confirmed tests were performed for catalase and coagulase. We selected only those colonies of bacteria that developed on agar S110 resulting golden yellow colonies, and Mannitol salt agar developed in the presence of yellow area, and catalase positive. Except for coagulase which was classified as S. aureus. Isolates of S. aureus were seeded in blood agar in order to classify the bacterial strain according to the type of hemolysis produced, morphology and grouping; then bacteria were plated in agar media S110, agar with mannitol and salt.
Bacterial colonies identified as S. aureus were seeded separately on petri dishes with Muller Hinton agar. We then, applied a Bio-Rad brand susceptibility test multi-disc, with specificity for Gram-positive cocci according to the manufacturer's instructions, in order to test resistance to the following antibiotics: amoxicillin, ceftriaxone, clindamycin, tetracycline, lincomycin, ciprofloxacin, ampicillin, penicillin, erythromycin, trimethoprim-sulfamethoxazole and dicloxacillin. Each bacterial colony was incubated at 37°C for 24 hours. We measured the halos of bacterial growth inhibition for each antibiotic and classified the bacteria as susceptible or resistant according to the instructions of commercial kit’s antibiogram.18
Since it was a descriptive and observational study, records about prevalence were calculated. However, in order to demonstrate statistically significant differences in results between preschool and elementary school children populations, we performed a nonparametric chi- squared statistical analysis.19
Prevalence of asymptomatic carriers of S. aureus is a risk factor for spreading infection of this bacterium in people of a community; therefore we found necessary to record the percentage of infected children. Table 1 shows that pre-school age girls have a lower prevalence of infection with S. aureus than boys with the same age. However, the difference was not statistically significant, as the prevalence for both boys and girls in pre-school was around 20% (X2 = 0.92, p> 0.05). On the other hand, table 1 shows that school age girls are significantly less frequently infected than boys (X2 = 12.74, p <0.04). It also shows that as children get older the rate for asymptomatic children infection increases.
Table 2 shows that half of the bacteria isolated from preschool children are resistant to any of the antibiotics studied, with more than 80% of bacteria isolated from boys being resistant. It also shows that bacteria isolated from girls are resistant to ciprofloxacin and mostly amoxicillin and to ciprofloxacin and tetracycline for boys. Four bacteria strains isolated from girls in elementary education were resistant to at least one antibiotic, erythromycin mainly as well as trimethoprim-sulfamethoxazole, while isolates from boys were resistant to 79% of antibiotics, in most cases to tetracyclines and ciprofloxacin.
According to our results, the prevalence of infection with S. aureus in children without symptoms in both pre-school and elementary education children is about 20%. Our results are similar to previous research conducted by Garcia Callejo et al. although his work has been carried out in other regions. Velázquez MME showed that 18.9% of children in a region of Spain has peritonsillar infection by S. aureus.16 Another study shows that the prevalence of asymptomatic nasal carriage of S. aureus in health personnel of various hospitals ranges from 27% to 38%, and 35 to 50% in tonsillar infections in the same population20-22 In another study of bacterial tracheitis, the prevalence of S. aureus infection was 42%.23 This may reflect that the susceptibility of different populations to infection with S. aureus is similar across different regions, and therefore, is not dependent on strain. To state this in a different manner, the virulence of strains of S. aureus is similar although the susceptibility of the host is different. It is important to note that the population studied in this paper was asymptomatic and was also demonstrated in the work of Sanabria and colleagues. However, peritonsillar populations and tonsillar symptoms was greater.21
The results of our study show some important facts. For example, it is noteworthy that in preschool children the figure is similar for boys and girls. However, there are significant genders differences in children aged 6 and 8 (10 % and nearly 30%, respectively). This outcome may be due to health care and gender related issues because of their own physiology. It must be noted that in girls the prevalence is about 9% at all age groups studied, but in boys the prevalence increases to nearly 30% as they age. The different cultural and personal hygiene habits in girls may result in less close social contact with their peers as compared to boys leading to a lower rate of infection among girls over time. Another explanation may be that girls have better personal hygiene practices, either because their mothers or girls are usually more attentive to personal hygiene as they grow older compared to boys. We must not forget that the main route of transmission is oral-nasal, and with advancing age the child becomes more independent from his parents and may neglect personal hygiene more frequently. We do know that with increasing age children have more contact with adults who could be a carrier of different infections increasing the likelihood of contact with S. aureus. Children may even have gone to hospital units where there are asymptomatic carriers among health personnel.24
Our data about hemolysis type support this explanation since strains of S. aureus isolated from children in primary school had particularly b-hemolysis , indicating thus more aggressive hemolysis and therefore that children were in contact with bacteria transmitted by more people.
With regard to antibacterial resistance by S. aureus, the greatest resistance was to tetracycline and secondly to ciprofloxacin, the latter in pre-school children which means that transmission occurs from an early age. It is noteworthy that strains isolated from girls have no resistance to tetracycline but rather to amoxicillin, and we know that is the most used antibiotic in pharyngeal bacterial infections in young children, which pressure bacteria to acquire resistance. On the other hand, the fact that children from 6 to 8 years old are infected with S. aureus resistant to tetracyclines may indicate that the excessive use of this antibiotic may cause resistance. However, we know that since it is not the most commonly used antibiotic, tetracycline resistance genes are probably present along with other resistance genes that are not commonly used by bacteria. Martin et al. showed resistance to ciprofloxacin in 43.8% and to amoxicillin in 20.3% of S. aureus isolated from Spain inhabitants.25 Furthermore Sanabria et al. found 26.6% of S. aureus strains in blood, and they were resistant to ciprofloxacin and 28.1% to amoxicilin.21 These results are consistent with those obtained in our work because we also found resistance to ciprofloxacin in a large percentage, and from preschoolers to amoxicillin, suggesting that the exaggerated use of antibiotics causes resistance to S. aureus. Vazquez et al. found similar results to ours (erythromycin resistance in 21%). Likewise, we found resistant bacteria in 17 % and 18% of children in pre-school and basic education children, respectively.23 This may suggest that the presence of S. aureus strains in a population depends on many factors such as phenotypic and genotypic characteristics of the bacteria, as well as other human factors such as measures of infection control, and above all antimicrobial treatment schedules.16 Administering antimicrobial treatment inappropriately may result in an exaggerated rise to the selection of strains that are resistant. Subsequent spread in population may lead to resistant S. aureus being the prevailing strain in a region. This may explain the resistance to amoxicillin and ciprofloxacin, two of the most commonly used antibiotics against gram-positive bacterial infections such as S. aureus. We suggest the use of antimicrobial susceptibility testing of throat infections before the administration of an antibiotic.26,27 Future research about the origins of resistant strains of S. aureus remains an important issue for further investigation, as previous work demonstrated that farm animals could be carriers of the bacteria.28-30
Table 1. Prevalence of infection with S. aureus in boys and girls, and the type of hemolysis presented by each strain of bacteria.
N: total number of boys and / or girls studied.
Table 2. Resistance to antibiotics of S. aureus bacteria isolated from the individuals studied.
No. of bacteria with the capacity of resistance / No. bacteria (percentage of resistant bacteria). Amoxicillin (AX), ceftriaxone (CX), clindamycin (CD), Tetracycline (TT), lincomycin (L), ciprofloxacin (CF), Ampicillin (AC), penicillin (P), erythromycin (E), trimethoprim-sulfamethoxazole (TS ) Dicloxacillin (DX).
Our results suggest that S. aureus is a bacterium with significant public health implications, especially since it has become resistant to multiple antibiotics and infects from early ages.
S. aureus has the potential to cause serious problems, both at an individual and a public level, such as in an epidemic with resistant bacteria.
Acknowledgements
We acknowledge the valuable collaboration of QTLC. Patricia Gonzalez Ramirez for her technical support during the implementation of techniques for identification of S. aureus.