Case Profile Analysis on Health- Care Associated Staphylococcal Infections and Community Acquired Sources: Demographic and Clinical Surveillance Study Ile – Ife South Western Nigeria

We looked into the frequent incidence of Staphylococcal infections among some clinical diagnosed infectious diseases reported cases at the Obafemi Awolowo University Teaching Hospital Complex, Ile – Ife, Nigeria and its community-based involvement. Eight hundred and fifty samples of different cultures were taken from hospital and community sources. The clinical sources were the routine specimens of wound swabs, urine, stool, blood and sputum from the Department of Microbiology and Parasitology laboratory of the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC) Ile–Ife. The non-clinical samples were obtained from the nasal cavity of apparently healthy food handlers at restaurants in Obafemi Awolowo University campus and food vendors in Ile–Ife central market. Samples were cultured on mannitol salt agar and incubated at 37C for 24-48 hours. Staphylococcus aureus were isolated and identified based on mannitol fermentation, Gram’s reaction, positive results for catalase, coagulase and DNAse tests. The data generated were subjected to statistical analysis using TTest. Two hundred and thirty (56.8%) of S. aureus isolates were recovered from the hospital sources and 175 (43.2%) from the community setting. Incidence rate was the highest in age range 21-30 among urine, wound, sputum and blood Case -based samples analyzed. Urine S. aureus Case-based infection in female (58.6%) was higher than (41.4%) in male, Sexually Transmitted Disease (STD) reported cases was about more than doubled of other infections follow by the urinary tract (UTI) independent infection. Wound associated casebased infections among Female (65.5%) was doubled that of Male which was (34.3%) and sepsis independent cases constituted 32.8%; Sputum based S. aureus infection in female was 56.8% higher than 43.2% in Male. 66.7% from Pulmonary Inflammation Casebased investigated constituted overwhelmingly more than double of other infections. In addition, (63.8%) Stool associated Case-based infections from Female was higher than 36.2% observed among Male. Diarrhea cases constituted majorly of 38.3% and it showed an exceptional incident rate of infections which was noticed to be higher among the age range 11-20. Blood S. aureus associated infections in female (53.3%) was higher than 46.7% in Male and bacteremia/ sepsis cases predominated about 63.3%. (T= 95% confidence interval of the difference). Community S. aureus isolates accounted for 43% of the total isolates from which cell phones and food handlers constituted 15%, and stethoscopes S. aureus isolates 13%. The carrier rate of S. aureus in the nose of apparently healthy individuals among the food handlers in the community was higher among Male (64.5%) than 35.5% Female. (T=95% confidence interval of the difference). Plan are underway to evaluate the relationship between antibiotics use in this hospital and the pattern of antimicrobial resistance observed.


Introduction
Staphylococcus aureus had been isolated from several clinical specimens from Nigeria (Esan et al., 2009). In 2003In -2004, approximately 29% (78.9 million persons) and 1.5% (4.1 million persons) of the US population was colonized in the nose with S. aureus and Methicillin Resistant Staphylococcus aureus (MRSA) respectively (Gorwitz et al., 2008). Over the past several decades, S. aureus has been a leading cause of hospital acquired infections (Lowy, 1998). It is also associated with wound sepsis, osteomyelitis and post surgical toxic shock syndrome with substantial rates of mobidity and mortality (Shopsin et al., 2001;Engemann et al., 2003). Methicillin-resistant S. aureus infections have become a common problem in the community acquired infections, and has also been associated with prolonged hospital stay and increased costs ( Kopp et al., 2004;Lodise and Mckinnon, 2005;Nixon et al., 2006).
In 2005, there was an estimated 478,000 hospitalization with a diagnosis of S. aureus infection in US hospitals, and of these, approximately 278,000 hospitalizations were related to MRSA. These include, people admitted to the hospital for treatment of an infection that was acquired or occurred outside the hospital. (Klein et al., 2007). In hospitals, the proportion of healthcare associated staphylococcal infections that are due to MRSA has been increasing and 2% of S. aureus infections in US intensive care units were MRSA in 1974, 22% in 1995, and 64% in 2004(Klevin et al., 2006. One of the reasons for the success of this human pathogen is its great variability in occurring at different periods and places with diverse clonal types and antibiotic resistance patterns within region and countries. `Infections caused by antibiotic-resistant S. aureus bring about serious problem in the general population, such infections can be particularly devastating for the very young, the elderly and the immunocompromised (American Society of Microbiology, 2007). Staphylococcus aureus strains colonise and establish infection in a wide range of body sites, including the blood, indwelling biomaterials, mucosa surfaces, bone, and other tissues. The mechanisms underlying the tropism of S. aureus for specific infection sites are unclear, associations have been demonstrated between the disease type, the pattern of toxin genes, and the genetic backgrounds of particular S. aureus strains (Jarraud et al., 2002). For example, a correlation has been found between unrelated cases of S. aureus infection and a single subset of strains, such as a clone producing TSST-1 responsible for most epidemiologically unrelated cases of urogenital toxic shock syndrome (Musser et al., 1990), a clone producing Panton Valentine Leukocidin (PVL) and causing neurotic pneumonia (Gillet et al.,2002), and clones producing Toxic Shock Syndrome Type 1 and enterotoxin C in neonatal toxic shock syndrome-like exanthematous disease (Kikuchi, 2003).
Patients and individuals that are at high risk of developing Staphylococcal infections include surgical patients, the ederly, neonates, diabetic patients and patients with chronic illness (Huda et al., 2011) and immune-compromised patients such as cancer and HIV/AIDS patients others include patients with indwelling devices such as catheters, trauma, burn patients and kidney dialysis patients (Klevens et al., 2007). S. aureus infections can be spread through contact with pus from an infected wound, skin to skin contact with an infected person by producing hyaluronidase that destroys tissues, and contact with objects such as towels, bed sheets, clothing, or athletic equipments used by an infected person. Deeply penetrating S. aureus infection can be severe. Prosthetic joints put a person at particular risk for septic arthritis, and staphylococcal endocarditis and pneumonia (Huda et al., 2011).
This study marked the cooperative effort for surveillance among health-care institution, academia and Ile -Ife central community and was designed to isolate and identify S. aureus in samples stocks of various patients' with related case histories in Academic Teaching Hospital sources viz, clinical ; (stool, blood, urine, sputum, wound), and nonclinical , viz food handlers and cell phones and doctors' stethoscope. Univariate analysis of correlated variables was done with T-test Statistical Significance.

Source of bacterial isolates
Staphylococcus aureus isolates were recovered from both clinical and non-clinical specimens. The clinical sources were from the routine specimens of wound swabs, urine, stool, and sputum of different diagnosed patients' samples submitted to the Microbiology laboratory of the Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), (Urban Centre), Ile -Ife. The non-clinical isolates were recovered as nasal swabs from food handlers at the Obafemi Awolowo University (OAU) campus restaurants, marketers at the Ile-Ife central market and also from fomites within the hospital, which comprised of doctors stethoscopes, and cell phones from the community dwellers and the Health care workers.

Samples collection
Samples were collected between the period of October 2007 and November 2009. Eight hundred and fifty (850) swab samples from clinical and nonclinical sources at Obafemi Awolowo University Teaching Hospital Complex; OAU Campus community and Ile-Ife environs were obtained. Sterile cotton-tipped applicators (Sterilin, England) appropriately moistened with sterile distilled water were used for swabbing sample surfaces.
Samples from both hospital ( hospitalized patients who were on different types of antibiotic treatment) and the community with different sexes, age ranges and of different diagnostic infections histories ranging from diabetic ulcers, cancers (breast cancer, prostrate carcinoma) obstructive uropathy, (e.g benign prostrate hypertrophy (BPH)) septicaemia, urinary tract infection, burnt injuries, gastroenteritis, pelvic inflammatory diseases, sexually transmitted infections, pneumonia and many other clinical diagnosis cases were considered.
The non-clinical samples were obtained from the nostrils of apparently healthy community food handlers, also from cell phones and stethoscopes. The swabs after collection were taken to the laboratory immediately for bacteriological analysis.

Microbiological analysis
Isolation of S. aureus was done by standard procedure in which the samples were inoculated on freshly prepared mannitol salt agar plates (Oxoid, Basingstoke, Hampshire, England) and incubated at 37 o C for 24h. Golden yellow colonies on Mannitol Salt agar (MSA) after the incubation period were taken presumptively for S. aureus.

Phenotypic and biochemical identification of the isolates
The isolates were Gram stained as described by Olutiola et al. (1991). Biochemical identification of the isolates was carried out using standard methods The following biochemical tests were carried out on the isolates namely catalase, tube coagulase test, and the DNase test using DNase agar based (Oxoid Ltd., Basingstoke, Hampshire, England). The confirmed isolates were stored as stock culture on nutrient agar slants and kept at about 4°C until further use.

Gram's reaction Test
Gram staining technique is a differential staining procedure that separate bacteria into two classes i.e Gram positive and Gram negative. A smear of an 18-24 hour old cuture on nutrient agar was prepared on a clean microscopic slides. The smear was then heat-fixed by passing the slide through a bursen burner flame. The smear was flooded with crystal violet and allowed to react for 1 minute after which the stained was poured of and the smear rinsed under gentle running tap water.
Thereafter, the slide was flooded with Gram's iodine solution ( a mordant) and then rinsed off under gentle running tap water. The smear was later decolorized with 95% ethanol, rinsed under gentle running tap water, and counterstained with safranin for about 30 seconds. The slide was then washed under gentle running tap, allowed to air dried and then examined under the oil immersion objective of the light compound microscope (Leica Gallen 111; Leica Inc, NY, USA). Gram positive staphylococci appeared as round clustered and purple in color.

Catalase test
This test was used to differentiate between staphylococci (catalase positive) and streptococci (catalase negative). Catalase positive bacteria are capable of producing an enzyme known as catalase which breaks down hydrogen peroxide to water and two drops of 3% hydrogen peroxide was placed on a clean grease-free slide. Colonies of the isolate similar to Gram positive cocci in clusters was emulsified in the drop. Rapid effervescence of gas was recorded as positive. A control slide containing only drop of hydrogen peroxide showed no gas bubbles.

Coagulase test
The isolates were inoculated into 1ml of nutrient broth in test tubes and incubated overnight at 37°C. One milliliter of fresh human plasma was added into the tubes previously incubated with the isolates and further incubated at 37°C and were examined at intervals for 4 hours. Formation of clot up to 4hr at 37 °C indicates positive coagulation (Olutiola et al., 1991). ATCC25923 serves as control strain.

DNase test
This is a confirmatory test for S. aureus based on its ability to produce DNase enzyme that can degrade nucleic acids. Thirty nine grams of the agar was suspended in 1 litre of distilled water and dissolved completely by boiling. The content was sterilized by autoclaving at 121ºC for 15 minutes. The plates were inoculated by spotting the bacterial culture onto the surface of the agar so that a thick plaque of growth became evident after 18 hours incubation. The plates were flooded with 1N HCl and allowed to stand for 2 minutes. The clear zones around the colonies were taken as positive result for the growth of S. aureus.
Staphylococcus aureus ATCC25923 and S. epidermidis served as positive and negative control.
The highest rate of isolation of S. aureus isolates from clinical sources was from wounds (14.3%) and stools samples (14.3%), while cell phones 15% and food handlers (15%) constituted the highest among nonclinical S. aureus isolates. Overall, the prevalence of S. aureus isolates recovered from clinical sources are of statistical different (T = 0.141). Table 2 shows the colonial morphology and biochemical identification of S. aureus isolates from clinical and non-clinical sources. The observation of golden yellow colouration showed fermentation of the mannitol salt agar by the Staphylococcus sp. The isolates appeared purple coloration with Gram stain (Gram positive cocci). Rapid effervescence of gas recorded showed positive catalase reaction coupled with positive tube coagulases. All the 405 S. aureus were DNase positive.  The frequency of urine S. aureus infections in relation to the age range and sex is presented in Table  3 Fifty eight samples were confirmed of S. aureus infections of which 24 (41.4%) were males and 34 (58.6%) were females.             Table 9. Distribution of Stool infections in relation to age and sex     (2008) in the work conducted on prevalence of MRSA among orthopaedic patients at large academic hospital that statistically significant increases in the incidence of MRSA per year occurred in women whereas the increase in MRSA incident was not significant for men. In this report, it is worth noting that incidence rate was the highest in age range 21-30 among urine , wound, sputum and blood samples analyzed which was in agreement with Harptuluoglu et al. (2005) which revealed that the rates of S. aureus carriage tends to decrease with age. Diarrheal stools analysis showed an exceptional incident rate of infections which was noticed to be higher among the age range 11-20 and this might likely be associated with infant diarrhea. The limitation of this finding was that the underlying risk factors of individual patients' from which clinical samples were obtained was not known.
Community source S. aureus isolates accounted for 43% of the total isolates from which cell phones and food handlers constituted 15%, and stethoscopes S. aureus isolates 13%. In this finding, the carrier rate of S. aureus in the nose of apparently healthy individuals among the food handlers in the community was higher among male (64.5%) than female 35.5% ( T=95% confidence interval of the difference). The present finding was at variance to the work conducted by Shanmugan et al. (2008) on the prevalence, antibiogram and characterization of S. aureus including MRSA among the healthy staff, and patients from Sri Manakula Vinayagar Medical College and hospital (SMVMCH) Pondicherry, that the carrier rate of S. aureus in the nose of male and female healthy carrier among medical students are nearly same which are 24.4% and 23.5%, respectively. The incident rates of nasal infections among the food handlers surveyed individual was higher among age groups 21-30. Plans are underway to evaluate the relationship between antibiotic use in the hospital and the pattern of antimicrobial resistance observed Periodic epidemiological studies must be encouraged to determine the interrelatedness of community/ hospital sources of isolates in Nigeria, establishing the pathogens biodiversity to assist infection control measures.