PREVALENCE OF BACTERIAL PATHOGENS ASSOCIATED WITH WOUND INFECTIONS FROM DIABETIC OUT-PATIENTS AT PLATEAU SPECIALIST HOSPITAL, JOS, NIGERIA

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INTRODUCTION
A wound is a type of injury which happens relatively quickly in which skin is torn, cut or punctured (an open wound) or where blunt force trauma causes a contusion (a closed wound) (Adegoke et al., 2010). In pathology, it specifically refers to a sharp injury which damages the dermis of the skin and therefore compromises its protective function, as a result wound can be contaminated by microorganisms like bacteria and infection occurs when such organisms multiply and cause damage (Bowler et al., 2001;Arabishahi and Koohpayezade, 2006). Infection also occurs when virulence factors produced by the microorganisms overwhelm the host natural resistance (Bowler et al., 2001). Wound infection is characterized by the presence of pus in lesions with pyrexia, pain and induration. The unbroken skin is the first line of defence and a barrier against microbial invasion which serves as host to a variable number of transient or contaminating bacteria (Stephan and Landis, 2008). Although its low surface pH, sebaceous fluid, and fatty acids produced inhibits the colonization and growth of pathogenic organisms, exposure of subcutaneous tissue following loss of skin integrity provides a moist, warm, and nutritious environment that is conducive for microbial colonization and proliferation (Bowler et al., 2001). The contamination or mere presence of pathogenic organisms in wound without local or systemic tissue involvement may not result in infection (Sule et al., 2002). The organisms can cause destruction if left untreated (AWMA, 2011). Bacterial wound infections are important because they can slow down the healing process, lead to wound breakdown, prolonged hospital stay and increase in the cost of treatment (Alexander, 1994;Sule et al., 2002). High index of wound contamination with bacteria are the third most frequent nosocomial infections (exposure to dirty environment, contaminated water or materials used for treatment and even from the hospital) (Dionigi, et al., 2001;Ducel et al., 2010).
However, the clinical significance of the type of microorganism present is reduced if there are limited signs of infection, which is common in people with infected diabetic ulcers (Bowler 2001). Delayed healing is more likely to occur in people with diabetic foot infections, even when less pathogenic microorganisms are present (Williams et al., 2018).
Epidemiological surveillance of infection is indispensable for effective management of diseases, and the creation and implementation of control measures. High prevalence of open wound infections has been reported as a matter of national health emergency (Mofikoya, 2009). Studies have shown that most hospitals in developing countries especially Africa, have rudimentary and highly compromised infection control programmes due to lack of awareness of the problem, lack of personnel, poor water supply, erratic electricity supply, poor laboratory back-up and funding, traumatic and surgical site infections (Fadeyi et al., 2008;Mehta et al., 2007;Anguzu and Olila, 2007;Samuel et al., 2010). These factors are rife in most rural health care centres in Nigeria, and underscore the need for this study. The etiology of wound infection, regional and local variations occur among causative micro-organisms from country to country and from hospital to hospital even within the same region (Wariso and Nwachukwu, 2003;Biadglegna et al., 2009;Egbe et al., 2011). With the surge of multi-drug resistant bacteria, health care providers have become increasingly challenged in effective wound management (AWMA, 2011). Accurate information of the incidence and etiology of infections acquired within a hospital is essential for articulation of effective preventive or management measures (Akinjogunla et al., 2009;Sanjay et al., 2010). Against this background, this study was aimed at determining the prevalence, causative organisms and profile of associated bacterial isolates among out-going patients in Plateau State Specialist Hospital, Plateau State, Nigeria.

STUDY AREA
Plateau is a state in the Middle-belt region of Nigeria with a population of about 3,206,531 people (NPC, 2006;NBS, 2009  South are farmers with few civil and public servants, teachers and students making less than 10% of the community.
Plateau Specialist Hospital is the only State-owned tertiary health care provider in Jos South, Plateau State. Some people from all neighbouring LGAs also attend the Hospital.

STUDY POPULATION AND DESIGN
Clinical samples of cases and symptoms of wound infections from both male and female volunteers with age range between 18-80 years in the out-patient departments (OPD) of Plateau State Specialist Hospital (PSH) were recruited for this study. The prospective, hospital-based parallel studies from clinical cases that suffered diabetic wound infections ran for 6 months from July 2019 to December 2019.
Subjects were recruited upon approval by the Ethical Research Committee of Jos University Teaching Hospital (JUTH) and Plateau State Specialist Hospital, Jos, Nigeria. Verbal informed consent was obtained from all participating subjects prior to specimen collection. A structured questionnaire was administered to study subjects for information on demographic data (age, gender, marital status, site of wound, wound type and the type of prophylactic antibiotics). Subjects who could read and write completed the forms while the semi literates and the illiterates were assisted following verbal response to questions.

CULTURE MEDIA AND PREPARATION
Media used include MacConkey agar, blood agar, Manitol salt agar, Cetrimade agar, Simmon citrate, Sabouraud dextrose agar, of Urease agar, Nutrient agar, Mueller Hilton Agar and Peptone water were prepared according to the method adopted by Microbiology Society (MS 2016).

COLLECTION OF SPECIMENS
Wound swabs and pus were obtained by doctors and nurses in the clinics and wards, and by Medical Laboratory Scientists in the laboratory using commercially available sterile cotton swabs from 220 subjects suspected to be infected or suffered diabetic clinical cases of wound infections were included in the study. Suspicion was dependent on the history of patients with delayed or inability of wounds to heal properly, swelling, foul odour, discharge, enlargement of wound lesions alongside exudation and painful wounds in Plateau Specialist Hospital (PSH), under standard universal aseptic precautions. Only two swabs per patient were collected. The samples collected were transported immediately to the Department of Clinical Microbiology Laboratory at the Faculty of Pharmaceutical Sciences, University of Jos to prevent drying (Pondei et al., 2013).

ISOLATION AND IDENTIFICATION OF ISOLATES
Bacterial isolates were identified using standard laboratory techniques (Collee et al., 1996;Sule et al., 2002;Barrow and Feltman, 2003;Cheesbrough, 2006;Cappuccino and Sherman, 1996;MS, 2016). One of the wound swabs was used to make film and stained by gram's stain. The second isolates from the swabs were immediately inoculated on blood, MacConkey, Centrimide and Mannitol Salt agar and incubated aerobically and in carbondioxide jar for 24 to 48 hours at 37°C (Pondei et al., 2013). Anaerobic cultures were not done due to lack of anaerobic jar in the laboratory at the time of this study. Bacterial isolates were isolated and identified based on the streak plate method and colonial characteristics, Gram staining and biochemical tests. Candidiasis was diagnosed by the presence of yeast-like cells as well as identification of isolates from culture. All yeast isolates were inoculated on CHROM Agar Candida™ and incubated for 48 hours at 37 o C. The colour produced by each colony was used to identify the yeast.

ANTIBIOTIC SUSCEPTIBILITY TESTING AND STANDARDIZATION OF ISOLATES
Antibiotic susceptibility testing was done using the Kirby-Bauer Disc Diffusion Method on Mueller-Hinton Agar (MHA) and Mueller-Hinton Broth (MHB) in distilled water at 15 lbs psi for 25-30 min. They were further maintained on the different prepared media at 37°C for 48 hours and stored at 4°C, and interpreted according to the recommendations of the Microbiology Society's Standards (MS 2016) and (Howe and Andrews, 2012). Exactly 0.2ml of overnight cultures of each organism was dispensed into 20ml of sterile nutrient broth and diluted to standard inoculum of 10 6 cfu/ml. A loopful of the standard inoculum was used for the antimicrobial assay (Collins et al., 1995).

STATISTICAL ANALYSIS
These parameters were tested in triplicates. Data obtained in the study were analysed with Epi Info 2012 software. Differences in infection rates between age groups and sex were analysed using χ2 test. A p-value ≤ 0.05 was considered significant. Percentage susceptibility was calculated.

Total number of patients and demographic data
A total of 220 (108 females and 112 males) were recruited from clinical cases and symptoms of wound infections from both male and female volunteer in the out-patient departments (OPD) of Plateau State Specialist Hospital (PSH).
The age range of the study population was from 18 -80 years.

PERCENTAGE FREQUENCY OF OCCURRENCE OF WOUND BACTERIAL ISOLATES
The result of the percentage frequency of occurrence of the organisms showed the presence of six (6)  ( Table 1).
The frequency and percentage distribution of wound bacterial infection among males and females' sample in out-patients from 2019 did not differ significantly (p>0.05). However, male patients had a slightly higher prevalence than female patients in the year 2019 as indicated in Table 1. Age and gender affected the prevalence of wound bacterial infection among male and female outpatients as shown in Table 1 &2.

SENSITIVITY TEST RESULT
The fluoroquinolones (antibiotics used in treatment of systemic infection by interfering with DNA replication) were the most active antibacterial agents against bacterial isolates from in and out patients studied. A generally higher resistance pattern was observed among nosocomial bacterial pathogens as indicated in Table 2. Gentamycin most effective against aerobic Gram-negative rods.    Pseudomonas aeruginosa (

DISCUSSION
In this study, the findings demonstrated the predominance of Pseudomonas aeruginosa Staphylococcus aureus and E.
coli. P. aeruginosa was the commonest isolate, accounting for 8 (26.7%), followed by coagulase negative Staphylococcus aureus and Escherichia coli with 7 (23.3%) and 6 (20.0%) respectively. These results were in agreement with results from previous studies conducted globally and in various parts of the country (Isibor et al, 2008).
However, the result of this study is at par with Oladeinde et al. (2013) and Imarenezor (2017) who reported that S. aureus was the most prevalent pathogen with the frequency of occurrence (62.7% and 52.8%) followed by P.
aeruginosa (23.1% and 9.9%) in male and female subjects when compared with the result of our investigation respectively.
Findings from Adegoke et al. (2010) carried out at a University hospital in Nigeria showed that the commonly isolated bacteria were S. aureus (25%) and P. aeruginosa (20%) which when compared with the result of our investigation are of lower prevalence rate. The high prevalence of P. aeruginosa among subjects may be attributed to contamination of wounds with soils, medical devices or the existence of locality variability. Ogba et al. (2014) also reported in their study of bacterial pathogens associated with wound infections in Calabar, Nigeria that Staphylococcus aureus was the most prevalent pathogen 91 (30.8%) among both category of patients, followed by Pseudomonas aeruginosa 51 (17.3%). Streptococcus species were the lowest isolated pathogens 4(1.3%) agreed with this result. Another study at a University teaching hospital in Iran, also reported Staphylococcus aureus to be the commonest bacteria isolated (43%). These slight variations may be due to the difference in study areas and populations. Although a number of studies have been conducted on wound infections in some hospital and clinics in Nigeria, to the best of our knowledge, none has been done in a tertiary health care facility (Plateau Specialist Hospital) on the prevalence of wound infections among diabetic out patients in Jos South LGA, Plateau State.
Jos South and other neighbouring communities are strictly urban settings with inhabitants being largely civil servants and farmers. In other words, the difference may be due to the fact that subjects enrolled for the study had wounds and are diabetic patients suspected to be infected as opposed to all wounds examined by many reported studies by scientists (Ogba et al., 2014).
The bacteria isolates obtained in this study includes pathogens such as E. coli known to cause food poisoning, diarrhea, abdominal pain, fever, nausea, vomiting and about 75% to 95% of urinary tract infections. E. coli infection is contagious and can be spread from person to person by faecal contamination. K. pneumonia that causes health careassociated infections in form of pneumonia, sepsis, wound and urinary tract infections (Long et al., 2017). It has caused a global explosion of drug-resistant and are increasingly difficult to treat because they are resistant to many of the available antibiotics (Dromigny et al., 2005;Long et al., 2017). Until now, K. pneumonia was thought to be the culprit in most Klebsiella infections. It is also capable of causing invasive and severe infections in patients with the same rate of mortality as other species of the Klebsiella. Our finding is in agreement with the work of Long et al. (2017), who stated that three Klebsiella species caused life threatening infections and shared drug resistance genes in a study in Houston, Texas.
Furthermore, any P. aeruginosa already present on a burn victim's skin before the injury can transform from an innocuous organism on the surface of the skin to a source of infection in the bloodstream and body tissues of the same individual (Fick, 1993;Lyczak et al., 2000). High rate of occupational, nutritional, unhealthy life styles related diseases, diabetics, poor hygiene and accessibility to health care facility may be responsible for the observed trend.
In this study, we observed that there was a link between age and gender of the diabetic wound infections in outpatients. Age did not significantly affect the prevalence of wound infection in our study. However, gender affects the prevalence of the wound infection from our study. These have been previously confirmed in two separate Nigerian studies by Ezebialu et al. (2010) and Egbe et al. (2011). This finding does not agree with the study by Ogba et al. (2014) who reported that age have no bearing on wound infections with people in their second to fourth decades of life being more prone since this is the most productive age range. This is inconsistent with the report of Pondei et al (2013), but different from the reports of Egbe et al (2011) in Bayelsa, Nigeria.
Generally, antibiotic susceptibility of isolates to commonly used antibiotics was low; the quinolones (Ciprofloxacin), Augmentin and Gentamycin were the most potent antimicrobial agents observed in our study.
Meanwhile, a high level of resistance of isolates tested with Doxycycline and Chloramphenicol was also observed.
This may be attributed to the fact that Doxycycline and Chloramphenicol derivatives have been widely abused and frequently implicated in self-medication in Nigeria. The multiple resistance of isolates, especially S. pneumonia, K.
pneumonia and P. aeruginosa to commonly used antibiotics in the locality of the study calls for an immediate action on the controlled use of antimicrobials in the hospitals and the need to monitor resistance. Good antimicrobial use is necessary for effective wound management. The susceptibility rate of bacterial isolates observed in this study, agrees with the reports of Ogba et al. (2014), Sule et al (2002), Oladeinde et al. (2013) and Pondei et al (2013) in Nigeria and Anguzu and Olila (2007) in Uganda. Odugbemi (1981) and Pondei et al. (2013) also reported that high level of antibiotic abuse in Nigeria arise from selfmedication which is associated with inadequate dosage and failure to comply with treatment regimen. These antibiotics are being sold over the counter with or without prescription (Anguzu and Olila, 2007;Oladeinde et al., 2013;Ogba et al., 2014).

CONCLUSION AND RECOMMENDATION
This study has shown a high index or prevalence of wound contamination of diabetes patients with P. aeruginosa and S. aureus being the most predominant etiologic agents of wound infection observed among out-patients with significantly higher prevalence of 8(26.7%) and 7 (23.3%) respectively. The flouroquinolones were the most active antimicrobial agents observed among bacterial isolates from out-patients studied. S. pneumonia, K. pneumonia and P.
aeruginosa showed low susceptibility to Doxycycline and Chloramphenicol which are commonly used in our locality.
However, a generally higher antimicrobial resistance pattern was observed among diabetes patients in our study.
In conclusion, P. aeruginosa was confirmed to be the most prevalent bacteria in diabetic wound infection of out-patient from this study. Contracting wound infection remains an ongoing problem when an injury is sustained and the rate of getting infection when a wound is contaminated is greatly increased, when risk factors are present. For example, diabetic patients are at high risk of getting infection because they have a weakened immune defence. To avoid wound infection in diabetic, one of the most importance things to do, is to practice careful foot care, in addition to wearing shoes and socks to avoid minor bumps and scrapes. the feet should be examined daily for any blisters, cuts, scrap, sore or other skin problems that could allow an infection to develop. Wound specimens should also be collected for culture and susceptibility testing to help the clinicians before appropriate antibiotic selection and chemotherapeutic management of wound infections is recommended. It is essential that people who have diabetes keep their blood sugar levels under control to reduce the risk of slow-healing wounds and complications, including foot ulcers. Hence good hygiene and proper care of wound infection plus in cooperation of antimicrobial drugs during treatment is recommended. The multiple antibiotic resistance of isolates calls for an immediate action on the control of antimicrobial usage and the need to monitor resistance.