Prevalence of Pathogenic Bacterial Isolates Infecting Wounds and their Antibiotic Sensitivity

Wound infection is one of the health problems that are caused by pathogenic bacteria and antibiotics resistance profiles of Gram negative bacteria increased treatment cost especially in diabetic patients. Thus, the objective of this work was to isolate, purification and identification of pathogenic bacteria from wound infection then determination of susceptibility testing against antibiotics. 41 isolated from wound infections were collected from microbiology laboratory of Zagazig University Hospitals and some private microbiology laboratory Cairo, Egypt. Bacterial isolates were identification by API 20E Enterobacteriaceae. These pathogenic bacterial isolates belong to these genera (Klebsiella, Enterobacter, Pseudomonas, Providencia, Serratia and Citrobacter). The frequency of the bacterial isolates was 24%, 20%, 14%, 12%, 6% and 6% of the bacterial pathogens isolates in this study respectively. Finally, the antimicrobial susceptibility was performed by disc diffusion method against 12 types of antibiotics covered all mode of action of bacteria.


Introduction
Wounds infection by bacteria and resistance to common antibiotics are the common post-surgical and medical challenges. Wounds bacterial contamination are the common hospital acquired infections causing more than 80% of the mortality [1]. The most common bacterial genera infecting wounds are Enterococci, Escherichia, Pseudomonas, Klebsiella, Enterobacter, Proteus and Acinetobacter [2,3]. Wounds infection have been a recognized as the most critical problem especially in the presence of foreign materials that increases the risk of serious infection even with relatively small bacterial infection [4]. Nosocomial infection is usually higher in burn patients that correlate with other factors like nature of burn injury, age of patient, extent of injury and burn depth. Other microbial factors such as type, number of organisms, enzymes, toxins production, colonization of the burn wound site, systemic dissemination of the colonizing organisms, have a strong effect on severity of bacterial wound infection [5,6]. As well as, widespread using of vast groups of antibiotics together with the length of time causes a significant development of antibiotic resistance to wound infecting bacteria [7], that subsequently increase the complications and costs of treatment [8].
Thus, the objective of this work was to isolate and characterize various bacterial isolates infecting wounds, and determination of their susceptibility to various common antibiotics.

Sample collections
Swabs from abscesses lesions or pus were collected from microbiology lab. It was cultivated immediately without delay. Each sample was inoculated into nutrient agar medium and McConkey agar media. The plate cultures were incubated for 24 h at 30-37°C [5] and the growth was observed thoroughly.

Identification of isolated pathogenic bacteria
Identification of Gram negative pathogenic bacterial isolates were carried out by API 20E Enterobacteriaceae (BioMérieux Co.). API system has been recognized as a rapid test for bacterial identification. The strips were inoculated by single colony in the suspension media and incubated at 35-37 °C for 18-24 h.The results were recorded based on the special chart, three tests were given a code number, the obtained seven digit numbers were expressed to the corresponding organisms regarding to the API index.
(Oxoid, ThermoFisher, Scientific, USA), were used. After incubation, the diameter of inhibition zone around each disk was measured and the organisms were nominated as sensitive (S), resistant (R) and intermediate resistant (MR).

Results and Discussion
Isolation, identification and prevalence of pathogenic bacterial isolates Bacterial wound contamination is a serious problem in the hospital and the treatment of wound infections remain a significant concern for surgeons. The risk of developing wound infection depends on the number of bacteria colonies on the wound. The problem has been magnified due to the unrestrained and rapidly spreading resistance to the available array of antimicrobial agents. Fifty cases from wound swabs were collected from different types of wounds, cultured on nutrient and MacConkey media. Fifty bacterial isolates were isolated from wounds infection out of the total cases. These bacterial isolates were identified based on the API 20E system. From the results, there was 41 cases (82%) recorded as a positive bacterial growth and only 9 samples were recorded as negative bacterial growth. Gram positive bacteria already isolated in nutrient agar media but the majority for Gram negative bacteria so that used the Gram negative bacteria for application. The identified forty-one pathogenic bacterial isolates were belonging to sex genera (Table 1). These genera were Klebsiella pneumoniae (12 isolates; 24%) followed by Pseudomonas fluorescens (10 isolates; 20%), Providencia stuartii (7 isolates; 14%) and Enterobacter cloacae (6 isolates; 12%) and Serratia rubidaea and Citrobacter freundii (3 isolates; 6%). Similarly, high percentage of microbial growth was reported by Mama et al. 2014 [11], 91.6% of culture was positive microbial growth and 12.7% had no bacterial growth. Coincident results for K. pneumonia (24%), P. fluorescens (20%), P. stuartii (14%) and E. cloacae (12%), S. rubidaea and C. freundii (6%) was reported [12]. Among the recovered isolates, Pseudomonas species were the most common isolates (48.9%) followed by Citrobacter spp (13.3%), Enterobacter spp (11.1%), Proteus vulgaris (6.6%), Klebsiella spp (2.2%) and Serratia rubidia (2.2%) infecting wounds [12].

Antibiotic susceptibility of the pathogenic bacterial isolates
The antibiotic sensitivity of isolated bacterial strains was carried out by Kirby-Bauer disk diffusion assay against 12 antibiotics (  The frequency antibiotic resistance of K. pneumonia isolates was summarized in Figure 1        Antimicrobial susceptibility pattern of bacterial isolates was tested against selected 12 antibiotics. From Table 2 The results obtained showed that the bacterial isolates varied in their susceptibility to all the antibiotics and showed that maximum sensitivity for  Table 3: Antibiotic sensitivity/Intermediate resistance pattern (%) of gram negative bacteria isolated from wound infection. Klebsiella pneumoniae was (100%) resistance to Rifampicin, (91.7%) in Sulphamethoxazole/ Trimethoprim, (83.3%) in both (Cefotaxim and Cefepime), (75%) in Ceftazidim, (58.3%) in Tobramycin, (50%) in Amoxicillin/ Clavulanic acid (41.7%) in Amikacin, (25%) in both of (Ciprofloxacin, Nitrofurantoin and Levofloxacin) and (16.6%) in Colistin Sulphate. Similar results were reported for sensitivity and resistance of Gram negative bacteria against selected antibiotics (Figure 7).
In conclusion, wound infection by pathogenic bacteria and increasing antibiotics resistance are of the most serious health threats facing the patients, especially diabetic foot patients. Thus, the objective of this work was to isolate and identify pathogenic bacteria infecting wounds. Antimicrobial susceptibility of the isolated pathogenic bacteria to different antibiotics covering all mode of action of antibiotics was conducted.