Detection of bacterial pathogens in surgical site infections and their antibiotic sensitivity profile

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Available online at ISSN No: International Journal of Medical Research & Health Sciences, 2016, 5, 5:75-82 Detection of bacterial pathogens in surgical site infections and their
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Available online at ISSN No: International Journal of Medical Research & Health Sciences, 2016, 5, 5:75-82 Detection of bacterial pathogens in surgical site infections and their antibiotic sensitivity profile * Ghaleb Adwan 1, Nael Abu Hasan 1, Ibrahim Sabra 2, Dalia Sabra 1, Shorouq Al-butmah 1, Shorouq Odeh 1, Zeinab Abd Albake 1 and Haneen Badran 1 Department of Biology and Biotechnology, An-Najah National University, Nablus, Palestine 2 Rafidia Hospital, Department of Gynecology ABSTRACT Surgical site infections considered as a major problem in health care centers, resulting in extended length of stay, substantial associated morbidity and mortality, and high excess hospital cost. Thirty wound swabs were collected from patients who had developed postoperative wound infections at Rafidia Hospital-Nablus, Palestine. Bacterial isolates were identified according to standard microbiological methods. Antibiotics susceptibility test was applied for all isolated bacterial species. ERIC-PCR was carried out to determine the identity between isolated clones. The results of this research showed that the prevalence of pathogens among surgical site infections was 56.7%, 30%, 6.7%, 3.3% and 3.3% for E. coli, S. aureus, Klebsiella sp., Enterobacter sp., and Acinatobacter sp., respectively. E. coli isolates showed high resistance against Nalidixic acid (88.2%), Trimethoprim/Sulfamethoxazole (76.5%), Tetracycline (70.6%), Norfloxacin (64.7%) and Ciprofloxacin (58.5%). S. aureus showed high resistance against Nalidixic acid (88.9%), Norfloxacin (77.8%), Amoxycillin/clavulanic acid (77.8%), Kanamycin (66.7%) and Ciprofloxacin (55.6%). Methicillin resistant S. aureus (MRSA) accounted for 33.3% of a total of S. aureus isolates. Resistant to 3 or more antibiotics were detected in 94.1% (16/17) and 77.8% (7/9) of E. coli and S. aureus isolates, respectively. ERIC-PCR typing E. coli and S. aureus isolates showed that each was consisted of 4 ERIC-PCR clusters at a 50% similarity level. Indistinguishable and closely related strains were detected for both microorganisms. Results of this study might be important in provoking awareness to postoperative wound infections and further studies are needed to identify other pathogens responsible for SSIs and the source of infections. Using effective antibiotic policy will restrict further spread of postoperative wound infections. Keywords: Surgical site infections, antibiotic resistance, ERIC-PCR profile, E. coli, S. aureus, MRSA. INTRODUCTION Surgical site infections (SSIs) are defined as infections that occur during one month after a surgical operation or one year after implant surgery and affecting either the injury site or near surgical injuries. [1] Since the publication of the guidelines for the prevention of surgical site infection in 1999 by the Center for Disease Control and Prevention (CDC), there has been a declining trend in SSI. According to the criteria put forth by the CDC, SSIs are classified as superficial incisional, deep incisional and organ/space SSIs. [2] Due to heterogeneous nature of these surgical infections, studies of the epidemiology of SSIs are very difficult. The incidence differs widely between surgical procedures, hospitals, patients and between surgeons. [3] Despite the technical advances in infection control and surgical practices, these infections still continue to be a major problem, even in hospitals with advanced modern facilities. The incidence of SSIs accounts as high as 20% among surgical patients, depending on the surgical procedure, the surveillance criteria used, and the quality of data collection. [4] Regardless the improvements in infection control techniques, surgical practices and substantial demands on healthcare resources, SSIs still considered as one of the major cause of morbidity and mortality. [2] Surgical site infections raise costs due to many 75 reasons such as extends the duration of hospitalization, additional diagnostic tests needed, more therapeutic antibiotic treatment, and other rarely reasons such as additional surgery. [5] In most SSIs, the responsible pathogens are part of the patient s own endogenous flora. The most commonly isolated organisms are Staphylococcus aureus (S. aureus), coagulase-negative staphylococci (CNS), Enterococcus sp. and Escherichia coli (E. coli); however, the pathogens isolated depend on the surgical procedure. [4] Due to emergence antibiotic-resistant pathogens such as methicillin-resistant S. aureus (MRSA), multidrug resistant pathogens and others, this led to increase in incidence of SSIs. Other type of SSI pathogens may arise from exogenous sources such as health care workers, operating theatre environment, instruments and used materials. Such pathogens are predominantly aerobic microorganisms, in particular Gram-positive organisms such as staphylococci and streptococci. [2] In a retrospective review, [6] it was shown that 67% of implant infections were due to S. aureus and 68% of these cases were MRSA, while the prevalence of Gram-negative bacteria was only 6% among infections. In recent study, [7] a total of 137 samples obtained from patients had SSIs, 132 (96.4%) yielded bacterial growth and 139 bacterial isolates were obtained. The commonest organism was S. aureus (50.4%) followed by E. coli (23.02%), Pseudomonas aeruginosa (P. aeruginosa) (7.9%) and Citrobacter sp. (7.9%). Antimicrobial profile of Grampositive isolates revealed maximum sensitivity to vancomycin, teicoplanin and linezolid, whereas among Gramnegative isolates meropenem, piperacillin-tazobactam, and amikacin were found to be most sensitive. Staphylococcus aureus strains showed a high degree of resistance for ampicillin (85.7%). Methicillin resistance was seen in 15.7% of all the S. aureus isolates. Gram-negative isolates showed high rate of resistance mainly to commonly prescribed agents like gentamicin, cotrimoxazole and ciprofloxacin. [7] Staphylococcus aureus, is considered as a major human pathogen and a predominant cause of SSIs worldwide with a prevalence rate ranging from 4.6% to 54.4%. [8] Infection with S. aureus is most likely associated with endogenous source as it is a member of the skin and nasal flora and also exogenous source with contamination from environment, surgical instruments or from hands of health workers. [9, 10] The prevalence of MRSA strains among SSIs ranged from 10%- 58.2%. [11-13 ] In other study, the prevalence of S. aureus, Proteus mirabilis, E. coli, P. aeruginosa and Proteus vulgaris among SSIs was 55.0%, 35.0%, 5.0%, 3.0% and 2.0%, respectively. [14] In other recent study, organisms associated with postoperative SSIs were S. aureus 22.4% followed by Klebsiella sp. 20.4% and Proteus sp. 18.4%, E. coli 12.2%, Enterobacter sp. and CNS each 8.2%, P. aeruginosa 6.1% and Citrobacter sp. 4.1%. [15] A total of 42 bacterial pathogens were identified of which 83.3% were from surgical sites and 16.7% were from blood stream infections. Staphylococcus aureus was the common pathogen accounting 26.2% followed by E. coli and CNS sp. Each represented by 21.4%. Approximately 100% of Gram-positive and 95.5% of Gram-negative bacterial isolates showed resistance against two or more antimicrobial agents. [16] In Palestine, no previous studies concerning SSIs, this current study aimed to determine the prevalence of bacterial pathogens among patients with postoperative wound infections and to evaluate the antibiotic susceptibility pattern of these pathogens from Rafidia Hospital, Nablus-Palestine. In addition, to determine the molecular epidemiology of these pathogens isolates using ERIC-PCR technique. MATERIALS AND METHODS Sample collection and identification A total of 30 bacterial isolates were collected using sterile cotton swabs from patients clinically diagnosed having SSIs at Rafidia Hospital-Nablus during February-April These samples were processed and identified as per standard microbiological techniques in Microbiology laboratory at An-Najah National University-Nablus, Palestine. The isolates were cultured on MacConkey and/or Eosin Methylene Blue agars, Mannitol salt agar, Blood agar and Triple sugar iron, Gram stain and biochemical tests were used as IMViC Tests (Indole production, Methyl red test, Voges-Proskauer test and Citrate utilization), catalse test, coagulase test, oxidase test, arginine hydrolysis, urea hydrolysis, H 2 S production and motility test. Antibiotic resistance Antimicrobial resistance was determined according to the Clinical and Laboratory Standard Institute (CLSI) using the disk diffusion method. [17] Antibiotic disks (Oxoid) used were Ceftriaxone (CRO) 30µg, Norfloxacin (NOR) 10µg, Gentamicin (CN) 10 µg, Nalidixic acid (NA) 30µg, Levofloxacin (LEV) 5µg, Ciprofloxacin (CIP) 5µg, Amikacin (AK) 30µg, Cefuroxime sodium CXM (30µg), Tetracycline (TE) 30µg, Meropenem (MEM) 10µg, Kanamycin (K) 30µg, Trimethoprim/Sulfamethoxazole (SXT) 25µg, Cefotaxime (CTX) 30 µg, Ceftazidime (CAZ) 30µg, Vancomycin (VA) 30 µg, Teicoplanin (TEC) 30 µg and Amoxycillin/clavulanic acid (AMC) 30 µg. The 76 plates were incubated at 37 C for hrs. Zones of inhibition were measured in millimeters using a caliper. Strains were classified as resistant or susceptible according to the criteria recommended by CLSI guidelines [17]. Oxacilllin (1µg) antibiotic disks (Oxoid) were used to detect MRSA. Staphylococcus aureus isolates were considered resistant if inhibition zones were 13 mm after incubation on 2% NaCl Mueller Hinton agar at 35 C for 24 hours. [17] DNA extraction and ERIC-PCR Bacterial DNA was prepared for PCR according to method described previously. [18] Briefly, cells were scraped off an overnight nutrient agar plate with a sterile loop, washed twice with 1 ml of 1X Tris-EDTA buffer (10 mm Tris- HCl, 1 mm EDTA [ph 8]), pellet was resuspended in 0.5 ml of sterile distilled H 2 O and boiled for min. The cells then were incubated on ice for 10 min. The debris pelleted by centrifugation at 11,500 X g for 5 min. DNA concentration was determined using spectrophotometer and samples stored at -20ºC until use for further DNA analysis. ERIC (Enterobacterial repetitive intergenic consensus) PCR was performed using Primer ERIC1: 5`-ATG TAA GCT CCT GGG GAT TCA C-3` and Primer ERIC2: 5`-AAG TAA GTG ACT GGG GTG AGC G-3`. Each PCR reaction mix was performed in a final volume of 25 µl containing 12.5 µl of PCR premix with MgCl 2 (ReadyMix TM Taq PCR Reaction Mix with MgCl 2, Sigma), 0.8 µm of each primer, ng of DNA template. In addition, the master mix was modified by increasing the concentration of dntps to 0.4 mm, 3mM MgCl 2 and 1.5U of Taq DNA polymerase. DNA amplification was carried out using the thermal cycler (Mastercycler personal, Eppendorf, Germany) according to the following thermal conditions: initial denaturation for 3 min at 94ºC was followed by 30 cycles of denaturation at 94 C for 50 s, annealing at 40 C for 50 s and extension at 72 C for 1 min, with a final extension step at 72 C for 5 min. The PCR products were analyzed by electrophoresis on 1.5 % agarose gel stained with Ethidium bromide. The gel image was scored using binary scoring system that recorded the presence and absence of bands as 1 and 0, respectively. A binary matrix was analyzed by the unweighted pair group method for arithmetic averages (UPGMA), using SPSS statistics software version 20 (IBM). The number of different bands in each fingerprint was considered for comparison of bacterial species as previously described, [19] based on the following criteria: Indistinguishable (No different band), Closely related (with 1 different band) Possibility different (with two different bands), Different (three or more different bands). RESULTS A total of 30 swab specimens were collected from patients with postoperative SSIs. In this study, the mean age of patients was 37.4 (1-80), and 63.3% (19/30) were males. Bacterial pathogens identified from these surgical site infections, age and sex of patients are presented in Table 1. Results of this research showed that the prevalence of pathogens among surgical site infections was 56.7%, 30%, 6.7%, 3.3% and 3.3% for E. coli, S. aureus, Klebsiella sp., Enterobacter sp., and Acinatobacter sp., respectively. E. coli isolates showed high resistance against Nalidixic acid (88.2%), Trimethoprim/Sulfamethoxazole (76.5%), Tetracycline (70.6%), Norfloxacin (64.7%), Ciprofloxacin (58.5%). S. aureus showed high resistance against Nalidixic acid (88.9%), Norfloxacin (77.8%), Amoxycillin/clavulanic acid (77.8%), Kanamycin (66.7%) and Ciprofloxacin (55.6%). Methicillin resistant S. aureus were 33.3% of a total of S. aureus isolates. Results of antibiotics resistance against bacterial pathogens isolated from patients who had postoperative surgical site infections are presented in Table 2. Resistant to 3 or more antibiotics were detected in 94.1% (16/17) and 77.8% (7/9) of E. coli and S. aureus isolates, respectively. ERIC-PCR typing of 17 E. coli isolates and 9 S. aureus isolates, which are believed to harbor different genes based on their antibiotic profiles, were genetically diverse and consisted of a heterogeneous population with a total of 4 ERIC-PCR profiles (clusters) at a 50% similarity level for both E. coli and S. aureus isolates. Results of ERIC-PCR profiles are presented in Figures 1, 2, 3 and 4. These results also showed that E. coli isolates numbered 16 and 17 were indistinguishable, while isolates 4 and15, 6 and 7, and 13 and 14 were closely related. For S. aureus isolates 2 and 9 were indistinguishable, while 1 and 5, and 6 and 8 were closely related. 77 Table 1: Surgical procedures and corresponding bacterial isolates from SSis patients Pathogen Operation (surgical site infection) sex Age No. of Isolates E. coli Laparoscopic cholecystectomy M 62 2 E. coli Incisional hernia repair F 57 1 E. coli Debridement and skin graft M 4 1 E. coli Debridement of heel ulcer F 65 1 E. coli Perianal fistula operation M 33 1 E. coli A bone knee amputation F 58 1 E. coli Abdominal laparotomy due to intestines obstruction due to sigmoid CA M 63 1 E. coli Appendectomy M 7 and 80 2 E. coli Rightleg skin graft M 48 1 E. coli Umbilical and incisional hernia repair F 57 1 E. coli Perianal surgery M 37 and 39 2 E. coli Perianal fistulectomy M 21 1 E. coli Perianal abscess M 1 1 E. coli Right gluteal abscess M 2 1 S. aureus Laparotomy due to perforated sigmoid tumor F 51 1 S. aureus Hand surgery F 62 1 S. aureus Vertebral fixation M 10 1 S. aureus Skin graft F 13 1 S. aureus Rightinguinal hernia repair F 39 1 S. aureus Back lipoma excision M 48 1 S. aureus Groin abscess incision and drainage F 1 1 S. aureus Right-forearm graft M 30 1 S. aureus Right- thigh operation F 1 1 Klebsiella sp. Sigmoidectomy due to sigmoid carcinoma M 61 1 Klebsiella sp. Below knee amputation F 22 1 Enterobacter sp. Foot skin graft M 18 1 Acinetobacter sp. Craniotomy - chronic subdural haematoma M 71 1 Figure 1: DNA fingerprints generated by ERIC-PCR analysis of 17 clinical E. coli isolates recovered from surgical site infections. Lanes L represent the ladder Table 2: Antibiotic resistance profile for recovered bacterial pathogens Microorganism Antibiotic resistance n (%) * SXT NOR CIP AK K TE NA CN MEM CTX CRO CAZ LEV CXM OXA VA TEC AMC E.coli (76.5) (64.7) (58.5) (23.5) (41.2) (70.6) (88.2) (35.3) (0.0) (35.3) (35.3) (35.3) N N N N N N Klebsiella sp (100) (0.0) (0.0) (0.0) (0.0) (100) (0.0) (100) (50) (200) (100) (100) (50) (50) N N N N Enterobacter sp. (100) (100) (100) (100) (100) (100) (100) (0.0) (0.0) (0.0) (0.0) (0.0) N N N N N N Acinatobacter sp. (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) (100) N N N N N N S. aureus N N N N N N N (22.2) (77.8) (55.6) (22.2) (66.7) (0.0) (88.9) (33.3) (0.0) (0.0) (77.8) *Trimethoprim/Sulfamethoxazole, SXT; Norfloxacin, NOR; Ciprofloxacin, CIP; Amikacin. AK; Kanamycin, K; Tetracycline, TE; Nalidixic acid, NA; Gentamicin, CN; Meropenem, MEM; Cefotaxime, CTX; Ceftriaxone: CRO; Ceftazidime, CAZ; Levofloxacin, LEV; Cefuroxime sodium, CXM; Oxacilllin, OXA; Vancomycin, VA; Teicoplanin, TEC; Amoxycillin/clavulanic acid, AMC. N: Not detected. 78 Figure 2: Dendrogram of 17 clinical E. coli isolates recovered from surgical site infections based on the UPGMA method using Ward s Method/ Squared Euclidean Distance by SPSS software version 20, derived from analysis of the ERIC-PCR-profiles at a 50% similarity level. C: Cluster Figure 3: DNA fingerprints generated by ERIC-PCR analysis of 9 clinical S. aureus isolates recovered from surgical site infections. Lanes L represent the ladder 79 Figure 4: Dendrogram of 9 clinical S. aureus isolates recovered from surgical site infections based on the UPGMA method using Ward s Method/ Squared Euclidean Distance by SPSS software version 20, derived from analysis of the ERIC-PCR-profiles at a 50% similarity level. C: Cluster DISCUSSION Postoperative hospital infections are considered a major problem in health care centers, resulting in extended length of stay, substantial associated morbidity and mortality, and high excess hospital cost. [14] These infections have been reported to be one of the major common causes of nosocomial infections and are accounting for 20% to 25% of all nosocomial infections worldwide. [20] It is estimated that SSIs develop in 2%-5% of the 16 million patients undergoing surgical procedures each year in the United States. [21] In spite of technological advances in surgery and wound management, wound infections still regarded as the one of the most common hospital infection mainly in patients undergoing surgery. [22] In the current study, E. coli isolates was the most common pathogen (63.3%) in postoperative wound infections. In other studies the incidence E. coli in postoperative wound infections ranged from 5%-23%. [7,14,15] It was also reported to be the commonest Gram-negative bacteria isolated in several other studies. [7,8,14,16] Infection with E. coli is most likely associated with endogenous source as it is a member of intestinal normal flora and this might explain the finding of this pathogen in most of operations related to the digestive system. Infection with S. aureus is most likely associated with endogenous source as it is a member of the skin and nasal flora and also exogenous source with contamination from environment, surgical instruments or from hands of health workers. [9,10,23] In previous study, it was shown that 4 out of 6 operating rooms in this hospital were contaminated with S. aureus, and the average number of S. aureus in these rooms ranged from CFU/m2 using passive air sampling. [24] Staphylococcus aureus is a major cause of infection in both healthcare and community settings. This pathogen was the only Gram-positive bacteria isolated from the postoperative wound infections in this study. Findings of the current study in this respect were in contrast to previously reported results, where S. aureus was a major cause of 80 SSIs. [6,7,14] In these studies prevalence of S. aureus ranged from 50%-67%, however, the findings of Gelaw et al. (2014) on the prevalence of this pathogen among SSIs was 22% and consistent with that found in the current study. [15] In the current study, around 30% (3/9) of S. aureus were MRSA and these constitute 10% (3/30) of the total SSIs isolated pathogens. In previous studies, the prevalence of MRSA strains among SSIs ranged from 10%- 58.2%. [11-13] In this study, most isolates of E. coli and S. aureus showed multi-drug resistant to the commonly prescribed antibiotics such as Nalidixic acid, Trimethoprim/Sulfamethoxazole, Tetracycline, Norfloxacin, Ciprofloxacin and Kanamycin. This is probably due to selective pressure resulting from uncontrolled, extensive incorrect and misuse of these antibacterial agents in hospitals as well as in the country as a whole. This is promoted by the lack of national antibiotic policy and over-the-counter antibiotic availability in Palestine. [25
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