TX_1~ABS:AT/ADD:TX_2~ABS:AT 6 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2021, 5 (2): 6-12 ReseaRch aRticle The Prevalence of Microorganisms and Antibiotics Activity Tests of Natural and Industrial Products in Surgical Site Infections in Referral Hospitals in Erbil City, Kurdistan Region – Iraq Raid D. Thanoon1*, Yara M. Mohammed2 1Department of Medical Biochemical Analysis, Cihan University-Erbil, Kurdistan Region, Iraq, 2Department of General Biology, Cihan University-Erbil, Kurdistan Region, Iraq ABSTRACT Background and Objectives: To study the prevalence of microorganisms and antibiotics activity tests of natural and industrial products, surgical site infection was found in referral hospitals in Erbil, Iraq’s Kurdistan Province. Methods: Pus samples were collected from 29 surgical patients using sterile swabs and primed for culture collection. Disk diffusion method was used to determine the bacterial activity of three types of plants (cinnamon, ginger, and radish) against three bacterial species including Staphylococcus aureus, Proteus spp., and Staphylococcus epidermidis. Results: Overall, 29 pus samples were processed for culture testing. One Gram-negative bacterium (7.14%) and 13 Gram-positive cocci (92.85%) were isolated from the 14 bacteria genera. S. aureus, Proteus spp., and S. epidermidis most potent isolates were found to be resistant to the variable of tested antibiotics, while they showed any sensitivity to the tested plant extracts. Cinnamon, ginger, and radish extracts were discovered to be among the few variable effective plant extracts against the tested pathogenic bacteria isolates. Conclusion: The data collected from this research showed a better way of explanation of the microorganism’s etiology of SSIs in hospitals where they might have epidemiological and healing insinuations. Keywords: Surgical site infections, bacteria isolated from wounds, antimicrobial resistance, pus, plant extract INTRODUCTION Surgical site infection occurs when surgical wounds get infected with germs from a previously diseased location. According to the report, it can result in illnesses, hospitalizations, and even death.[1] The third most often observed nosocomial infection is surgical site infections.[2] The most prevalent causes of these contagions are exogenous and endogenous germs that reach the operating wounds during surgery.[2] The occurrence of infected operational injuries might be affected by such factors as pre-operative therapy, theater, post-operative care, and surgery.[2] Staphylococcusaureus is a naturally occurring bacterium that colonizes about 30% of the human population’s nares and a new study indicates that very diverse nasal microbiosis is required if colonizations of S. aureus are to be encouraged or prevented. It spreads easily from person to person, onto health care workers’ hands and clothing, as well as onto surfaces and into the air.[2,3] Staphylococcus epidermidis,once considered a comparatively harmless pathogen, is the biggest and best investigated member of the CoNS group.[4,5] It is considered as an unsafe commensal of the human skin and is becoming known for its useful role in skin immunity and microbiota.[4] The active metabolites of cinnamon bark (Cinnamomum verum) belonging to the Lauraceae family were cinnamon aldehyde and trans-cinnamic acid. The nutritional content, phenolic profile, and antioxidant activities of cinnamon were studied. According to a compositional study, this species is a great source of protein and minerals. The nutritional content, phenolic profile, and antioxidant activities of cinnamon were studied. According to a compositional study, this species is a great source of protein and minerals.[6] Radish (Raphanus sativus L.), belonging to the Brassicaceae family, is an annual or biennial root crop globally of economic importance. There are many resources of radish germplasm in China, and for conservation and use of the radish resources, cultivars and genetic diversity studies are very important.[7] Since antiquity, ginger (Zingiber officinale) belonging to the Zingiberaceae Corresponding Author: Raid D. Thanoon, Department of Medical Biochemical Analysis, Cihan University-Erbil, Kurdistan Region, Iraq. E-mail: raid.duraid@ cihanuniversity.edu.iq Received: May 30, 2021 Accepted: July 18, 2021 Published: August 10, 2021 DOI: 10.24086/cuesj.v5n2y2021. pp 6-12 Copyright © 2021 Raid D. Thanoon, Yara M. Mohammed. This is an open- access article distributed under the Creative Commons Attribution License (CC BY-NC-ND 4.0). Cihan University-Erbil Scientific Journal (CUESJ) Thanoon and Mohammed: Antibiotics Activity Tests of Natural and Industrial Products in Surgical Site Infections 7 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2021, 5 (2): 6-12 family for more than 200 years, traditional Chinese medicine has been utilized as a spice and medication. Ginger is a major herb with numerous nutritional and therapeutic properties that are utilized in traditional Asian and Chinese medicine.[8] Ginger and its general components, such as Fe, Ca, Vitamin C, flavonoids, phenolic compounds, paradols, and so on, are used for a wide variety of symptoms, including vomiting, pain, and other herbal medications.[8] Objectives 1. Isolation of microorganisms from surgical wounds 2. Natural and industrial products were tested for antibiotic activity. MATERIALS AND PROCEDURES Population Research The research was carried out at Cihan University-Erbil’s Biology Department. Specimens were collected from four hospitals between November 2019 and March 2020. Specimen Collection and Transport Under aseptic conditions, two sterile swabs were used to extract pus samples from each patient. Swabs were taken to the Department of Biology, Cihan University-Erbil. The specimens can be stored in the sample tube at 4°C for 72 h. Types of Antimicrobials Identification The bacterial growth of the incubated plates was identified on the basis of cultural, morphological, and biochemical characteristics (Table 1).[9] Production of Specimens Inoculated specimens of pus have been immediately inoculated on MacConkey, chocolate, and mannitol salt agar dishes. They were incubated aerobically at 37°C for 24–48 h with a CO2 concentration of 7–10%. After this, the specimens was inoculated on the plates using four flame methods.[10,11] Procedure of catalase testing Fill a slide with a small amount of bacterial growth. Drops of H2O2 were added to the smear and blended with a toothpick.[12] Gram staining Overflow slide with mineral violet – 60 s by Gram – 180 s, iodine floods. Prudently decolor with 95% ethanol until the thinnest part of the smear is colorless. Flood with safranin (pink color) (10% fuchsine) – 60 s (wash on the water). Air dehydrated or spot by permeable paper.[13] Slide coagulase test Identify where the test strain (T) and the control (C) are placed by marking the slide. Set up positive and negative control organisms on the same slide for simultaneous testing. Emulsified test strain to obtain a homogeneous thick suspension. Observed self-agglutination. Within 10 s, there will be a visible cluster. Negative result: No visible clustering within 10 s.[14] Testing for Antimicrobial Sensitivity Drugs have been chosen on the basis of their impact on a specific organism, as well as hospital antibiotic policies.[20] The diameter of the inhibitor was measured on a scale of 24 h after 18–24 h of incubation. Each antimicrobial disk’s area size was used to determine if it was susceptible, intermediate, or immune (CLSI, 2006). Mueller-Hinton agar and antibiotic disks were used.[15] Ginger Extraction Ginger was bought from local marketplaces in Erbil City, using ginger for this investigation. In boiling water bath for 30 min, 10 g of ginger powder combined with 40 ml soy oil was heated. Before disk diffusion testing, extracts were kept at 4°C to retain their antibacterial activities. The plates were dried in the air flow chamber of the laminar for 15 min. Extracts of ginger (20 μl) have been applied individually for 18–24 h on each disk.[16] Radish Extraction A local supermarket provided Raphanus sativus (white radish variety). Radish was lyophilized and crushed into a fine powder before ethanol extraction. Powder root was extracted for 24 h with 300 ml of ethanol at room temperature and 150 rpm.A filter paper disk diffusion test was used to assess the extract’s antibacterial function.[17] The research bacteria inoculums were prepared according to McFarland standard 0.5. Uniform bacterial lawns were developed, using 100L of inoculum on a Mueller-Hinton agar plate. The plates were incubated at 37°C for 24 h.[18] Cinnamon Extraction After cleaning the spices, they were dried in sunlight for 2 days. The dried material was pulverized by a grinder into a Table 1: Antimicrobials and their concentrations No. Name Dose Manufactures 1 Amikacin 10 mcg Amikin 2 Amoxicillin 25 mcg Trimox 3 Ampicillin/cloxacillin 25/5 mcg AMPOXIN 4 Ceftazidime 30 mcg Ceptaz 5 Ceftriaxone 10 mcg Rocephin 6 Chloramphenicol 10 mcg Fenicol 7 Ciprofloxacin 10 mcg Ciloxan 8 Gentamicin 10 mcg Gentasol 9 Imipenem 10 mcg Primaxin 10 Netilmicin 30 mcg Netmicin 11 Oxacillin 10 mcg Bactocill 12 Rifampin 5 mcg Rifadin 13 Tetracycline 10 mcg Declomycin Figure 1: The prevalence rate of surgical site wound infections Figure 2: Number of isolated bacteria according to Gram-positive and Gram-negative bacteria Figure 4: Staphylococcus aureus organism control Thanoon and Mohammed: Antibiotics Activity Tests of Natural and Industrial Products in Surgical Site Infections 8 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2021, 5 (2): 6-12 fine powdered substance. Twenty grams of cinnamon powder (weighted by electric balance) were transferred to 100 ml of conical flask where 40 ml of ethanol was added. The ethanol crude was filtered using Whatman No. 1 filter paper. Cinnamon bark extract was made using dimethyl sulfoxide as a solvent at concentrations of 2.5%, 5%, and 10%. With a sterile cotton bud, the bacterial solution was spread on the whole surface of Mueller-Hinton agar. At 37°C, the Petri dish was incubated overnight.[19] RESULTS Specimens of Surgical Wound Infection A total of 29 pus specimens were processed for culture testing. The present study being conducted between November (2019) and March (2020). The overall percentage of infection was (48.27%) while the remaining (51.72%) was negative [Figure 1]. Two patients from the maternity hospital were cultivated with MacConkey agar and the mannitol salt agar had a negative result and a positive result of S. aureus bacteria and specimens were taken from females. Ten patients from the cardiac center were sampled, two males and eight females, and nine of the 10 were negative on MacConkey agar, with Proteus spp. bacteria being the only positive result. Eight patients from Sardam Hospital, two specimens from females and six specimens from males, five of whom were cultivated with mannitol salt agar, and all of whom had a positive result were S. aureus bacteria and three of them cultivated with chocolate agar, had a positive result, two of whom were S. aureus bacteria, and one of them was S. epidermidis bacteria. Nine patients from Jamhuri Hospital, specimens taken from males, cultivated mannitol salt agar, five of whom were negative, and four of whom were positive, were S. aureus and S. epidermidis bacteria [Table 2]. Out of the 14 isolated bacteria, 1 was Gram-negative bacteria (7.14%) and 13 were Gram-positive cocci (92.85%). S. aureus was the most common bacteria 10 isolates (71.42%). Other isolated bacteria were Proteus sp. (7.14%) and S. epidermidis (21.42%) [Figure 2]. Antimicrobial Activity of Plant Extracts andAntibacterial Susceptible of S. aureus Sensitivity pattern for S. aureus to the following antibiotics; RF (20 mm), TE (16 mm), NET (27 mm), and the other antibiotics resistance for S. aureus such as C, OX, CRO, IPM, CAZ, APX, and CRO [Figure 3a and b]. The result presented in Figure 4 was S. aureus control bacteria. Each extract was tested against S. aureus isolated. The results showed cinnamon resistance to antibacterial activity against S. aureus [Figure 5]. Antimicrobial Activity of Plant Extracts and Antibacterial Susceptible of S. epidermidis Antibacterial resistant and susceptible of S. epidermidis isolated from patients. The results show that S. epidermidis isolated from patients were susceptible to NET (34 mm) and APX (28 mm), and other antibiotic resistant for S. epidermidis such as TE, C, and CRO [Figure 6]. The result presented in Figure 7 exhibited S. epidermidis control bacteria. The results given in Figure 8 show that ginger, cinnamon, and radish root extracts are not effective against S. epidermidis. Figure 3: (a and b) Antibacterial inhibition zoneagainst Staphylococcus aureus ba Figure 5: Inhibition zone of plants against Staphylococcus aureus Figure 6: Antibacterial inhibition zone against Staphylococcus epidermidis Figure 7: Staphylococcus epidermidis bacteria control Thanoon and Mohammed: Antibiotics Activity Tests of Natural and Industrial Products in Surgical Site Infections 9 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2021, 5 (2): 6-12 Antimicrobial Activity of Plant Extracts and Antimicrobial Susceptible of Proteus spp. The results show that Proteus spp. isolated patients were susceptible to NET (17 mm), RF (19 mm), AK (25 mm), TE (26 mm), and CN (20 mm). Moreover, other antimicrobial is resistant to Proteus spp. OX, APX, CAZ, and AX [Figure 9a-c]. Results given in Figure 10 showed that ginger and radish root extract are not effective against Proteus species. The results showed that cinnamon had very little (5 mm) effect on Proteus species [Figure 10]. DISCUSSION Surgical site infection is a prevalent kind of disease related to health-care research conducted by the way.[20-22] SSIs Table 2: The patients number, gender, isolated bacteria, media cultured from the surgical site of infection, and the name of the hospitals Serial no. of patient Media Isolated bacteria Hospitals Sex 1 MacConkey agar Negative (–) Cardiac center ♀ Female 2 MacConkey agar Negative (–) Cardiac center ♀ Female 4 MacConkey agar Negative (–) Cardiac center ♂ Male 5 MacConkey agar Positive (+) Proteus sp. Cardiac center ♀ Female 6 MacConkey agar Negative (–) Maternity hospital ♀ Female 7 MacConkey agar Negative (–) Cardiac center ♀ Female 8 MacConkey agar Negative (–) Cardiac center ♀ Female 9 MacConkey agar Negative (–) Cardiac center ♀ Female 10 MacConkey agar Negative (–) Cardiac center ♀ Male 11 MacConkey agar Negative (–) Cardiac center ♀ Female 12 Mannitol salt agar Positive (+) S. aureus Maternity hospital ♀ Female 13 Mannitol salt agar Positive (+) S. aureus Sardam Hospital ♀ Female 14 Mannitol salt agar Positive (+) S. aureus Sardam Hospital ♂ Male 15 Mannitol salt agar Positive (+) S. aureus Sardam Hospital ♂ Male 16 Mannitol salt agar Positive (+) S. aureus Sardam Hospital ♂ Male 17 Mannitol salt agar Positive (+) S. aureus Sardam Hospital ♂ Male 18 Chocolate agar Positive (+) S. aureus Sardam Hospital ♂ Male 19 Chocolate agar Positive (+) S. aureus Sardam Hospital ♀ Female 20 Chocolate agar Positive (+) S. epidermidis Sardam Hospital ♂ Male 21 Mannitol salt agar Positive (+) S. aureus Jamhuri Hospital ♂ Male 22 Mannitol salt agar Positive (+) S. epidermidis Jamhuri Hospital ♂ Male 23 Mannitol salt agar Negative (–) Jamhuri Hospital ♂ Male 24 Mannitol salt agar Negative (–) Jamhuri Hospital ♂ Male 25 Mannitol salt agar Positive (+) S. aureus Jamhuri Hospital ♂ Male 26 Mannitol salt agar Positive (+) S. epidermidis Jamhuri Hospital ♂ Male 27 Mannitol salt agar Negative (–) Jamhuri Hospital ♂ Male 28 Mannitol salt agar Negative (–) Jamhuri Hospital ♂ Male 29 Mannitol salt agar Negative (–) Jamhuri Hospital ♂ Male S. aureus: Staphylococcus aureus, S. epidermidis: Staphylococcus epidermidis Figure 10: Antimicrobial disk diffusion test against Proteus spp. Figure 8: Antimicrobial disk diffusion test against Staphylococcus epidermidis Thanoon and Mohammed: Antibiotics Activity Tests of Natural and Industrial Products in Surgical Site Infections 10 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2021, 5 (2): 6-12 (infections at surgical site) were found to be 48.27 percent prevalent in the present specimens, with S. aureus and S. epidermidis being the most common etiological agents among Gram-positive bacteria. Gram-positive bacteria while the Gram-negative bacteria were Proteus spp. respectively.[23] These findings support that previous research S. aureus was the most common bacteria found in post-operative wound infections.[24,25] S. aureus was found in 71.42% of surgical site infections. The most common Gram-positive S. aureus isolate (71.42%) followed by S. epidermidis (21.42%) and Proteus spp. (7.14 percent of the total). In the current study, a shorter post- operative stay was seen in patients with acquired infection, who had a roughly triple longer patient without operational site infection. In hospitals, neonatal infections, operative care for the elderly and malnutrition, and individuals with diabetes and other chronic diseases, S. aureus is an important source of infection.[26] The minimal number of S. aureus sensitivity found in the results of this investigation against TE and C was compatible with studies published in Eritrea by certain Nigerian employees.[27,28] Sensitivity to RF, TE, NET, and the other antibiotics resistance for S. aureus such as C, OX, CRO, IPM, CAZ, APX, and CRO did not match the findings of Chowdhury et al.,[29] however, resistance to other antibiotics did. CAZ and CIP were consistent with the report published by the commission.[29] Moreover, S. aureus was sensitive to TE and resistance to CRO and C, which is not in agreement with the report published by the commission.[29] Pattern of antibiotic resistance of S. epidermidis isolated from hospital infectious specimens S. epidermidis resistance against: C, CRO, and TE were consistent with reports published by Chabi and Momtaz,[30] Wijesooriya et al.,[31] and S. epidermidis sensitive to APX and NET.[32] Results show that Proteus spp. isolated from patients were susceptible to NET, RF, AK, TE, and CN and resistant to Proteus spp.;OX, APX, CAZ, and AX.[33] Current results showed that cinnamon, radish, and ginger were resistant to S. aureus. When opposed to the previous findings,[18,34] the present findings show a higher level of activity. Raphanus extract had the best activity against a multiresistant strain of P. aeruginosa, while P. mirabilis was the most resistant to the extract’s influence.[35] Piper nigrum and Zingiber officinale are active in S. aureus and K. pneumoniae. Cinnamon essential oil shows maximum activity against E. coli and S. aureus.[36] The results show that ginger extract and root radish extract are not effective against S. epidermidis.[37] The current results showed that cinnamon and ginger extract are not effective against Proteus species. The different concentrations of radish root extract had no effect on Proteus spp.[18] The antibacterial action of cinnamon was ascribed to the existence or long- term storage of specific active components which have been declined by melting and exposure to air.[38] The negative result of cultured pus specimens may not have been effective due to the transfer of the swabs, and the bacteria may have been dead. It could be the species of bacteria that may not grow in these media. They need special media or anaerobic bacteria could not grow aerobically in the 37°C incubator. CONCLUSION The findings of this research will aid in our understanding of the microbiological etiology of SSIs in our hospitals, which could have epidemiological and therapeutic implications. S. aureus was the most common surgical site infection we found (71.42%). This study demonstrates that RF, TE, and NET are sensitive to S. aureus, and NET and APX are sensitive to S. epidermidis and NET, RF, TE, AK, and CN are affective against Proteus spp. and C, OX, CIP, IPM, CAZ, APX, and CRO Figure 9: (a-c) Antimicrobial inhibition zone on Proteus spp. c ba Thanoon and Mohammed: Antibiotics Activity Tests of Natural and Industrial Products in Surgical Site Infections 11 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2021, 5 (2): 6-12 are resistant to S. aureus. TE, C, and CRO are resistant to S. epidermidis. OX, APX, CAZ, and AX are resistant against Proteus spp. Plant extracts have no antimicrobial activity against S. aureus, S. epidermidis, or Proteus ssp., which typically cause surgical wound infections, according to the findings of this report. The findings showed that cinnamon, radish root, and ginger are ineffective at inhibiting the growth of wound pathogenic bacteria. RECOMMENDATION The findings of this study can be used to launch an effort to strengthen hospital antimicrobial policy and antimicrobial prescribing guidelines. It is difficult to choose the best drug for any condition, particularly in infectious diseases. Since it is performed in vitro, the susceptibility pattern cannot be used as the sole criterion because it ignores the patient’s immunological status and clinical condition. This research may be useful in determining which plant extracts to use for further isolation of constituents responsible for the studied species’ behavior. ACKNOWLEDGMENTS I’d want to offer my heartfelt gratitude to Dr. Raid Duriad Thanoon, the research supervisor, for assisting me through each step of my research paper it would have been impossible to write such a significant and fascinating work without his help. And a special thanks to Ahmed Majeed Mohammed, who inspired us to achieve many goals during this research. Many thanks to biolaboratory for their help and guidance. Finally, many thanks to all the other people and teachers for their help and advice. REFERENCES 1. T. Anggrahita, A. Wardhana and G. Sudjatmiko. Chlorhexidine- alcohol versus povidone-iodine as preoperative skin preparation to prevent surgical site infection:A meta-analysis. Medical Journal of Indonesia, vol. 26, no. 1, pp. 54-61, 2017. 2. K. S. Bhaskarrao, R. Vaishali and V. J. Katkar. Study of bacteriological profile of post-operative wound infections in obstetrics and gynaecological surgeries in a tertiary care hospital. 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