Article Information

SM Ali Ahmed, Ahmed Abu Saleh, Ismet Nigar, Rehana Razzak Khan, Sharmeen Ahmed, Abu Naser Ibne Sattar, Chandan Kumar Roy, Sanjida Khondakar Setu, Shaheda Anwar^*

Bangabandhu Sheikh Mujib Medical University, Department of Microbiology & Immunology, Dhaka, Bangladesh

*Corresponding author: Dr. Shaheda Anwar, Department of Microbiology & Immunology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh

Received: 13 August 2024; Accepted: 20 August 2024; Published: 28 August 2024

Citation: SM Ali Ahmed, Ahmed Abu Saleh, Ismet Nigar, Rehana Razzak Khan, Sharmeen Ahmed, Abu Naser Ibne Sattar, Chandan Kumar Roy, Sanjida Khondakar Setu, Shaheda Anwar. Retrospective Study of Bacterial Profile in Wound Swab and Their Susceptibility Pattern in A Tertiary Care Hospital in Dhaka, Bangladesh. Archives of Microbiology and Immunology. 8 (2024): 383-389.

Abstract

Keywords

wound infection, bacterial pathogens, and antimicrobial resistance

Article Details

1. Introduction

Wound infection is defined as the presence of replicating microorganisms within a wound, leading to host or tissue injury. Agents that cause wound infection can be classified based on the depth of the wound, and they serve as the carriers for organisms that cause infection [1]. The presence of pathogenic bacteria in the wound does not imply infection. Infection occurs when one or more than one contaminant evades the host defenses, replicating in large numbers, attacks, and harms the host tissue. Different microbial organisms can infect wounds [1]. They are likely Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella spp, Acinetobacter spp, Escherichia coli, Proteus spp, and Enterobacter spp [2,3]. Wound infection is a significant problem in Bangladesh. Complications of wound infection are very common because of poor hospital management and poor aseptic techniques used in the hospitals during surgical procedures and other hospital procedures. It is the most acquired infection in hospitals, which has contributed the majority to prolonged hospitalization and higher costs and is associated with considerable morbidity and mortality rates, especially in the developing world [3,4,5]. Regional and local variations occur among causative microorganisms of wound infection. Thus, clinicians should be aware of common causative agents and their antimicrobial susceptibility profile in their locality [6].

This study was conducted to identify bacterial pathogens associated with wound infections and determine their resistance to commonly used antibiotics among patients with wound infection isolates.

2. Materials and Methods

A retrospective study was done in the Department of Microbiology and Immunology at Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, from January 2023 to December 2023 for one year. All samples were collected from outpatients and inpatients of BSMMU. A total of 1630 wound swabs were collected. The skin around the surgical wound was sterilized with 70% ethyl alcohol using a sterile cotton-wool swab to avoid touching the surrounding tissues to prevent swab contamination with endogenous skin flora. The wounds were carefully cleaned using sterile gauze moistened with sterile physiological saline.  Each sample was collected using two sterile swabs from the wound ground and edge using the Levine technique. The sample was placed in an Amies transport medium, labeled, and transported to the clinical microbiology laboratory without any delay. The smear was prepared directly from the first swab and stained with gram stain. Wound swab samples were received in non-sterile containers; dry samples and samples from patients on antibiotics were rejected. All samples were cultured in blood agar and MacConkey agar media, and incubated overnight at 37⁰C for 24-48 hours. Organisms were identified by a standard microbiological procedure, including colony characters and gram staining.

All the isolates were tested for antimicrobial susceptibility by disc diffusion methods according to the Clinical Laboratory Standard Institute (CLSI) guidelines [7,8]. The following antibiotics were used for gram-negative bacteria: amoxicillin, amoxicillin-clavulanic acid, ciprofloxacin, ceftriaxone, gentamicin, cefotaxime, ceftazidime, cotrimoxazole, cefuroxime, amikacin, aztreonam, meropenem, netilmicin, tazobactam piperacillin, cefepime and colistin. For gram-positive bacteria, the following antibiotics are used: amoxicillin, ciprofloxacin, cefradine, cloxacillin, erythromycin, gentamicin, cotrimoxazole, cefoxitin, vancomycin and linezolid. P. aeruginosa ATCC 27853, E. coli ATCC 25922, and S. aureus ATCC 25923 were included as control strains.  Cefoxitin 30 microgram was used as a surrogate marker for identifying MRSA.  Staphylococcus aureus, which showed a zone of inhibition < 21 mm with cefoxitin on Mueller-Hinton Agar after overnight incubation at 37⁰C, was considered MRSA [9].

3. Results

A total of 1630 wound swabs were collected, of which 786 (48.22%) yielded bacterial growth (Table 1). Among them, gram-negative bacteria were 645(82.02%), and gram-positive bacteria were (17.93%) (Figure 1).

Table 1: Frequency of Bacterial isolates in wound swabs (n=1630)

Out of culture-positive cases, the majority 424(53.94%) were in the age group of 21-40 years (Table 2), and male 476(60.55%) were more commonly affected than female 310(39.44%) patients.

Table 2: Characteristics of the study population with wound swab culture-positive patients (n=786)

Out of 786 isolated organisms, the most common isolate was Pseudomonas aeruginosa, which accounted for 257(32.69%) of all the bacterial isolates, followed by Klebsiella spp 230 (29.26%), Staphylococcus aureus 141 (17.93%), Acinetobacter spp 91 (11.57%), Escherichia coli 41(5.21%). The least isolated organisms were Proteus spp 17(2.16%), Enterobacter spp 8 (1.01%), and Serratia spp 1(0.12%) respectively (Table 3).

Table 3: Distribution of Bacterial isolates from wound swabs (n=786)

Figure 1: Shows distribution of gram positive and gram-negative bacteria

Among the isolated Staphylococcus aureus, 16.31% showed resistance to cefoxitin,73.75% were resistant to cepharadine, and 65.24% to gentamicin. S. aureus showed 40.42% resistance to cotrimoxazole, followed by 41.84% resistance to cloxacillin. However, 84.39% of isolates were resistant to amoxicillin, and 82.26% and 78.72% were resistant to ciprofloxacin and erythromycin, respectively. At the same time, the isolated Staphylococcus aureus were 100% sensitive to vancomycin and linezolid (Table-4).

Table 4: Antibiotic resistance pattern of gram-positive bacteria to different antibiotics (n=141)

The antibiotic resistance pattern of the two hundred fifty-seven Pseudomonas spp. isolated from the wound swab is shown (Table 5). Among isolated gram-negative bacteria, Pseudomonas spp. was highly resistant to ciprofloxacin (83.65%), aztreonam (70.03%), gentamicin (65.76%), and moderately resistant showed against cefepime (64.20%), and ceftazidime (61.08%) and netilmicin (59.92%). The highest sensitivity was exhibited for colistin, which had only 10.11% resistance among the isolates. However, it is the least resistant to meropenem (45.91%) and piperacillin-tazobactam (47.85%) and amikacin (47.85%) respectively.

Table 5: Antimicrobial resistance pattern of Pseudomonas spp.(n=257)

Resistance was higher for cephalosporins like ceftriaxone, cefotaxime, and ceftazidime for all the gram-negative isolates, between 61%-83% except for Proteus spp, which showed 23.52% resistance to cefepime whereas 50% of Enterobacter spp were resistant to cefepime.  All isolated Proteus and Enterobacter exhibited resistance to amoxicillin (100%), but Klebsiella and E.coli showed almost similar resistance patterns (94.34%) and (95.12%) against amoxicillin respectively. Only one Serratia spp was isolated which was sensitive to ciprofloxacin, gentamicin, amikacin, ceftriaxone, and meropenem but resistant to amoxicillin, cotrimoxazole, ceftazidime, cefotaxime, and cefuroxime. The most sensitive antibiotic against all other gram negatives was colistin 100%, against Klebsiella, Acinetobacter, E. coli, and Enterobacter.  Proteus was 100% resistant to it because Proteus is intrinsically resistant to colistin (Table 6).

Table 6: Antimicrobial resistance patterns of isolated gram-negative bacteria (n=786) in wound infections.

Nt= Not Tested

Discussion

Wound infection remains a significant concern among healthcare practitioners worldwide, owing to associated morbidity and mortality [10]. It is an important cause of illness that results in prolonged hospital stays and increased treatment costs. It is also likely to play an important role in the development of antimicrobial resistance [11]. Therefore, correctly identifying organisms and determining antimicrobial susceptibility patterns is crucial for appropriately managing wound infection.

In our study, out of 1630 samples from wound infection, 48.22% of samples showed growth. A study by Maharjan, Kartik, Shrestha, and Basnet showed similar results of 50.95%, 47%, and 50% growth from wound infection [1]. In contrast to this study, a higher isolation rate was reported by Mohammed et al. (83.9%) [12]. On the contrary, the rate was lower (61.8%) in the study by Khanam et al. [13]. This difference in bacterial isolation rate may be due to differences in the types of wounds, specimen collection procedures, specimen quality, antibiotic intake of the patient, or microbiological techniques used.

A higher wound infection rate was recorded in males 1120(68.72%) than in females 510(31.28%) of which culture-positive in males were 476(60.56%) and females 310(39.44%) respectively. Similar male predominance was also reported in other studies [14,15].  The reason might be attributed to the fact that male employment is higher in this country. They are involved in occupations such as construction work, farming, transportation, and industry work and were exposed to trauma. In our study, the majority (53.94%) of wound infection cases were within 21-40 years of age group. This agrees with other studies, where it was reported that people in their second to fourth decades of life are more prone to wound infection [12,16]. This is the most vulnerable age group; people are involved in different types of work and have a higher risk of exposure to a variety of wounds.

In our study, out of total bacterial isolates, 82.02% were gram-negative and 17.93% were gram-positive bacteria.  In a similar study conducted by Giri et al. (2008), Iregbu et al. (2013), and Eselbelhie et al. (2013), gram-negative bacteria were found to be predominant, which was 56.79%,66%, and 65.45% respectively [17,18,19]. Most hospital-based studies showed that gram-negative bacteria were more prevalent than gram-positive bacteria. Banjara et al. (2002) showed that a higher rate of gram-negative bacteria was found in the HAI (Hospital-acquired infection) [20]. A similar study was conducted by Acharya [21] and Yakha et al. [22] where gram-negative bacteria were predominant. The higher number of gram negative isolates in our study may be attributed to the inclusion of hospitalized patients only as it is well known that hospitalization and the procedure undertaken after hospitalization increase the risk of acquiring gram negative infections, The other causes may include the regional variations in geographic locations and socioeconomic status of the study population [22].

Among these isolated bacteria, P. aeruginosa was most predominant (32.69%) among total gram-negative isolates followed by Klebsiella 29.26%, Acinetobacter 11.57%, E.coli 5.21%, Proteus spp 2.16%, Enterobacter spp 1.01% and Serratia spp 0.12%. At the same time, S. aureus was (17.94%) predominant among total gram-positive isolates. In a similar study conducted by Thanni et al. 2003, P. aeruginosa was the most predominant one (29.9%) among the total isolates, while S. aureus was predominant (27.5%) among the isolated gram-positive bacteria. In a study conducted by Irebgu et al. (2013) [19], gram-negative bacilli constituted 66% of all pathogens detected, and P. aeruginosa was the most frequent (19%). In another study by Pondei K (2013) in Nigeria, P. aeruginosa was the predominant pathogen in wound infection [23], which differed from other studies in Nigeria reporting S. aureus as the predominant bacteria isolated [24,25]. This disparity might be due to the endogenous infection source or wound contamination from the environment or skin surface.

In our study. S aureus showed 100% sensitivity to vancomycin and linezolid, followed by gentamicin (58.86%), whereas amoxicillin and ciprofloxacin were more resistant (84.39% and 82.62%, respectively).  Another two studies had shown 100 % sensitivity to Linezolid and vancomycin, followed by gentamicin 78.75% and 73.35%, [26,27], whereas organisms showed maximum resistance to amoxicillin, ciprofloxacin, and erythromycin [28,29]. The above two findings are nearly like our study findings.

In our study, 16.31% of S. aureus isolates were MRSA and emerged as a multidrug-resistant pathogen worldwide. Studies on MRSA have shown their wide variation. Naik and Deshpande (2011) showed 8.0% MRSA, consistent with our study [30]. Another study done by Pant et al. (2018) detected 30.70% MRSA, which was higher than our study [31]. Similarly, higher detection was also observed in other studies by Balchandra et al. and Giri et al. 67.6%, and 53.06%, respectively [23,32]. This finding shows that the prevalence of MRSA is increasing.  The most effective drugs for MRSA were linezolid and vancomycin, which were 100% sensitive among those isolates, and the finding was similar to the study done by Harsan et al [31].

The remarkable susceptibility of S aureus to vancomycin, linezolid, and gentamicin might be due to the lesser use of these antibiotics owing to their low availability, cost, and adverse effects. Low activities of commonly used antibiotics such as cefradine, erythromycin, and ciprofloxacin might be due to increased consumption of these antibiotics, which leads to selection pressure, giving rise to the multiplication of resistant organisms. Increasing resistance might also result from mutation at drug target sites or the disturbance of drug accumulation in the cytoplasm due to cell wall or membrane rearrangement [33]. As a result, they have lost their efficacy in treating wound infections.

The antibiotic susceptibility pattern of isolates revealed high resistance to selected antimicrobials. Bacterial isolates were mainly resistant to amoxicillin (94-100%) and cephradine (73-100%). Similar results were also reported in other studies [12,16,27]. Widespread and non-judicious use of antibiotics without sensitivity testing and self-medication, availability of antibiotics, and low cost might promote the development of resistance to these antibiotics. Similarly, resistance to third-generation cephalosporin like ceftriaxone, cefotaxime, and ceftazidime was higher (60-80% vs 70-100% vs 60-74% respectively). These findings agreed with Sultan et al. [27]. The resistance pattern may be due to the widespread and frequent overuse of third generation cephalosporins for an extended period in this country. Similar studies by Khanam et al. and Sultana et al. supported these findings [13,27]. In our study among gram-negative bacteria, ciprofloxacin resistance was (71-89%). However, other studies reported higher sensitivity to ciprofloxacin (81.2%), (91.8%) and (75.3%) respectively  [12,14,15]. This reduced sensitivity in the present study might result from extensive use of these drugs in clinical practice without susceptibility testing. The most effective antibiotics in our study were colistin, meropenem, amikacin, and gentamicin. Bacterial isolates were reasonably sensitive to these antimicrobial agents, which agrees with other studies [13,27]. This may be attributed to the fact that these antibiotics are less commonly prescribed for empirical treatment and are only used in hospitalized patients, according to susceptibility reports.

Among isolated gram-negative bacteria, Pseudomonas showed the lowest resistance to colistin, meropenem, piperacillin+tazobactam, and amikacin (10.11%,45.91%, and 47.85% respectively).  However, almost all other drugs were resistant. The study done by Albumani et al. [34] showed variable susceptibility patterns with meropenem, piperacillin plus tazobactam, ciprofloxacin, and ceftazidime (100%, 87.71%, 85.71%, and 71.42% respectively) for P. aeruginosa. P. aeruginosa has a high intrinsic and acquired resistance mechanism to counter most antibiotics.

In our study, P. aeruginosa was less resistant to imipenem/meropenem (45.91%) and colistin (10.11%) and maximum resistant to ciprofloxacin and ceftazidime. Rajput et al. [35] agreed, in which Pseudomonas isolates from wound swabs were less resistant to imipenem/meropenem (26%) and showed maximum resistance to ceftazidime (70%).

Due to the retrospective nature of this study, we were unable to present detailed clinical data on a patient to identify predictors of all forms of wound infection and antimicrobial resistance. This calls for improvements in patient documentation and record keeping.

Conclusion

The present study identified a high frequency of bacterial isolates from wound infections. The predominant isolates were Pseudomonas, Klebsiella, Acinetobacter, and Staphylococcus. Most of the isolates were found to be resistant to commonly used drugs. Hence, periodic monitoring and surveillance of antimicrobial susceptibility testing are essential for proper wound infection management.

Funding:

Not applicable.

Data availability:

The data is contained within the manuscript and supplementary material.

Competing Interests:

The authors declare no conflict of interest.

Author’s Contribution

SMA and SA drafted the manuscript. SMA provided statistical analysis of the data. IN, RRK, CKR, AAS, NIS, and SKS validated the results, and revised the manuscript.

References

1. Maharjan N. Bacteriological Profile of Wound Infection and Antibiotic Susceptibility Pattern of Various Isolates in a Tertiary Care Center. Ast in Wound Infection. 2020;8(2): 218-224.
2. Tarana MN, Fardows J, Farhana N, Khatun R, Akter S. Bacteriological Profile of Wound Swab and Their Antimicrobial Susceptibility Pattern in Shaheed Suhrawardy Medical College, Dhaka. J Shaheed Suhrawardy Med Coll. 2019;11(1):65–8.
3. Pushpa M. Bacteriological Profile of Wound Infection and Antibiotic Susceptibility Pattern of the Isolates. J Microbiol Exp. 2017;4(5):1–6.
4. Pallavali RR, Degati VL, Lomada D, Reddy MC, Durbaka VRP. Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections. PLoS One. 2017;12(7):1–16.
5. Puca V, Marulli RZ, Grande R, Vitale I, Niro A, Molinaro G, et al. Microbial species isolated from infected wounds and antimicrobial resistance analysis: Data emerging from a three-years retrospective study. Antibiotics. 2021;10(10).
6. Mls B, Onemu S, Phil M. Microbiology of Wound Infections and its Associated Risk Factors among Patients of a Tertiary Hospital in Benin City, Nigeria. JRHS 2011; 11(2): 109-113.
7. Clinical and Laboratory Standards Institute. Institute CaLS. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animalsCLSI Supplement VET08. Edited by Pennsylvania. CLSI Suppl M100. 2018;4:282.
8. Weinstein MP, Lewis JS. The clinical and laboratory standards institute subcommittee on Antimicrobial susceptibility testing: Background, organization, functions, and processes. Journal of Clinical Microbiology. 2020.
9. Limbago B. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 29th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2019. Clin Microbiol Newsl. 2019;23(6):49.
10. Mayhall CG. The epidemiology of burn wound infections: Then and now. Clin Infect Dis. 2003;37(4):543–50.
11. Sader HS, Jones RN, Silva JB. Skin and soft tissue infections in Latin American medical centers: Four-year assessment of the pathogen frequency and antimicrobial susceptibility patterns. Diagn Microbiol Infect Dis. 2002;44(3):281–8.
12. Mohammed A, Seid ME, Gebrecherkos T, Tiruneh M, Moges F. Bacterial Isolates and Their Antimicrobial Susceptibility Patterns of Wound Infections among Inpatients and Outpatients Attending the University of Gondar Referral Hospital, Northwest Ethiopia. Int J Microbiol. 2017;2017.
13. Khanam RA, Islam MR, Sharif A, Parveen R, Sharmin I, Yusuf MA. Bacteriological Profiles of Pus with Antimicrobial Sensitivity Pattern at a Teaching Hospital in Dhaka City. Bangladesh J Infect Dis. 2018;5(1):10–4.
14. Azene MK, Beyene BA. Bacteriology and antibiogram of pathogens from wound infections at Dessie Laboratory, North-east Ethiopia. Tanzan J Health Res. 2011;13(4):68–74.
15. Mama M, Abdissa A, Sewunet T. Antimicrobial susceptibility pattern of bacterial isolates from wound infection and their sensitivity to alternative topical agents at Jimma University Specialized Hospital, South-West Ethiopia. Ann Clin Microbiol Antimicrob. 2014;13(1).
16. OM Ogba OOGA. Bacterial pathogens associated with wound infections in Calabar, Nigeria. J Med. 2014;13(1):26–33.
17. Giri K, Gurung S, Subedi S, Singh A, Adhikari N. Antibiotic Susceptibility Pattern of Bacterial Isolates from Soft Tissues Infection among Patients Visiting Birendra Military Hospital, Chhauni, Kathmandu. Tribhuvan Univ J Microbiol. 2019;6(July):119–26.
18. Reviews ID. Bacteriology of Wound Infections in Nigeria and its Effect on Antimicrobials Selection during Management. Infect Dis Rev. 2022;1–19.
19. Esebelahie N, Newton-Esebelahie F, Omoregie R. Aerobic bacterial isolates from infected wounds. African J Clin Exp Microbiol. 2013;14(3):155–9.
20. Adhikari K, Basnyat S, Shrestha B. Prevalence of Multidrug-Resistant and Extended-spectrum Betalactamase Producing Bacterial Isolates from Infected Wounds of patients in Kathmandu Model Hospital. Nepal J Sci Technol. 2020;19(1):171–9.
21. Yakha JK, Sharma AR, Dahal N, Lekhak B, Banjara MR. Antibiotic Susceptibility Pattern of Bacterial Isolates Causing Wound Infection Among the Patients Visiting B & B Hospital. Nepal J Sci Technol. 2015;15(2):91–6.
22. Fisman D, Patrozou E, Carmeli Y, Perencevich E, Tuite AR, Mermel LA, et al. Geographical variability in the likelihood of bloodstream infections due to gram-negative bacteria: Correlation with proximity to the equator and health care expenditure. PLoS One. 2014;9(12):1–18.
23. Pondei K, Fente BG, Oladapo O. Current microbial isolates from wound swabs, their culture and sensitivity pattern at the Niger Delta University Teaching Hospital, Okolobiri, Nigeria. Trop Med Health. 2013;41(2):49–53.
24. B. A Wariso et al. A survey of common pathogens in wound in patients at the University of Port Harcourt Teaching Hospital (U.P.T.H), Port Harcourt. WAJM. 2003;22: 50-54.
25. Kawai M, Yamada S, Ishidoshiro A, Oyamada Y, Ito H, Yamagishi JI. Cell-wall thickness: Possible mechanism of acriflavine resistance in meticillin-resistant Staphylococcus aureus. J Med Microbiol. 2009;58(3):331–6.
26. KC R, Shrestha A, Sharma V. Bacteriological Study of Wound Infection and Antibiotic Susceptibility Pattern of the Isolates. Nepal J Sci Technol. 2014;14(2):143–50.
27. Sultana S, Mawla N, Kawser S, Akhtar N, Ali MK. Current Microbial Isolates from Wound Swab and Their Susceptibility Pattern in a Private Medical College Hospital in Dhaka city. Delta Med Coll J. 2015;3(1):25–30.
28. Kwong SM, Ramsay JP, Jensen SO, Firth N. Replication of staphylococcal resistance plasmids. Front Microbiol. 2017;8(NOV):1–16.
29. Memmi G, Filipe SR, Pinho MG, Fu Z, Cheung A. Staphylococcus aureus PBP4 is essential for β-lactam resistance in community-acquired methicillin-resistant strains. Antimicrob Agents Chemother. 2008;52(11):3955–66.
30. Naik G, Deshpande SR. A study on surgical site infections caused by staphylococcus aureus with a special search for methicillin-resistant isolates. J Clin Diagnostic Res. 2011;5(3):502–8.
31. Shrestha D. Antibiogram of bacterial species causing skin wound infection. Nov Res Microbiol J. 2018;2(3):53–60.
32. Harshan KH, Chavan SKD. Prevalence and Susceptibility Pattern of Methicillin Resistant Staphylococcus aureus (MRSA) in Pus Samples at Tertiary Care Hospital in Trivandrum, India. Int J Curr Microbiol Appl Sci. 2015; 4(11):718-723.
33. Barker KF. Antibiotic resistance: A current perspective. Br J Clin Pharmacol. 1999;48(2):109–24.
34. Giacometti A, Cirioni O, Schimizzi AM, Prete MSDEL, Barchiesi F, Errico MMD, et al. Epidemiology and Microbiology of Surgical Wound Infections. 2000;38(2):918–22.
35. Mengesha RE, Kasa BG, Saravanan M, Berhe DF. Aerobic bacteria in post surgical wound infections and pattern of their antimicrobial susceptibility in Ayder Teaching and Referral. 2014;4–9.