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ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 4  |  Page : 261-268

Bacteriological profile of postoperative wound infection in LSCS patients in MKCG Medical College, Berhampur


1 Department of Microbiology, MKCG Medical College, Berhampur, Odisha, India
2 Department of Microbiology, SCB Medical College, Cuttack, Odisha, India

Date of Submission19-Dec-2020
Date of Decision28-Aug-2021
Date of Acceptance07-Sep-2021
Date of Web Publication22-Mar-2022

Correspondence Address:
Dr. Swetalina Dash
Department of Microbiology, 3rd year PG, MKCG Medical College, Berhampur, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdrntruhs.jdrntruhs_202_20

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  Abstract 


Background: Post-lower segment cesarean section (LSCS) infection is a common complication and is mainly responsible for a longer hospital stay, higher treatment cost, and maternal mortality.
Aim: To isolate and identify the different bacterial spp. in patients with postoperative LSCS wound infection and to determine their antimicrobial susceptibility.
Material and Methods: Pus samples were collected from 30 patients with infected LSCS wounds using two sterile swabs from each patient from May 2019 to July 2019. One was used for Gram stain and the other inoculated into blood agar and MacConkey agar. The bacterial isolates were identified using a standard protocol. An antimicrobial susceptibility test was carried out using the Kirby Bauer disk diffusion method. Double-disk diffusion and E-test using Cefotaxime (CTX)/CTX+and Ceftazidime (CAZ)/CAZ+ were done for Extended Spectrum beta lactamases (ESBL) producer. All isolates were put in Congo red agar to see the biofilm production.
Results: Out of 30 samples, 76% (22) were culture positive. The predominant age group was 20–30 years. Gestational diabetes and hypertension were the common risk factors. Pond bathing was a major predisposing factor. Of the 23 isolates, 65.2% (15) were gram-positive and 34.8% (8) were gram-negative bacteria. Among the gram-positive isolates, Staphylococcus aureus was the predominant isolate (80%) and the other being Enterococcus and M. tuberculosis. Among the gram-negative isolates, Acinetobacter was predominant (50%) followed by Pseudomonas, Escherichia coli, and Klebsiella. All the gram-positive isolates were sensitive to linezolid and vancomycin while among the gram-negative isolates, Escherichia coli, Pseudomonas spp., and Klebsiella spp. were 100% sensitive to piperacillin-tazobactam. A case of multidrug resistance Acinetobacter was found. Of all the isolates, two were ESBL producers, and five were biofilm producers. Those were also methicillin-resistant Staphylococcus aureus (MRSA).
Conclusions: A majority of the LSCS wound infection was due to gram-positive bacteria. Educating the patients about personal hygiene and antimicrobial prophylaxis is thought to decrease the incidence of LSCS wound infection.

Keywords: Biofilm, ESBL, Staphylococcus aureus


How to cite this article:
Dash S, Paty BP, Sahu SK. Bacteriological profile of postoperative wound infection in LSCS patients in MKCG Medical College, Berhampur. J NTR Univ Health Sci 2021;10:261-8

How to cite this URL:
Dash S, Paty BP, Sahu SK. Bacteriological profile of postoperative wound infection in LSCS patients in MKCG Medical College, Berhampur. J NTR Univ Health Sci [serial online] 2021 [cited 2022 May 28];10:261-8. Available from: https://www.jdrntruhs.org/text.asp?2021/10/4/261/339818




  Introduction Top


The surgical site infection often develops after a surgical procedure and is defined as an 'infection that develops at the surgical site within 30 days of surgery.'[1] Postoperative lower segment cesarean section (LSCS) wounds are the common surgical site infection (SSIs) among other SSIs in the Department of Obstetrics and Gynecology, resulting in higher treatment costs and longer hospital stays. The diagnosis of the infected wound requires evidence of clinical signs and symptoms and confirmation by microbiological examination.[2] Depending upon the degree of microbial contamination, the wounds are classified into four types.[3] The LSCS wound belongs to type class Ⅱ that is known as the clean-contaminated type.[3] Post-LSCS wound infection is often caused by endogenous as well as exogenous bacteria. The endogenous sources are from the patients' flora, i.e., from the skin and mucosa of the gastrointestinal tract (GIT) or respiratory tract. The exogenous sources are based on contact with the operation room personnel, instrument, and/or environment involving Staphylococcus, Pseudomonas, and different gram-negative bacilli.[4] In general, most cesarean section infections depend on various risk factors: multiparity, preterm labor, prolonged labor, previous LSCS history, and lack of antibiotic prophylaxis.[5]

Antimicrobials are the mainstay treatment that results in a better prognosis for the patient. However, nowadays antimicrobial resistance like methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β lactamases (ESBL)-producing bacteria are found to exhibit a rising trend that hampers with the treatment.[6],[7] Remarkably, bacteria that produce biofilms are reported to be significantly virulent and are more resistant to antimicrobials.[8] This is due to the complex structure of the biofilm that prevents the access of antibiotics into the bacteria. However, the mechanism of biofilm production, and the relation to antimicrobial resistance, are not clear yet. Enzymes like alginate lyase and DNase prevent biofilm production and that dissolves the extracellular polymeric substance (EPS) matrix, resulting in an increase in the susceptibility toward an antibiotic.

For effective control of wound infection and proper use of antimicrobials, knowledge of the causative organism and its sensitivity pattern is mandatory.[9] There are many recent studies[5],[10] available on this topic, however, there is no clear evidence regarding the antimicrobial resistance related to biofilm production.

In the present studies, our main aims and objectives are the following:

  • To isolate and identify different bacterial species causing postoperative LSCS wound infection
  • To perform their antibiotic susceptibility test and look for any special correlation
  • To know the prevalence of LSCS wound infection in our hospital
  • To correlate the antimicrobial resistance with biofilm production



  Materials And Methods Top


The prospective study was conducted in the Department of Microbiology, MKCG Medical College, Berhampur, Odisha.

The study period was from May 2019 to July 2019. Informed consent was taken from all patients with infected LSCS wounds.

Sample size

A total of 1,450 cases of LSCS were conducted in the Department of Obstetrics and Gynaecology, MKCG Medical College, Berhampur, Odisha, out of which 30 patients developed SSIs. The details of the patients were recorded as per Performa.

Ethical approval

Consents were taken from the patients. The institutional ethical committee clearance was also taken.

Inclusion criteria

All women having infected LSCS wound during the hospital stay or 30 days following the LSCS surgery.

Exclusion criteria

Patients having infected wound due to normal vaginal delivery.

Sample collection

Pus was collected using two sterile swabs from all the 30 patients having infected LSCS wounds. The swab was put deep inside the wound and the pus was collected to reduce contamination with skin commensals. One swab was used for staining and the other for culture.

Staining

The Gram stain and ZN stain were done for each patient by taking appropriate control.

Culture and sample processing

The sample processing and culture are presented schematically below. The pus samples were inoculated into blood agar and MacConkey agar plates. The plates were incubated at 37°C overnight for 18–24 h. In case, when the Ziehl-Neelsen (ZN) stain was positive, the sample was inoculated into an Lowenstein Jensen (LJ) medium.



(Schematic of samples processing and their culture.)

Identification of the isolates was done using the standard identifying protocol.[11] The antimicrobial susceptibility was done by the Kirby Bauer disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI)[12] by using gentamicin (10 μg), cefotaxime (30 μg), piperacillin-tazobactam (100/10 μg), ciprofloxacin (5 μg), imipenem (10 μg), and cefepime (30 μg) disks for gram-negative isolates. On the other hand, the antimicrobial susceptibility test for the gram-positive isolates such as Staphylococcus aureus Scientific Name Search  and Enterococcus spp. was carried out using two separate sets of disks. For Staphylococcus aureus, cotrimoxazole (1.25/23.75 μg), clindamycin (5 μg), vancomycin (5 μg), azithromycin (15 μg), cefoxitin (30 μg), and linezolid (30 μg) disks were used, and for Enterococcus spp., vancomycin (5 μg), linezolid (30 μg), high-level gentamycin (30 μg), ampicillin-sulbactam (10/10 μg) and ampicillin (10 μg) were used.

Double-disk diffusion using cefotaxime and cefotaxime + clavulanic and ceftazidime and ceftazidime + clavulanic disk were done for ESBL detection and confirmed by Epsilometer test (E-test) using Enz minimum inhibitory concentration (MIC) detection E-strips (HIMEDIA). In the E-strip containing CTX+/CTX, the gradient of cefotaxime + clavulanate (0.016–1), and that of cefotaxime alone (0.25–16) were used.

MRSA was detected using the cefoxitin disk (30 μg).

For the detection of biofilm, Congo red agar plates were inoculated with all the test organisms and incubated at 37°C for 24–48 h aerobically. Black-colored colonies on the plate suggested that the bacteria were biofilm producers.[13]

The Congo red agar is a medium composed of brain heart infusion broth (37 gm/L), sucrose (5 gm/L), agar-agar type 1 (10 gm/L), and Congo red dye (0.8 gm/L). The Congo red stain was prepared as a concentrated aqueous solution and autoclaved at 121°C for 15 min. The solution was then added to the autoclaved brain heart infusion agar with sucrose at 55°C.[13]


  Results Top


A total of 1,450 cases of LSCS operation were carried out in our hospital of which 30 patients developed infected LSCS wounds (2.06%). The pus samples were collected from all the 30 patients, out of which 22 were Gram stain positive, well correlated with the culture report (100%). Out of those 30 samples, 22 were culture positive (73.33%) and 8 were culture-negative (26.66%). Among these patients, 66% of the females were multiparous and 60% of them were staying in rural areas. Anemia, gestational diabetes, and hypertension are noticed to be the other risk factors that cause a delay in wound healing [Table 1].
Table 1: Factors predisposing to post-lscs wound infection

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Out of 30 samples, 22 samples were culture positive: 21 samples were monomicrobial and the remaining 1 sample was polymicrobial in which two bacteria were isolated. In total, 23 bacteria were isolated out of which 15 isolates were gram-positive and 8 were gram-negative.

The most common isolate was Staphylococcus aureus[12] followed by Acinetobacter spp.[4] [Table 2].
Table 2: Bacteria isolated are listed

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A case of Mycobacterium tuberculosis was isolated which was confirmed by its growth in the LJ media which took more than a month to grow. The growth of Mycobacteria in the LJ medium and the ZN stain showing acid-fast bacilli are depicted in [Figure 1] and [Figure 2], respectively.
Figure 1: Mycobacterium tuberculosis on the LJ medium is shown

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Figure 2: ZN stain showing acid-fast bacilli is depicted

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  Antimicrobial Susceptibility Test (AST) Results Top


It was observed that 100% of the gram-positive isolates were susceptible to linezolid and vancomycin [Table 3] and [Table 4]. [Figure 3] and [Figure 4] depict the antimicrobial susceptibility pattern of Staphylococcus aureus and Enterococcus spp., respectively.
Table 3: Antimicrobial susceptibility test results of staphylococcus aureus using different antimicrobial disks

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Table 4: Antimicrobial susceptibility test results of enterococcus spp.

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Figure 3: The AST plate of Staphylococcus aureus

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Figure 4: Shows the AST plate of Enterococcus spp

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A 100% of Klebsiella spp.,  Escherichia More Details coli, and Pseudomonas spp. were found to be sensitive to piperacillin-tazobactam [Table 5]. A multidrug-resistant Acinetobacter baumanii was isolated as shown in [Figure 5]. All the gram-negative isolates showed 100% resistance to cefotaxime and imipenem.
Table 5: Antimicrobial susceptibility test results of gram-negative organisms

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Figure 5: Isolated multidrug-resistant Acinetobacter baumanii is clearly shown

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  Detection Of Extended-Spectrum β Lactamase Producers Top


It was noticed that 50% of Acinetobacter spp. and 100% of Enterobacteriaceae were ESBL producers as seen by the more than 5 mm difference in cefotaxime (CTX) and cefotaxime + clavulanic acid or ceftazidime and ceftazidime + clavulanic acid disk [Figure 6]. This was subsequently confirmed by the E-test results: more than eight-fold decrease in the MIC was found when tested with cefotaxime + clavulanate (CTX +) than that obtained from the test using CTX alone. In our study, it was observed that for a given bacteria, the MIC for CTX was 16 and that for CTX+ was 0.64, hence, the decrease in MIC after the addition of clavulanate was observed to be twenty-five-fold. Thus, the bacteria were confirmed to be ESBL producer [Figure 7].
Figure 6: Double-disk diffusion test results are shown

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Figure 7: E-test showing ESBL production is depicted

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  Methicillin-Resistant Staphylococcus Aureus (MRSA) Detection Using The Cefoxitin Disk Top


Out of 12 Staphylococcus aureus, 5 isolates were found to be MRSA. [Figure 8] shows a case of MRSA.
Figure 8: Detection of MRSA using cefoxitin disk is shown

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  Biofilm Production On Congo Red Agar Top


All the isolates were inoculated onto the Congo red agar and it was observed that all the bacteria that showed biofilm production were MRSAs. The biofilm production was confirmed by a black color colony on Congo red agar as shown in [Figure 9].
Figure 9: Congo red plates with black color colonies indicating biofilm production are illustrated

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  Discussion Top


Developing SSIs is a traumatic experience[4] for the patients on surgery. Despite many aseptic measures and antimicrobial prophylaxis, there are still some cases of SSIs which remain a serious problem.

In India, the incidence of postoperative wound infection ranges from 10 to 25% (ref. 16, Vijayan et al.). In our study, out of the 1,450 cases of LSCS conducted, 30 (2.06%) patients were infected, similar to the prevalence of SSIs reported by the others. For instance, Kochhal et al.[10] reported 3.33%, Anvikar et al.[14] observed 10.06%, and Mah et al.[15] reported 2.8%. The low rate of infection of the wound was probably because the post-LSCS wound infection was a clean-contaminated type of wound, and in some cases, a patient could not be followed up after being discharged from the hospital as they visit local facilities near them.[16]

In our study, multiparity was the main predisposing factor. This may be because of repeated surgery at the same site. People living in rural areas were more prone to SSIs due to their low socioeconomic status and a habit of pond bathing. In our study, anemia was a predisposing factor (50%), consistent with the study of Vijayan et al.[16] where it is shown that anemia decreases the resistance to infection. Another important factor was gestational diabetes that led to a delay in wound healing.

SSIs are caused by exogenous as well as endogenous bacteria. The pathogens from exogenous sources may come from the environment, personnel, and instruments of the operating room, and these mostly include aerobic gram-positive organisms.[4] The most common pathogenic organism isolated was found to be Staphylococcus aureus followed by the observation of Acinetobacter spp. as the second most common isolates, similar to the findings of Morhason-Bello et al.[17] and Zejnullahu et al.[18] The present observation is contrary to the earlier reports[4],[19] that inferred Escherichia coli as the common isolates.

In comparison to pulmonary TB, extrapulmonary TB is difficult to diagnose. In our present study, a case of Mycobacterium tuberculosis was also isolated, similar to the observation of Salam et al.,[20] but is different from Islam et al.'s[21] study, where non-tubercular Mycobacteria were isolated. In our present study, the case with Mycobacterium tuberculosis is presented with the non-healing wound for more than 1 month despite many uses of antimicrobial therapy and surgical debridement. The growth of the Mycobacterium tuberculosis in LJ media took more than a month while that had not grown on the MacConkey agar plate. It is known that some rapid growers such as M. fortuitum and M. abscessus, causing infection in humans, can grow in LJ medium over a short period (nearly 1 week) and can also grow on the MacConkey agar plate.[22] The growth of the Mycobacteria on the LJ medium took a long period suggesting that is likely to be Mycobacterium tuberculosis. The patient recovered after the anti-tubercular drug therapy.

All the gram-positive isolates were observed to be sensitive to linezolid and vancomycin; 100% of gram-negative isolates showed resistance to imipenem, in contrast to the earlier report[5] that reported that most of the isolates were sensitive to carbapenems. The observed resistance to imipenem can be due to the recent excessive use of carbapenems during treatment. All the Pseudomonas isolates were found to be 100% sensitive to piperacillin-tazobactam. Five out of twelve Staphylococcus aureus were observed to be methicillin-resistant (41.66%), in accord with the earlier report.[4]

ESBLs are enzymes produced by gram-negative bacteria that mediate resistance to penicillin, cephalosporins, and monobactams. They are commonly found in different members of the Enterobacteriaceae and Pseudomonas spp.[23]

The gram-negative isolates showing cefotaxime resistance were tested for ESBL production by the double-disk diffusion test. It was observed that 100% of Escherichia coli, Klebsiella spp., and 50% of Acinetobacter spp. were ESBL producers. The ESBL-producing organisms were frequently associated with wound infection which took more time to cure.

Biofilm production is a serious threat to developing antibiotic resistance and is believed to be associated with the surgical suture used to stitch operation sites.[24] Biofilm formation alters the growth rate and produces many transcribing genes which are argued to be the reasons[25] for inherent antimicrobial agents. In our present study, all the isolates were put on Congo red agar to observe biofilm production and it was found that all biofilm-producing isolates were MRSAs.


  Conclusions Top


Even though the rate of the LSCS procedure is increasing day by day, the rate of SSIs related to LSCS is still constant. Most of the infections were caused due to gram-positive isolates. Multiparity, anemia, and lifestyle in rural areas were found to be the predisposing factors that aggravate SSIs. Educating patients on personal hygiene, proper nutritional intake, and prior antimicrobial prophylaxis can decrease the incidence of LSCS wound infection.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Centres for Disease control and prevention's National Healthcare Safety Network (NHSN) Patient Safety Component Manual. CDC, Atlanta, 2019.  Back to cited text no. 1
    
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Gur R, Duggal SD, Rongpharpi SR, Srivastava R, Kumar A, Gupta V, et al. Post caesarean surgical site infections. Arch Clin Microbiol 2015;6:1-6.  Back to cited text no. 2
    
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24.
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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