An Increasing Trend in the Antimicrobial Resistance of Bacterial Isolates from Skin and Soft Tissue Infections in a Tertiary Care Hospital

Skin and soft tissue infections (SSTIs) are commonly occurring infections with mild to serious clinical manifestations. The incidence of wound sepsis in India ranges from 10-33%1,2. It is important to know the potential microbial pathogens causing wound infections for clinicians to start empirical treatment for patients, while laboratory culture reports are awaited. To identify the common microorganisms and their antimicrobial resistance pattern in pus samples. A total of 8656 pus samples were received in the Microbiology department from various OP and IP departments of Stanley Medical College Hospital, Chennai. The samples were processed in the laboratory for aerobic culture to isolate the pathogens and to perform antibiotic sensitivity testing as per standard protocol31. This prospective study was done for a period of twelve months (Jan. 2018 to Dec. 2018). Growth was observed in 5793 samples (66.92%), while growth was absent in 2863 samples (33.07%). Of the culture positive samples, 250 (4.31%) showed mixed infection, while 5543 samples (95.68%) yielded a single isolate. In this study, among the isolates (6043 in number), 5965 (98.70%) were bacterial and 78 (1.29%) were fungal. The most common bacterial isolate was Pseudomonas species (27.42%), followed by Staphylococcus aureus (15.60%), Klebsiella pneumoniae (11.95%), Escherichia coli (9.53%), Coagulase negative Staphylococci (9.22%) and Acinetobacter spp. (8.65%). Among the S.aureus isolates, 59% were Methicillin resistant and 41% were Methicillin sensitive. The fungal isolates were Candida spp. ( 80.76%) and Aspergillus spp. (19.24%). The common pathogens isolated in this study were Pseudomonas species (27.42%), Staphylococcus aureus (15.60%) and Klebsiella pneumoniae (11.95%). The increased incidence of antimicrobial-resistant microorganisms like Methicillin-resistant S. aureus, ESBL and MBL producers causes great global concern leading to more difficult to treat infections and death.


InTRODuCTIOn
Skin and soft-tissue infections (SSTIs) encompass a variety of pathological conditions that involve the skin and underlying subcutaneous tissue, fascia, or muscle, ranging from simple superficial infections to severe necrotizing infections 3 . They are caused by microbial pathogens in wounds due to trauma, burns, and surgical procedures and result in the production of pus, a yellowish white fluid formed as a part of an inflammatory response, composed of exudate containing dead WBCs, cellular debris, necrotic tissues and pathogenic bacteria. 10. 26 Uncomplicated infections are often superficial and can be treated by incision and drainage alone or along with oral antibiotics The complicated SSTIs extend to subcutaneous tissue, fascia, or muscle and require a combination of antimicrobials with surgical intervention. 29 According to the definitions of Centers for Disease control and Prevention, Criteria for Skin Infections include purulent drainage, pustules, vesicles or boils with tenderness, swelling or erythema. Criteria for Soft tissue infections include Purulent discharge from affected site, positive culture from tissue or drainage from affected site, abscess or gross evidence of infection. 30 Among hospitalized patients, the estimated prevalence of SSTIs is 7%-10% 29 . In South India, incidence is about 2% 2 and the mortality rate ranges from 4% (Singh et al, North India) 8 to 14% (Abhilash KP et al, South India) 2 Even with advances in infection control practices like improved operating room ventilation, sterilization methods, surgical technique and availability of antimicrobial prophylaxis, Surgical Site Infections (SSIs) remain a substantial cause of morbidity, prolonged hospitalization and mortality rate of 3% globally 24 . Surgical-site infection (SSI) is an infection of the skin or deep-space occurring at the incision or in the field of an invasive procedure within 30 days after operation (1 year for an implant) 24 . SSIs are the most common healthcareassociated infection (HAI), accounting for 31% of all HAIs among hospitalized patients. 29 Skin and soft tissue infections: open sores (ulcers, burns and bedsores) encourage bacterial colonization and may lead to systemic infection 24  Successful management of patients with severe SSTIs involves prompt recognition, appropriate antibiotic therapy, timely surgical debridement or drainage, and resuscitation when required 3 . Rapid emergence of antimicrobial resistant strains necessitates periodic evaluation of antimicrobial resistance patterns of potential pathogens to frame an antimicrobial policy for implementation in the health care setting.
The objective of this study is to identify the pyogenic bacteria from pus samples and to determine their antibiotic susceptibility to various antibiotics commonly used in therapy.

Study group
This is a cross-sectional study conducted at Stanley Medical College Hospital in Chennai, South India. In this study, patients of all age groups and both genders from out-patients and in-patients presenting with signs and symptoms of SSTI were included. Patients who were admitted in the hospital for more than 3 days ,on prior antibiotic treatment, those with infected burns were excluded from this study. A total of 8656pus samples collected from out-patients and in-patients were studied during the period January 2018 to December 2018.

Specimen collection and processing
Number of pus samples collected from general surgery department were 3909, orthopaedics department 2555, medicine department 761, ENT department 543,other surgical specialities 487 and Obstetrics and gynaecology department 401.
Pus samples were collected using sterile cotton swabs placed in sterile tubes prepared and sterilized in-house by the lab and pus aspirates were collected using sterile disposable syringes (Paras syringes). Tissue specimens were obtained from wound margins, ulcers and deep seated infections after surgical debridement of the wound and collected in Brain heart infusion broth.
Samples were immediately transported to the Microbiology laboratory and processed as per standard protocol. 31 Samples were inoculated on to Blood agar (BA) and MacConkey agar (MA) and the plates were incubated at 37°C for 24 to 48 hrs. Gram staining of the samples were done for microscopy. Tissues inoculated in BHI broth were subcultured after 24 hours. Bacterial culture isolates were identified by standard microbiological techniques like Gram staining and biochemical reactions such as catalase test , coagulase test, Urease test, Mannitol fermentation, Bile esculin hydrolysis and heat tolerance test for Gram positive cocci .
Gram staining, motility testing, Oxidase test, Indole test, Citrate utilization, Urease test, Triple sugar iron test, Mannitol motility medium test and phenyl pyruvic acid test were done for identification of gram negative bacilli.

MIC testing
To

Quality Control
Quality control (QC)for antimicrobial susceptibility testing is done in the laboratory once a week and, in addition,every time a new batch of Mueller-Hinton agar or a new batch of discs is used. Performance testing of media used for culture and biochemical reactions are done once a week using control strains. The standard strains used for QC are Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). 27

Fungal culture
Fungal growth observed in Blood agar plates were sub-cultured onto Sabouraud Dextrose Agar and identified by colony characteristics, Germ tube test, Gram staining and Lactophenol Cotton blue mount.

Statistical Analysis
Data were collected and analyzed using SPSS software by descriptive statistical methods by computing means and proportion with 95% confident interval. A p < 0.05 was considered statistically significant.
Among the 5965 bacterial isolates, the most common bacteria isolated was Pseudomonas species (1636 isolates, 27.     The isolates of Pseudomonas showed high sensitivity to Piperacillin-Tazobactam and Carbapenems in all wards. (Fig. 5) In this study ,78% of Enterobacteriaceae were detected to be ESBL producers by double disc ESBL test. 59% of Staphylococcus aureus isolates were methicillin resistant. 15% of isolates of Pseudomonas spp. and Acinetobacter spp. were MBL producers.

DISCuSSIOn
Skin and soft tissue infection is a common medical condition and the disease burden is high. In this study,a total of 8656 pus samples were collected from out-patients and in-patients with the aim of isolation of pathogenic micro-organisms and to study the antibiotic resistance pattern.
Among the samples collected, 7642 were pus swabs (88.3 %) and 961 were aspirates (11.1%). This is similar to a study done by Upreti et al where 85.7% of the samples were pus swabs and 14.3% were aspirates. 28 Pus samples were received maximally from general surgery department (3909, 45.15%), followed by orthopaedics department (2555, 29.51%). Higher number of samples from surgery department has been observed in many studies (Roopa et al) 7 . The most common age group affected by pyogenic infection in our study was 41-50 years and there was a predominance of males(75.7%) over female patients(18.9%).This was comparable to a study by Singh et al 8 and Roopa et al 7 .
Growth was observed in 5793 samples (66.92%). Isolation rate correlates with several studies done on pus cultures in developing countries like India and Nepal ranging between 60-75% (Esposito et al. 14 16 show polymicrobial growth in 20% and 8.6% respectively. Open wounds get easily colonized and invaded by numerous bacteria as they provide a warm and moist environment for bacterial colonization and proliferation. This might be the reason for polymicrobial growth  34 Antibiotic sensitivity testing of the isolated Gram negative bacilli showed that most of them were sensitive to Carbapenems, Piperacillin-tazobactam, Amikacin, Aztreonam and Levofloxacin but resistant to Ampicillin and Amoxycillin-Clavulanic acid. All the multidrug resistant strains were sensitive to Colistin and Tigecycline. These findings are correlating with several pus culture studies (Roopa et al. 7 ).
Most of the isolates of Staphylococcus aureus and CoNS were highly sensitive to Linezolid, Levofloxacin and Gentamicin and moderately sensitive to Cotrimoxazole and Clindamycin. All the resistant strains were sensitive to Vancomycin. The Streptocooci isolates were highly sensitive to all the antibiotics. Enterococci spp were 100% sensitive to Linezolid and Vancomycin and moderately sensitive to Ciprofloxacin(58%)and High level Gentamicin (59%). In the study by Mudassar et al S.aureus isolates were highly sensitive to Gentamicin(86%)and Clindamycin(79%) 33 Extended spectrum Beta-lactamase production as determined by double disk test was around 78%. This is comparable to a study done in India which gives ESBL rate of 68% by Agrawal et al 18 and 84% by Perumal et al 19 .

COnCLuSIOn
In this study, Pseudomonas spp. was the most common pathogen isolated (27.42%) followed by Staphylococcus aureus(15.6%)%), Klebsiella pneumoniae (11.95%) and Escherichia coli (9.53%). The study indicates gram-negative bacteria as significant emerging causative agents of SSTI in our setting. Antibiotic susceptibility pattern of the isolated pathogens shows high sensitivity to higher antibiotics like Carbapenems, Piperacillin-Tazobactam and Linezolid. MBL production was observed in 15% of the Pseudomonas strains.78% of the Enterobacteriaceae isolates were ESBL producers and 59% of Staphylococcus aureus strains were methicillin resistant. This study shows the common pathogens causing Skin and soft tissue infections and their antibiotic sensitivity to plan empiric treatment for patients in our center.
Prolonged hospital stay, indiscriminate use of antibiotics and lack of awareness are the possible predisposing factors of emergence of ESBL and MRSA. This study highlights the importance of infection control practices in health care settings to prevent the spread of resistant strains and avoiding indiscriminate and irrational use of antibiotics.