Pyogenic infections are one of the most common out patient’s encountered health problems in which the bacterial infection causes accumulation of pus in a tissue or organ. Microorganisms breach in to human body either through skin or blood vessels where they evade the host defense, replication of a large numbers of microorganisms and attack the host tissues by releasing cellular or toxic metabolites and leukocidins, which results in local inflammation of skin, soft tissue and bodily parts. Anyone can have pyogenic infections, but it tends to be observed predominantly in males. The risk of pyogenic infections continues to grow as the age advances. The evaluated root cause behind the poor wound healing include age where poor wound healing increases by 34% for each additional year of age, association with injuries to the lower limbs and trunk and perineum, outdoor injuries, co-morbidities like chronic diseases, immunosuppression, malnutrition, radiation therapy and vascular insufficiencies [1], large wounds and stress [2]. Pyogenic infections can be cause by either Gram positive or Gram negative or aerobic or facultative aerobes. The bacterial strain most commonly associated with pyogenic infections are Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Streptococcus pneumoniae, Proteus species [3,4]. Hemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitides pyogenic infections have been drastically reduced in the general population due to vaccination [5]. Pyogenic infections clinical manifestation varied from mild symptoms like carbuncles, abscess, furuncles, venous ulcers, diabetic ulcers, traumatic injuries to severe diseases like osteomyelitis, pyelonephritis, loss of range of motion, spondylitis, meningitis and septic shock. As it is a bacterial pyogenic infection the first and foremost management of the disease is by prescribing antibiotics and other treatment modalities are aspiration or surgical drainage along with antibiotics. Antibiotic therapy is becoming a challenging aspect now-a-days as the resistance has been emerging in various microorganisms; both inherent and acquired resistance plays a vital role in curing the infections especially in vulnerable patients. To avoid the prolonged hospital stay, financial loss, temporary and permanent disability it is strongly recommended to do microbiological surveillance to start the effective therapy [6]. Microbiological surveillance by appropriate sample culture and sensitivity will definitely aid in the earliest accurate antibiotic therapy. So in this study we aimed to evaluate the prevalence of bacterial pathogens in pyogenic infections and to identify the antibiotic susceptibility profile of pyogenic isolates in our community.

Study Design: This is a retrospective cross-sectional study conducted in a Microbiology department of Government General Hospital/Medical College, Nandyal, Andhra Pradesh. Institutional ethics approval was obtained before conducting the study. This study was performed for the period of one year (i.e., December 2023 to November 2024). Sample size: We have monitored 460 pus samples registered for pus culture & sensitivity testing of all the outpatient and inpatient departments to monitor the various pathogens and its susceptibility to antimicrobials. Inclusion criteria: All the participants during the study period irrespective of age and sex were included in this study. Participants with suspicious of bacterial wound infections such as venous ulcers, diabetic ulcers, abscesses and traumatic injuries were included. Exclusion criteria: Patients who were on antibiotics in the last 3 weeks were excluded from this study. Sample collection: A total of 460 pus samples were processed in culture section of Microbiology department. Pus samples were collected under aseptic precautions using a sterile syringe aspiration or sterile swabs by following standard protocols. If it is swab then two pus specimens were collected from each patient, one for gram stain and another one for bacterial culture and sensitivity, these pus samples were transported immediately to the laboratory under aseptic precautions at appropriate temperature. Study Procedure: Standard procedures as per microbiological guidelines were followed for processing of the culture specimens under aseptic precautions. Samples were streaked on media such as nutrient afar, macconkey agar, 5% sheep blood agar and incubated at 37 °C for 24h. Direct microscopic examination of Gram-stained smears of isolates was done. Additional tests included Coagulase test, Sorbitol fermentation, Arabinose fermentation test, other sugar fermentation test, species specific identification tests, Optochin and Bacitracin sensitivity test, and specific biochemical tests to identify Enterobacteriaceae members. After 24 hours of incubation, the bacteria were identified by colony characteristics and biochemical reactions. Antimicrobial susceptibility of the isolates was assessed on Muller Hinton Agar plates using Kirby-Bauer disc diffusion method according to the Clinical Laboratory Standards institute (CLSI) guidelines. The list of antibiotics tested include amikacin (Amk-30 μg), bacitracin (Bac-10 μg), cefipime (Cef-30 μg), ceftazidime (Cez-30 μg), cefoxitin (Cex-30 μg), cefotaxime (Cet-30 μg), ceftriaxone (Ceo-30 μg),cefuroxime (Ceu-30 μg), ciprofloxacin (Cip-5 μg), clindamycin (Cli-2 μg), erythromycin (Ery-15 μg), gentamycin (Gen-10 μg), gentamycin high (GeH-120 μg), levofloxacin (Lev-5 μg), linezolid (Lin-30 μg), meropenem (Mer-10 μg), penicillin (Pen-10 μg), piperacillin (Pip-10 μg), cotrimoxazole, tazobactum (Taz -10 μg), tetracycline (Tet-30 μg), teicoplanin (Tei-30 μg), and vancomycin (Van-30 μg). Data Collection: All the data pertaining to patient demographic characteristics, microbiology data including the type of sample, ward or hospital unit, culture details and their antibiotic susceptibility pattern was collected from microbiology registers and hospital charts. Patient details were kept unlinked anonymously. All the results were calculated as numbers and percentages.

All the patients presenting with pyogenic infections were scrutinized properly and suggested them to undergo culture and sensitivity testing for accurate treatment. A total of 460 pus samples were processed as per the microbiology standard textbooks and references as mentioned in materials and methods. Among these 8 (17.3%) samples showed polymicrobial growth and 269 (58.4%) samples showed mono microbial growth. Out of 285 bacterial isolates, 172 (60.3%) were gram negative bacteria and 113 (39.6%) were gram positive bacteria. The most common age group presented with pyogenic infections was 31-50 years; it was about 64.5% (184 out of 285). Male population was predominantly noted the percentage was 62.4% (178 out of 285). Out of 460 pus cultures, 285 (61.9%) bacterial isolates were noticed. Bacterial distribution among pyogenic infections suggested Escherichia coli (23.8%) as the predominant pathogen followed by Klebsiella species (21.7%) and Staphylococcus aureus (20.3%) (Table 1). Table 1. Distribution of pathogens in pyogenic infections Organism No. of patients Percentage E.coli 68 23.8% Klebsiella species 62 21.7% Staphylococcus aureus 58 20.3% CONS 42 14.7% Pseudomonas aeruginosa 33 11.5% Enterococcus species 13 4.56% Proteus species 9 3.15% Total 285 100% Antibiotic susceptibility testing method of Gram negative bacilli showed higher susceptibility to tetracycline, levofloxacin, meropenem, minocycline, gentamicin, cotrimoxazole, piperacillin+tazobactum, cefaperazone+sulbactum, ceftazdiime +avibactum. Escherchia coli and Klebsiella isolates showed above 80% sensitive to meropenem, imipenem, levofloxacin, tetracycline, ceftazidime+avibactum, 60-80% were sensitive to cotrimoxazole, piperacillin+tazobactum, cefaperazone+sulbactum, gentamicin. Table 2. Antibiotic susceptibility pattern of Gram negative bacilli Organism No. of isolates AMC AS CAZ CTR LE TE G COT PIT CFS CAV MRP MI E.coli 68 42.6% 47.05% 44.1% 44.1% 85.2% 97.0% 76.4% 63.1% 63.1% 63.1% 100% 83.8% 83.8% Klebsiella 62 37.0% 41.1% 41.1% 37.0% 85.4% 85.4% 77.4% 61.2% 61.2% 64.5% 100% 83.8% 80.6% Proteus 33 IR 45.4% 45.4% IR 60.6% 75.7% 72.7% IR 63.6% 63.6% 100% 75.7% 72.7% Pseudomonas 9 44.4% 44.4% 44.4% 44.4% 100% IR 100% 88.8% 88.8% 88.8% 100% 100% 100% *AMC - Amoxiclav, AS - Ampicillin+Sulbactum, CAZ - Ceftazidime, CTR - Ceftriaxone, LE - Levofloxacin, TE - Tetracycline, G - Gentamicin, COT - Cotrimoxazole, PIT - Piperacillin+ tazobactum, CFS - Cefaperazone+sulbactum, CAV - Ceftazidime+Avibactum, MRP - Meropenem, MI - Minocycline. All the Gram positive isolates were 100% susceptible to linezolid, teicoplanin, vancomycin. MRSA and MRCONS were noted as 55.2% and 40.5% respectively. 69.2% of Ampicillin sensitive Enterococcal isolates was observed. No vancomycin resistant isolates were observed. Table 3. Gram positive cocci isolates susceptibility pattern Organism No. of isolates AMP AMC AZM CTX CIP CD CX LE COT DO VA LZ TEI S.aureus 58 32.7% 51.7% 60.3% 44.8% 67.2% 56.8% 44.8% 77.5% 77.5% 79.3% 100% 100% CONS 42 14.2% 47.6% 59.5% 59.5% 73.8% 71.4% 59.5% 80.9% 85.7% 85.7% 100% 100% 100% Enterococci 13 69.2% 76.9% - IR 84.6% IR - 84.6% IR 92.3% 100% 100% 100% *AMP-Ampicillin, AMC - Amoxiclav, AZM - Azithromycin, CTX - Cefotaxime, CIP - Ciprofloxacin, CD - Clindamycin, CX - Cefoxitin, LE - Levofloxacin, COT - Cotrimoxazole, DO - Doxycycline, VA - Vancomycin, LZ-Linezolid, TE-Tetracycline.

Pyogenic infection is a typical inflammatory response which constitutes both living and dead neutrophils, bacteria, local damaged tissue and blood protein [7]. Pus can be localized in deeper soft tissue or spreading infection like cellulitis or necrotizing fasciitis. Wound infections can be endogenous infection in which pus accumulated by patients own flora or exogenous infections in which infection occurs by outside environment either by direct or indirect contact. In the present study out of 285 culture positive pus isolates, 16 (5.61%) samples showed polymicrobial growth and 269 (94.3%) samples showed monomicrobial growth. Out of 285 bacterial isolates, 172 (60.3%) were Gram negative bacteria and 113 (39.6%) were Gram positive bacteria. Roopa C et al [8] did a retrospective study on pyogenic infections Out of 293 samples, 177 (60.40%) samples were positive for growth. Out of 177 samples, 11(6.21%) samples showed polymicrobial growth. The total number of isolates was 188 isolates and gram negative bacteria were isolated more compared to gram positive pathogens. Pyogenic infections were predominantly observed in adult age group of 31-50 years which is similar to few studies done in India [8,9]. The isolation rate of pus samples of this study is in line with Biradar A et al [9], Duggal S et al [10], Rugira Trojan et al [11]. Escherichia coli (23.8%) as the predominant pathogen followed by Klebsiella species (21.7%) and Staphylococcus aureus (20.3%), CONS (14.7%), Pseudomonas aeruginosa (11.5%), Enterococcus species (4.56%), and Proteus species (3.15%). An Uganda study in 2007 noted isolates from surgical site operated septic infections incidence as Staphylococcus aureus (45.1%), Coliforms (16.9%), Proteus mirabilis (11.3%), Pseudomonas aeruginosa (9.9%), Klebsiella pneumoniae (7%) and Enterobacter species (2.8%) [12]. A study on automobile accidental wounds in 2009 reported Staphylococcus aureus (37.5%), Pseudomonas aeruginosa (27%), Streptococcus pyogenes (12.2%), Klebsiella pneumoniae (9.64%) and Escherichia coli (14.9%) [13]. Roopa C et al observed the most common pathogen isolated as E.coli (36, 19.14%) followed by Staphylococcus aureus (31, 16.48%), Klebsiella pnuemoniae (30, 15.95%) and Coagulase negative Staphylococcus (23, 12.23%) and other pathogens were less than 10% of total cultures [8]. Wajid M et al noted the most common isolate observed was Escherichia coli 70/302 [23.1%] followed by Staphylococcus aureus 59/302[19.5%], Klebsiella pneumoniae [14]. A study from western Rajasthan in India observed Staphylococcus aureus (30.9%) as a predominant pathogen followed by Escherichia coli (24.76%), Pseudomonas aeruginosa (16.68%), and Klebsiella (14.4%) [15]. Staphylococcus aureus predominance among pyogneic infections was noted in some studies [16,17]. Most of the above mentioned studies noted Gram negative bacteria isolation was more compared to gram positive bacteria in pus microbiotia. Escherichia coli was the most commonest pathogen observed which might be responsible for causing infections due to colonization of wounds by acquiring bacteria from gut which is a normal habitat for Escherichia coli. Antibiotic susceptibility testing method of Gram negative bacilli showed higher susceptibility to tetracycline, levofloxacin, meropenem, minocycline, gentamicin, cotrimoxazole, piperacillin+tazobactum, cefaperazone+sulbactum, ceftazidime +avibactum. Oral Beta lactam and beta lactamase inhibitors and 3rd generation cephalosporins susceptibility to all isolates was around 45%, which indicates still physicians or surgeons can opt for these antibiotics on empirical or curative therapy. Shama M et al [18] noted among the Gram negative pathogen E.coli shown maximum resistance towards Penicillin (95.25%), Ampcillin (85.71%), Cotrimoxazole (76.19%), Cefotaxime (80.95%), Netilmicin (61.9%) and Levofloxacin (57.14 %) where as Proteus species have maximum resistance towards Cotrimoxazole (76.25%). In case of gram positive bacteria, Streptococcus pyogenes were resistance towards Cotrimoxazole (62.5%), Cefotaxime (75%), Netilmicin (62.5%) and Gentamicin (87.5%). Kalita JM et al noted [15] that most of the Gram negative isolates showed high resistance towards cephalosporin, cotrimoxazole and quinolones and Gram positive cocci showed high resistance towards penicillin and quinolone group of drugs. Among gram negative bacterial isolates, 74.79% were multidrug resistant Klebsiella and 74.32% were MDR Acinetobacter spp. Methicillin resistant Staphylococcus aureus percentage was 13.26%, inducible clindamycin resistance among S.aureus isolates was 16.19%. 16.98% of total Enterococci isolates were Vancomycin resistant. Rijal BP et al [16] studied microbiota in pyogenic infection and noted 51.9% and 48.7% of high levels of drug resistance among gram positive bacteria and gram negative bacteria respectively. Gram positive isolates were resistant to ampicillin, ciprofloxacin, cotrimoxazole, erythromycin, and cloxacillin. Gram negative isolates were resistant to cephalosporins but were well susceptible to amikacin and imipenem. Wajid M et al [14] noted among the 197 Gram-negative isolates, ESBL production was observed in 58 isolates [29.5 %]. The aforementioned isolates E.coli 23/70 [32.8 %] and K.pneumoniae 24/40 [60 %] were ESBL producers. Carbapenemase production was observed in 30.1 % [59/197] of the isolates. 10/34 [29.4 %] isolates of Pseudomonas aeruginosa were Carbapenemase producers, and 16/34 [47.1 %] were MDR strains. All the Gram positive isolates were 100% susceptible to linezolid, teicoplanin, vancomycin. MRSA and MRCONS were noted as 55.2% and 40.5% respectively. 69.2% of Ampicillin sensitive Enterococcal isolates was observed. No vancomycin resistant isolates were observed. Wajid M et al [14] concluded Methicillin resistance was observed in only 49.1 % [29/59] of Staphylococcus aureus isolates, whereas 50 % [13/26] of CONS were resistant to Methicillin. All the Staphylococcus aureus, CONS and Enterococcus species were sensitive to Vancomycin. MRSA percentage in studies conducted by Sanjana et al was 39.6% [19] and Kshetry et al was 37.6% [20]. Shama M et al [18] documented that among gram positive bacteria, Streptococcus pyogenes were resistance towards Cotrimoxazole (62.5%), Cefotaxime (75%), Netilmicin (62.5%) and Gentamicin (87.5%). Isolation of bacteria from pyogenic samples is much needed to avoid the delay in wound recovery by starting accurate and prompt therapy to patients. The ability to inhibit the growth of bacterial isolates indicates effective use of antibiotics as antibacterial agents depending on their antimicrobial activity, efficacy in infections and low toxicity. Isolation of bacteria is fairly depends on correct sample collection and transportation, and prior antibiotic exposure by patient. Further studies on pathogenesis of multidrug resistance bacteria, antibiogram in various communities and very importantly transmission of gut flora into wounds which are playing vital role in causing infections will help to reduce the rate of pyogenic infections.

From this study we conclude that bacterial isolation rate among pus samples is about 60% and the gram negative bacteria were predominantly isolated. The most common population affected was male in the age group of 31-60 years. Escherichia coli, Klebsiella species, Pseudomonas aeruginosa and Staphylococcus aureus are the majority organisms responsible for pyogenic infections of various clinical scenarios. The percentage resistance of different isolates against different antibiotics varies; most of the isolates were sensitive to broad spectrum antibiotics, aminoglycosides and beta lactam and beta lactam inhibitor antibiotics. MDR pathogens can be treated by penems, fluoroquinolones along with aminoglycosides to add synergistic effect. This study will help physicians or surgeons to start empirical therapy accurately. Effective infection control measures and knowing antibiogram of the community will definitely aid to in regulating the pyogenic infections.

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