Mechanical ventilation is a life-saving procedure for many patients in Intensive care unit (ICU), but it is associated with a high risk of acquiring respiratory infections and a high morbidity and mortality in critically ill patients1. Bacterial colonisation of the pharynx and upper airways is initial portal of entry into generally sterile lower respiratory tract. Therapeutic techniques like Endotracheal intubation aid colonisation which is further enhanced by the formation of microbial biofilms around Endotracheal tubes and their dislodgement following suctions and repeated intubations leading ultimately to Ventilator Associated Pneumonia (VAP)2. To add to the trouble, the statistical data and evidences from research prove that multi drug resistant bacteria are rapidly emerging across the world and pose a big challenge to health care system. Extensive and non-specific use of broad-spectrum antibiotics in hospitalized patients has led to both increased carriage and the development of multi drug resistant strains3. Endotracheal (ET) aspiration helps in the evaluation for the occurrence of the febrile episodes in the mechanically ventilated patients to assess the patient’s risk to develop Ventilator associated pneumonia (VAP) or Hospital acquired pneumonia (HAP)4. Knowledge of local antimicrobial resistance patterns are important to initiate the empirical antimicrobial therapy. This will help in preventing overuse and misuse of antimicrobial agents which has arisen as a serious challenge to clinicians in choosing the most effective therapeutic option5. Hence, the present study was conducted to isolate and identify the bacteria from endotracheal aspirates and determine their antimicrobial susceptibility pattern from endotracheal aspirate specimens. Objective: 1. To study the common bacterial isolates in endotracheal aspirates of mechanically ventilated patients. 2. To analyse the antibiotic susceptibility pattern of the isolates.

Study Design: Prospective cross-sectional was carried out on endotracheal aspirates of mechanically ventilated patients. Study Population: The study population included the patients who were on mechanical ventilation in various ICUs of our hospital. Sample Size: The study was carried out on endotracheal aspirates of 75 patients who were on mechanical ventilation. Inclusion Criteria: Patients on mechanical ventilation for more than 48 hours were included in the study. The following data were collected at the start of patient’s admission into the ICUs that were put on mechanical ventilation like age, gender, primary diagnosis at admission, underlying disease, date of initiation of mechanical ventilation, any surgical procedure during hospital stay and current antibiotic therapy. The approval of the institutional ethical board was also obtained (Ethical no. 2022-01353) during the planning phase of study and informed consent of each patient was taken before their participation in this study. Exclusion criteria: Exclusion criteria for study were patients already with pneumonia or other respiratory disorders like (ARDs), cystic fibrosis, cavitary lung disease, primary or metastatic lesion in the lungs based on chest X-Ray findings. Study Area: The study was carried out in 2022 for a period of 6 months in the department of Microbiology and various ICUs of Heritage institute of medical college, Varanasi, Uttar Pradesh. Data Collection: During the study period 75 patients who were on mechanical ventilation for more than 48 hours were analysed and diagnosis of pathogenic infection was made. The endotracheal aspirates collected from patients were processed by direct microscopy using gram’s stain technique in which >25 neutrophils/low power field and <10 epithelial cells/ low power field were considered significant. Each sample was inoculated on Blood agar, MacConkey’s agar and Chocolate agar. All plates were incubated overnight at 37ºC in presence of 5% carbon dioxide. All plates were checked for growth after overnight incubation and no growth was considered after 48 hours of incubation. Plates with growth were subjected to analysis for bacterial counts and colony count of >105 colony forming units (CFU) was considered as significant. Culture with lower colony count was considered as colonisation or contamination. Identification of isolate was done by colony characteristics, gram stain from culture and various arrays of biochemical tests. Antimicrobial sensitivity testing of the bacterial isolates was made by Kirby-Bauer disc diffusion method done on Mueller Hinton agar. Interpretation was made according to the Clinical and Laboratory Standard’s Institute (CLSI) guidelines. Statistical analysis-: Latest statistical package for the social sciences “SPSS” was used for analysis of the data and the results were presented in terms of percentages and frequencies.

Bacteriological examination of 75 patients who were admitted to various ICU’s were done. 61 patients (81.3%) showed positive culture results. Out of 75 patients 48 (64%) were male and 27 (36%) were female (Chart 1). After microbiological analysis, 40 males and 21 females showed positive culture. Amongst 75 patients, 5 (6.7%) were less than 20 years of age, 15 (20.0%) patients were of 21-40 years of age, 31(41.3%) patients of 41-60 years of age, 22 (29.3%) patients were of 61-80 years of age and 2 (3.0%) patients were greater than 80 years of age. So Maximum number of patients were in the age group of 41-60 years (Chart 2). Amongst the 61 samples showing growth on culture, 57 (93.4%) samples have shown monomicrobial growth and 04 (6.6%) samples were polymicrobial (Table 1). In the 04 samples of polymicrobial growth Acinetobacter baumannii was the predominant organism present in 3 samples with Enterobacter spp, Pseudomonas aeruginosa and Klebsiella pneumoniae. One sample showed the mixed significant growth of Escherichia coli and Enterococcus faecium (Table 2). So, the total number of isolates found were 65 including monomicrobial and polymicrobial growth in 61 samples. (Table 1) Growth on culture media Number of samples (n=61) Percentage (%) Monomicrobial 57 93.4 Polymicrobial 04 6.6 (Table 2) Polymicrobial growth on culture Total No. of Samples = 04 Isolates = 08 Acinetobacter baumannii + Enterobacter spp. 01 + 01 Acinetobacter baumannii + Pseudomonas aeruginosa 01 + 01 Acinetobacter baumannii + Klebsiella pneumoniae 01 + 01 Escherichia coli + Enterococcus faecium 01 + 01 The order of prevalence of organism in the study was found to be 52.3% isolates were of Acinetobacter baumannii followed by 20.1% isolates of Klebsiella pneumoniae, 9.2% isolates of Pseudomonas aeruginosa, 4.6% isolates of Escherichia coli, 3.1% isolates of Enterobacter spp, 3.1% isolates of Corynebacterium spp, 3.1% isolates of Staphylococcus aureus (MRSA), 1.5% isolates of Citrobacter spp, 1.5% isolates of Proteus spp and 1.5% isolates of Enterococcus faecium (Table 3). (Table 3) Organisms isolated from Endotracheal tube Total number = 65 (%) Acinetobacter baumannii 34 (52.3) Klebsiella pneumoniae 13 (20.1) Pseudomonas aeruginosa 06 (9.2) Escherichia coli 03 (4.6) Enterobacter spp 02 (3.1) Corynebacterium spp 02 (3.1) Methicillin resistant Staphylococcus aureus (MRSA) 02 (3.1) Citrobacter spp 01 (1.5) Proteus spp 01 (1.5) Enterococcus faecium 01 (1.5) Antibiotic sensitivity pattern of Acinetobacter baumannii has shown 100% resistance to 3rd and 4th generation cephalosporins, ciprofloxacin, piperacillin, ticarcillin-clavulanate and tobramycin. High resistance pattern has also been seen for imipenem (97.6%), meropenem (97.6%), gentamicin (92.7%), levofloxacin (92.7%), piperacillin-tazobactam (90.2%), amikacin (88%), ampicillin-sulbactam (85.4%) and tetracycline (78.0%). Antibiotics like colistin (66.0%), doxycycline (46.4%), cotrimoxazole (34.2%) have shown good susceptibility. (Chart 3) Pseudomonas aeruginosa has shown maximum resistance of 90.9% to ceftazidime, imipenem, levofloxacin and piperacillin followed by resistance of 81.8% to cefepime, ofloxacin, and ticarcillin-clavulanate. Ciprofloxacin (73.0%), gentamicin (73.0%), Meropenem (72.7%), netilmicin (63.6%) and tobramycin (63.6%) have also shown high resistance pattern. Amikacin has shown maximum susceptibility of 73.0% followed by piperacillin-tazobactam (45.6%) and aztreonam (36.4%). (Chart 4) Antibiotic susceptibility pattern of organisms belonging to Family Enterobacteriaceae i.e. Klebsiella pneumoniae, Escherichia coli, Enterobacter spp, Proteus spp, Citrobacter spp have shown 100% resistance to ampicillin, aztreonam, azithromycin, cefepime, cefuroxime, ceftriaxone, cefotaxime, ceftazidime, ciprofloxacin, levofloxacin, ofloxacin, piperacillin, ticarcillin-clavulanate, trimethoprim followed by Amoxycillin-clavulanate and Cotrimoxazole which have shown resistance of 92.0%. Ampicillin-sulbactam (88.0%), Imipenem (88.0%), Meropenem (80.0%), Piperacillin-tazobactam (80.0%), Tobramycin (76.0%), Amikacin (72.0%) and Gentamicin (68.0%) have also shown high resistance pattern. Antibiotic like Doxycycline has shown high susceptibility (64.0%) followed by Tetracycline (40.0%) and chloramphenicol (36.0%). (Chart 5). Amongst Gram positive isolates, single isolate of Enterococcus faecium was susceptible to linezolid, high level gentamicin whereas it was found to be resistant to penicillin, ampicillin, chloramphenicol, ciprofloxacin, levofloxacin, moxifloxacin, doxycycline, erythromycin, teicoplanin and vancomycin. For Methicillin resistant Staphylococcus aureus (MRSA) (n=2) linezolid, levonadifloxacin, doxycycline, chloramphenicol, cotrimoxazole has shown 100% susceptibility whereas clindamycin, erythromycin, gentamicin, levofloxacin, moxifloxacin, ofloxacin and tetracycline has shown 50% susceptibility. Penicillin and azithromycin have shown complete resistance (100%).

Ventilator associated pneumonia (VAP) is difficult to diagnosis in ICU patients with an endotracheal tube or a tracheostomy. It is associated with high mortality and morbidity rates. Thus, early and accurate diagnosis and appropriate empirical antibiotic treatment are important outcome variables. In our study 61(81.3%) out of 75 patients sample showed positive culture results which was similar to the study done by Malik et al 6. Amongst the 61 samples showing growth on culture, 57 (93.4%) samples have shown monomicrobial growth and 04 (6.6%) samples were polymicrobial with 2 isolates in each polymicrobial sample so total number of isolates were 65 altogether. The most common bacteria found in polymicrobial growth was Acinetobacter baumannii in which it was found as Acinetobacter baumannii and Enterobacter spp, Acinetobacter baumannii and Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae making Acinetobacter a predominant bacterium in polymicrobial growth samples. This pattern of mixed growth has also been reported in a study done by Malik et al6.The reason for mixed growth could be that ICU patients had a prolonged stay at hospital and they underwent multiple invasive procedures like central venous lines or catheterization. These invasive procedures allow introduction of multiple microbial pathogens, which can flourish in these immunocompromised patients. This observation was also reported by Saddique et al 7. In the present study, out of 75 patients, 48 (64%) were males and 27 (36%) were females. This is in accordance with study conducted by Panda et al.8, Hoque MM. et. Al9 where number of males were more as compared to females. In addition to a higher prevalence of the smoking habit among males, reproductive hormones such as testosterone and oestrogen have been linked to a greater prevalence of males developing VAP. Testosterone tends to reduce the body's immune response to infectious agents, while estrogen increases the intensity and number of immune cells. Higher estradiol levels in women allow for better protection against pathogens.10 Maximum numbers of positive cultures were found in the age group of 40-60 years (41.3%) which is in accordance with study done by Sharma RS et al11 however in other studies preponderance of different age groups have been reported as in the study conducted by Bhumika et al6 where maximum patients were in the age group of 0-10 years, Hoque MM et al9 have reported the maximum number of patients in the age group of 61-70 years whereas in a study conducted by Panda et al8 most of the patients were from age group of >70years. The causative organisms vary with the patients' demographics in the ICU, the method of diagnosis, the duration of hospital stays, and the institutional antimicrobial policies. In the present study, gram negative bacteria were the most common pathogens which was in accordance to the study done by Bhumika et al5 and Panda et al8. Amongst Gram negative bacilli the most common pathogen was Acinetobacter baumannii followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterobacter spp, Citrobacter spp and Proteus spp. Similar findings have been seen in studies done by Natham et al2, Panda et al8, Patadia et al12, also where Acinetobacter is the predominant bacteria in endotracheal tubes whereas in studies done by Malik et al6 and Hoque et al9 the predominant Gram-negative bacteria was found to be Klebsiella pneumoniae followed by Pseudomonas aeruginosa and Acinetobacter. In Gram positive cocci Methicillin Resistant Staphylococcus aureus (MRSA) and Enterococcus faecium were isolated along with Corynebacterium spp in Gram positive bacilli. As Endotracheal tubes are quickly colonized by biofilm-forming bacteria, which have been reported to be better than planktonic cells at surviving environmental stress, antimicrobial therapy and immune system responses, it has been estimated that biofilms may be implicated in approximately 65% of all infections, including those associated with the use of medical devices, which are strongly related to higher rates of morbidity and mortality. Most of these infections are caused by ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp), a group of pathogens that is also recognized for their ability to develop resistance to antibiotics, which undermines treatment and increases the rates of morbidity and mortality.13 In our study 2 isolates of Corynebacterium spp were also seen which are gram-positive bacilli and non-diphtheria agents famously known as Diphtheroids. They are part of the normal skin microbiota and mucous membranes. These well-known contaminants of clinical materials have been increasingly recognized as causing opportunistic infections. Non-diphtheria Corynebacterium spp. has been implicated in community-acquired, hospital-acquired and ventilator associated pneumonia in immunocompromised patients or whose upper airways were bypassed (e.g., mechanical ventilation, tracheostomy). An increased number of Corynebacterium pneumonia has been identified in the ICU during the COVID-19 pandemic in immunocompromised and ventilated patients.14 In the present study, Acinetobacter baumannii has shown 100% resistance to 3rd and 4th generation cephalosporins, ciprofloxacin, piperacillin, ticarcillin-clavulanate and tobramycin. High resistance pattern has also been seen for Imipenem (97.6%), Meropenem (97.6%), Gentamicin (92.7%), levofloxacin (92.7%), Piperacillin-tazobactam (90.2%), Amikacin (88%), Ampicillin-sulbactam (85.4%) and Tetracycline (78.0%) which is in concordance to study done my Bhumika et al5. Colistin which used to be the last resort drug for Acinetobacter baumannii has shown susceptibility of only 66.0% which is alarming. Similar to our finding, the emergence of colistin resistant Acinetobacter baumannii has been reported in study done by Elham B15. Pseudomonas aeruginosa has also shown high resistance pattern to cephalosporins, fluoroquinolones, carbapenems, aminoglycosides. Amikacin has shown maximum susceptibility of 73.0% followed by Piperacillin-tazobactam (45.6%) and Aztreonam (36.4%) which is similar to study done by Gowda et al16. For the organisms belonging to Family Enterobacteriaceae high resistance has been seen against Penicillin, cephalosporins, monobactam, fluoroquinolones, macrolides, aminoglycosides Piperacillin, Ticarcillin-clavulanate, Amoxycillin-clavulanate, Cotrimoxazole. In our study Doxycycline has shown high susceptibility of 64.0% followed by Tetracycline (40.0%). In a study done by Lai CC et al Doxycycline combinations therapy has been found to be efficacious in multidrug resistant infections.17

Acinetobacter baumannii and Klebsiella pneumoniae were the most common organism isolated in our study. Colistin, doxycycline, amikacin have shown promising results whereas penicillin, cephalosporins, fluoroquinolones, carbapenem etc were associated with high resistance. The high mortality and morbidity seen in VAP can be reduced by identifying the common causative organisms and starting the antibiotic treatment according to the antibiotic susceptibility pattern. Implementation of strict infection control practices can play a major role in the prevention of infection and spread of these multidrug-resistant pathogens.

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