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ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 3  |  Page : 151-157

Bacteriological profile and antimicrobial sensitivity pattern of endotracheal tube aspirates of patients admitted in ICU


1 2nd Year PG Student Department of Microbiology, MKCG MCH, Berhampur, Odisha, India
2 Department of Microbiology, SBB MCH, Balangir; Department of Microbiology, MKCG MCH, Berhampur, Odisha, India
3 Department of Microbiology, MKCG MCH, Berhampur, Odisha, India

Date of Submission23-Jul-2020
Date of Decision03-Sep-2020
Date of Acceptance24-Aug-2020
Date of Web Publication30-Sep-2020

Correspondence Address:
Dr. Sanghamitra Padhi
HOD and Professor, Department of Microbiology, BB MCH, Balangir, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_118_20

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  Abstract 


Background: Ventilator-associated pneumonia (VAP) in critically ill patients are associated with high morbidity and mortality as they are at a high risk of acquiring respiratory infections, due to complex interplay between the endotracheal tube, host immunity and virulence of invading bacteria. Several studies reported multidrug-resistant bacteria like Acinetobacter spp., Pseudomonas spp., Gram-positive bacteria like S. aureus. For prompt initiation of empirical antimicrobial treatment, knowledge of local antimicrobial resistance patterns is essential.
Aims: To study antimicrobial sensitivity among organisms isolated from endotracheal aspirates of patients with VAP and examine their various resistance pattern and look for biofilm production.
Materials and Methods: ET aspirates were taken from 140 patients who were mechanically ventilated for various reasons in ICU of our hospital and were subjected to Gram stain and semiquantitative cultures. Organism identification and antimicrobial susceptibility testing were performed according to standard guidelines. Various resistance patterns and biofilm production on Congo Red Agar were observed.
Results: Out of 140 ET aspirates processed, 120 samples (85.7%) were culture positive; most common isolate being Acinetobacter spp. (45.8%), followed by Pseudomonas spp. and Klebsiella spp. (16.6% each), and Gram-positive isolate Staphylococcus aureus (12.5%). All Staphylococcus aureus were sensitive to linezolid and resistant to cefoxitin (MRSA). Most of the Gram-negative isolates were sensitive to imipenem. ESBL resistance was seen in 25% of Klebsiella spp. and Amp C resistance was seen in 27% of Acinetobacter spp. Biofilm was produced in 62.5% of the isolates. Mortality was maximum in patients whose ET aspirates showed biofilm production.
Conclusion: A local antibiogram pattern for each hospital, based on bacteriological profile and susceptibilities, is essential, to initiate empiric therapy and help in framing the appropriate institutional antibiotic policy.

Keywords: Acinetobacter, biofilm, endotracheal aspirate, mechanical ventilation


How to cite this article:
Samal N, Padhi S, Paty BP. Bacteriological profile and antimicrobial sensitivity pattern of endotracheal tube aspirates of patients admitted in ICU. J NTR Univ Health Sci 2020;9:151-7

How to cite this URL:
Samal N, Padhi S, Paty BP. Bacteriological profile and antimicrobial sensitivity pattern of endotracheal tube aspirates of patients admitted in ICU. J NTR Univ Health Sci [serial online] 2020 [cited 2020 Nov 26];9:151-7. Available from: https://www.jdrntruhs.org/text.asp?2020/9/3/151/296825




  Introduction Top


Mechanical ventilation is a life-saving procedure for many patients in the intensive care unit, but it is associated with a high risk of acquiring respiratory infections and a high morbidity and mortality in critically ill patients.[1] This is due to the complex interplay between the endotracheal tube (ET tube), host immunity and virulence of invading bacteria. Pneumonia that occurs 48–72 h or thereafter following endotracheal intubation, characterized by the presence of any new or progressive infiltrate, with signs of systemic infection (fever, altered white blood cell count), changes in sputum characteristics, and detection of a causative agent is called as ventilator-associated pneumonia (VAP).[2] Three factors are critical in the pathogenesis of VAP: colonization of oropharynx with pathogenic organisms, aspiration of these organisms from oropharynx into lower respiratory tract, and compromise of the normal host defense mechanism.[3] The etiologic agent of VAP may vary according to the onset of VAP, type of ICU, preexisting illness, age and sex of the patients, and prior antimicrobial therapy. The procedure of ET aspirate collection is easily performed at the bedside, relatively simple, minimally invasive and inexpensive, has a proven acceptable accuracy and requires minimal investment for the training of health professionals.[4]

Several studies demonstrate the ET culture as an additive diagnostic tool along with the routine tests in detection of plausible pneumonia pathogen.[5],[6],[7] Bronchoalveolar lavage and protected specimen brush have been reported to have high sensitivity and specificity for the diagnosis of VAP, but are invasive and difficult to perform.[8] Infectious Disease Society of America recommends (IDSA) to use noninvasive sampling (ET aspirate) with semiquantitative culture to diagnose VAP rather than invasive or noninvasive sampling with quantitative cultures. Moreover endotracheal aspirate is a relatively noninvasive method and also easily performed.

ET tube also acts as a reservoir for infecting microorganisms and it harbors them especially in the interior of the 1st distal third of the tube; making them an important risk factor for pulmonary infection. These organisms are potent biofilm producers as they are encased in a polymeric matrix and can show multidrug-resistance patterns, leading to high mortality rates.

The present study has been undertaken to determine the local bacteriological profile of ET aspirates, their resistance pattern, and virulence by testing for their biofilm production.


  Material and Methodology Top


Design of the Study

Prospective Study

Study place

MKCG Medical College and Hospital, Berhampur

Ethical approval

Consent was taken for collection of ET aspirates of mechanically ventilated patients from their respective attendants.

Time frame

Over a period of 1 year from January 2019 to December 2019

Inclusion criteria

Patients under mechanical ventilation for more than 48 h in the ICU.

Exclusion criteria

Patients having pneumonia prior to mechanical ventilation.

Patients having pulmonary edema.

Patient whose ET aspirate's gram staining showed more than 10 epithelial cells per low-power field.

Sample size

140 samples.

Methodology

The endotracheal aspirates were collected from the patients who were mechanically ventilated for more than 48 h for various reasons in different ICUs of our hospital. It was collected by nonbronchoscopic method by using a 22-inch Ramson's 12-F suction catheter which was gently introduced through the endotracheal tube (for a distance of approximately 25–26 cm). Gentle aspiration was then performed without instilling saline, and the catheter was withdrawn from the endotracheal tube. After suctioning the tip of the suction catheter, along with the secretions were transferred to a sterile container containing BHI broth. The samples were immediately brought to the Microbiology laboratory where they were subjected to Gram Staining and semiquantitative culture. Endotracheal aspirate samples were considered valid for culture if <10 squamous epithelial cells and >25 neutrophils were present (per low power field).[4],[9] All samples were plated on Blood agar (BA) and MacConkey agar (MA) by semiquantitative culture method, i.e., 4 quadrant streak technique using a 1 μl calibrated loop and incubated for 24 h. Only those plates which showed moderate to heavy growth were suggestive of colony count >105 CFU/mL, then the bacteria were identified by standard biochemical methods and they were further subjected to antimicrobial susceptibility testing by Kirby-Bauer Disk Diffusion method on MHA.

Antimicrobials tested for Gram-negative isolates were gentamicin (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefepime (30 μg), levofloxacin (5 μg), tobramycin (10 μg), piperacillin-tazobactam (100/10 μg), cefotaxime-clavulanic acid (30/10 μg), and imipenem (10 μg).

Antimicrobials tested for Gram-positive isolates were gentamicin (10 μg), azithromycin (15 μg), cotrimoxazole (25 μg), moxifloxacin (5 μg), linezolid (30 μg), and cefoxitin (30 μg).

Interpretation of the zone diameters was done as per clinical laboratory and standards institute (CLSI) guidelines 2017.

For extended-spectrum beta-lactamase (ESBL) detection, disc diffusion method was performed on Muller Hinton agar (MHA) with cefotaxime (30 μg) and cefotaxime- clavulanic acid (30/10 μg). A ≥5 mm increase in zone diameter for either antimicrobial agent tested in combination with clavulanate vs. zone diameter of the agent when tested alone was identified as ESBL producers.

For AmpC β-lactamases detection, disc diffusion method was performed on MHA, which was 1st swabbed by freshly prepared inoculum of  Escherichia More Details coli ATCC 25922 (indicator organism). Plate was allowed to dry and cefoxitin disc (30 μg) was placed at the center. A sterile filter paper disc moistened with 10 μL sterile saline was taken and inoculated with several colonies of the test organism picked from a blood agar plate. Inoculated disk was then placed beside the cefoxitin disk almost touching it, with the inoculated side in contact with the agar plate and incubated overnight at 35°C. Cefoxitin disc being a potent AmpC inducer, a flattening or an indentation of the zone of inhibition of cefoxitin disk indicated a positive test (AmpC producer). A negative test had an undistorted zone.

For detection of methicillin resistance in Staphylococcus spp., 30 μg cefoxitin disc was placed on the lawn culture of the test organism on MHA and the plate was incubated for 16–18 h. For Staphylococcus aureus, zone of inhibition (ZOI) <21 mm was considered as resistant (mecA positive) and ≥22 mm was considered sensitive (mecA negative).

All the isolated organisms were tested for biofilm production by testing their growth on Congo Red Agar medium (prepared with brain-heart infusion broth, sucrose, agar No. 1 and sucrose indicator). Biofilm producing organism showed black colonies after 24 h incubation at 37°C.


  Results Top


A total of 140 endotracheal aspirates were received during 1 year of study period. Samples were collected from various ICUs, the maximum case load being from the neonatal ICU [Table 1].
Table 1: Case Distribution From Different ICUS

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The occurrence of VAP was common in men (54%) than women (46%) among the patient studied. Maximum number of cases were between 0 and 14 years of age group [Table 2].
Table 2: Age and Sex Distribution of Cases

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[Table 3] shows that the occurrence of VAP was more common in patients suffering from severe pneumonia, followed by hollow viscous perforation and cerebrovascular accidents. The most common predisposing factor was general anesthesia followed by type 2 diabetes mellitus [Table 4].
Table 3: Comparison of Disease With Vap

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Table 4: Associated Risk Factors With Vap

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Out of 140 samples processed, 120 samples (85.7%) were culture positive with significant count ≥105 CFU/mL [Figure 1].
Figure 1: Processing of samples

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Gram-negative bacilli were the most predominant isolate (n = 105), followed by Gram-positive cocci (n = 15).

Among the GNB, Acinetobacter spp. was the most common isolate (46%) followed by Pseudomonas spp. (17%), Klebsiella spp. (17%), and Citrobacter spp. (8%). Gram-positive isolate was Staphylococcus aureus (12%) [Figure 2] and [Figure 3].
Figure 2: Isolation rates of ET aspirates

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Figure 3: (a): Organism isolated. (b) Gram-negative coccobacilli showing honeycombing- Acinetobacter spp.

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Most of the Gram-negative isolates were sensitive to carbapenems (Imipenem), followed by Piperacillin-tazobactam combination [Table 5]. Some multidrug-resistant strains were also found [Figure 4].
Table 5: Antibiogram Of Gram-Negative Isolates (% Of Susceptible Isolates)

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Figure 4: AST of multidrug-resistant organism

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All the isolated Staphylococcus aureus were sensitive to Linezolid and none of them were sensitive to Cefoxitin [Figure 5]. Thus, all of them were methicillin-resistant Staphylococcus aureus (MRSA).
Figure 5: (a): Antibiogram of Gram-positive isolates. (b) MRSA detection by cefoxitin disc diffusion method

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By double disc diffusion method, ESBL was detected in 25% of Klebsiella spp. [Figure 6], while Amp C resistance was seen in 33% and 27% of Pseudomonas spp. and Acinetobacter spp., respectively [Figure 7].
Figure 6: ESBL detection by double disc diffusion method

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Figure 7: Amp C resistance detection

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Biofilm production was detected by inoculation on Congo red agar, where 62.5% of biofilm producers were noticed. Considering bacterial isolates, biofilm production was seen in 100%, 81.8%, and 75% of Staphylococcus aureus, Acinetobacter spp., and Pseudomonas spp. respectively [Figure 8]. [Figure 8]a shows biofilm production by isolates.
Figure 8: (a): Biofilm production by isolates. (b) Biofilm production on Congo Red Agar

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An important correlation was noticed between biofilm production and mortality. Mortality was high among the patients whose ET tube aspirates showed biofilm-producing organisms [Table 6].
Table 6: Biofilm Production Vs. Mortality In Patients

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  Discussion Top


Ventilator-associated pneumonia is associated with high rate of mortality and morbidity rates, due to the common complications among patients requiring mechanical ventilation. Invasive procedures including protected specimen brush and bronchoscopy are done in late stages of VAP and may lead to complications like cardiac arrhythmia, hypoxemia, and bronchospasm. Thus, there is a need for noninvasive technique that can be carried out in early VAP state. The estimated prevalence of VAP ranges from 10% to 65%, with a 20% case fatality. It accounts for 13%–18% of all hospital-acquired infections.[10] Causative organisms of VAP vary from nonmultidrug-resistant to multidrug-resistant pathogens leading to increased complications and treatment cost. As per a recent interview in Morehead andPinto, the incidence of ventilator-associated pneumonia was 9% to 24% for patients intubated longer than 48 h.[11] They found that culture positivity was more common in elderly male patients who were smokers, and who were admitted for respiratory causes or patients who had preexisting lung disease which was similar with the study of Ferrer et al.[12] Another study has also shown that the majority of patients are >70 years of age group.[13] But, most common age group affected in our study was 0–14 years, majority being the patients from Neonatal ICU. As our hospital is a tertiary care center, cases generally come at the terminal stage of a disease, so longer duration of stay on mechanical ventilation is required. Although most of the patients were male in the study of Ferrer et al., gender had no significant role in the development of VAP, and they were equally affected in our study.

In our study, the most common risk factor was general anesthesia; the reason being sedative state of patient (delay in ambulation), leading to pooling of secretions and increase chances of aspiration. Whereas other studies showed Chronic obstructive pulmonary disease the most common risk factor followed by CVA and diabetes mellitus.[4],[13] In consistency with our study, the highest number of patients enrolled in Dey and Bairy study was also from postoperative wards.[14]

Most common isolate in our study was Acinetobacter spp. (46%) followed by Pseudomonas spp. Other studies showed similar findings.[4],[13],[15],[16],[17] In a study by Malik et al., the commonest bacterium isolated from tracheal secretions was Klebsiella pneumoniae (35.4%).[18]

In our study, most of the Gram-negative bacteria were sensitive to carbapenems which is in concordance with Dey and Bairy.[14] Only 9% of Acinetobacter spp. were sensitive to cephalosporins, while 81.8% were sensitive to carbapenems. But, 85% sensitivity to carbapenems was shown in a study by Patel et al.,[19] while 7.9% sensitivity to carbapenem was shown in a study by Gowda et al.[17] In our study, all isolates (100%) of Staphylococcus aureus were susceptible to linezolid while 25% were sensitive to linezolid in the study by Patel et al.

ESBL producing organisms, AmpC resistance, and MRSAs are of increasing clinical concern; thus, they have to be documented for epidemiological and infection control point of view, as they are challenging to the clinicians (limited therapeutic options). In our study, ESBL producers were seen in 25% of Klebsiella spp., AmpC resistance was seen in 27% of Acinetobacter spp. and 33% of Pseudomonas spp. But ESBL producers were 64% in Klebsiella spp. in a study by Swati et al., while ESBL-producing Enterobacteriaceae colonization was identified among 5% to 30% of ICU-admitted patients in different studies.[14],[19],[20] Dey and Bairy showed that 30.43% of Acinetobacter spp. were AmpC beta-lactamase producing strains.[14] MRSA producers in our study was 100%, while it was 86% in a study by Swati et al.[4] and 75% in Patel et al.[19]

Biofilm plays an important role in the development of VAP among patients with endotracheal tube in-situ. The multidrug-resistant ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) play a dominant role in VAP etiology, and these organisms were frequently identified in ET biofilms.[21] 62.5% of the isolated organisms in our study showed biofilm production which was checked on CRA medium. But the study by Gil-Perotin et al. has shown that 95% of the isolates were biofilm producers.[22] Acinetobacter spp. and Pseudomonas spp. were the most prevalent Gram-negative bacteria in our study that produced biofilm, i.e., 81.8% and 75%, respectively. All the MRSA were biofilm producers. But in the study by Devi and Gomathi, Acinetobacter was the most common organism associated with biofilm formation (64.29%) followed by Klebsiella pneumoniae (21.43%).[23] We also noticed an important correlation between biofilm production and mortality of patients, i.e., mortality was high in the patients whose ET aspirates showed biofilm-producing organisms.


  Conclusion Top


The most common isolate for the endotracheal aspirate culture was Acinetobacter spp. followed by Pseudomonas spp. and Klebsiella spp., most of them being sensitive to carbapenems. Biofilm producers were very predominant in the isolates which lead to an increase in mortality of the patients. Microbial persistence and impaired response to treatment (treatment failure and relapse) were more frequent when multidrug-resistant microorganisms were present in ETT biofilm. A local empirical antibiogram of the respective hospital will help in the early start of the course of treatment. Combined approaches of rotational antibiotic therapy, proper handwashing and decontamination techniques with education programs might be beneficial to fight against these MDR pathogens and will also help to decrease the incidence of VAP.

Declaration of patient

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for 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.



 
  References Top

1.
Chandra D, Laghawe A, Sadawarte K, Prabhu T. Microbiological profile and antimicrobial sensitivity pattern of endotracheal tube aspirates of patients in ICU of a Tertiary Care Hospital in Bhopal, India. Int J Curr Microbiol App Sci 2017;6:891-5.  Back to cited text no. 1
    
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American Thoracic Society, Infectious Diseases Society of America. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005;171:388-416.  Back to cited text no. 2
    
3.
Harrison's Principles of Internal Medicine. 20th ed. New York: McGraw-Hill Education; 2018. p. 908.  Back to cited text no. 3
    
4.
Swati A, Yamini K, Rajkumar RV. Microbiological spectrum and antimicrobial susceptibility patterns of various isolates from endotracheal tube aspirates in a tertiary care hospital, Hyderabad, Telangana. Indian J Microbiol Res 2018;5:202-7.  Back to cited text no. 4
    
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El-Ebiary M, Torres A, González J, Puig de la Bellacasa J, García C, Jiménez de Anta MT, et al. Quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia. Am Rev Respir Dis 1993;148:1552-7.  Back to cited text no. 5
    
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McCauley LM, Webb BJ, Sorensen J, Dean NC. Use of tracheal aspirate culture in newly intubated patients with community-onset pneumonia. Ann Am Thorac Soc 2016;13:376-81.  Back to cited text no. 7
    
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Miles RS, Amyes SG. Laboratory control of antimicrobial therapy. Mackie and McCartney practical medical microbiology. 1996;14:151-78.  Back to cited text no. 8
    
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Shastry AS, Deepashree R. Essentials of Hospital Infection Control. 1st ed.. Jaypee Brothers Medical Publishers Nepal; 2019. p. 44-27.  Back to cited text no. 9
    
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Garner JS, Jarvis WR, Emori TG, Horan TC, Hughes JM. CDC definitions for nosocomial infections, 1988. Am J Infect Control 1988;16:128-40.  Back to cited text no. 10
    
11.
Morehead RS, Pinto SJ. Ventilator-associated pneumonia. Arch Intern Med 2000;160:1926-36.  Back to cited text no. 11
    
12.
Ferrer M, Ioanas M, Arancibia F, Marco MA, de la Bellacasa JP, Torres A. Microbial airway colonization is associated with noninvasive ventilation failure in exacerbation of chronic obstructive pulmonary disease. Critical Care Med 2005;33:2003-9.  Back to cited text no. 12
    
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Panda G, Mohapatra BP, Routray SS, Das RK, Pradhan BK. Organisms isolated from endotracheal aspirate and their sensitivity pattern in patients suspected of ventilator associated pneumonia in a tertiary care hospital. Int J Res Med Sci 2018;6:284-8.  Back to cited text no. 13
    
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14. Dey A and Bairy I. Incidence of multidrug resistant organisms causing ventilator associated pneumonia in a tertiary care hospital: A nine months prospective study. Ann Thorac Med 2007;2:52-7.  Back to cited text no. 14
    
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Ahmad H, Sadiq A, Bhatti HW, Bhatti AA, Tameez-Ud-Din A, Ibrahim A, Chaudhary NA. Bacteriological profile and antibiogram of cultures isolated from tracheal secretions. Cureus 2019;11:e4965  Back to cited text no. 15
    
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Ranjitha Shankare Gowda, Sowmya G.S., Pavithra N., Raghavendra Rao M., Krishna Karthik M. and Satya Sai B., Bacteriological Profile of Endotracheal Aspirates and their Antibiotic Susceptibility Pattern, J Pure Appl Microbiol., 2018;12:2283-7. http://dx.doi.org/10.22207/JPAM.12.4.69.  Back to cited text no. 17
    
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Malik M, Malik MI, Sajjad A. Pattern of bacterial pathogens isolated from endotracheal secretions in Intensive care unit (ICU) patients of a tertiary care hospital of Lahore. Pak J Pathol 2018;29:46-8.  Back to cited text no. 18
    
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Patel A, Lakhani S, Khara R. Microbiological profile of ventilator associated pneumonia at ICU of rural based teaching hospital. Int J Biol Med Res 2015;6:4732-6.  Back to cited text no. 19
    
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Pilmis B, Zahar JR. Ventilator-associated pneumonia related to ESBL-producing gram negative bacilli. Ann Transl Med 2018;6:424.  Back to cited text no. 20
    
21.
Diaconu O, Siriopol I, Poloşanu LI, Grigoraş I. Endotracheal tube biofilm and its impact on the pathogenesis of ventilator-associated pneumonia. J Crit Care Med (Targu Mures) 2018;4:50-5.  Back to cited text no. 21
    
22.
Gil-Perotin S, Ramirez P, Marti V, Sahuquillo JM, Gonzalez E, Calleja I, et al. Implications of endotracheal tube biofilm in ventilator-associated pneumonia response: A state of concept. Critical Care 2012;16:R93.  Back to cited text no. 22
    
23.
Nanthini Devi P, Gomathi S. Biofilm production by organisms causing ventilator associated pneumonia. Int J Curr Microbiol App Sci 2018;7:486-93.  Back to cited text no. 23
    


    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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