DETECTION OF EXTENDED SPECTRUM BETA LACTAMASES-PRODUCING KLEBSIELLA PNEUMONIAE ISOLATES IN CHRONIC OBSTRUCTIVE PULMONARY DISEASES PATIENTS

Objective: Chronic obstructive pulmonary disease (COPD) is the leading cause of morbidity and mortality worldwide which includes chronic bronchitis and emphysema. 80% of acute exacerbation of COPD (AECOPD) are triggered by respiratory viruses, atypical bacteria, and aerobic Gram-positive and Gram-negative bacteria. The aim of our study was to analyze the prevalence and antimicrobial susceptibility testing of Klebsiella pneumoniae and detection of extended spectrum beta lactamases (ESBL)-producing K. pneumoniae isolates from COPD patients. Methods: Sputum samples received from seventy-five diagnosed cases of COPD from the department of General Medicine and Chest Medicine, TSRM MCH and RC, Trichy, from March 2022 to August 2022. Direct Gram stain was done for all sputum samples. The suitable sputum samples were cultured. The identification of organism and antimicrobial susceptibility testing was done by standard microbiological techniques. Results: Out of seventy-five cases, 64% were males and 36% were females. Forty-seven were sputum positive, and the prevalence of Gram-negative bacteria was 64%.


INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is a spectrum of airway diseases that includes chronic bronchitis and emphysema [1]. The World Health Organization says it is the third leading cause of death worldwide [2]. COPD is defined as a preventable and treatable disease characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases, exacerbation is the acute worsening of clinical condition of COPD patients [3].
Acute exacerbation of COPD (AECOPD) is defined as a sustained worsening of the patient's condition, from normal day-to-day activities. The prevalence of AECOPD varying from 1% in urban nonsmoker to 21% in rural smokers and mortality rate of 24% of the patients required intensive care unit admission [4]. This mortality rate increased to 30%, if the patient was above 65 years [5]. The clinical guidelines have included "The Winnipeg criteria," which are based on increased breathlessness, sputum purulence, and sputum volume, to diagnose the patients and grade the severity of acute exacerbations of COPD [6].
AECOPD is triggered by respiratory viruses (Influenza, Para influenza, Rhinovirus, Coronavirus, Adenovirus, and RSV), atypical bacteria (Mycoplasma pneumoniae and Chlamydia pneumoniae), and aerobic Gram-positive and Gram-negative organisms. Early diagnosis and knowledge of local bacteriological profile and antibiogram help us to reduce the number of failure cases recorded with empirical treatment during AECOPD. The present work was done to find out the aerobic bacteria and their antibiotic sensitivity pattern in AECOPD. The aim and objective of the present study were to analyze the prevalence and antimicrobial susceptibility testing of Klebsiella pneumoniae and also to detect the extended spectrum beta lactamases (ESBL) producers of K. pneumoniae isolates from AECOPD patients.

Seventy-five sputum samples of AECOPD patients from General
Medicine and Chest Medicine were collected and processed over a period of 6 months (March 2022 to August 2022) after getting the approval from the ethical committee of our institute.
Variables included for the study were age, sex, smoking, comorbidities, signs, and symptoms of the patient. The information regarding these variables was collected using a pretested questionnaire.

Exclusion criteria
• Bronchial asthma/lung abscesses/Interstitial lung disease/lung cancer • Known case of pulmonary tuberculosis • Ischemic heart disease • Subjects who were recently started on antibiotic therapy.

Ethical considerations
Before the commencement of the study, ethical approval was obtained from the institutional review board. Written consent was obtained from the study participants or their guardians. Confidentiality of individual patient's information was maintained during data collection, analysis, and interpretation.

Specimen collection
Early morning samples were obtained from clinically diagnosed cases of AECOPD. Patients were instructed to collect deep coughed sputum into a sterile wide mouth container with a screw cap after rinsing the mouth twice with plain water. Material is transported and processed immediately within 30 min from the time of collection [8].

Culture methods
Direct Gram stain was done from sputum sample and reported according to Bartlett's grading system. A score of one and above was considered suitable sample. The suitable sputum samples were inoculated onto Nutrient agar, Mac-Conkey's agar, chocolate agar, and two blood agar plates. On one blood agar streaking with Staphylococci was done to facilitate growth of Haemophilus influenzae. All the plates were incubated at 37°C for 24 h in 7-10% CO 2 concentration. The isolated organisms were identified by standard microbiological techniques [8]. All the confirmed isolates of K. pneumoniae were tested for antimicrobial susceptibility by Kirby-Bauer disk diffusion method on Muller-Hinton Agar plates. The suspension of the isolated organism's broth was adjusted to 0.5 MacFarlands standard, and lawn culture was done and incubated at 37°C overnight. The sensitivity and resistant patterns were reported according to the latest CLSI guidelines [9]. The detection of ESBL producers of K. pneumoniae was done by combined disc test method [9].

RESULTS
A total of seventy-five sputum samples of AECOPD were included in the present study, 51 were males and 24 were females. Among them, 49 (65.3%) belong to the age group of 51-70 years and 17 (22.6%) were above 70 years ( Table 1).

DISCUSSION
It was observed that AECOPD was prevalent in 51-70-year age group. Thus, AECOPD was common in advanced age group as respiratory tract is more susceptible due to the impairment of immunological defense mechanism, associated comorbid illness, increased duration of seasonal variation, and tobacco smoking. This is concordance with the study conducted in Bhubaneswar by Rao et al., [10] which states 70% belongs to the age group of 61-75 years. Males were affected more than females because they were more involved in smoking and start it in younger age group; therefore, more chance of inhalation and increased    environmental exposure or temperature variation. In non-smokers, especially among women, exposure to indoor air pollution was an important factor. This is concordance with a study done by Mythri et al. [11] and Sharan [12], respectively, and both states 60-70% were smokers. Out of seventy-five patients, 72% were diabetic and the remaining 28% were non-diabetic. This is concurrent with the study done at Thanjavur by Mary et al., stating 60% of diabetic patients are involved acute exacerbation.
In our study, Gram staining findings were in correlation with the culture findings in 89.4% of cases; aerobic culture positivity was 62.7% which is similar to other studies. The prevalence of Gram-negative isolates was 76.5%, as compared to 23.4% of Gram-positive isolates corresponding to other studies. This corresponds to the study done at Karimnagar by Bannaravuri et al. [13] and Mussema et al., in Southwest Ethiopia [14], in which >70% of culture positive were Gram-negative bacilli.
K. pneumoniae and P. aeruginosa were the common bacteria in our study. The bacterial isolates depend on the prevalence of bacteria in hospital environment; in the community, antibiotic prophylaxis and severity of exacerbation. H. influenzae was not isolated in our study which can be explained by the fact of temperature variations and the use of antibiotics either self or by prescription. This corresponds to the study done at Bangalore by Narayanagowda et al., [15] and also in concordance with the study done by Bannaravuri et al., [13] showed >40% prevalence of K. pneumoniae in COPD patients sample. A study done at Egypt by Ghanem et al., [16] which states the prevalence of K. pneumoniae is 58% followed by P. aeruginosa 14% which is concurrent with our study. AltThough few study done by Sharma et al., [17] it is not concurrent which states the prevalence of S. pneumoniae is 13%.
AST pattern of K. pneumoniae was found to be sensitive to Imipenem, Aminoglycosides, Amoxycillinclavulinic acid, Ciprofloxacin, and Piperacillin-Taxobactam. This is similar to the study conducted in Indore by Sharan [12], which showed 40% resistant to amoxicillin and clavulinic acid, 30% resistant to aminoglycosides, and 5% resistant to Carbapenams.
Among these, 68% isolates were ESBL producers and 21% were carbapenemase producers. This corresponds to the study done at Nagpur by Hansotia et al., [18] which showed 57% were ESBL producers.

CONCLUSION
The optimal usage of antibiotics and effective antimicrobial therapy can significantly diminish health-care costs and maintain the quality of life, especially in the elderly patients. In our study, Imipenem, Aminoglycosides, Amoxycillin clavulinic acid, and Ciproflaxacin were the most active antibacterial agents and the emergence of ESBL and Carbapenemase-resistant cases are highly increasing nowadays. More studies are required to be conducted on antibiotic susceptibility pattern at regular interval, to formulate the antibiotic policy for improvement in patients' conditions mainly in acute exacerbations of COPD, which in turn reduces the morbidity and mortality.

Recommendation
• Proper hand hygiene measures, health education of the patients regarding strict compliance to the prescribed antibiotics regimen • Mandating judicious usage of antibiotics by the clinical practitioners to overcome ESBL and carbapenemase-resistant strains • Formulation of antibiotic policies for health institutions and antibiotic stewardship programs can aid to a great extent in proper antibiotic usage and the prevention of further spread of resistance, thereby paving a way for a healthy community.

Limitations of the study
• Test sample size is low • It is a single-center-based study • Phenotypic test has to be confirmed by genotyping.

ACKNOWLEDGMENT
I would like to thank the Chairperson and Dean of the institute for providing laboratory facilities and healthy working atmosphere during the study period. I am also thankful for my HOD and professor's and the technical staff of the institute for providing necessary helping hand during the endeavor.

AUTHORS CONTRIBUTIONS
R. Saraswathi and R. Shangamithra conceptualized the study. R. Shangamithra, A. Anupriya performed the experiments and analyzed the data and R. Saraswathi and R. Shangamithra wrote the manuscript.

CONFLICTS OF INTEREST
All the authors declare that they have no conflicts of interest.

FUNDING
No external funding.