Gram Negative Bacilli (GNB) Isolated from Sputum Samples of Patients with Lower Respiratory Tract Infections and the Prevalence of Extended Spectrum Β Lactamase (ESβl) Enzyme Producing Strains

Introduction

Lower Respiratory Tract Infections (LRTI) are a common infectious disease of the respiratory tract that are prevalent worldwide affecting all age groups.¹ This includes a broad description of infections like pneumonia, acute bronchitis and has been one of the important cause of morbidity and mortality.¹ The common gram negative bacilli (GNB) isolated from sputum samples were Klebsiella pneumonia, E. coli, Pseudomonas aeruginosa, Acinetobacter baumanii, Haemophilus influenzae.² Extended Spectrum β Lactamase (ESβL) is an enzyme produced by GNB like E. coli, K. pneumoniae, K. oxytoca, P. mirabilis that acquire the ability to degrade third and fourth generation cephalosporins and monobactams.³ Infections with ESβL producing strains not only decreases the broad spectrum of antibiotics used in empirical treatment, but can also lead to worse outcome of the disease including longer hospital stay, increased morbidity and cost escalation in the treatment of disease.^1,4 ESβL enzymes were first discovered in the US during the late 1980s. These enzymes are plasmid mediated, capable of inactivating penicillins, third generation cephalosporins and Aztreonam.⁵

Extended Spectrum β Lactamase enzymes can be classified based on two systems:

a)  Ambler’s Classification - This classification is based on the amino acid molecule present on the ESβL enzyme. The classification is termed as A, B, C, D and so on, as depicted in Figure 1.⁶

b) Bush & Jacobi-Madeiros Classification - This classification is based on the effect of  inhibitors and degradation of β Lactam substrates. The classification is termed as Group1, 2, 3.⁶

Bush-Jacoby-Medeiros Classification⁶

Group 1: Cephalosporinases (Ambler Class A & D)

Group 2: Serine β Lactamase (Ambler Class A & D)

Group 3: Metallo β Lactamases (Ambler Class B)

Lower respiratory tract infections (LRTI) have a nonspecific presentation in the community being responsible for 4.4% of the total hospital admissions and 6-5% deaths among adults.⁸ It  includes a broad spectrum of conditions like bronchitis, pneumonia, acute exacerbations of  chronic obstructive pulmonary disease, chronic bronchiectasis.⁹ Some of the common GNB isolated from LRTIs include Haemophilus, Klebsiella, Pseudomonas and Acinetobacter species, which usually respond to antibiotics. However, overuse of antibiotics in developing nations can lead to the emergence of Drug Resistance.¹⁰ The airway irritation following an infection results in the production of mucous by the lining epithelium of the respiratory tract which is coughed out of the respiratory tract as phlegm (mixture of mucous and saliva).¹¹ The sputum characteristics may vary depending upon the infection.¹²

 LRTI occurs as a result of failure of eliminating a pathogen from the alveoli and lower airways that results in exudates filling up the airway passages with fluid and inflammatory exudates of leucocytes.¹³ This directly affects the oxygen exchange causing respiratory imbalance and appearance of symptoms like cough, chest pain, sputum production, dyspnoea and sometimes shock in severe cases.¹³ Gram negative bacteria are predominantly demonstrating antimicrobial drug resistance making the treatment of LRTI complicated. Hence there is need for studies related to the emergence of drug resistance among the GNB isolated from LRTI.¹⁴

Materials and Methods

Sample collection

• Early morning sputum sample was collected from the patients diagnosed with LRTI.
• A sterile container was provided and the patient was advised to disinfect the oral cavity with chlorhexidine mouth wash 2-3 times after brushing the teeth, as soon as they wake up in the morning and the sputum was expectorated into the container.

Transport and Storage

The sample was transported immediately to the laboratory and subjected to processing. If any delay was expected, the sample was stored in the refrigerator to prevent overgrowth of commensal bacteria.

Sample Processing

The sample was subjected to gross examination and macroscopic details (if any) were recorded related to colour, mucopurulent and any other visible conditions of the sputum.

Staining

• The sputum sample was subjected to Gram’s Stain and ZN Stain. The microscopic observation related to the epithelial cells, inflammatory cells, type of bacteria and acid fastness were also recorded.
• The stained smear was graded according to the criteria.
• The sputum sample was further homogenized by N-Acetyl –L cysteine method (NALC) for a better yield.

Culture

• The homogenized sputum sample was plated on to Blood Agar, MacConkey Agar and Chocolate Agar. The inoculated plates were placed in an incubator at 37^oC for 24- 48 hrs. The plates were examined for the colony growth at the end of the incubation period.
• The colonies were identified and further subjected to biochemical reactions like, Indole, Urease, Citrate and triple sugar ion (TSI) for presumptive identification of the bacteria upto the species level.
• The colony growth obtained was further subjected to Antibiotic Susceptibility Testing (AST) on Mueller Hinton Agar by Kirby-Bauer method using antibiotics for GNB.
• The sensitivity and resistance pattern along with ESβL enzyme production among the strains were recorded
• ESβL detection was performed by Disc approxi- mation method. Antibiotic discs Ceftriaxone (CTR), Piperacillin-Tazobactam (PIT), Ceftazidime (CAZ) in the same order were prepared during the AST along with other antibiotics depending on the species of the organism.

 Results

Table 1 shows that out of the total 50 samples processed, 18% showed growth i.e., nine samples, whereas 41 samples showed no growth (82%). K. pneumoniae was the most frequently isolated organism followed by Pseudomonas aeruginosa. 71% of K. pneumoniae were ESβL strains, while there were none in Pseudomonas aeruginosa.

Table 2 shows the prevalence of LRTI and the age distribution of the samples collected from various patients. ESβL was isolated from 26% of the patients in the age category of 25-45 years, 45-65 years age category showed 40% isolation followed by above 65 years age category that showed 34% isolation. Out of the total ESβL isolated, 58% were from males followed by 42% from females.

Table 2 depicts the age and gender wise distribution of LRTI and samples collected from the patients.

Discussion

Out of total 50 samples, 18% yielded growth with 82% showing no growth. In a study conducted by Wun Yan Huang et al., to assess the diagnostic performance of sputum gram stain in predicting sputum culture, 22.51% samples exhibited positive sputum culture, while 77.49% were negative for culture.²

The same study also found that out of the total samples tested, 63% were from males and 44% were from females. In the present study, the positive culture growth was found to be 58% in males and 28% in females as compared to the study by Wun Yan Huang et al.²

The present study isolated Klebsiella pneumoniae and Pseudomonas aeruginosa species as the common ESβL producers which was quite similar to a study by Horie H, et al. which isolated Klebsiella pneumoniae and Proteus mirabilis as the most commonly isolated Gram negative ESβL producers.³

The incidence of LRTI with Pseudomonas and Klebsiella pneumoniae as the predominant organisms in the age group 25-45 years was approximately 30%, whereas the present study noted an incidence of 26% in the same age group. This was very similar to the study conducted by Khan et al. A study by Mishra et al. showed 44.4% pathogen recovery rate in contrast to  the present study which showed 18% recovery rate indicating a decline in the recovery of pathogens in LRTI.¹⁰

The ESβL production among the total isolates of K. pneumoniae was found to be 71.4% showing high chance of ESβL isolation in contrast to a study conducted in Ethiopia by Teklu et al. which showed 84.4% prevalence of multidrug resistant organisms.

Conclusion

The present study isolated Klebsiella pneumoniae as the most common organism from the family of Enterobacterales and Pseudomonas aeruginosa as the most common organism from the family of Non fermenter Gram Negative Bacilli.

Klebsiella pneumoniae showed almost 71.42% ESβL production rate among the total organisms isolated, limiting the available treatment options and leading to prolonged hospital stays for the patients. Pseudomonas aeruginosa was the next frequently isolated organism in LRTI; however ESβL strains were not isolated in the present study.

The strains that were identified as ESβL producers were found to be resistant to all third and fourth generation cephalosporins leaving very few options for the treatment. The treatment for ESβL infections should consider

1. In vitro susceptibility of the organism

2. The location of infection

3. The degree of source control of infection

4. Clinical condition of the patient

Carbapenem antibiotics, a combination of β Lactam antibiotic with β Lactamase inhibitor like piperacillin tazobactam, amoxicillin clavulanic acid can be used in the treatment after considering the above mentioned factors.

LRTI prevalence was highest in the age group 45-65 years, followed by >65 years showing a prevalence rate of 40% and 34%, respectively. This indicates that timely detection of LRTI and appropriate antibiotic therapy in the respective age groups can reduce hospital admissions and morbidity.

Increased incidence of multi drug resistant and ESβL strains necessitates bacteriology research, diagnostic capacity and surveillance of antibiotic resistance. Routine screening for ESβL production and strong infection prevention strategies are very important in health care settings.

Conflict of Interest

None

Funding

Rajiv Gandhi University of Health Sciences (RGUHS) Short Term Research Project for Undergraduates.

Acknowledgement

Thankful to Rajiv Gandhi University of Health Sciences (RGUHS). Thankful To St. Alphonsa Institute of Allied Health Sciences.