Culture identification and antibiogram of Klebsiella pneumoniae isolated from a sputum sample

doi:10.21203/rs.3.rs-2975095/v1

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Culture identification and antibiogram of Klebsiella pneumoniae isolated from a sputum sample

https://doi.org/10.21203/rs.3.rs-2975095/v1

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Gram-negative pathogens are an important cause of community and hospital-acquired infections throughout the world. Klebsiella pneumoniae has become one of the more common causes of these infections and one of the important aspects of Klebsiella associated infections. It is the emergence of multi-drug resistant strains particularly those involved in nosocomial diseases. This study was done to determine the isolation rate of Klebsiella, their antibiogram and for the presence of resistant strains from sputum samples.

A total number of 16 sputum samples were included in the present study. Sixteen clinical isolates were collected from Mardan Medical Complex ,Khyber Pakhtunkhwa, Pakistan. Klebsiella were identified by standard microbiological techniques and antibiotic susceptibility testing was done by Kirby-Bauer disc diffusion method and interpreted as per CLSI guidelines. Through KBDD (Kirby Bauer Disk Diffusion Method), four verified K.pneumoniae isolates were subjected to further testing for antimicrobial drug sensitivity. It was discovered that roughly 50% of them were multidrug resistant. As per CLSI guidelines results of the present study revealed that confirmed K.pneumoniae isolates were resistant to Vancomycin 30(µg) , Amikacin 30(µg), Chloramphenicol 30(µg), Amoxicillin 30(µg) , Ticarcillin 75(µg) and were susceptible to Meropenem 10(µg),Piperacillin 100(µg) and Tazobactam 110(µg). These are capable of acting as an effective antibacterial agent in the treatment of serious bacterial infections caused by K. pneumoniae.

The present study reveals the isolation of Klebsiella from sputum samples and their tendency towards antibiotic resistance. K. pneumonia was found to be most sensitive to Meropenem 10(µg), Piperacillin 100(µg) and Tazobactam 110(µg).

The data of this study may be used to determine trends in antimicrobial susceptibilities to formulate local antibiotic policies and overall to assist clinicians in the rational choice of antibiotic therapy.

Klebsiella pneumoniae

Antibiotic resistance

Antibiotic susceptibility testing

Sputum

Klebsiella pneumoniae is gram-negative, rod-shaped, aerobic and facultative anaerobic, nonmotile, nitrate positive, and has large polysaccharide capsules (Brooks et al., 2004). It belongs to the Enterobacteriaceae family and the genus Klebsiella. It is also an opportunistic pathogen, capable of infecting humans and other animal species with a variety of diseases. It belongs to the ESKAPE group of bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter specie) that effectively 'escape' the effects of antibacterial drugs (Von Gottberg et al., 2001).

In terms of habitat associations, the genus Klebsiella is diverse. It is the second most common member of the human intestine's aerobic bacterial flora. It is flora that is either resident or transient in the gastrointestinal system (Seaton, 2000). In humans, K. pneumoniae is mostly found in the gastrointestinal tract, with a few isolated cases in the nasopharynx, where it can reach the bloodstream or other tissues, causing illness (Chiang et al., 2010). Humans, animals, sewage, drinking water, surface waterways, polluted water, industrial effluents, and vegetation are examples of other habitats (Ferragut et al., 2004).

Bacteria identification is very important in microbiology and pathology as it serves a basis of understanding diseases. Klebsiella was first identified as a cause of pneumonia by pathologist Karl Friedlander in 1882, and capsular polysaccharide has been identified as the species' most distinguishing feature and most studied virulence component. Friedlander isolated a capsulated bacillus from the lungs of a patient who had died of pneumonia in 1883, and the microorganism was given its name Friedlander’s bacillus .It was later identified as Klebsiella and is now reported worldwide (Eisenstadt and Crane,1994).

Clinical strains of K. pneumoniae can be separated into two groups based on the accessory genome: the classical group (cKp), which includes MDR strains, and the hypervirulent (hvKp) group (Martin 2018). cKp strains are commonly found in the gastrointestinal (GI) tract of patients with intestinal. HvKp strains are considered serious pathogens and can cause community-acquired, aggressive, and metastatic infections, as well as liver abscesses, pneumonia, endophthalmitis, meningitis, and septic arthritis (Russo 2019).

K.pneumoniae is the most common cause of liver swelling, Gram-negative bacillary meningitis, brain abscess, lung abscess, thoracic empyema, prostatic abscess, deep neck infection, and complicated skin and soft tissue infections, accounting for 4.8 percent of community acquired pneumonia (CAP) pathogens (Chang, 2008). It is one of the most common bacteria that causes severe epidemic and endemic nosocomial infections in hospitals. Septicaemia, neonatal septicaemia, intra-abdominal infections, and bloodstream infections (BSIs) are all common causes (Ramphal and Ambrose, 2006). It is an opportunistic bacterium that can colonize the mucosal epithelium of the stomach and nasopharynx, then move to vulnerable patients' deep tissues and bloodstreams, causing serious infections including endophthalmitis (Donskey, 2004). These infections are difficult to cure because of the pathogen's high inherent antibiotic resistance. Infection with multidrug-resistant gram-negative bacteria is linked to a high rate of morbidity and mortality (Patel et al., 2009). It is caused by the microbiota of the patient. The loss of microbiota colonization resistance causes a bloom of K. pneumoniae cells in the digestive tract, which is thought to be an early stage in the progression of the disease. Indwelling systems such urine catheters, feeding tubes, and central blood catheters are well-known risk factors for Klebsiella infection. Antibiotic resistance in Klebsiella species spread due to the use of ineffective antibiotics. Furthermore, widespread use of broad-spectrum antibiotics in hospitalized patients has resulted in increased Klebsiella carriage as well as the emergence of multidrug-resistant strains. Antimicrobial resistance caused by K. pneumoniae can have serious consequences, including increased morbidity, mortality, and even longer hospital stays. (Li & Webster, 2018 and Pacios et al., 2020). In order to implement effective control measures to prevent rapid spread of drug resistance, it is critical to understand the antimicrobial susceptibility pattern of Klebsiella, which differs in different geographical settings (Namratha, 2015). Following that, the use of third-generation cephalosporins like ceftriaxone, cefotaxime, and ceftazidime was limited to the treatment of K .pneumoniae infections. K. pneumoniae carbapenemases (KPC) is resistant to carbapenem, amino glycosides, cephalosporins, and fluoroquinolones, despite the fact that it is usually sensitive to the antibiotic colistin. This makes it a highly resistant bacterium that currently requires the use of three different antibiotics to treat the infection successfully (Johnson et al., 2011). Colistin, tigecycline, and gentamicin are the current antibiotic combinations used to treat K. pneumoniae infections. The indiscriminate use of colistin has resulted in several cases of K. pneumoniae resistance (Feizabadi and Etemadi, 2007).

The present study was done to identify different strains of K. pneumoniae through morphological and biochemical characterization using several assays and to screen the antibiotic susceptibility pattern of K. pneumoniae.

Total 16 samples (sputum samples) were collected from Mardan Medical Complex, KPK, Pakistan. These samples were subjected to Microbiology Research Laboratory for analysis of antibiotic sensitivity. This research was conducted between October, 2021 to April, 2022. The samples were collected in sterile leak proof container and stored at 37℃ for 24 hours. Samples were processed according to antiseptic methods recommended by Clinical and Laboratory Institute and the isolates were identified by standard biochemical tests according to standard conventional methods.

Table 2.1 Overview of methodology

2.1 Characterization of bacterial isolates

Sputum samples were aseptically inoculated on to MacConkey agar plates and incubated overnight at 37℃. K. pneumoniae isolates were identified by their morphology and biochemical characteristics. Morphology of Klebsiella identified were large dome shaped colonies and lactose fermenting mucoid colonies on MacConkey agar. In Gram staining fig.(a) ,Gram negative, short, stout, blunt rods were seen .The biochemical characters identified were negative oxidase test fig.(b), Positive catalase test fig.(c), negative Indole test fig.(d) and abundant gas production from glucose, lactose, sucrose, maltose sugar fermentation tests fig(e).

2.2 Antimicrobial susceptibility testing

On Muller Hinton agar, total of four positive isolates were screened for antimicrobial susceptibility testing. Isolated broth culture inoculums were prepared and lawn cultured on Mueller-Hinton agar before drying. Using sterile forceps, antibiotic discs were applied to the Mueller Hinton agar surface as shown in Fig. 2.2. The plates were then incubated for 24 hours at 37°C. Using a calibrated scale, the diameter of the inhibition zones was measured in millimeters. Zone Measurement was checked according to CLSI guidelines. The antibiotics chosen for the study were Gentamicin 10(µg), Vancomycin 30(µg), Meropenem 10(µg), Ticarcillin 75(µg), Tazobactam 110(µg), Chloramphenicol 30(µg), Ampicillin 10(µg), Piperacillin 100(µg), Amikacin 30(µg), Amoxicillin 30(µg) and Doxycycline 30(µg). A panel of antibiotics which was selected as per the CLSI guidelines, but only the antibiotics which were showing higher degree of sensitivity were included in this study. The plates were then incubated at 37°C for 24 hours. Antimicrobial activity was indicated by an inhibition zone. The diameter of the inhibition zones was measured in millimeter using a calibrated scale. An organism was interpreted as susceptible if the diameter of inhibition zone was more than 19 mm, intermediate if diameter was 15-18mm and resistant if the diameter was less than 13 mm, which were compared with the zones of control strains as demonstrated in the Kirby-Bauer chart. Antimicrobial susceptibility of K. pneumoniae to different antibiotics is obtained. From antimicrobial susceptibility, antibiogram for K. pneumoniae is prepared and results are interpreted, considering the antimicrobial susceptibility probable drug of choice is selected for K. pneumoniae.

During the 6-month period, a total of 128 sputum samples were processed for culture and sensitivity testing. Antimicrobial susceptibility testing revealed isolates were sensitive to Amikacin, Piperacillin, Meropenem and Tazobactam. Isolates were highly resistant to Amoxicillin, Vancomycin, Ticarcillin, Amoxicillin, and Chloramphenicol.

Antimicrobial susceptibility pattern of K. pneumoniae is shown in figure;

Table 2.2

Antibiotic susceptibility pattern against NKP 1
Zone of inhibitions
Antibiotics	Disc potency (µg)	Disc diameter (mm)	Zone of inhibition	Susceptible	Intermediate	Resistant
Amikacin	30	6mm	19	≥ 14	15–16	≤ 17
Piperacillin	100	6mm	23	≥ 17	18–20	≤ 21
Vancomycin	30	6mm	18	≥ 12	10–11	≤ 9
Amoxicillin	30	6mm	13	≥ 13	14–17	≤ 18
Doxycycline	30	6mm	15	≥ 14	11–13	≤ 10
Gentamicin	30	6mm	18	≥ 12	13–16	≤ 17

Table 2.2

Antibiotic Susceptibility pattern against NKP 2
Zone of inhibitions
Antibiotics	Disc potency (µg)	Disc diameter (mm)	Zone of inhibition	Susceptible	Intermediate	Resistant
Amikacin	30	6mm	18	≥ 14	15–16	≤ 17
Piperacillin	100	6mm	20	≥ 17	18–20	≤ 21
Vancomycin	30	6mm	18	≥ 12	10–11	≤ 9
Amoxicillin	30	6mm	15	≥ 13	14–17	≤ 18
Doxycycline	30	6mm	17	≥ 14	11–13	≤ 10
Gentamicin	30	6mm	17	≥ 12	13–16	≤ 17

Table 2.3

Antibiotic Susceptibility pattern against NKP 3
Zone of inhibitions
Antibiotics	Disc potency (µg)	Disc diameter (mm)	Zone of inhibition	Susceptible	Intermediate	Resistant
Meropenem	10	6mm	18	≥ 18	15–17	≤ 14
Ticarcillin	75	6mm	0	≥ 14	15–19	≤ 20
Vancomycin	30	6mm	11	≥ 12	10–11	≤ 9
Chloramphenicol	30	6mm	12	≥ 18	13–17	≤ 12
Amoxicillin	30	6mm	0	≥ 13	14–17	≤ 18

Table 2.4

Antibiotic Susceptibility pattern against sample 4
Zone of inhibitions
Antibiotics	Disc potency (µg)	Disc diameter (mm)	Zone of inhibition	Susceptible	Intermediate	Resistant
Vancomycin	30	6mm	15	≥ 12	10–11	≤ 9
Meropenem	10	6mm	20	≥ 18	15–17	≤ 14
Ticarcillin	75	6mm	12	≥ 14	15–19	≤ 20
Chloramphenicol	30	6mm	17	≥ 18	13–17	≤ 12
Tazobactam	110	6mm	26	≥21	18–20	≤17

K. pneumoniae is one of the most prevalent bacteria that infects patients .K. pneumoniae is quickly becoming known for its resistance to the majority of commonly used antibiotics. It is particularly problematic in hospitals, where it can lead to a variety of serious infections. For the treatment of nosocomial infections caused by K. pneumoniae, selecting the best antibiotic is critical. Antibiotic resistance in Klebsiella is spread due to the use of ineffective antibiotics.

The present study highlights the most alarming situation of highly diverse antibiotic resistance. In our study, 91.66% of the isolates showed resistance to Amoxicillin. Aminoglycosides such as Amikacin (66.66%) and Gentamicin (62.50%) were the most effective antibiotic agents against K.pneumoniae. An alarming finding seen in this study was that resistance was shown to Ticarcillin/clavulanic acid. Overall resistance was high on account of the production of extended-spectrum β-lactamases (ESBLs) by the K. pneumoniae. The resistance may also be due to the production of Metallo-β-lactamases (MBL). These drugs are proposed to be an alternative and better treatment of K. pneumoniae infection in this part of the country.

In conclusion, high antibiotic resistance of K. pneumoniae towards commonly used antibiotics are the major reasons for prolonged infections, increased hospitalization, increased cost of therapy and enhanced morbidity and mortality rates. K. pneumoniae was found to be most sensitive to Amikacin, Gentamicin, Piperacillin, Ticarcillin, Vancomycin. Considering the antibiogram, Amikacin, Gentamicin, Piperacillin, Ticarcillin, Vancomycin should be preferred drugs for K. pneumoniae infection isolated from sputum. Regular surveillance of antibiotic susceptibility pattern may help to overcome the indiscriminate use of antibiotics a major cause of emergence of drug resistance among pathogens and to develop antibiotic policies. The data of this study may be used to determine trends in antimicrobial susceptibilities to formulate local antibiotic policies and overall to assist clinicians in the rational choice of antibiotic therapy.

There is no financial backing organization involved in this research, which was put together from prior material. I would like to acknowledge that all the authors equally participated in this article.

Ethical Approval and Consent to Participate: The study was conducted with ethical approval and informed consent as per the institutional guidelines.

Consent to Publish: Not applicable (NA) for consent to publish as all identifying information has been kept confidential.

Availability of Data and Materials: The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Funding: The authors received no financial support for the research, authorship, and/or publication of this article.

Competing Interests: No competing interests.

Authors' Contributions: All authors contributed equally to the manuscript. The authors are Ruby Khan, Saima Wali, Bakht Pari, and Nimra Amir.

Acknowledgements: We would like to thank the participants for their involvement in the study. Additionally, we would like to express our gratitude to our colleagues who provided helpful feedback during the writing process. No further acknowledgments are applicable.

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No competing interests reported.

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Culture identification and antibiogram of Klebsiella pneumoniae isolated from a sputum sample

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