INTRODUCTION:

Klebsiella is a gram negative, non-motile, rod shaped, facultative aerobic and anaerobic, fermentative, catalase positive, nitrate reducing, lactose negative bacteria showing mucoid growth and bearing a prominent polysaccharide-based capsule.¹ These species are found everywhere in nature. They can be found easily in water, soil of plants, insects, animals and humans.²

Being an opportunistic pathogen Klebsiella spp. can primarily attack immunocompromised individuals, but it can equally attack immune-competent and cause the damage. These bacteria frequently cause human nosocomial infections.^3,4It is also responsible to cause hospital acquired urinary tract infections, pneumonia, septicaemia, and soft tissue infections.^2,5The principal pathogenic reservoirs for transmission are gastrointestinal tract and the hands of hospital personnel.

Antibiotics are an important drug to treat the microbial infection. But due to extensive use of broad-spectrum antibiotics in hospitalised patients, it is now leading to the development of multi drug resistant strain and this can become leading cause of morbidity and mortality all over the world.^6,7 In the 21^st century, the world health organization has resolute the antibiotic resistance as one of the greatest significant public health risks.^8,9Current knowledge on antimicrobial resistant pattern is limited and it is essential to be updated for choosing appropriate therapy by medical professionals for effective patient treatment. This study specifically aimed for studying the antimicrobial resistance pattern among Klebsiella species isolated from different clinical specimens obtained from C. U. Shah Hospital, after getting permission from Ethical Committee.

MATERIALS AND METHODS:

Sample Collection:

In present study total 856 numbers of clinical isolates in different form like (urine, blood, sputum, CSF, swab, pus etc.), were collected from different wards (medicine, surgery, orthopaedics, pulmonary medicine, ENT, paediatrics) of the hospital during January 2016 to December 2018. The samples were processed and examined for isolating the needed strain. The primary work of this study was carried out in microbiology department of C.U. Shah Hospital, Surendranagar.

Isolation of Klebsiella from samples:

By morphological characteristics on MacConkey agar (Hi-Media) clinical isolates Klebsiella (large mucoid colonies) were isolated and identified. Further gram staining was performed for primary confirmation of the isolates.

Biochemical characterization:

Selected isolates were further confirmed through Urease production, citrate utilization, sugar fermentation, indole test, oxidase, catalase, H₂S production on TSI agar for Klebsiella sp.

Antibiotic sensitivity testing:

Antibiotic susceptibility testing was performed for all bacterial isolates by modified Kirby Bauer disk diffusion method as per Clinical and Laboratory Standards Institute (CLSI) guidelines. A suspension equivalent to 0.5 McFarland standards was prepared from single isolated colony. A swab was dipped in suspension and streaked over surface of a Mueller-Hinton agar plate.¹⁰ Obtained Isolates were subjected to susceptibility testing for the following antibiotics: Amikacin, Amoxycillin, Amoxycillin/Clavulanic Acid, Cefepime, cefixime, cefoperazone, cefoperazone/Sublactum, cefotaxime, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, co-trimoxazole, colistin, gentamycin, imipenem, levofloxacin, ofloxacin, piperacillin/ tazobactam, tetracycline, tobramycin.

RESULTS:

Results of year 2016:

Total number of 856 clinical specimens were processed and obtained colonies of Klebsiella sp. were isolated and tested during year 2016 to 2018. These strains were isolated from different samples and different wards of hospital.

During year 2016, Klebsiella sp. Obtained from various specimens had shown the highest resistance towards the combination of Amoxycillin and clavulanic acid and lowest resistance to colistin. Almost all variant of beta-lactam became less sensitive towards the organisms (Table -I).

Table-I: Antibiotic resistance pattern of the clinical specimens isolated and obtained during year 2016.

Antibiotic	No. of resistant isolates (%)	Antibiotic	No. of resistant isolates (%)
Amikacin	22.79	Ciprofloxacin	44.65
Amoxycillin	87.67	Co-trimoxazole	53.95
Amoxycillin/Clavulanic Acid	91.39	Colistin	3.48
Cefepime	35.58	Gentamicin	64.18
Cefixime	70.93	Imipenem	18.13
Cefoperazone	62.09	Levofloxacin	27.44
Cefoperazone/Sublactum	27.44	Ofloxacin	22.55
Cefotaxime	50.46	Piperacillin/Tazobactum	10.69
Ceftriaxone	49.30	Tetracycline	31.86
Chloramphenicol	17.20	Tobramycin	25.58

Figure-I: Percentage resistance of Klebsiella isolates to different antibiotics

Figure-II: Clustering of 20 antibiotics obtained by the results of the resistance patterns of isolates of klebsiella species during 2016.

Result of Year 2017:

During year 2017, Klebsiella sp. Obtained from various specimens had shown the highest resistance towards the combination of Amoxycillin and Clavulanic acid as well as Cefixime. Moderate resistance towards Cefotaxime, Ceftriaxone and Co-trimoxazole. Lowest resistance against Colistin (Figure-III, Table-II).

Table-II Antibiotic resistance pattern of the clinical specimens isolated and obtained during year 2017.

Antibiotic	No. of resistant isolates (%)	Antibiotic	No. of resistant isolates (%)
Amikacin	21.65	Ciprofloxacin	35.61
Amoxycillin	74.92	Co-trimoxazole	57.26
Amoxycillin/Clavulanic Acid	82.05	Colistin	5.98
Cefepime	44.15	Gentamycin	23.64
Cefixime	74.92	Imipenem	11.68
Cefoperazone	49.85	Levofloxacin	26.21
Cefoperazone/Sulbactum	43.01	Ofloxacin	35.04
Cefotaxime	52.70	Piperacillin/Tazobactum	23.36
Ceftazidime	48.43	Tetracycline	15.84
Ceftriaxone	50.42	Tobramycin	31.05
Chloramphenicol	27.63

Figure-III: Percentage resistance of Klebsiella isolates to different antibiotics.

Figure-IV: Clustering of 21 antibiotics obtained by the results of the resistance patterns of isolates of klebsiella species during 2017.

Result of Year 2018: -

During year 2018, Klebsiella sp. Obtained from various specimens had shown the highest resistance towards Amoxycillin and lowest resistance against Colistin (Figure-V, Table-III)

Table-III: Antibiotic resistance pattern of the clinical specimens isolated and obtained during year 2018.

Antibiotic	no. of resistant sample (%)	Antibiotic	no. of resistant sample (%)
Ampicillin	72	Cefepime	33.33
Amoxicillin/clavulanic acid	35.13	Nitrofurantoin	16
Amoxicillin	92	Gentamicin	24
Amikacin	13.33	Imipenem	18.66
Ciprofloxacin	45.33	Meropenem	29.33
Ceftriaxone	58.66	Nalidixic acid	50.66
Colistin	9.3	Cefoperazone/sublactum	28
Cefotaxime	57.33	Trimethoprim/sulfamethoxazole	40
Cefuroxime	64	Tigecycline	17.33
Cefuroxime Axetil	64	Piperacillin/tazobactum	37.33
Ertapenem	29.33

Figure-V: Percentage resistance of Klebsiella isolates to different antibiotics.

Fugure-VI: Clustering of 21 antibiotics obtained by the results of the resistance patterns of isolates of Klebsiella species during 2018.

Figure-VII: Comparison of result obtained of antibiotic resistance for 3 years.

Statistical Analysis:

Since p-value is greater than the conventionally accepted significance level of 0.05 (i.e., p > 0.05) we fail to reject the null hypothesis. In another words, there is no statistically considerable difference. (Accept the hypothesis). This means that p < 0.05 and we reject the null hypothesis in favour of the alternative hypothesis. So, when p < 0.05 we generally refer to this as a significant difference. (Reject the hypothesis).¹¹

Results (Table-IV) of chi square in red colour indicate there is increase in the percentage of resistance of the year 2016-2017 or 2017-2018. As per antibiotic policy - Antibiotic Stewardshipand Chennai Declaration¹² for AMR guideline go with discontinuation or alteration of that antibiotic.Results (Table-IV) in black colour indicate no major difference in resistance developed against the antibiotic.

Table-IV: Χ² (chi square) result (p < 0.05, df =1)

Antibiotic	2016-2017	2016-2018	2017-2018
Amikacin	0.144	2.894	2.223
Amoxicillin	21.22	9.515	3.963
Amoxicillin/Clavulanic acid	15.11	108.2	48.49
Cefepime	5.954	0.019	2.177
Cefixime	1.558
Cefoperazone	11.78
Cefoperazone/Sublactum	20.77	0.438	3.168
Cefotaxime	0.388	3.838	2.546
Ceftriaxone	0.097	6.219	5.242
Chloramphenicol	12.28
Ciprofloxacin	6.547	0.164	3.433
Co-trimoxazole	0.857
Colistin	2.735	5.891	1.432
Gentamicin	57.81	15.15	0.094
Imipenem	6.242	0.233	3.963
Levofloxacin	0.149
Ofloxacin	14.89
Piperacillin/Tazobactum	22.62	39.00	7.486
Tetracycline	28.39
Tobramycin	2.869

PCA (Principle Component Analysis):

Figure-VIII: Plot of the principal component analysis (PCA) of the 12 antibiotics for 3 years’ patients’ resistance data represented as -2016 (group 1 in red), 2017 (group 2 in green) and 2018 (group 3 in pink).

DISCUSSION:

The control of Klebsiella sp. is seriously at risk by the steady rise in the number of these microorganisms that show resistance to various ranges of antibiotics. These undesirably affect death rate, treatment costs, disease spread and duration of disorder.¹³ So, a thorough in-depth study is required from different places which can reveal detail about its pattern of resistance.

Antibiotic Resistance profile from year 2016 to 2018, we found that the Klebsiella developed high resistance for latest fifth generation penicillin-based drug Amoxicillin which is from the class of Penam antibiotic. In the present study from year 2016 to 2018 resistance against Amoxicillin got increased (87.67%-2016, 74.92%-2017, 92%-2018) (Table-I, II and III) which is comparable with the study which was carried out in MGM Medical College and LSK Hospital, Kishanganj, Bihar, India- and noted same resistance pattern. In their study, 95.2% isolates had developed resistance against Amoxicillin.¹⁴ In our study ampicillin resistance (72%- 2018) (Table-III) while 86.7% resistant isolates against Ampicillin also noted in the study of Government Medical college, Srinagar during year 2007 and 2008,¹⁰ 0.19% in Government Medical college, Kota during 2018¹⁵and 66.66% at Hakim Abdul Hameed Centenary Hospital New Delhi, India.¹⁷

It is observed that Klebsiella is slowly developing the resistance to latest drug colistin (class polymyxin) also. In our study It had shown an increase in resistance (3.48%- 2016, 5.98%- 2017, 9.3%- 2018) (Table-I, II and III), while study carried out in Khorramabad Shohadaye Ashayer hospital, in which 17.5% resistance was noted during 2014 against the colistin.² It is now less prescribed to patients due to its more risk linked to side effects rather than benefits.

Antibiotic class cephalosporin is a type of beta lactam antibiotic. Newer generation cephalosporins when compared to the previous generation have a better action against gram negative bacteria.¹⁸ In this study we found there was a gradual development of resistance against its third-generation ceftriaxone, cefotaxime and forth generation cefepime. That we can study from Table– (I, II and III) as well as from figure no (I), (III) and (V). From past two decades due to over use of these class of antibiotics, which is generally used in UTI treatment in patients, bacteria have developed multi drug resistance.¹⁹These MDR bacteria developed multiple antimicrobial resistance mechanism like boosting its natural resistance through drug efflux, alteration of drug target and produced plasmid mediated beta lactamase, making it not effective in treatments.^14,20

In our study, we find Klebsiella sp., were susceptible to amikacin (22.79%-2016, 21.65%-2017, 13.33%-2018) (Table-I, II and III) and gentamicin (64.18%-2016, 23.64%-2017, 24%- 2018) (Table-I, II, and III) (aminoglycoside class) which is showing similar results like the study which was carried out at Bai Jerbai Wadia Hospital, Mumbai, ²¹ Amikacin - 61.5%, Gentamycin - 37.1%. And 31.69% resistant isolates for amikacin and 59.2% resistance against Gentamicin noted during January 2015 to December 2017 in Dr.V.M.Govt. Medical college, Solapur.¹ These antibiotics are generally used for nosocomial infections.

As per study of Rajesh KS et al. in tertiary care hospital Klebsiella pneumonia was found to be highly resistant to most of the classes of cephalosporins, which shows more than 90% resistance, then ciprofloxacin with 80% resistance and nitrofurantoin with 72.2% resistance. Ertapenem was got to be utmost active drug against Klebsiella which indicated 91.66% sensitivity followed by amikacin of 80% sensitivity. In our result we also found development of resistance in different cephalosporin class antibiotics and 92% resistance found against latest antibiotic Amoxicillin. But susceptibility is noted with amikacin and colistin. ^22,23

Hospital-acquired infections have raised worldwide, causing significantly to morbidity of the hospitalized patients and boosting clinical occurrence of antibiotic resistance Klebsiella is becoming a major health care issue and evolve in the world as a most dangerous superbug.^14,15,24 So, in order to understand more about antibiotic resistance and the responsible genes further study is also carried out with help of NGS by doing the whole genome sequencing of selected isolates. Samples will be selected out of total isolates based on different criteria and will be proceeded with the WGS to understand resistance pattern.

CONCLUSION:

It is quite alarming to notice that nearly all of the isolates included during this study were found resistant to five or more antibiotics. A large amount of data from researches across the world reveals that the MDR (Multi Drug Resistance) category bacteria are arising world-wide at an alarming rate and causing many public health problems and questions to healthcare. Extensive use of broad-spectrum antibiotics led to the multidrug resistance strains. As we know Antibiotic susceptibility patterns can be varying, it is important to update the susceptibility pattern so as per that can prescribe the drug to the patient. The rate of the prevailing problem of antimicrobial resistance should be taken as main issue and balanced prescription of antibiotics essentially strictly trailed.

The major limitation of the study is that it is a single hospital specific study and the sample size might be relatively small to study and understand more about antibiotic resistance pattern. The large specimen study from varied places will be useful for precise results and conclusion.

ABBREVIATIONS:

AMR- Antimicrobial resistance

CLSI- Clinical and Laboratory Standards Institute

CSF- Cerebrospinal fluid

ENT- Ear, Nose and Throat

MDR- Multi drug resistance

NGS- Next genome sequencing

UTI- Urinary tract infection

WGS- Whole genome sequencing

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

INFORMED CONSENT:

For this type of study, informed consent is not required.

ETHICAL APPROVAL (RESEARCH INVOLVING HUMAN PARTICIPANTS AND/OR ANIMALS)

This article does not contain any studies with human participants or animals performed by any of the authors.

AVAILABILITY OF DATA AND MATERIALS:

The datasets used and /or analysed during the current study are available from the corresponding author on reasonable request.

REFERENCES:

1. Pathak S, Ingole K, Gawande R. (2018) Antibiotic sensitivity pattern of Klebsiella species isolated from various clinical samples, International Journal of Scientific Research. 7(1):21-22 doi: 10.36106/IJSR

2. Babakhani S, Shokri S, Reza M, Kazem M. (2014) Comparison frequency and Determination antibiotic resistance pattern of Klebsiella SPP. isolated from Nosocomial infection in Khorramabad Shohadaye Ashayer hospital. Bull. Env. Pharmacol. Life Sci.3:149-154

3. Akram M, Shahid M, Khan A. (2007) Etiology and antibiotic resistance patterns of community acquired urinary tract infections in JNMC Hospital, Aligarh, India. Annals of Clinical Microbiology and Antimicrobials. 6:4 doi.org/10.1186/1476-0711-6-4

4. Sreeja. M.K, Gowrishankar N.L, Adisha. S, Divya. K.C. Antibiotic Resistance-Reasons and the Most Common Resistant Pathogens – A Review. Research J. Pharm. and Tech. 2017; 10(6): 1886-1890. doi: 10.5958/0974-360X.2017.00331.6

5. Sikarwar A, Batra H. (2011) Prevalence of antimicrobial drug resistance of Klebsiella pneumonia in India. International Journal of Bioscience, Biochemistry and Bioinformatics. 1(3):211-215 doi: 10.7763/IJBBB. 2011.V1.38

6. Chakraborty S, Kaniz M, Sarker P, Zahangir M, Ismail M, Abu S. (2016) Prevalence, antibiotic susceptibility profiles and ESBL production in Klebsiella pneumoniae and Klebsiella oxytoca among hospitalized patients. Periodicum Biologorum. 118(1):53-58. doi: 10.18054/pb.2016.118.1.3160

7. Sara H. Mohamed, Mary S. Khalil, Mona I. Mabrouk, Mahmoud S.M. Mohamed. Prevalence of antibiotic resistance and biofilm formation in Klebsiella pneumoniae carrying fimbrial genes in Egypt. Research J. Pharm. and Tech. 2020; 13(7): 3051-3058. doi: 10.5958/0974-360X.2020.00542.9

8. Kishnani Khushboo, Bhandari Saloni, Rathore Kamal Singh. A Briefing of a Global Crisis: Antibiotic Resistance. Asian J. Res. Pharm. Sci. 2020; 10(4):264-272. doi: 10.5958/2231-5659.2020.00047.8

9. Bezlon G., Shanmugha Sundhar D., Rinu Edwin R.E. Design and Stabilization of Natural Antibacterial Compound Allicin against Methicillin-Resistant Staphylococcus Aureus for Treatment as a Novel Antibiotic. Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 179-181.

10. CLSI Performance standards for antimicrobial susceptibility testing. 28th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute. (2018)

11. Chi square- Mathbeans Project. David, Jim Ryan. National Science Foundation DUE-9950473.

12. Step-by-step approach for development and implementation of hospital antibiotic policy and standard treatment guidelines- World Health Organization, Regional Office for South-East Asia, Indraprastha Estate, Mahatma Gandhi Marg, New Delhi-110 002, India

13. Sachin H. Rohane, Ashlesha G. Makwana. A Review on Hydrazone, the fascinating field of investigation in medicinal chemistry. Asian J. Research Chem. 2017; 10(4):417-430. doi: 10.5958/0974-4150.2017.00070.0

14. Saha A. (2019) Pattern of antimicrobial susceptibility of Klebsiella pneumoniae isolated from urinary samples in urinary track infection in a Tertiary Care Hospital, Kishanganj, and Bihar. MGM Medical College and LSK Hospital, Bihar, India. IJCMR. 6(12): L25-L28. doi.org/10.21276/ijcmr.2019.6.12.42

15. Wani R, Ahmed N, Irshad M, Ashraf M, Bashir A. (2019) Clinicobacteriological profile, antibiotic sensitivity patterns and mortality of neonatal sepsis in a tertiary care hospital in Kashmir, Jammu and Kashmir, India. 6(3):917-921 doi:10.18203/2349-3291.ijcp20191994

16. Dr. Bhupendra Kumar. (2019) Detection and antibiogram of extended spectrum beta lactamase (esbl) producing klebsiella pneumoniae in south–east Rajasthan, Government Medical College, Kota (Rajasthan). Wipmr. 5(9):130-137

17. Rizwan M, Akhtar M, Kalam A, Singh K, Hakim A. (2018) Escherichia coli and Klebsiella pneumoniae sensitivity/resistance pattern towards antimicrobial agents in primary and simple urinary tract infection patients visiting university hospital of jamia hamdard New Delhi, India. Epub . 68(7):415-420. doi: 10.1055/a-0576-0079

18. Aishwarya J. Ramalingam. History of Antibiotics and Evolution of Resistance. Research J. Pharm. and Tech. 8(12): Dec., 2015; Page 1719-1724. doi: 10.5958/0974-360X.2015.00309.1

19. Ashish Vyas, Karamjeet Singh, Gaurav Kumar. Prevalence and drug resistance among bacteria of urinary tract infections in females in Punjab, India. Research J. Pharm. and Tech. 2017; 10(2): 575-578. doi: 10.5958/0974-360X.2017.00114.7

20. Nanda Anima, Dhamodharan S, Nayak B. K. Antibiotic Resistance Pattern Exhibited by Esbl (Extended Spectrum β-Lactamases) in Multidrug Resistant Strains, Escherichia coli. Research J. Pharm. and Tech 2017; 10(11): 3705-3708. doi: 10.5958/0974-360X.2017.00672.2

21. Srinivasan S, Saldanha S, Abhyankar S, Modi N, Patil A, Vartak A. (2017) Antibiotic sensitivity pattern of Klebsiella species in burn wounds at Bai Jerbai Wadia hospital for children, Mumbai, India-a 21year study. Int J Burn Trauma. 7(5):64-71.

22. Rajesh KS, Honey VS, Ullas Prakash D’souza, Ragava Sharma, Bharath Raj KC. Study of Antibiotic resistance pattern in uropathogens at a Tertiary Care Hospital. Research J. Pharm. and Tech 2020; 13(3): 1253-1256. doi: 10.5958/0974-360X.2020.00231.0

23. Woldu M. (2016) Klebsiella pneumoniae and Its Growing Concern in Healthcare Settings. Addis Ababa University. Clin Exp Pharmacol. 5(6). doi: 10.4172/2161-1459.1000199

24. Niveditha, Srikanth, Rathai Rajagopalan, Shivamurthy M.C. A Retrospective Evaluation of Compliance in Various Surgical Departments with Respect to Surgical Antibiotic Prophylaxis in a Tertiary Care Hospital. Research J. Pharm. and Tech 6(7): July 2013; Page 749-752.

Received on 07.02.2021 Modified on 28.06.2021

Research J. Pharm. and Tech. 2022; 15(5):1989-1994.

DOI: 10.52711/0974-360X.2022.00330