CORRELATION OF DRUG RESISTANT PATTERN WITH BIOFILM PRODUCTION BY USING MICROTITRE PLATE METHOD IN CLINICAL ISOLATES OF KLEBSIELLA PNEUMONIAE

CORRELATION OF DRUG RESISTANT PATTERN WITH BIOFILM PRODUCTION BY USING MICROTITRE PLATE METHOD IN CLINICAL ISOLATES OF KLEBSIELLA PNEUMONIAE

Kanimozhi Devanathan *, Joshy M. Easow, Umadevi Sivaraman and Rajkumar Chinnadurai

Department of Microbiology, Mahatma Gandhi Medical College & Research Institute, Pillayarkuppam, Pondicherry, India.

ABSTRACT: Health care associated infections (HAIs) are frequently caused by Klebsiella pneumoniae (K. pneumoniae) which has a high level of antibiotic resistance pattern and the ability to form biofilms. In this research work, 105 K. pneumoniae was isolated from both inpatients and outpatients between January 2021 and January 2022 at the tertiary care hospital viz, Mahatma Gandhi Medical College and Research Institute (MGMCRI), India. The K. pneumoniae isolates were further analysed using morphological analysis of the colonies, microscopic examination, and biochemical testing. The Kirby Bauer disk diffusion method and adhesion quantitative assays were utilized for testing antibiotic susceptibility and biofilm- producing capacity. K. pneumoniae isolates were mostly derived from the urine specimens (42.9%) and pus (19.0%). Most of  the K. pneumoniae were resistant to a wide range of antibiotics and is also well known to the ability to produce biofilm. This study shows that the biofilm producing K. pneumoniae has been a good resistant to Ceftriaxone/Cefotaxime (80.9%), Amikacin (78.7%), Piperacillin+ Tazobactam (79.8%), and Meropenem (79.8%). On the contrary among non- biofilm producing K. pneumoniae also showed good resistant to Gentamicin (87.5%), ciprofloxacin+ Norfloxacin (87.5%), Amikacin 15 (93.8%) and Cefoperazone+ sulbactam 12 (75.0%) of resistance.  Thus, in this study, it was shown that among 105 K. pneumoniae isolates were tested, there were 89 (84.76%) isolates were found to be biofilm producer and 16 (15.2%) isolates were non-biofilm producers. Among biofilm producers, it was shown that there were 36(34.28%) isolates as strong, 42 (40%) isolates as moderate, and 11 (10.47%) isolates identified as weak biofilm producers. The majority of the K. pneumoniae isolates showed resistance to a variety of antibiotics and were capable of producing biofilms.

Keywords: Klebsiella pneumoniae, Biofilm, Antibiotics, Clinical and laboratory standards institute

INTRODUCTION: Communities of bacteria known as biofilms form when they stick to a surface and create an extracellular polymeric substance (EPS) matrix.

Because the EPS matrix shields the micro-organisms from environmental stressors like antibiotic therapy, biofilm associated illnesses are more challenging to cure ¹. Biofilms prevent the entry of antibiotics, inhibit the growth of bacteria, promote the development of persisted cells, and allow for genetic exchange ^{2, 3}. Bacterial resistance to antibiotics has been steadily rising as a result of the extensive usage  of  antibiotics  worldwide. The frequency with which functional genes are acquired through mobile components has increased, leading to a rise in drug resistance and virulence in K. pneumoniae ⁴. K. pneumoniae is a gram-negative bacillus that can cause urinary tract infections (UTIs), pneumonia, bacteremia, and liver abscesses in young people and other healthy individuals ⁵. The two  pathogenic  varieties  of  K. pneumoniae that  are now  known  to  exist  are   Ckp  and hvkp. Because of their genes for antibiotic resistance, ckp and hvkp are more challenging to treat. Furthermore, K. pneumoniae has a strong propensity to build biofilms, which exacerbates the already difficult illness ⁶.

K. pneumoniae is linked to a significant amount of ventilator-acquired pneumonia and hospital acquired pneumonia, which usually affects susceptible patients in intensive care units ⁷.

The K. pneumoniae strain is frequently colonized in hospitalized patients and is more prevalent in immunocompromised people, such as diabetics, the elderly, and children ⁸. Hospital infections are commonly associated with highly biofilm- forming bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae ⁹. The misuse of antibiotics has led to treatment challenges for K. pneumoniae and reduced alternatives for the efficient control of this bacterial infection ^10-11. Treatment for infections resulting from K. pneumoniae strains that develop biofilms is more challenging than for other  strains ¹².

The present study was designed to evaluate biofilm development and drug resistant pattern among K. pneumoniae clinical isolates.

MATERIALS AND METHODS:

Bacterial Isolation and Identification: In the present study, 105 K. pneumoniae   strains were isolated at Mahatma Gandhi Medical College & Hospital in Jan 2021 to Jan 2022. The K. pneumoniae strains were recovered from blood, sputum, surgical wound swabs, and urine samples of hospitalized patients (Outpatients & Inpatients).

Definitive identification of isolates was confirmed using colony morphology, gram staining, manual biochemical testing ¹³. After recognition, the K. pneumoniae strains were stored in tryptic soy broth (TSB) (Merck Co., Germany) with 10% glycerol at -80 degree Celsius. All isolates were freshly subculture on brain-heart infusion (BHI) agar prior to every analysis.

Antibiotic Susceptibility Tests of K. pneumoniae: Antibiotic susceptibility tests were conducted by using the Kirby Bauer disc diffusion method. According to the Clinical and Laboratory standards Institute 2020 ¹⁴, K. pneumoniae were categorised as resistant, intermediate, or susceptible. To this study, correlate biofilm production under resistant pattern ¹⁴.

Biofilm Formation Assay: The Biofilm formation test was conducted using a quantitative adherence assay. For every isolate, an overnight culture in Trypticase Soy Broth (TSB) at 37 degrees Celsius was carried out. Following this, 198 microlitre of TSB was enclosed in 96 well flat bottom polystyrene microtitre plates that were sterile and inoculated with 2 microliter of cell suspension. Each test also included negative control wells containing 200 microlitre of uninoculated TSB. For twenty-four hours, incubation was maintained at 37 degrees Celsius. The wells were gently rinsed three times with 200 microlitre of phosphate buffered saline (PBS). The wells were dried with the bottoms facing up. Mass of biofilm-stained utilising 50 microlitres of 0.1% crystal violet.

The wells were gently cleaned three times using 200 microlitres of distilled water, and   then they were dried inverted. Ultimately, the stained biofilm mass was solubilized by dissolving the wells in 200 microliters 5% isopropanol. An Optical Density (OD) measurement was performed at 570 nm using a microplate reader. By examining each isolate or negative control for eight to twelve wells, the mean OD was calculated. Optical Density Cut-off (ODC) was assigned as an average OD of negative controls + (3* Standard Deviation (SD) of negative controls). Isolate with OD ≤ ODC categorized as non-Biofilm producer. Meanwhile, the isolate was categorized as biofilm producer consisting of weak biofilm producer if 2*ODC < OD ≤ 4*ODC; moderate 2* ODC* OD ≤ 4*ODC; and strong biofilm producer if OD > 4* ODC.¹⁵.

Statistical Analysis: The collected data from the K. pneumoniae isolates and their biofilm production and antibiotic drug   resistant pattern were further analysed and compared using the chi-square test and Fischer’s exact test. The statistical analyses had been performed using SPSS (Statistical Package for the Social Sciences) statistics software for Windows, version 15 (which is available from SPSS (statistical package for the social sciences) Inc., Chicago, USA). The p - value ≤0.05 had been taken as a statistically significant.

Ethics Statement: The study was approved by the IHEC (MGMCRI/RAC/02/2020/XX/IHEC/137) of the MGMCRI of Sri Balaji Vidyapeeth University, Pondicherry, India.

RESULTS: Demographics of patients from which K. pneumoniae were isolated is given in the following table: Table 1 Demographics.

TABLE 1: DEMOGRAPHICS

TABLE 2: ANTIBIOTIC RESISTANT PATTERN AND BIOFILM PRODUCING CAPACITY OF K. PNEUMONIAE CLINICAL ISOLATES

* Indicates statistically significant

TABLE 3: ANTIBIOTIC DRUG RESISTANT PATTERN WITH BIOFILM AND NON- BIOFILM PRODUCERS

* -Indicates  statistically  significant.

FIG. 1: BIOFILM PRODUCTION AND BIOCHEMICAL TESTS. In the Fig. 1 the following are displayed as: (A) shows MacConkey Agar produces dome shaped, smooth, Lactose fermenting colonies (B) shows Mueller Hinton Agar produces the antibiotic Susceptibility pattern (C) shows the biochemical tests for K. pneumoniae (D) shows the biochemical tests for subspecies K. pneumoniae

FIG. 2: BIOFILM CHARACTERIZATION FORMATION FROM WEAK TO MODERATE AND TO STRONG ADHERENCES

Characteristics of Clinical Samples: From Jan 2021 to Jan 2022, 105 K. pneumoniae isolates were examined from total clinical bacterial isolates at Mahatma Gandhi Medical College & hospital, India. K. pneumoniae isolates were isolated from 59 male (56.2%) and 46 (43.8%) female patients Table 1. Most of K. pneumoniae were isolated from patients aged 41- 60 years old. K. pneumoniae samples were mostly isolated from urine specimens (42.9%) and pus (19.0%).

Antibiotic Resistant Pattern: Most of K. pneumoniae were resistant to a wide range of antibiotics. Among biofilm producer isolates, K. pneumoniae had only a good resistant to ceftriaxone/cefotaxime (80.9%), Amikacin (78.7%), and Piperacillin+ Tazobactam (79.8%), Meropenem (79.8%). In contrast, among non- biofilm producer isolates, K. pneumoniae showed good resistant to Gentamicin (87.5%), ciprofloxacin+ Norfloxacin (87.5%), Amikacin 15 (93.8%) and Cefoperazone+ sulbactam 12 (75.0%) of resistance respectively Table 2.

Biofilm Formation Detection: In this study, among the 105 K. pneumoniae isolates tested, there were 89 (84.76%) isolates as biofilm producer and 16 (15.2%) isolates that were not biofilm producers. Among biofilm producers, there were 36(34.28%) isolates as strong, 42 (40%) isolates as moderate, and 11 (10.47%) isolates identified as weak biofilm producers Table 2.

Association between type of Patients and Biofilm Producing Capacity: In this study, inpatients show more biofilm production than outpatients, because of immunocompromised and long term of hospitalization. In subspecies, K. pneumoniae subspeciespneumoniae shows more biofilm production than K. pneumoniae subspecies ozaenae.

DISCUSSION: The percentage of   K. pneumoniae isolates in the present study was 17.36% of all clinical bacterial isolates from January 2021 to January 2022. Considering that K. pneumoniae is one of the major global sources of MDR infections, this proportion be cause for significant anxiety. These bacteria are frequently linked to HAIs and extremely contagious outbreaks that have longer hospital stays and high fatality rates, all of which drive up healthcare expenses ¹⁶. Male patients provided the majority of the K. pneumoniae isolates used in this investigation. This outcome was consistent with the findings of Osagie et al ¹⁷ who obtained samples from five primary healthcare facilities in Nigeria and said that males were more likely than females to be infected with K. pneumoniae. Additionally, Akter et al ¹⁸ noted that male patients were more likely than female patients to be infected with Klebsiella. Gender and the occurrence of K. pneumoniae were linked to unhealthy lifestyle choices, such as alcohol consumption and smoking. Nevertheless, those investigations did not disclose any statistically significant differences between male and female subjects ¹⁷.

The majority of the K. pneumoniae used in this investigation came from patients between the ages of 41-60 years of age. This conclusion deviates from a prior study that found the majority of K. pneumoniae isolates were from patients who were older than 70. However, a different recent study revealed that individuals between the ages of 40 and 65 accounted for a higher proportion of K. pneumoniae isolates ¹⁹. The variations in the age distribution of patients may be associated with the immune system response strength, which is predicted to decrease with ageing.  Since patients under 40 often have stronger immune systems, K. pneumoniae is under more pressure to combat the host’s immunity. On the other hand, because concurrent illnesses become increasingly common as people age, they are more likely to contract K. pneumoniae. K. pneumoniae is linked to a significant percentage of ventilator-acquired pneumonia and hospital-acquired pneumonia, which usually affects susceptible patients in intensive care units.

Urine specimens were the primary source of K. pneumoniae isolates. Ashurst and Dawson ¹⁵ highlighted that K. pneumoniae usually colonized the gastrointestinal tract and oropharynx mucosal surfaces in humans. Because of this, K. pneumoniae   is thought to be the most frequent cause of hospital acquired pneumonia in the US. In contrast, wang et al. ²⁰ found that the Republic of China’s dominant site of K. pneumoniae infection was the respiratory system.

Similar findings were made by Seifi et al. ²¹ who obtained samples from two hospitals in Tehran. They found that the proportion of K. pneumoniae in the urine, surgical wounds, sputum and blood were 61.7%, 18.1%, 11.7% and 8.5% respectively.

The majority of K. pneumoniae was resistant to several antibiotics; the most effective combination against K. pneumoniae was Cefoperazone+ sulbactam, followed by Fosfomycin. The least combination was cotrimoxazole, ceftriaxone+ cefotaxime. The research done by Madahiah et al. ²² which indicated that the isolates of K. pneumoniae were 100% responsive to amikacin and 100% resistant to ampicillin, lends evidence to this account. The resistant percentages for ciprofloxacin and amoxicillin - clavulanic acid were 38.75% and 36.69%, respectively. This result is comparable to that of Cepas et al. ²³ who found that 40% of K. pneumoniae strains were resistant to amoxicillin - clavulanic acid, as well as Ciprofloxacin.

The most important determinant in the development of antibiotic resistance is antibiotic exposure. Numerous causes contribute to the rise in antibiotic resistance, including the use of antibiotics in the population, in hospitals, in agriculture, and in the environment. Because they are frequently purchased, antibiotics are over prescribed. The key underlying cause responsible for the widespread transmission of nosocomial infections that are resistant to antibiotics and are difficult to cure in the health service context is most likely the extensive and continuous use of antibiotics ²⁴.

In this study, among the 105 K. pneumoniae isolates tested, there were 89 (84.76%) isolates as biofilm producer and 16 (15.2%) isolates that were not biofilm producers. Among biofilm producers, there were 36 (34.28%) isolates as strong, 42 (40%) isolates as moderate, and 11 (10.47%) isolates identified as weak biofilm producers. Out of 110 K. pneumoniae studied, 70 isolates were found to be strong or moderate biofilm producers and 40 isolates to be weak biofilm producers, according to a similar study performed by Hassan et al. ²⁵ A different study by Cepas et al.²³ found that 37.6% of K. pneumoniae strains were capable of producing biofilm. According to Yang and Zhang, 62.5 % of the K. pneumoniae isolated from blood, sputum, urine, and wound swabs were biofilm producers. According to Seifi et al. ²¹ 93.6% of K. pneumoniae were biofilm producers, while the remaining 6.4% were not. Of the strains that produced biofilm, 33% were classified as strong producers, 52.1% as moderate producers, and 8.5% as weak biofilm producers. In a related investigation, Nirwati et al. ²⁶ reported that biofilm production accounted for 85.63% of the K. pneumoniae strains identified from an Indonesian hospital ²⁶. Every isolate had a different capacity to form biofilms because, in simple terms, a number of factors influence this capacity, including the physicochemical characteristics of K. pneumoniae, the physical interactions between constituents, the type of surface to which the biofilm adheres, temperature, pH, and so on. The ability of K. pneumoniae to form biofilms and exhibit extensive drug resistance (XDR) has been shown by Vuotto et al. ¹³ to be correlated with a profile of antibiotic resistance.

According to this study, K. pneumoniae, a biofilm - producing bacteria, had higher levels of antibiotic resistance than non- biofilm producing bacteria. Numerous studies have reported on this conclusion. According to a study by Saha et al. ²⁷ all of the isolates that produced biofilms showed more resistant pattern than isolates that produced not an isolated event; however, the defensive mechanisms found in biofilms are distinct from those that cause conventional antibiotic resistance.

The protective layer of the sticky biomaterial in biofilms, which prevents antibiotics from penetrating, the adaptive reactions to stress, and the development of persistent cells are thought to form a multi-layered defence that makes eradication more difficult, particularly when paired with the bacteria’s resistance. Although it seems that bacterial biofilm formation and antibiotic resistance are major contributing factors  to  the  worldwide spread  of K. pneumoniae, a different study by Alcantar  Curie et  al.²⁸ suggested that  the  precise nature  of  this association  remains  unclear. De Campos et al. ²⁹ corroborate this conclusion by stating that there was no discernible correlation between the ability to create biofilms and clonal types of MDR bacteria.

Numerous studies have demonstrated that, in the majority of circumstances, a single antibiotic treatment is insufficient to eradicate biofilm forming infections. Consequently, for the successful treatment of infections linked to biofilms, controlling infections with currently available antibiotics and assessing the results have become crucial and necessary tasks. Due to their strong antibiofilm activity both inside and outside of living organisms, a number of studies suggest combining antibiotic therapy with macrolides like azithromycin, clarithromycin, and erythromycin as the primary antibiotics for biofilm associated infections caused by gram negative bacteria. Wu et al.³⁰proposed that, in addition to the administration of combined antibiotics, removal of infected foreign bodies and the source of infection as well as the quorum sensing inhibitors or biofilm dispersal agents would result in a more effective management for biofilm infections, taking into account the currently known environmental and bioecological aspects.

Our investigation demonstrated the issues with antibiotic - resistant bacteria in hospital settings, which have previously been shown in another investigations. Taking into account the quantity and quality of antibiotic prescriptions expressed in the majority of hospitals, this scenario is concerning. In 2012, hospital surveillance in Surabaya revealed that 30.6% of antibiotics were given without indications validated by susceptibility testing. As a result, prescribing antibiotics continues to be a difficult task   everywhere, even in Indonesia. According to Vander Meer, prescription antibiotic guidelines are not the best in Netherlands, a country with low rates of antibiotic resistance in bacteria and utilisation of antibiotics. According to their research, 15% of antibiotic therapy in wards dedicated to internal medicine and surgery was deemed adequate.

Penicillin, Cephalosporins (including third generation cephalosporins), and   aztreonam are among the drugs whose   resistance is mediated by ESBLs. In order to determine which gram-negative bacteria, create biofilms, Dumaru et al. conducted a study in which they also determined the antibiograms of these bacteria and detected the development of metallo-beta-lactamases (MBLs) and EBLs, A statistically significant correlation has been detected between the formation of biofilm and MBL. But there was no significant association between ESBL and biofilm formation. Lack of knowledge about infections and the administration of antibiotics is the main factor contributing to the incorrect prescription of antibiotics. A crucial phase in the prescription of antibiotics is modifying the first course of treatment on the basis of the clinical microbiology findings. Testing for antibiotic susceptibility is therefore necessary. Another crucial step is gathering   clinical samples prior to giving antibiotics. Numerous medical professionals who provide antibiotic prescriptions are unaware of the potential effects of their improper recommendations on the emergence of bacterial resistance. The selection pressure on the pathogenic bacteria entrusted with hospital-based infections will be reduced by modifying the first antimicrobial therapy in accordance with the clinical microbiology results. Therefore, based on the most recent microbiological data, it is crucial that every hospital implement an antibiotic guideline or stewardship programme for all pharmacists and clinicians. To combat the fast spread of antibiotic resistant bacteria, ongoing efforts in hospital surveillance, infection control, and clinical audits are required in addition to these recommendations.

CONCLUSIONS: The majority of the K. pneumoniae isolates have shown resistance to a variety of antibiotics and are capable of producing biofilms.  In our study, we compared the biofilm producing capacity with subspecies of K. pneumoniae. The  development  of  biofilms  is a crucial step in the  cause  and  effect  of  Klebsiella pneumoniae  disease, as  it  increases resistance  to a environmental  stresses  and  acts  as  a  reservoir for  the  spread  of  the  bacteria and  further  gene exchange  with  antibiotic  drugs.  The  production of  biofilms  by  bacterial  competitors  in  their colonizing  environment  is  facilitated by  many virulence factors. Numerous  of   these  compounds have  been  investigated  as  potential  vaccination candidates  or  as  targets  for  novel  antibacterial medicines.

ACKNOWLEDGEMENT: The author (Kanimozhi Devanathan) acknowledges ICMR, India for providing Senior Research Fellowship (file no. OMI- Fellowship /2/2022- ECD (Project ID: 2021- 15257). The author would also acknowledge the MVK Iyer Fellowship from Sri Balaji Vidyapeeth (Deemed to be University) for the research work.

Financial Support and Sponsorship: This study was supported by M.V.K Iyer fellowship from Sri Balaji Vidyapeeth (Deemed to be university).

CONFLICTS OF INTEREST: The author declares no competing of interest.

REFERENCES:

1. Guerra MES, Destr OG, Vieira B, Lima AS, Ferraz LFC, Hakansson AP, Darrieux M and Converso TR: Klebsiella  pneumoniae biofilms and their role in Disease  pathogenesis. Front Cell Infect Microbiol 2022; 12: 555.
2. Wang G, Zhao G, Chao X, Xie L and Wang H: The characteristics of virulence, biofilm and antibiotic resistance of Klebsiella pneumoniae. Int J Environ Res Public health 2020; 17: 6278.
3. Mahto KU and Das S: Bacterial biofilm and extracellular polymeric substances in the moving red biofilm reactor for wastewater treatment: A review Bioresour Technol 2022; 345: 126476.
4. Cai M, Pu B, Wang Y, LV L, Jiang C and Fu X:  A plasmid  with  conserved phage  genes  helps  Klebsiella  pneumoniae  defend  against  the  invasion  of  transferable  DNA  elements  at  the  cost  of  reduced  virulence. Front Microbiol 2022; 13: 827545.
5. Ahmadi  Z, Noormohammadi  Z and Ranjbar  R: Prevalence  of  tetracycline  resistance  genes  test (A,B,C,39)  in  Klebsiella  pneumoniae  isolated  from  Tehran, Iran. Iranian J Med Microbiol 2022; 16(2): 141-147.
6. Cusumano JA, Caffrey AR and Daffinee KE: Weak biofilm formation among carbapenem- resistant Klebsiella pneumoniae. Diagn Microbiol Infect Dis 2019; 95(4): 114877.
7. Assoni L, Girardello R, Converso TR and Darrieux M: Current stage in the development of Klebsiella pneumoniae vaccines. Infect Dis Ther 2021; 70: 2157-2175.
8. Ripabelli G, Sammarco ML, Salzo A, Scutella M, Felice V and Tamburro M: New Delhi  metallo- beta- lactamase (NDM-1) producing  Klebsiella pneumoniae  of  sequence  type  ST11: first  identification in  a  hospital  of  central  Italy’’ Letters  in Applied  Microbiology 2020; 71(6): 652-659.
9. Khan F, Pham DTN and Oloketuyi SF: Antibiotics application strategies to control biofilm formation in pathogenic bacteria. Curr Pharm Biotechnol 2020; 21(40): 270-286.
10. Ding Y, Wang  H, pu   S, Huang  S and  Niu  S: Resistance  trends  of  Klebsiella  pneumoniae  causing  urinary  tract  infections  in  chongging, 2011-2019. Infection and Drug Resistance 2021; 14: 475-481.
11. Tella Di, Tamburro  M,  Guerizio G, Fanelli  I, Sammarco  ML and Ripabelli  G: Molecular  epidemiological  insights  into  colistin - resistant  and  carbapenemases  producing  clinical  Klebsiella  pneumoniae Isolates,. Infection and Drug Resistance 2019; 12: 3783-3795.
12. Shadkam S, Goli  HR,  Mirzaei  B, Gholami M and Ahanjan M: Correlation  between antimicrobial  resistance and biofilm formation capability among Klebsiella pneumoniae, strains  isolated  from hospitalized  patients in Iran’’, Annala of Clinical Microbiology and  Antimicrobials 2021; 20(1): 13-17.
13. Vuotto C, Longo F, Balice MP, Donelli G and Varaldo PE: Antibiotic  resistance  related  to biofilm formation in Klebsiella  pneumoniae. Pathogens 2014; 3(3): 743-58.
14. Clinical and Laboratory Standards Institute (CLSI) Performance standards for Antimicrobial Susceptibility Testing: Twenty- Fifth informational Supplement. CLSI document M100-S25 2020.
15. Ashurst JV and Dawson A: Klebsiella pneumonia. In: Stat Pearls [Internet]. Treasure Island (FL) 2019.
16. Kidd TJ, Mills G, Sa- Pessoa J, Dumigan A, Frank CG, Insua JL, Ingram R, Hobley L and Bengoechea JA: A Klebsiella pneumoniae antibiotic resistance mechanism that subdues host defences and promotes virulence. EMBO Mol Med 2017; 9(4): 430-47.
17. Osagie RN, Eyaufe AA, Iserheinrhein O, Okodua M, Onuabonah F and Daibo OO: Antibiotic Susceptibility profile of Klebsiella pneumoniae isolated from sputum samples amongst hospitalized adults in parts of Edo state, South-South. Nigeria.  Merit Res J 2017; 5(8): 378-83.
18. Akter J, Chowdhury AMMA and Forkan MAI: Study on prevalence and antibiotic resistance pattern of Klebsiella isolated from clinical samples in southeast region of Bangladesh. American J Drug Discovery and Development 2014; 4: 73-9.
19. Zheng JX, Lin ZW, Chen C, Chen Z, Lin FJ, Wu Y, Yang SY, Sun X, Yao WM, Li DY, Yu ZH, Jin JL, Qu D and Deng QW: Biofilm Formation in Klebsiella pneumoniae bacteraemia strains was found to be associated with CC23 and the presence of wcaG. Front Cell Infect Microbiol 2018; 8: 21.
20. Wang C, Yuan Z, Huang W, Yan L, Tang J and Liu CW: Epidemiologic analysis and control strategy of Klebsiella pneumoniae infection in intensive care units in a teaching hospital   of people’s Republic of China. Infect Drug Resist 2019; 12: 391-8.
21. Seifi K, Kazemian H, Heidari H, Rezagholizadeh F, Saee Y, Shirvani F and Houri H: Evaluation of biofilm formation among Klebsiella pneumoniae isolates and molecular characterization by ERIC- PCR. Jundishapur J Microbiol 2016; 9(1): 30682.
22. Madahiah BM, Noor US, Abdul S and Dan Ali AQ: Klebsiella pneumoniae urinary tract infections Associated with long term characterization and spinal cord Injuries. J Med Sci 2002; 2: 227-9.
23. Cepas V, Lopez Y, Munoz E, Rolo D, Ardanuy C, Marti S, Xercavins M, Horcajada JP, Bosch J and Soto SM: Relationship between biofilm formation and antimicrobial resistance in gram- negative bacteria. Microb Drug Resist 2019; 25(1): 72-9.
24. Prestinaci F, Pezzotti P and Pantosti A: Antimicrobial resistance: a global multifaceted phenomenon. Pathog Glob Health 2015; 109(7): 309-18.
25. Hassan A, Usman J, Kaleem F, Omair M, Khalid A and Iqbal M: Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis 2011; 15(4): 305-11.
26. Nirwati H, Sinanjung K and Fahrunissa F: Biofilm formation and antibiotic resistance of Klebsiella pneumoniae isolated from clinical samples in a tertiary care hospital, Klaten, Indonesia. BMC Proc 2019; 13.
27. Saha A, Devi KM, Damrolien S and Devi KS:  Krossnunpuii, Sharma KT.  Biofilm production and its correlation with antibiotic resistance pattern among clinical isolates of   Pseudomonas aeruginosa in a tertiary care hospital in north-East India.  Int J Adv Med 2018; 5(4): 964-8.
28. Alcantar- Curiel MD, Ledezma- Escalante  CA,  Jarillo- Quijada  MD, Gayosso-Vázquez C, Morfin-Otero R, Rodriquez-Noriego E, Cedillo-Remirez ML, Santos-Preciadio JI and Giron JA   Al: Association of Antibiotic Resistance, Cell Adherence, and Biofilm Production with the Endemicity of Nosocomial Klebsiella pneumoniae, Biomed Res Int 2018; 7012958.
29. Campos PAd, Royer S, Batistao DWdF, Araujo BF, Queiroz LL, Britto CSd, Gontijo- Filho PP and Ribas RM:  Multidrug Resistance Related to Biofilm Formation in Acinetobacter baumannii and Klebsiella pneumoniae Clinical Strains from Different Pulsotypes. Curr Microbiol 2016; 72(5): 617-27
30. Wu H, Moser C, Wang HZ, Hoiby N and Song ZJ: Strategies for combating bacterial biofilm infections. Int J Oral Sci 2015; 7(1): 1-7.
    

    ---

    How to cite this article:

    Devanathan K, Sivaraman U, Chinnadurai R and Easow JM: Correlation of drug resistant pattern with biofilm production by using microtitre plate method in clinical isolates of Klebsiella pneumoniae. Int J Pharm Sci & Res 2024; 15(9): 2807-15. doi: 10.13040/IJPSR.0975-8232.15(9).2807-15.

    ---

    All © 2024 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.