INTRODUCTION:

The microbial confrontation, particularly opposition to the cephalosporins, is an increasingly public health difficulty internationally among pathogenic Enterobacteriaceae are specifically; Escherichia coli and Klebsiella pneumoniae¹. Compare to further countries, recurrent overdo of antibiotics, crowd of patients through elevated levels of sickness perception in comparatively diminutive, dedicated hospice areas in Indian subcontinent discriminating strength to constrain this contagion like antimicrobial struggle improvement amongst all most important microbial pathogens. Pathogenic bacteria obtain these confrontation possessions unpaid to achievement of group A 𝛽-lactamase to facilitate hydrolyze 𝛽-lactam sphere of cephalosporins^1,2.

The ever seeing as antibiotics discovery, numerous pathogens measured beaten, constant to extend confrontation to these drugs. Appearance of microorganisms resistant to the antibiotics internationally is a difficulty that has unlimited². Occurrence is prevalent in hospital mostly since the huge and frequently unsuitable utilize of antimicrobials. The bacteria’s capability to replace their environmental, genetically information and nosocomial pathogens enclose urbanized the identical confrontation mechanism, because the majority of conflict genes have their credible source in ecological microbes³. Resistant pathogens to most antibiotics have already been described, but in particular it is of great collision the opposition to β-lactams. Fighting to these antimicrobial agents is owing to the manufacture of β-lactamases, and genes indoctrination these enzymes establish global mostly in Gram -tive bacteria, commonly in Enterobacteriaceae⁴.

Among β-lactamases, AmpC-type -lactamases, comprehensive spectrum β-lactamases (ESBLs), and carbapenemases are enormous distress. In meticulous ESBLs the majority plentiful in Enterobacteriaceae, with extra 600 natural variants, popularity of CTX-M relations that became biggest greater than SHV and TEM throughout the primary decade of the 21^st century^5,6. In particular, ESBLs group of plasmid sencoded enzymes bestow confrontation to third invention cephalosporins⁷. ESBLs are alienated hooked on groups are 3 encoding of CTX-M, TEM and SHV genes⁸. CTX-M enzymes the majority common and supplementary into major five groups are phylogenetic based on genetic material sequences specifically, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25⁸. ESBL producing E. coli and K. pneumoniae are largest organisms in babyhood infections, they pretense important danger to human health⁹, these are listed surrounded by pathogens for which nearby are hardly any potentially successful drugs. About 57% of bloodstream infections are through ESBL produce Enterobacteriaceae, which are additional probable to consequence in passing away compared to the infections caused strains through a non ESBL producing strains¹⁰.

The present study was to investigate the different characterizations like clinical samples for the bacterial isolates, determine the ESBL frequency in clinical isolates of β-lactamase in Escherichia coli and Klebsiella spp.

MATERIALS AND METHODS:

Sample collection:

The clinical isolates of Escherichia coli and Klebsiella spp. were obtained 520 samples from patients in prakasam (DT), Coastal Andhra, Andhra Pradesh, India. The samples were collected in 2012 to 2015.

Identification of E. coli and Klebsiella Spp. in clinical lab:

Selected clinical organisms are a Gram -tive, facultative anaerobic bacteria, it’s in rod shape, while, these dangerous strains can origin urinary tract infections (UTI), gastroenteritis and neonatal meningitis, peritonitis, hemolytic-uremic syndrome, septicemia mastitis, and Gram -tive pneumonia.

Preparation of media:

Blood, MacConkey and HiCrome UTI agar method:

HiChrome UTI agar, MacConkey agar and base for Blood agar media were obtained as a dehydrated powder from HiMedia. All culture petri-plates were equipped in dwelling through succeeding manufacturer’s recommendation and instructions. Blood agar preparation (5%) defibrinated sheep blood was used. Plates prepared, stored at 2-8°C for a month. Each unmarked consignment of media was experienced for its capability to carry the enlargement of Escherechia coli and Klebsiella spp to ensure the quality of the media¹¹.

Culture of urine:

All the urine samples were inoculated aseptically on to HiChrome UTI agar, Blood agar and MacConkey agar media through using a calibrate cable loop of 28G with an interior diameter (3.26mm) holding urine (0.004ml). The petri-plates were incubated at 37°C aerobically and subsequent to suddenly incubation they were checkered for noteworthy bacteriuria as under by means of enumeration of colonies¹².

Presumptive identification:

Presumptive identification of the bacterial development was done on HiCrome UTI agar according to settlement morphology and colour as depict through the producer. Colony on the BA and MAC agar is also recognized subsequent colony individuality alongside every one of the uropathogens. The concluding discovery of isolates was completed using typical detection protocol such as motility test, gram’s staining, catalase test, oxidase test, coagulase test and other appropriate biochemical tests as suitable for the isolates¹³.

RESULT AND DISCUSSION:

The clinical isolates of E. coli and Klebsiella spp. were obtained 520 samples from patients in prakasam (DT), Coastal Andhra, Andhra Pradesh, India. Identified under clinical laboratory condition, for microbial culture Blood agar and MacConkey agar (Fig. 1) used and identified for selected pathogens. Different biochemical test was tested against microbes (Fig. 2), indole teste showed +ve and –ve, methyl red, VP test, citrate utilization and also motility tested (Fig. 3).

The urine sample examined for this study number of samples 520 screened, among them culture +ve are 250, β-lactamase 33, E. coli 15, Klebsiella 18 and others 217 (Fig. 4). Blood samples was collected and analyzed 250 samples are collected among them culture +ve are 25, β-lactamase 13, E. coli 6, Klebsiella 4 and others 15 (Fig. 5). There are 10 pleural fluids are evaluated among them culture +ve are 3, β-lactamase 2, E. coli didn’t observed, Klebsiella 2 and others 1 (Fig. 6). Synovial fluid samples are 10 screened and evaluated among them culture +ve are 3, β-lactamase 3, E. coli didn’t observed, Klebsiella 3 and others didn’t find out (Fig. 7).

In antibiotic sensitivity test, observed that sensitive to Piperacillin tazobactum and Sensitive to imepenam (Fig. 8, 9). The commonness of ESBL produce strains at Korea was greatly higher than that in Netherlands, anywhere roughly 2% of isolates produced the ESBLs^13,14. The clinical implication of these isolates, which were pervasive in Korea, is of enormous consequence, as clinicians are advised alongside utilize of extended spectrum cephamycins, cephalosporins and aztreonam^15,16.

Fig. 1: Microbial cutlure growth on MacConkey and Blood agar

a. E. coli, b. Klebsiella spp.

Fig. 2: Biochemical tests identification of E. coli IMVIC tests

Fig. 3. Biochemical Tests identification of Klebsiella

a. Indole test, b. Methyl red test, c. VP test,

d. Citrate utilization, e. Motility test

Fig. 4. Urine samples examined

Fig. 5. Blood samples examined

The identification of presumptive bacterial species is troubled, through matching with standard colours considerably elevated proportion of bacterial species were probable to be identified on HiCrome UTI agar method as contrasting to conservative culture system. lofty velocity of classification might be connected with effortlessness of recognition method through considering the dissimilar and perceivable colour of the colony fashioned through every bacterial species on medium of chromogenic agar and the conclusion are unswerving with intelligence available elsewhere^17,18nearby was important dissimilarity in speed of identification of presumptive particularly in E. coli, Klebsiella spp.,

Enterobacter spp. and Enterococci spp. on HiCrome UTI agar medium and method comparable results were also experimental through other investigators^19,20. In information, this discrepancy colour construction through personality²¹ of bacterial species is amongst the majority exhilarating features of chromogenic agar advocated to be worn as prime urine culture medium^22,23.

Fig. 6. Pleural fluid samples examined

Fig. 7: Synovial fluid samples examined

Fig. 8: Antibiotic sensitivity test

Fig. 9. Antibiotic Tests

a. Sensitive - Piperacillin tazobactum

b. Sensitive - Imepenam

CONCLUSION:

The urine samples collected and examined in different methods β-lactamase 3, E. coli didn’t observed, Klebsiella identified and characterized in different confirmatory analysis, use of these antibiotics is compulsory to decrease the extend of these challenging strains.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of PRIST Deemed University, Thanjavur for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Bradford P. A., Extended-spectrum 𝛽-lactamases in the 21^st century: characterization, epidemiology, and detection of this important resistance threat. Clinical Microbiology Reviews, 2001; 14 (4): 933–951.

2. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug resistant, extensively drug-resistant and pan drug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18: 268–81.

3. Yogesh Joshi, Punam Gurung, Siddhi Jain. Evaluation and Assessment of Drug Utilization in Patients of Urinary Tract Infections. Asian Journal of Pharmaceutical Research, 2018; 8(3): 167-169.

4. Paterson DL, Bonomo RA. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev.2005; 18: 657–86.

5. Jones RN, Guzman-Blanco M, Gales AC, Gallegos B, Castro AL, Martino MD, Vega S, Zurita J, Cepparulo M, Castanheira M. Susceptibility rates in Latin American nations: report from a regional resistance surveillance program (2011). Braz J Infect Dis.2013; 17: 672–81.

6. Dasari Venkateswarlu, Arjun P, Ramesh S. Phenotypic Detection of β-lactamase extended spectrum in Clinical Samples of Enterobacteriaceae. Waffen-und Kostumkunde Journal.; 2020: XI (IV): 111-119.

7. Akhilesh Gupta. Plazomicin: A step toward next generation aminoglycosides. Review. Asian Journal of Research in Pharmaceutical Sciences. 2017; 7(3): 173-180.

8. Lahlaoui H, Khalifa A, Haj B, Moussa MB. Epidemiology of Enterobacteriaceae producing CTX-M type extended spectrum β-lactamase (ESBL). Medecine et maladies infectieuses. 2014; 44(9): 400–4.

9. Bakshi R, Walia G, Shikha J. Prevalence of extended spectrum β-lactamases in multidrug resistant strains of gram negative bacilli. J Acad Indus Res. 2013; 1: 558–60.

10. Dasari Venkateswarlu, Arjun P, Ramesh S. Molecular characterization of β-lactamase producing bacterial pathogens of clinical samples. Waffen-und Kostumkunde Journal. 2020; XI (III): 182-193.

11. CDC. Antibiotic resistance threats in the United States, 2013. In: Centers for disease control and prevention USA.

12. Zain Baaity, Iyad Almahmoud, Atiea Khamis. Prevalence of Extended Spectrum β-Lactamases (ESBL) in E. coli at Al-Assad Teaching Hospital. Research Journal of Pharmacy and Technology. 2017: 10(7): 2433-2466.

13. Collee JG, Marr W. Specimen collection, culture containers and media. In: Collee JG, Fraser AG, Marmion BP, Simmons A. eds. Mackie & McCartney Practical Medical Microbiology, 14^th edition New York, Churchill Livingstone. 1996: 85-111.

14. Stobberingh, E. E., J. A. Hoogkanp-Korstanje, J. Arends, W. H. Goessens, M. R. Visser, and A. G. Buiting. 1999. Occurrence of extended-spectrum beta-lactamases (ESBL) in Dutch hospitals. Infection 27: 348–354.

15. Ashish Vyas, Karamjeet Singh, Gaurav Kumar. Prevalence and drug resistance among bacteria of urinary tract infections in females in Punjab, India. Research Journal of Pharmacy and Technology 2017: 10(2): 575-578.

16. Parveen R, Saha SK, Shamshuzzaman SM, Rashid AL, Chowdhury A, Muazzam N. Detection of Uropathogens by Using Chromogenic Media (Hicrome UTI agar), CLED agar and other Conventional Media. Faridpur Med Coll J. 2011; 6(1): 46-50.

17. Rani L, Pinnelli VBK, Hemavathi, Belwadi S, Rajendran R. Utility of HiChrome Urinary Tract Infection (UTI) Agar Medium for Identification of Uropathogens: A Comparative Study with Other Conventional Media. J Chem Pharm Res. 2012; 1(4): 95-105.

18. Shireen Rana, Siddesh Basawaraj Sirwar, Vijayaraghavan. Prevalence and Antibiogram of Extended Spectrum beta- Lactamase Producing Klebsiella pneumoniae and Proteus mirabilis in UTI. Research Journal of Pharmacy and Technology. 2015: 8(11): 1465-1468.

19. Ali I. Prevalence of multi-drug resistant uro-pathogenic Escherichia coli in Potohar region of Pakistan. Asian Pacific Journal of Tropical Biomedicine. 6 (1); 2016: 60-66.

20. Ashish Vyas, Karamjeet Singh, Gaurav Kumar. Prevalence and drug resistance among bacteria of urinary tract infections in females in Punjab, India. Research Journal of Pharmacy and Technology. 2017: 10(2): 1-4.

21. Dinesh Mishra, Anuja Mishra, Vaibhav Dubey, Karunakar Shukla. Transdermal Immunization: A Recent Tool for Immunization. Research Journal of Pharmacy and Technology. 2010: 3(4): 964-969.

22. Gowtham R, Gopinath P. Detection of Cell Surface Hydrophobicity among Uropathogenic isolates of Escherichia coli. Research Journal of Pharmacy and Technology, 2016;9(11): 1883-1885.

23. Shireen Rana, Siddesh Basawaraj Sirwar, Vijayaraghavan. Prevalence and Antibiogram of Extended Spectrum beta- Lactamase Producing Klebsiella pneumoniae and Proteus mirabilis in UTI. Research Journal of Pharmacy and Technology. 2015;8(11): 1465-1468.

Received on 21.03.2020 Modified on 09.07.2020

Research J. Pharm. and Tech. 2021; 14(6):2995-2998.

DOI: 10.52711/0974-360X.2021.00524