Isolation, Identification, Speciation and Antibiotic Susceptibility Pattern of Klebsiella Species among Various Clinical Samples at Tertiary Care Hospital

To isolate, identify and speciate Klebsiella from various clinical samples and their Antimicrobial susceptibility pattern at tertiary care hospital, Chennai. Out of this 76 ESBL producing Klebsiella pneumoniae, 64(84%) were positive by Double Disc Synergy Test (DDST), 71(93%) by phenotypic confirmatory disc diffusion test (PCDDT) and 69(90.7%) by Etest strip method. Sensitivity was found to be maximum (93%) by PCDDT method, compared to other phenotypic methods such as DDST and Etest strip method. The Present study also highlights the need for the continued monitoring of Antimicrobial susceptibility patterns of important bacterial pathogens, so that rational antibiotic policies can be formulated.


INTRODUCTION
Enterobacteriaceae are a group of gram negative bacilli most commonly colonizing the gastrointestinal tract causing various human infections. Few genera are recognized as environmental habitats or colonizers of animals. The term "Enterobacteriaceae" was first put

Original Research Article
forward by Rahn in 1937, which comprises organisms that share subsequent properties. They are gram negative, rod shaped bacilli or coccobacilli, catalase positive, oxidase negative, motile by peritrich flagella or non-motile; capsulated or non-capsulated; mostly nonsporing; and are facultative anaerobes.
The Enterobacteriaceae family confers resistance to frequently used and prescribed antibiotics that share common structures and mechanism of action, inhibition of synthesis of bacterial peptidoglycan of the cell wall. β-lactam antibiotics groups include penicillin, its derivatives and cephalosporins. Enterobacteriaceae are capable of producing beta-lactamase enzymes that hydrolyzes penicillin and cephalosporin and monobactams but not carbapenems. The beta-lactamase enzymes produced by various bacteria cleave the amide b and in the β-lactam ring, thereby rendering β-lactam antibiotics ineffective. These Extended spectrum beta-lactamases producing strains are treated using beta-lactamase inhibitors.
The Enterobacteriaceae family embraces seven tribes, of which Klebsiella fits into the fifth tribe. The genus Klebsiella is the second most common organism among Enterobacteriaceae family which is a gram negative, lactose fermenting, non-motile, rod shaped, and facultative anaerobic bacilli. Klebsiella spp. cause 3 -8% of all nosocomial infections and are widely recognized as important pathogens in genitourinary infections, bronchopneumonia, wound, soft tissue and blood stream infections (BSI) [1]. The most common opp ortunistic nosocomial Klebsiella infections are caused by K. pneumoniae and K. oxytoca [2].
Depending on the site of infections, klebsiella species were isolated from different a clinical sample that causes various infections. K. pneumoniae subspecies pneumoniae is the most common hospital acquired pathogens causing urinary tract infection, wound infections, meningitis, abscesses, lung infections and stated to cause sepsis and death of newborn in the intensive care unit [3]. They account for about 75% to 80% of all Klebsiella species [4]. The rarely encountered species are K. pneumoniae subspecies ozaenae and K. pneumoniae subspecies rhinoscleromatis, which are still retained as separate species producing specific diseases [2].

Methodology
This prospective study was conducted in Sree Balaji Medical College and Hospital, Chennai. The period of study was from (January 2017 to December 2017). Various clinical samples such as sputum, urine, wound swab/pus and blood received in the central diagnostic laboratory from General surgical wards were processed in microbiology section. Samples were then subjected to microscopy, bacteriological culture, biochemical identification and Antimicrobial susceptibility testing [11]. The specimens of pus and wound exudates were collected in disposable sterile swab stick/sterile syringe.

Collection of specimen
Coughed out sputum was collected in sterile container

RESULTS
The

Fig. 13. E Test strip method
Two sides strip containing ceftazidime and ceftazidime clavulanic acid; The ratio of ceftazidime with and without clavulanic acid is ≥ 8 indicating ESBL producer A B C  [12] showed 53% sensitivity to Nitrofurantoin in their study [17,18].
This study shows, out of total 189 Klebsiella isolates, 76(49%) of 155 K. pneumoniae alone were resistant and out of 34 Klebsiella oxytocanone (0%) showed resistance to cefotaxime and ceftazidime by disc diffusion method. A study done by sadaf guldin et al.
showed 44.5% of Klebsiella pneumoniae were resistance to both cefotaxime and ceftazidime which was similar to the present study [19]. 76(49%) of Klebsiella pneumoniae which was resistant to both ceftazidime and cefotaxime were further tested for ESBL production by various phenotypic methods. Out of 76 ESBL positive Klebsiella pneumoniae(by screening method), 64(84%) were positive by DDST, 71 (93%) by PCDDT and 69(90.7%) by Etest strip method.Sensitivity were found to be maximum (93%) by PCDDT method [20]. This study shows out of 76 ESBL screening positive isolates of Klebsiella pneumoniae, same 64 isolates were found to be ESBL producer by DDST, PCDDT and Etest, 5isolates which werepositive by PCDDT and Etest were not detected by DDST and 2 isolate that were positive by PCDDT was not detected by both DDST and Etest [21].
A study by Khalid et al. isolated 46% ESBL producers in Klebsiella pneumoniae by DDST method, whereas 51% were detected by PCDDT method which shows PCDDT is more sensitive than DDST [12]. A study done by Singh et al. [22] showed sensitivity to DDST to be (89%), Etest for cefotaxime and ceftazidime 83.6% and 88.52% respectively. PCDDT was most sensitive (93.44%) in their study also.A study done by Ashok kumar et al. [23] reported 47% of ESBL producing Klebsiella pneumoniae by both DDST and PCDDT [24,25,22]. Dalela et al. [26] inhis study detected ESBL in 90% isolates by DDST and 100% by PCDDT method. The ability of PCDDT method to detect ESBL is satisfactory with sensitivity of 93% in his study. A study by singh et al. [22] demonstrated that PCDDT achieved the highest sensitivity 93.44% among all the phenotypic tests applied. All the above studies correlated well with this study [23].
The PCDDT test was verified with DDST and it was found to be more sensitive and alternative to DDST for the detection of ESBL producers. The DDST lacks sensitivity because of the problem in optimal disc space and storage of Clavulanicacid containing discs. According to CLSI guidelines, PCDDT is recommended for confirmatory method to detect ESBL producing Klebsiella species [26,27,11]. The limitation of this study was that PCR were not performed due to cost factor. Hence, we performed various phenotypic methods for ESBL detection and compared the sensitivity of each with reference to disc diffusion screening method.

CONCLUSION
In conclusion, this study shows klebsiella is most commonly encountered in wound/pus samples, among which K.pneumoniae isolates were maximum than K. oxytoca. Imipenem and meropenem is the drug of choice for the above Klebsiella isolates and also for ESBL producing Klebsiella pneumoniae among hospitalized patients. High rates of resistance to most classes of antimicrobials, except carbapenems were reported in our study. The first line drugs used in the treatment of infections caused by a member of the family Enterobacteriaceae is Cephalosporins. The use of third generation cephalosporins extensively has resulted in the increased prevalence of extended spectrum beta-lactamases (ESBLs) and plasmid mediated AmpC among Klebsiella isolates. ESBL production is frequently accompanied by drug resistance to commonly used 3 rd and 4th generation cephalosporins and aztreonem making therapeutic options limited, resulting in need for new measures for management of Klebsiella species. Capsular polysaccharides (CPS) have been the obvious vaccine candidates due to its high immunogenic and nontoxic properties. A serious disadvantage of a Klebsiella CPS vaccine is the great number of K antigens different antigens). However, in a study of the incidence of the capsule types among bacteremia Klebsiella isolates, Cryz et al. [26] observed that only 25 serotypes made up 70% of all bacteremia strains. Based on their epidemiological findings, they formulated a 24 -valent Klebsiella CPS vaccine that subsequently was proven to be safe and immunogenic. To date, this vaccine seems to be the most promising approach for preventing sepsis caused by Klebsiella and has already passed phase I human trials.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
As per international standard, respondents' (parents & patients) written consent has been collected and preserved by the authors.