Phenotypic Screening of Aminoglycoside Resistance and theirTransferability in Clinical Isolates of Klebsiella pneumoniae from India

Method: A total of 177 consecutive, non-duplicate, clinical isolates of Klebsiella pneumonia were collected from patients from a period of September 2013 to February 2014. Screening for aminoglycoside resistance was performed. Transferability of aminoglycoside resistance was done by transformation assay. Genetic stability was checked by consecutive serial passage of 70 days. Incompatibility types were determined by PCR based replicon typing.


Introduction
Klebsiella is known to be the second most important multidrug resistant organism within enterobacteriaceae family after Escherichia coli. Klebsiella pneumoniae often reported to produce carbapenemase (KPC) leaving the carbapenem antibiotics inactive on them. The organism too often exhibit resistance towards quinolone and aminoglycoside group of antibiotics [1] that continue to play an important role in antimicrobial therapy against both gram negative and gram-positive pathogens, usually in combination with β-lactam agents [2]. Resistance to aminoglycosides can be widespread and has primarily been the result of aminoglycoside inactivation through the chemical processes of acetylation, phosphorylation, and/or adenylation, with varying effects depending upon the particular agent [3,4]. Recently another mechanism i.e, acquired 16S methyl transferase is also known to play a significant role in aminoglycoside resistance. These mechanisms, when combined with additional coexisting multidrug resistant trait, pose severe threat to therapeutic regime. Till now there is a paucity of data regarding aminoglycoside resistance among gram negative pathogen from this geographical part of the world. Thus, this study was undertaken to screen aminoglycoside resistance and their transferability within member of enterobacteriaceae family.

Sample size
A total of 177 consecutive, non-duplicate, clinical isolates of Klebsiella pneumoniae were collected from indoor and outdoor patients of Silchar Medical College and Hospital, India for duration of six month from September 2013 to February 2014. Isolates were identified based on conventional microscopical observation, cultural characteristics and biochemical testing methods.

Transferability
Isolated plasmids were tested for their ability of horizontal transfer by transformation assay. The recipient strain used was E. coli JM107. Transformation was carried out by heat shock method [6].

Discussion
In recent years K. pneumoniae is 84 a major organism which is said to be an etiological agent in hospital and community acquired infection [9]. However, increasing resistance to aminoglycosides is becoming a serious clinical problem. Resistance to aminoglycosides is frequently due to the acquisition of modifying enzymes that vary in their substrate ranges, such as acetyltransferases, phosphorylases and adenylyltransferases. Aminoglycoside resistant klebsiella pneumonia was reported in 2009 [10].
In this study high antibiotic resistance pattern was observed within study isolates family whereas, in this study moderate resistance was observed compared to data of previous studies Jones Goosen, Scwaber. This study underscores the presence and persistence of multidrug resistance within single phenotype, with implications on clinical outcome.
Earlier it was described that plasmid transfer happens due to possible high frequencies present in host, original host carrier, which are not compatible and permits the entry of plasmids. The range of pathogens in which multi-drug resistance or virulence plasmids are stably maintained could thus expand in the future and a better understanding of the evolutionary mechanisms of plasmid host range shifts [11]. In the current study it was observed various multidrug resistance plasmids were highly stable and retained the resistance phenotype till 47 consecutive serial passages. This implies the ability of pathogens to maintain the resistance gene even there is no antibiotic exposure. Thus, therapeutic alternative becomes limited which may lead into treatment failure. Two different studies in India have contrast in their finding where Potron in 2011 has found that blaKPC-2 encoding plasmid was non typeable while performing PCR based replicon typing [12]. Though later Kumaraswamy in 2011 described that enterobacteriaceae (Klebsiella and E. coli) were harboring some resistance gene within inc A/C type plasmid [13]. In this study it was observed that this centre, F inc type was more predominant followed by I and Y inc type. Also, it is evident from current investigation F inc type plasmids were encoding multiple resistance gene and could propagate within broad host range. Thus, this inc group could possibly responsible for horizontal transmission of multidrug resistance, their maintenance, persistence within hospital isolates under antibiotic stress.
Klebsiella pneumoniae that produced 16S rRNA methylases 113 were reported in 2003 [14]. These enzymes were found to Transformants were selected on LB Agar plates containing gentamicin (0.25 μg/ml).

Analysis of stability of plasmid encoding multidrug resistant phenotype
The experiment was performed to determine the vertical transmission dynamics of resistant genes along with their plasmid in subsequent generations. Plasmid stability of screened multi drug resistant (MDR) isolates as well as their transformants was analyzed by serial passages method for consecutive 75 days at 1:1000 dilutions without antibiotic pressure [7].

Determination of plasmid incompatibility typing
Plasmid incompatibility was determined for wild type as well as transformants by PCR based replicon typing using 18 pairs of different basic replicon primers, targetting FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA replicons [8].

Result
On susceptibility testing, 94 (53.6%) organisms out of 177, all of which were isolated from urine and pus (Table 1) were found to be resistant against one of the aminoglycosides, (gentamicin, netilimicin, amikacin, kanamycin) ( Table 2) or more antibiotic. All the isolates showed multidrug resistance (MDR) phenotypes. Aminoglycoside resistance was horizontally transferrable. The plasmid could be selected on E. coli JM 107 on Luria Bertani agar containing gentamicin. Most of the transformants showed co-resistance to other representatives of aminoglycoside antibiotics (Table 3).
Transformants retained resistance till 70th passage. Gentamicin resistance was lost after 47th passages.
Incompatibility typing could reveal that plasmid encoding aminoglycoside resistance was carried within diverse type of inc group. Among them F inc type (n = 17) was more predominant,

Type of Clinical specimen
Urine 94 Pus 83     confer extraordinarily high levels of resistance to clinically useful aminoglycosides, such as amikacin, tobramycin, and gentamicin [15].

Conclusion
Aminoglycosides are used to treat both gram positive and gram negative bacterial infections. This group of antibiotic is considered to be a second line drug which is prescribed in combination with other groups in clinical settings. Carriage of aminoglycoside resistance and their horizontal transferability in hospital setting demands urgent need to devise proper antibiotic policy and to slow down their expansion from hospital to communityenvironment.