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Asian J. Exp. Sci., Vol. 21, No. 2, 2007, 435-443 Intra and Interspecies Variations among Environmental Klebsiella
Isolates
Anjana Sharma*, Susheel Kumar Singh and Sunita Patra
Bacteriology Laboratory,
Department of P.G. Studies and Research in Biological Science,
R.D. University, Jabalpur (M.P.) 482001, India Abstract : Majority of the study of Klebsiella is restricted to clinical isolates and its occurrence
in fresh water environment is sporadically reported. Biochemical and molecular diversity of 40
Klebsiella isolates, isolated from 180 water samples of river Narmada from eleven stations
(Amarkantak to Dahej) during 2005-2006 was assessed by employing Biotyping, SDS-PAGE
profiling, Randomly Amplified Polymorphic DNA (RAPD) analysis, and Amplified Ribosomal
DNA Restriction Analysis (ARDRA). The Antibiogram analysis suggested that most of the
isolates were resistant against Ampicillin, Amoxycillin, Tetracycline, Chloramphenicol and
Trimethoprim and all isolates were found to be sensitive against Norflaxcin, Cefotaxime and
Gentamycin. RAPD and ARDRA prevailed over Biotyping and Protein profiling for identification,
and Intra and interspecies variations among environmental isolates of Klebsiella spp.
Key words : Klebsiella, Fresh water, Antibiogram, SDS-PAGE, Molecular typing.
Introduction
electrophoretic profiling, multilocus enzyme Klebsiella spp. are found in surface water, sewage, soil and on plants, and also amplified polymorphic DNA (RAPD) analysis on the mucosal surfaces of mammals such as (Wong et. al. 1993). The association between humans, horses, or swine, which they colonize Jemeyey et.al. (2006). Clinically, the most transmissibility of Klebsiella strains is not well understood, but there is clear evidence for pneumoniae and K. oxytoca, while K.
differential behavior of Klebsiella strains.
ornithinolytica, K. terrigena, and K.
planticola are rarely isolated from human infections are often multidrug resistant and an increasing proportion of strains produce (1998). K. planticola and K. terrigena are considered to be environmental species, as enzymes, that confer resistance to penicillins reflected in their species designations. K. (such as ampicillin or amoxicillin), to first pneumoniae and K. oxytoca exhibit a high demonstrated by capsular typing (Orskov and ceftazidime, cefoxitin and ceftiofur, and to Orskov, 1984), O-antigen variation, biotyping prevalence of antimicrobial resistance in Corresponding author : Dr. Anjana Sharma, Bacteriology Laboratory, Department of P.G. Studies
and Research in Biological Science, R.D. University, Jabalpur (M.P.) 482001, India; Phone No : +
91761-2416667 (R); + 919425155323 (M); Fax : + 91761-2603752; E-mail : anjoo_1999@yahoo.com
Sharma A. et al. (2007) Asian J. Exp. Sci., 21(2), 435-443 environmental Klebsiella isolates is poorly Identification of Bacteria) computer kit Bryant (2003), maintained in the Bacteriology lab, countries use river water directly not only for Department of Biological Sciences, R.D.
drinking but also for various recreational University, Jabalpur (MP), India and were purposes. Narmada is the fifth largest river of India and originates from Amarkantak (M.P.) Collection Centre) numbers. All the cells (n = 40), were grown to the stationary phase in (Gujarat). The diversity studies of Klebsiella Luria-Bertani (LB) broth. These cultures were are restricted to the clinical isolates and to our knowledge the present study is the first report on the diversity of Klebsiella, an immense clinically significant organism, in the following the method of Sambrook, et. al.
(1989). For ARDRA, oligonucleotideprimers were derived from conserved regions Material and Methods
present at the edges of the l6S rDNA. The sequences of primers were 16S rRNA F-5'– eleven stations of river Narmada (viz.
Dahej), over one year period from July 2005 49 ml aliquots of PCR mixture containing 5 to June 2006. 500ml water samples, collected ml of l0X buffer supplied with enzyme Taq aseptically under ice-cold condition were brought to the Bacteriology lab, Dept. of (i.e.12.5 picomol of each primer), 2.5 ml of University, Jabalpur and concentrated on 0.45 10 mM (each) deoxynucleotide triphosphates mm pore diameter filters, enriched in 1.5% and 0.5 ml of DNA polymerase (3 unit/ ml), and final volume was made 50 ml by adding sterile distilled water. After initial denaturation for isolation of Klebsiella spp (APHA, 1985).
at 94°C for 5 min, the reaction mixture was run through 35 cycles of denaturation at 94°C following biochemical characteristics; Arginine for 1 min, annealing at 51°C for 1 min and dihydrolase, lysine decarboxylase, ornithine extension at 72°C for 1 min, finally a 10 min extension period at 72°C was carried out.
urease, MR, VP, gluconate, malonate, Acid from arabinose, dulcitol, glucose, lactose, digestion of the amplicons with HindIII A/ sorbitol, sucrose and xylose following method Genei, India) and analysed by electrophoresis on 1.5% agarose gel with staining by ethidium were identified with the help of Bergey’s Manual of Systematic Bacteriology (Krieng amplification of isolated template DNA wascarried out with a primer [5’ – CAT TCG Intra and Interspecies Variations among Environmental Klebsiella Isolates ACC 3’] by following the method of Lopes (Exeter software, New York) and calculating dendrogram using “Unweighted pair group (Eppendroff master cycler gradient, Humburg, method using arithmetic - average clustering Germany). The reaction was performed in 25 ml reaction mixture containing 2.5 ml of 10X Results and Discussion
PCR buffer [500 mM KCl, 100 mM Tris (pH8.3)], 25 mM MgCl ], 3ml of template DNA, 2.0 ml primer, 0.3 ml of Taq DNA polymerase, these groups of aquatic bacteria deserve in 2.5 ml of 2.5 mM dNTP’s (Bangalore Genei, depth investigation, in order to understand India) . The volume was raised to 25 ml with better, their ecological role and function in the sterile triple distilled water. A negative control investigation of intra and interspecies diversity denaturation at 94°C for 5 min the reaction of Klebsiella spp from riverine environment, denaturation at 94°C for 1 min, annealing at 36°C for 1 min and elongation/extension at 72°C for 2 min followed by a 10 min final extension period at 72°C and the expected size of the amplicons were ascertained by isolates of Klebsiella from fresh water of river electrophoresis in 1.5% Agarose gel with an DNA ladder, Bangalore Genei, India). Protein conventional methods using biochemical tests.
Biochemical characterization, which involved a number of biochemical reactions important assay was followed to assess the antibiotic resistance/ sensitivity pattern as described ornithine decarboxylase, arginine dihydrolase, (CLSI, 2002). Using commercially available as the decarboxylase activity is an important diagnostic tool in identifying Klebsiella spp.
25mcg), Ampicillin (A, 30mcg), Amoxicillin (Trevison, 1987). Five different biotypes distributed among the 40 Klebsiella spp Cefotaxime (Ce, 30mcg), Nalidixic acid (Na, isolated from river Narmada on the basis of biochemical characteristics (Table 1).
Klebsiella pneumoniae isolates were bands were scored as either present (1) or Urease positive, MR negative), B4 (VP and absent (0). All binary data were entered and genetic distances were calculated through (Lysine positive and Indole negative). All K. oxytoca and K. terrigena were grouped into biotypes B2 (Malonate and VP negative) and Sharma A. et al. (2007) Asian J. Exp. Sci., 21(2), 435-443 Source, phenotype, genotype and antibiotype of 40 Klebsiella spp isolated fromriver Narmada.
Intra and Interspecies Variations among Environmental Klebsiella Isolates bp for each species (Fig. 1). ARDRA using HindIII appears to be a promising tool for biochemical differentiation among various identification of environmental Klebsiella spp.
species by generating different biochemical profiles among Klebsiella isolated from the (2001), ARDRA with EcoRI did not appear suitable for cluster identification in the present have limitations of being time consuming, study, but its higher discriminatory power is which could be unrealistic for the identification useful to subdivide HindIII ARDRA groups.
of genus Klebsiella during any outbreak to discriminate K. pneumoniae strains in previous investigations (Lopes et. al. 2005).
extremely useful in defining relationships of Klebsiella spp (Boye and Hanson, 2003).
Klebsiella spp studied, which demonstrates In this study, the 16S rRNA gene of each of the high discriminatory power of RAPD.
the 40 Klebsiella isolates was amplified and restricted with the enzymes EcoRI and in the molecular weight of 300 bp. However HindIII. This resulted in three separate no species-specific band was observed. The restriction patterns (assigned as AR1, AR2, AR3, and BR1, BR2, BR3, respectively) for run. The high number of serotypes in this each species. Each enzyme generated different could explain the relevant degree of genetic Fig. 1 : Representative 16S rRNA PCR-RFLP profiles obtained for Klebsiella isolates using
restriction enzymes (EcoRI and HindIII). Lane M: DNA marker (100 bp Ladder).
ARDRA types AR1, AR2 and AR3 correspond to K. pneumoniae, K. oxytoca and K.
terrigena restricted by EcoRI respectively and ARDRA types BR1, BR2 and BR3correspond to K. pneumoniae, K. oxytoca and K. terrigena restricted by Hind IIIrespectively.
Sharma A. et al. (2007) Asian J. Exp. Sci., 21(2), 435-443 Fig. 2 : UPGMA Cluster Analysis of Klebsiella isolated from river Narmada on basis of RAPD.
Intra and Interspecies Variations among Environmental Klebsiella Isolates diversity highlighted by RAPD analysis. The found that most of the isolates (72.5%) were resistant to amoxycillin and 62.5% isolates similarity at a coefficient of 2.08. At 1.97 were resistant against ampicillin. Sixteen coefficient, two clusters were formed (A and isolates (44%) were resistant to three or more B). The cluster A was subdivided into two antibiotics. All Klebsiella isolates found to be sub divisions (A1 and A2) at 1.73 coefficient sensitive against gentamycin, cefotaxime and nature of the population structure and high showed about 20 different bands, ranging in level of diversity of Klebsiella in the river Narmada. Little is known about the ecology bands the 40 isolates were grouped into 8 these organisms in fresh water environments different patterns designated as Pr1 to Pr8 (Table 1). In the present study, out of 40 isolates, 16 isolates belonged to group PrI, 3 significant source of fresh water for Central isolates belonged to group PrII, 10 isolates India sustaining millions of populace, and also belonged to group PrIII, 3 and 5 isolates used for recreational purposes, the occurrence belonged to group PrVII and PrVIII, and only of antibiotic resistant Klebsiella spp. raises a single isolate belonged to group PrIV, PrV question regarding potential risk of human and PrVI respectively. Protein analysis was exposure; hence it’s indispensable to monitor found less efficient to differentiate K. pneumoniae strains than antibiotype, plasmid possibility of any epidemic. Moreover, many analysis and RAPD (Lopes, et. al. 2005).
The role of antibiotics in the management exclusively with clinical isolates; while there of human infections caused by Klebsiella has aspects of environmental strains. Since the resistance could be an important problem for therapy directed against these organisms.
Klebsiella isolates are naturally resistant to ampicillin, due to a constitutively expressed transmission in humans, it is obvious that detailed studies on the pathogenic potential of the environmental Klebsiella strains will Amoxycillin, Ceftazidime, Trimethoprim or certainly contribute to understanding the virulence properties of these bacteria and to associated with multidrug resistance is due to establish the importance of these organisms.
the acquisition of mobile elements carrying several resistance genes. The 40 Klebsiella grouped into 12 antibiotypes (designated A1 (biotyping, protein analysis and antibiogram to A12) depending upon their resistance to analysis) as a useful technique in distinguishing different antimicrobial drugs (Table 2). It was Klebsiella spp. There currently seems to be Sharma A. et al. (2007) Asian J. Exp. Sci., 21(2), 435-443 Distribution of 40 Klebsiella spp according to antibiotypes isolated from riverNarmada.
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