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Advances in Animal and Veterinary Sciences

AAVS_MH20161001061016_Sahay et al

Short Communication

 

 

Isolation and Characterization of Klebsiella pneumoniae from Respiratory Infection of Yak (Bos grunniens), India

 

Swati Sahay1, Juwar Doley2, Natesan Krithiga1, Gundallahalli Bayyappa Manjunatha Reddy1, Siddanna Sharangouda Patil1, Bibek Ranjan Shome1, Rajeswari Shome1*

1ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), Yelahanka, Bengaluru–560064, India; 2ICAR-National Research Centre on Yak, Dirang-790 101, Arunachal Pradesh, India.

 

Abstract | Yak (Bos grunniens) is multipurpose long haired ruminant reared by the poor tribal farmers for wool, milk, meat, etc in the high altitudes of Himalayan region of Indian Territory. There is a serious concern about increasing morbidity and mortality of yak due to respiratory diseases. Nasal swab sample was collected from pneumonic yak in Amies charcoal transport media from Arunachal Pradesh, India and processed for bacterial isolation and identification and direct PCR detection in 18h BHI enriched broth sample. Based on biochemical characteristics and multiplex PCR, the culture was identified as K. pneumoniae (isolate No. KP1) and in in-vitro antibiotic sensitivity test, the isolate was resistant to ampicillin. The 16S rRNA sequence analysis and 16S rRNA secondary structure prediction revealed close geographical relatedness to environmental K. pneumoniae PB12 from River Mahananda from East India and K. pneumoniae JPR 9 isolated from soil samples of Assam. This study describes pneumonia in yak due to K. pneumoniae from Arunachal Pradesh, India by direct PCR detection from enriched clinical sample, isolation, PCR and 16S rRNA sequence, phylogenetic and secondary structure relation studies.

 

Keywords | Isolation, Klebsiella pneumoniae, Pneumonia, 16S sequencing, Yak

 

Editor | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.

Received | November 02, 2016; Accepted | November 22, 2016; Published | December 07, 2016

*Correspondence | Rajeswari Shome, ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Yelahanka, Bengaluru–560064, India; Email: rajeswarishome@gmail.com

Citation | Sahay S, Doley J, Krithiga N, Reddy GBM, Patil SS, Shome BR, Shome R (2016). Isolation and characterization of Klebsiella pneumoniae from respiratory infection of yak (Bos grunniens), India. Adv. Anim. Vet. Sci. 4(12): 637-641.

DOI | http://dx.doi.org/10.14737/journal.aavs/2016/4.12.637.641

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2016 Sahay et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Yak (Bos grunniens) is domesticated and multipurpose long haired ruminant found throughout the Himalayan region of South Central Asian countries like China, Nepal, India and Tibet and North Asian countries like Russia and Mongolia (Dubal et al., 2013). In India, yak is reared mostly by the poor tribal farmers for wool, milk, leather and meat in the high altitudes of Himalayan region of Indian Territory. Recently there is a growing concern of infectious and non-infectious diseases affecting yaks causing high morbidity and mortality (Bandyopadhyay et al., 2012).

 

Klebsiella spp. are mostly associated with sepsis, infections of urinary and respiratory tracts, mastitis and meningitis etc. (Brisse and van Duijkeren, 2005). Pulmonary infections due to K. pneumoniae are often characterized by a rapid progressive clinical course complicated by lung abscesses and cause high mortality and morbidity if remain untreated (Cortés et al., 2002). K. pneumoniae has been isolated from bovines, small ruminants (Suguna et al., 2012), camels (Sharma et al., 2015), foals (Boguta et al., 2002) and non-diarrheic yaks (Goswami et al., 2009).

 

Isolation of bacteria is gold standard diagnostic approach though various serological and molecular diagnostic methods are gaining importance because of high specificity and sensitivity. Among bacterial identifications, 16S rRNA sequence analysis and 16S rRNA secondary structure prediction modeling (Clarridge, 2004) have proven to be a stable and specific molecular marker for the identification of bacteria. This short communication describes pneumonia in yak due to K. pneumoniae from Arunachal Pradesh, India by direct PCR detection from enriched clinical sample, isolation, PCR and sequence phylogeny and secondary structure relation studies.

 

Female yak of 5 years age suffering from respiratory distress, off feed, pyrexia, severe nasal discharge, depression during peak winter season where temperature ranges from 4°C to 18°C was reported to the institute. The yak belonged to Arunachali breed from a Shyro village of district Tawang, Arunachal Pradesh maintained at altitudes above 9750 feet and not vaccinated against FMD, HS and BQ diseases. Nasal swab sample was collected from pneumonic yak in Amies charcoal transport media (Hi media, India) and airlifted on cold chain to the laboratory within 48 h. After initial enrichment in brain heat infusion (BHI) broth for 18-24 h, sample was streaked onto 7% sheep blood agar (BA) and incubated for 24-36 h at 370C. Pure mucoid colonies observed were purified on Nutrient Agar (NA) later on Mac Conkey’s Lactose agar (MLA) for detection of lactose fermentation.

 

The culture was examined for gram’s staining, oxidase, catalase, urease, indole, H2S production on triple sugar iron agar (TSI) and sugar fermentation with sucrose, glucose, mannitol, lactose, adonitol, dulcitol, melibiose and esculin (Cruickshank, 1980). DNA was extracted from 18 h BHI enriched broth sample and pure culture by QIAamp DNA mini Kit and quantified by nanodrop. Multiplex genus (Brisse and Verhoef, 2001) and species (Kovtunovych et al., 2003) PCRs were performed for target genes gyrA (441 bp) and ropB (108 bp), respectively. DNA of K. pneumoniae ATCC BAA 1706 and E. coli ATCC 700336 were used as positive and negative control templates, respectively.

 

In vitro antibiotic sensitivity was carried out using 10 antibiotics (Bauer et al., 1966) [tetracycline (30mcg), cotrimoxazole (25mcg), cefotaxime (30mcg), piperacillin/Tazobactam (100/10mcg), chloramphenicol (30mcg), gentamicin (10mcg), cefepime (30mcg), ampicillin (10mcg), imipenem (10mcg) and meropenem (10mcg)].

 

The 16S rRNA sequences were aligned with 10 published sequences from India and other neighboring countries (Table 1) to compare phylogenetic relatedness (www.genebee.msu.su.). Similarly, 16S rRNA secondary structure prediction for complimentary region was analysed using Mfold and RNA fold softwares (Zuker, 1989) and the structure with minimum free energy was considered the most stable structure (Hofacker, 2003; Mathews et al., 2004).

 

Respiratory diseases cause economic losses to the producers in terms of post-infection loss of condition, reduced milk production and inhibited development (Fels-Klerx et al., 2002). The respiratory pathogens/diseases reported in yak are 41% seroprevalence of BHV-1 by virus neutralization test (VNT) and AB-ELISA (Bandyopadhay et al., 2008); bovine viral diarrhoea (BVD) (Mishra et al., 2004) and 36-89% mortality due to Pateurella multocida type I (Pal, 1993). In respiratory infections, it is difficult to arrive at specific diagnosis because of the involvement of large number of pathogens. The pneumonic yak was sampled to rule out the etiology as many of the yaks in the region had similar symptoms. The highly mucoid, non-hemolytic and lactose fermenting pure colonies were observed on NA, BA and MLA media, respectively from the processed sample.

 

Table 1: List of K. pneumoniae sequences used in the study

 

S. No.

Strains

Accession numbers

Source of strain

Place

Minimum Free energy (Kcal/mol)

1

K. pneumoniae KP1/yak

KP866814

Yak

Arunachal Pradesh, India

-524.4

2

K. pneumoniae QLR-18

KM096439

Cattle

China

-517.7

3

K. pneumoniae AU45

EF032681

Goat

Tamil Nadu, India

-524.9

4

K. pneumoniae -sheep

AY963633

Sheep

China

-524.6

5

K. pneumoniae

M.D.K.NA1-13

JF690874

Deer

China

-527.7

6

K. pneumoniae LXY

KM272989

Swine

China

-517.9

7

K. pneumoniae KP1513

KJ746503

Mice

China

-519.2

8

K. pneumoniae Amm2

KJ950284

Human

Cambridge, USA

-526.8

9

K. pneumoniae PB12

KF192506

River Mahananda

West Bengal, India

-523.9

10

K. pneumoniae JPR9

KM083804

Soil, East India

Assam, India

-516.4

11

K. pneumoniae CF-S9

JX680326

Waste water

Odisha, India

-529.2

 

The culture was Gram negative short rod reacted positive in catalase, urease and sodium citrate tests and negative in indole and H2S production on TSI agar. Based on biochemical tests and multiplex PCR, the culture was identified as K. pneumoniae (isolate no. KP1) (Figure 1). The isolate was resistance to only ampicillin antibiotic.

 

In the present study, K. pneumoniae was detected in 18 h BHI enriched broth by PCR. The direct detection appears to be of great value in diagnosis and epidemiological mapping of K. pneumoniae as an alternate to time consuming isolation method in difficult geographical terrain where yaks are inhabited. The 16S rRNA (1456 bp) sequences (NCBI accession number KP866814) and RNA secondary structure prediction revealed close geographical relatedness to environmental K. pneumoniae PB12 from River Mahananda from East India and K. pneumoniae JPR 9 isolated from soil samples of Assam. However, sequence divergence was observed with animals isolates from India and other neighboring countries (Figure 2 and 3). There are records of respiratory diseases in yaks in the region but various etiological agents have been implicated other than K. pneumoniae (Mishra et al., 2004).

 

 

 

 

Identification of K. pneumoniae from respiratory infections reiterates as a respiratory pathogen (Alves et al., 2006; Hansen et al., 2004) and further studies involving large number of isolates and genotyping are essential to understand geographical, pathogencity and physio-chemical factors on the distribution and divergence of K. pneumoniae.

 

Acknowledgments

 

We are thankful to the Director and scientists, ICAR-National Research Centre on Yak, Dirang, Arunachal Pradesh for their support & collaboration for sharing samples. This work is funded by Department of Biotechnology Govt. of India through twinning project entitled “Aetio-Pathology and molecular epidemiology of bacterial and viral diseases associated with the respiratory problems of yak in the North Eastern Regions of India (Grant No: BT/391/NE/TBP/2012)”.

 

Conflict Of Interest

 

None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

 

Authors’ Contribution

 

RS designed the study and corrected the manuscript. JD provided the sample for study. SS, KN carried out the experimental work, analyzed the data and drafted the manuscript. GBMR,SSP, BRS provided guidance and support. All authors read and approved the final manuscript.

 

References

 

  • Alves MS, Dias RCS, Castro ACD (2006). Identification of clinical isolates of indole-positive and indole-negative Klebsiella spp. J. Clin. Microbiol. 44: 3640-3646. https://doi.org/10.1128/JCM.00940-06
  • Bandyopadhyay S, Lodh C, Rahaman H, Bhattacharya D, Bera AK, Ahmed FA, Mahanti A, Samanta I, Mondal DK, Bandyopadhyay S, Sarkar S, Dutta TK, Maity S, Paul V, Ghosh MK , Sarkar M, Baruah KK (2012). Characterization of shiga toxin producing (STEC) and entero-pathogenic Escherichia coli (EPEC) in raw yak (Poephagusgrunniens) milk and milk products. Res. Vet. Sci. 93(2): 604-610. https://doi.org/10.1016/j.rvsc.2011.12.011
  • Bandyopadhyay S, Sasmal D, Ghosh MK, Borah BD, Bora M, Biswas TK (2008). Collibacillosis in newborn yak calves and antibiogram profile of rectal flora isolated from the same. Indian J. Anim. Sci. 78: 1101-1102.
  • Bauer AW, Kirby WMM, Sherhis JC, Turck M (1966). Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45: 493-496.
  • Boguta L, Gradzki Z, Borges E, Maruin F, Kodjo A, Winiarczyk S (2002). Bacterial flora in foals with upper respiratory tract infections in Poland. J. Vet. Med. 49(6): 294-297. https://doi.org/10.1046/j.1439-0450.2002.00570.x
  • Brisse S, Duijkeren VE (2005). Identification and antimicrobial susceptibility of 100 Klebsiella animal clinical isolates. Vet. Microbiol. 105: 307-312. https://doi.org/10.1016/j.vetmic.2004.11.010
  • Brisse S, Verhoef J (2001). Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC gene sequencing and automated ribotyping. Int. J. Syst. Evol. Microbiol. 51: 915-924. https://doi.org/10.1099/00207713-51-3-915
  • Clarridge EJ (2004). Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious disease. Clin. Microbiol. 17: 840-862. https://doi.org/10.1128/CMR.17.4.840-862.2004
  • Cortés G, Álvarez D, Saus C, Albert S (2002). Role of Lung epithelial cells in defense against K. pneumonia. Infect. Immun. 70 (3): 1075–1080. https://doi.org/10.1128/IAI.70.3.1075-1080.2002
  • Cruickshank R (1980). Medical Microbiology, 12th eds. (revised reprint) Churchill Livingstone, Edinburg. Pp. 170-189.
  • Dubal ZB, Khan MH, Dubal PB (2013). Bacterial and viral zoonotic diseases of yak. Int. J. Bioresour. Stress Manage. 4(2): 288-292.
  • Fels-Klerx HJ, Saatkamp HW, Verhoeff J, Dijkhuizen AA (2002). Effects of bovine respiratory disease on the productivity of dairy heifers quantified by experts. Livest. Prod. Sci. 75(2): 157-166. https://doi.org/10.1016/S0301-6226(01)00311-6
  • Goswami A, Saikia GK, Sharma RK (2009). Isolation and characterization of bacteria recovered from diarrheic and non-diarrheic yaks. Indian J. Comp. Microbiol. Immunol. Infect. Dis. 30(1): 29-30.
  • Hansen DS, Aucken HM, Abiola T (2004). Recommended test panel for differentiation of Klebsiella species on the basis of a trilateral inter laboratory evaluation of 18 biochemical tests. J. Clin. Microbiol. 42: 3665-3669. https://doi.org/10.1128/JCM.42.8.3665-3669.2004
  • Hofacker IL (2003). Vienna RNA secondary structure server. Nucl. Acids Res. 31: 3429–3431. https://doi.org/10.1093/nar/gkg599
  • Kovtunovych G, Lytvynenko T, Negrutska V (2003). Identification of Klebsiella oxytoca using a specific PCR assay targeting the polygalacturonase pehX gene. Res. Microbiol. 154: 587-592. https://doi.org/10.1016/S0923-2508(03)00148-7
  • Pal RN (1993). Domestic yak (Poephagusgrunniens L.): a research review. Indian J. Anim. Sci. 63: 743-753.
  • Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH (2004). Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc. Natl. Acad. Sci. 101: 7287–7292. https://doi.org/10.1073/pnas.0401799101
  • Mishra N, Pattnaik B, Vilcek S, Patil SS, Jain P, Swamy N, Bhatia S, Pradhan HK (2004). Genetic typing of bovine viral diarrhoea virus isolates from India. Vet. Microbiol. 104: 207-212. https://doi.org/10.1016/j.vetmic.2004.08.003
  • Sharma SK, Mudgal NK, Sharma P, Shrngi BN (2015). Comparison of phenotypic characteristics and virulence traits of Klebsiella pneumoniae obtained from pneumonic and healthy camels (Camelus dromedarius). Adv. Anim. Vet. Sci. 3(2): 116-122. https://doi.org/10.14737/journal.aavs/2015/3.2.116.122
  • Suguna M, Bhat R, Nadiah WAW (2012). Microbiological quality evaluation of goat milk collected from small-scale dairy farms in Penang Island, Malaysia. Int. Food Res. J. 19: 1241-1245.
  • Zuker M (1989). On finding all suboptimal foldings of an RNA molecule. Science. 244: 48–52. https://doi.org/10.1126/science.2468181
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