Identification and Molecular Characterization of Antimicrobial Resistance and Genetic Characteristics of Zoonotic Klebsiella pneumoniae Isolates from a Dairy Farm in Laguna, Philippines Flor Marie Immanuelle R. Pilapil 561 01180 31 (Master in Veterinary Science) Research Methodology February 19, 2014 1 Dr. Rungtip Chuanchuen - Program Adviser Associate Professor Department of Veterinary Public Health Faculty of Veterinary Science, Chulalongkorn University Dr. Hope Rovira - Thesis Adviser Associate Professor Department of Paraclinical Sciences College of Veterinary Medicine, University of the Philippines
Transcript
Identification and Molecular Characterization
of Antimicrobial Resistance and
Genetic Characteristics of Zoonotic
Klebsiella pneumoniae Isolates
from a Dairy Farm in Laguna, Philippines
Flor Marie Immanuelle R. Pilapil561 01180 31 (Master in Veterinary Science)
Methyl red (-) D-glucose, acid & gas production (+)
Voges- Proskauer (+) Tartrate, Jordans (+)
Citrate (Simmons) (+) Esculin hydrolysis (+)
H2S production (-) Acetate utilization (+)
Urea hydrolysis (+) Nitrate reduction (+)
Phenylalaline deaminsae (-) Lipase (-)
Lysine decarboxylase (+) Pigment (-)
Arginine dihyrolase (-) Flagella arrangement (-)
Ornithine decarboxylase (-) Catalase production (+)
Motility (-) Oxidation-fermentation F
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(Holt , JG., et al., 1994)
Table 1. Phenotypic characterization of Klebsiella pneumoniae
Introduction (4)
Research problem and its significance
Klebsiella pneumoniae
most common Klebsiella species infecting animals & causing mastitis further
imposing a higher economic loss in terms of milk production & survival.
Infections have developed multi-drug resistance (MDR) otherwise known as
multiple antibiotic resistant Klebsiella spp. (MRKs) due to production of
‘extended-spectrum’ β-lactamases (ESBLs).
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(Munoz, MA., et al.. 2006)
(Macrae, et al., 2001)
Hypothesis:
Klebsiella pneumoniae in bovine mastitic milk is
zoonotic & has developed multiple antimicrobial
resistant properties against human & animal
antibiotics.
Introduction (5)
Objectives of the study
The study aims to understand the antimicrobialresistance & genetic relatedness of Klebsiella pneumoniaebetween human & bovine milk isolates.
Specific Objectives
1. To establish the prevalence of Klebsiella pneumoniae inrelation to season in mastitic and bulk tank milk in a dairyfarm in Laguna,
2. To determine the antibiotic resistance patterns of K.pneumoniae & characterize its mechanisms, distribution &transfer among Klebsiella sp. isolated from humans & milk.
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Literature Review (1)
# Antibiotic Humans Antibiotic Bovine milk
1 Ampicillin X Penicillin/Cloxacillin X
2 Ceftazidime/Ceftiofur X Ceftiofur X
3 Imipenem X
4 Aztreonam X
5 Gentamicin X Gentamicin X
6 Tetracycline X Tetracycline X
7 Trimethoprim/
Sulfonamide
X Trimethpprim/
Sulfonamide
X
8 Ciprofloxacin X Enrofloxacin X
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Table 2. Comparison of antimicrobial resistance of Klebsiella pneumoniae
between clinical and bovine milk isolates.
(Mena, et al., 2006) (Macrae, et al., 2001)
(CLSI, 2012) (Brissea & Duijkerenb, 2005)
Literature Review (2)
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(Timofte, D., et al.. 2013)
Fig 3. Schematic diagrams of environments around blaCTX-M-15 in common human isolates
(a to c) and bovine mastitis isolate (d) in United Kingdom.
UPLB
CU
Dr. HR
Dr. RC
Information & dissemination of prevalence, antimicrobial resistance & genetic relatedness of clinical & bovine milk K. pneumoniaeisolates
Technologytransfer
Implementationand systems/policymanagement onmastitis & prudentantibiotic use
Literature Cited• Hogan, J. and Smith, KL. Coliform mastitis. Vet. Res. 34 (2003) 507–519.
• Munoz, MA., Ahlstrom C., Rauch, BJ., and Zadoks, RN. Fecal Shedding of Klebsiella pneumoniaeby Dairy Cows. J. Dairy Sci. 89 (2006):3425–3430.
• Zadoks , RN., Griffiths , HM., Munoz , MA., Ahlstrom , C., Bennett ,GJ., Thomas , E., andSchukken, YH. Sources of Klebsiella and Raoultella species on dairy farms: Be careful where youwalk. J. Dairy Sci. 94 (2011) :1045–1051.
• Holt, JG., Krieg NR., Sneath, PHA., Staley, JT., and Williams, ST. Bergey’s Manual ofDeterminative Bacteriology. 9th edition. 1994. Lippincott Williams & Wilkins, Maryland, USA.p.211.
• Timofte, D., Maciuca, IE., Evans, NJ., Williams, H., Wattret, A., Fick, JC., and Williams, NJ.Detection and Molecular Characterization of Escherichia coli CTX-M-15 and Klebsiellapneumoniae SHV-12 b-Lactamases from Bovine Mastitis Isolates in the United Kingdom.Antimicrob. Agents Chemother. 2014, 58(2):789.
• Mena, A., Plasencia, V., Garci, L., Hidalgo, O., Ayestara, JI., Alberti, S., Borrell, N., Perez, JL.,and Oliver, A. Characterization of a Large Outbreak by CTX-M-1-Producing Klebsiellapneumoniae and Mechanisms Leading to In Vivo Carbapenem Resistance Development. Journalof CLINICAL Microbiology, Aug. 2006, p. 2831–2837 Vol. 44, No. 8.
• Macrae, MB., Shannon, KP., Rayner, DM., Kaisery, AM., Hoffmanz, PN and French, GL. Asimultaneous outbreak on a neonatal unit of two strains of multiply antibiotic resistant Klebsiellapneumoniae controllable only by ward closure. Journal of Hospital Infection (2001) 49: 183±192.
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Thank you!!!
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Kyeizu tin ba de!!!
Introduction (4)Molecular characterization
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E.coli E.coli
Figure 2. Phylogenetic clusters in Klebsiella pneumoniae in clinical isolates.
(Brisse and Verhoef , 2001)
Materials & Methods
MIC.pptx
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Workers’ rectal swab
• Clinical
• Subclinical
Mastiticmilk
Bulk tank milk
Bacterial isolation• Ciprofloxacin
• Ampicillin
• ImepenemHumans
• Cloxacillin
• Ceftiofur
• Gentamicin
• Tetracycline
• Enrofloxacin
AnimalsAntibiotic
Susceptibility
testing
Molecular characterization
Class 1 integron & transferability
AMR mechanicams
ESBLs
characterization
Virulence genes
characterization
August –November 2014n= 184/season (368 samples)
7% prevalence rate w/ 95% CI
n= 50/season (100 samples)
17% prevalence rate w/ 95% CI
Genetic
relatedness
(CLSI, 2012)
(CLSI, 2008)
Cow handling:
• RA 8485 “The Animal Welfare Act of 1998”
• Good Agricultural and Husbandry Practices (GAHP) set by
the Bureau of Agriculture and Fisheries Product Standards
