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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)
Research MethodologyFebruary 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
2
1• Background
2• Conceptual Framework
3• Materials and methods
2
OUTLINE
4• Results
5• Plan of Study
6 • Budget
7 • Advantages of the Study
8• References
Laguna, Philippines
3
https://www.google.co.th/search?q=laguna,+philippines+image
Fig 1. Philippine map showing Southern Tagalog region
Introduction (1)
Introduction (2)
Klebsiella pneumoniae
- facultative anaerobic G(-) bacteria
- source: environment; mucosal surfaces of humans & animals
Family: Enterobacteriaceae
Genus: Klebsiella
Blood agar: gray-brown 3-5mm diameter colonies; fecal odor;
non-hemolytic
McConkey agar: small to large (1-7mm) wet, glistening, dome-shaped
pink-yellow mucoid colonies,
w/ smooth edges,
w/o precipitate in the surrounding agar
4
(Zadoks , et al.,2011)
(Hogan and Smith, 2003)
(Munoz, MA., et al..,2006)
(Macrae, et al., 2001)
(Holt , et al., 1994)
https://www.google.co.th/search?q=klebsiella+pneumoniae+milk+image
Fig 2. Klebsiella pneumoniae
Introduction (3)
Test Result Test Result
Gram stain (-) Gelatin hydrolysis (-)
Oxidase (-) TSI A/A, (-) gas
Indole production (-) Malonate utilization (+)
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
5
(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).
6
(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.
7
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
8
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)
9
(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
Outputs
Researchassistantship &
Scientific writingprograms
Procurement &laboratory set up
Bacterialisolation,
MIC, PCR &Conjugationstudies
Thesis drafting
Process
Competent lab &research writingskills
Adequatefunding & labequipment
Feasible &effective labmethods
Adequatesupervisoryassistance
Inputs
Conceptual Framework
10
HMI
DOST
PCAARD
HMI
NDA, BAI
DA, DOH
Materials & Methods
MIC.pptx
11
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
n= 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)
n = 30/season (60 samples)
63% prevalence rate w/ 95% CI
n= 12/season (24 samples)
38% prevalence rate w/ 95% CI
Results
• Table 3. List of bacteria isolated from mastitic animals in a dairy
farm in Laguna.
Results ID Table 1.xlsx
• Figure 4. Graph showing antimicrobial resistance of Klebsiella
pneumoniae isolates.
• Table 5. List of antimicrobial resistance genes in Klebsiella
pneumoniae isolates.
Results AMR genes Table 3.xlsx
• Figure 5. Graph showing virulence genes of Klebsiella pneumoniae
isolates.
12
Study plan
Time and place of the study
• Time: January 2014 to June 2015
Gantt chart.docx
• Location:
Department of Para-clinical Sciences,
College of Veterinary Medicine,
University of the Philippines Los Banos, Laguna, &
Commercial dairy cattle farm in Laguna
13
Budget
Expenses Amount (Baht)
Scientific activities 187,000
Documentation 13,000
Miscellaneous 5,000
TOTAL 205,000
14
Advantages of this Study
• Data such as antimicrobial resistance genes of
Klebsiella pneumoniae isolates from milk &
humans will be made available.
• This study will provide vital information to
various industry players, academicians, drug
companies & policy makers.
• As a pioneering work, it will serve as a
benchmark for further researches.
15
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.
16
Introduction (4)Molecular characterization
18
gyrAparC
E.coli E.coli
Figure 2. Phylogenetic clusters in Klebsiella pneumoniae in clinical isolates.
(Brisse and Verhoef , 2001)
Materials & Methods
MIC.pptx
19
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
(BAFPS).
n = 30/season (60 samples)
63% prevalence rate w/ 95% CI
n= 12/season (24 samples)
38% prevalence rate w/ 95% CI