This document outlines a study on the identification and characterization of antimicrobial resistance and genetic traits of zoonotic Klebsiella pneumoniae isolates from a dairy farm in Laguna, Philippines. The study aims to determine the prevalence of K. pneumoniae in mastitic and bulk tank milk, and characterize the antibiotic resistance patterns and mechanisms. Isolates will be collected from cow milk and human workers from the dairy farm and tested for antibiotic susceptibility. Genes conferring antimicrobial resistance and virulence factors will be identified using molecular techniques. Results will provide data on antimicrobial resistance genes in K. pneumoniae from animals and humans to inform industry and policy.

1. 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

2. 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

3. Laguna, Philippines
3
https://www.google.co.th/search?q=laguna,+philippines+image
Fig 1. Philippine map showing Southern Tagalog region
Introduction (1)

4. 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

5. 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

6. 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.

7. Introduction (5)
Objectives of the study
The study aims to understand the antimicrobial
resistance & genetic relatedness of Klebsiella pneumoniae
between human & bovine milk isolates.
Specific Objectives
1. To establish the prevalence of Klebsiella pneumoniae in
relation to season in mastitic and bulk tank milk in a dairy
farm 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

8. 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)

9. 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.

10. UPLB
CU
Dr. HR
Dr. RC
Information &
dissemination of
prevalence,
antimicrobial
resistance &
genetic
relatedness of
clinical & bovine
milk K.
pneumoniae
isolates
Technology
transfer
Implementation
and systems/policy
management on
mastitis & prudent
antibiotic use
Outputs
Research
assistantship &
Scientific writing
programs
Procurement &
laboratory set up
Bacterial
isolation,
MIC, PCR &
Conjugation
studies
Thesis drafting
Process
Competent lab &
research writing
skills
Adequate
funding & lab
equipment
Feasible &
effective lab
methods
Adequate
supervisory
assistance
Inputs
Conceptual Framework
10
HMI
DOST
PCAARD
HMI
NDA, BAI
DA, DOH

11. Materials & Methods
MIC.pptx
11
Workers’
rectal
swab
• Clinical
• Subclinical
Mastitic
milk
Bulk
tank
milk
Bacterial isolation
• Ciprofloxacin
• Ampicillin
• Imepenem
Humans
• Cloxacillin
• Ceftiofur
• Gentamicin
• Tetracycline
• Enrofloxacin
Animals
Antibiotic
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

12. 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

13. 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

14. Budget
Expenses Amount (Baht)
Scientific activities 187,000
Documentation 13,000
Miscellaneous 5,000
TOTAL 205,000
14

15. 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

16. 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 pneumoniae
by Dairy Cows. J. Dairy Sci. 89 (2006):3425–3430.
• Zadoks , RN., Griffiths , HM., Munoz , MA., Ahlstrom , C., Bennett ,GJ., Thomas , E., and
Schukken, YH. Sources of Klebsiella and Raoultella species on dairy farms: Be careful where you
walk. J. Dairy Sci. 94 (2011) :1045–1051.
• Holt, JG., Krieg NR., Sneath, PHA., Staley, JT., and Williams, ST. Bergey’s Manual of
Determinative 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 Klebsiella
pneumoniae 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 Klebsiella
pneumoniae and Mechanisms Leading to In Vivo Carbapenem Resistance Development. Journal
of CLINICAL Microbiology, Aug. 2006, p. 2831–2837 Vol. 44, No. 8.
• Macrae, MB., Shannon, KP., Rayner, DM., Kaisery, AM., Hoffmanz, PN and French, GL. A
simultaneous outbreak on a neonatal unit of two strains of multiply antibiotic resistant Klebsiella
pneumoniae controllable only by ward closure. Journal of Hospital Infection (2001) 49: 183±192.
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17. Thank you!!!
17
Kyeizu tin ba de!!!

18. Introduction (4)
Molecular characterization
18
gyrAparC
E.coli E.coli
Figure 2. Phylogenetic clusters in Klebsiella pneumoniae in clinical isolates.
(Brisse and Verhoef , 2001)

19. Materials & Methods
MIC.pptx
19
Workers’
rectal
swab
• Clinical
• Subclinical
Mastitic
milk
Bulk
tank
milk
Bacterial isolation
• Ciprofloxacin
• Ampicillin
• Imepenem
Humans
• Cloxacillin
• Ceftiofur
• Gentamicin
• Tetracycline
• Enrofloxacin
Animals
Antibiotic
Susceptibility
testing
Molecular characterization
Class 1 integron &
transferability
AMR
mechanicams
ESBLs
characterization
Virulence genes
characterization
August –November 2014
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)
 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