klebsiella pneumoniae dairy

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

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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

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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

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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

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(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

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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

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

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HMI

DOST

PCAARD

HMI

NDA, BAI

DA, DOH

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

n= 184/season (368 samples)

7% prevalence rate w/ 95% CI



Genetic

relatedness

(CLSI, 2012)

(CLSI, 2008)

n = 30/season (60 samples)




MIC.pptx

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.

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Results ID Table 1.xlsx

Results AMR genes Table 3.xlsx

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

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Gantt chart.docx

Budget

Expenses Amount (Baht)

Scientific activities 187,000

Documentation 13,000

Miscellaneous 5,000

TOTAL 205,000

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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.

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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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gyrAparC

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)




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)




MIC.pptx

klebsiella pneumoniae dairy

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