Antimicrobial Resistance and Klebsiella pneumoniae in Bovine Mastitis

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INTRODUCTION
1.1 Background
Antimicrobials are medically important in the prevention, control and treatment of
infections and disease. They are mainly used for therapy, metaphylaxis, prophylaxis and in
the case of the animal industry, growth promotion. In fact, the Center of Disease Control
(CDC) in the United States of America says that 80% of the antimicrobial use is for the farm
animals which help microorganisms become resistant.
The World Health Organization (WHO) defines antimicrobial resistance (AMR) as
the resistance of a microorganism to an antimicrobial previously effective for treatment of
infections and diseases caused by it. The growing concern on AMR involves the over use of
antimicrobials (Younes, A.M., 2011) which include non-observance of the withdrawal period
for meat and milk. Moreover, extra-label or off-label use of antimicrobials is also rampant
(Barlow, 2011). The care for extra-label drug use in food animals relate to residue avoidance,
and its use requires documentation of adequate withholding period for milk and slaughter to
ensure food safety. Extra-label use is prohibited if the use results in the presence of drug
residue in food or if presents a public health risk (CDC). WHO lists the major causes of
antibiotic resistance below.
1. Over-prescription of antibiotics
CDC states that in humans up to half of the time, antibiotics are not properly
prescribed in terms of dosage and duration and often done so when not needed. Also in
animals, there are instances when some minor clinical signs can be alleviated with mere
vitamin and/or mineral supplementation, rest and isolation, but some are still prescribed with

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antimicrobials. Over-prescription of antibiotics can be prevented by also using alternative
methods such as herbal medicine.
2. Patient’s non-completion of treatment regimen and period
Due to expensive medical costs, some patients, farmers and pet owners opt to
discontinue the treatment. Moreover, as in the animal industry, medication may also be quite
difficult to perform.
3. Over-use of antibiotics in livestock and fish farming
It has been a wide practice in the animal sector to give antibiotics as prophylaxis and
more especially to promote growth by suppressing bacterial load that hinders optimum
growth and production of the animals and not necessarily intended for those microorganisms
already causing infection and disease. CDC further mentions that the use of antibiotics in
food animals increases resistance for some microbes.
4. Inadequate infection control in hospital and clinics
An effective sanitation program in hospitals and clinics helps prevent infection and
spread of disease. Poor infection control helps microorganisms thrive in the environment
enabling them to adapt well even after exposure to sanitizers and disinfectants thus leading to
resistance.
5. Lack of hygiene and inadequate sanitation
Basic hand washing and safe food preparation are essential elements in good hygiene,
optimum health and prevention of spread of disease.

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6. Lack of new antibiotics being developed
The growth and spread of microorganisms is exponentially fast however due to lack
of funds and manpower, research has been difficult and slow in the development of a new
drug. Aside from this, the development and validation of methods to quantify and document
antimicrobial use and the effect of prudent antimicrobial use practices have continue to be a
challenge (Barlow, 2011).
As an effect to AMR, CDC declares that at least 2 million people in the United States
become infected with antibiotic resistant bacteria annually and at least 23,000 people die each
year as a direct result of these infections.
Moreover, bovine mastitis is an economically significant disease due to the high
veterinary costs, extra labor, decreased fertility, decrease in milk production let alone the
discarded milk, and death or culling of infected animals thus affecting the daily income of the
local dairy farmers (Paulin-Curlee, et al., 2007). Specifically, Klebsiella pneumoniae is a
facultative anaerobic Gram negative bacterium (Holt, et al., 1994) that is present in the
environment, mucosal surfaces of humans and animals (Macrae, et al., 2001; Brisse, et al.,
2009). Mastitis caused by Klebsiella pneumoniae can be more severe than the other mastitis
pathogens due to its poor antimicrobial response, rapid progress to toxic shock and death. It
has been reported to be more pathogenic and cause higher losses than infections due to
Eschericia coli (Paulin-Curlee, et al., 2007).
This study will help the dairy animals in the Philippines especially the cattle which is
estimated to be 46, 363 heads (NDA, 2015) as the data and recommendations that will be
generated already fit the local conditions. Identifying Klebsiella pneumoniae as the cause of
mastitis and the risk factors leading to it will help provide worthy recommendations to the
local dairy farmers on its prevention, management and treatment. The whole dairy animal

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industry which comprises of dairy cattle farmers, fresh milk processors, farm workers, dairy
products consumers and government officers will also benefit from this research through the
increase of food source.
AMR can also be acquired through the consumption of untreated or inadequately
treated milk (Timofte et al., 2014). Furthermore, studying about the antimicrobial properties
of Klebsiella pneumoniae will help prevent or at least lessen the occurrence of their drug
resistance which is essential in combating mastitis and of not incurring high treatment costs.
This will also help diminish or avoid transfer of such resistance properties to other pathogens
that could infect humans. Through this study, data such as antimicrobial resistance genes of
Klebsiella pneumoniae from bovine milk 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.
1.2 Objectives of the study
The study aims to understand the antimicrobial resistance and its associated risk
factors, and genetic characterization of Klebsiella pneumoniae isolates from bovine milk.
Specific Objectives
1. To establish the prevalence of Klebsiella pneumoniae in mastitic cows from dairy
cattle farms in Batangas;
2. To determine the antibiotic resistance patterns and virulence factors of Klebsiella
pneumoniae and characterize its mechanisms, distribution and transfer among bacteria
isolated from bovine milk;

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3. To establish the risk factors present in each farm in relation to Klebsiella pneumoniae
antimicrobial resistance in bovine mastitis; and
4. To formulate recommendations for each farm involved in terms of prevention, control
and management of Klebsiella pneumoniae antimicrobial resistance bovine mastitis.
1.3 Time and place of the study
The study will be conducted at the Department of Paraclinical Sciences, College of
Veterinary Medicine, University of the Philippines Los Banos, Laguna, and in dairy cattle
farms in Batangas from December 2015 to August 2016.

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REVIEW OF RELATED LITERATURE
2.1 Mastitis
Mastitis is the inflammation of the mammary gland caused by several bacteria (Oliver
& Murinda, 2012; Zadoks et al., 2011) but it is also a response to intramammary
mycoplasmal, fungal, or algal infections. Microorganisms may escape the natural defense
mechanisms by multiplication along the streak canal (especially after milking), or by
propulsion into the teat cistern by vacuum fluctuations at the teat end during milking.
Mechanical trauma, thermal trauma, and chemical insult predispose the gland to
intramammary infection (IMI) as well. Occurrence of mastitis depends on the interaction of
host, agent, and environmental factors (Zhao & Lacasse, 2008).
The two classifications of mastitis according to severity are subclinical and clinical.
Subclinical mastitis depicts mild non-visible inflammation of the mammary gland and the
milk and quarter still appear normal. It is the main form of mastitis in dairy herds, exceeding
50% of cows in given herds (Oliver & Murinda, 2012). Subclinical mastitis may be
identified by bacteriological culture of milk or by the measurement of indicators of
inflammation such as Somatic cell count (SCC) and California Mastitis Test (CMT) (Oliver
& Murinda, 2012 and Barlow, 2011). The culture of milk from cows postpartum or cows
with high SCC may be used as a surveillance tool to identify common organisms causing
subclinical mastitis during lactation or as a component of a mastitis control program to
identify cows for treatment, segregation, or culling during lactation (Barlow, 2011).
Subclinical mastitis can be self-limiting and could heal spontaneously or it could develop
within hours up to several months to clinical mastitis (Oliver & Murinda, 2012). The cost of
subclinical mastitis is very difficult to quantify, but most experts agree that subclinical

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mastitis costs the average dairy farmer more than does clinical mastitis (Zhao & Lacasse,
2008).
On the other hand, clinical mastitis is manifested through visible abnormal, clotty or
flaky appearance of the milk even if the udder may appear normal (Oliver & Murinda, 2012).
It is in dairy cattle in as many as 40% of samples (Barlow, 2011). Furthermore, clinical
mastitis can be categorized to mild, moderate and severe. In moderate clinical mastitis, the
udder is already visibly inflamed caused by the clots blocking the milk passage preventing
drainage from the alveoli. Consequentially, the alveoli swell leading to lower milk
production. Lastly, severe clinical mastitis poses a systemic threat to the animal as it
becomes ill inclusive of dull, sunken eyes, drooping cold ears, weakness, loss of appetite,
depression, dehydration, shivering, increased rectal temperature, increased pulse rate and
respiratory rate, reduced rumen contraction rate and diarrhea (Oliver & Murinda, 2012).
The two types of mastitis in terms of causative agent are the contagious and the
environmental type. The contagious type includes that of Staphylococus aureus,
Streptococcus agalactiae and Mycoplasma sp. which may spread from cow to cow (Oliver &
Murinda, 2012; Zhao & Lacasse, 2008) through the milkers’ hands, milking machine, and
flies (Levesque et al, 1995). Milking time hygiene is the basis for control of contagious
mastitis (Hogan & Smith, 2012). Antibiotic treatment of clinical mastitis caused by the
gram-positive cocci (e.g. Staphylococcus aureus, Streptococcus uberis, Streptococcus
dysgalactiae, and Streptococcus agalactiae) is often recommended. Treatment decisions
should be guided by culture results (Barlow, 2011).
The environmental type includes that of Streptococcus uberis, Streptococcus
dysgalactiae and coliforms such as Escherichia coli and Klebsiella pneumoniae (Oliver &
Murinda, 2012; Zhao & Lacasse, 2008) which have increased in relative importance as a

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cause of both clinical and subclinical mastitis (Barlow, 2011). They are of fecal origin or
may come from the surroundings such as the beddings, feed and soil. Rates of environmental
mastitis are directly proportional to the temperature and moisture and are greatest during the
dry period and early lactation compared with other stages of lactation. Bulk tank and
monthly cow somatic cell counts (SCCs) are poor milk quality indicators of environmental
mastitis. Approximately 85% of coliform and 50% of environmental streptococcal infections
will cause clinical mastitis. The severity of clinical mastitis brought about by environmental
pathogens ranges from mild local signs to death. The vast majority of clinical coliform and
environmental streptococcal clinical cases are characterized by only abnormal milk and a
swollen gland. During the dry period, susceptibility to intramammary infections is greatest at
the 2 weeks after drying off and the 2 weeks prior to calving. Research has shown that 65%
of coliform clinical cases that occur in the first 2 months of lactation are intramammary
infections that originated during the dry period. Coliforms are skilled at infecting the
mammary gland during the transitional phase from lactating to fully involuted mammary
gland. Management include frequent manure removal, eliminating standing water in the
cow’s walking lanes and loafing areas, and avoiding overcrowding of animals in barns and
pastures (Hogan & Smith, 2012).
Culture negative results have been attributed to infectious bovine mastitis where
concentrations of pathogens are beneath the limit of detection using standard techniques, the
presence of endogenous inhibitory substances in milk decreases the viability of bacteria in
vitro, or the bacteria from the mammary gland were effectively cleared by the host immune
response prior to obtaining milk samples for culture. Less commonly isolated organisms
such as Mycoplasma spp., Serratia spp., Pseudomonas spp., Arcanobacterium pyogenes
(formerly Actinomyces pyogenes), Nocardia spp., Prototheca spp., Bacillus spp., yeasts and
fungi are unlikely to respond to treatment (Barlow, 2011). Escherichia coli, Klebsiella

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pneumoniae, Streptococcus agalactiae and Staphylococcus aureus also occur as commensals
or pathogens of humans whereas other causative species, such as Streptococcus uberis,
Streptococcus dysgalactiae subsp. dysgalactiae or Staphylococcus chromogenes, are almost
exclusively found in animals (Zadoks et al., 2011).
Mastitis is recognized as the most costly disease in dairy cattle. Decreased milk
production accounts for approximately 70% of the total cost of mastitis (Zhao & Lacasse,
2008). As it is caused by several bacteria, it is difficult to control and massive economic loss
is to be expected. In the United States, the national mastitis council estimates that the annual
economic loss due to mastitis amounts to more than $2 billion (Oliver & Murinda, 2012).
Mammary tissue damage reduces the number and activity of epithelial cells and consequently
contributes to decreased milk production. Mammary tissue damage has been shown to be
induced by either apoptosis or necrosis (Zhao & Lacasse, 2008).
Segregation and culling is often the most prudent response for persistently infected
animals. It influences prevalence of mastitis pathogens in dairy herds and selective culling of
cows with mastitis may influence the prevalence of specific species or strains. Pathogen
genotype and host-restriction may influence the probability of infection persistence and cure
following treatment. Moreover, acquired resistance of species and strains through horizontal
gene transfer can be influenced by its bacterial genotype. Non-antibiotic control options such
as culling, segregation, hygiene and biosecurity will be important to limit transmission within
and between farms. In the past, when milk was bought largely for volume, the main aim of
treatment was to restore milk production and the failure to eliminate infection was not of
major priority. This likely brought about the use of short duration treatment regimens such as
2 days of therapy, targeting resolution of clinical signs but not bacteriological cure, although
the importance of bacteriological cure has long been recognized (Barlow, 2011).

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Antimicrobial agents remain a component of infectious mastitis treatment and control
(Barlow, 2011). Antibiotic therapy of clinical mastitis involves detection of the infected
quarter, immediate treatment, administration and completion of recommended treatments,
recordkeeping, identification of treated cows, and strict observance of milk withdrawal
periods (Oliver & Murinda, 2012). The success of the therapy depends on the treatment
product, length of treatment and whether treatment was administered during lactation or
during the dry period, or in the case of heifers, shortly before calving, increasing cow age,
increasing SCC, increasing persistence of infection, increasing bacteria counts, and
increasing numbers of mammary quarters infected. Of these factors, the most important
affecting cure is treatment duration (Middleton, 2012). Antibiotics such as penicillin,
cephalosporin, non-cephalosporin beta-lactam, streptomycin, tetracycline and macrolide-
lincosamide drugs are used to combat mastitis. Additionally, penicillin is combined with
either novobiocin or dihydrostreptomycin (Barlow, 2011; Oliver & Murinda, 2012).
Treatment of clinical IMI caused by coliform organisms with IMM (intramammary) or
systemic formulations is not recommended due to the short duration of infection and high
spontaneous cure rates. Supportive care such as fluid therapy and treatment with steroidal or
non-steroidal anti-inflammatory drugs has been recommended for cases of acute clinical
coliform mastitis. Frequent milk-out is a popular recommendation in the dairy industry for
treatment of acute clinical coliform mastitis.
Cure of IMI following treatment of either clinical or subclinical mastitis is generally
higher for lower parity, lower number bacterial colonies in the pre-treatment sample, a
shorter duration of infection or lower number of positive pre-treatment samples, and a lower
pre-treatment milk somatic cell count. Bacterial genetic factors also affect clinical properties
of infection and the response to treatment. Cases of subclinical mastitis are commonly

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treated at the end of a lactation cycle such as dry-cow therapy administered at the start of the
dry period. Dry cow therapy is an established mastitis control practice that is applied to 100%
of cattle on an estimated 73% of U.S. dairy farms (Barlow, 2011).
2.2 Antimicrobials and its stewardship
Antimicrobial drugs (Appendix 1) function by targeting different parts of the bacterial
cell. Various mechanisms include interference with cell wall synthesis; interference of
protein synthesis through the 30S and 50S subunit; interference with nucleic acid (DNA)
synthesis; inhibition of Ribonucleic acid (RNA) synthesis; inhibition of a metabolic pathway;
and disruption of bacterial membrane structure.
In detail, interference with cell wall synthesis happens through synthesis of uridine
diphosphate (UDP)-N-acetylglucosamine and uridine diphosphate (UDP)-N-acetylmuramyl
pentapeptide; peptidoglycan formation (UDP-N-acetylglucosamine, UDP-N-acetylmuramyl-
pentapeptide and pentapeptide of glycine); and cross-linkage of peptidoglycans by enzyme
transpeptidase (PBPs) also known as “transpeptidation”. Antimicrobials of this mode of
action include ß-lactam antibiotics such as penicillinase-resistant aminopenicillins and first-
to fifth-generation cephalosporins. Antimicrobials that interfere the protein synthesis through
the 30S subunit are aminoglycosides and aminocyclitols which interfere with the recognition
between amino-acyl tRNA and codon causing incorporation of incorrect amino acids,
formation of abnormal and non-functional protein and rapid cell death; and tetracyclines
which prevent the binding of aminoacyl tRNA to the A site of the ribosome and suppress the
movement of tRNA along the ribosome. On the other hand, interference through the 50S
subunit happens through binding to the domain V of 23S rRNA (peptidyl transferase center)
and inhibiting the formation of peptide bond between amino acid on aminoacyl t-RNA and

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growing peptide chain. They also bind to the A site and prevent the transfer of peptide chain
form the A site to the P site. Antimicrobials having this mechanism are chloramphenicol,
macrolides, lincosamides and streptogramins.
Intervention with nucleic acid (DNA) synthesis occurs by interfering with DNA
gyrase (topoisomerase II) for gram-negative bacteria and topoisomerase IV for gram-positive
bacteria. Antimicrobials having this mode of action are quinolones, nitroimidazoles and
nitrofurans. While on ribonucleic acid (RNA), synthesis is inhibited by rifamycins by
binding on DNA directed beta subunit RNA polymerase disabling bacterial DNA to transfer
its information to RNA and inhibiting protein synthesis. Furthermore, inhibition of a
metabolic pathway ensues by acting on the synthesis of tetrahydropholic acid specifically on
the dihydropteroate synthetase and dihydrofolate reductase by the sulphonamides and
diaminopyrimidines respectively. Lastly, disruption of bacterial membrane structure takes
place as manifested by polymyxins through interaction with the phospholipids of cell
membrane of gram-negative bacteria by increasing its permeability thus disrupting and
destabilizing the membrane (Younes, A.M., 2010).
Aspects of antimicrobial use to consider in the development of farm specific strategies
may include pathogen identification causing specific health problems, determination of the
most appropriate drug classes to use for treatments, ensuring appropriate treatment regimens
including dosage, route of administration, and duration of therapy, and pathogen
susceptibility testing and monitoring. Strategies should be reviewed regularly and revised to
meet changing circumstances. Use minimum inhibitory concentration (MIC) test methods,
report results at the species level, and present MIC data as the proportion of isolates
susceptible or resistant for each dilution tested in complete tabular form or using histograms.
Eliminating unnecessary antibiotic treatments would be beneficial for economic and prudent
drug use purposes. Treatment of culture negative mastitis is not recommended. Selective

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dry-cow therapy can also be implemented to only treat cows at high risk for infection at the
end of lactation as opposed to doing blanket dry cow therapy which entails treating all cows
at the end of lactation regardless of infection status. The former appears to be an option in
herds with low prevalence of infection, but the potential impact on net drug use still remains
unknown. Non-antibiotic alternatives to dry cow therapy such as internal teat sealants may
provide an alternative which contributes to reduced drug use in dairy herds. It has been
estimated that antibiotics would not be justified for treatment of at least 50% of clinical
mastitis cases (Barlow, 2011).
Benefits of antimicrobial usage include healthier, more productive cows; lower
disease incidence; reduced morbidity and mortality; decreased pathogen loads; and
production of abundant quantities of nutritious, high-quality, longer shelf-life milk for human
consumption. However, there is controversy on its wide usage which may have led to the
occurrence of antimicrobial resistance. It may also lead to presence of antibiotic residue in
milk. These are two public health and food safety issues but also an economic issue for the
farmer to be penalized of having poor quality milk (Oliver & Murinda, 2012).
2.3 Antimicrobial Resistance
Issues related to antimicrobial use in dairy production systems include antimicrobial
agents such as cephalosporins, lincosamides, non-cephalosporin beta-lactams and
aminoglycosides relating to their availability ‘over-the-counter’ (OTC) at the disposal of
producers without veterinary supervision; the use of antimicrobial agents in an extra-label
manner; the relationship between antimicrobial use practices and the risk for development of
antimicrobial resistance; the development and validation of methods to quantify and
document antimicrobial use and the effect of prudent antimicrobial use practices.

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Specifically, key conditions for extra-label drug use in food animals relate to residue
avoidance and documentation of adequate milk and slaughter withholding times to ensure
food safety. Extra-label use is not permitted if the use results in a violative drug residue in
food or if the use presents a public health risk, if another drug exists equivalent to what is
needed, and if no evidence is available on any approved antibiotic product establishing its
efficacy. Injectable products approved for use in beef or dairy cattle less than 20 months of
age are strictly prohibited for IMM extra label use. Such drugs are the macrolides or
flouroquinolones labelled for treatment of bovine respiratory disease.
U.S. Food and Drug Administration (FDA) Center for Veterinary Medicine (CVM)
has prohibited the extra-label veterinary use of flouroquinolones and glycopeptides in food
animals due to their importance in human medicine and the risk that extra-label use may
increase the antimicrobial resistance of bacteria that can cause human illness. Systemic use
of an antimicrobial drug such as ceftiofur or ampicillin to treat severe acute coliform mastitis,
especially when bacteremia is suspected or documented, represents an extra-label drug use
that maybe justified as there are no antimicrobials labelled for systemic administration for
mastitis and a significant proportion of coliform mastitis cases have been demonstrated to
progress to bacteremia where inclusion of antimicrobial therapy in treatment regimens
improves cow survival. Improved surveillance of antimicrobial use in food-producing
animals, including standardized class specific estimates of dosing per animal unit such as per
kilogram live weight, per time period such as the animal daily dose, is required to accurately
attribute risk to specific production systems (Barlow, 2011).

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2.3.1 Transmission of resistance genes
AMR started from antimicrobial-producing organisms such as fungi or soil bacteria.
Through selective pressure, a few bacteria emerge after exposure to a given antimicrobial
with development of antimicrobial resistance mechanisms. Resistance genes can either be
transmitted vertically or horizontally via mobile genetic elements such as plasmids,
transposons and integrons. Specifically, plasmids are single stranded DNA in gram-positive
bacteria called the “jumping genes”. They vary widely in size from 1,000 to 10,000 base
pairs. They occur most often as closed covalently circular (CCC) with no free ends. They
replicate as the cell grows and encode RNA and protein. Secondly, transposons are small
pieces of DNA that insert itself into another place in the genome. Lastly, integrons, with nine
classes, are genetic units characterized by their ability to capture and incorporate gene
cassettes by site-specific recombination. Moreover, a gene cassette is a type of
mobile genetic element that contains a gene and a recombination site of 57-141 base pairs.
They often carry antibiotic resistance genes. They may vary considerably in total length from
262 to 1,549 base pairs. They exist incorporated linearized form into an integron or at a non-
specific location or freely as closed covalently circular DNA molecules which are important
intermediates in the dissemination of the cassettes. The second unit of transfer is the vector
which is a DNA molecule used as a vehicle to artificially carry foreign genetic material into
another cell, where it can be replicated and/or expressed (Synder and Champness, 1997).
Lastly, bacteria itself through zoonosis can transfer resistance genes from man to animals and
vice versa and through the different species of animals.

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2.3.2 Types of Antimicrobial resistance
There are two types of AMR such as endogenous and exogenous AMR. Endogenous
AMR is the genetic change in bacterial genome also known as mutation while exogenous
AMR is the horizontal acquisition of foreign genetic information. In the latter, gene transfer
is classified as transformation or acquisition of free DNA, transduction via bacteriophages,
and conjugation or cell-to-cell transfer.
Transformation is the transfer of free or “naked” DNA into competent recipient cells.
It requires homology between donor and recipient DNA for recombination to happen. It only
plays a limited role in the transfer of resistance genes due to a rapid degradation of free DNA
from lysed bacteria. Only a few bacteria, such as Streptococcus pneumoniae and Bacillus
spp. exhibit a natural ability to take up free DNA from environment. On the other hand,
transduction is a bacteriophage-mediated transfer process. A bacteriophage is a virus that
infects and replicates within a bacterium. Transduction does not require viability of the
donor cell. It is also limited only to closely related bacteria carrying the same receptors
(specific receptor) for phage attachment. It is commonly observed between bacteria of the
same species particularly in gram-positive bacteria such as the spread of β-lactamase genes in
Staphylococcus aureus or multiple resistance phenotypes in Salmonella Typhimurium phage
type DT104. Lastly, we have conjugation which is a self-transfer of conjugative plasmid or
transposon from donor to recipient cells. It requires close contact between donor and
recipient cells via the conjugation bridge and it is an important means for the spread of
resistance genes between bacteria of different species and genera (Synder and Champness,
1997).

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2.3.3 Mechanisms of AMR
The major mechanisms of AMR are enzymatic drug inactivation, reduced intracellular
accumulation of antimicrobials, and protection, alteration or replacement of the cellular target
sites. Enzymatic drug activation happens through resistance to β-lactams and
aminoglycosides via the enzymes β-lactamases and aminoglycoside-modifying enzymes
respectively. Decreased drug uptake through decreased cell wall permeability is how
intracellular accumulation of antimicrobials is reduced. This is an important mechanism of
resistance to β-lactams and fluoroquinolones in gram-negative bacteria, especially in
Pseudomonas aeruginosa and in Enterobacteriaceae. The outer membrane of gram-negative
bacteria may represent a permeability barrier to certain antibiotics. Mutations leading to
reduced expression, structural alteration or even loss of porins have been associated with
reduced permeability to antimicrobial drugs.
Aside from that, there could be increased removal of the drugs through an active efflux
which is an energy-dependent transmembrane protein mechanism. Furthermore, it is a
channel that actively exports antimicrobials and other compounds out of the cell. It prevents
intracellular accumulation necessary to exert the lethal activity inside the cell (Wannaprasat,
2012). The last mechanism is modification or replacement of the drug target and target
protection so the drug can no longer bind and exert its activity on the cell. This is important
for resistance to penicillin and glycopeptides in gram-positive and to quinolones in both
gram-positive and gram-negative bacteria. Structural changes of the binding sites of the
drugs targeting the bacterial ribosome in aminoglycosides are usually due to methylation.
Other changes are modification of DNA gyrase enzyme due to gene mutation causing
quinolone resistance and glycopeptide resistance in enterococci and methicillin resistance in
Staphylococcus aureus (MRSA) are due to drug target replacement. Target replacement is

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also the main mechanism of acquired resistance to sulfonamides and trimethoprim followed
by increasing production of the drug target or another molecule with affinity for the drug
while target protection is the one mainly associated with tetracycline resistance.
2.3.4 Antibiotic sensitivity test (AST)
ASTs act as an epidemiologic tool and as a guide for treatment as it is a diagnostic
procedure being done to detect the extent of AMR in common pathogens and to assure
susceptibility to antimicrobials of choice for treatment of particular infections. The ideal
AST has low detection limit, high sensitivity and validity, ease of usage, storage and
longevity, no need for expensive equipment, and has scientific support. It should also be fast,
economical and environmental-friendly. However, its limitations include none mimicry of in
vivo environment and its results cannot predict outcome such as diffusion in tissue and host
cells, serum protein binding, drug interactions, host immune status & underlying illness,
organism virulence and site and severity of infection.
ASTs can fall under two types of methods such as the diffusion and the dilution
method. The diffusion method detects AMR through zone diameter breakpoint but still
considered qualitative since measurement of resistance through zone of inhibition (diameter
in mm) as compared with a standard table can only be categorized as susceptible,
intermediate and resistant. Intermediate results can further be characterized as moderate
susceptible for low toxic antibiotics and a buffer zone between resistant and susceptible for
high toxic antibiotics. The diffusion method is further classified into two types of tests such
as the disk diffusion test also known as the Kirby- Bauer test and the Epsilometer test also
known as the E-test. The former uses antibiotic-impregnated filter discs with set

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concentration and measures AMR against more than one antibiotic through measurement of
the size of the zone of inhibition. The result depicts a direct relationship between the sizes of
the zone of inhibition to the antibiotic effectivity. Three possible AST results can occur such
as susceptible with wide zone of inhibition, intermediate with medium zone of inhibition and
resistant without zone of inhibition. The latter uses a plastic strip instead with a predefined
gradient of fifteen antibiotic concentrations. It measures an approximate-MIC value. Results
are read directly on the strip where the elliptical zone of inhibition intersects with the strip.
This is good for slow-growing or nutritionally deficient microorganisms and is used on
antimicrobials not used routinely or on a new antimicrobial. Additionally, it can
confirm/detect a specific resistance phenotype and can detect low levels of resistance.
On the other hand, the dilution method is a quantitative type which detects AMR
through minimum inhibitory concentration (MIC) which is the lowest concentration of the
antimicrobial completely inhibiting visible growth of the microbial isolate being tested. It is
also further classified into three tests such as agar dilution test, broth microdilution and broth
macrodilution. The first test gives visible growth of the microbial isolate on agar plates with
a series of antimicrobials. It is the method of choice for a large number of bacterial isolates
as multiple isolates are tested on each plate and it is not good to use if susceptibility to a wide
range of different antimicrobial is to be tested. It uses a replicator, be it 96-teeth manual
applicator with a rod handle or 64-teeth semi-automatic applicator with a knob handle in a
64-well plate. Final concentration of organism is at 1 x 104
CFU/mL. Secondly, broth
microdilution uses various concentrations of antimicrobial in broth of which the range varies
depending on the antimicrobial used. Testing volume is at 0.05-0.1mL. Final concentration
of organism is at 5 x 105
CFU/mL. The disadvantages of this test include test limiting to only
one antimicrobial & one organism to be tested each time and it being time consuming. It uses

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96-well plates that are manually or commercially prepared. The broth macrodilution uses the
same principle as that of broth microdilution. Testing volume is rather at >1.0 mL. Final
concentration of organism is at 5 x 105
CFU/mL (CLSI, 2012).
2.4 Klebsiella pneumoniae
2.4.1 General characteristics
Klebsiella pneumoniae is a facultative anaerobic Gram negative bacterium (Holt, et
al., 1994), named after Edwin Klebs, a German microbiologist and recognized over a century
ago as a source of community-acquired pneumonia (Younes, A.M., 2011). It is present in the
environment, mucosal surfaces of humans and animals (Macrae, et al., 2001; Brisse, et al.,
2009). It belongs to the family Enterobacteriaceae and under the genus Klebsiella. It appears
gray-brown 3-5mm diameter colonies, non-hemolytic with the characteristic fecal odor on
blood agar (Hogan and Smith, 2003) while on McConkey agar, it appears small to large (1-
7mm) wet, glistening, dome-shaped, pink-yellow mucoid colonies with smooth edges
(Younes, A.M., 2011) and without precipitate in the surrounding agar (Munoz et al., 2006;
Zadoks, et al., 2011).
It is oxidase and methyl red negative and does not produce indole and H2S. It is
catalase, Voges-Proskauer (VP), Simmons citrate and lysine decarboxylase positive. It
produces acid but not gas on Triple Iron Sugar and is negative to arginine dihydrolase and
ornithine decarboxylase. It is not motile and does not hydrolyze urea and gelatin. It ferments
using D-glucose and reduces nitrates (Holt, et al., 1994). Clinical isolates of Klebsiella
pneumoniae are categorized according to the nucleotide variations of the gyrA, parC, and

21
rpoB genes into four phylogenetic groups called KpI, KpII-A, KpIIB, and KpIII (Younes,
A.M., 2011).
Klebsiella spp. populates soils, grains, water, and intestinal tracts of animals (Brisse,
et al., 2009). It is more capable than Escherichia coli at surviving in the mammary gland
from the onset of involution until calving as E. coli intramammary infections will only last
for less than 10 days on the average during lactation while intramammary infections caused
by K. pneumoniae would endure about 21 days on the average. The prevalence of coliform
mastitis in a herd seldom exceeds 5% of lactating quarters because coliform infections tend to
be short duration during lactation. They rarely cause chronic infections of greater than 90
days (Hogan & Smith, 2012). The most common Klebsiella species causing bovine mastitis
is K. pneumoniae. The presence of Klebsiella in used bedding is due to contamination with
bovine feces or with milk from Klebsiella infected cows (Zadoks et al., 2011).
There are three layers composing the cell wall of Klebsiella namely the cytoplasmic
membrane, the peptidoglycan layer and the outer membrane consisting of a complex of
lipopolysaccharide (LPS) forming the O antigen, phospholipid and protein. Additionally, the
LPS has three parts, viz region I, which is the outermost part called O-specific polysaccharide
composed of oligosaccharide repeating units to which the O-antigen is chemically based,
region II, the middle area termed core oligosaccharide which expresses the rough (R) antigen
specificity and region III, the innermost part which is the lipid moiety of the molecule named
lipid A where the hydrophobic reaction is attached to the lipoprotein of the outer cell
membrane of the bacterial cell. Aside from this, Klebsiella is covered by a thick
polysaccharide capsule forming glistening mucoid colonies of viscid consistency (Bergan,
1984) which becomes the basis for serotyping in reference to the 77 known antigenic capsular
or K-antigen strains of which serotypes K1 and K2 are the virulent types due to resistance to

22
serum killing (Pan et al, 2008). Conventional serotyping through slide agglutination for O
antigens and capsular swelling tests for K antigens yielded cross reactivity between serotypes
(Bergan, 1984; Podschun and Ullman, 1998) that is why molecular serotyping has gain its
popularity over the years since polymerase chain reaction is more sensitive and specific.
Further classification of Klebsiella pneumoniae isolates would be the three phylogenetic
groups called KpI which represents more than 80% of Klebsiella pneumoniae human clinical
isolates and has higher antimicrobial resistance rates to the remaining groups KpII and KpIII
(Brisse and Duijkere, 2005).
Due to imprudent use of antibiotics, Klebsiella pneumoniae 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) (Macrae, et al.,
2001) which are enzymes contributing to resistance to penicillins, aztreonam, first generation
cephalosporins and to newer ones like cefotaxime, ceftazidime, cefoxitin and ceftiofur
(Brisse and Duijkeren, 2005). Klebsiella pneumoniae is also the most common Klebsiella
species infecting animals and causing mastitis further imposing a higher economic loss in
terms of milk production and survival (Munoz et al., 2006). It also carries potential public
health implications through the consumption of untreated or inadequately treated milk
(Timofte et al., 2014). However, not much research has still been done on the prevalence of
antimicrobial resistance in animal Klebsiella isolates (Brisse and Duijkeren, 2005).
2.4.2 Pathogenesis
Klebsiella pneumoniae is the most medically important amongst the Klebsiella
species (Younes, A.M., 2011). It is an opportunistic pathogen both shared by humans and

23
animals. It can be spread horizontally through the gastrointestinal tract, personnel hands and
devices and environmental contamination (Parasakthi et al., 2000; Brisse, et al., 2009).
Klebsiella pneumoniae causes bacteraemia, respiratory and urinary tract infection particularly
in immunocompromised patients (Cortes et al., 2002; Brisse, et al., 2009) and community-
acquired pyogenic liver abscess and septic metastatic complications like meningitis and
endophthalmitis (Yeh et al., 2006; Pan et al., 2008; Brisse, et al., 2009). It has the ability to
spread rapidly in the hospital environment causing intense nosocomial outbreaks (Podschun
and Ullman, 1998; Brisse, et al., 2009; Younes, A.M., 2011). In animals, it causes similar
clinical signs to hospital patients and mastitis specifically on bovine (Brisse and van
Duijkere, 2005; Younes, A.M., 2011) and metritis in mares after transmission from an
infected stud especially capsular serotype K1, K2, K5 and K7. It can further cause infection
in dogs, monkeys, guinea pigs, muskrats, birds and fox (Younes, A.M., 2011). Adhesins,
siderophore (Koczura and Kaznowski, 2003), lipopolysaccharide (LPS), and the capsular
polysaccharide (CPS) are factors adding to its virulence (Brisse, et al., 2009; Younes, A.M.,
2011).
2.4.2.1 Capsular antigens
Capsular polysaccharide (CPS) gives the characteristic mucoid appearance of the
colony and is deemed to be one of the primal virulence factors of Klebsiella pneumoniae. It
is composed of four to six sugars such as glucose, galactose, mannose, fucose and rhamnose,
and very often, uronic acids (Podschun and Ullman, 1998; Younes, A.M., 2011). It is
incorporated by the horizontal transfer of the cps operon (Brisse, et al., 2009). Now with 77
serotypes, it is involved in resistance to macrophage phagocytosis and to the complement
system (Cortes et al., 2002; Brisse, et al., 2009) especially C3b and serum resistance due to

24
the bulky bundles of fibrillous structures covering the bacterial surface in extensive layers
(Podschun and Ullman, 1998; Younes, A.M., 2011).
Being the predominantly virulent strains, Klebsiella pneumoniae K1 capsular serotype
isolates cause liver abscess (Younes, A.M., 2011), endophthalmitis and acute pneumonia
(Chuang, et al., 2006; Brisse, et al., 2009). K2, K4 and K5 isolates can also be involved in
the latter aside from causing metritis in mares (Brisse, et al., 2009). They have also started to
develop resistance to neutrophil phagocytosis as opposed to non-K1/K2 isolates such as K3,
K4, K5 and K6 (Struve et al., 2005; Yeh et al., 2006).
2.4.2.2 Adhesins
Adhesins are almost always hemagglutinins that may be located on fimbrae or pili
protruding on the bacterial cell surface. Majority of the Klebsiella pneumoniae isolates have
fimbrae which display either one or both adhesive properties such as “mannose-sensitive
(MS) adhesion”, linked to the common type 1 thick fimbrae (MSHA) and susceptible to
inhibition by D-mannose, and “MR adhesion”, involved with type 3 thinner fimbrae
(mannose-resistant, Klebsiella-like hemagglutination or MR-K/HA) and resistant to mannose
(Bergan, 1984; Podschun and Ullman, 1998 and Yousen, A.M., 2011). In addition, type 3
fimbriae are set by the mrk gene cluster composing the major fimbrial subunit mrkA gene and
the mrkD fimbrial adhesin in charge of the mannose resistant Klebsiella-like
hemagglutination. They are also believed to help in the establishment of extended
extracellular structures known as biofilms which serve as structural anchors and barriers to
contact with host defenses thus protecting against antibiotics (Yousen, A.M., 2011).

25
Other types of Klebsiella adhesins include Type 6 pili (Yousen, A.M., 2011),
nonfimbrial CF29K, aggregative adhesion and KPF-28 fimbriae (Koczura and Kaznowski,
2003). The non-fimbrial R-plasmid-encoded CF29K adhesin is known to mediate adherence
to the human intestinal cells lines Intestine-407 and CaCo-2. Non-fimbrial adhesin consists
of capsule-like extracellular material that mediates adherence pattern described by
aggregative adhesion to intestinal cell lines. Lastly, the fimbrial KPF-28 produces the CAZ-
5/SHV-4 type ESBL (Podschun and Ullman, 1998).
2.4.2.3 Lipopolysaccharide
Three distinguishable sections such as the lipid A, the core polysaccharide and the
side chain O-antigen (O-Ag) polysaccharide comprise the lipopolysaccharide (LPS) molecule
which is with eight serotypes and are associated with resistance to complement-mediated
killing. Particularly, the lipid A attaches the LPS molecule into the outer membrane. It also
serves as an endotoxin which stimulates the immune system through agonism of Toll-like
receptor 4 (TLR4) present on macrophages, dendritic cells and other cell types inducing
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) mediated production
of cytokines. The negatively charged core polysaccharide likewise links the O-Ag onto the
lipid A molecule. Finally, the O-Ag forms a polysaccharide layer covering up to 30 nm into
the surrounding media (Younes, A.M., 2011).
2.4.2.4 Other factors
Siderophores are high-affinity, low-molecular-weight iron chelators that solubilize
and import the required iron bound to host proteins. Phenolates or enterochelin/enterobactin

26
and hydroxamates or aerobactin are the two different groups of siderophores prominent in the
genus Klebsiella. The former is found to be produced by all strains as opposed to the few
that can only produce the latter (Koczura and Kaznowski, 2003; Younes, A.M., 2011).
2.4.3 Typing
Typing is being done to obtain information about endemic and epidemic outbreaks of
Klebsiella infections and to determine the clonality of the strains. The two typing methods
include the phenotypic or molecular typing. Explicitly, phenotypic typing can be done
through biotyping, phage typing, bacteriocin typing or serotyping. On the other hand,
molecular typing methods are used to determine bacterial strains or clones and are further
subdivided to protein based method such as sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) which is proven effective by Costas, et al (1990) when
comparable to capsular serotyping or nucleic acid based methods such as PCR amplification
and sequencing, pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic
DNA (RAPD), restriction fragment length polymorphism (RFLP), multilocus sequence
typing (MLST) and repetitive sequence-based PCR (rep-PCR) (Younes, A.M., 2011).
Additionally, biotyping is based on biochemical reactions and environmental
tolerance with the use of automated instruments such as API 20E systems with macrotube
tests. Unfortunately, identification to the species level is often difficult due to the similarity
of biochemical profiles making it of little use to epidemiological studies and only appropriate
for smaller laboratory setups. Phage typing is based on the receptiveness of bacterial strains
to a group of bacteriophages. It has never been used extensively starting from its
establishment in 1964 because of its poor typing rate due to the lack of standardization and

27
inoculum concentration, the limited availability and stability of bacteriophages needing
maintenance and evaluation from time and again. Supplementary, bacteriocin typing makes
use of protein-based bactericidal substances produced by bacteria to inhibit the growth of
other bacteria of the same species through inhibition of protein and nucleic acid synthesis and
uncoupling of electron transport from active transport of thiomethyl-ß-Dgalactoside and
potassium. Lastly, seroptying is the reaction of the surface-exposed antigen determinant such
as the capsule to a specific antiserum (Younes, A.M., 2011). Although it is the predominant
method in typing Klebsiella species now, it has disadvantages such as the occurrence of large
number of serological cross-reactions among the 77 capsule types, the weak reaction due to a
weak antigen which affects interpretation, the huge amount of time consumed, the scarcity of
commercially available anti-capsule antisera, and the occurrence of non-typable isolates
(Podschun and Ullmann 1998).
Molecular methods were developed to address the numerous concerns regarding
phenotypic typing. PFGE, which may be used for genotyping or genetic fingerprinting, is
considered the gold standard in epidemiological studies of pathogenic organisms as it can
detect chromosomal rearrangements by mobile elements with swift evolutionary rates. To
establish taxonomic identity, evaluate kinship relationships, investigate mixed genome
samples, and generate specific probes, RAPD is the method of choice as it makes use of low-
stringency PCR amplification with single primers of random sequence to produce strain-
specific arrays of anonymous DNA fragments. gyrA PCR-RFLP using restriction enzymes
HincII, TaqI and HaeIII of the 441-bp fragment of the gyrA gene, and the 940-bp fragment of
the RNA polymerase beta subunit gene (rpoB) can be used as well to confirm identified
isolates of Klebsiella pneumoniae. Moreover, MLST is set to describe the genetic
relationships among bacterial isolates and is more appropriate for strain phylogeny and large-
scale epidemiology. Last of all, rep-PCR is a quick method for strain typing and description

28
of bacteria by using primers targeting noncoding repetitive elements interspersed throughout
the bacterial genome (Younes, A.M., 2011).
2.4.4 Antimicrobial resistance
Emergence of nosocomial multidrug-resistant Klebsiella pneumoniae (MRKP) and
ESBL-producing strains have been observed since 1983 followed by the emergence of
resistant strains to third-generation cephalosphorins since 1990 (Parasakthiet al., 2000 and
Younes, A.M., 2011). Extended-spectrum β-lactamases (ESBL) are plasmid-mediated
multiple antimicrobial resistance enzymes that can be spread horizontally to recipient
microorganisms. It can hydrolyze broad-spectrum cephalosporins and monobactams and
cannot be detected on routine antimicrobial susceptibility testing resulting to poor clinical
outcome (Mosqueda-Gomez et al., 2008) although can be hindered by β-lactamase inhibitors
such as clavulanic acid (Younes, A.M., 2011).
Its molecular classification depends on their amino acid homology namely classes A,
B, C and D as proposed by Russell Ambler (Jeong et al., 2004; Younes, A.M., 2011) or on
substrate and inhibitor profile namely groups 1, 2, 3 and 4 as proposed by Bush-Jacoby-
Medeiros as listed on Table 1 (Younes, A.M., 2011).
Table 1. β-lactamase classification schemes.
Ambler
class
Bush-
Jacoby
group
Distinctive
substrates
Inhibited by Representative
enzymes
CA /
TZB
EDTA
C 1 Cephalosporins - - AmpC, P99, ACT-1,
CMY-2, FOX-1, MIR-
1
C 1e Cephalosporins - - GC-1, CMY-37
A 2a Pencillins + - PC1
A 2b Pencillins,
early
+ - TEM-1, TEM-2,
SHV-1

29
cephalosporins
A 2be Extended-
spectrum
cephalosporins,
monobactams
+ - TEM-3, SHV-2, CTX-
Ms, PER, VEB
A 2br Penicillins - - TEM-30, SHV-10
A 2ber Extended-
spectrum
cephalosporins,
monobactams
- - TEM-50
A 2c Carbencillin + - PSE-1, CARB-3
A 2ce Carbencillin,
cefepime
+ - RTG-4
D 2d Cloxacillin V - OXA-1, OXA-10
D 2de Extended-
spectrum
cephalosporins
V - OXA-11, OXA-15
D 2df Carbapenems V - OXA-23, 0XA-48
A 2e Extended-
spectrum
cephalosporins
+ - CEPA
A 2f Carbapenems V - KPC-2, IMI-1, SME-1
B 3a (B1) Carbapenems - + IMP-1, VIM-1, IND-
1, CcrA
(B2) L1, CAU-1, GOB-1,
FEZ-1
B 3b (B3) Carbapenems - + CphA, Sfh-1
Unkown 4 -
ESBLs have various types such as those of class A like TEM and SHV types which
are more associated to hospital-acquired infections and have evolved from narrow-spectrum
β-lactamases such as TEM-1, -2 and SHV-1; PER type which denotes resistance to
oxyimino-β-lactams and are mostly restricted to South America and Europe so far (Paterson
et al., 2003), and CTX-M type enzymes identified mainly as ciprofloxacin resistant causing
community-acquired urinary tract infections (Pitout et al., 2005).

30
The TEM family of ESBLs which name came from the patient Temoniera, is the
largest and widely spread. Its plasmid mediated TEM-1 was first discovered in 1965 and is
the most prevalent in enteric bacilli such as Klebsiella pneumoniae and in other Gram-
negative bacteria. It is encoded by a series of gene alleles, blaTEM-1A to blaTEM-1F, differing
from each other by specific silent mutations. Although not as common, TEM-2 being the
first derivative of TEM-1, encoded by blaTEM-2 possesses a stronger promoter than that of the
blaTEM-1 gene giving a higher enzymatic activity as compared to TEM-1 producing strains
(Younes, A.M., 2011).
Moreover, the SHV (sulfhydryl variable) enzymes are categorized in Ambler class A
and in groups 2b and 2be of the Bush-Jacoby-Medeiros classification scheme. Specifically,
SHV-1 was first reported in 1972 and named Pit-2 after its discoverer Pitton. It denotes
resistance to ampicillin, amoxicillin, carbenicillin and ticarcillin and encoded by gene alleles
blaSHV-1 or blaSHV-11 which are prevalent in Klebsiella pneumoniae strains and is behind
approximately 20% of the plasmid-mediated ampicillin resistance in this species. Such genes
are possibly mobilized from genome to plasmid as facilitated by IS26 insertion which was
identified into the blaSHV promoter particularly in plasmid-mediated SHV-2a, SHV-11 and
SHV-12. There are only a few SHV that signify resistance to ß-lactamase inhibitors as
opposed to TEM ß-lactamases (Younes, A.M., 2011). The β-lactamases of ceftazidime-
resistant Klebsiella pneumoniae strains are usually of the SHV-5 type in Europe and TEM-10
and TEM-12 types in the United States (Podschun and Ullman, 1998).
Last of those belonging to class A, the CTX-M type ß-lactamases (active on
cefotaxime) were first discovered in Japan in 1986. They are further subcategorized in 5
subgroups namely CTX-M-1, CTX-M-2, CTXM-8, CTX-M-9 and CTX-M-25. Over time,
they have become more predominant than TEM and SHV type ß-lactamases in Africa,
Europe, South America and Asia mainly due to their mode of acquisition of horizontal gene

31
transfer from other bacteria and to the ability of insertion sequences such as ISEcp1, ISCR,
IS26, IS10 and IS903, phage-related elements and plasmids, to facilitate and induce the
expression of ß-lactamase genes (Pitout et al., 2005 and Younes, A.M., 2011). The genes
responsible for CTX-M ß-lactamases are encoded by plasmids belonging to the narrow host-
range incompatibility types (IncFI, IncFII, IncHI2 and IncI) or the broad host-range
incompatibility types (IncN, IncP1, IncL/M and IncA/C). The CTX-M enzymes depict
higher level resistance to cefotaxime, ceftriaxone and aztreonam than to ceftazidime and are
susceptible to ß-lactamase inhibitors, although a low-level of resistance to the combination of
clavulanic acid with amoxicillin and ticarcillin could be experienced (Younes, A.M., 2011).
Furthermore, class B enzymes termed ‘metallo-ß-lactamases’ were first distinguished
in 1980 again by Russell Ambler. They hydrolyse penicillin, cephalosporins and
carbapenems but not monobactams. They are EDTA-inhibited enzymes and are resistant to ß-
lactamase inhibitors. They are further subdivided on the basis of sequence alignments into
three subclasses B1, B2 and B3. AmpC type enzymes belonging to class C are named
according to the resistance produced, type of enzyme, site of discovery or patient’s name.
These include CMY-1 (cephamycin resistance), MOX-1 (moxalactam resistance), FOX-1
(cefoxitin resistance), LAT-1 (latamoxef resistance), ACT-1 (AmpC type enzyme), MIR-1
(Miriam Hospital, Providence) and ACC-1 (Ambler class C enzyme) (Jeong et al., 2004;
Younes, A.M., 2011). They have emerged due to the ongoing use of 7--methoxy-
cephalosporins (cefoxitin and cefotetan) and ß-lactamase inhibitor combinations (clavulanate,
sulbactam or tazobactam) with amoxicillin, ticarcillin, ampicillin, or piperacillin (Younes,
A.M., 2011) which lead to the resistance to many β-lactam antibiotics like cephamycins,
extended-spectrum cephalosporins (Jeong et al., 2004) and ß-lactamase inhibitor-ß-lactam
combinations. They are usually chromosomal such as FOX-1 and MOX-1 but can also be
plasmid-encoded such as MIR-1, CMY-1 and CMY-2. The continued spread of AmpC

32
enzymes globally may be attributed to the association of mobile elements such as ISEcp1,
ISCR1 or IS26 to the latter. Finally, OXA ß-lactamases belong to Ambler class D (2d) which
attack the oxyimino-cephalosporins and have a high hydrolytic activity in opposition to
oxacillin, methicillin and cloxacillin more than benzylpenicillin. They are inhibited by NaCl
and less efficiently by clavulanalic acid. Contrary to class C, OXA ß-lactamases are typically
plasmids incorporated as gene cassettes in integrons than chromosomal encoded. They are
often not considered as ESBLs as they do not hydrolyze the extended-spectrum
cephalosporins (Younes, A.M., 2011).
As presented on Table 2, nosocomial Klebsiella pneumoniae isolates are resistant to
ampicillin, gentamicin, amikacin, trimethoprim-sulfamethoxazole, cefuroxime, cefotaxime,
ceftriaxone, cefoperazone, and ceftazidime but susceptible to imipenem and ciprofloxacin
(Parasakthi et al., 2000). Studies of Macrae et al (2001) and Mena et al (2006) showed
similar results on human isolates adding resistance to tetracycline but still showed differently
as it was susceptible to imipenem, aztreonam and ciprofloxacin. In bovine milk, their isolates
were resistant to penicillin, cloxacillin, ceftiofur, gentamicin, tetracycline, trimethoprim-
sulfonamide and enrofloxacin. In addition, those isolates of Timofte et al (2014) taken from
bovine milk showed resistance to penicillin G, amoxicillin-clavulanic acid, co-trimoxazole,
neomycin, streptomycin, tylosin, ceftiofur, cefquinome and cefpodoxime and were
susceptible only to framycetin. On the other hand, the animal isolates of Brisse and Duijkere
(2005) showed susceptibility to ceftazidime, ceftiofur, tetracycline, enrofloxacin, gentamicin
and trimethoprim-sulfamethoxazole but were resistant also to ampicillin and cephalexin.
Most of their isolates also showed multi-drug resistance. Moreover, Mosqueda-Gomez et al
(2008) demonstrated that there are higher resistance rates in ESBL-Kp to aminoglycosides,
quinolones, ticarcillin/clavulanate, and piperacillin/tazobactam but susceptible to imipenem.

33
ESBL production can be determined through the use of ESBL E-test screen strips
impregnated with ceftazidime and ceftazidime-clavulanate wherein a positive test denotes
ceftazidime MIC/ceftazidime-clavulanate MIC ratio to be ≥8 (Podschun and Ullman, 1998;
Afifi, 2013). Disc diffusion method using the discs impregnated with the aforementioned
antibiotics can be used as well although double disc synergy method is more widely
employed to detect synergy between cefotaxime and clavulanate exemplified by a clear-cut
extension of the edge of cefotaxime inhibition zone toward the disc containing clavulanic
acid. This is done by placing a disc of amoxicillin-clavulanic acid and a disc of cefotaxime
30 mm apart strategically placed center to center and is considered ESBL positive when there
is decreased susceptibility to cefotaxime combined with synergy between cefotaxime and
amoxicillin-clavulanic acid (Younes, A.M., 2011).
Fig 1. Combination disc method showing synergy
between cefotaxime, ceftazidime and amoxicillin-
clavulanate (amoxiclav). The right disc is
cefotaxime, the left is ceftazidime. Amoxiclav
disc is in middle.
Fig. 2. Confirmation of ESBLs production by
double disc diffusion method. The plate shows
that the inhibition zone around cefotaxime-
clavulanate (left disc) is more than 5 mm of
cefotaxime (right disc).
33
disc is in middle.
33
disc is in middle.

34
Table 2. List of more recent AMR cases of Klebsiella pneumoniae
Country Samples AMR Authors
China Cooked meat
products
tetracycline
Jiang and Shi, 2013
trimethoprim
sulphonamide
India
hospital
patients
cephalosporins
Parasakthi et al., 2000ampicillin
aminoglycosides
trimethoprim
sulfamethoxazole
Australia hospital
patients
gentamicin
Jones et al., 2005tobramycin
kanamycin
streptomycin
spectinomycin
Mexico
hospital
patients
aminoglycosides
Mosqueda-Gomez et al
(2008)
quinolones
ticarcillin/clavulanate
piperacillin/
tazobactam
Italy
human
samples;
bovine milk
tetracycline
Macrae et al., 2001;
Mena et al., 2006
penicillin
cloxacillin
cephalosporins
aminoglycosides
trimethoprim
sulfamethoxazole
fluoroquinolone
UK bovine milk
penicillin
Timofte et al., 2014
amoxicillin-
clavulanate
co-trimoxazole
neomycin
streptomycin
tylosin
cephalosporins
France animal
ampicillin Brisse and Duijkere,
2005
cephalosporins

35
2.4.5 Genetics of Antimicrobial resistance
Horizontal transfer through the mobile gene cassettes enhances the spread of
antimicrobial resistance genes through mobilization of individual cassettes by the integron-
encoded integrase, migration of the cassette in the integron probably by targeted tranposition,
distribution of larger transposons such as Tn21 carrying integrons and relocation of
conjugative plasmids with integrons among different bacterial species (Levesque et al., 1995;
White, et al., 2001). There are four classes of integrons namely classes 1, 2, 3 and 4 which
are differentiated by their respective integrase (int) genes (White, et al., 2001). They possess
two conserved segments that is the 5’ and the 3’, separated by a variable region with
integrated antibiotic resistance genes or cassettes. The 5’ conserved segment contains the int
gene while the 3’ conserved segment contains an open reading frame (ORF) termed orf5 and
the qacE∆1 and sulI which establish resistance to ethidium bromide and quaternary
ammonium compounds and to sulfonamide, respectively (Levesque et al., 1995).
Genes found on the mobile genetic elements such as the bacterial chromosome, plasmids,
transposons or integrons, encode ESBLs enabling the spread of β-lactamases to other
members of the Enterobacteriaceae family and increasing the incidence of multi-drug
resistant bacteria with complex resistance patterns to aminoglycosides, trimethoprim,
sulphonamides, tetracyclines, chloramphenicol and recently, to quinolones specifically
nalidixic acid (Pitout et al., 2005 and Younes, A.M., 2011). The most common ESBL
phenotypes come from point mutations in the blaTEM, blaSHV or blaCTX genes which happen
regularly at position 104 (TEM), 146 (SHV), 156 (SHV), 164 (TEM), 167 (CTX-M), 169
(SHV), 179 (SHV and TEM), 205 (TEM), 237 (TEM), 238 (SHV and TEM) and 240 (TEM,
SHV and CTX-M), leading to changes in the primary amino acid sequence of the enzyme
(Younes, A.M., 2011). blaCTX-Mgenes are usually involved with sul1-type class 1 integrons

36
known to harbor antimicrobial resistance gene casettes resistant to β-lactams,
aminoglycosides, chloramphenicol, sulphonamides and in a lower level, rifampicin.
Specifically, blaCTX-M-14gene is linked with insertion sequence ISEcp1 which is responsible
for mobilization and high-level expression of the β-lactamase gene (Pitout et al., 2005).
Other Klebsiella pneumoniae ESBL genes include blaoxa, blaAMPC (Timofte et al., 2014),
blaPER, blaVER, (Nobrega et al., 2013), blaCMY-1, blaFOX, blaMox, blaMIR, blaACT, blaToho (Lee et
al., 2000) and blaNDM genes which are associated with metallo-β-lactamase 1 (NDM-1)
(Yong et al., 2009 and Giske et al., 2012).
A study by Paterson et al. (2003) identified CTX-M-type ESBL-producing Klebsiella
pneumoniae isolates in Taiwan, Australia, South Africa, Turkey, Belgium and Argentina but
not United States. SHV and TEM type β-lactamases were seen in Australia, South Africa,
Turkey, Argentina and United States but not Taiwan and Belgium. Lastly, PER-1-type β-
lactamases were found in isolates from Turkey alone although previous study denotes its
detection in South America. In milk, Nobrega et al. (2013) noted that earlier studies done by
Hammad et al. (2008) and Locatelli et al. (2009) already detected TEM and SHV enzymes in
ESBL bacteria causing intramammary infections in dairy herds while his study was the first
to report detection of blaCTX-M gene in Klebsiella pneumoniae isolated from bulk tank milk.
It can be then noted that SHV and TEM type β-lactamases are already predominant
worldwide and CTX-M and PER-1 types are increasingly emerging in various countries
(Paterson et al., 2013).
Aminoglycoside resistance genes include aadB gene, which denotes resistance to
gentamicin, tobramycin and kanamycin and aadA1 and aadA2 genes which relate resistance
to streptomycin and spectinomycin (Jones et al., 2005). The Klebsiella pneumoniae isolates
of Jiang and Shi (2013) obtained dfrA6 and dfrA12 and sul1 genes associated with

37
trimethoprim and sulphonamide resistance respectively. The same study also discovered
tetracycline resistance genes such as tetA which is linked with ribosomal protection and/or
efflux pump mechanism, tetB and tetM which are associated with efflux pump mechanism
only (Ng, et al., 2001).
Quinolone resistance genes which are plasmid-mediated include qnr gene, composed
of qnrA, qnrB, qnrS, qnrC and qnrD, which encodes a protein protecting type II
topoisomerase increasing its MICs to nalidixic acid and flouroquinolones by four to eight
times (Nazik et al, 2011; Younes, A.M., 2001; and Ruiz et al, 2012). qnrA and qnrB genes
had been located in complex In4 family class 1 integrons In36 and In37 also known as
complex sul1-type integrons which may serve as a recombinase for mobilization of CTX-M
and ampC (Wang et al., 2004 and Younes, A.M., 2011). They were first reported in 1998
from Klebsiella pneumoniae clinical isolates in the USA (Cattoir, et al., 2007) followed by
Canada, Asia, Australia, Turkey and Europe. On the other hand, qnrS genes were reported to
be connected to Tn3-like blaTEM-1-containing transposon and not like as a gene cassette in a
common class 1 integron. They were found in Shigella flexnri isolates in Japan. Lastly,
qnrC and qnrD genes were discovered in China in isolates of Proteus mirabilis and
Salmonella enterica respectively (Younes, A.M., 2011). Other plasmid-mediated quinolone
resistance genes include aac(6’)-Ib-cr gene which encodes an aminoglycoside
acetyltransferase convening reduced susceptibility to aminoglycosides and ciprofloxacin
(Nazik et al, 2011 and Ruiz et al, 2012) and qepA gene which involves active efflux pumps
namely OqxAB multidrug efflux pump related to reduced fluoroquinolone susceptibility, and
QepA efflux pump pertaining to decreased susceptibility to hydrophilic flouroquinolones
such as norfloxacin and ciprofloxacin (Nazik et al, 2011).

38
2.4.6 Virulence genes
Mucoviscosity-associated gene A (magA) is an important virulence gene present only
in serotype K1 K. pneumoniae. It is associated the hypermucoviscosity phenotype and also
played an important role in resistance to serum and phagocytosis (Chuang, et al., 2006;
Nadasy, et al., 2007). Contrary to previous knowledge as suggested by Fang et al (2004), it
is the capsular serotype K1 and not the magA gene that is responsible for the majority of the
clinical K. pneumoniae liver abscess cases observed by Yeh et al (2006) and Brisse, et al
(2009). Figure 3 shows us the gene clusters found in serotype K1 K. pneumoniae. The
regulator of mucoid phenotype A (rmpA) is plasmid-mediated managing the extracapsular
polysaccharide synthesis (Nadasy, et al., 2007; Brisse, et al., 2009; Giske et al., 2012). It
was first described in 1989 but was only established recently to be involved with the
hypermucoviscosity phenotype and with the invasive clinical syndrome (Nadasy, et al.,
2007).
Fig. 3. Gene cluster for K1 capsular polysaccharide (GenBank accession no. AY762939),
indicating genes with known and unknown functions (Yeh et al2006)

39
Other than that, wzy gene family inputs an O-polysaccharide polymerase that
identifies and expands the O-antigen polysaccharide-repeating units. This was also thought
responsible for lipid-linked repeat unit polymerization in the capsular synthesis process of
K57 of whose deletion would lead to diminished mucoviscosity. galF, ORF2 and gnd are
regarded to be associated with carbohydrate metabolism; wzi (orfX), wza , wzb and wzc are
deemed responsible for the translocation and surface assembly of the capsule (Chuang, et al.,
2006; Pan et al., 2008). Other virulence genes include allS which stimulates growth in iron-
deficient media, codes for activator of the allantoin regulon and specific for K1 pyogenic
liver abscess (PLA) (Brisse, et al., 2009), wcaG which synthesizes fucose needed to escape
phagocytosis (Brisse, et al., 2009; Giske et al., 2012), mrkD coding for the type 3 fimbriae
adhesin responsible for the adhesion to the basement membranes of several human tissues
(Brisse, et al., 2009; Younes, A.M., 2011), kfu being the iron uptake marker, cf29a, fimH,
uge, wabG, and ureA (Brisse, et al., 2009).

40
MATERIALS AND METHODS
3.1 Study Area
The Bureau of Agricultural Statistics (BAS) states that in 2009, there are already
about 15,073 dairy cattle and cow fresh milk production amounted to 8.6 million liters
(Villareal, 2009). Presently, the national cow fresh milk production is now at 20.01 million
liters. Specifically last year, South Luzon produced 40.2% valued at Php162.3 million. In
particular, Batangas produced more than half of South Luzon’s milk production. Milk
producers vary from the cooperative farms (63.0%), individual farms (19.3%), commercial
farms (12.1%), and institutional farms (5.6%) (NDA, 2015).
The study shall be carried out in dairy cattle farms in Batangas and the laboratory
work shall be done in the microbiology and molecular biology laboratories of Department of
Paraclinical Science, College of Veterinary Medicine, University of the Philippines Los
Banos from December 2015 to August 2016. The cows will be handled according to RA 8485
“The Animal Welfare Act of 1998” (Appendix 2) and the Animal Welfare Code (2011) Good
Agricultural and Husbandry Practices (GAHP) set by the Bureau of Agriculture and Fisheries
Product Standards (BAFS). The laboratory work shall conform to the standards of the
National Mastitis Council (NMC) and Performance Standards for Antimicrobial
Susceptibility Testing; 22ND
Informational Supplement of the Clinical and Laboratory
Standards Institute (CLSI).
The list of dairy cattle farms in Batangas and their respective herd population will be
obtained from the National Dairy Authority. The selected South Luzon dairy zone (Fig. 4)
was selected due to its greatest contribution to the national dairy industry in terms of highest
density of cattle, greatest number of high producing cattle, and highest milk production
(NDA, 2015). The altitude of Batangas ranges from approximately 80 m to 360 m. The

41
average ambient temperature and relative humidity in Batangas are approximately 25 °C and
78 % respectively. The annual average rainfall is 1767 mm being climate type I having only
two seasons such as the dry season from November to April and wet season from May to
September (PAGASA, 2016). Farms and farm associations to be included in the study will
be selected randomly. Individual farms in each included farm association will be selected
randomly (Furgasa, et al., 2010). To be qualified, set inclusion criteria for each farm include
good record-keeping and history of recurrent bovine clinical mastitis cases.
Records of daily milk production and clinical mastitic cases and their respective
treatment for at least a year prior to the start of the study will be examined. Government
standardized milking protocols, post milking teat disinfection, pre-dipping or pre-wiping
factors (Furgasa, et al., 2010; Swinkels, et al., 2013) and mastitic cases monitoring including
mastitis diagnostic tests, treatment and antibiotic sensitivity tests will be inspected if being
practiced in the farm in all cows throughout the lactation (Swinkels, et al., 2013). Moreover,
other factors such as farmers’ education, frequency of personnel and environment cleaning
and disinfection will also be looked into (Gunawardana, et al, 2014). As much as possible,
milking procedures and equipment management will not change during the study period
(Swinkels, et al., 2013).
3.2 Study Animals (Lactating cattle)
Holstein-Friesian crossbred lactating cattle suffering from subclinical and clinical
mastitis in at least one teat will be used in this study and will be chosen randomly. A
combination of concentrates and forage feed will be made available to feed the study animals.
Drinking water will be made available ad libitum. The cows will be managed under either a
small scale or a semi-intensive management system (Furgasa, et al., 2010). Source animals

42
will be pooled in one pen so cleaning and feeding will be organized so as to prevent cross
contamination effectively throughout the course of the study. Pertinent data to be taken for
each cow include age, average milk production (L), lactation number, days in milk, present
lactation total, past milk production average (L), past lactation total, mastitis history, mastitis
therapy, other disease treatment history, dry cow therapy and other relevant clinical data.
These will be recorded onto the respective form or logbook, electronic report or on-farm
software (Swinkel et al, 2013).
3.3 Research design
This study is of a cross-sectional study design that mainly aims in assessing the
prevalence of Klebsiella pneumoniae (Tenhagen, et al., 2006) in bovine milk and
understanding its antimicrobial resistance, genetic characterization and risk factors.
Fig. 4 Philippine map showing Batangas and its various cities
and municipalities

43
3.3 Sample size
The sample size was identified using the OpenEpi version 2.3.1. The total number of
sample units (lactating animals) to be used in this study will be calculated based on 37% cow
prevalence of mastitis (Gunawardana, et al, 2014) with 5% confidence limit and 95%
confidence level. To avoid confounding and to further increase its power, an additional of
20% will be added to have a sample size of 233. Assuming that mortality rate of 4.8% is
expected (McConnel, et al., 2008), an additional of 4.8% will be added to have a final sample
size of 244 (Israel, D.G. 2013).
3.4 Clinical mastitis screening
California mastitis test (CMT) together with physical examination will be done to
screen mastitis and differentiate patients from subclinical to clinical cases (Ruegg, P.L, 2005;
Safi, et al., 2009; Furgasa, et al., 2010; Gunawardana, et al, 2014) (Appendix 3). The
severity of mastitis shall be classified with the following scores as listed on Table 3: Negative
(N), no infections due to no thickening of the mixture which is estimated to be 100,000
somatic cell count (SCC); Trace (T), possible infections due to slight thickening of the
mixture with estimated 300,000 SCC which seems to disappear with continued paddle
rotation. If all quarters sampled read trace, there is no infection but if one or two quarters
read trace, there is possible infection. Other scores include Grade 1 (weak positive), mild
infection with only clots in the milk due to distinct thickening of the mixture but no tendency
of gel formation; Grade 2 (distinct positive), moderate infection indicative of immediate
thickening of the mixture, with a slight gel formation estimated to be 2,700,000 SCC leading
to milk changes also in colour and/or presence of clots, heat, pain and/or swelling of the

44
udder; and Grade 3 (strong positive), severe infection indicative of gel formation and
elevation of surface of mixture with central peak remaining projected even after the rotation
of CMT paddle has stopped further leading to milk changes in colour and/or presence of clots
and systemic signs such as fever, depression, anorexia and very swollen udder (Ruegg, P.L,
2005; Furgasa, et al., 2010; Swinkel et al., 2013).
Table 3. California Mastitis Test (CMT) scores (Ruegg, P.L;, 2005)
CMT score Somatic Cell Range Interpretation
N (negative) 0-200,000 Healthy quarter
T (trace) 200,000-400,000 Subclinical mastitis
1 400,000-1,200,000 Subclinical mastitis
2 1,200,000-5,000,000 Serious mastitis infection
3 Over 5,000,000 Serious mastitis infection
3.5 Sample Collection
10 mL milk samples shall be obtained according to the standards of National Mastitis
Council (1999) a day after CMT screening from identified subclinical and clinical mastitic
cows which did not receive any antibiotic treatment at least one week prior to collection in
accordance to the respective milk withdrawal period of each antibiotic being used at the farm
(Furgasa, et al., 2010). Samples shall be collected aseptically and stored in sterile 10 mL
glass tubes with screw cap and kept in ice at approximately 4ºC during transport to the
laboratory. Pertinent data shall be obtained.
3.6 Bacterial isolation and identification
Samples will be cultured and bacteria that had grown will be identified using rapid
laboratory techniques (NMC, 1999). 10µL of milk will be inoculated onto trypticase soy

45
agar plate supplement with 5% defribinated bovine blood (Gillespie, B.E. and Oliver, S.P.,
2005; Furgasa, et al., 2010) and onto McConkey agar (Younes, A.M., 2011) before
incubation at 37ºC overnight. Growing bacteria will be identified by colony morphology and
by using a microtube identification system API Rapid 20 E® (API System, France) which is
a useful first stage in determining Gram negative bacteria (Younes, A.M, 2011). Individual
identified bacterial isolates of Klebsiella pneumoniae will be streaked in nutrient dish agar
before incubation at 37ºC overnight. 3 separate colonies will be chosen, suspended in Luria-
Bertani (LB) broth with 20% glycerol and will be stored in Eppendorf tubes at -80ºC (Paulin-
Curlee, G.G. et al., 2007; Yamane, K, et al., 2008)).
3.7 Molecular serotyping
Serotyping of K1, K2, and K5 will be done through multiplex Polymerase Chain
Reaction (PCR). 3 colonies from each positive sample will be taken to determine the various
serovars. As recommended by EU, one Klebsiella isolate will be collected for each serotype
from each positive sample which then will give the actual number of isolates. DNA
extraction of the Klebsiella isolates will be done through the boiling method (Appendix 4) as
done by Yeh et al (2007) and described by Levesque et al (1995). The PCR reactions will be
composed of 5µL of 2X Reddymix® PCR MasterMix (0.625 units Taq polymerase, 1.5nM
MgCl2 and 0.2mM each of dNTP/reaction), 0.5µL of each primer (10µM), 2.5µL of DNA
template and 4µL of double distilled water. The PCR conditions for K1 (1283 bp), K2 (641
bp) and K5 (280 bp) will be an initial denaturation at 94ºC for 1 minute, and 30 cycles each
of denaturation at 94ºC for 30 seconds, primer annealing at 59ºC for 45 seconds and

46
extension at 72ºC for 1 minute & 30 seconds and one cycle of final extension at 72ºC for 6
minutes (Turton et al., 2008).
Non-K1/K2 isolates will be serotyped by determination of the prevalence of rmpA
(516 bp) through PCR of which conditions will be an initial denaturation at 95ºC for 5
minutes, and 40 initial cycles each of denaturation at 95ºC for 60 seconds, primer annealing
at 50ºC for 60 seconds and extension at 72ºC for 2 minutes and one cycle of final extension at
72ºC for 7 minutes (Yeh et al., 2007). PCR amplification will be performed using a PCR
Swift Maximodl (Esco®, South Yorkshire, UK). PCR amplicons will be separated using
1.5% agarose gel electrophoresis (Major Science, Saratoga, CA, USA) in 1X Tris-
acetate/EDTA (TAE) buffer. Gel staining will be done by soaking in an ethidium bromide
solution (Sigma-Aldrich®) for 10 minutes and destaining in distilled water for 5 minutes.
The gels will be digitally photographed under UV light. The primers used in typing are listed
in Appendix 5.
3.8 Antibiotic susceptibility & ESBL production testing
To determine the minimum inhibitory concentration (MIC), microbroth dilution as the
method of choice (Tenhagen, et al., 2006) (Appendix 6) will be done conforming to the
Performance Standards for Antimicrobial Susceptibility Testing of the Clinical and
Laboratory Standards Institute. Various classes of antibiotics being used in bovine mastitis
(Appendix 7) and in humans such as amoxicillin-clavunalate (AMC), ampicillin (AMP),
ceftiofur (CEF), ciprofloxacin (CIP), cloxacillin (CLX), enrofloxacin (ENR), gentamicin
(GEN), penicillin (PEN), streptomycin (STR), sulfamethoxazole (SUL), tetracycline (TET)
and trimethoprim (TRI) will be used for this study (CLSI, 2012). Reference strain used to

47
serve as quality control will be Klebsiella pneumoniae ATCC 700603 (Mosqueda-Gomez, et
al., 2008), Staphylococcus aureus NCTC 6571, Escherichia coli NCTC 10418 and
Pseudomonas aeruginosa NCTC 10662 (Younes, A.M., 2011).
ESBL production will be done using microbroth dilution compliant to the guidelines
from CLSI (2012). Any isolate with a ceftazidime/ ceftiofur MIC >1µg/mL will be suspected
of having ESBLs thus E-test will be done to ceftazidime alone and in combination with
clavulanic acid (AB Biodisk, Solna, Sweden). A decrease of >3-fold in the MIC value for
ceftazidime in combination with clavulanic acid versus the MIC value for ceftazidime alone
will be considered as confirmation of ESBL production (Mosqueda-Gomez, et al., 2008).
3.9 Characterization of class 1 integron and test for transferability
All isolates will be screened for the presence of the integrase gene, intI1 (254 bp)
using polymerase chain reaction (PCR). The PCR reactions composed of 5µL of 2X
Reddymix® PCR MasterMix (0.625 units Taq polymerase, 1.5nM MgCl2 and 0.2mM each of
dNTP/reaction), 0.5µL of each primer (10µM), 1µL of DNA template and 8µL of double
distilled water. The PCR conditions will be an initial denaturation at 94ºC for 4 minutes, and
10 cycles each of denaturation at 94ºC for 60 seconds, primer annealing at 65ºC for 30
seconds (decreasing 1ºC/cycle) and extension at 70ºC for 2 minutes, 24 cycles of 94ºC for 60
seconds, 55ºC for 30 seconds and 70ºC for 2 minutes, and one cycle of final extension at
70ºC for 5 minutes (Murinda et al., 2005). PCR amplicons will be separated using 1%
agarose gel electrophoresis (Esco®, South Yorkshire, UK) in 1X Tris-acetate/EDTA (TAE)
buffer.

48
Gene cassettes (1000 bp) will be screened on any of the isolates containing int1 gene
using PCR with a specific primer pair 5’CS and 3’CS. The PCR reactions composed of 5µL
of 2X Reddymix® PCR MasterMix (0.625 units Taq polymerase, 1.5nM MgCl2 and 0.2mM
each of dNTP/reaction), 0.5µL of each primer (10µM), 1µL of DNA template and 8µL of
double distilled water. The PCR conditions will be an initial denaturation at 94ºC for 12
minutes, and 35 cycles each of denaturation at 94ºC for 60 seconds, primer annealing at 55ºC
for 60 seconds and extension at 72ºC for 5 minutes with five seconds to be added to the
extension time at each cycle, and one cycle of final extension at 72ºC for 5 minutes
(Levesque et al, 1995). The PCR products will be subjected to purification using Nucleospin
Gel Extension Kit (Nucleospin®, Gutenberg, France) and sent for DNA sequencing to
Macrogen, South Korea. DNA sequences will be compared with the published sequence
using NCBI blast search available at the National Center for Biotechnology Information
website (www.ncbi.nlm.nih.gov). Restriction enzymes such as EcoRI, Alul and Taql will be
used to digest any PCR products with the same size and will be considered identical if they
show the same restriction patterns (Wannaprasat, 2012). The primers used are listed in
Appendix 8.
Conjugation studies as described by Wang et al (2004) (Appendix 9) will be done to
all isolates carrying class 1 integrons with resistance gene casettes which are to be used as
donors and E. coli J53 AzR
derivatives to be used as recipients. Transconjugants will be
screened on presence of blaCTX-M, blaSHV,blaTEM through PCR. All PCR products obtained
for this screening will be sent for DNA sequencing on both 5’ and 3’ strands and will be
BLAST compared with those of GenBank (Timofte et al., 2014).

49
3.10 Characterization of quinolone resistance mechanisms
All non-susceptible Klebsiella isolates to ciprofloxacin will be tested for the
presence of three types of PMQR determinants which includes qnr family (qnrA, qnrB,
qnrS), quinolone efflux pump (qepA) and aac(6’)lb-cr using PCR. The PCR reactions
composed of 5µL of 2X Reddymix® PCR MasterMix (0.625 units Taq polymerase, 1.5nM
MgCl2 and 0.2mM each of dNTP/reaction), 0.5µL of each primer (10µM), 1µL of DNA
template and 8µL of double distilled water. The PCR conditions for qnrA (516 bp), qnrB
(469 bp), and qnrS (417 bp) genes will be an initial denaturation at 94ºC for 4 minutes, and
32 cycles each of denaturation at 94ºC for 45 seconds, primer annealing at 53ºC for 45
seconds and extension at 72ºC for 60 seconds and one cycle of final extension at 72ºC for 5
minutes (Stephenson., et al., 2010). On the other hand, the PCR conditions for qepA gene
(617 bp) will be an initial denaturation at 96ºC for 1 minute, and 30 cycles each of
denaturation at 96ºC for 60 seconds, primer annealing at 60ºC for 60 seconds and extension
at 72ºC for 60 seconds and one cycle of final extension at 72ºC for 5 minutes (Yamane, et al.,
2008). Lastly, the PCR conditions for aac(6’)lb-cr gene (482 bp) will be an initial
denaturation at 94ºC for 4 minutes, and 34 cycles each of denaturation at 94ºC for 45
seconds, primer annealing at 55ºC for 45 seconds and extension at 72ºC for 45 seconds and
one cycle of final extension at 72ºC for 5 minutes (Park, et al., 2006). PCR amplicons will be
separated using 1.5% agarose gel electrophoresis (Esco®, South Yorkshire, UK) in 1X Tris-
acetate/EDTA (TAE) buffer. The primers used are listed in Appendix 10.
3.11 Detection and characterization of extended-spectrum β-lactamases (ESBLs) and
other non-integron borne antibiotic resistance genes
Only the main groups of ESBL genes like blaCTX-M (variable size), blaPER-1(7-301 bp),
blaAMPC (141-311 bp), blaTEM (799bp) and blaSHV (862bp) will be tested on all of the Klebsiella

50
isolates. Resistance genes for other antibiotics such as gentamicin (aadB – 300bp),
streptomycin (aadA1 – 631 bp and aadA2 – 500 bp), sulfamethoxaole (sul1 – 331 bp),
tetracycline (tetA – 372bp, tetB – 228bp and tetM – 406 bp) and trimethoprim (dfrA6 – 419
bp and dfrA12 – 395bp) will also be investigated. The PCR reactions composed of 6.25µL of
2X Reddymix® PCR MasterMix (0.625 units Taq polymerase, 1.5nM MgCl2 and 0.2mM
each of dNTP/reaction), 0.5µL of each primer (10µM), 2.5µL of DNA template and 2.75µL
of double distilled water. The multiplex PCR conditions for blaTEM and blaSHV will be an initial
denaturation at 94ºC for 5 minutes, and 35 cycles each of denaturation at 94ºC for 30
seconds, primer annealing at 60ºC for 30 seconds and extension at 72ºC for 3 minutes and
one cycle of final extension at 72ºC for 10 minutes (Afifi, 2013). PCR amplicons were
separated using 1.5% agarose gel electrophoresis (Esco®, South Yorkshire, UK) in 1X Tris-
acetate/EDTA (TAE) buffer. The PCR conditions for blaCTX-M will be an initial denaturation at
94ºC for 2 minutes, 35 cycles each of denaturation at 95ºC for 20 seconds, primer annealing
at 51ºC for 30 seconds and extension at 72ºC for 30 seconds and one cycle of final extension
at 72ºC for 3 minutes (Edelstein et al., 2003). PCR amplicons were separated using 1%
agarose gel electrophoresis (Esco®, South Yorkshire, UK) in 1X Tris-acetate/EDTA (TAE)
buffer.
The multiplex PCR conditions for aadB (300bp), aadA1 (631 bp) and aadA2 (500
bp) will be an initial denaturation at 94ºC for 5 minutes, and 30 cycles each of denaturation at
94ºC for 45 seconds, primer annealing at 54ºC for 45 seconds and extension at 72ºC for 60
seconds and one cycle of final extension at 72ºC for 5 minutes (Chuanchuen et al., 2008).
The PCR conditions for dfrA6 (419 bp), dfrA12 (406 bp) and sul1 (331 bp) genes will be an
initial denaturation at 95ºC for 10 minutes, and 30 cycles each of denaturation at 95ºC for 30
seconds, primer annealing at 55ºC for 60 seconds and extension at 72ºC for 60 seconds and

51
one cycle of final extension at 72ºC for 7 minutes. PCR amplicons will be separated using
1% agarose gel electrophoresis (Esco®, South Yorkshire, UK) in 1X Tris-acetate/EDTA
(TAE) buffer (Chen et al., 2004). The PCR conditions for tetA (210 bp) and tetB (659 bp)
will be an initial denaturation at 94ºC for 5 minutes, and 35 cycles each of denaturation at
94ºC for 60 seconds, primer annealing at 55ºC for 60 seconds and extension at 72ºC for 1.5
minutes. PCR amplicons will be separated using 1% agarose gel electrophoresis (Esco®,
South Yorkshire, UK) in 1X Tris-acetate/EDTA (TAE) buffer (Ng et al., 2001). The primers
used in detection of antibiotic resistance genes are listed in Appendix 11.
3.12 Detection and characterization of plasmid-borne virulence genes
PCR will be done to detect the presence of virulence gene rmpA gene (regulator of
mucoid phenotype A). The PCR reactions will be composed of 5µL of 2X Reddymix® PCR
MasterMix (0.625 units Taq polymerase, 1.5nM MgCl2 and 0.2mM each of dNTP/reaction),
0.5µL of each primer (10µM), 2.5µL of DNA template and 4µL of double distilled water.
The PCR conditions for rmpA gene (516 bp) will be the same that of molecular serotyping
with an initial denaturation at 95ºC for 5 minutes, and 40 initial cycles each of denaturation at
95ºC for 60 seconds, primer annealing at 50ºC for 60 seconds and extension at 72ºC for 2
minutes and one cycle of final extension at 72ºC for 7 minutes (Yeh et al., 2007). To detect
magA gene (1283 bp), the PCR conditions will be an initial denaturation at 94ºC for 1
minute, and 30 cycles each of denaturation at 94ºC for 30 seconds, primer annealing at 59ºC
for 45 seconds and extension at 72ºC for 1 minute & 30 seconds and one cycle of final
extension at 72ºC for 6 minutes (Turton et al., 2008). PCR amplicons will be separated using
2% agarose gel electrophoresis (Esco®, South Yorkshire, UK) in 1X Tris-acetate/EDTA
(TAE) buffer. The primers in this study are listed in Appendix 5.

52
3.13 Risk factor analysis
A pretested standardized questionnaire will be used to collect information on each
farm’s clinical history, use of antimicrobials for bovine mastitis, use of disinfectants, farmer
knowledge especially on antimicrobial resistance, farm demographics, and farm-level
management including post milking teat disinfection, pre-dipping or pre-wiping, mastitic
cases monitoring, frequency of personnel cleaning and disinfection factors (Furgasa, et al.,
2010) and environmental factors. Additional records will be gathered on any cases of
misdiagnosis of mastitis by non-veterinary staff including farmer, treatment with indigenous
and/or herbal medicine, delay in seeking veterinary service, treatment without laboratory
diagnosis, non-adherence to set treatment protocol due to economic constraints, unavailability
of recommended drugs, and access to limited laboratory diagnostic facilities and veterinary
services (Gunawardana, et al, 2014).
On a cow level, data will be gathered relating to average milk production (L),
lactation number, days in milk, present lactation total, past milk production average (L), past
lactation total, mastitis history, mastitis therapy, other disease treatment history, dry cow
therapy and other relevant clinical data. All interviews will be conducted in the farmers’
native language (Pilipino). Both the clinical examination and the survey will be conducted
by the same investigator (Gunawardana, et al, 2014). The introductory letter for the survey is
presented in Appendix 12.
Data that will be coming from the questionnaires will be encoded into a Microsoft
Excel worksheet. The prevalence of mastitis will be computed. Association between
antimicrobial resistance and the various factors will be known by calculating Pearson’s chi-
square value, and the degree of association will be calculated via the odds ratio (OR) using
SPSS 12.0 statistical software, SPSS, Inc. (Munich, Germany) for Windows. Logistic

53
regression by means of p<0.05 will be used to identify potential risk factors (Furgasa, et al.,
2010; Afifi, 2013; Gunawardana, et al, 2014). All descriptive and inferential analyses will be
executed using SPSS 12.0 statistical software for Windows (Gunawardana, et al, 2014).

54
RESULTS
Table 1. List of serotypes and virulence genes found in Klebsiella pneumoniae isolates
# Serotype Virulence genes
Number (%)
1 K1 magA 5 (1)
2 K2
3 K5
4 Non-K1/K2 rmpA
Table 2. Antibiotic resistance genes in Klebsiella pneumoniae isolates
# Antibiotic
Resistance
# (%)Gene
1 Streptomycin
aadA1 1 (3)
aadA2
2 Sulfamethoxazole sul1
3 Gentamicin aadB
4 Tetracycline
tetA
tetB
5 Trimethoprim
dfrA6
dfrA12
6 β-lactamase
blaTEM
blaSHV
blaCTX-M

55
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Afifi, M.M. 2013. Detection of extended spectrum beta-lactamase producing Klebsiella
pneumoniae and Escherichia coli of environmental surfaces at upper Egypt. Int J of
Biological Chem. 7(2): 58-68.
Barlow, J., 2011: Mastitis therapy and antimicrobial susceptibility: a multispecies review
with a focus on antibiotic treatment of mastitis in dairy cattle. J Mammary Gland Biol
Neoplasia,16, 383-407.
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