ABSTRACT
Pseudomonas
aeruginosa is an ubiquitous, Gram-negative, rod-shaped, monoflagellated
bacterium, capable of causing serious infections in immunocompromised host. It
is one of the most difficult multidrug resistant microorganisms capable of
producing metallo-β-lactamase (MBL). Though a notorious organism, its
identification should not be limited to pigment production alone, as some
strains do not produce pigments. This study was aimed at evaluating the
increasing prevalence of multidrug resistance profile and MBL production in P. aeruginosa isolated from nasal cavity
of cattle from Kara market, Ogun state, Nigeria.
In this study, a total of 570
samples were randomly collected from the nasal cavity of healthy male and
female cattle using sterile swab sticks. They were tested for the presence of P.
aeruginosa on cetrimide agar, observing the agar plates for growth and
pigmentation. Further biochemical test and molecular analysis were carried out
to further confirm their identity as P. aeruginosa. The antibiogram was determined using ceftazidime,
cefroxime, gentamicin, ciprofloxacin, ofloxacin, augumentin, nitrofurantouin,
ampicillin, imipenem and meropenem discs. Carbapenems (meropenem and imipenem)
discs combined with EDTA were used to test for the production of MBL in P. aeruginosa.
A total of 439 samples were found
to be positive for Pseudomonas out of
the 570 samples collected. Of these positive samples, 229 isolates were from
female cattle (31.0% showing pigment production and 68.9% non-pigment producers)
and 210 isolates were from the male cattle (48.57% showing pigment production
and 51.43% non-pigment producers). Isolates obtained from the female cattle
showed 69.01% (pigment producers) and
82.28% (non-pigment producers) multidrug resistance while 75.49% (pigment
producers) and 84.26% (non-pigment producers) were multidrug resistant among
the isolates from the male cattle. In determining MBLs production, 47.84% were
positive for MBL
production using the IMP+EDTA and 75.50% were positive for MBL using the
MER+EDTA.
From this study, it was
concluded that multidrug resistant P. aeruginosa is present in the nasal
cavity of cattle and its presence could be detrimental to the cattle, cattle
herders, beef handlers and consumers. The level of resistance was high in both
pigment and non-pigment producing isolates, though the resistance of
non-pigment producing P. aeruginosa was higher. Drugs of choice to be
considered first for the treatment of infections caused by multidrug resistant P.
aeruginosa are gentamicin, ofloxacin, ciprofloxacin and meropenem.
Nitrofurantoin is expensive and not easily purchased hence they are to be
considered as the last drug of choice at extreme cases.
CHAPTER ONE
INTRODUCTION
1.1 Background
to the Study
Cattle are the most common and
largest domesticated animal in the world (Bollongino,2012). They are reared for meat, milk and other dairy products. According to an
estimate from 2011, there are about 1.4 billion cattle in the world. In 2009,
cattle became one of the first livestock animals to have a fully mapped genome(Brown, 2009). They are herbivores because they feed on
grasses, legumes and roughage. They are also known as ruminants because they
have one stomach with four compartments. In Nigeria (West Africa), commercial
beef cattle production is common especially in the Northern part of the country
(Bollongino, 2012).
Pseudomonas
aeruginosa (P. aeruginosa) is a
bacterium capable of causing serious infections in cattle e.g. mastitis. It is
found in the milk of cattle because it requires few nutrients to grow and
multiply. Water supplies, contaminated drugs and infusion equipment are the
major sources of this organism. It has also been isolated from waste feed,
soil, manure and animal skin. Cows that are immunologically compromised due to
other infectious diseases or are nutritionally deficient are also more
susceptible to P. aeruginosa
infections. The bacterium is resistant to antibiotics (John & Roger, 2011).
In 2005,
Haydar was able to isolate some bacteria causing pneumonia from the nasal cavity of healthy cattle especially in animals suffering from defects in their
immune status or stressed (Haydar, 2005). About 2.43% of the isolates were Pseudomonas species. This showed that Pseudomonas
aeruginosa can be an infectious
agent in cattle and can be transmitted as zoonotic infection (Haydar, 2005).
1.1.1
Pseudomonas
aeruginosa
Pseudomonas aeruginosa is a Gram-negative, rod-shaped,
monoflagellated bacterium ranging from about 1-5 µm long and 0.5-1.0 µm wide (Lederberg, 2000). They are ubiquitous microorganism that can be isolated from
soil, water, humans, animals, plants, sewage, and hospitals (Lederberg, 2000). Itis an opportunistic human pathogen which often colonizes
immunocompromised patients such as those with cystic fibrosis, cancer or AIDS (Botzenhardt et
al., 1993). Itis the
second leading cause of Gram-negative nosocomial infections (Carmeli et al., 1999) carrying a 40-60%
mortality rate (Fick, 1993). It has a natural resistance mechanism
to many antibiotics because of a resistance
transfer plasmid, extra genetic material carried in the cells
with genes that code for proteins that destroy antibiotic substances (Madigan
& Martinko 2006).
1.1.2 Multi-drug
resistantPseudomonas
aeruginosa
Multi-drug resistant (MDR) P. aeruginosa are organisms resistantto one
antimicrobial agent in three or moreantipseudomonal antimicrobial
classes(carbapenems, fluoroquinolones, penicillins/cephalosporins and
aminoglycosides)(Magiorakos et al., 2011). Multi-drug
resistance in P. aeruginosa arises
from low outer membrane permeability, multidrug efflux systems which accounts
for its intrinsic mechanisms of resistance, enzyme production, target mutations
(Kotra et al., 2000) and biofilm
formation (Carmeli et al., 2002).
In addition to these factors, other bacterial exoproducts contributing to
multidrug resistance in P. aeruginosa arelipopolysaccharides and elastase which induce harmful
pathogenesis resulting in tissue destruction.
Apart from enabling motility, the flagellum of P. aeruginosa plays
an indirect role in membrane permeabilization and surfactant protein-mediated
bacterial clearance (Zhang, 2007). MDR P. aeruginosa are very
problematic because of its inherent resistance to many drug classes andability
to acquire resistance to all effectiveantimicrobial agents (Gad et
al., 2007).
1.1.3 Carbapenems
Carbapenems are β-lactam group of
drugs that are usually used as antibiotics of last resort for treating
infections due to multiple-resistant Gram-negative bacilli. Often times, the
stable response of P. aeruginosa to extended-spectrum β-lactamases has
changed due to the emergence of metallo-β-lactamase (MBL)-producing strains
(Jesudason et al., 2005). They bear a penemic together with
the beta-lactam ring inhibiting bacterial cell wall synthesis by binding to and
inactivating Penicillin Binding Proteins (PBPs).
Multi-drug
resistant (MDR) P. aeruginosa are capable of producing enzymes that can
inactivate beta-lactams such as metallo-β-lactamase (MBL) which is responsible
for a significant proportion of carbapenem resistance in these bacteria (Moya et al., 2009, Borgianni et al., 2010). These enzymes can
hydrolyse all classes of β-lactam drugs and withstand neutralization by
β-lactamase inhibitors (Wan Nor Amilah et
al., 2012).
Imipenem, panipenem, meropenem, biapenem, ertapenem, doripenem and tebipenem
belong to the carbapenem family. Each one present different characteristics
that influence their way of administration and their usefulness as
anti-pseudomonal agents.Carbapenem resistance mechanisms in P. aeruginosa may be classified as
enzymatic, mediated by carbapenemases (beta-lactamases hydrolyzing carbapenems
among other beta-lactams). Carbapenem resistance, however, develops frequently
due to the concomitant presence of more than one mechanism (El Amin et al., 2005; Hammami et al., 2009). Another
resistant mechanism of P. aeruginosa to carbapenem is the
reduction of outer membrane (OM) permeability through alterations in or
decreased production of outer membrane porin D (OprD). This porin allows the
cellular entry of carbapenems (Farra et
al., 2008).
1.1.4 Metallo-Beta-Lactamase
Metallo-beta-lactamases (MBLs) are enzymes that make bacteria resistant to a broad range of beta-lactam
antibiotics one of which includes the cabapenem family (Kumarasamy et
al., 2010). They belong to class B of the structural
classification of β-lactamases and are able to efficiently hydrolyze all
β-lactams with the exception of monobactams (Yan et al., 2006; Gutierrez et al.,
2007; Palzkill, 2013). The enzymes require divalent cations, usually zinc, as
metal cofactors for enzyme activity and are inhibited by metal chelators such
as ethylenediamine tetra acetic acid (EDTA) (Maltezou, 2009). MBLs are encoded
either by genes that are part of the bacterial chromosome in some bacteria or
by heterologous genes acquired by transfer of mobile genetic elements.
Therefore, acquired MBL can be spread among various strains of bacteria such as
P. aeruginosa (Cornaglia et al.,
2011).
1.2 Statement
of the Problem
Scientifically, P. aeruginosa is known to be a notorious
organism because it is highly resistant to virtually all antibiotics.
Infections to which it is implicated are always difficult to treat. This may be
as a result of most virulence factors and mechanisms of resistance in P. aeruginosa. Although P. aeruginosa is ubiquitous, pathogenic
and possibly a zoonotic agent, there is a death of information on its isolation
from domesticated animals especially asymptomatic cattle and the susceptibility
of these isolates from cattle to different antibacterial agents. Though cattle
are not carriers of P. aeruginosa,
they are often infected by them (mastitis being the most common infection). The
presence of multidrug resistant P.
aeruginosa poses a threat not only to the cattle but also to the cattle
herders, beef retailers, beef handlers and beef consumers.
Consequently, this study will
determine
1. The
possibility of isolating pathogenic pseudomonas from the nasal cavity of
healthy cattle as against the expected Staphylococcus
aureus which are usually considered natural microflora of the nasal
cavity.
2. Whether the different antibiotics readily
available would be effective in inhibiting the growth of P. aeruginosa strainsisolated from the nasal cavity of cows or not
for effective therapy in infections.
3. The
multi-drug resistant profile of P.
aeruginosa isolated from cattle
4.
If the isolates are capable of producing
metallo-beta-lactamase which is a resistance mechanism and to what extent or
degree.
1.3 Objective of the Study
The
general objective of this study is to educate the public on the presence of P.
aeruginosa if isolated and to evaluate the increasing prevalence of
multi-drug and carbapenem resistant P. aeruginosa isolated from cattle
in Kara market, Ogun State, Nigeria. The specific objectives are to:
1. isolate
P. aeruginosa from the nasal cavity
of male and female cattle;
2. identify P.
aeruginosa from the nasal cavity of male and female cattle;
3. determine whether all strains of P. aeruginosa are pigment producing or
non-pigment producing P. aeruginosa ;
4. determine
the antibiogram of the isolates and compare the antibiotic resistant pattern
between the pigment producing P.
aeruginosa and non-pigment producing P.
aeruginosa;
5. determine
metallobeta-lactamase production in the isolated P. aeruginosa from nasal cavity of asymptomatic cattle.
1.4 Research Questions
1.
Can P.
aeruginosa be isolated from the nasal cavity of healthy cattle?
2.
How is P. aeruginosa identified when isolated from the nasal cavity of
healthy cattle?
3.
Are non-pigmented Pseudomonas truly P.
aeruginosa
4.
How can the isolated pigment producing P. aeruginosa bedifferentiated from the
non-pigment producing P. aeruginosa?
5.
How are the multiple antibiotic
resistance index of the isolate calculated using the antibiogram of the
isolate?
6.
How is the test for
metallo-beta-lactamase carried out?
1.5 Significance of the Study
This study would possibly indicate
cattle as a nasal carrier of Pseudomonas
aeruginosa and indicate the susceptibility profile of the isolates. It
would also, educate the general public on the danger of negligence of multidrug
resistant P. aeruginosa in cattle and
its effect in eating undercooked beef as well as providing a more recent data
on the increasing prevalence of resistance in multidrug resistant P. aeruginosa.
1.6 Justification for the Study
This
study will most importantly provide baseline information and a more recent
epidemiological data on the increasing prevalence of multi-drug resistant P. aeruginosa resulting from cattle.
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