ABSTRACT
Extended-spectrum
beta-lactamases (ESBL) are enzymes that confer resistance to most beta-lactam
antibiotics, including penicillin, cephalosporin, and the aztreonams. The aim
of this present study is to phenotypically identify and establish the presence
of ESBL-producing organism among students in the university community. Within
the University community of Godfrey Okoye University, Enugu, early morning
urine samples of midstream-catch were collected into sterile bottles from sixty
(60) students between ages 18 and 25years from the 2ndMay to 31st
May. Thirty (30) male students and thirty (30) female students were sampled.
Eighteen (18) isolates were identified after the following biochemical test
were carried out: Gram staining, IMViC test (Indole test, methyl red test,
Vogesproskauer test and citrate utilization test), and coagulase test. Twelve
(12) isolates were from female students and six (6) isolates were from male
students. The organisms identified were: Streptococcus
spp, Corynebacteriumspp, Staphylococcus spp, and Escherichia coli. All theisolates were
Gram positive except for one which was Gram negative. The double disc synergy
test (DDST) was also carried out to phenotypically confirm the presence of ESBL
producing organisms. All isolates were sensitive to the test drugs in the
antimicrobial susceptibility test but there was no obvious DDST zones of
inhibition. The result of the study suggests the absence of ESBL producing
organisms among the students involved in this study.
CHAPTER ONE
1.1 Introduction
Extended-spectrum
beta-lactamases (ESBL) are enzymes that confer resistance to most beta-lactam
antibiotics, including penicillin, cephalosporin, and aztreonams (Bush and
Jacoby, 2010).
Extended-spectrum
Beta(β)-lactamases (ESBLs) are a group which are mostly plasmid-mediated,
diverse, complex and rapidly evolving enzymes that are posing a major
therapeutic challenge today in the treatment of hospitalized and
community-based patients. Infections due to ESBL producers range from
uncomplicated urinary tract infections (UTI) to life-threatening sepsis. These
enzymes share the ability to hydrolyze third-generation cephalosporin and
aztreonam and yet, are inhibited by clavulanic acid. In addition,
ESBL-producing organisms exhibit co-resistance to many other classes of
antibiotics, resulting in limitation of therapeutic option. Because of inoculum
effect and substrate specificity, their detection is also a major challenge
(Deepthiet al 2010).
Numerous
studies have barbed towards high incidence rate of UTI associated with Escherichia coli (E.
coli) and antibiotic
resistance. The emergence of Multi Drug Resistant (MDR) variant of E.
colihas
been accounted. MDR is defined as resistance to at least two antibiotics of
different classes including aminoglycosides, chloramphenicol, tetracycline
and/or erythromycin. MDR in many bacteria is due to the action of multi-drug
efflux pumps and by the accumulation on Resistance (R) plasmids or transposons
of genes with each coding for resistance to a specific agent. Nowadays, in UTIs,
ESBL -expressing Gram-Negative Bacilli (ESBL-GNB) generally cause
community-acquired infections. The resistance of Gram-negative bacteria is
typically owed to plasmid mediated enzymes of ESBL. ESBL producing bacteria are
typically associated with multi-drug resistance (MDR) and antibacterial choice
is often complicated by multi-drug resistance (Prakash and Yadav, 2017).
1.2 CLASSIFICATION OF
ESBL
There
are two major classification systems for β-lactamases:
1.2.1
Molecular
classification is based on the amino acid sequence and
divides β-lactamases Ambler classes into A (serine penicillinases), C
(cephalosporinases), and D (oxa-cillinases) enzymes which utilize serine for
β-lactam hydrolysis and class B metalloenzymes which require divalent zinc ions
for substrate hydrolysis (Bush and Jacoby, 2010).
1.2.2
Functional
classification scheme was initially proposed by Bush
in 1989 and then expanded in 1995. It takes into account substrate and
inhibitor profiles in an attempt to group the enzymes in ways that can be
correlated with their phenotype in clinical isolates (Bush and Jacoby, 2010).
1.3
DIVERSITYOF
THE TYPES OF ESBL
1.3.1
TEM beta-lactamases
The first plasmid-mediated
beta-lactamase in gram-negative bacteria was discovered in Greece in the 1960s.
It was named TEM after the patient from whom it was isolated (Temoniera). Although TEM-type
beta-lactamases are most often found in Escherichia coli and
Klebsiellapneumoniae,
they are also found in other species of Gram-negative bacteria with increasing
frequency (Clark et al., 1990).
1.3.2
SHV beta-lactamases
Sulfhydryl variable, (SHV) shares 68 percent of its amino
acids with TEM and has a similar overall structure. The SHV beta-lactamase is
most commonly found in Klebsiellapneumoniae and is responsible for up to 20% of
the plasmid-mediated ampicillin resistance in this species. ESBLs in this
family also have amino acid changes around the active site. More than 60 SHV
varieties are known (Chow et al., 2010).
1.3.3
CTX-M beta-lactamases
Cefotaximase Munich (CTX-M), these enzymes were named for
their greater activity against cefotaxime than other oxyimino-beta-lactam
substrates (e.g., ceftazidime, ceftriaxone, or cefepime). Rather than arising by mutation, they represent examples
of plasmid acquisition of beta-lactamase genes normally found on the chromosome
of Kluyvera species, a group of rarely pathogenic commensal organisms.
These enzymes are not very closely related to TEM or SHV beta-lactamases in
that they show only approximately 40% identity with these two commonly isolated
beta-lactamases. More than 80 CTX-M enzymes are currently known. Despite their
name, a few are more active on ceftazidime than cefotaxime. They have mainly been found in
strains of Salmonella entericaserovartyphimurium and E. coli, but have also been described in other species of Enterobacteriaceae(Chow et al., 2010).
1.3.4
OXA beta-lactamases
The OXA-type β-lactamases are so
named because of their oxacillin-hydrolyzing abilities. OXA beta-lactamases
were long recognized as a less common but also plasmid-mediated beta-lactamase
variety that could hydrolyze oxacillin and related anti-staphylococcal penicillin.
These beta-lactamases differ from the TEM and SHV enzymes in that they belong
to molecular class D and functional group 2d. The OXA-type beta-lactamases
confer resistance to ampicillin and cephalothin and are characterized by their high
hydrolytic activity against oxacillin and cloxacillin and the fact that they are poorly
inhibited by clavulanic acid. Amino acid substitutions in OXA enzymes can also give the
ESBL phenotype. While most ESBLs have been found in E. coli, Klebsiellapneumoniae, and other Enterobacteriaceae, the OXA-type ESBLs have been found
mainly in Pneumoniaeaeruginosa. The OXA beta-lactamase family was
originally created as a phenotypic rather than a genotypic group for a few
beta-lactamases that had a specific hydrolysis profile. Therefore, there is as
little as 20% sequence homology among some of the members of this family.
However, recent additions to this family show some degree of homology to one or
more of the existing members of the OXA beta-lactamase family. Some confer
resistance predominantly to ceftazidime (Chow et al., 2010).
1.3.5
PER
type
The PER-type ESBLs share only
around 25–27% homology with known TEM- and SHV-type ESBLs. PER-1 β-lactamase
efficiently hydrolyzes penicillin and cephalosporin and is susceptible to
clavulanic acid inhibition. PER was first detected in Pseudomonas
aeruginosa, and later in Salmonella entericaserovarTyphimurium
and Acinetobacter isolates as well. In Turkey, as many as 46% of
nosocomial isolates of Acinetobacter spp. and 11% of P. aeruginosa
were found to produce PER, which shares 86% homology to PER-1, has been
detected in S. entericaserovarTyphimurium, E. coli, K.
pneumoniae, Proteus mirabilis, and Vibrio cholerae
(Danish et al 2015).
1.3.6
GES type
GES was initially described in a
K. pneumoniae isolate from a neonatal patient just transferred to
France from French Guiana. GES has hydrolytic activity against penicillin and
extended-spectrum cephalosporin, but not against cephamycin or carbapenem, and
is inhibited by β-lactamase inhibitors. These enzymatic properties resemble
those of other class A ESBLs; thus, GES was recognized as a member of ESBLs
(Danish et al 2015).
1.3.7
VEB-1,
BES-1, and other ESBL type
Other unusual enzymes having
ESBL have also been described (e.g. BES, CME, VE-B, PER, SFO, and GES). These
novel enzymes are found infrequently (Danish et al 2015).
1.4
GLOBAL
EPIDEMIOLOGY OF ESBL
Antimicrobial
resistance has been declared a global threat to public health, as a massive
increase in this problem has been observed in different parts of the world (Kang and Song 2013). The reported frequency of
MDRs is increasing, putting strain on the public health organizations that are
attempting to control this issue in many countries. The alarming increase in
the prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae
has serious consequences for treatment outcomes (Pitout 2010). E. coli and Klebsiella species
are important pathogens isolated from community-acquired and
nosocomial-acquired infections, and have been studied extensively. The ESBL
enzymes produced by these bacteria make them resistant to the first-choice
antibiotic therapies that are commonly used. ESBL-positive strains are
associated with a delay in the commencement of suitable antibiotic therapy,
which consequently lengthens hospital stay and raises hospital costs. Failure
of antibiotic therapy is responsible for higher mortality rates in patients
infected with these bacteria. MDRs are posing a treatment challenge, and are
emerging as a major cause of morbidity and mortality worldwide. Unfortunately,
proper surveillance and documentation of such pathogens is very limited,
especially in developing countries like Nigeria (Hayat et al, 2018).
The
epidemiology of health-care associated infections has been characterized by the
emergence of gram-negative multi drug resistant organisms, including
ESBL-producing Enterobacteriaceae during the past decade. While nosocomial
transmission was initially considered by their principal cause of spread,
earlier report points to the importance of the food-chain as a continuous
source of dissemination (Kluytmanset al 2013).
In addition to a growing body of literature regarding the detection of ESBL-
producing Enterobacteriaceae in retail meat and food worldwide, food has been
reported as a vector for transmission of ESBL- producing Klebsiellapneumoniae in a hospital outbreak (Calboet al 2011). This leads to the conclusion
that control teams should consider extending their surveillance towards food as
it is a vector of ESBL.
1.4.1 AFRICA
In
Africa, the prevalence of ESBL in Enterobacteriaceae has been researched
at local levels in various countries, but there is no summarizing research on
how prevalent ESBL is on the continent, what type of genes are involved, and
where research is missing (Victor, 2014).
In
patients treated in African hospitals, the prevalence of ESBL-producing Enterobacteriaceae
has been shown to vary between countries and the type of specimen studied.
There is a trend of higher prevalence of ESBL in stool samples than in other
specimens. There is also a trend of increasing prevalence over time. This is
noticeable in the Tunisian setting, where a large amount of studies are
available. In two hospitals studied (study periods: 1999–2005 and 2010), ESBLs
have increased from 11.7 to 77.8% among K. pneumoniae. (Aouniet al,
2010). In other settings, the trend is not noticeable among the few
studies available. In the studied countries in Africa, the prevalence is widely
different: in Algeria, it was between 16.4 and 31.4% in mainly urine samples (Barguiguaet al, 2012) and even 99% among Salmonella
enterica in stool samples (Bentchoualaet al, 2011) 19 and 42.9%,
respectively, in urine and stool samples in Egypt (Domanyet al, 2012);
32.6% among stool samples in Guinea-Bissau (Giskeet al, 2012); 11.7–77.8% in
mainly urine, blood, and stool samples from Tunisia (Kechridet al, 2011);
62.8% in stool and blood samples from Ethiopia (Asratet al, 2011); 38.3% in urine
samples from Rwanda (Bayinganaet al, 2011); 55.3 and 82.8% in stool samples from Cameroon
(Assoumouet
al, 2013); 10.3–27.5% in mainly urine and stool samples from Nigeria
(Aibinuet
al, 2012); and 8.8–13.1% in urine, nasopharyngeal, and wound samples
from South Africa (Dubeet al, 2009).
1.4.1.1 Northern Africa
In Algerian hospitals, ESBLs existed in 16.4–31.4% of the samples.
Class A ESBLs were most common, but plasmid-encoded AmpC (pAmpC) was also present
(Canicaet al, 2011).
In Egypt, ESBLs were found in 11–42.9% of samples in both hospitals
and communities; the genes involved were class A ESBLs (
In Guinea-Bissau and Libya, class A and D ESBLs and a carbapenemase
were found in 32.6 and 16%, respectively, in rectal or stool samples (Giskeet al, 2012).
In Morocco, class A and D ESBLs, pAmpC, and carbapenemases were
found in hospital settings (In the community setting, class A and D ESBLs were
found in between 1.3 and 7.5% of acquired urine samples (
In Tunisia, class A and D ESBLs, pAmpC, and carbapenemases were
present, and the prevalence ranged from 11.7 to 77.8% in hospitals and was 0.7
and 7.3% in two communities (Kechridet
al, 2011).
1.4.1.2 Eastern Africa
In Ethiopia and Kenya, 62.8 and 37.4%, respectively, of hospital
and community samples were ESBLs (Asratet al, 2011).
Class A ESBLs and pAmpC were present in the Kenyan sample (et
al, 2012). In samples taken from Kenya and Malawi, class A and D ESBLs were
found (Boyle et al, 2011).
In Rwanda, ESBLs were found in 38.3% of hospital urine samples and
in 5.9% of community urine samples (
In Tanzania, class A ESBLs were found in various samples from hospital
settings (et al, 2011).
1.4.1.3
Central
Africa
In Cameroon, class A and D ESBLs were found in 55.3 and 82.8% of
hospital stool samples and in 17.2% of community stool samples (Assoumouet
al, 2013).
In the Central African Republic, ESBLs were found in 11.3% of
community urine samples (
1.4.1.4 Southern Africa
In South Africa, class A and D ESBLs and pAmpC were present, and
the prevalence ranged from 8.8 to 13.1% in hospitals and was 0.3 and 4.7% in
two communities (
1.4.1.5 Western Africa
In Ghana and Mali, class A ESBLs were found in 49.4 and 63.4–96%,
respectively, in hospital and community samples (Bougoudogoet al, 2009).
In Niger, 40% of hospital samples carried class A ESBLs or pAmpC (et
al, 2011).
In Senegal, class A and D ESBLs were found in 10% of community
stool samples. (Andremontet al, 2009)
1.4.1.6NIGERIA (West Africa)
In Nigeria, class A and D ESBLs
and pAmpC were found in hospital settings, and the prevalence ranged from 10.3
to 27.5% (Aibinuet al, 2012)In a mixed
sample from a hospital and a community, the prevalence was 11.7%. (
In Nigeria, an ESBL prevalence of
9.25% was recorded in a study conducted to screen for ESBLs production among
isolates of Enterobacteriaceae (Aliyuet al, 2010).
In another study conducted in a
tertiary health center in Nigeria to determine ESBL prevalence in Escherichia coli and Klebsiella Species; an ESBL prevalence
of 2.5% for Escherichia coli and 5%
for Klebsiellapneumoniae were
recorded (Aboderin and Olowe, 2010).
1.5 PHENOTYPIC
IDENTIFICATION OF ESBL
Extended-spectrum β-lactamase
(ESBL) detection tests should accurately discriminate between bacteria
producing these enzymes and those with other mechanisms of resistance to
β-lactams, e.g., broad-spectrum β-lactamases, inhibitor-resistant β-lactamases
and cephalosporinase overproduction. Several phenotypic detection tests, based
on the synergy between a third-generation cephalosporin and clavulanate, have
been designed: the double-disk synergy test (DDST), ESBL E-tests, and the
combination disk method. These tests often need to be refined in order for them
to detect an ESBL in some bacterial strains, such as those that also
overproduce a cephalosporinase. The sensitivity of the DDST can be improved by
reducing the distance between the disks of cephalosporins and clavulanate. The
use of cefepime, a fourth-generation cephalosporin that is less rapidly
inactivated by cephalosporinase than by ESBL, improves the detection of synergy
with clavulanate when there is simultaneous stable hyperproduction of a
cephalosporinase; alternatively, the cephalosporinase can be inactivated by
performing phenotypic tests on a cloxacillin-containing agar. Some β-lactamases
can hydrolyze both third-generation cephalosporins and carbapenems, such as the
metallo-β-lactamases, which are not inhibited by clavulanate, but rather by
Ethylenediaminetetraacetic acid (EDTA). The production of an ESBL masked by a
metallo-β-lactamase can be detected by means of double inhibition by EDTA and
clavulanate. Since extended-spectrum Ambler class D oxacillinases are weakly
inhibited by clavulanate and not inhibited by EDTA, their detection is difficult
in the routine laboratory (Danish et al
2015).
1.6 DESCRIPTION OF THE ESBL DETECTION TESTS
1.6.1 DOUBLE-DISK SYNERGY TEST
The first test specifically
designed to detect ESBL production in Enterobacteriaceae
was the double disk synergy test (DDST) (Jarlier et al, 1988). It was initially designed to differentiate between
cefotaxime-resistant strains, that is, those overproducing cephalosporinase,
and those producing ESBLs. The test is performed on agar with a 30-μg disk of
cefotaxime (and/or ceftriaxone and/or ceftazidime and/or aztreonam) and a disk
of amoxicillin–clavulanate (containing 10 μg of clavulanate) positioned at a
distance of 30 mm (center to center). The test is considered as positive when a
decreased susceptibility to cefotaxime is combined with a clear-cut enhancement
of the inhibition zone of cefotaxime in front of the clavulanate-containing
disk, often resulting in a characteristic shape-zone referred to as
‘champagne-cork’ or ‘keyhole’. The DDST was first used in epidemiological
studies to assess the spread of ESBL-producing Enterobacteriaceae in French hospitals (Brossieuret al, 2008). It has been shown to work
well with a wide range of Enterobacteriaceaespecies
and ESBL types, and it is generally regarded as a reliable method for the
detection of ESBLs, although it is sometimes necessary to adjust the disk
spacing. It is important to note that reducing the distance between the
clavulanate-containing disk and the third-generation cephalosporin disk (e.g.,
to 20 mm) significantly improves the test sensitivity (Brossieuret al, 2008).
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