Major Enzymatic Factors Involved in Bacterial Penicillin Resistance in Burkina Faso
Odile Germaine Nacoulma
Many clinical species of bacteria were isolated from biological samples
such as urines, blood and wound in Saint Camille medical centre of Ouagadougou.
Among the concerned species, the most important members were Escherichia
coli and Klebsiella pneumoniae. These β-lactamases producing
isolates were directly screened by PCR to identify the nature of the amplified
genes responsible for penicillin destroying activity. Therefore specific
TEM and SHV primers were used. The PCR products were sequenced.
The sequencing results indicated that the parental forms blaTEM-1
and blaSHV-1 were the most common determinants of β-lactamase
found, respectively in Escherichia species and Klebsiella pneumoniae.
The bacterial susceptibility analysis by MICs measurement clearly correlated
the presence of concerned β-lactamase determinants and their resistance
patterns. This study is part of a set of investigations carried out by
our laboratory to assess the β-lactamase incidence in the failure
of β-lactam therapy. In particular, the purpose of this study was
to determine the precise nature of β-lactamase supporting the low
susceptibility of host bacteria towards penicillins.
Antibiotics are widely used to fight pathogenic bacteria. Penicillin
was the first antibiotic discovered seventy-eight years ago (Fleming,
1929). Penicillin belongs to a vast family of molecules including cephalosporins,
carbapenems and monobactams. All these compounds bear the β-lactam
ring which is their reactive centre. The appearance of enzymes from bacteria
(Abraham and Chain, 1940) able to destroy penicillin and related molecules
was the beginning of a severe war between pathogenic bacterial cells and
man. These penicillin inactivating enzymes known as β-lactamases
secreted by bacteria were subdivided into four classes A, B, C and D on
the basis of sequence characteristics (Ambler, 1975, 1980; Ambler and
Scott, 1978).The class A enzymes are numerically the most important group
of β-lactamases found in many clinical isolates of Enterobacteriaceae
worldwide. These enzymes are often inhibited by suicide inactivators such
as clavulanic acid (Bush, 1988) which is clinically used to protect β-lactamase
susceptible antibiotics. This class comprises two main representatives
named TEM and SHV. The term TEM comes from Temoniera, the name of the
patient in Greece from which the host bacteria of the first enzyme of
this species was isolated (Medeiros, 1984). TEM-1 is the parental form.
The word SHV comes from Sulphydryl Variable the biochemical properties
(Barthelemy et al., 1986) of this species whose parental form is
SHV-1. TEM-1 and SHV-1 share 68% homology in amino-acid sequence (Du Bois
et al., 1975; Majiduddin et al., 2003). The two β-lactamases
have also similar substrate profiles in that both can readily hydrolyse
β-lactams belonging to penicillin group such as benzylpenicillin,
ampicillin, amoxicillin piperacillin etc. However, it was reported that
SHV-1 achieves better activity against ampicillin (Bush et al.,
Resistance to β-lactam antibiotics has been a problem for
as long as these drugs have been used in clinical practice. Throughout
the world, the problem of resistance to β-lactam antibiotic via the
production of β-lactamase has become a serious public health preoccupation.
Until now, no significant information on the nature of β-lactamase
involved in the clinical isolates resistance to penicillin in Burkina
Faso (West Africa) was available. This study was a real attempt to fulfil
this gap in order (1) to characterize the type of Escherichia coli
and Klebsiella pneumoniae β-lactamase directly being involved
in penicillin resistance in Burkina Faso (2) to eventually highlight derivatives
of existent enzymes.
MATERIALS AND METHODS
Bacterial isolates: The isolates analysed in this study were all β-lactamase producing
enterobacteriaceae collected at the Saint Camille Medical Centre (SCMC),
a central institution for care and medical analysis in the capital city-Ouagadougou.
The sources of the isolates were mainly faeces, urines, pu etc. They were
identified by Api 20 and Vitek 2 Systems (BioMerieux France). One hundred
organisms were selected solely because of the substrate profile of their
β-lactamase and their resistance to penicillins as reported by the
laboratory analysis. Thirteen representative isolates were retained for
further investigation. They include: 2 Escherichia fergusonii (N°672
and 681) 5 Escherichia coli (N°685, 1004, 1181, 1190, 1204
1220) and 5 Klebsiella pneumoniae (N°Z54, 291, 312, 392, 1201).
Antibiotic susceptibility: Bacterial species analysed were phenotypically characterized by determining
their susceptibility towards different penicillins. Therefore, the Minimum
Inhibitory Concentrations (MICs) were determined using the automatic method
VITEK 2 (BioMerieux In., St Louis, Mo.)
Molecular study: Considering that Escherichia species and Klebsiella pneumoniae
often carry TEM or SHV-β-lactamase, the genes encoding these enzymes
were targeted by PCR amplification directly on bacterial cells using TEM
and SHV specific primers. The different Primers used for amplification
are shown in Table 1. These oligonucleotides were obtained
from Eurogentec Bel. S.A. Cycling conditions were as follow for blaSHV
genes amplification: 24 cycles of denaturation at 94°C for 1 mn, annealing
at 37°C for 1 mn and extension of primers at 72°C for 1 mn. Cycling
conditions for blaTEM amplification were the same except
that anneling temperature was raised to 45°C. All reactions were carried
out in a final volume of 50 μL containing 1U of Taq or GoTaq DNA
polymerase, 8 pM of Each primer, 200 μM of deoxynucleotide triphosphate
and 2 μL of crude bacterial lysate (obtained by boiling bacterial
suspensions in distilled water for 20 mn).
Oligonucleotides used as primers for amplification of
blaSHV and blaTEM genes
This crude bacterial lysate was used as template. PCR amplicons
were immediately purified by Wizard purification kit of PCR products.
Antibiotic susceptibility: All the clinical isolates analysed were β-lactamase-positive and
the corresponding enzymes were active on benzylpenicillin, ampicillin,
amoxicillin, carbenicillin, oxacillin and piperacillin. The results obtained
from the analysis of their susceptibility towards these β-lactams
and some combinations are reported in Table 2.
PCR results: A total of 13 penicillin resistant and β-lactamase producing isolates
including Escherichia species and Klebsiella pneumoniae
were screened by PCR to elucidate the determinants of enzymes involved
in major penicillins (benzylpenicillin, ampicillin, amoxicillin) resistance.
From the 13 isolates, 3 Klebsiella pneumoniae (N°291, 312 and
392) were analysed in another experiment (results not shown). The PCR
amplification was successful with all the isolates screened as shown in
Fig. 1. The amplicons generated were good in term of
quantity and quality. The fragments amplified using TEM and SHV
primers were all about 800 base pairs (Fig. 1). These
sizes correspond approximately to those of TEM and SHV nucleotide
sequences. Genes encoding TEM-types were generated from Escherichia
coli, while SHV genes were obtained from Klebsiella pneumoniae
isolates. Klebsiella pneumoniae isolate N°1201 was found to
carry both TEM and SHV types of genes.
Sequence analysis: All the amplicons obtained were sequenced after purification. As shown
from Fig. 1, isolate N°1201 appeared twice because
of carrying simultaneously SHV and TEM genes. The complete
nucleotide sequences of most of the fragments were obtained (Table
3) except 2 amplicons where the whole sequence could not be determined.
These amplicons were from Klebsiella pneumoniae N°291 and Escherichia
coli N°1004. But amplicon N°291 was identified to be a SHV
determinant and amplicon 1004 was a TEM one. The lack of full sequence
did not enable the exact assignment of the determinant type (parental
or variant in the two cases).
Analysis of purified PCR fragments from cell lysate
by agarose 1% gel electrophoresis amplicons N°672 and 681 were
obtained from Escherichia fergusonii
. Amplicons N° 685,
1004, 1181, 1190, 1201, 1220 were obtained from Escherichia coli
amplicons N°1201 and Z54 were generated from Klebsiella pneumoniae
Two fragments bear the same number (1201) on the figure because of
the generation of 2 amplicons from SHV and TEM primers using the same
template (lysate from Klebsiella pneumoniae
Ladder (SL) is the DNA marker. The size of the markers in base pairs
(bp) is shown on the right
Various isolates (Escherichia species and Klebsiella pneumoniae)
from human biological samples at Saint Camille Medical Centre of Ouagadougou
and supposed to be responsible for infections (urinary, intra abdominal
disease, wound etc.) were analysed. Their potency to resist to penicillin
was investigated and correlated to their contents of β-lactamase
determinants. Most the isolates were found to have low susceptibility
towards the three major penicillins as shown by the values of their MICs
(Table 2). Isolate N°1204 was quite different from
the others and appeared to be a negative control owing to its overall
susceptibility to penicillin molecules. This behaviour could suggest the
very low level of the β-lactamase expression in this isolate. Sequencing
of blaSHV and blaTEM genes isolated
by PCR amplification from the corresponding isolates identified the exact
type of β-lactamase determinant. All the Escherichia species
(coli and fergusonii) carried blTEM-1,a
while most of the Klebsiella pneumoniae carried blaSHV-1.
Nevertheless one Klebsiella pneumoniae was found to carry the derivative
blaSHV-11 while another appeared to carry
both blaSHV-11 and blaTEM-1.
||MICs of β-lactams for clinical isolates analysed
Abbreviations used in Table 2, AMX-CA = Amoxicillin-clavulanic
acid, TIC-CA = Ticarcillin-clavulanic acid, E. coli
, E. fergu
= Escherichia fergusonii
= Klebsiella pneumoniae
= Not Determinated
||Types of β-lactamase encoding genes
the presence of two determinants would confer an exceptional high level of resistance
to this isolate. Indeed, it was quite resistant to the three penicillins
reported above but the profile was not exceptional probably because of
low constitutive expression level of the two genes. About the isolates
N°291 and 1004 where detection of SHV and TEM gene was
obtained respectively but no full sequences, it is improbable for these
organisms to carry other determinants than blaSHV-1
or blaTEM-1 owing to the substrate profiles of their
extracts and their susceptibility patterns. It should be noted that there
has been few data about β-lactamase investigation in Africa, except
South Africa where substantial data on the subject were available. So
blaSHV-1 and many derivatives (-2, -5, -19, -20, -21
and -22) and blaTEM-1 with some derivatives (-53, -63)
were reported in South Africa (Pitout et al., 1998; Essack et
al., 2001). South African data include an important number of Extended
Spectral β-Lactamase (ESBL) determinants among blaSHV
genes as well as blaTEM genes isolated from Klebsiella
pneumoniae and Escherichia coli species. The list of African
countries where ESBL are emerging grows with a recent report from Cameroon/Central
Africa (Gangoué-Pieboji et al., 2005). However, the current
data obtained in Burkina Faso do not show the presence of ESBL into the
two types of β-lactamase encoding genes. The results obtained from
the screened isolates strongly suggest the occurrence of SHV-1 and TEM-1
enzymes and their probable role in penicillins resistance. These conclusions
are consistent with evidence that SHV-1 and TEM-1 enzymes generally demonstrate
greater turnover numbers (kcat) for benzylpenicillin, ampicillin and amoxicillin
(Hujer et al., 2001; Maveyraud et al., 1996; Dubois et
al., 2004). Hitherto no ESBL (efficient derivative from SHV-1 or TEM-1
enzyme) has been identified in our investigations. But this does not mean
that ESBL are absent in our country. In the local antibiotherapy background
a rare enzyme of high clinical relevance and multiple β-lactam resistance
factor such as metallo-β-lactamase was reported in Burkina Faso (Zeba
et al., 2005). Before this evidence, the hypothesis was that the
antibiotherapy conditions were not favourable for the emergence of this
type of enzyme. This means that ESBL existence is not impossible. However,
the small number of isolates analysed could also explain why none of ESBL
was not encountered yet. Investigations are underway to establish the
possible ESBL existence and the extent of their involvement in bacterial
In summary, this study shows the existence of SHV-1 and its variant SHV-11
that were carried by Klebsiella pneumoniae in Burkina Faso. Likewise,
TEM-1 was found in Escherichia species. SHV-1, its derivative SHV-11
and TEM-1 are probably the major enzymatic factors involved in penicillins resistance
of the concerned species.
This study was supported by the Coopération Universitaire
au Développement (CUD) from Belgium Kingdom through a project named
Etude des enzymes de la résistance bactérienne aux antibiotiques.
We greatly thank this institution. We also thank Professor Jean-Marie
Frère Head of CIP (Protein engineering Center) in University of
Liège/Belgium where the molecular study was carried out. A great
thank was also addressed to Dr. Colette Duez and Pierrette Melin for their
useful technical contribution.
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