3.7. Compatibility of biochemical and gene analyses and
PCA results of some CFEC-ESBL
These results from MALDI-TOF MS-based PCA analyses pointed to important
clues that some of the 35 ESBL-CFEC isolates had significant
differences. In this respect, the
biochemical characteristics of the isolates with the highest affinity
(CFEC-ESBL-68) and the lowest (CFEC-ESBL-38) and low affinity
(CFEC-ESBL-90) to all isolates and findings for beta-lactam resistance
genes were detected. The data is
given in Figure 3F confirm all the PCA-based analysis results given
above. Unlike the other 34 isolates, bla CTX-M-15is one of the beta-lactam resistance genes in only CFEC-ESBL-38
isolates. It is considered that the presence of 41% variance causes the
scattering profile to be located farthest in the scattering profile and
with a different line in the dendrogram. Besides, in both (CFEC-ESBL-38
and CFEC-ESBL-90) isolates, bla CTX-M,bla CTX-M-1, bla TEM,bla OXA-10 The absence of their genes may be a
reason for the 87% affinity between them, as well as leading to their
separation from the main cluster (n=33).
Discussion
In the present study, 122 isolates were identified as E. coli by
two methods, traditional (biochemical tests) and the new-generation
method (MALDI-TOF MS). The
identification scores obtained from MALDI-TOF MS are between ≥2.000,
16% were between 1.700-1.999 and there was no definition below the
cut-off value (≤1.699). Within the scope of phyloproteomic analysis,
firstly, PCA analyses of all E. coli isolates were carried out
and it was determined that there are isolates with different
characteristics. Then, more detailed analyzes were carried out on a
smaller group of E. coli with ESBL characteristics. With the
support of all the analyzes performed, it was concluded that there were
significant differences in the light of the data on variance, CCI index
values, dendrogram, and scattering profile placements, even though hints
on bacteria belonging to the same species are provided.
ESBL was detected in almost one of the 3 isolates. It is a very high
rate especially according to the scales of industrial production
facilities. Because E. coli is a very important strain in terms
of food pathogens and antibiotic resistance profile. Unfortunately,
similar or even higher rates of ESBL were detected in some studies. For
example, Yang et al. [38] similarly reported that 22.9% of the
bacteria which were ESBL- E. coli. In other studies, Badr et al.
[39] determined the rate of ESBL producing E. coli isolated
from chickens is 46.7%, while Gazal et al. [40] detected 66% and
Fournier et al. [41] detected ESBL 84%. Cormier et al. [1]
reported that this rate reached up to 90%. According to the findings of
the present study, predominantly bla CTX-M-1resistance genes were detected. In previous studies, the most frequently
reported resistance genes were CTX-Ms and their derivatives [42].
These genes, which are reported to be common on a global scale, may be
an indication that bacteria are in contact with each other [2,39].
CFEC-ESBL-38 is a strain that came to the fore with its difference in
the study as the only atypical
ESBL strain that shows only indole (-) and lac (-) among 122 isolates.E. coli is lac (+) commonly and lactose permease enzyme (LacY
protein) is a very important protein that enables the use of lactose inE.coli. However, in some bacteria, lac (-) variants occur due to
deficiencies in the level of this enzyme encoded by this LacY gene
[43,44]. Stępień-Pyśniak et al. [8] showed thatEnterecoccus faecalis and E. mundtii isolates were
separated in the dendrogram with phyloproteomic analysis by using the
spectral profiles of the isolates. In the same study, it was also noted
that there was clustering of very similar strains in terms of phenotype
and genotype according to galactosidase and mellobiose characteristics,
and it was stated in the study that a single gelatinase negative isolate
gave a different peak. Unlike the other 34 CFEC-ESBL isolates, it is
thought that the presence of bla CTX-M-15 only in
CFEC-ESBL-38 causes it to settle furthest in the scattering profile and
with a different line in the dendogram with a variance of 41%. The
expression of a peptide/protein that is directly or indirectly related
to phenotypic resistance might be cause the difference of this strain
[45-47].
In the I. Phyloproteomic study, the CFEC-ESBL-90 was completely excluded
from the large cluster with 14% variance in the full dendrogram (n=122,
for CFEC) profile while it was located on a separate line in the
dendrogram connected to the large cluster with 15% variance in the II.
Phyloproteomic study (n=35, for CFEC-ESBL). Additionally, it was
determined that the closeness ratios (CCI) to large cluster members,
except for a few, were at a low level. Biochemically, this strain had
weak catalase ability. The presence of catalase enzyme is very
characteristic of E. coli. In general, E. coli harbors two
different catalase genes: katG encodes hydroperoxidase I (HPI) and katE
encodes HPII. The activity of both catalases increases when both are
present together and the expression level of genes increases [48].
In the dendrogram profile, the difference in common with the members of
the cluster including CFEC-ESBL-90 (CFEC-19 and CFEC-ESBL-38) is that it
does not have hemolysis activity. The presence of the hemolysis enzyme
and the observed hemolysis ability are mostly variable within the
species in E.coli [49,52]. On the other hand, another
possible feature makes this strain different from others is that the
indole test is negative. Because the indole test is an indicator of the
tryptophanase and tryptophan permease enzymes of the tryptophanase
operon (TNA Operon), and the indole test of E. coli (90-95%) is
mostly positive [51]. Deficiency of these proteins was also
determined as a possibile affecting all PCA analysis results.
Torres-Corral and Santos [52] pointed out that Lactococcus
garvieae isolates gave characteristic 3 peaks and as the reason for
grouping of isolates, enzymes such as epimerase, methyltransferases, and
acetylphophatases possessed by the isolates. There are some studies
suggesting that changes in enzyme structures may be affect results
because protein analyses of biological structures are performed with
MALDI-TOF MS [53,54]. On the other hand, according to the present
study, catalase, tryptophanase, and tryptophan permease enzymes ofE . coli may be the reasons for the differences, but it must be
supported by further studies. Because bacteria produce many specific and
non-specific proteins [55].
Besides, other genes (which were not tested in the study) are considered
to responsible for ESBL in CFEC-ESBL-90. The majority of genes
responsible for ESBL appear as CTX-M, SHV, and TEM variants. However,
there are also other genes responsible for resistance [39, 56-58].
In the study of Laudy et al. [59], similar to the present study,
although ESBL-producing Pseudomonas aeruginosa strains obtained
from phenotypic test results, they could not detect all the genes it
screened at the same rate. They also reported new 3 different
ESBL-producing genes with their further studies. Because many genes
responsible for beta-lactamases were reported and continue to be
reported [2,39,59]. Further studies can be carried out to detect
ESBL genes in CFEC-ESBL-90.
In the present study, as well as the detection of differences betweenE.coli and ESBL -E.coli, it was observed that similar ones
were numerically higher. For example, the greatest affinity was detected
in the CFEC-ESBL-68 isolate. Although the ratio of CFEC-ESBL-68 to 13
cluster members was 49-69%, the closeness ratio to the remaining 21
cluster members was 70-99%. This might be because CFEC-ESBL-68 has
typical biochemical features for E.coli like most isolates in the
study (strong catalase property, alpha hemolysis ability, indole and
lactose positive, etc.). However, performing further tests (other simple
sugar fermentation, gelatinase, nitrate, arginine, biofilm, multidrug
resistance profile, etc.) are needed to understand.
The present study was conducted to determine the differences and
similarities between E. coli isolates with all PCA analysis
(Dendrogram, scatter plotting, variance, and CCI). It was also found
that the ESBL group generally differed from susceptible strains and
there were some heterogeneities and homogeneities in the isolates.
Alharbi et al. [17] showed that MSSA and MRSA can be separated in
dendrogram cluster analysis. Similarly, in another study, it was shown
that MSSA and MRSA were distinguished by peaks of different masses, and
it was emphasized that MALDI TOF MS saves time according to molecular
studies [60]. All these data suggest that ESBL producing E.
coli are phylogenetically separated and may differ greatly in natural
ecosystems. Previous studies have shown the feasibility of MALDI-TOF-MS
for the clonal identification of bacteria. Our study is also an example
for that.
In conclusion, phyloproteomic
analyses with MALDI-TOF MS may be useful for characterization of
phenotypic behaviors. Unfortunately, the high cost of analysis of
multiple samples with high-cost methods such as Whole Genome Analysis is
a major challenge for analytical studies. Thus, the most important
result was found in the present study is that performing advanced
analyzes as well as identification with the less costly MALDI-TOF MS
contributes significantly to the validation of traditional analysis
results. Also, this study represents a first in terms of ESBL screening
and characterization in broiler chickens for the region (Duzce,
Türkiye). There is no previous study that was conducted or published in
the region. In recent years, epidemiological studies have focused on the
spread of resistant strains which are extremely important in terms of
clinically and food safety. In this sense, it is anticipated that this
study will contribute to the monitoring of the data in the region.
Although important clues were obtained, further analyzes are planned to
make sense of the effect of biochemical characteristics on variance
values.