Abstract
E.coli from broiler is a reservoir for ESBL (extended spectrum
beta-lactamase) and presence of ESBL is a growing concern for antibiotic
resistance. The aim of the study was to investigate and characterize
ESBL and AmpC beta-lactamases in E. coli with traditional and
new-generation methods.
As well as biochemical analyses, the identification of isolates was
performed with the MALDI-TOF MS. Within the scope of phyloproteomic
analysis, all components of MALDI-TOF MS-based Principal Component
Analysis (PCA) (dendrogram, scatter plotting, composit corelation index
(CCI) and variance,) were applied. In the present study which is the
first report for Duzce (Türkiye), 28.6% of 122 CFEC (chicken fecesE. coli ) isolates were identified as CFEC -ESBL.bla CTX-M, bla CTX-M-1,bla CTX-M-15, bla SHV,bla TEM, bla OXA-10, AmpC,bla CIT, bla MOX,bla SHV, bla CIT, andbla MOX genes were explored with PCR andbla CTX-M-1 gene was detected with the highest
rate (68.5%). At least one of the resistance genes was detected in the
phenotype screening tests, except one of the isolates (CFEC-ESBL-90). On
the other hand CFEC-ESBL-38 contained only blaCTX-M-15 and the fact that this isolate was the only
atypical ESBL strain with indole (-) and lac (-) characteristics among
all isolates explains the highest variance (41%) and the most different
from other PCA components. Also, this isolate had a high degree of
similarity (87%; CCI) with the other isolate (CFEC-ESBL-90), which had
low similarity to CFEC-ESBLs.
As a result, phyloproteomic analyses with MALDI-TOF MS are considered to
be beneficial in the characterization of phenotypic bacterial behavior.
Keywords: Antibiotic resistance, Extended-spectrum
beta-lactamase, E. coli , MALDI-TOF MS, Principal component
analysis
Introduction
Escherichia coli (E. coli ) is a common member of
the gut microbiota in humans and animals and it is characterized as an
opportunistic pathogen. The bacterium is considered an important source
of many antibiotic resistance genes in the ecosystem [1].
Antimicrobial resistance (AMR) is a very important growing crisis in the
entire world. It is predicted by some experts that there will be 10
million deaths related to AMR every year in the world by 2050, and it is
considered that the gene reservoir of E. coli is decisive for
solution of the crisis [3]. Because E. coli is a zoonotic and
spreads easily in food-environment-human interaction, which leads to a
potential change in the microbiome at the global level. It was reported
that E. coli plays an important role, especially in the spread of
Extended-Spectrum Beta-Lactamases (ESBL), acquired AmpC beta-lactamases
[4,5].
E. coli can hydrolyze almost all penicillin and cephalosporins
via its ESBL encoding genes. The uninterrupted transmission and spread
of these resistance genes, which can pass from one bacterium to another
bacterium with their fast and easy mobility features, depend on
hierarchically organized systems such as integron or it depends on their
interactions with the network between ecologically related bacterial
populations. In other words, the distribution of resistant bacteria is
an ecological evolutionary process [5]. Understanding this can only
be possible by examining the phylogenetic and phyloproteomic
relationships between bacteria with various analytical methods. As well
as the expensive and laborious sequence analyses (whole genome or
multilocus sequencing etc.), the analysis of 16S ribosomal proteins,
which are relatively inexpensive and effortless, has made a significant
contribution to this field in recent years [6].
The use of the Matrix-Assisted Laser Desorption/Ionization
Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) method, which analyzes
the proteins of bacteria with the help of validated databases created
with reference spectra of standard strains, became widespread in the
past 10 years. It allows sensitive and specific applications in food
safety and many clinical studies and it is also a method approved by the
FDA (Food and Drug Administration) for microbial identification
[7-9]. In a comparative study was conducted in 2010, it was reported
that MALDI-TOF MS had a very high rate of identifying the bacteria in
cases (99.1%) compared to routinely used biochemical methods [10].
In another study, it was reported that Gram (-) bacteria were directly
detected as a species in 99.2% of blood and urine samples [11].
MALDI-TOF MS identify after comparing highly conserved ribosomal
proteins of microorganisms with reference proteomic profiles of standard
strains that are abundantly available in the database. At the same time,
it is possible to perform phyloproteomic analyzes in the case of working
with multiple isolates. In this respect, it is possible to compare
fingerprints with unique mass spectra created for each bacterium by
MALDI-TOF MS, and then compare them with each other. Thus, with this
series of analyses that can be performed with MALDI-TOF MS, besides
microbial identification, contributes to an idea of similarities or
differences in some processes operating in metabolism [9,12]. For
example, Suen et al., (2019) identified pathogenic Staphylococcusspecies from indoor samples with MALDI-TOF MS and analyzed the
multi-drug resistance profiles of isolates with MALDI biotyper software
[13]. In another study, it was reported that Mycobacteriumspp. identification takes 7-21 days after colony formation with
tradinational biochemical methods, but it can be accurately identified
in as little as 1 hour with MALDI-TOF MS [14]. Elbehiry et al.
(2019) performed species discrimination in Aeromonas strains with
MALDI-TOF MS-based Principle Component Analysis (PCA) and single peak
analysis methods [15]. In another study, a new species ofStaphylococcus edaphicus is identified with MALDI-TOF MS from
sandy soil [16]. These studies show analytical identification
potential of MALDI-TOF MS, along with the specificity of peptide and
protein mass fingerprinting, and the identification of new species, with
a constantly developed and updated database. Alharbi et al. [17]
identified S. aureus and coagulase-negative staphylococci at
100% from 400 samples in their study comparing MALDI-TOF MS with
conventional methods. There are other studies conducted on MALDI-TOF MS
in which high rates of bacteria are identified from different sources
(wastewater, rural areas, etc.) [18-20]. It is increasingly
preferred for the identification of Gram (-) bacteria of fecal origin
from food and farm animals [21,22].
The present study was designed as food safety research and broiler
chickens were preferred. In general, there are microorganisms with very
different characteristics in poultry, and many studies have shown that
MALDI-TOF MS can reliably identify these bacteria [23]. Therefore
this study was aim (i) to investigate presence of ESBL, AmpC
beta-lactamase in E. coli obtained from the gut contents of
broiler chickens and (ii) to characterize the isolates by
phenotypic-proteomic analyses.
Materials and Methods
Chicken feces samples and chemicals
A total of 130 chicken feces samples were obtained fresh from 4
different slaughterhouses in the city centre of Duzce (Türkiye). Mac
Conkey Agar (MAC) (Merck, Germany), Tryptic Soy Agar (TSA) (Merck,
Germany) and Tryptic Soy Broth (TSB) (Condalab, Spain) were used for
isolation and culture and α-Cyano-4-hydroxycinnamic acid (CHCA; Bruker,
Germany) was used as a MALDI-TOF MS matrix. Acetonitrile (ACN, HPLC
grade; Sigma-Aldrich, Missouri, USA), trifluoroacetic acid (TFA;
Sigma-Aldrich, Missouri, USA), 0.1 µm filtered ultrapure water
(Sigma-Aldrich, Missouri, USA) free of DNAse and RNAse, and a Bruker
Bacterial Test Solution (BTS) containing E. coli , RNAase and
myoglobin protein profiles were also used.
Isolation of Escherichia coli
The feces were inoculated directly into the medium with a sterile swab
and incubated at 37°C for 24 hours and the identification was done by
conventional methods (Gram stain, catalase, oxidase,
oxidation/fermentation (OF), indole, methyl red (MR), voges Proskauer
(VP), citrate, urease, triple sugar iron, and H2S)
[24]. Also, the isolates’ ability to ferment simple sugars
(inositol, lactose, xylose, and mannose) and hemolysis on an agar medium
containing 5-10% defibrinated
sheep blood were also recorded.
Phenotypic Determination of ESBL and AmpC
All isolates were passaged into MAC mediums containing 1 mg/L
Cefotaxime, and growth was recorded [25]. Then, all isolates were
subjected to double disk phenotype screening and confirmation test. The
antibiotic susceptibility tests were performed according to the Kirby
Bauer method recommended by the Clinical Laboratory Standards Institute
[26]. Ceftazidime (30 µg), Cefotaxime (30 µg), Aztrenoam (30 µg),
Ceftriaxone (30 µg), Cefpodoxime (10 µg), Ceftazidime-Clavulanic acid
(40 µg), Cefotaxime-Clavulanic acid (40 µg), Cefoxitin (30 µg), Cefepime
(30 µg) discs were used. The evaluation of zone diameters was made
according to CLSI directives and the threshold values were determined
according to CSLI 2018 and CTX: R ≤27, ATM: R ≤27, CPD: R ≤27, CAZ: R
≤22, CRO: R ≤25, FOX: R ≤14, FEP: R ≤18 were accepted. Also, Cefotaxime
and Ceftazidime zone diameters progressing more than 5 mm in zone
diameters with Clavulanic acid were evaluated as positive
[26].
Investigation of Beta-Lactamase Genes
Genomic DNA was extracted by using boiling method (boiling in distilled
water at 95ºC for 10 minutes). After boiling, it was centrifuged at
12000 rpm for 5 minutes and used as supernatant DNA. For beta-lactamase,bla CTX-M, bla CTX-M-1,bla CTX-M-15,blaS HV,bla TEM,blaO XA-10, with conventional PCR,
and bla CIT, bla MOX genesfor AmpC beta-lactamase were investigated with multiplex PCR. The
primers used in PCR reactions are given in Table 1. Each PCR reaction
was run in 35 cycles with a total volume of 25 µl. For each gene, PCR
mix (K0171 Thermo Scientific) 10 pmol reverse and forward primers and
PCR water were used. In each reaction, pre-denaturation at 95ºC for 5
min, denaturation at 95ºC for 30 sec. bla CTX-Mat 57ºC, bla CTX- M-15 at 48ºC, bla CTX-M-1 andbla OXA-10 at 45ºC, bla TEMat 44ºC, bla SHV primer bonding temperature at
42ºC, 45 sec synthesis at 72ºC and 7 min final synthesis at 72ºC. The
primers for bla CIT andbla MOX were added in half and the same PCR cycle
was run with an annealing temperature of 53ºC. E. coli NCTC
13461-NCTC 13462-NCTC 13463, E. coli ATCC 35218, Klebsiella
pneumoniae ATCC 700603 strains were used as positive controls.
Identification of bacterial colonies from chicken feces
by MALDI-TOF MS
For the identification of the peptide and protein spectra, the updated
IVD database containing 10694 MSPs (Bruker Daltonics, respectively) was
applied. For microbial biomass analysis using the MALDI-TOF MS method, a
single colony was placed onto a special steel 96 micro scout plate (MSP)
(Bruker Daltonics), which was spread onto the wells in the plate in the
form of a thin film. After drying, 1 µL CHCA matrix solution (12.5 mg/mL
CHCA in a 50% ACN and 2.5% TFA mixture) was added and allowed to dry
completely at room temperature. The MALDI 96 MSP was placed in the
MALDI-TOF MS Device, and the system was operated by using the optimized
method for the identification of micro-organisms in linear positive ion
mode at a 2.000-20.000 Dalton (Da) mass range. A 60 Hz Nitrogen laser
was used at 337 nm as the ion source. The laser pulses consisting of 40
packets of 240 were applied in the measurement of each colony to obtain
the spectra. Each sample was studied in triplicate, and the highest
readings were included in the analysis. The internal quality control for
MALDI-TOF MS in general bacteriology is in part achieved by using a
Bruker BTS, consisting of an extract of E. coli proteins for mass
calibration of the instrument [34]. Mass spectrum calibration was
completed with seven peaks in the present study (m/z, 5095.39141 Da;
5381.28948 Da; 6265.88537 Da; 7254.94790 Da; 10289.99287 Da; 13692.32900
Da and 16962.67711 Da) assigned with a standard deviation of 58.52 ppm
and maximum peak error of 78.19 ppm.
The Use of PCA in MALDI-TOF MS
The spectra were analyzed using Bruker Daltonics MALDI Biotyper Flex
Analysis version 3.4 automation-controlled Biotyper Compass Explorer 1.4
software and the MALDI Biotyper 3.1 database. The identification score
criteria used were applied following the recommendations of the
manufacturer (Bruker). MALDI-TOF MS biotyping analysis elicits the
characteristic mass and peak density distribution of ribosomal 16S
proteins in the sample. Since this mass spectrum is species-specific for
many microorganisms, it represents a “molecular fingerprint” [35].
The spectra were massed using the PCA method supported by external
MATLAB software integrated into the MALDI Biotyper.
Based on the unique peptide and protein peaks within each spectrum, PCA
helped to create clustered groups of spectra with similar variational
properties and visualization of the differences among them. With
phyloproteomic-PCA, the data were given on a three-dimensional (3D)
coordinate system, and the dimensionality of the data set was reduced,
preserving the original information. Optimized preliminary procedures
(correction method: Savitski-Golay; subtraction method: multi-polygon;
normalization method) were applied for each spectrum to increase the
speed of the analysis and reduce the size of the data body [36]. The
variance among the bacteria was automatically calculated with software
support. In addition, virtual gel images (VGI) containing the projection
of the peaks within the bacteria spectra were created. Vertical traces
of VGI ranging from red to light blue corresponded to each peak within
the spectrum and were given by a color scale ranging from low relative
abundance (light green) to high relative abundance (red). For cluster
analyses, PCA dendrograms and 3D or 2D scatter plots representing the
relationship and closeness of each spectrum were created [37].
Finally, the similarity (proximity) and difference (distance)
relationships of each bacteria to the others, whose composite
correlation index (CCI) was calculated statistically using the software,
were determined.
Results