Antimicrobial Resistance and Biofilm Formation of Escherichia 2023

microorganisms
Article
Antimicrobial Resistance and Biofilm Formation of Escherichia
coli Isolated from Pig Farms and Surroundings in Bulgaria
Mila D. Kaleva 1 , Yana Ilieva 1 , Maya Margaritova Zaharieva 1 , Lyudmila Dimitrova 1 , Tanya Chan Kim 1 ,
Iva Tsvetkova 1 , Yordan Georgiev 1 , Petya Orozova 2 , Krasimir Nedev 3 and Hristo Najdenski 1, *
1 Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy
of Sciences, 1113 Sofia, Bulgaria; milakalevavet@abv.bg (M.D.K.); illievayana@gmail.com (Y.I.);
zaharieva26@yahoo.com (M.M.Z.); lus22@abv.bg (L.D.); tanya_85@abv.bg (T.C.K.); i.likovska@abv.bg (I.T.);
y.georgiev001@gmail.com (Y.G.)
2 National Reference Laboratory for Fish, Mollusks and Crustacean Diseases, National Diagnostic Research
Veterinary Institute, 1000 Sofia, Bulgaria; petyorozova@gmail.com
3 Swine Complex (Svinekompleks) Krumovo Gradishte, Boni Holding AD, 1527 Sofia, Bulgaria;
k.nedev@boniholding.com
* Correspondence: hnajdenski@gmail.com; Tel.: +359-2979-3161
Abstract: Escherichia coli (E. coli) is a ubiquitous microorganism with pathogenic and saprophytic
clones. The objective of this study was to evaluate the presence, virulence, antibiotic resistance
and biofilm formation of E. coli in three industrial farms in Bulgaria, as well as their adjacent sites
related to the utilization of manure (feces, wastewater in a separator, lagoons, means of transport,
and soils). The isolation of single bacterial cultures was performed via standard procedures with
modifications, and E. coli isolates were identified via matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR). The disk
diffusion method was used to assess antimicrobial resistance, and PCR was used to detect genes for
antibiotic resistance (GAR) (qnr, aac(3), ampC, blaSHV/blaTEM and erm) and virulence genes (stx,
Citation: Kaleva, M.D.; Ilieva, Y.; stx2all, LT, STa, F4 and eae). The protocol of Stepanović was utilized to measure the biofilm formation
Zaharieva, M.M.; Dimitrova, L.; Kim, of the isolates. A total of 84 isolates from different samples (n = 53) were identified as E. coli. Almost
T.C.; Tsvetkova, I.; Georgiev, Y.; all demonstrated antimicrobial resistance, and most of them demonstrated resistance to multiple
Orozova, P.; Nedev, K.; Najdenski, H.
antibiotics from different classes. No virulence genes coding the Shiga toxin or enterotoxins or
Antimicrobial Resistance and Biofilm
those associated with enteropathogenicity were detected. No GAR from those tested for quinolones,
Formation of Escherichia coli Isolated
aminoglycosides and macrolides were found. However, all isolates that were resistant to a penicillin-
from Pig Farms and Surroundings in
class antibiotic (56) had β-lactamase-producing plasmid genes. All of them had ampC, and 34 of them
Bulgaria. Microorganisms 2023, 11,
1909. https://doi.org/10.3390/ had blaTEM. A total of 14 isolates formed strongly adherent biofilms. These results in a country where
microorganisms11081909 the use of antibiotics for growth promotion and prophylaxis in farms is highly restricted corroborate
that the global implemented policy on antibiotics in human medicine and in animal husbandry
Academic Editor: Todd Riley
needs revision.
Callaway
Received: 25 April 2023 Keywords: Escherichia coli; pigs; antibiotic resistance; resistance genes; β-lactamase; ESBL;
Revised: 13 July 2023 MALDI-TOF-MS; biofilm
Accepted: 25 July 2023
Published: 27 July 2023
1. Introduction
A famous and widely cited report stated that 10 million people will die each year
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
by 2050 due to antimicrobial resistance (AMR), popularly known as antibiotic resistance [1].
This article is an open access article
However, scientists, who have devoted more studies to predictive modeling, have empha-
distributed under the terms and sized that, fortunately, this is not to be expected with the current rate of increasing mortality
conditions of the Creative Commons due to AMR but will occur under a very specific worst scenario if no action is taken [2].
Attribution (CC BY) license (https:// According to our observations, such misunderstandings are to be expected, because there
creativecommons.org/licenses/by/ are not many research articles on that topic, and we rely on reports for citing. However, a
4.0/).
Microorganisms 2023, 11, 1909. https://doi.org/10.3390/microorganisms11081909 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 1909 2 of 22
recent and also much cited article estimated that, in 2019, there were 1.27 million deaths at-
tributable to bacterial AMR and 4.95 million deaths associated (indirectly attributable) with
it [3]. Given these numbers, it is absolutely justified that The World Health Organization
recognized AMR as a top-priority health threat [4].
Besides unnecessary prescriptions in medicine, antibiotics are used in animal hus-
bandry to specifically promote the growth and/or health maintenance of livestock. This
can lead to the development of drug-resistant bacteria in the gut of animals and is con-
sidered to be one of the main reasons for the rapid exacerbation of AMR worldwide [5,6].
That phenomenon limits the therapeutic options for severe livestock infectious diseases.
The antimicrobials used for food-producing animals are frequently the same or belong
to the same classes as those used in human medicine. In addition, the use of one class
of antimicrobial may result in the selection of resistance against another, unrelated class
(co-resistance) [7].
Drug-resistant bacteria from the intestines of farm animals are considered a potential
source of genes for antibiotic resistance (GAR) that can spread horizontally to zoonotic and
other bacteria through the food chain but also through water, manure and direct animal
contact, and cause illness [7,8]. On a global scale, about 73% of all antimicrobials sold
are used in food animals [9], and that figure is increasing. The domestic pig (Sus scrofa
domesticus) is one of the main sources of meat for human consumption, as over 40% of all
meat consumed in the world comes from this animal species [10]. Some antibiotics, such as
fluoroquinolones and tetracyclines, cannot be fully metabolized in the pig intestine, and
these residues are found in dust, feces, sewage, soil, surface water and crops. These different
groups of antibiotic residues are suitable breeding grounds for resistant bacteria [11,12].
Escherichia coli (E. coli) represents a major reservoir of antimicrobial resistance genes
that can spread horizontally to zoonotic and other bacteria. It is actually intrinsically sus-
ceptible to almost all clinically relevant antimicrobial agents, but this bacterial species has a
great capacity to accumulate resistance genes, mostly through horizontal gene transfer [13].
E. coli also produces extended-spectrum beta lactamase (ESBL), which is also a global health
problem. These enzymes are coded by genes such as blaTEM, blaSHV, blaNDM-1, etc. The
blaTEM gene is expressed with a high value in livestock nurseries, pigs for fattening, and
manure. E. coli has also been found to have an important role in the spread of mcr and/or
tet(X3)/(X4). These genes collectively mediate resistance in Gram-negative bacteria to drugs
such as penicillins, carbapenems, polymyxins and tigecycline, which may lead to a lack of
choice of antimicrobials in both human and veterinary medicine [14–17]. Plasmid-mediated
quinolone resistance (PMQR) genes and 16S rRNA methylases are other problematic ge-
netic determinant classes of AMR in E. coli [18]. It is recommended to monitor commensal
E. coli (and enterococci) as a biomarker to monitor AMR in livestock farms, from randomly
selected healthy animals, in food and/or in hospitals because their resistance is an indicator
of the selective pressure exerted by the use of antimicrobials on intestinal populations of
bacteria in food animals [7,19–21].
In addition, in 2019, E. coli was considered one of the major pathogens responsible for
deaths associated with AMR [3]. Shiga toxin-producing E. coli (STEC) is the fourth most
commonly reported foodborne gastrointestinal infection in humans in the EU [22], which
determines the importance of testing for virulence genes.
Although E. coli is not as ubiquitous of a pathogen in biofilms found in healthcare
as methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa are, it can still
cause sepsis [23]. Biofilms are one of the mechanisms of AMR, because they protect the
inner bacterial cells as a result of reduced permeability. It is established that bacterial AMR
(including resistance to host immune factors) increases up to several hundred times in the
biofilm. Persister cells also contribute to reduced sensitivity [24]. For those reasons, we
included tests for biofilm formation in our research.
In order to continue our work in the field of antimicrobial resistance in Bulgarian
farm swine samples, we tested feces and lagoons in the same way as that in our previous
work [25], but this time, we include three farms as well as wastewaters, transport vehicles
and soils in addition. The results concerning the antimicrobial susceptibility, GAR and
biofilm formation of E. coli are presented here.
2. Materials and Methods

2.1. Swine Farms and Sample Collection
The first pig farm for this research was the same as that in our previous study [25]
(near Kostinbrod town). Samples were taken in May 2021. Three fecal samples (FK1–FK3),
three samples from lagoons (LK1–LK3), three samples from wastewaters from pigs for
fattening (WK1–WK3) and five samples from soils from fields adjacent to the farm/lagoons
(SK1–SK5) were collected. Additionally, nine soil samples were taken from soils from
fertilized fields adjacent to the farm again in November 2022 (SK6–SK14), adding to a total
of 23 samples. Sample FK3 was from young pigs, sample FK2 was from lactating sows, and
sample FK3 was from pigs for fattening. Samples LK1, LK2 and LK3 were taken at a depth
of 10 cm, 50 cm and 70 cm, respectively. WK1, WK2 and WK3 were taken at a depth of
5, 25 and 40 cm, respectively. SK1, SK2, and SK3 were taken at a distance of 20 cm, 1 m and
4 m from a lagoon, respectively, and at a depth of 16 cm, 20 cm and 26 cm. SK4 and SK5
were taken from a surface layer and at a 16 cm depth, respectively, from a fertilized field.
The second swine farm was in Samovodene village (near Veliko Turnovo town). Four
fecal samples (FV1–FV4), four samples from wastewaters drained from a separator and
at a depth of 1 m (WV1–WV4), one sample from a lagoon with a depth of 1 m (LV1) and
three samples from a 25 cm depth from agriculture fields (with tomatoes) within the limits
(vicinity) of the farm (S1–S3) were collected (a total of 12 samples). FV1 and FV2 were from
pregnant and lactating sows, respectively. FV3 was from pigs for fattening, and FV4 was
from young pigs. WV1, WV2, WV3 and WV4 were from pigs for fattening, young pigs,
lactating sows and pregnant sows, respectively.
The third pig farm was in Krumovo Gradisthe village (near Karnobat city). The farm
was established before 1980 to meet the needs of meat in the Burgas Province, which is the
largest province by area and is privately owned. A total of 2560 sows, about 7000 suckling
pigs and 8000 growing pigs are reared on the farm. They are raised until the age of 60 days
from birth and are then fattened in another pig-rearing complex—the largest in the Burgas
region. At the cultivation premises, the fertilizer mass enters a collective shaft, and from
there, it is taken to separators for separating the liquid from the solid phase. The solid
phase is separated in concrete basins, and after one month, it can be used for fertilizing.
The liquid phase goes into clarifiers, and from there, it goes into “earth fill”-type lagoons,
where it stays for about six months. From there, it is spread by a tanker as fertilizer after the
harvest period. Six fecal samples (F1–F6), two samples from wastewaters (W1–W2), two
samples from lagoons (L1–L2), one sample from a transport vehicle (T1) and seven samples
from soils from fields adjacent to the farm (S1–S7) were collected in October 2022 (a total of
18 samples). Samples F1 and F2 were from the feces of lactating sows, samples F3 and F4
were from suckling piglets, and samples F5 and F6 were from young pigs. The samples
from the wastewaters were a liquid fraction from the border, with a thicker fraction (W1)
and a fresh hard sample from a separator (W2). Lagoon samples were from the surface
layer at 5 cm (L1) and from the deep layer at 25 cm (L2). T1 was a hard dry sample from
a tractor. Soil samples were from different depths and 3 different fields: S1 was taken at
10 cm from field 1; S2–S4 were taken at 10, 30 and 50 cm, respectively, from field 2; and
S5–S7 were taken at the same depths from field 3. All samples were collected according to
ISO 5667-3:2018 with the permission of the farm owners.
The farms comprise western, eastern and central Bulgaria, and the total number of
samples was 53 (n = 53).
2.2. Isolation of Single Bacterial Cultures

Single colonies, suspected to consist of E. coli, were isolated according to ISO
16654:2001/Amd 1:2017 with some modifications. The enriched samples were cultured on
HiCrome™ Chromogenic Coliform Agar (CCA) (M1991I) or Endo agar (M029) (HiMedia,
Mumbai, India). Because this study was part of research that included culturing in the
search for other bacterial genera, single colonies, suspected of having Salmonella spp., were
isolated according to ISO 65791:2017. Enriched samples were cultured on XLD agar (M031,
HiMedia, Mumbai, India).
For positive controls, we used E. coli ATCC 35218 (American Type Cell Culture Col-
lection, Manassas, VA, USA), as well as E. coli O:157 and E. coli 41 (Collection of the
Stephan Angeloff Institute of Microbiology). All isolated colonies were morphologically
characterized with the automatic HD colony counter Scan 1200 (INTERSCIENCE, Saint-
Nom-la-Bretèche, France).
2.3. Identification of E. coli via Spectral Methods (Matrix-Assisted Laser Desorption/Ionization

Time-of-Flight Mass Spectrometry) (MALDI-TOF-MS)
All isolated colonies from Endo and XLD agar were identified via MALDI-TOF mass
spectrometer (Bruker Daltonics, Billerica, MA, USA). The essence of the technique is the
identification of microorganisms by mapping their unique protein pattern. A small amount
of an overnight bacterial mass with a density of 104 to 106 CFU/mL was mixed with 1 µL
of a matrix solution—α-cyano-4-hydroxycinnamic acid (HCCA)—and was placed on the
corresponding well of the matrix. The mixture thus made was allowed to dry and was
later loaded into the apparatus. Mass spectrometry occurred under constant high vacuum
values, and each sample was exposed to short pulses of laser rays (acceleration voltage of
20 kV, mass range of 2.6–20 kDa, laser frequency of 60 Hz and pulsed ion extraction delay
of 170 ns). With the energy created from the laser ray, ribosomal proteins were ionized. The
molecular fingerprints were comparted with a reference database for ID using the MALDI
Biotyper software (Bruker Daltonics, Billerica, MA, USA). The strains identified as E. coli
were used for further analysis.
2.4. Isolation of DNA of the Bacterial Colonies of E. coli

The strains confirmed for E. coli via MALDI-TOF were recultured, and the total DNA
was extracted from single colonies with either the GeneMATRIX Tissue & Bacterial DNA
Purification Kit (E3551, EURx Ltd., Gdańsk, Poland) or the GenElute Bacterial Genomic
DNA Kit (Sigma-Aldrich, St. Louis, MO, USA) or through crude lysate preparation. The
lysates were made by dissolving one bacterial colony in 100 µL of lysis buffer of 0.05 M
NaOH and 0.125% sodium dodecyl sulfate (final concentrations), and samples were in-
cubated for 17 min at 90 ◦ C. The DNA concentration and purity were determined with
NanoDrop Lite (Thermo Fisher Scientific Inc., Waltham, MA, USA).
2.5. Polymerase Chain Reaction (PCR) Analysis

The extracted DNA from the isolated E. coli strains was subjected to conventional and
multiplex PCR with the following:
• Gene-specific primers for E. coli (genes uidA, coding β-glucuronidase and yccT, coding
a conserved protein with an unknown function);
• Primers linked to virulence genes—Shiga toxin (verotoxin)-producing (STEC/VTEC)
(stx and stx2all), enterotoxigenic (ETEC) (LT, STa, and F4) and enteropathogenic
(EPEC) (eae);
• Primers linked to genes for antibiotic resistance (GAR)—quinolones (qnr), aminogly-
cosides (aac(3)), β-lactamase-producing plasmid genes (ampC and blaSHV/blaTEM)
and macrolides (erm) (Table 1);.
• BlaSHV/blaTEM codes ESBL, and ampC codes AmpC beta-lactamase.
For PCR amplification, we used the Color perpetual Taq PCR Master Mix (2×) protocol
(E2745, EURx Ltd., Gdańsk, Poland) optimized in our laboratory, as follows: 1 cycle of
initial denaturation running at 95 ◦ C for 5 min; a total of 30 cycles of denaturation (at
94 ◦ C for 30 s), annealing (depending on the melting temperature of the primer for 30 s)
and extension (at 72 ◦ C for 1 min); and 1 cycle of final extension (at 72 ◦ C for 7 min) and
cooling (at 4 ◦ C). Where lysates were used, Tween-20 and gelatine were added to the
reaction mix to final concentrations of 0.5% and 0.01%, respectively. The PCR products
were visualized in 1.5 or 2% agarose gels. For positive controls, we used the following
strains: E. coli ATCC 35218 for the detection of E. coli strains (uidA and yccT), E. coli O:157
containing LT and E. coli 41 (Collection of the Stephan Angeloff Institute of Microbiology)
for the detection of eae genes. For the other different E. coli, strains from the Collection of
the Stephan Angeloff Institute of Microbiology were used.
Table 1. List of primers with their sequences and temperature of melting (annealing) (Tm).
Primers Sequences Tm Amplicon Reference

E. coli uidA F 50 -AAA ACG GCA AGA AAA AGC AG-30
55 ◦ C 147 bp 1 [26]
E. coli uidA R 50 -ACG CGT GGT TAC AGT CTT GCG-30
E. coli yccT F 50 -GCA TCG TGA CCA CCT TGA-30
56 ◦ C 59 bp [27]
E. coli yccT R 50 -CAG CGT GGT GGC AAA A-30
stx1-1 F 50 -TTA GAC TTC TCG ACT GCA AAG-30
60 ◦ C 531 bp [28]
stx1-1 R 50 -TGT TGT ACG AAA TCC CCT CTG-30
stx2all F 50 -TTA TAT CTG CGC CGG GTC TG-30
60 ◦ C 327 bp [28]
stx2all R 50 -AGA CGA AGA TGG TCA AAA CG-30
LT F 50 -TTA CGG CGT TAC TAT CCT CTC TA-30
60 ◦ C 275 bp [28]
LT R 50 -GGT CTC GGT CAG ATA TGT GAT TC-30
STa F 50 -TCC CCT CTT TTA GTC AGT CAA CTG-30
60 ◦ C 163 bp [28]
STa R 50 -GCA CAG GCA GGA TTA CAA CAA AGT-30
F4 F 50 -ATC GGT GGT AGT ATC ACT GC-30
60 ◦ C 601 bp [28]
F4 R 50 -AAC CTG CGA CGT CAA CAA GA-30
eae (Intimin) F 50 -CAT TAT GGA ACG GCA GAG GT-30
60 ◦ C 791 bp [28]
eae (Intimin) R 50 -ATC TTC TGC GTA CTG CGT TCA-30
qnrA F 50 -GGG TAT GGA TAT TAT TGA TAA AG-30
50 ◦ C 670 bp [29]
qnrA R 50 -CTA ATC CGG CAG CAC TAT TTA-30
qnrB F 50 -GAT CGT GAA AGC CAG AAA GG-30
54 ◦ C 469 bp [30]
qnrB R 50 -ACG ATG CCT GGT AGT TGT CC-30
aac(3)-IV F 50 -CTT CAG GAT GGC AAG TTG GT-30
55 ◦ C 286 bp [31]
aac(3)-IV R 50 -TCA TCT CGT TCT CCG CTC AT-30
blaSHV F 50 -TCG CCT GTG TAT TAT CTC CC-30
58 ◦ C 768 bp [32]
blaSHV R 50 -CGC AGA TAA ATC ACC ACA ATG-30
blaTEM F 50 -TCG GGG AAA TGT GCG CG-30
55 ◦ C 972 bp [33]
blaTEM R 50 -TGC TTA ATC AGT GAG GCA CC-30
ampC F 50 -AAT GGG TTT TCT ACG GTC TG-30
58 ◦ C 191 bp [34]
ampC R 50 -GGG CAG CAA ATG TGG AGC AA-30
ermB F 50 -GAA AAA GTA CTC AAC CAA ATA-30
45 ◦ C 639 bp [35]
ermB R 50 -AAT TTA AGT ACC GTT AC-30
1 Base pairs.
2.6. Disk Diffusion Method

Antimicrobial susceptibility testing was performed via a standard disk diffusion
method, also known as the Kirby–Bauer method, according to the protocols of the CLSI [36].
We again used antibiotics applicable to the treatment of patients, namely meropenem
(10 µg, MEM10C Oxoid ltd, Basingstoke, Hampshire, UK), ampicillin (10 µg, SD002-
1PK), amoxycillin (25 µg, SD129-1PK), amoxycillin/clavulanic acid (20/10 µg, AUG30C),
carbenicillin (100 µg, SD004-1PK), cefamandole (30 µg, SD200-1PK), erythromycin (15 µg,
SD013-1PK), streptomycin (10 µg, SD031-1PK), tetracycline (30 µg, SD037-1PK), doxycycline
hydrochloride (30 µg, SD012-1PK), chloramphenicol (30 µg, SD006-1PK), nalidixic acid
(30 µg, SD021-1PK), ciprofloxacin (5 µg, SD060-1PK), pefloxacin (5 µg, SD070-1PK) and co-
trimoxazole (25 µg, SD010-1PK) from HiMedia, India. The results were evaluated according
to the cut-off breakpoint values of EUCAST version 12.0, 2022 [37], CLSI, 31st edition [36]
and the Manual of BBL Products and Laboratory Procedures [38]. Breakpoint values of
erythromycin for other bacterial species were taken for E. coli.
2.7. Test for Biofilm Formation

We used the protocol of Stepanović et al. [39] with small modifications, as described in
Dimitrova et al. [25]. The biofilms were photodocumented with a microscopic configuration
Nikon Eclipse-Ci-L (Nikon Instruments Europe BV, Amstelveen, The Netherlands), and the
optical density (OD) was measured at 570 nm by using an ELISA reader ELx800 (BioTek
Instruments, Winooski, VT, USA). The classification of Christensen et al. (Table 2) was used
again to determine the adherence potential [40].
Table 2. Correlation between the optical density of samples and bacterial adherence [40].
Formula Adherence
ODsample ≤ ODblank non-adherent
ODblank < ODsample ≤ 2 × ODblank weakly adherent
2 × ODblank < ODsample ≤ 4 × ODblank moderately adherent
4 × ODblank < ODsample strongly adherent
3. Results
3.1. Isolation of Single Bacterial Cultures
Selected colonies from CCA, Endo or XLD agar were used for the spectral identification
of the bacterial species. Endo agar is recommended for the confirmation of suspected
members of the coliform group. E. coli are expected to have a metallic sheen on this agar.
As XLD agar is a selective medium for Salmonella spp., no colonies were suspected to
be E. coli.
3.2. Identification by MALDI-TOF-MS and PCR

A total of 85 colonies from different samples were identified as E. coli via MALDI-TOF-
MS. Later, 84 of them were confirmed with PCR. Isolates that were positive for either the
uidA or the yccT gene were accepted as E. coli. Some colonies that did not have a metallic
sheen on Endo and that were not suspected for E. coli turned out to be this bacterial species
(F4.2 and T1.1), and not all colonies that had a metallic sheen on this agar turned out to be
this species. Generally, not all strains of a species isolated with a certain selective nutrient
medium have all the expected typical morphological characteristics; therefore, this is not a
new phenomenon.
It is interesting that six of the identified colonies were previously suspected to be
Salmonella spp., as they were isolated from XLD agar (W2.4, T1.2, T1.3, S6.1, S6.2 and S7.2).
Moreover, when recultured on Endo agar, two of them had a metallic sheen (W2.4 and
T1.3), but the rest of them did not.
3.3. Antibiotic Resistance from the Disk Diffusion Method

Although erythromycin is not used for E coli, we tested it, because there is the potential
horizontal transfer of erythromycin GAR to other bacterial species. As can be seen from
Tables 3–8, all isolates, except six (WV1.7, WV2.1, WV2.2, WV2.6, SV3.2 and SV3.4), had
resistance to at least one agent, and many of them had resistance to multiple antibiotics. The
resistance varied in wide ranges—from 0% for cephalosporins to 81% for tetracyclines and
other agents. E. coli was isolated from all types of samples. Our results show that almost
all isolates had resistance to multiple antibiotic agents, in line with the global tendency of
increases in AMR, including in farm animals [5,6]. The antibiotic class that was associated
with the greatest developed resistance was tetracycline (81%), followed by penicillins (56%).
The percentage of chloramphenicol resistance was very high in the Karnobat farm and
much lower in the other two, averaging 42.9%. The resistance to aminoglycoside was
39.3%. The resistance to trimethoprim/sulfamethoxazole followed the same pattern as
that of the other unsorted agent, chloramphenicol, averaging 27.4%. Fluoroquinolones, on
the contrary, showed much lower resistance in the Karnobat farm, in comparison to the
others, and the average was 20.2%. Resistance to the class of macrolides was low (6%), and
resistance to the class of carbapenems and cefamandole was absent.
Table 3. Antibiotic resistances from the disk diffusion method of the isolated E. coli strains from the
farm near Kostinbrod (FK3.1–SK3.1).
Drug
Antibiotic/Strain FK3.1 FK3.2 FK3.3 FK3.4 FK3.5 WK1.3 WK1.4 WK2.5 WK2.6 WK3.3 LK1.1 LK1.3 LK1.6 LK2.1 LK2.2 LK3.1 LK3.4 SK3.1
Class
Tetracycline R R R R R R R R I I R I R R R R R R
Tetracy- Doxycycline
clines R R R I R I I R R I I S S I S S R R
hydrochloride
Macrolides Erythromycin I I I I I I I I I I I I I S I I I I
Cephalo- S S S S S S S S S S S S S S S S S S
Cefamandole
sporins
Nalidixic acid S S S S S R R R R S S S S S S S S S
Fluoroqu- Pefloxacin S S S S I R R R R S I R I R R I R S
inolones Ciprofloxacin S S S S S R I I I S S S S S S S S S
Ampicillin R R R R R R R R R S S S S S S S S R
Amoxicillin R R R R R R R R R R S S S S S S S R
Penicillins Amoxicillin/ R R R R R R R R R S S S S S S S S R
clavulanic acid
Carbenicillin I S S S I R I R I S S S S S S S S I
Carbapen- Meropenem I S S S I S S I S S S S S I S S S I
ems
Aminogly-
Streptomycin R R R R R R R R R S S S S S S S S R
cosides
Chloramphenicol S S S S S S S S S S S S S S S S S S
Other
agents Trimethoprim/
S S S S S R R R R R S S S S S S S S
sulfamethoxazole
Legend: R, Resistant; I, Intermediate; S, Sensitive; F, Feces; W, Wastewater; L, Lagoon; SK, Soil. The first number is
the number of the sample, and the second number is the number of the isolate.
Table 4. Antibiotic resistances from the disk diffusion method of some of the isolated E. coli strains
from the farm near Veliko Turnovo (FV1.1–FV4.3).
Drug Class Antibiotic/Strain FV1.1 FV2.1 FV2.2 FV2.3 FV2.4 FV2.5 FV2.6 FV2.7 FV3.1 FV3.2 FV3.3 FV4.1 FV4.2 FV4.3
Tetracycline R R R R R I R I R R R I R R
Tetracyclines Doxycycline
R R R I I R I I I R I I R R
hydrochloride
Macrolides Erythromycin I I I I I I I S I I I I I R
Cephalosporins Cefamandole S S S S S S S S S S S S S S
Nalidixic acid S S S I S S S I S S S S S R
Fluoroquinolones Pefloxacin S S I R S S I R S S S S R R
Ciprofloxacin S S S S S S S S S S S S S I
Ampicillin R R S R R R R R S S S S R R
Amoxicillin S R S R R R R R S S S S R R
Penicillins Amoxicillin/ S S S S S S S S S S S R R S
clavulanic acid
Carbenicillin S I I I I I I I S S S S I I
Carbapenems Meropenem S S S S S S S S S S S S I S
Aminoglycosides Streptomycin S S S R S S S S I I R R I R
Chloramphenicol S S R S S R R R S S S S S R
Other agents Trimethoprim/
S S R R S S R R I S R S S R
sulfamethoxazole
Legend: R, Resistant; I, Intermediate; S, Sensitive; F, Feces. The first number is the number of the sample, and the
second number is the number of isolate.
from the farm near Veliko Turnovo (FV4.4–WV4.2).
Drug Class Antibiotic/Strain FV4.4 WV1.2 WV1.3 WV1.4 WV1.5 WV1.7 WV2.1 WV2.2 WV2.3 WV2.5 WV2.6 WV4.1 WV4.2
Tetracycline R R R R I S I S R R S R R
R R R R S S I S S R S R R
hydrochloride
Macrolides Erythromycin R I I I I I I I I I I I I
Cephalosporins Cefamandole S S S S S S S S S S S S S
Nalidixic acid R S S S S S S S S S S S S
Fluoroquinolones Pefloxacin R I I I S S S I S R I S S
Ciprofloxacin I S S S S S S S S S S S S
Ampicillin R R R R R S S S S R S R S
Amoxicillin R R R R R S S S S R S R S
Penicillins Amoxicillin/ R S S S S S S S S S S R S
clavulanic acid
Carbenicillin I S I I S I I S S S S I I
Carbapenems Meropenem S S I S S I S S S S I S S
Aminoglycosides Streptomycin R S S S S S S S S S S I I
Chloramphenicol R S S S S S S S S R S S S
R S S R I S S S S R S S S
sulfamethoxazole
Legend: R, Resistant; I, Intermediate; S, Sensitive; F, Feces; W, Wastewater. The first number is the number of the
sample, and the second number is the number of the isolate.
from the farm near Veliko Turnovo (WV4.3–SV3.4).
Drug Class Antibiotic/Strain WV4.3 WV4.4 WV4.5 WV4.6 LV1.1 LV1.2 LV1.3 LV1.5 LV1.6 SV3.1 SV3.2 SV3.3 SV3.4
Tetracycline I R R R R R R R R R S R I
I R R R R I R R S R S R S
hydrochloride
Macrolides Erythromycin I I I I I I I I I I I I I
Nalidixic acid S S I S S S S S S S S S S
Fluoroquinolones Pefloxacin S I R I I S I S S S S S S
Ciprofloxacin S S S S S S S S S S S S S
Ampicillin R S R R S S R S S S S S S
Amoxicillin R S R R S S R S S S S S S
Penicillins Amoxicillin/ S S R I S S S S S S S S S
clavulanic acid
Carbenicillin S S R S I S I S S S I S I
Carbapenems Meropenem S S S S I S I S S S S S S
Aminoglycosides Streptomycin S R R S R S S S S S S S S
Chloramphenicol R S R R S R S S S S S S S
S S R S S R S S S S S S S
sulfamethoxazole
Legend: R, Resistant; I, Intermediate; S, Sensitive; W, Wastewater; L, Lagoon; SV, Soil. The first number is the
number of the sample, and the second number is the number of the isolate.
Multidrug resistance (MDR), defined as resistance to three or more antimicrobial

classes of the panel tested, was found for 25 isolates (29.8%).
Although there was a variation in the patterns between farms, fecal samples were
resistant to the greatest number of antibiotics (e.g., tetracyclines, penicillins and strepto-
mycin). Likely, the fecal bacteria were subjected to more selective pressure due to the direct
consumption of antibiotics, and/or the environmental factors could play a role in losing
GAR in some other environments. The fewest E. coli were isolated from lagoons and soils,
and they had resistance to fewer antibiotics. However, some of them still showed consistent
patterns, such as resistance to tetracyclines or, in the case of Karnobat, to chloramphenicol
in addition.
from the farm near Karnobat (F1.1–W2.4).
Drug Class Antibiotic/Strain F1.1 F2.1 F2.2 F3.1 F4.1 F4.2 F5.1 F6.1 F6.2 W1.1 W1.2 W2.1 W2.2 W2.3 W2.4
Tetracycline R R R R R R R R R R I S R R R
R R R R R R R R R R I R S R R
hydrochloride
Macrolides Erythromycin I I I R I I I R R I I I I I I
Cephalosporins Cefamandole S S S S S S I S S S S S S S S
Nalidixic acid S S S S S S S S S S S S S S S
Fluoroquinolones Pefloxacin S S S S S S S S S S S S R S S
Ciprofloxacin S S S S S S S S I S S S S S S
Ampicillin S R R S R R R R R R S R S R S
Amoxicillin S R R S R R R R R R S R S R S
Penicillins Amoxicillin/ R S R S R R R R R S S S S S S
clavulanic acid
Carbenicillin I I R S S S I I I S S I S S S
Carbapenems Meropenem S S S S S S S S S S S S S S S
Aminoglycosides Streptomycin R R R R R R R S I S S R I I R
Chloramphenicol R R R R S S R R R R R R R R S
S R S S R S R S S S R S S S S
sulfamethoxazole
Legend: R, Resistant; I, Intermediate; S, Sensitive; F, Feces; W, Wastewater. The first number is the number of the
sample, and the second number is the number of the isolate.
from the farm near Karnobat (F1.1–S7.2) and the controls.
E. coli
L1.1 L1.2 L2.1 L2.2 T1.1 T1.2 T1.3 S6.1 S6.2 S6.3 S7.2 E. coli
Drug Class Antibiotic/Strain ATCC
O:157
35218
Tetracycline S S R R I I R R R R R R S
S I R R R R R R R R R S S
hydrochloride
Macrolides Erythromycin I S I I I I I I I I I S S
Nalidixic acid S S S S R S S S S I S S R
Fluoroquinolones Pefloxacin S S S S R S S S S S S S S
Ciprofloxacin S S S S R S S S S S S S S
Ampicillin S S S R S R R S S S R S R
Amoxicillin S S S R S R R S S S R - R
Penicillins Amoxicillin/ S S S S S R R S S S R S S
clavulanic acid
Carbenicillin S S S S I S S S S I S S S
Carbapenems Meropenem S S S S S S S S S S S S S
Aminoglycosides Streptomycin I R R R S S I R R R I S R
Chloramphenicol R R R R R R R R R R R R R
S S S S R S R S S R S S S
sulfamethoxazole
Legend: R, Resistant; I, Intermediate; S, Sensitive; L, Lagoon; T, Transport vehicle; S6 and S7, Soil. The first number
is the number of the sample, and the second number is the number of isolate.
3.4. Detection of Antibiotic Resistance Genes

We sought GAR for a certain antibiotic class only in the isolates that showed resistance
to an antibiotic from this class (including to antibiotics not presented in this study). The
results (Figure 1) show that, from the group of GAR in this study, the isolates were positive
only for β-lactamase-producing genes. They were ampC and blaTEM. Out of 56 tested
isolates, all the samples had the ampC GAR, and there were 34 isolates that were positive
for blaTEM. These results corroborate that ESBL and AmpC β-lactamase production are
important resistance mechanisms in members of the Enterobacteriaceae family [18].
Microorganisms
Microorganisms 2023,
2023, 11,
11, x1909
FOR PEER REVIEW 1110ofof 23
22
Figure
Figure 1.
1. Gel
Gel electrophoresis
electrophoresis for
for blaTEM
blaTEM β-lactam
β-lactam resistant
resistant gene.
gene. The
The gel
gel electrophoresis
electrophoresis from
from the
the
farm
farm near Karnobat is confirmative. Black lines designate non-adjacent samples. Legend: M,marker;
near Karnobat is confirmative. Black lines designate non-adjacent samples. Legend: M, marker;
(+),
(+), positive
positive control;
control; (−),
(−),negative
negativecontrol.
control.
3.5.
3.5. Detection
Detection of
of Virulence
Virulence Genes
Genes
No
No virulence
virulence genes from the panel STEC/VTEC
STEC/VTEC(stx (stxand
andstx2all),
stx2all),ETEC
ETEC(LT,
(LT, STa,
STa, and
and
F4) and
F4) and EPEC
EPEC (eae)
(eae) were detected among the isolates.
3.6. Test
3.6. Test for
for Biofilm
Biofilm Formation
Formation
Strongly adherent E.
Strongly adherent E.coli
coli(14
(14isolates)
isolates)was
wasfound
foundamong
among allall
types of samples
types except
of samples the
except
transport vehicle ones (Tables 9–12). Moderately adherent E. coli was present
the transport vehicle ones (Table 9, Table 10, Table 11 and Table 12 ). Moderately adherent among all
types of samples. The Karnobat farm had the most strongly adherent isolates
E. coli was present among all types of samples. The Karnobat farm had the most strongly (8), whereas
the Velikoisolates
adherent Turnovo (8),farm had the
whereas the least adherent
Veliko Turnovo ones
farm(2).had
Apart from adherent
the least that, thereones
was(2).
no
correlation in concern to the type of sample and the farm.
Apart from that, there was no correlation in concern to the type of sample and the farm.
Table
Table
Microorganisms 2023, 11, x FOR PEER 9. Adherence
9. Adherence
REVIEW of isolated
of isolated E. from
E. coli coli from the farm
the pig pig farm
nearnear Kostinbrod,
Kostinbrod, compared
compared withwith
that that
of12 ofofthe
the 24
controls.
controls.
Microorganisms
Strain
Strain 2023,OD
11, OD
x550FOR
550PEER
nm
REVIEW
Adherence
Adherence
nm Biofilm
Biofilm Strain
Strain ODOD Adherence
Adherence
550 nm
550 nm
12 of
Biofilm
Biofilm 24
Microorganisms
Microorganisms 2023,2023,
11, x11,
FOR x FOR
PEERPEERTable 9. Adherence of isolated E. coli from the pig farm near Kostinbrod, compared with12that
REVIEW
REVIEW ofofthe
of1224 24
Microorganisms
Microorganisms
2023,
Microorganisms 11, x
11,
2023, x11,
FOR
FOR
11, PEER
xx FOR
PEER
FOR REVIEW
PEER REVIEW
REVIEW
PEER REVIEW 12 of
12 of1224of
1224 of 24
24
Microorganisms
2023, x 11,
11, 11,
FOR x FOR
PEERPEER controls.
REVIEW
REVIEW 1224
12 of11 of2224
Microorganisms 2023, 1909 of
Microorganisms 11, x11,
FOR PEER
x FOR REVIEW
PEER REVIEW 12 of1224of 24
ATCC
ATCC 35218 0.676
35218
Strain OD0.676
550 nm
SA SA
Table 9. Adherence of isolated E. coli from
Adherence Biofilm WK2.6
the
WK2.6
Strain farm0.711
pig 0.711
ODnear
550 nm
SA SA compared with
Kostinbrod,
Adherence that of the
Biofilm
controls.
TableTable 9. Adherence
9. Adherence of isolated
of isolated E. from
E. coli coli from the farm
the pig pig farm
nearnear Kostinbrod,
Kostinbrod, compared
compared withwith
that that of the
of the
Table
Table 9.
9. Adherence
9. Adherence
Table of
of isolated
of isolated
Adherence E.
E. coli
isolated coli
coli from
E. from the
the pig
the pig
from pig farm
farm nearnear
farm Kostinbrod,
Kostinbrod,
near compared
compared
Kostinbrod, withwith
compared thatthat
with of the
that of
of the
the
Strain Table
OD550 nm Adherence controls.
controls.
Table
9.
Table 9.
9. Adherence
Adherence of of
Biofilm
Adherence of isolated
isolated E.
isolated E.
coli coli
from from
the
from the
pig
the pig
farm
pig farm
near
Strain OD550 nm Adherence
coli farm near Kostinbrod,
Kostinbrod,
near compared
compared
Kostinbrod, with
comparedwith
that
Biofilm that
withof of
the
that the
of
controls.
controls.
controls.
Table
Table
9. Adherence
9.
controls.
controls. Adherence
of isolated
of isolated
E. coli
E. from
coli from
the pig
the farm
pig farm
nearnear
Kostinbrod,
Kostinbrod,
compared
compared
withwith
that that
of the
of the
ATCC
Strain 35218ODOD
Strain 0.676 theSA
Adherencecontrols.
Adherence Biofilm
Biofilm StrainWK2.6
StrainODOD 0.711 SA
Adherence
Adherence Biofilm
Biofilm
Strain ODOD
550 nm
550 nm controls.
controls.
Adherence Biofilm Strain
550 nm
550 nm
Strain 550 nm
550 nm550 Adherence
550 nm
nm Biofilm Strain ODOD550 nm
550 nm550
550 nm
nm Adherence
Adherence Biofilm
Biofilm
Strain
Strain
O157O157 OD OD
550 nm550 nm
0.321
0.321 Adherence
Adherence
MAMA Biofilm
Biofilm Strain
Strain
WK3.3
WK3.3 OD OD0.239
0.239 Adherence
Adherence
550 nm550 nm
WA WA Biofilm
Biofilm
ATCCStrain
Strain
Strain
35218 ODODOD
550
0.676
550 nm
550
nm nmnm
550
550 nm Adherence
Adherence
Adherence
SA Biofilm
Biofilm
Biofilm Strain
Strain
Strain
WK2.6 OD OD
OD
550 nm
0.711
550 nm
550
550 nm
550 nm
nm Adherence
Adherence
Adherence
SA Biofilm
Biofilm
Biofilm
ATCC
ATCC 35218 0.676
35218 0.676 SA SA WK2.60.711
WK2.6 0.711 SA SA
ATCC
ATCC 35218 0.676
35218 0.676 SA SA WK2.60.711
WK2.6 0.711 SA SA
ATCC
ATCC 35218 0.676
35218 0.676 SA SA WK2.60.711
WK2.6 0.711 SA SA
ATCC
ATCC
ATCC35218
35218
O157 0.676
35218 0.676
0.676
0.321 SASA
SA
MA WK2.6
WK2.6
WK2.6
WK3.30.7110.711
0.711
0.239 SA
SA WA
SA
O157
Blank
Blank 0.321
0.156
0.156 MA
- - WK3.3 0.239
LK1.1 0.224
LK1.1 0.224 WAWA
WA
O157O157 0.321
0.321 MAMA WK3.30.239
WK3.3 0.239 WAWA
O157
O157 0.321
0.321 MAMA WK3.3
WK3.30.239
0.239 WAWA
O157
O157 0.321
0.321 MAMA WK3.30.239
WK3.3 0.239 WAWA
O157
O157
O157 0.321
0.321
0.321 MA MA
MA WK3.3
WK3.30.239
WK3.3 0.239
0.239 WAWA
WA
Blank 0.156 - LK1.1 0.224 WA
Blank
FK3.1
FK3.1 0.156
0.196
0.196 WA- WA LK1.1 0.224
LK1.3 0.188
LK1.3 0.188 WAWA
WA
Blank
Blank 0.156
0.156 - - LK1.1 0.224
LK1.1 0.224 WAWA
Blank
Blank 0.156
0.156 - - LK1.1 0.224
LK1.1 0.224 WAWA
Blank
Blank
Blank 0.156
0.156
0.156 - -- LK1.10.224
LK1.1
LK1.1 0.224
0.224 WAWA
WA
Blank
Blank 0.156
0.156 - - LK1.1
LK1.1 0.224
0.224 WAWA
FK3.1 0.196 WA LK1.3 0.188 WA
FK3.2
FK3.2 0.237
0.237 WAWA LK1.6 0.174
LK1.6 0.174 WAWA
FK3.1 0.196 WA LK1.3 0.188 WA
FK3.1
FK3.1 0.196
0.196 WAWA LK1.3 0.188
LK1.3 0.188 WAWA
FK3.1
FK3.1
FK3.1 0.196
0.196
0.196 WAWA
WA LK1.3
LK1.3 0.188
LK1.3 0.188
0.188 WAWA
WA
FK3.1
FK3.1 0.196
0.196 WAWA LK1.30.188
LK1.3 0.188 WAWA
FK3.1
FK3.1
FK3.2 0.196
0.196
0.237 WAWA LK1.3
LK1.3
LK1.6 0.188
0.188
0.174 WAWA
FK3.2
FK3.3
FK3.3 0.237
0.254
0.254 WAWA
WA LK1.6 0.174
LK2.1 0.242
LK2.1 0.242 WAWA
WA
FK3.2
FK3.2 0.237
0.237 WAWA LK1.6 0.174
LK1.6 0.174 WAWA
FK3.2
FK3.2
FK3.2 0.237
0.237
0.237 WAWA
WA LK1.6
LK1.6 0.174
LK1.6 0.174
0.174 WAWA
WA
FK3.2
FK3.2 0.237
0.237 WAWA LK1.60.174
LK1.6 0.174 WAWA
FK3.2
FK3.2 0.237
0.237 WAWA LK1.6
LK1.6 0.174
0.174 WAWA
FK3.3 0.254 WA LK2.1 0.242 WA
FK3.3 0.254 WA LK2.1 0.242 WA

FK3.3
FK3.3
FK3.3 0.254
0.254
0.254 WAWA
WA LK2.1 0.242
LK2.1
LK2.1 0.242
0.242 WAWA
WA
FK3.3
FK3.3 0.254
0.254 WAWA LK2.1 0.242
LK2.1 0.242 WAWA
FK3.3
FK3.3 0.254
0.254 WAWA LK2.10.242
LK2.1 0.242 WAWA
FK3.3
FK3.3
FK3.4
FK3.4 0.254
0.254
0.229
0.229 WA
WAWA
WA LK2.1
LK2.1
LK2.2 0.242
LK2.2 0.242
0.222
0.222 WA
WAWA
WA
FK3.4
FK3.4 0.229
0.229 WA
WA LK2.2
LK2.2 0.222
0.222 WA
WA
FK3.4 0.229 WA LK2.2 0.222 WA

FK3.4
FK3.4 0.229
0.229 WAWA LK2.2 0.222
LK2.2 0.222 WAWA
FK3.4 2023,
Microorganisms
FK3.4 0.229
11,
0.229 x FOR PEER WA
WAREVIEW LK2.2 0.222
LK2.2 0.222 WAWA 13 of 24
FK3.4
Microorganisms
FK3.4 2023, 11, x 0.229
0.229 FOR PEER WA WA
REVIEW LK2.20.222
LK2.2 0.222 WAWA 13 of 24
FK3.4
FK3.4
FK3.5
FK3.5
FK3.5 0.229
0.229
0.293
0.293
0.293 WA
WAWA
WA
WA LK2.2
LK3.1 0.222
LK2.2
LK3.1
LK3.1 0.222
0.215
0.2150.215 WA
WAWA
WA
WA
FK3.5 0.293 WA LK3.1 0.215 WA

WK1.3 0.859 SA LK3.4 0.287 WA
WK1.3
WK1.3
FK3.5 0.859
0.859
0.293 SA
SA
WA LK3.4
LK3.4
LK3.1 0.287
0.287
0.215 WA WA
WA
FK3.5
FK3.5 0.293
0.293 WAWA LK3.1 0.215
LK3.1 0.215 WAWA
FK3.5
FK3.5 0.293
0.293 WAWA LK3.1
LK3.1 0.215
0.215 WAWA
FK3.5
FK3.5 0.293
0.293 WAWA LK3.10.215
LK3.1 0.215 WAWA
FK3.5
FK3.5 0.293
0.293 WAWA LK3.1
LK3.1 0.215
0.215 WAWA
WK1.4 0.864 SA SK3.1 0.580 MA

WK1.4 0.864 SA SK3.1 0.580 MA
FOR PEER
x FOR REVIEW
PEER REVIEW 13 of
13 24
of 24
WK1.3 0.859 SA LK3.4 0.287 WA
WK1.3 0.859 SA LK3.4 0.287 WA
WK1.3
WK1.3 0.8590.859 SA SA LK3.40.2870.287
LK3.4 WA WA
WK1.3
WK1.3
WK1.3
WK1.3 0.859
0.8590.859
0.859 SA SA
SA SA LK3.4
LK3.4
LK3.4 0.287
LK3.40.2870.287
0.287 WAWA
WA WA
WK1.3 0.859 SA LK3.4 0.287 WA
WK1.3
WK1.3 0.859
0.859 SA SA LK3.4
LK3.4 0.287
0.287 WAWA
WK1.4 0.864 SA9. Cont.
Table SK3.1 0.580 MA
WK1.4 0.864 SA SK3.1 0.580 MA
Strain OD550 nm Adherence Biofilm Strain OD550 nm Adherence Biofilm
WK1.4
WK1.4 0.8640.864 SA SA SK3.10.5800.580
SK3.1 MA MA
WK1.4
WK1.4
WK1.4
WK1.4 0.864
0.8640.864
0.864 SA SA
SA SA SK3.1
SK3.1
SK3.1 0.580
SK3.10.580 0.580
0.580 MAMA
MA MA
WK1.4 0.864 SA SK3.1 0.580 MA
WK1.4
WK1.4
WK1.4 0.864
0.864
0.864 SASA
SA SK3.1
SK3.1 0.580
SK3.1 0.580
0.580 MAMA
MA
WK2.5 0.680 SA
WK2.5 0.680 SA
WK2.5
WK2.5 0.6800.680 SA SA
WK2.5
WK2.5
WK2.5
WK2.5 0.680
0.6800.680
0.680 SA SA
SA SA
WK2.5
WK2.5 0.680
0.680 SASA
WK2.5
WK2.5 0.680
0.680 SATable
SA
10. Adherence of a part of the isolated E. coli from the pig farm near Veliko Turnovo, compared
Tablewith that of the controls.
10. Adherence of a part of the isolated E. coli from the pig farm near Veliko Turnovo, compared
with that of the controls.
TableTable 10. Adherence
10. Adherence of a of
of a part part
theofisolated
the isolated
E. coliE.from
coli from
the
OD the
pig pig farm
550farm near near Veliko
Veliko Turnovo,
Turnovo, compared
compared
Strain OD550 nmwithTable 10. Adherence
Table
Adherence
Table 10.
Table Adherence
10.
10. Adherence
Adherence of aa part
of part
of of the
aa part
Biofilm
of of
partthe
of isolated
the
ofisolated E.Strain
the isolated
E.
isolatedcoli
E.
coli from
coli
E.from the
coli from
the
from pig
the
pig farm
pig
thefarm nearnear
farm
Adherence
pig near
farm Veliko
Veliko
near Turnovo,
Veliko
Veliko compared
Turnovo,
Biofilm
Turnovo, compared
compared
Turnovo, compared
with
that 10.
Table that
of the ofcontrols.
the controls.
Adherence of a part of the isolated E. coli
OD from the pig farm near Veliko Turnovo, compared
with
with that
with
Table that
with of
10. that theof
Adherence
of
thatthe controls.
the
the controls.
of a part of the isolated
ofcontrols.
controls.
550
E. coli from the pig nm farm near Veliko Turnovo, compared
Strain OD550 nm Adherence with that Biofilm Strain Adherence Biofilm
with that of of
thethe controls.
controls. OD nm
OD 550 pig
Strain OD550
OD Table 10. Adherence
Table
Adherence 10. Adherence of aof
part of the
a part
Biofilm isolated
of the E.
isolated coli from
E. coli
Strain OD the pig
550
from
OD the farm nearnear
farm
Adherence Veliko Turnovo,
Veliko compared
Turnovo,
Biofilmcompared
Strain
Strain OD Adherence
nm550 nm
Adherence Biofilm
Biofilm Strain
Strain OD OD550
550
550 Adherence
550
Adherence Biofilm
Biofilm
Strain
Strain
Strain
Strain OD OD
OD
550
550 nm550
nm
OD550 nm
550 nm
nm Adherence
Adherence
with that
Adherence
with
Adherence of
that the
of controls.
the Biofilm
Biofilm
Biofilm
controls.
Biofilm
Strain
Strain
Strain
Strain ODnm
OD
Adherence
nm Adherence
Adherence
Adherence
Biofilm
Biofilm
Biofilm
Biofilm
nm 550 550
nmnm
ATCC 35218OD5500.676
Strain nm SA
Adherence Biofilm FV3.1
Strain 0.208nm WA
nm Adherence Biofilm
nm
ATCC 35218 OD 0.676 SA FV3.1 OD OD
Strain0.208 Adherence
WA
550 550
Strain
Strain OD 550 nmAdherence
550 nm Adherence Biofilm
Biofilm Strain Adherence Biofilm
Biofilm
nm nm
ATCC
ATCC 35218 0.6760.676
35218 SA SA FV3.10.2080.208 WA WA
FV3.1
ATCC
ATCC 35218
ATCC
ATCC 35218
35218
35218
ATCC 0.6760.676
0.676
35218 0.6760.676 SA
SASA
SA SA FV3.1
FV3.1
FV3.1
FV3.1 0.208
0.208
0.208
FV3.10.208 WAWA
0.208 WA WA
WA
ATCC 35218 0.676 SA FV3.1 0.208 WA
ATCC 35218
ATCC 35218 0.676
O157 0.676
0.321 SA SA
MA FV3.1
FV3.20.208
FV3.1 0.143 WAWA
0.208 NA
O157 0.321 MA FV3.2 0.143 NA
O157
O157 0.3210.321 MA MA FV3.20.1430.143 NA NA
FV3.2
O157
O157
O157
O157
O157 0.321
0.3210.321
0.321
0.321 MA
MA
MAMA
MA FV3.2
FV3.2
FV3.2 0.143
FV3.20.143
FV3.2 0.143
0.143
0.143 NA NA
NA NA
NA
O157 0.321 MA FV3.2 0.143 NA
O157
O157 0.321
0.321 MAMA FV3.2
FV3.2 0.143
0.143 NANA
Blank 0.156 - FV3.3 0.262 WA
Blank 0.156 - FV3.3 0.262 WA
Blank
Blank
Blank 0.1560.156
0.156 -- - - FV3.30.262
FV3.3
FV3.3 0.262
0.262 WA WA
WA
Blank
Blank
Blank
Blank 0.156
0.1560.156
0.156 - -- FV3.3
FV3.3
FV3.3 0.262
FV3.30.2620.262
0.262 WAWA
WA WA
Blank 0.156 - FV3.3 0.262 WA
Blank
Blank 0.156
0.156 - - FV3.3
FV3.3 0.262
0.262 WAWA
Microorganisms
11, x 11,
FOR x FOR
PEERPEER REVIEW
REVIEW 14 of1424of 24
Microorganisms 11, x 11,
FOR PEERPEER
x FOR REVIEW
FV1.1
FV1.1 0.382
0.382 MAMA FV4.1FV4.1 0.193
0.193 WA
WA
FV1.1 0.382 MA FV4.1 0.193 WA
FV1.1
FV1.1 0.3820.382 MA MA FV4.10.1930.193 WA WA
FV4.1
FV1.1
FV1.1
FV1.1
FV1.1 0.382
0.3820.382
0.382 MAMA
MA MA FV4.1
FV4.1
FV4.1 0.193
FV4.10.1930.193 WAWA
0.193 WA WA
FV1.1
FV2.1
FV2.1 0.382
0.2690.269 MAWA
WA FV4.1
FV4.2
FV4.2 0.193
0.293
0.293 WA
WAWA
FV1.1
FV1.1
FV2.1
FV2.1
FV2.1 0.382
0.2690.382
0.269
0.269 MA
WAMA
WAWA FV4.1
FV4.1
FV4.2 0.193
FV4.20.293
FV4.2 0.193 WAWA
0.293 WA
0.293 WA
WA
FV2.2
FV2.2
FV2.2 0.246
0.246
0.246 WAWA
WA FV4.30.204
FV4.3
FV4.3 0.204
0.204 WAWA
WA
FV2.2
FV2.2 0.2460.246 WA WA FV4.3
FV4.30.2040.204 WA WA
FV2.3
FV2.3 0.313
0.313 MAMA FV4.40.323
FV4.4 0.323 MAMA
FV2.3
FV2.3 0.3130.313 MA MA FV4.4
FV4.40.3230.323 MA MA
FV2.1
FV2.1 0.269
0.269 WAWA FV4.2
FV4.20.293
0.293 WAWA
FV2.2
FV2.2 0.246
0.246 WA WA FV4.3
FV4.30.204
0.204 WA WA
FV2.2
FV2.2 0.2460.246 WA WA FV4.3
FV4.30.2040.204 WA WA
FV2.2
FV2.2 0.246
0.246 WAWA FV4.30.204
FV4.3 0.204 WAWA
FV2.2
FV2.2 0.246
0.246 WA WA FV4.30.204
FV4.3 0.204 WA WA
FV2.2
FV2.2 0.246
0.246 WAWA FV4.3
FV4.30.204
0.204 WAWA
FV2.3
FV2.3 0.313
0.313 MA MA
Table 10. Cont. FV4.4
FV4.40.323
0.323 MAMA
FV2.3
FV2.3 0.3130.313 MA MA FV4.4
FV4.40.3230.323 MA MA
FV2.3
FV2.3 0.313
0.313 MAMA FV4.40.323
FV4.4 0.323 MAMA
FV2.3
FV2.3 0.313
0.313 MAMA FV4.40.323
FV4.4 0.323 MAMA
FV2.3
FV2.3
FV2.3 0.3130.313
0.313 MAMA
MA FV4.4
FV4.40.323
FV4.4 0.323
0.323 MAMA
MA
FV2.4
FV2.4 0.238
0.238 WA WA WV1.2
WV1.20.192
0.192 WA WA
FV2.4
FV2.4 0.2380.238 WA WA WV1.2
WV1.20.1920.192 WA WA
FV2.4
FV2.4 0.238
0.238 WAWA WV1.20.192
WV1.2 0.192 WAWA
FV2.4
FV2.4
FV2.4 0.238
0.238
0.238 WAWA
WA WV1.20.192
WV1.2
WV1.2 0.192
0.192 WA WA
WA
FV2.4
FV2.4 0.238
0.238 WAWA WV1.2
WV1.20.192
0.192 WAWA
FV2.5
FV2.5 0.330
0.330 MAMA WV1.3
WV1.30.283
0.283 WA WA
FV2.5
FV2.5 0.3300.330 MA MA WV1.3
WV1.30.2830.283 WA WA
FV2.5
FV2.5
FV2.5 0.330
0.330
0.330 MAMA
MA WV1.30.283
WV1.3
WV1.3 0.283
0.283 WAWA
WA
FV2.5
FV2.5 0.330
0.330 MAMA WV1.30.283
WV1.3 0.283 WA WA
FV2.5
FV2.5 0.330
0.330 MAMA WV1.3
WV1.30.283
0.283 WAWA
FV2.6
FV2.6 0.351
0.351 MAMA WV1.4
WV1.40.253
0.253 WA WA
FV2.6
FV2.6
FV2.6 0.3510.351
0.351 MA
MAMA WV1.4
WV1.40.253
WV1.4 0.253
0.253 WA WA
WA
FV2.6
FV2.6 0.351
0.351 MAMA WV1.40.253
WV1.4 0.253 WAWA
FV2.6
FV2.6 0.351
0.351 MAMA WV1.40.253
WV1.4 0.253 WA WA
FV2.6
FV2.6 0.351
0.351 MAMA WV1.4
WV1.40.253
0.253 WAWA
FV2.7
FV2.7
FV2.7 0.200
0.200
0.200 WA
WAWA WV1.5
WV1.50.229
WV1.5 0.229
0.229 WA WA
WA
FV2.7
FV2.7 0.2000.200 WA WA WV1.5
WV1.50.2290.229 WA WA
FV2.7
FV2.7 0.200
0.200 WAWA WV1.50.229
WV1.5 0.229 WAWA
FV2.7
FV2.7 0.200
0.200 WA WA WV1.50.229
WV1.5 0.229 WA WA
Microorganisms
Microorganisms 2023, 2023, 11, x PEER
11, x FOR Table
FOR PEER
Table 11.11.
REVIEW
REVIEW
Table Adherence
Adherence
11. Adherenceofofthe
of the theother
other part
other
part ofofthe
part
of the theisolated
isolated E.E.coli
isolated
E. coli colifrom
from thethe
from thepig
pig pigfarm
farm farmnear
nearnearVeliko
Veliko 15 Turnovo,
Veliko of1524of 24
Turnovo,
Turnovo,
Microorganisms
FV2.7FV2.7
Microorganisms
Microorganisms 11,
2023,2023, xx 11,
FOR
11,0.200
2023, x PEER
x FOR
0.200
FOR
11, FOR REVIEW
PEERPEER
WAREVIEW
REVIEW
PEER WA
REVIEW WV1.5 WV1.5 0.2290.229 WA WA 15
15 of 1524
of1524of
of 24
24
compared with that of the controls.
compared
compared
withwith
that that
of the
ofcontrols.
the controls.
Table 11. Adherence
Table of the
11. Adherence ofother part part
the other of the
ofisolated E. coli
the isolated E.from the pig
coli from thefarm nearnear
pig farm Veliko Turnovo,
Veliko Turnovo,
Strain OD550 nm comparedAdherence
Table
Table
withwith
compared that that
11. Adherence
of the
ofcontrols.
Biofilm
11. Adherence
of the
the controls. Strain
of other
the other
part part OD
of isolated
of the OD
the isolatedOD E. nm
E. coli
550
550 Adherence
coli from
from
550 the farm
the pig pig farm
nearnear Biofilm
Veliko
Veliko Turnovo,
Turnovo,
Strain
Strain OD550 OD Adherence
nm550 nm Adherence Biofilm
Biofilm StrainStrain Adherence
Adherence Biofilm
Biofilm
compared
compared withwith
that that ofcontrols.
of the the controls. OD nm550 nm
Table
Table 11. Adherence
11. Adherence ofother
of the the other
part part ofisolated
of the E. OD
the isolated E.from
coli coli from
550 the farm
the pig pig farm
nearnear Veliko
Veliko Turnovo,
Turnovo,
Strain
Strain OD550 OD Adherence
Biofilm StrainStrain Adherence
Adherence Biofilm
Biofilm
compared
compared with with
that that
of ofcontrols.
the the controls. OD nm OD nm
ATCCATCC
35218 35218OD 0.676 Table 11.
SAAdherence
Table of the
11. Adherence of other part part
the other ofWV4.4
the isolated
ofWV4.4 E. 550
the isolatedcoli
E. from
0.275 the WA
coli from
550 pig
the farm nearnear
pig farm Veliko Turnovo,
Veliko Turnovo,
Strain
ATCC
ATCC
ATCC 35218
ATCC
ATCC 35218 0.676
Strain
35218
3521835218 OD
0.6760.676
550
0.676
0.676 nm550 nm
0.676
SA
Adherence
Adherence
SA
SA SA withwith
SASA
compared
compared that that
of the
Biofilm
Biofilm Strain
WV4.4
the controls. WV4.4
ofcontrols. WV4.4 Strain0.275
WV4.4
WV4.4 0.275 0.275
0.275
0.275
nm 0.275
nm
WA
Adherence
Adherence
WA
WA WA
WAWA Biofilm
Biofilm
OD OD550
550
Strain OD550
Strain OD Adherence
Adherence
nm550 nm Biofilm
Biofilm Strain
Strain Adherence
Adherence Biofilm
Biofilm
OD550
nmOD nm550
Strain
Strain OD550
OD Adherence
Biofilm Strain
Strain Adherence
Adherence Biofilm
Biofilm
nm nm
O157
O157
O157 0.321
0.3210.321 MA
MA MA WVT4.5
WVT4.5
WVT4.5 0.231
0.2310.231 WA WA
O157
O157
O157
O157 0.3210.321
0.321
0.321 MA
MAMA
MA WVT4.5
WVT4.5
WVT4.5 0.231
WVT4.5 0.231 WA
0.231
0.231 WA WA
WA
WA
Blank
Blank
Blank
Blank 0.1560.156
0.156
0.156 -- - -- WV4.60.264
WV4.6
WV4.6
WV4.6 0.264 WA
0.264 WA
WA
Blank
Blank
Blank 0.1560.156
0.156 - - WV4.60.264
WV4.6
WV4.60.2640.264 WA
0.264 WA WA
WA
WV1.7
WV1.7
WV1.7 0.168
0.168
0.168 WA
WA WA LV1.10.300
LV1.1
LV1.1 0.300 WA WA
WV1.7
WV1.7
WV1.7 0.168
0.168
0.168 WA
WA WA LV1.10.300
LV1.1
LV1.1 0.300 WA
0.300
0.300 WA WA
WA
Blank
O157O157 0.156
0.321
0.321 -
MAMA WV4.60.231
WVT4.5
WVT4.5 0.264
0.231 WAWA
WA
Blank 0.156 - WV4.6 0.264 WA
Blank
Blank 0.156
0.156 - - WV4.60.264
WV4.6 0.264 WA WA
Blank
Blank 0.156
0.156 - - WV4.6
WV4.60.264
0.264 WA WA
Blank
Blank 0.156
0.156 - - WV4.6
WV4.60.264
0.264 WA WA
Blank
Blank 0.156
0.156 - - WV4.6
WV4.60.264 0.264 WAWA
WV1.7 0.168 WA LV1.1 0.300 WA
WV1.7 0.168 WA
Table 11. Cont. LV1.1 0.300 WA
WV1.7
WV1.7 0.168
0.168 WA WA LV1.10.300
LV1.1 0.300 WA WA
WV1.7
WV1.7 0.168
0.168 WA WA LV1.1
LV1.10.300
0.300 WA WA
WV1.7
WV1.7 0.168
0.168 WA WA LV1.1
LV1.10.300
0.300 WA WA
WV1.7
WV1.7
WV1.7 0.168
0.168
0.168 WAWA
WA LV1.1
LV1.1 0.300
LV1.1 0.300
0.300 WAWA
WA
WV2.1 0.499 MA LV1.2 0.442 MA
WV2.1 0.499 MA LV1.2 0.442 MA
WV2.1
WV2.1 0.499
0.499 MAMA LV1.20.442
LV1.2 0.442 MAMA
WV2.1
WV2.1 0.499
0.499 MAMA LV1.2
LV1.20.442
0.442 MAMA
WV2.1
WV2.1
WV2.1 0.499
0.499
0.499 MA
MAMA LV1.2
LV1.20.442
LV1.2 0.442
0.442 MAMA
MA
WV2.1
WV2.1 0.499
0.499 MAMA LV1.3
LV1.2
LV1.2 0.442
0.442 MAMA
WV2.2 0.314 MA 0.202 WA
LV1.3
WV2.2 0.314 MA 0.202 WA
LV1.3
LV1.3
WV2.2
WV2.2 0.314
0.314 MAMA 0.202 WA WA
0.202
LV1.3
LV1.3
WV2.2
WV2.2
WV2.2 0.314
0.314
0.314 MAMA
MA LV1.3 0.202
0.202 WA WA
0.202 WA
LV1.3
LV1.3
WV2.2
WV2.2 0.314
0.314 MAMA 0.202
0.202 WA WA
LV1.3
LV1.3
WV2.2
WV2.2 0.314
0.314 MAMA 0.202
0.202 WAWA
WV2.3 0.302 WA LV1.5 0.232 WA
WV2.3 0.302 WA LV1.5 0.232 WA
WV2.3
WV2.3
WV2.3 0.302
0.302
0.302 WAWA
WA LV1.50.232
LV1.5
LV1.5 0.232
0.232 WA WA
WA
Microorganisms
Microorganisms
2023,2023,
11, x 11,
FOR x FOR
PEERPEER
REVIEW
WV2.3
WV2.3 0.302
0.302 WA WA LV1.5
LV1.50.232
0.232 WA WA
FOR PEERPEER
x FOR REVIEW
WV2.3
WV2.3 0.302
0.302 WA WA LV1.5
LV1.50.232
0.232 WA WA
FOR PEERPEER
x FOR REVIEW
WV2.3
WV2.3 0.302
0.302 WAWA LV1.5
LV1.5 0.232
0.232 WAWA
FOR PEERPEER
x FOR REVIEW
WV2.5
WV2.5 0.175
0.175 WA
WA LV1.6
LV1.6 0.261
0.261 WAWA
WV2.5 0.175 WA LV1.6 0.261 WA
WV2.6
WV2.6 0.258
0.258 WAWA SV3.1
SV3.1 0.550
0.550 MAMA
WV2.5
WV2.5 0.175
0.175 WA WA LV1.60.261
LV1.6 0.261 WA WA
WV2.6
WV2.6 0.258
0.258 WA WA SV3.1
SV3.10.5500.550 MAMA
WV2.5
WV2.5 0.175
0.175 WA WA LV1.6
LV1.60.2610.261 WA WA
WV2.6
WV2.6 0.258
0.258 WAWA SV3.1
SV3.1 0.550
0.550 MAMA
WV2.5
WV2.5 0.175
0.175 WA WA LV1.6
LV1.60.2610.261 WA WA
WV2.6
WV2.6
WV2.6 0.258
0.258
0.258 WA
WAWA SV3.1
SV3.10.550
SV3.1 0.550 MAMA
0.550 MA
WV2.5
WV2.5 0.175
0.175 WAWA LV1.6
LV1.6 0.261
0.261 WAWA
WV4.1
WV4.1 0.207
0.207 WAWA SV3.2
SV3.2 0.747
0.747 SA SA
WV4.1
WV4.1 0.207
0.207 WA WA SV3.2
SV3.20.747
0.747 SA SA
WV4.1
WV4.1
WV4.1 0.207
0.207
0.207 WAWA
WA SV3.2
SV3.2 0.747
SV3.2 0.747
0.747 SA SA
SA
WV4.1
WV4.1 0.207
0.207 WA WA SV3.2
SV3.20.747
0.747 SA SA
WV4.2
WV4.2 0.192
0.192 WAWA SV3.3
SV3.3 0.996
0.996 MAMA
WV4.2
WV4.2
WV4.2 0.192
0.192
0.192 WA
WAWA SV3.3
SV3.30.996
SV3.3 0.996
0.996 MAMA
MA
WV4.2
WV4.2 0.192
0.192 WAWA SV3.3
SV3.3 0.996
0.996 MAMA
WV4.2
WV4.2 0.192
0.192 WA WA SV3.3
SV3.30.996
0.996 MAMA
WV4.3
WV4.3
WV4.3 0.406
0.406
0.406 MA
MAMA SV3.4
SV3.4 0.780
SV3.4 0.780
0.780 SA SA
SA
WV4.3
WV4.3 0.406
0.406 MAMA SV3.4
SV3.40.780
0.780 SA SA
WV4.3
WV4.3 0.406
0.406 MAMA SV3.4
SV3.4 0.780
0.780 SA SA
WV4.3
WV4.3 0.406
0.406 MAMA SV3.4
SV3.40.780
0.780 SA SA
TableTable
12. Adherence
12. Adherence
of the
of isolated
the isolated
E. coli
E. from
coli from
the pig
the farm
pig farm
nearnear
Karnobat,
Karnobat,
compared
compared
withwith
that that
of of
the controls.
Tablethe
Tablecontrols.
12. Adherence of the
12. Adherence ofisolated E. coli
the isolated E.from the pig
coli from the farm nearnear
pig farm Karnobat, compared
Karnobat, withwith
compared that that
of of
the controls.
the12.
controls.
Strain
Strain OD550
OD Adherence
nm550 nm Adherence
TableTable AdherenceBiofilm
12. Adherence Biofilm
of the
of isolated E. Strain
the isolatedcoli Strain
E. from OD OD
the pig
coli from farm
thenm
550 pig Adherence
Adherence
near
farm
550 nm Karnobat,
near Biofilm
compared
Karnobat, Biofilm
withwith
compared that that
of of
Strain the controls.
the controls.
Strain OD550
OD Adherence
nm550 nm Adherence
Table 12.
Table Adherence Biofilm
12. Adherence Biofilm
of the
ofisolated E. Strain
the isolatedcoli Strain
E.from OD OD
the pig
coli from farm
thenm
550 pig Adherence
Adherence
near
farm
550 nm Karnobat,
near Biofilm
compared
Karnobat, Biofilm
withwith
compared that that
of of
Strain
Strain ODOD
550 nm
550 nm
theAdherence
controls.
the controls.
Adherence Biofilm
Biofilm Strain ODOD
Strain 550 nm Adherence
550 nm Adherence Biofilm
Biofilm
WV4.3
WV4.3 0.4060.406 MA MA SV3.4
SV3.40.7800.780 SA SA
WV4.3
WV4.3 0.406
0.406 MAMA SV3.4
SV3.40.780
0.780 SA SA
WV4.3
WV4.3 0.406
0.406 MAMA SV3.40.780
SV3.4 0.780 SA SA
Table
Microorganisms 2023, 11, 1909 Table
12. Adherence
12. Adherence
of theofisolated
the isolated
E. coli
E.from
coli from
the pig
thefarm
pig farm
near near
Karnobat,
Karnobat,
compared
compared
withwith
that15of
that
of 22of
the controls.
the controls.
TableTable
12. Adherence
12. Adherence
of the
ofisolated
the isolated
E. coli
E.from
coli from
the pig
the farm
pig farm
nearnear
Karnobat,
Karnobat,
compared
compared
withwith
that that
of of
Strain
Strain OD550
OD nm550 nm theAdherence
controls.
the controls.
Adherence Biofilm
Biofilm StrainStrain OD OD
550 Adherence
Biofilm
TableTable 12.Adherence
12.12.
Table Adherence
Adherence ofofthe
of the theisolated
isolated
isolated E.E.coli
E. coli colifrom
from from thepig
thethe
pig pigfarm
farm farm
near near Karnobat,
Karnobat,
near compared
compared
Karnobat, comparedwithwith that
of ofof
thatthat
with
Strain
Strain OD550
OD nm550 nm the the
Adherence controls.
controls.
Adherence Biofilm
Biofilm StrainStrain OD550
OD Adherence
Biofilm
the controls.
Strain OD550
Strain OD Adherence
Adherence
nm550 nm Biofilm
Biofilm Strain
Strain OD
OD550 Adherence
Adherence
nm550 nm Biofilm
Biofilm
Strain OD550 nm Adherence Biofilm Strain OD550 nm
ATCC
ATCC
3521835218 0.6800.680 SA SA W2.2W2.20.6450.645 SAAdherence
SA Biofilm
ATCC
ATCC
35218
35218 0.680
0.680 SA SA W2.2
W2.2 0.645
0.645 SA SA
ATCC
ATCC
ATCC 35218 0.680
35218
35218 0.680
0.680 SASA
SA W2.2 0.645
W2.2
W2.2 0.645
0.645 SA SA
SA
Microorganisms
Microorganisms 2023, 2023, 11, x PEER
11, x FOR FOR PEER REVIEW
Microorganisms
Microorganisms
O157O157 2023,2023,
11,0.317
x FOR
11,0.317
x PEER
FOR PEER
REVIEW
MA REVIEW
MA W2.3W2.30.9750.975 SA SA 17 of1724of 24
Microorganisms
O157
O157 2023,2023,
Microorganisms
O157 11,0.317
x0.317
FOR
11, PEERPEER
x FOR
0.317 REVIEW
MA REVIEW
MAMA W2.3
W2.3 0.975
W2.3 0.975
0.975 SA SA
SA 17 of1724of 24
Microorganisms
O157 O1572023,2023,
Microorganisms 11,0.317
x 11,
FOR PEERPEER
x FOR
0.317 REVIEW
MA REVIEW
MA W2.3 0.975
W2.3 0.975 SA SA 17 of1724of 24
Microorganisms
11, x 11,
FOR x FOR
PEERPEER REVIEW
F1.1 F1.1 0.4690.469 MA MA L1.1L1.1 0.7370.737 SA SA
Microorganisms 2023, 2023,
Microorganisms 11, x FOR
11, x PEER REVIEW
FOR PEER REVIEW 17 of1724of 24
F1.1F1.1 0.4690.469 MA MA L1.1L1.1 0.7370.737 SA SA
Blank
Blank
Blank 0.1560.156
0.156 -- - W2.4W2.40.5320.532
W2.4 0.532 MA MA
MA
F1.1F1.1 0.4690.469 MAMA L1.1L1.1 0.7370.737 SA SA
Blank
Blank 0.1560.156 - - W2.4W2.4 0.5320.532 MAMA
F1.1F1.1 0.4690.469 MA MA L1.1L1.1 0.7370.737 SA SA
Blank
Blank 0.1560.156 - - W2.4W2.4 0.5320.532 MAMA
F1.1F1.1 0.4690.469 MA MA L1.1L1.1 0.7370.737 SA SA
F2.1 F2.1 0.4610.461 MA MA L1.2L1.2 0.7590.759 SA SA
F1.1 F1.1
F1.1 0.4690.469
0.469 MA
MAMA L1.1L1.1 0.7370.737
L1.1 0.737 SA SA
SA
F2.1F2.1 0.4610.461 MA MA L1.2L1.2 0.7590.759 SA SA
F2.1F2.1 0.461
0.461 MAMA L1.2L1.2 0.759
0.759 SA SA
F2.1F2.1 0.4610.461 MA MA L1.2L1.2 0.7590.759 SA SA
F2.1 F2.1
F2.1 0.4610.461
0.461 MA
MAMA L1.2L1.2 0.7590.759
L1.2 0.759 SA SA
SA
F2.2 F2.2 0.2350.235 WA WA L2.1L2.1 1.0841.084 SA SA
F2.1 F2.1 0.4610.461 MA MA L1.2L1.2 0.7590.759 SA SA
F2.2F2.2 0.2350.235 WA WA L2.1L2.1 1.0841.084 SA SA
F2.2F2.2 0.235
0.235 WA WA L2.1L2.1 1.084
1.084 SA SA
F2.2F2.2
F2.2 0.2350.235
0.235 WA
WAWA L2.1L2.1 1.0841.084
L2.1 1.084 SA SA
SA
F2.2F2.2 0.2350.235 WA WA L2.1L2.1 1.0841.084 SA SA

F3.1 F3.1 0.2780.278 WA WA L2.2L2.2 0.1900.190 WA WA
F2.2 F2.2 0.2350.235 WA WA L2.1L2.1 1.0841.084 SA SA
F3.1F3.1 0.2780.278 WA WA L2.2L2.2 0.1900.190 WA WA
F3.1F3.1
F3.1 0.2780.278
0.278 WA
WAWA L2.2L2.2 0.190
L2.2 0.190
0.190 WA WA
WA
F3.1F3.1 0.2780.278 WA WA L2.2L2.2 0.1900.190 WA WA

F3.1F3.1 0.2780.278 WA WA L2.2L2.2 0.1900.190 WA WA
F4.1 F4.1 0.2290.229 WA WA T1.1T1.1 0.4230.423 MA MA
F3.1 F3.1 0.2780.278 WA WA L2.2L2.2 0.1900.190 WA WA
F4.1 F4.1
F4.1 0.2290.229
0.229 WA
WAWA T1.1T1.1 0.4230.423
T1.1 0.423 MA MA
MA
F4.1F4.1 0.229
0.229 WA WA T1.1T1.1 0.4230.423 MAMA
F4.1F4.1 0.2290.229 WA WA T1.1T1.1 0.4230.423 MA MA
F4.1F4.1 0.2290.229 WA WA T1.1T1.1 0.4230.423 MA MA
F4.2 F4.2
F4.2 0.3230.323
0.323 MA
MAMA T1.2T1.2 0.4610.461
T1.2 0.461 MA MA
MA
F4.1 F4.1 0.2290.229 WA WA T1.1T1.1 0.4230.423 MA MA
F4.2F4.2 0.3230.323 MA MA T1.2T1.2 0.4610.461 MA MA
F4.2F4.2 0.323
0.323 MAMA T1.2T1.2 0.4610.461 MAMA
F4.2F4.2 0.3230.323 MA MA T1.2T1.2 0.4610.461 MA MA
F4.2F4.2 0.3230.323 MA MA T1.2T1.2 0.4610.461 MA MA
F5.1 F5.1 0.3060.306 WA WA T1.3T1.3 0.5020.502 MA MA
F4.2 F4.2 0.3230.323 MA MA T1.2T1.2 0.4610.461 MA MA
F5.1F5.1 0.3060.306 WA WA T1.3T1.3 0.5020.502 MA MA
F5.1F5.1 0.306
0.306 WA WA T1.3T1.3 0.5020.502 MAMA
Microorganisms 11, 1909
F4.2F4.2 2023,0.3230.323 MAMA T1.2T1.2 0.461
0.461 MAMA 16 of 22
F4.2F4.2 0.323
0.323 MAMA T1.2T1.2 0.4610.461 MAMA
Table 12. Cont.
F5.1F5.1 0.306
0.306 WA WA T1.3T1.3 0.502
0.502 MAMA
Microorganisms
F5.1
F5.1 2023, 11,0.306
F5.1 x0.306
FOR PEER REVIEW
0.306 WAWAWA T1.3T1.3 0.5020.502
T1.3 0.502 MAMA
MA 18 of 24
FOR PEERPEER
x FOR REVIEW
Microorganisms 11, x FOR
11, x PEER REVIEW
FOR PEER REVIEW 18 of1824of 24
Microorganisms
11, x 11,
FOR x FOR
PEERPEER REVIEW
F6.1 F6.1
F6.1 0.6500.650
0.650 SASA
SA S6.1
S6.1S6.1 0.266
0.266
0.266 WA WA
WA
F6.2 0.686 SA S6.2 0.295 WA
F6.1F6.1 0.6500.650 SA SA S6.1S6.1 0.2660.266 WA WA
F6.2F6.2 0.6860.686 SA SA S6.2S6.2 0.2950.295 WA WA
F6.2F6.2 0.6860.686 SA SA S6.2 S6.2 0.2950.295 WA WA
F6.2F6.2
F6.2 0.6860.686
0.686 SASA
SA S6.2S6.2 0.295
S6.2 0.295
0.295 WA WA
WA
W1.1 0.317 MA S6.3 0.455 MA

W1.1
W1.1 0.317
0.317 MAMA S6.3S6.3 0.455
0.455 MAMA
W1.1
W1.1
W1.1 0.3170.317
0.317 MA
MAMA S6.3
S6.3S6.3 0.4550.455
0.455 MA MA
MA
W1.1
W1.1 0.3170.317 MAMA S6.3S6.3 0.4550.455 MAMA
W1.2 0.568 MA S7.2 0.546 MA

W1.2
W1.2
W1.2 0.5680.568
0.568 MAMA
MA S7.2S7.2 0.546
S7.2 0.546
0.546 MAMA
MA
W1.2W1.2 0.5680.568 MA MA S7.2 S7.2 0.5460.546 MA MA

W1.2
W1.2 0.5680.568 MAMA S7.2S7.2 0.5460.546 MAMA
W2.1
W2.1 0.949
0.949 SA
SA
W2.1
W2.1 0.949
0.949 SA SA
W2.1W2.1 0.9490.949 SA SA NA, non-adherent confirmed E. coli strains; WA, weakly adherent confirmed E. coli strains; MA, moder-
Legend:
Legend: NA, non-adherent
ately adherent confirmed E.confirmed
coli strains; E.
SA,coli strains;
strongly WA, weakly
adherent adherent
confirmed confirmed E. coli strains;
E. coli strains.
W2.1
W2.1 0.949 MA,
0.949 SA
SAmoderately adherent confirmed E. coli strains; SA, strongly adherent confirmed E. coli strains.
Legend: NA, NA,
Legend: non-adherent confirmed
non-adherent confirmedE. coli
E.strains; WA,WA,
coli strains; weakly adherent
weakly adherentconfirmed E. coli
confirmed E.strains;
coli strains;
4. moderately
MA,MA,
Discussion adherent
moderately confirmed
adherent E. coli
confirmed E.strains; SA, strongly
coli strains; adherent
SA, strongly adherentconfirmed
confirmedE. coli
E.strains.
coli strains.
4. Discussion
Legend: NA, NA,
Legend:
The non-adherent confirmed
use non-adherent
of antibiotics E. coliE.strains;
confirmed
as growth WA, WA,
coli strains;
promoters weaklyforadherent
and weakly adherent
prophylaxis confirmed E. coliE.
confirmed
in farms isstrains;
coli strains;
a highly
MA, MA,
moderately
The use of adherent
moderately confirmed
adherent
antibiotics as E.
confirmed
growth coli
E. strains;
coli
promoters SA,
strains; strongly
andSA, adherent
strongly
for adherent
prophylaxis confirmed E.
confirmed
in farms coli
is E.
a strains.
coli strains.
highly
debated
Legend: NA,issue.
4. Discussion
4. Discussion
Legend: NA, However,
non-adherent
non-adherent there is
confirmed
confirmed E. enough
E.strains;
coli evidence
coli strains;
WA,WA, not only
weakly
weakly for the
adherent
adherent spread E.of
confirmed
confirmed AMR
E.strains;
coli from
coli strains;
debated
MA, MA, issue.such
livestock, However,
moderately
moderately as there
swine,
adherent
adherent to is enough
humans
confirmed
confirmed E. E.
coli evidence
(pig
coli feces
strains;
strains; SA, notstrongly
and
SA, only
strongly for
wastewater theconfirmed
adherent
adherent spread
are one ofE.
confirmedofAMR
the
E.
coli from
hotspots
coli strains.
strains.
4.The The
use use
of antibiotics
Discussion as growth
of antibiotics
4. Discussion as growthpromoters
promoters andandfor prophylaxis
for prophylaxis in farms is a is
in farms highly
a highly
livestock,
for such
theissue.
spread as swine,
and to humans
circulation ofis(pig
AMR feces and
andevidence
GAR) wastewater are one of the hotspots(clonal for
debated
debated However,
issue. However, there is enough
there enough evidence notbut only
not alsoforfor
only forgenetic
the spread
the spread similarity
of AMR of AMR fromfrom
the
4. The
spread
4. use
The and
Discussion
Discussion
types) of
use
between antibiotics
ofresistant
antibiotics
circulation asbacteria
of growth
AMR as growth
and promoters
in GAR)
animals butand
promoters alsoand
and for prophylaxis
for
forhumans,
in prophylaxis
genetic in farms
similarity
some inwhich
of is a are
farms
(clonal highly
is a given
types) highly
livestock,
livestock,suchsuch as swine,
as swine, to humans
to humans (pig(pig
fecesfeces
andand wastewater
wastewater are one
are oneof theof hotspots
the hotspots for for
debated
betweena issue.
indebated resistant
comprehensive However,
issue. However,
bacteria there
review in is
there enough
animals
by is enough
andevidence
in
Sirichokchatchawanevidence not only
humans, not
et some for2021
only
al., the
for
of spread
the
which
[41]. spread
are
For of example,
AMR
of
given AMR from
in afrom
four
the spread
the The
Thespread
use anduse
ofand of circulation
antibiotics
antibiotics
circulation as AMR
of as AMR
growth
of growth
and GAR)
and promoters
promoters butand
GAR) alsoand
but for for
forprophylaxis
forprophylaxis
also genetic in (clonal
in farms
similarity
genetic similarity farms a is
is(clonal a highly
highly
types) types)
livestock,
livestock,
comprehensive
sequence suchtypesas
such swine,
as ESBL
review
of swine,
by toSirichokchatchawan
humans
to humans
producing (pigE. feces
(pig
coli and
feces
et
shared wastewater
and
al., 2021 wastewater
between[41]. areexample,
For
humans one of
are oneand the
of hotspots
four
pigsthesequence
hotspots
were for for
found
debated
debated
between
betweenissue. issue.
resistant
resistantHowever,
However,
bacteria there
bacteria inthereanimalsis enough
isinenough
animals and inevidence
evidence
and in not
humans,humans,not
only only
some for
some forwhich
the
of thewhich
spread
of spread
areof AMR of AMR
given
are in afrom
from
given in a
thein
spread
typestheofspread
ESBL
Thailandandproducing
circulation
and
[41]. PlasmidE.ofhumans
circulation coliAMRof AMR
shared andbetween
(sub)types GAR)
and and, but
GAR) also
but
humans
again, for
also genetic
and
ESBL forpigs similarity
genetic
genes were similarity
such found
as (clonal
bla types)
(clonal
inCTX-M-1
Thailand types)
were
livestock,
livestock,
comprehensive such
comprehensive such
as as
review swine,
swine,
review to to humans
by Sirichokchatchawan (pig
by Sirichokchatchawan (pig
feces feces
and
et al.,and wastewater
wastewater
et2021 [41].[41].
al., 2021 are are
one
For example, one
of
For example, of
the the
hotspots
fourfour hotspots
sequence for
sequence for
between
between
found
[41]. Plasmidresistant
to beresistantbacteria
shared
(sub)types bacteria
between
and, inagain,
animals
in animals
Dutch
ESBL and
pigs in
and
genesand humans,
inpig
such humans,
as some
farmers
bla some
CTX-M-1 ofwere
[42]. which
of whicharetoare
Therefore,
found given
bethe in ain a
given
general
shared
the
typestheofspread
spread
types ofand
ESBL ESBL and circulation
circulation
producing
producing E.ofcoliE.ofshared
AMR AMR
coli and
sharedand
GAR)
between GAR)
but
between but
also
humans also
forand
humans forand
geneticgenetic
pigs similarity
similarity
were
pigs found
were in(clonal
(clonal
found intypes)
Thailand types)
Thailand
comprehensive
comprehensive
agreement
between Dutch review
among
pigs bypig
review
policy
and Sirichokchatchawan
byfarmers
Sirichokchatchawan
makers andand
[42]. societyet al.,
Therefore, 2021
isetthat
the [41].
al., 2021
the Foragreement
[41]. example,
For example,
disadvantage
general four
of sequence
four
creating
among sequence
policybacte-
between
between
[41].[41].
Plasmid
Plasmid resistant
resistant
(sub)types bacteria
bacteria
(sub)types and,in inagain,
again,
and, animals
animals ESBL ESBL and
ingenes
genes in
humans,
such humans,
as bla
such some
as blasome
CTX-M-1 ofwere
ofwere
which
CTX-M-1 which
found are
foundtoarebetogiven
given ain a
beinshared
shared
types
rial
makers ofAMR
types ESBL
and of ESBL producing
outweighs
society producing
is by E. coli
the
that E. shared
coli
benefits
the ofbetween
shared
disadvantage ofhumans
between
antibiotics. humans and
Therefore,
creating pigs
and were
pigs
even
bacterial AMR found
were
though in Thailand
found
outweighs in sequence
subclinical Thailand
thean-
comprehensive
comprehensive
between Dutch review
pigs review
and pig by Sirichokchatchawan
Sirichokchatchawan
farmers [42]. et
Therefore, et
al., al.,
2021
the
between Dutch pigs and pig farmers [42]. Therefore, the general agreement among policy 2021
[41].
general [41].
For For example,
example,
agreement four
among four
sequence
policy
[41]. Plasmid
[41].
tibiotic Plasmid (sub)types
(sub)types
concentrations and, again,
and, ESBL
again, genes
ESBL such
genes as bla
such asCTX-M-1 werewere
blareduce found foundto betoshared
benotshared
benefits of antibiotics. E.not
Therefore, E.only promote
even though growth but
subclinical also antibiotic animal morbidity
concentrations and
CTX-M-1
typestypes
makers of and
makers of
ESBL ESBL
society
and producing
producing is that
society is coli
the
that coli
shared sharedbetween
disadvantage
the between
disadvantage ofhumans
ofhumans
creating and
creating and
pigs
bacterial pigs
were were
AMR
bacterial found
AMR found in Thailand
inoutweighs
outweighs Thailand the the
between
betweenDutch
mortality,
only promote Dutchpigspigs
numerous
growth andbut pig
and
bansalso farmers
pig farmers
orreduce [42].
restrictions
animalTherefore,
[42].forTherefore,
antibiotic
morbidity the and
general
the
feedgeneralagreement
additives
mortality, agreement
haveamong
numerous among
been policy
bans policy
adopted
or
[41].[41].
benefits Plasmid
Plasmid
of
benefits (sub)types
(sub)types
antibiotics.
of antibiotics. and,
Therefore,and,
again,
Therefore, again,
ESBL
even ESBLgenes
though
even genes
though such such
as bla
subclinical asCTX-M-1
subclinical blaCTX-M-1
werewere
antibiotic
antibioticfound foundto betoshared
concentrations
concentrations be not
sharednot
throughout
makers
makersandfor
restrictions and the
society world
is that
society
antibiotic is
feed[41,43].
the
that the
additives The
disadvantage dilemma
disadvantage
have been is
of creating
of excellently
creating
adopted bacterial described
AMR
bacterial
throughout AMR
the in outweighs
the[41,43].
outweighs
world workthe by the
between
between
onlyonly Dutch
promote
promote Dutchpigs
growth pigs
growthbutand
and pigalso
but pig
farmers farmers
reduce
also [42].
reduce [42].
animal Therefore,
Therefore,
morbidity
animal morbidity the and
the and general
general agreement
agreement
mortality,
mortality, numerousamong
numerousamong policy
bans policy
or or
bans
TheChattopadhyay,
benefits of antibiotics.
benefits
dilemma 2014
ofis antibiotics.
excellently [43].
Therefore,
Therefore,
described even though
ineven
the though
work subclinical
by subclinical antibiotic concentrations
antibiotic concentrations not not
makers
makers andfor
restrictions
restrictions and
societysociety
is that
antibiotic
for antibioticis
feedthat
the the
additives
feed disadvantage
disadvantage
additives havehave of been
been ofChattopadhyay,
creating
creating
adoptedadopted bacterial
bacterial
throughout
2014
AMR
throughout AMR
the
[43].
theoutweighs
outweighs
world world the the
[41,43].
[41,43].
onlyonly
promote
The promote growth
legislation growth
in but also
Bulgariabut reduce
also
is reduce
strict, animal
and morbidity
animal
since morbidity and and
approximately mortality,
mortality,
the year numerous
numerous
2000, bansbans
antibiotics or or
The benefits
benefits of
dilemma of isantibiotics.
antibiotics.
excellently Therefore,
Therefore,
described even ineven
though
the though
work subclinical
subclinical
The dilemma is excellently described in the work by Chattopadhyay, 2014 [43]. by antibiotic
antibiotic
Chattopadhyay, concentrations
concentrations
2014 [43]. not not
restrictions
in restrictions
animal for antibiotic
for antibiotic
husbandry feedfeed
have additives
been additives havehave
allowed been
only adopted
been as adoptedthroughout
throughout
therapeutics the world
under [41,43].
theveterinary
world [41,43].
onlyonly promote
promote
The The growth
legislation growth
legislation but
in Bulgariabut
also
in Bulgaria also
reducereduce
is strict, animalanimal
andand
is strict, since morbidity
morbidity and
approximately
since and mortality,
mortality,
approximately the year numerous
numerous
the year2000, bans
antibiotics
2000, bans
or
antibiotics or
The The
dilemma
supervisiondilemma is excellently
and isantibiotic
with excellently
thefeed described
demand described in the
of reportingin work
theitwork bythe
to Chattopadhyay,
by Chattopadhyay,
competent 20142014
authorities. [43].[43].
In poultry
restrictions
restrictions
in animal
in animal for for
antibiotic
husbandry
husbandry havehave feed additives
additives
beenbeen have
allowed have
been
allowed been
adopted
onlyonly adopted throughout
throughout
as therapeutics
as therapeutics the
under the
world
under world [41,43].
[41,43].
veterinary
veterinary
The legislation
The legislationin Bulgaria
in Bulgaria is strict,
is and
strict, since approximately the in
year 2000,farms
antibiotics
farms,
The The most
dilemma
dilemma
supervision
supervision
antibiotics
andis excellently
with
and
are
is excellently
the demand
with
banned
described
the demand
even
described inofthe
of reportinginand
for work
reporting
since
therapy.
the itwork
approximately
byitthe
to
An exception
bycompetent
Chattopadhyay,
Chattopadhyay,
to the competent
the
2014
year
swine2014
authorities.
2000,
[43].[43].
authorities.
antibiotics
is the
In poultry
In poultry
in animal
in animal
prophylactic husbandry
use husbandry
of colistinhave have
against beenbeen
postallowed
allowed
weaning only onlyas therapeutics
diarrhea. as therapeutics under under veterinary
veterinary
farms,The The
most
farms, legislation
legislation
antibiotics
most antibiotics in Bulgaria
in Bulgaria
are are is
banned banned is
strict,strict,
even andforand
even forsince
since
therapy.
therapy.approximately
approximately
An exception
An exceptionthe intheswine
year year
in2000, 2000,
swine antibiotics
antibiotics
farms farmsis the is the
supervision
supervision and and withwiththe demand
the demand of reporting
of reporting it to ittheto competent
the competent authorities.
authorities. In poultry
In poultry
The legislation in Bulgaria is strict, and since approximately the year 2000, antibiotics
in animal husbandry have been allowed only as therapeutics under veterinary supervision
and with the demand of reporting it to the competent authorities. In poultry farms, most
antibiotics are banned even for therapy. An exception in swine farms is the prophylactic
use of colistin against post weaning diarrhea.
Nevertheless, in 2010–2016, even newborn suckling pigs still carried as part of their
normal intestinal microflora coliforms that had resistance to most tested antibiotic classes,
especially to tetracycline and ampicillin. Lactating sows and young pigs had high resis-
tance to streptomycin. Moreover, despite legislative bans, if we compare our results with
those from previous studies of swine farms in Bulgaria, an increase in AMR is observed.
In the period of 2010–2016, resistance toward tetracycline, ampicillin and streptomycin
(approximately 70%, 60% and 65%, respectively) doubled in comparison to the period
of 2000–2004 [44]. After a transient decrease in 2020 [25], resistance to tetracycline rose
even more to 77.8% and to 81% for the drug class as a whole in our study. Resistance
to pefloxacin, carbenicillin and chloramphenicol rose slightly, and there was not a clear
correlation regarding the other tested agents [25,44,45].
It is interesting that blaTEM was relatively rare in the study of the period of
2010–2016 [44], whereas in this work, blaTEM was a very prevalent gene with 34 posi-
tive samples from 56 tested. This marks an increase from our last time tested in 2020 [25].
AmpC was the only other GAR detected by us with all samples positive out of 56 tested.
Last time, it had a similar pattern, because the most numerous positive samples were for
that gene [25].
Indeed, there is decreased resistance for some agents, but it still remains relatively high.
For example, ampicillin values showed growth in 2021 to 75% but have now decreased to
53.6%. Similarly, streptomycin resistance decreased in 2020 (12.5%) [25] and in this study
(39.3%) but still remains high. Amoxicillin resistance decreased in comparison with the
period of 2012–2020 [25,45], from 75% to 52.4%. There is very low resistance in farm pigs to
third-generation cephalosporins [44] and even to the second-generation agent cefamandole
in 2020 [25] and in our study.
As a summary for Bulgarian farm swine, there is high resistance of resident and
pathogenic strains to tetracyclines, penicillins and aminoglycosides. Therefore, the high
prevalence and increase in AMR in farms with highly restricted antibiotic use (and only
for therapy) could indicate residual AMR from past times, the overuse of antibiotics for
therapy in farms and/or the high circulation of AMR in the environment due to the high
use of antibiotics by humans. This is enhanced by travel and transport in our global society,
raising the spread of resistant strains.
The rate of antibiotic resistance differs considerably from country to country globally,
depending upon the amount of usage. In the European Union (EU), the lowest levels
of AMR E. coli isolates have been found in countries where lower antimicrobial usage is
practiced, such as in Norway, Sweden and Finland, whereas countries with high levels of
use, such as Spain, Portugal and Belgium, have relatively higher levels of AMR E. coli [46].
For instance, a clear spatial pattern was detected for tetracycline resistance, with high
resistance levels reported for southern and western European countries and much lower
levels reported for northern and eastern countries in 2004–2007 [7].
In 2019, some antimicrobial classes were assigned the highest priority, with critically
important antimicrobials for human medicine only being available for food animals through
veterinary prescription [41]. These are fluoroquinolones, third- and fourth-generation
cephalosporins, carbapenems, macrolides and polymyxins (colistin). An increase in resis-
tance to these antibiotics in E. coli in animals may indicate a general resistance trend of
concern among Gram-negative bacteria originating from the animal reservoir. Carbapen-
ems may not be used in food-producing animals in the present, but it is alarming that
resistance genes have been found in pigs, chickens and other livestock [47].
The monitoring and reporting of resistance data from indicator organisms (commensal
E. coli and enterococci) to the European Food Safety Authority (EFSA) is voluntary. EFSA
reports show that, for a timeframe of 2004–2020, resistance to nalidixic acid is, in general,
low, as it was in our studies. However, in the past (2004–2007), large variability was ob-
served in the reported ciprofloxacin resistance median levels (4–24%), with the highest
occurrence of 74% reported by Estonia in 2007. In Denmark, legal restrictions have been
in place for the use of fluoroquinolones in food animals since 2002, and as a consequence,
ciprofloxacin resistance decreased from 3% in 2004 to 0% in 2007 [7]. Wider legal restrictions
likely led to the low overall resistance to ciprofloxacin now (median approximately 5%) [48].
It is noteworthy that resistance to third-generation cephalosporins is very low, usually
below 1%, in European as well as in local Bulgarian pig farms; therefore, resistance to that
agent is still not a threat in animal husbandry, unlike in clinical settings [7,48]. Regarding
human infections with E. coli, the European Antimicrobial Resistance Surveillance Network
(EARS-NET) reported in 2017 that the highest population-weighted mean resistance per-
centage in the European Union and the European Economic Area for E. coli that causes
serious infections was to aminopenicillins (58.7%), followed by fluoroquinolones (25.7%),
third-generation cephalosporins (14.9%) and aminoglycosides (11.4%). In 2017, resistance
to carbapenems remained rare in E. coli [49].
In 2019–2020, reports of farm swine from 30 countries showed that high or very high
resistance to ampicillin, sulfamethoxazole, trimethoprim and tetracycline was the most
common resistance trait observed. MDR was observed in 34.2% (versus 29.8% in our
study) of commensal E. coli isolates from pigs. A wide variety of resistance patterns were
observed, but mostly to tetracycline, ampicillin, sulfamethoxazole and trimethoprim, often
in combination with other substances but rarely with quinolones. About half of the porcine
MDR isolates (52.3%) were resistant to all these antimicrobials. Meropenem resistance was
not detected in any isolate of indicator E. coli, in line with the results from Bulgaria [48].
Complete susceptibility to 14 antimicrobials tested was higher than that in our study
without a statistically significant difference between countries. The aminoglycoside gen-
tamicin had low median levels of resistance through 2004–2020, unlike in Bulgaria. The
median levels of chloramphenicol resistance for all reporting countries were moderate in
pigs, unlike the high resistance in Bulgaria [48].
There were positive trends (decreases in the level of resistance) in several countries
that were possibly due to the documented overall decline in sales of antimicrobials. Most
notably, tetracycline resistance has decreased in 15 countries and increased in only two.
In 13 countries, there were only decreasing trends, notably in the Netherlands for four
agents and in Cyprus for three agents [48]. Indeed, countries such as Denmark and
the Netherlands, which both have had massive swine production in recent years, have
achieved tremendous reductions in antimicrobial usage while sustaining peak production.
Comparable results have been accomplished in Belgium, France, Sweden and the United
Kingdom [7,46,48,50]. In contrast, in six countries, there were only increasing trends, and
there were increasing trends in Belgium (despite the reduction in antibiotic use), Poland
and Romania for three antimicrobials [48].
In reference to the genetic profile, unlike our results, presumptive ESBL producers
were more common than AmpC-producers, and isolates with a combined phenotype
(ESBL + AmpC) were uncommon. The occurrence of presumptive ESBL, AmpC or
ESBL + AmpC producers in commensal E. coli was 1.3% in fattening pigs. In pork, the
prevalence of presumptive E. coli ESBL and/or AmpC-producers in meat was less variable,
ranging from 0% (Finland and the Netherlands) to 24.4% (Portugal) [48].
It is interesting that, in the period of 2004–2007, AMR to commonly used antimicrobials
was higher in porcine E. coli than that in isolates from chickens and cattle, and in most
cases, the countries that reported a high occurrence of AMR in E. coli from chickens also
had a high occurrence of resistance in E. coli from pigs [7]. This was likely due to the fact
that the global average annual consumption of antibiotics for swine (172 mg/kg) is greater
than that for cattle (45 mg/kg) and chickens (148 mg/kg) [51].
As a summary for the EU, the EFSA Animal Health and Welfare Panel (2021) revealed
clinical swine E. coli isolates with a high proportion of resistance to numerous antibiotics,
with a prevalence from 63% to 70% (to aminopenicillins, sulfonamides and tetracycline).
However, lower rates of resistance to clinically critical antibiotics (fluoroquinolones and
third-generation cephalosporins) were detected [50]. Likely, the latter was the first fruit of
the recent Regulation (EU) 2019/61 on Veterinary Medicines and Regulation (EU) 2019/4 on
Medicated Feed, stating that antibiotics shall not be applied routinely, nor for prophylaxis,
unless in exceptional cases. They should only be applied for metaphylaxis (treatment of
animals without signs of disease, which are in close contact with animals that do have
evidence of infectious disease) when the risk of spreading infection is very high and there
are no other options, as Barros et al., 2023, described [18]. Differences in resistance between
countries might reflect the dissemination of certain E. coli types within animal populations
and/or differences in the consumption of antimicrobials in animals among countries [7,48].
The recommendations that could make the use of antibiotics as a feed additive un-
necessary are several, including improved hygiene and vaccines (although their efficacy
varies considerably), biosecurity (measures taken to prevent disease introduction, such as
monitoring animals or plant materials that enter the property, as well as sources of water
and feed). Numerous alternatives to antibiotics exist—prebiotics, probiotics, phytogenic
substances, bacteriophages, etc. However, they have their limits for therapy and are mostly
used for prophylaxis [18]. The development of alternatives for the clinical management
of the infections of livestock appears to be the need of the hour [43]. There is a need
to establish national surveillance programs and effective policies, particularly in certain
world regions, to curtail the threat of the evolution of resistant isolates in swine or other
livestock production [33,52].
It is clear that more unhygienic farms (e.g., in the developing world) demand more
antibiotics in their feed. However, antimicrobials as a feed additive reduce morbidity and
mortality in most hygienic farms in the developed world [43]. Whether farm animals are
exposed to more infectious agents than animals in their natural habitat is a question beyond
the scope of this work. However, it is clear that wildlife has access to more open ventilated
spaces and disinfecting sun beams. Therefore, our hypothesis is that, as the number
of natural conditions that livestock lives in increases, fewer antibiotics are needed for
growth promotion.
Biofilm formation by foodborne pathogens is a serious threat to food safety and public
health [53]. As a foodborne pathogen, E. coli can adhere to and form biofilms on most
materials and under almost all environmental conditions in food production plants [54]. In
this context, this is of importance for irrigation installations and meat processing plants,
given the fact that E. coli can survive for months on dry surfaces [55]. Viable pathogens in
detached biofilms from contact surfaces can lead to cross-contamination. Environmental
biofilms are most often composed of multispecies microorganisms, and mixed biofilm
formation can enhance the sanitizer tolerance of foodborne pathogens. E. coli is capable of
forming biofilms with other bacterial species, and that could enhance its pathogenic clones’
survival in the biofilm community. Biofilm formation in commercial meat plants may be a
source of product contamination with no identifiable cause [53].
Even after the cleaning and disinfection processes, the biofilm could still persist. For
example, in nursery units in a pig facility after an extensive washing protocol that included
disinfection and being kept empty for two weeks, for E. coli and fecal coliforms, reductions
of 41% and 51% were observed, respectively; however, they were still found on floors,
drinking nipples and feeding troughs [55].
Although we did not find pathogenic clones in the present research, the ability to form
strongly adherent biofilms for approximately 17% of the isolated E. coli commensals that
were resistant to commonly used antimicrobials and that were MDR strains is alarming,
because a detached biofilm can lead to the spread of the AMR to the environment and
bacteria in humans through horizontal gene transfer.
As future directions for our research, colistin resistance could be studied because of its
prophylactic use against post weaning diarrhea.
5. Conclusions
Although some antimicrobial agents show a lower level of resistance in Bulgarian
porcine farms in comparison to European ones (e.g., ciprofloxacin), the higher prevalence of
resistance to aminoglycosides in Bulgaria is alarming, and so is the higher level of resistance
to tetracyclines, because it is already high in the European Union. Antibiotic stewardship
is the effort to improve how antibiotics are prescribed by clinicians and used by patients.
In terms of antimicrobial stewardship programs, national action plans in many countries
cover both human and animal health sectors [41]. Legal restrictions lead to positive trends,
but our study is an example of the relatively high prevalence and increase in AMR in farms
with banned antibiotics as feed additives and prophylaxis. This is likely the result of their
overuse for therapy in farms and/or the high circulation of AMR in the environment due to
the high usage of antibiotics by humans. Antimicrobial utilization should be more correctly
structured as a dosage and course of treatment.
Author Contributions: Conceptualization, H.N.; methodology, H.N. and M.M.Z.; software, not
applicable; validation, H.N. and M.M.Z.; formal analysis, H.N., M.M.Z. and Y.I.; investigation,
M.D.K., I.T., Y.I., T.C.K., P.O., M.M.Z. and Y.G.; resources, H.N. and K.N.; data curation, not applicable;
writing—original draft preparation, Y.I., Y.G., M.M.Z., M.D.K., H.N. and L.D.; writing—review and
editing, Y.I. and H.N.; visualization, Y.I., T.C.K. and M.M.Z.; supervision, H.N.; project administration,
H.N.; funding acquisition, H.N. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the National Fund for Scientific Research, Republic of Bulgaria
(grant number: KΠ-06-H36/7 from 13 December 2019).
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Biofilm absorbance was measured on equipment donated by the Alexander
von Humboldt Foundation to Maya Margaritova Zaharieva in the frame of the Alumni program
“Equipment subsidies”.
Conflicts of Interest: The authors declare no conflict of interest.
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microorganisms

Microorganisms 2023, 11, 1909. https://doi.org/10.3390/microorganisms11081909 https://www.mdpi.com/journal/microorganisms

2. Materials and Methods

2.2. Isolation of Single Bacterial Cultures

2.3. Identification of E. coli via Spectral Methods (Matrix-Assisted Laser Desorption/Ionization

2.4. Isolation of DNA of the Bacterial Colonies of E. coli

2.5. Polymerase Chain Reaction (PCR) Analysis

Primers Sequences Tm Amplicon Reference

2.6. Disk Diffusion Method

2.7. Test for Biofilm Formation

3.2. Identification by MALDI-TOF-MS and PCR

3.3. Antibiotic Resistance from the Disk Diffusion Method

Multidrug resistance (MDR), defined as resistance to three or more antimicrobial

3.4. Detection of Antibiotic Resistance Genes

FK3.3 0.254 WA LK2.1 0.242 WA

FK3.4 0.229 WA LK2.2 0.222 WA

FK3.5 0.293 WA LK3.1 0.215 WA

WK1.4 0.864 SA SK3.1 0.580 MA

F2.2F2.2 0.2350.235 WA WA L2.1L2.1 1.0841.084 SA SA

F3.1F3.1 0.2780.278 WA WA L2.2L2.2 0.1900.190 WA WA

Strain OD550 nm Adherence Biofilm Strain OD550 nm Adherence Biofilm

W1.1 0.317 MA S6.3 0.455 MA

W1.2 0.568 MA S7.2 0.546 MA

W1.2W1.2 0.5680.568 MA MA S7.2 S7.2 0.5460.546 MA MA

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