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Article

Virulence, Antimicrobial Resistance and Biofilm Production of Escherichia coli Isolates from Healthy Broiler Chickens in Western Algeria

by
Qada Benameur
1,
Teresa Gervasi
2,*,
Filippo Giarratana
3,
Maria Vitale
4,
Davide Anzà
4,
Erminia La Camera
5,
Antonia Nostro
5,
Nicola Cicero
2 and
Andreana Marino
5
1
Nursing Department, Faculty of Nature and Life Sciences, University of Mostaganem, Mostaganem 27000, Algeria
2
Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, 98100 Messina, Italy
3
Department of Veterinary Sciences, University of Messina, 98100 Messina, Italy
4
Istituto Zooprofilattico Sperimentale della Sicilia “Adelmo Mirri”, 90141 Palermo, Italy
5
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98100 Messina, Italy
*
Author to whom correspondence should be addressed.
Antibiotics 2021, 10(10), 1157; https://doi.org/10.3390/antibiotics10101157
Submission received: 26 August 2021 / Revised: 20 September 2021 / Accepted: 22 September 2021 / Published: 24 September 2021
(This article belongs to the Special Issue Antimicrobial Resistance and Virulence - 2nd Volume)
Versions Notes

Abstract

:
The aim of this study was to assess the virulence, antimicrobial resistance and biofilm production of Escherichia coli strains isolated from healthy broiler chickens in Western Algeria. E. coli strains (n = 18) were identified by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry. Susceptibility to 10 antibiotics was determined by standard methods. Virulence and extended-spectrum β-lactamase (ESBL) genes were detected by PCR. The biofilm production was evaluated by microplate assay. All the isolates were negative for the major virulence/toxin genes tested (rfbE, fliC, eaeA, stx1), except one was stx2-positive. However, all were resistant to at least three antibiotics. Ten strains were ESBL-positive. Seven carried the β-lactamase blaTEM gene only and two co-harbored blaTEM and blaCTX-M−1 genes. One carried the blaSHV gene. Among the seven strains harboring blaTEM only, six had putative enteroaggregative genes. Two contained irp2, two contained both irp2 and astA, one contained astA and another contained aggR, astA and irp2 genes. All isolates carrying ESBL genes were non-biofilm producers, except one weak producer. The ESBL-negative isolates were moderate biofilm producers and, among them, two harbored astA, two irp2, and one aggR, astA and irp2 genes. This study highlights the spread of antimicrobial-resistant E. coli strains from healthy broiler chickens in Western Algeria.

1. Introduction

One of the most important global challenges to public health is represented by foodborne illnesses. Healthy food-producing animals can be vectors for a wide range of commensal and pathogenic bacteria, as well as Escherichia coli. This microorganism can contaminate the food chain at each step, from the slaughterhouses to the food processing phases [1,2,3,4]. To date, although several E. coli strains are commensals, which colonize the gastrointestinal tract of humans and warm-blooded animals, and are not often disease-causing, E. coli represents one of the most frequent causes of several common infections in humans and animals [5]. E. coli clones acquiring specific virulence factors (VFs), including adhesins, toxins, invasins, etc., can cause intestinal and extra-intestinal diseases such as enteric/diarrheal disease, urinary tract infections (UTIs) and sepsis/meningitis in human hosts [6,7,8,9,10].
VFs are generally carried on phages, plasmids or pathogenicity islands (PAIs) [11] and, among microbial strains, can be vastly interchanged via horizontal transfer [12]. Given the presence of definite virulence genes, E. coli strains can be classified as pathogens [13,14], in particular as zoonotic intestinal pathogenic E. coli pathotypes (IPEC) or extraintestinal pathogenic E. coli pathotypes (ExPEC) based on the type of VFs present and the host’s clinical symptoms [15]. The specific “pathotypes” are grouped into enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC), and can cause intestinal diseases [8,16].
Moreover, the emergence of antibiotic resistance in food pathogens represents further complications.
The wide use of antibiotics, in both animals and humans, is responsible for an increased antibiotic resistance not only in pathogenic bacteria, but also in the endogenous microflora. Resistant animal bacteria can be transmitted to humans through several routes, such as direct contact with the animal or its manure, and through contact with or the consumption of uncooked meat [17,18,19,20,21]. Given the development of combined resistance to multiple antibiotics such as the β-lactam group, including cephalosporins and carbapenems, over the last few years, the chemotherapeutic choices for enterobacteria are becoming strictly limited [22]. Resistance to cephalosporin is the result of the production of one or more types of β-lactamases, the so called extended spectrum-β-lactamases (ESBLs) [23]. ESBLs are categorized into several classes, among which the most important include Temoniera (TEM), sulfhydryl variable (SHV) and cefotaximase (CTX-M) types [24,25]. Thus, nowadays ESBL-producing Gram-negative bacteria represent a growing concern and an important challenge for chemotherapy [26]. In addition, another issue is represented by the fact that, in food factory environments, some biofilm-forming bacteria are human pathogens. Biofilms are ecosystems made up of one or more bacterial species submerged in an extracellular matrix, whose composition varies according to the colonizing species and the food manufacturing environment [27,28,29].
The zoonotic potential of E. coli from chicken food products is important for public health purposes [30,31]. Meat harbors different bacteria as inherent contamination and is further contaminated during handling, improper dressing, cleaning, unsanitary conditions and unhygienic practices during its commercialization [32]. Considering the factors described, the objective of this preliminary study was to examine virulence and antimicrobial resistance (AMR) gene profiles, and the ability of biofilm formation of E. coli strains isolated from healthy broiler chickens in Western Algeria. The Algerian poultry industry, consisting of 20,000 farmers, every year yields an average of 340,000 tons of white meat and over 4.8 billion eggs. The present Algerian poultry industry structure is the result of government development policies, which were initiated in the 1980s [33].

2. Results

A total of 18 presumptive E. coli strains were isolated from 32 fecal samples, collected from different broiler chicken farms situated in five geographic areas of Western Algeria: Mostaganem (n = 8, 25.0%), Oran (n = 6, 18.75%), Mascara (n = 6, 18.75%), Relizane (n = 6, 18.75%) and Tiaret (n = 6, 18.75%). MALDI-TOF-MS analysis confirmed the identification of all the 18 presumptive E. coli strains (Table 1). All the isolates were negative for the major virulence/toxin genes tested, including shiga-like toxin 1 (stx1), O157:H7 serotype (rfbE), flagellar gene (fliC) and attaching and effacing gene (eaeA), except for one E. coli strain positive for the shiga-like toxin 2 (stx2) gene (Table 1), coming from 1/7 broiler houses located in the Mostaganem area.
However, in contrast to the low percentage of virulence genes detected, all strains were shown to be resistant to at least three antibiotics most frequently used in poultry (Table 1). They were resistant to nalidixic acid (NA) (100%), neomycin (N) (100%), tetracycline (TE) (94%), ciprofloxacin (CIP) (89%), amoxicillin–clavulanic acid (AUG) (83%), trimethoprim–sulfamethoxazole (SXT) (78%), amoxicillin (AML) (72%), cefotaxime (CTX) (50%) and chloramphenicol (C) (17%). Among the strains, 10 were phenotypically confirmed to be ESBL-positive isolates. Genotypic analyses revealed that nine strains (CTX-resistant 50%) carried the blaTEM gene and one harbored the blaSHV gene (5.55%). Among the blaTEM-producing E. coli isolates, two co-harbored the blaCTX-M−1 gene (11%) (Table 2). The distribution of the percentages of ESBL isolates and the geographical area visited was 67% in Oran, 57% in Mostaganem, 67% in Tiaret, 50% in Mascara and none in Relizane.
Furthermore, an association between biofilm production and the presence of enteroaggregative genes was evaluated.
Enteroaggregative genes were detected in the ESBL-producing strains (Table 1). Among the seven strains harboring only blaTEM−1, two strains contained iron regulatory protein 2 (irp2) (28.5%), two both irp2 and the heat-stable enterotoxin-1 (astA) (28.5%), one astA (14%) and another the transcription factor (aggR), astA and irp2 (14%) genes. Two strains contained blaTEM/blaCTXM−1, and one had the irp2 (50%) gene. Among all ESBL-producing strains, only one isolate, harboring blaTEM and irp2 genes, was a weak biofilm producer (14%) (Table 2). The remaining strains (86%) were regarded as non-biofilm producers (specific biofilm formation (SBF): 0.16–0.29).
Among the eight non-ESBL-producing strains, five (62.5%) harbored putative enteroaggregative genes: astA (n = 2, 25%), irp2 (n = 2, 25%) and astA–irp2–aggR (n = 1, 12.5%). Moreover, one isolate (12.5%) expressed the stx2 gene (Table 1). The non-ESBL-producing isolates were more likely to produce biofilm than ESBL-producing strains (p ≥ 0.001; r = 0.85). Among the non-ESBL-producing isolates, three strains were classified as moderate biofilm producers (Table 2). One harbored astA (12.5%), another stx2 (12.5%) and yet another contained no virulence gene (12.5%), with SBF: 0.81, SBF: 0.76 and SBF: 0.84, respectively. The remaining strains (62.5%) were regarded as weak biofilm producers (SBF: 0.39–0.65). E. coli ATCC 25922 was a moderate biofilm producer (SBF: 0.81).

3. Discussion

The majority of E. coli strains are commensals inhabiting the intestinal tract of humans and warm-blooded animals and rarely cause diseases, unless they acquire VFs carried by mobile genetic elements such as bacteriophages, pathogenicity islands and plasmids [34]. Additionally, E. coli can form a reservoir of AMR genes that may be transferred among different bacterial species, including pathogenic bacteria for both humans and animals.
In this study, the E. coli strains, isolated from fecal samples of apparently healthy chickens, showed a low percentage of virulence genes, which are characteristic of shiga toxin-producing E. coli (STEC O157:H7) (rfbE, fliC, eaeA and stx1). Indeed, except for one E. coli strain, which was positive for the stx2 gene detected at one Mostaganem farm, all the isolates were negative for the major genes encoding VFs. This finding is in accordance with previous Algerian studies describing a low prevalence of stx genes in E. coli isolates from poultry origin, i.e., a recent Algerian study showed the presence of stx2 in only one E. coli isolate from broiler chickens, which had just died [35]. Another study conducted in the north of Algeria revealed the total absence of the stx2 gene and the presence of the stx1 gene in only two E. coli strains isolated from diarrheic hens and chickens [36]. Our results are also in agreement with several previous studies conducted in other countries, which concluded that the prevalence of STEC O157:H7 in broiler chickens is relatively low compared with other animal species [37,38,39,40].
However, in contrast to the low percentage of STEC virulence genes detected, all isolated strains were shown to be resistant to at least three antibiotics most frequently administered to poultry. Antimicrobial agents are being used in many countries in veterinary practice for the treatment of disease, disease prevention and growth promotion [41]. However, the indiscriminate use of antimicrobials can result in bacterial selection pressure of the intestinal microbiota of animals [19,42,43].
The high levels of resistance of the isolated strains against more than three antibiotics were not surprising given the uncontrolled use of these antibiotics in poultry in Algeria and their use without prior antimicrobial susceptibility tests. It must also be noted that the lack of legislative restrictions on antibiotic use in the poultry industry could also lead to a build-up of antibiotic resistance, i.e., they are still used in poultry feeds at sub-therapeutic dosages for growth promotion purposes (to reduce bird mortality and improve production performance). In contrast, this practice is banned in many countries, including those of the European Union, to avoid AMR diffusion in pathogenic bacteria in food-producing animals [44]. The high level of resistance recorded in this study for CTX (50%) is troubling as third-generation cephalosporins (ceftiofur) are not used in Algerian poultry production. These results are in agreement with those reported in other studies [45,46], which highlighted the emergence and persistence of ESBL-producing E. coli in the poultry production pyramid in many countries despite the absence of third-generation cephalosporin usage. This might be linked to the abuse and misuse of other antimicrobials (i.e., aminoglycosides, β-lactams, quinolones, macrolides, nitrofurans, phenicols, polypeptides, sulphonamides and tetracyclines) in broiler breeding or to the selection of ESBL-producing E. coli in broiler breeders and their vertical transmission in the poultry production pyramid [47,48,49,50]. The high levels of ESBL-producing E. coli isolates in Mostaganem, Oran, Mascara, Relizane and Tiaret could be explained by their horizontal transmission in broiler farms and hatcheries, as previously suggested [51], during broiler chicken transfer and likewise through national trade to several regions of the country. In addition, encoding cephalosporin resistance genes are generally placed on self-transmissible plasmids [52], which can be promiscuous and are capable of circulating among a wide variety of hosts. Despite the fact that third-generation cephalosporins are not used in Algerian poultry production, several studies highlighted their colonization in broiler chickens in the last few years [53,54,55]. The genetic background for cephalosporin resistance was diverse in those studies. Benameur et al. [54] reported the presence of the blaCTX-M−1 gene and Meguenni et al. [55] showed the presence of blaCTX-M−1 and blaCTX-M−15. Furthermore, Belmahdi et al. [53] detected the presence of blaCTX-M−1, blaSHV−12 and blaTEM−1.
However, in our study, the most prevalent group was blaTEM followed by blaTEM and blaCTX-M−1 gene combinations and blaSHV, like the findings in a study in Turkey that demonstrated blaTEM as the most frequent gene, followed by blaCTX-M and blaSHV [56].
In many other studies, multiple occurrences of the genes were also common [57], given that these genes frequently exist in large plasmids [58]. SHV and TEM were the main types of ESBL until 2000, while, in recent decades, CTX-M enzymes took their place [59].
All genes encoding resistance to macrolides, quinolones, tetracyclines, aminoglycosides, trimethoprim, chloramphenicol and sulfonamides have been associated with plasmids containing the blaCTX-M type [60]. The association of antibiotics, and the coexistence of blaCTX-M genes with blaTEM or other resistance determinants, could contribute to the spread of CTX-M enzymes. Nowadays, enzymes of the CTX-M-1 group are frequently identified in North Africa [61].
This issue is further worsened by the formation of biofilm, which promotes an additional bacterial tolerance or resistance to antimicrobial agents [29,62] and represents an advantage in the survival against host defense factors, antibiotics, physical and chemical stress as well as disinfectants [63,64].
In this study, the ability of biofilm formation was found to be significantly higher in negative ESBL strains of E. coli than in strains carrying the blaTEM gene. However, despite the small number of strains used in this study, the results align with those of other authors who demonstrate that the expression of the blaTEM gene can negatively impact biofilm formation in E. coli [65].
The production of biofilm is also regulated by putative enteroaggregative genes such as the transcription activator known as “aggR”, the master regulator of EAEC virulence, which controls the expression of adherence factors, and several other genes including yersiniabactin operon (irp2) and EAST1 toxin (astA) [61].
However, according to other authors, no correlation was reported between aggR alone or in association with irp2 and astA and biofilm formation in producing isolates, indicating that there are additional factors involved in biofilm production in EAEC [9,66,67].

4. Materials and Methods

4.1. Study Area and Sampling

A total of 16 broiler farms were randomly selected to carry out this study. All the farms were located within five geographic areas of Western Algeria, namely Mostaganem, Oran, Mascara, Relizane and Tiaret, representing the major broiler poultry producing sites in Algeria. Each broiler farm comprised several houses. Two poultry houses were sampled from each farm and one sample per house was collected. The poultry houses were chosen by considering their capacities (at least 3000 birds per house). All the farms included in this study were under control by official veterinary services. Broilers were commonly kept for a short period, which is generally less than two months. All broiler farms were visited once and sampling was carried out a few days before submission of the birds to slaughter. Fresh (still soft and warm) poultry feces was sampled from the poultry houses and transported to the laboratory for isolation of E. coli. All sampled broiler flocks were apparently healthy on the day of sampling.

4.2. Escherichia coli Isolation

Between March and September 2020, a total of 32 fecal samples, collected from different broiler chicken farms situated in five geographic areas of Western Algeria (Mostaganem, Oran, Mascara, Relizane, Tiaret), were analyzed in this study. To isolate E. coli, one gram of fecal specimens was mixed with 9 mL of buffered peptone water and incubated for 18 h at 37 °C. A drop was then streaked on MacConkey agar (MAC, Oxoid, Hampshire, UK) plates and incubated for 18 h at 37 °C. E. coli ATCC 25922 and ATCC 10536 (American Type Culture Collection, Rockville, MD, USA) were used as reference strains. Single colonies were stored in glycerol at −80 °C until further testing.

4.3. Identification of Colonies by MALDI-TOF-MS

The presumptive E. coli colonies were identified by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Briefly, strains were cultured on tryptic soy agar (TSA; Oxoid, Hampshire, UK) supplemented with 5% of sheep blood and incubated at 37 °C for 24 h.
A single bacterial colony was deposited on FlexiMass MALDI target plates, with 48-well sample spots (bioMérieux, Firenze, Italy), followed by the addition of 1 μL of matrix of alpha-cyano-4-hydroxycinnamic acid matrix in 50% acetonitrile and 2.5% trifluoroacetic acid (Vitek MS-CHCA, bioMérieux, Firenze, Italy).
E. coli ATCC 8739, used as a calibrator and internal ID control, grown on TSA, which was supplemented with 5% of sheep blood (according to the constructor procedure) and incubated at 37 °C for 24 h, was inoculated on the calibration spots as well as the test strains. The prepared plate, after the complete crystallization of the microbial matrix complex, was inserted in a Vitek MS Axima Assurance linear mass spectrometer (bioMérieux, Firenze, Italy) set with a laser frequency of 50 Hz, an acceleration voltage of 20 kV, an extraction delay time of 200 ns and mass spectra from 2000 to 20,000 Da. Every single strain was analyzed three times in three separate runs at different times.
The obtained mass spectra for each microorganism were analyzed by SARAMIS software (Spectral ARchive And Microbial Identification System—Database version 4.10—Software year 2010, bioMérieux, Firenze, Italy) by comparing them with the database bacteria reference spectra. The result of this comparison, calculated by the software algorithm, is a percentage probability (confidence level) that represents the similarity (presence or absence of specific peaks) among the obtained spectra and the reference spectra.
A perfect match reported as “excellent ID” corresponded to a percentage probability of identification (confidence level) of 99.9%, a “good ID” from >60% to 99.8%, while for <60% “no identification” (no ID) was given.

4.4. Genes Encoding VF Detection by Polymerase Chain Reaction

All E. coli isolates were tested for the genes encoding VFs characteristic of pathogenic E. coli O157:H7: stx1, stx2, rfbE, fliC and eaeA, using specific primers [68]. Each polymerase chain reaction (PCR) reaction was performed in a 50 μL amplification mixture consisting of 10 μL 5 × PCR buffer (1.5 mM MgCl2), 5.0 μL dNTPs (2.5 mM), 1 μL of each primer (10 μM), 0.25 μL of GoTaq DNA polymerase (5 unit/μL) and 10 μL of template. E. coli ATCC 43894 was used as a reference strain (E. coli O157:H7). The sequence of the used primers and the conditions of PCR were performed according to Tabashsum et al. [68]. Amplification products were separated by electrophoresis on 1.5% agarose gel, on 1 × Tris-Acetate-EDTA (242 g/L trizma base; 57.1 mL/L glacial acetic acid; 100 mL/L EDTA 0.5 M pH 8.0) at 100 V for 1 h and then visualized by GelRed staining, illuminated by UV transilluminator and visualized by a gel reader (Bio Rad Gel DOC XR+, Hercules, CA, USA). A 100 bp DNA ladder was used as a marker for PCR assay. The expected sizes of products for eaeA, rfb O157 and fliC H7 gene amplification were 150, 259 and 625 bp, and for stx1 and stx2 genes were 348 and 584 bp, respectively [68].

4.5. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility of the isolates was tested using the Kirby Bauer method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [69]. The following antibiotics were tested: NA, 30 µg; CIP, 5 µg; AML, 25 µg; AUG, 20/10 µg; CTX, 30 µg; TE, 30 µg; SXT, 1,25/23,75 µg; N, 30 µg; C, 30 μg; CT, 50 μg (Oxoid, Hampshire, UK). Briefly, the isolates were grown on TSA for 24 h at 37 °C. Subsequently, each bacterial suspension was adjusted to McFarland 0.5 in normal saline and uniformly spread onto Mueller–Hinton agar (MHA; Oxoid, Hampshire, UK). Paper disks impregnated with antibiotics were placed on the surface of agar plates and incubated for 24 h at 37 °C aerobically. Then, the diameters of the inhibition zones were measured by using a Vernier caliper and the values were interpreted according to the CLSI guidelines [69]. E. coli ATCC 25922 and ATCC 10536 (American Type Culture Collection, Rockville, MD, USA) were used as quality control strains.

4.6. Phenotypic Confirmation of ESBL Production

Phenotypic confirmation of ESBL production was performed by double-disk synergy test according to the CLSI guidelines [69], by positioning an AUG disk at a distance of 30 mm to third-generation cephalosporin disk (CTX) on MHA. The test was considered as positive when a synergy (champagne cork aspect) between AUG and CTX disks was observed in combination with resistance or reduced susceptibility to third-generation cephalosporin. Isolates showing decreased susceptibility to third-generation cephalosporin without clear synergy were subjected to a Combination Disk Test, by applying disks containing third-generation cephalosporin alone and in combination with clavulanic acid, following CLSI guidelines [69].

4.7. ESBL Gene Identification by PCR

DNA of the isolated E. coli strains was prepared by boiling methods. Briefly, for each strain, 2 or 3 colonies were dissociated in 1 mL of distilled sterile water and centrifuged for 5 min at 13,000 rpm. The supernatant was eliminated, and the pellet was suspended in 200 μL of distilled sterile water and heated at 100 °C for 10 min, cooled on ice for 5 min, and the DNA was removed from the supernatant after 5 min of centrifugation (13,000 rpm) to pellet the cellular debris and stored at −20 °C until use. Genetic characterization of ESBLs was performed on phenotypically confirmed E. coli isolates by PCR. The sequence of primers and the conditions of PCR for the detection of blaESBL genes were performed as described previously for blaCTX-M genotype groups 1, 2, 8 and 9, blaSHV [70] and blaTEM [71]. Amplification products were separated by gel electrophoresis using a 2% agarose gel.

4.8. Putative Enteroaggregative Gene Detection by PCR

The isolates were also investigated for the detection of various enteroaggregative putative genes: aggr, astA and irp2. The sequence of the used primers and the conditions of PCR were performed according to Mohamed et al. [9].

4.9. Biofilm Formation Assay

All E. coli isolates were evaluated for their ability to form biofilm by staining assay, as described by Cramton et al. [72] with some minor modifications. Briefly, overnight cultures in tryptic soy broth (TSB) were adjusted in culture medium to 5 × 105 CFU/mL and then 200 μL was dispensed into all the wells of the microtiter plate. The biofilm biomass formed in each well, after incubation for 24 h at 37 °C, was washed twice with phosphate-buffered saline (PBS), dried at room temperature, stained with aqueous 0.1% safranin solution (200 μL) for 1 min and then washed with water. The stained biofilms were dissolved in 30% (v/v) acetic acid and measured at OD 492 nm using a microplate reader. The following formula was applied to classify the biofilm formation: SBF = (AB − CW)/G, where AB is the stained attached bacteria (OD 492 nm), CW is the stained control wells containing bacteria-free medium only (OD 492 nm) and G is the cell growth in suspended culture (OD 540 nm) [73]. E. coli ATCC 25922 served as a positive control. TSB without bacteria was included as medium control.
The degree of biofilm formation of the isolates was classified into 4 categories: negative (SBF < 0.35), weak (SBF ≥ 0.35–0.69), moderate (SBF ≥ 0.70–1.09) and strong (SBF ≥ 1.10) [74].

4.10. Statistical Analysis

All experiments were performed in triplicate. Statistical data analysis was carried out using MATLAB_R2020a (MatWorks, Inc. Natick, MA, USA). A two-tailed Student’s t-test was applied to evaluate the mean ± standard deviation and the significant differences in the grade of biofilm formation among different strains. For each comparison between virulence or resistance genes and biofilm formation, a correlation coefficient (r) was determined via Pearson’s analysis. p-values of ≤0.05 were considered significant in all experiments.

5. Conclusions

In conclusion, our results reported a low frequency of virulence-associated genes of STEC O157:H7 in E. coli strains isolated from different poultry farms in Western Algeria. However, all isolates were shown to be resistant to at least three antibiotics most frequently used in poultry, and among these more than half were ESBL-positive E. coli despite no use of third-generation cephalosporins in Algerian poultry production. The ability of biofilm formation, which is considered a further virulent factor in pathogenic bacteria, was instead found to be higher among non-ESBL-producing strains of E. coli. Given that E. coli in chickens represents one of the major opportunistic pathogens and that it can be easily transferred from animals to humans, ESBL-producing E. coli represents an important risk factor for the poultry industry and human health. This study emphasizes the importance of monitoring the spread of the E. coli isolates that harbor virulence and antibiotic resistance genes in poultry farms, including the ones with healthy chickens, in order to prevent and control the spread of resistant bacteria and their virulence genes.
In Algeria, antimicrobials are not only used for therapeutic reasons but also for growth promotion and disease prevention. Consequently, the Algerian authorities should enforce AMR rules in order to guarantee a wise use of antimicrobials that will limit the risk of transmission along the food chain.

Author Contributions

Conceptualization, A.M., Q.B. and T.G.; Methodology, Q.B., T.G., F.G., D.A. and E.L.C.; Investigation, F.G., M.V., A.N., E.L.C. and N.C.; Writing—original draft preparation, A.M., Q.B. and T.G.; Data curation, A.N., N.C. and M.V.; Writing—review and editing, A.M. and T.G.; Funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Table
Table 1. Characteristics of E. coli isolates.
Table 1. Characteristics of E. coli isolates.
StrainsAlgerian AreaVirulence Gene 1MALDI-TOF
Mean Value		bla Gene 2AMR 3
S13/15OranastA90.0%NoneNA, CIP, AML, SXT, TE, N
S14/15Oranirp287.4%CTX-M-1, TEMNA, CIP, AUG, SXT, TE, N, CTX
S2/15Oranirp285.1%TEMNA, CIP, AML, AUG, SXT, TE, N, CTX
S4/15Mostaganemstx287.5%NoneNA, AML, AUG, SXT, TE, N
S19/15MostaganemNone93.0%CTX-M-1, TEMNA, CIP, AML, AUG, SXT, TE, C, N, CTX
S12/15MostaganemaggR, astA, irp285.8%TEMNA, CIP, AML, AUG, SXT, TE, N, CTX
S25a/16MostaganemastA88.7%NoneNA, CIP, AUG, TE, N
S1/16MostaganemastA, irp297.7%TEMNA, CIP, AML, AUG, SXT, TE, N, CTX
S22/15MostaganemastA, irp289.4%TEMNA, CIP, AML, AUG, SXT, TE, N, CTX
S16/15Mostaganemirp292.6%NoneNA, CIP, AML, AUG, SXT, TE, N
S34/16Relizaneirp293.3%NoneNA, CIP, N
S31/16RelizaneNone91.2%NoneNA, CIP, TE, N
S33/16RelizaneaggR, astA, irp290.3%NoneNA, CIP, AML, AUG, SXT, TE, C, N
S47/16Tiaretirp295.2%TEMNA, CIP, AML, AUG, SXT, TE, N, CTX
S6/15TiaretNone96.5%NoneNA, CIP, AML, AUG, TE, N
S48a/16TiaretastA93.4%TEMNA, CIP, AML, AUG, SXT, TE, C, N, CTX
S19a/16MascaraNone92.7%TEMNA, CIP, AML, AUG, SXT, TE, N, CTX
S61a/16MascaraNone95.0%SHVNA, AUG, SXT, TE, N
E. coli
ATCC 259222		 99.9% AML, AUG
1 astA, heat-stable enterotoxin-1; irp2, iron regulatory protein 2; stx2, shiga-like toxin 2; aggR, transcription factor; 2 TEM, temoniera; CTX-M-1, cefotaximases; SHV, sulfhydryl variable; 3 AMR, antimicrobial resistance; NA, nalidixic acid; N, neomycin; TE, tetracycline; CIP, ciprofloxacin; AUG, amoxicillin–clavulanic acid; STX, trimethoprim–sulfamethoxazole; AML, amoxicillin; CTX, cefotaxime; C, chloramphenicol.
Table
Table 2. Enteroaggregative and ESBL genes and biofilm production of E. coli isolates.
Table 2. Enteroaggregative and ESBL genes and biofilm production of E. coli isolates.
StrainsaggRirp2astATEMCTX-M-1SHVSBFBiofilm Grade
S13/15 + 0.81M
S14/15 + ++ 0.16N
S2/15 + + 0.26N
S4/15 stx2 0.76M
S19/15 ++ 0.20N
S12/15++++ 0.18N
S25a/16 + 0.62W
S1/16 +++ 0.19N
S22/15 +++ 0.29N
S16/15 + 0.45W
S34/16 + 0.52W
S31/16 0.84M
S33/16+++ 0.65W
S47/16 + + 0.39W
S6/15 0.59W
S48a/16 ++ 0.22N
S19a/16 + 0.24N
S61a/16 +0.27N
E. coli ATCC 25922 0.76M
aggR, transcription factor; irp2, iron regulatory protein 2; astA, heat-stable enterotoxin-1; TEM, temoniera; CTX-M-1, cefotaximases; SHV, sulfhydryl variable; SBF, specific biofilm formation; + gene presence; M, moderate (SBF ≥ 0.70–1.09); N, negative (SBF < 0.35); W, weak (SBF ≥ 0.35–0.69).
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Benameur, Q.; Gervasi, T.; Giarratana, F.; Vitale, M.; Anzà, D.; La Camera, E.; Nostro, A.; Cicero, N.; Marino, A. Virulence, Antimicrobial Resistance and Biofilm Production of Escherichia coli Isolates from Healthy Broiler Chickens in Western Algeria. Antibiotics 2021, 10, 1157. https://doi.org/10.3390/antibiotics10101157

AMA Style

Benameur Q, Gervasi T, Giarratana F, Vitale M, Anzà D, La Camera E, Nostro A, Cicero N, Marino A. Virulence, Antimicrobial Resistance and Biofilm Production of Escherichia coli Isolates from Healthy Broiler Chickens in Western Algeria. Antibiotics. 2021; 10(10):1157. https://doi.org/10.3390/antibiotics10101157

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Benameur, Qada, Teresa Gervasi, Filippo Giarratana, Maria Vitale, Davide Anzà, Erminia La Camera, Antonia Nostro, Nicola Cicero, and Andreana Marino. 2021. "Virulence, Antimicrobial Resistance and Biofilm Production of Escherichia coli Isolates from Healthy Broiler Chickens in Western Algeria" Antibiotics 10, no. 10: 1157. https://doi.org/10.3390/antibiotics10101157

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Benameur, Q., Gervasi, T., Giarratana, F., Vitale, M., Anzà, D., La Camera, E., Nostro, A., Cicero, N., & Marino, A. (2021). Virulence, Antimicrobial Resistance and Biofilm Production of Escherichia coli Isolates from Healthy Broiler Chickens in Western Algeria. Antibiotics, 10(10), 1157. https://doi.org/10.3390/antibiotics10101157

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