1. Introduction
The intestinal microbiota of animals is a dynamic community of microorganisms, among which bacteria of the species
Escherichia coli are of particular importance. Pathogenic strains of
E. coli cause a wide range of diseases in livestock: intestinal pathogenic
E. coli (IPEC) cause outbreaks of intestinal infections and uropathogenic
E. coli (UPEC), sepsis-causing
E. coli (SePEC), and neonatal meningitis-associated
E. coli (NMEC) lead to infectious processes in extra-intestinal areas [
1,
2]. Of special significance are avian pathogenic
E. coli (APEC) which cause colibacillosis [
3,
4]. Commensal
E. coli produce vitamins, are involved in modulating the host immune system, strengthen the intestinal barrier, and provide colonization resistance of the intestinal mucosa against pathogenic and opportunistic bacteria competing for a place in this ecological niche [
5,
6].
One of the main mechanisms of the antagonistic activity of commensal
E. coli is the secretion of antimicrobial peptides/bacteriocins [
7].
E. coli produce two types of bacteriocins: colicins and microcins [
7,
8]. Bacteriocins have highly specific activity against phylogenetically related species and unique mechanisms of action [
9]. Some colicins form pores (colicins A, E1, K, N, U, S4, B, Ia, Ib), some have nuclease activity, for example, DNase (E2, E7, E8 and E9), 16S RNase (E3, E4, E6), and tRNase (E5, D), and colicin M inhibits the biosynthesis of peptidoglycan [
7,
10]. Microcins are able to form pores in the bacterial membrane (microcins V and L), inhibit DNA gyrase (B17), RNA polymerase (J25), and disrupt ATP synthase (H47) [
10,
11].
Since the widespread use of antimicrobials, including in agriculture, has led to the spread of antibiotic resistance, studies are being conducted on the antimicrobial activity of bacteriocins and bacteriocin-producing strains as an alternative to antibiotic therapy [
7,
12,
13,
14]. On the one hand, bacteriocin-producing strains can be used as producers of new antimicrobial substances/bacteriocins; on the other hand, bacteriocin-producing strains themselves can be used as probiotics. The main staples in the search for new useful bacteriocin-producing
E. coli strains are the isolation of bacteriocin-producing bacteria from natural sources, the comparative assessment of their activity, and the selection of the most promising strains [
15,
16].
E. coli from the normal gut microbiota of healthy animals are one of the safe sources of bacteriocin-producing strains.
The aim of the study was to determine the prevalence of bacteriocin-encoded genes among E. coli strains from the feces of healthy farm animals and to characterize the presence of virulence-associated genes, the possibility of prophage induction, and the hemolytic and bacterial antagonistic activity of the bacteriocin-producing E. coli in order to reveal their potential for application.
4. Discussion
In this work, screening of bacteriocin-producing E. coli strains isolated from healthy farm animals was performed as well as further characterization of the bacteriocin-producing E. coli strains found: the detection of bacteriocin genes and virulence-associated genes. In addition, CFS of the bacteriocin-producing strains were tested for antagonistic activity against APEC and IPEC, including multidrug-resistant strains.
The percentage of bacteriocin-producing
E. coli strains isolated from different biotopes and ecological niches varied in different studies. In the study by Cameron et al., 2019 [
15], 15.6% of isolates isolated from wastewater and feces of cattle inhibited the growth of the test culture. Mazurek-Popczyk et al., 2020 [
7], showed that among isolates obtained from the feces of healthy humans, 37.1% were bacteriocinogenic. Micenková et al., 2016 [
25], reported much higher numbers: 54.2% of all tested fecal
E. coli isolates were found to be bacteriocin producers. An even higher prevalence of bacteriocin producers was shown by Budič et al., 2011 [
20], in the population of
E. coli isolated from patients with bacteremia: 61% of the strains carried at least one bacteriocin. In our study, the prevalence of bacteriocin-producing
E. coli strains among representatives of the microbiota of healthy animals was 23.6%, while among cattle the frequency of occurrence of such strains was 26.5%. The relatively low prevalence of bacteriocinogenity in the population of
E. coli obtained from healthy farm animals might be explained by the conditions in agricultural enterprises—the animals are artificially isolated from the environment (mostly kept indoors, use of standard feeding). Indirectly, this assumption could be confirmed by the fact that mainly bacteriocin-producing strains were isolated from cows, grazing outdoors in the summer–autumn period, while bacteriocin-producing strains were not isolated from turkeys, rabbits, and pigs living in boxes/cages all year round. Another fact that limits the biodiversity of strains, including bacteriocinogenity, may be the use of multiple antibiotic therapy regimens at the enterprises.
It is known that encoding multiple bacteriocins gives the cell a selective advantage since such strains have a broader inhibitory effect spectrum on competing organisms [
11], including bacteria with multiple resistance to bacteriocins [
26,
27]. In a study by Gordon O’Brien, 2006 [
8], it was shown that among the bacteriocin-producing strains, 42% produced one type of bacteriocin, 41% produced two, 16% produced three, and one strain produced four different types of bacteriocins. In our study, more than one bacteriocin gene was detected in all 17 strains, while about 30% of the strains had four or more bacteriocin genes.
Among the
E. coli strains isolated from different sources, most often genes of the following colicins E1, Ib, K, and M and microcins H47, M, and V were found [
7,
15,
20]. A number of researchers have reported correlations of encoding microcins H47 and M as well as colicins B and M [
8,
28]. In our study, the majority of strains (94.1%) encoded several bacteriocins with different modes of action. The most prevalent were strains encoding pore-forming bacteriocins (colicin E1, Ia, B) and those encoding colicin M with the mode of action of peptidoglycan degradation as well as microcin M (
Table 2,
Figure 1). It should be noted that out of 30 bacteriocin genes, 12 were not detected, including the E7 colicin gene, which, according to Mazurek-Popczyk et al., 2020 [
7], is a rare colicin among the commensal microbiota.
According to some studies,
E. coli bacteriocins are effective against IPEC strains, including the O157:H7 serotype [
29]. The development of probiotics based on bacteriocin-producing strains that eliminate STEC in farm animals can prevent zoonotic transmission of resistant bacteria to humans. Taking into account the problem of bacterial antibiotic resistance, bacteriocins are considered promising candidates for use in agriculture, especially in relation to multidrug-resistant bacteria [
14]. Mazurek-Popczyk et al., 2020, showed that bacteriocin-producing
E. coli strains showed the same antagonistic activity against antibiotic-resistant and antibiotic-sensitive zoonotic
E. coli strains [
7]. Our study also showed that CFS of bacteriocin-producing strains inhibited the growth of both sensitive and antibacterial-drug-resistant, even multidrug-resistant, pathogenic
E. coli belonging to different pathotypes.
Interestingly, we did not find a significant difference in antagonistic activity against APEC strains that differ in sensitivity to various antibiotics and bacteriocins (
Table 5). The CFS of
E. coli strains C32, C40, and C41, encoding the colicin E9 with the DNase activity, inhibited growth of all APEC strains by 50% or more. The growth of strains BR4, BR35, and BR37, which differed in sensitivity to bacteriocins, was significantly suppressed in the presence of CFS of bacteriocin-producing
E. coli from farm animals. This could be due to the action of other bacteriocins with different modes of action preventing the appearance of insensitive strains. It is known that the use of only one bacteriocin can quickly give rise to insensitive strains [
20].
The main primary requirements for the selection and verification of probiotic strains of microorganisms are the absence of virulence-associated genes (the strain should not produce enzymes related to virulence) and mechanisms associated with horizontal transmission of genetic information [
30]. Preliminary characterization of the strains in terms of hemolytic activity, lysogenicity, and the presence of virulence-associated genes allowed us to select three potentially probiotic strains with high antagonistic activity against pathogenic and conditionally pathogenic
E. coli from the 17 bacteriocin-producing strains, i.e., studied strains Q5, C23, and C41, whose probiotic potential can be further explored. For the development of a probiotic product, this primary study should be followed up in the future with the evaluation of the strains’ ability to actively colonize and persist long-term in the biotope as well as their biological safety. It is also necessary to select the optimal growth medium for the biotechnological process of producing the potential probiotic drug and to evaluate the survival of the microorganisms in the form in which the drug will be stored.