Antibacterial Activity of Bacteriocinogenic Commensal Escherichia coli against Zoonotic Strains Resistant and Sensitive to Antibiotics

Antibiotic resistance concerns various areas with high consumption of antibiotics, including husbandry. Resistant strains are transmitted to humans from livestock and agricultural products via the food chain and may pose a health risk. The commensal microbiota protects against the invasion of environmental strains by secretion of bacteriocins, among other mechanisms. The present study aims to characterize the bactericidal potential of bacteriocinogenic Escherichia coli from healthy humans against multidrug-resistant and antibiotic-sensitive strains from pigs and cattle. Bacteriocin production was tested by the double-layer plate method, and bacteriocin genes were identified by the PCR method. At least one bacteriocinogenic E. coli was detected in the fecal samples of 55% of tested individuals, adults and children. Among all isolates (n = 210), 37.1% were bacteriocinogenic and contained genes of colicin (Col) Ib, ColE1, microcin (Mcc) H47, ColIa, ColM, MccV, ColK, ColB, and single ColE2 and ColE7. Twenty-five E. coli carrying various sets of bacteriocin genes were further characterized and tested for their activity against zoonotic strains (n = 60). Strains with ColE7 (88%), ColE1-ColIa-ColK-MccH47 (85%), MccH47-MccV (85%), ColE1-ColIa-ColM (82%), ColE1 (75%), ColM (67%), and ColK (65%) were most active against zoonotic strains. Statistically significant differences in activity toward antibiotic-resistant strains were shown by commensal E. coli carrying MccV, ColK-MccV, and ColIb-ColK. The study demonstrates that bacteriocinogenic commensal E. coli exerts antagonistic activity against zoonotic strains and may constitute a defense line against multidrug-resistant strains.


Introduction
The intestinal microbiota is a highly diverse and dynamic community. It consists of indigenous flora and autochthonous microorganisms from the environment, which compete for niches and nutrients to colonize the intestinal habitat [1,2]. Protection of commensal species against invasion by exogenous microorganisms is a part of colonization resistance and involves the production of inhibitory molecules such as bacteriocins [3,4]. Bacteriocins are antimicrobial peptides, ribosomally produced with narrow killing spectra, produced by most bacteria [5].
Escherichia coli produces two classes of bacteriocins, colicins and microcins, which differ in their molecular weight, synthesis induction, mechanisms of secretion, and cytotoxicity [6]. Colicins are plasmid-encoded bacteriocins. Their operons, under the LexA promoter, include one to three genes responsible for the unique action of the system-toxin, immunity, and lysis. Colicin production is induced in stressful conditions that stimulate SOS responses or by nutrient depletion, but small amounts leading to the expression and activity of bacteriocins in these environments, especially in the processes of combating pathogens and antibiotic-resistant strains.

Results
In total, from 100 fecal samples, 210 nonidentical E. coli bacteria were isolated (120 from adults, 90 from children). Phenotypic studies with the indicator strain revealed that 78 strains (37.1%) were bacteriocinogenic, including 42 strains from 28 adults and 36 strains from 27 children.
The presence of one bacteriocin gene was detected in 32 strains, while co-occurrence of two or more colicin genes was found in 37 strains. In nine tested strains, none of the tested bacteriocin genes were detected. Among bacteriocinogenic strains, eight colicin genes-E1, E2, E7, Ib, Ia, M, B, K-and two microcin genes-H47 and V-were detected ( Table 1). The colicin Ib gene (n = 23) and E1 (n = 22) were the most frequently detected, then microcin H47 (n = 17), colicin Ia (n = 16) and M (n = 15), microcin V (n = 11), colicin K (n = 10), B (n = 8), and single ColE2, ColE7. Colicins E1, Ib and Ia were detected more frequently in tested isolates from adults, while microcin H47 and colicin M were more frequently detected in E. coli from children. Twenty-five strains (12 from adults and 13 from children) carrying various bacteriocins and bacteriocin sets (Table 2) were further characterized and tested for their activity against strains derived from animals.
Most of the commensal E. coli showed similar activity against antibiotic-resistant and antibiotic-sensitive strains; however, there was a statistically significant difference in the bactericidal activity toward antibiotic-resistant strains for four E. coli with detected bacteriocins: MccV, ColK-MccV, ColIb-ColK, and ColE1-ColIa-ColB-ColM. These strains showed effective bactericidal activity from 18.3% to 43.3%. Strains carrying varied sets of bacteriocin genes did not show clear synergistic effects. Commensal E. coli carrying two or three bacteriocins did not inhibit the growth of a larger number of zoonotic strains than E. coli with one bacteriocin. E. coli with four bacteriocins, despite a shared bacteriocin type, revealed totally different results (e.g., E1-Ib-K: 0%; E1-Ia-K-H47: 85%).
Taking into account the bactericidal potency of bacteriocinogenic strains, based on the inhibition zone size, strains with single bacteriocins (E1, E7, Ia, Ib, M, MccV, MccH47) revealed low (1-4 mm) or medium (5-9 mm) antagonistic activity against zoonotic strains ( Figure 1). The only exception was colicin K, where 13% of strains showed large (10-15 mm) inhibition zones. Strains with two and three bacteriocins more often revealed medium and large growth inhibition zones of zoonotic strains. No statistically significant difference in the bactericidal potency against antibiotic-sensitive and antibiotic-resistant strains was observed.

Discussion
The scale of antibiotic resistance in the population is vast and much effort is put into its limitation [58]. High prevalence of antibiotic resistance among farm animals is the result of widespread and excessive use of antibiotics during animal husbandry [57,59,60]. Zoonotic strains transmitted to humans by food can induce a disease process or try to settle in the intestines of the host. E. coli is an important species transmitted from livestock and agricultural products [26,27,[61][62][63]. An element of the natural protective barrier against infections or colonization by pathogenic and antibiotic-resistant bacteria is the commensal microbiota [39,51,64]. Production of bacteriocins plays an important role in the defense. These inhibitory molecules affect phylogenetically related bacteria and can be produced by commensals such as E. coli strains. In our study, bacteriocinogenic E. coli strains were detected in 55% of individuals of the tested healthy group. The frequency of bacteriocin production among fecal E. coli strains varied, but according to many researchers, it does not exceed this value and is most often around 50% [6,[65][66][67]. Bacteriocin genes detected among E. coli from the study E7

Discussion
The scale of antibiotic resistance in the population is vast and much effort is put into its limitation [58]. High prevalence of antibiotic resistance among farm animals is the result of widespread and excessive use of antibiotics during animal husbandry [57,59,60]. Zoonotic strains transmitted to humans by food can induce a disease process or try to settle in the intestines of the host. E. coli is an important species transmitted from livestock and agricultural products [26,27,[61][62][63]. An element of the natural protective barrier against infections or colonization by pathogenic and antibiotic-resistant bacteria is the commensal microbiota [39,51,64]. Production of bacteriocins plays an important role in the defense. These inhibitory molecules affect phylogenetically related bacteria and can be produced by commensals such as E. coli strains. In our study, bacteriocinogenic E. coli strains were detected in 55% of individuals of the tested healthy group. The frequency of bacteriocin production among fecal E. coli strains varied, but according to many researchers, it does not exceed this value and is most often around 50% [6,[65][66][67]. Bacteriocin genes detected among E. coli from the study population encode quite homogeneous bacteriocins in terms of the mechanism of action concerning the cell membrane. Pore-forming colicins ColIb, ColE1, ColIa, ColK, ColB, microcin MccV, and cellular proton channel disruption MccH47 prevailed. ColM, inhibiting peptidoglycan synthesis, was most frequently detected in strains from children. The difference in the prevalence of bacteriocins between the groups of adults and children was also in regard to the presence of ColE2 and ColE7 DNA cleavage colicins. The structure of the microflora is variable over time and showed greater genetic diversity in adults [68], but as we can observe, the bacteriocin-producing strains, which promote competitive advantage, are present from an early age.
The study revealed that commensal bacteriocinogenic E. coli strains have antagonistic activity against zoonotic strains. This activity is mainly caused by ColE7-, ColE1-, ColM-, and ColK-positive strains. The largest numbers of E. coli zoonotic strains were killed by DNA-degraded colicin ColE7, although without an advantage toward multi-resistant strains. The high sensitivity of strains from animals can be explained by the low prevalence of colicin producers in the populations. Sensitivity to the bacteriocins results from the presence of the appropriate receptors on the outer membrane, function of the bacteriocin transfer system, and finally, from the production of immune proteins when strains are able to produce specific bacteriocins themselves [7]. The availability of mechanisms enabling the introduction of colicin into the target cell in the absence of immune proteins results in high antagonistic activity in the population. ColE7 is a rare colicin among commensal flora. In our study, it was detected only in one strain (0.5% of all those tested); in literature data, it was found in 2.3% [6] and 1.7% [71] of fecal isolates. This colicin was also found in small amounts among E. coli causing asymptomatic bacteriuria [72], urinary tract infection [71], and in fecal samples from patients with different diseases [65], not exceeding 4%. Confirmation of the bactericidal effectiveness of this colicin was presented by Budič, where purified colicin inhibited the growth of 87% of tested E. coli strains from patients with bacteremia [41].
The most commonly identified in our tested set of strains, colicin Ib, showed low bactericidal activity (13.3%). It was similar when it occurred in sets with other bacteriocins, except co-occurrence with ColE1 (60%) and microcin V (60%). It can be assumed that the production of many bacteriocins that use different receptors, translocation systems, or mechanisms of action will allow a stronger bactericidal effect. Colicins Ib and ColE1 use different receptors (Cir receptor for catecholate siderophores and BtuB-cyanocobalamin transporter, respectively) and translocation systems (TonB-ExbBD and TolCAQ, respectively) [7], although they are pore-formation colicins. Their co-occurrence results in greater bactericidal activity, observed as larger diameters of the growth inhibition zone, but not as an increase in the number of destroyed zoonotic strains, compared to the activity of the monoproducers (Col E1-75%). Greater potency was also observed for the strain with the set of ColIb and MccV, despite these bacteriocins not only having a similar mechanism of action but also the same receptor (Cir protein receptor) and TonB transport pathway into the cell [73]. Another combination occurs in the strain carrying ColIb and ColM. They use different receptors (Cir and FhuA), but the same TonB transport systems and ColM inhibits cell wall synthesis. E. coli with ColIb and ColM acted against only 10% of zoonotic strains, whereas the monoproducers of ColIb and ColM were able to inhibit the growth of 13.3% and 66.6% of animal strains. It appears that colicin Ib does not have major antagonistic importance in the studied population.
E. coli strains carrying single genes of microcins MccH47 and MccV showed low antimicrobial activity (5% and 25%, respectively), while the strain carrying the genes of both of these microcins was able to inhibit 85% of the zoonotic strains. MccV, despite a low rate of growth inhibition, was mostly effective against multidrug-resistant strains, also in co-occurrence with ColK. Different indications regarding the activity of microcins against antibiotic-resistant strains were presented by Palmer et al. [52], who showed that purified MccH47 has broad and potent activity against medically relevant multidrug-resistant members of Enterobacteriaceae.
As can be observed, the bacteriocin activity detected in monoproducers did not correspond to their activity in the multiproducer. In many cases, no synergistic effect was noted. The results can be different when purified colicins are studied. Thus, for example, Budič observed that a colicin mix (E1, E6, E7, K, and M) acted better and was effective against 98% of E. coli from bacteremia [41]. It is likely that in the bacteriocin operons of naturally occurring strains, some mutation prevents the effective synthesis, secretion, or attack of the target cell. Another issue is the question of whether the strain carrying numerous bacteriocin genes produces all bacteriocins at once to launch a "massive attack" on the invader's cell. Bacteriocin expression is triggered by stress conditions, but not all mechanisms are the same. Bacteriocin expression is a costly trait, so perhaps not all of them are expressed simultaneously. There are no studies showing whether all bacteriocins of a multiproducer are secreted into the environment. Bacteriocin activity studies are usually conducted on purified colicins or strains with operons of one bacteriocin, introduced by genetic engineering methods [41]. The abovementioned colicin M, occurring with ColIa and ColE1 (they differ in receptors and import system), shows 82% activity against zoonotic strains. Colicin M occurring with colicin Ib (closely related to ColIa) shows low activity (10%), although they also use different receptors and there was no competition for the binding site. Similarly, in the presented results, the strain with bacteriocins ColE1-ColIb-ColK (all pore-forming but with individual receptors) did not affect any of sixty tested animal strains (although it was able to inhibit the growth of the control strain), but when microcin MccH47 was "added" in the other strain, 85% of zoonotic strains were eliminated. Therefore, in the case of multibacteriocin producers, their antimicrobial effect appears to be the result of many factors affecting the sensitivity of the target cell.
The growth of multidrug-resistant zoonotic strains was inhibited by the bacteriocins with similar frequency as antibiotic-sensitive strains in most cases. Three strains inhibited the growth of antibiotic-resistant E. coli more frequently than antibiotic-sensitive E. coli, with a highly statistically significant difference: MccV, ColK-ColV, and ColIb-ColK. Taking into account the activity of E. coli with microcin V, the above sets of bacteriocins, and the activity of individual bacteriocins in the collection, one can identify two bacteriocins that are most active against multidrug-resistant bacteria: MccV and ColK. This indicates the potential of commensal E. coli strains to defend against the invasion of zoonotic, antibiotic-resistant strains, which should be further investigated.
The antagonistic activity of a bacteriocinogenic strain in a complex population does not depend only on its activity but also on the susceptibility of other strains and the environment. As was already mentioned, the reason for the insensitivity of the cells to the bacteriocins may result from the lack of appropriate receptors or a disorder of the bacteriocin transfer system that makes the cells tolerant. In vivo, prevailing conditions may lead in an additional way to the strains' antagonism. In the natural intestinal environment, other factors such as competition for the receptors of bacteriocin and the natural ligands may be relevant [7]. It has been shown that colicins compete with the corresponding natural ligand for the receptors. For example, cobalamin protects the BtuB receptor against E-type colicins or using ferrichrome blocks the FhuA receptor and action of colicin M [7]. According to the latest research, the density of the O-antigen also influences colicin sensitivity [13]. It has been shown that minor changes in growth conditions influence the density of LPS and hence sensitivity toward colicin. It was suggested that colicin could be more effective than is presented [13]. Colicin activity in vivo depends primarily on the colicin synthesis activated by the SOS system. In in-vitro studies, the SOS system is activated by nutrient depletion or the use of mitomycin, but other inducing factors are achievable in the natural intestinal environment. SOS can be triggered by antibiotics, which affect DNA replication such as quinolones or induce membrane stress such as b-lactams [74][75][76]. Additionally, oxidative stress induced by reactive oxygen [77] and some nutrient conditions appear prone to producing and releasing colicins [78]. The main argument indicating bacteriocin activity in the natural habitats are in vitro studies showing that bacteriocin-producing strains can enhance colonization resistance and protect against pathogens.
A practical approach to using direct competition of bacteriocinogenic commensals in therapy has been recently tested on Gram-positive enterococci by Kommineni [39]. The in vivo studies on mice have shown that bacteriocin expressed by commensals can influence niche competition in the gastrointestinal tract and eliminate intestinal colonization by multidrug-resistant bacteria. These studies indicate the possibility of a novel therapeutic strategy to eliminate antibiotic-resistant enterococci from the human gastrointestinal tract and, thereby, prevent the emergence of resistant enterococcal infections [39]. Another example is the study of Bosák et al., revealing an increase of colonization capacity by strains producing colicin F Y against Yersinia enterocolitica infection [50].
The in vitro studies have shown the activity of bacteriocins produced by Gram-negative bacteria under culture conditions in agar [6,41,65,69,71] or liquid solution media [41,45,48,49], but in vivo [37] approaches can prove the effectiveness of bacteriocin antagonistic actions. Therefore, our research should be continued for a full understanding of the expression and activity of the bacteriocins, which play a role in the colonization resistance, protecting against multidrug-resistant strains of animal origin.

Bacterial Strains
Commensal E. coli bacteria were isolated from healthy individuals, fifty adults (aged 18-56 years) and fifty children (aged from 6 months to 3 years) from Lubuskie Province in Poland. The people were from separate households, not related. Strains were isolated and identified from stool samples, as described earlier [68]. Briefly, samples were inoculated on mFC medium and incubated in 44 • C; next, characteristic blue colonies were transferred to MacConkey medium and identified in biochemical tests: Indole production, Methyl red reaction, Voges-Proskauer reaction, and Citrate utilization (IMVC). Five random E. coli strains were isolated from a single sample and then distinguished at the bases of BOX-PCR profiles [3,79]. As a result, 210 genetically diverse strains were selected for further studies. This study was approved by the bioethics committee at the District Medical Council in Zielona Góra (No. 03/85/2018).
E. coli bacteria from animals were isolated from fecal samples from pigs and cattle from two farms in Lubuskie Province in Poland. E. coli was isolated and identified using biochemical tests, as described above. One strain from one animal was further characterized. Antibiotic sensitivity was tested for ampicillin, cefotaxime, ceftazidime, tetracycline, streptomycin, gentamicin, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, and chloramphenicol, using the broth dilution method as described previously [60]. From over one hundred strains with different resistance profiles, thirty E. coli strains susceptible to antibiotics, and thirty multidrug-resistant strains were randomly selected for further research.

Detection of Bacteriocins Producers among E. coli from Humans
The tests were conducted using the double-layer method. The agar plates were inoculated by spots of 10 µL of overnight LB cultures of tested E. coli isolates from humans. Cultures were incubated at 37 • C for 48 hours. Bacteria were killed using chloroform vapors. Next, plates were overlaid with a soft agar containing 100 µL of an indicator strain E. coli K12-Row (PCM 827) that is sensitive to bacteriocins (OD 600 approximately 0.05). The cultures were incubated at 37 • C overnight and examined for inhibition zones, which were measured in millimeters. In order to detect the activity of microcins H47 and M, which are sensitive to chloroform [65,80], tests were repeated without killing the bacteriocin producers. In such tests, the resulting growth inhibition zones are slightly less clear and sharp compared to the use of chloroform but sufficient for detection.

Identification of Bacteriocins Genes
The presence of the bacteriocin genes was detected by the PCR method developed by Smajs using multiplex and uniplex reactions [71]. The presence of the colicin genes A, B, D, El, E2, E3, E6, E7, Ia/Ib, K, and M was detected, as well as microcins B17, H47, J25, M, and V.

Evaluation of Antibacterial Activity of Bacteriocinogenic E. coli against Zoonotic Strains
The test was carried out as described above, with the difference that bacteriocin producers were grown as spots on the first agar layer and were overlaid with a second agar layer containing the zoonotic strain.

Statistical Analysis
Pearson's chi-squared test was used to assess the significance of differences in bactericidal activity against resistant and antibiotic-sensitive strains, with the significance level set at p < 0.05. The chi-square calculations were conducted for expected values equal to 5 or higher. The statistical analyses were performed using the program GraphPad (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
Antibiotic resistance is a serious health problem and arises from the high antibiotic consumption in different areas such as medicine, veterinary, and husbandry. Taking into account the epidemiological threat arising from the transmission of resistant strains of animal origin to humans, we undertook preliminary research on using natural interactions between microorganisms to tackle this problem. Colonization resistance involves the production of bacteriocins that destroy related strains, and we wanted to estimate the potential of commensal strains from healthy humans to defend against zoonotic strains. The study revealed that bacteriocinogenic commensal E. coli strains show antagonistic activity against strains of animal origin and some of them, particularly those carrying microcin V and colicin K, can be active against multidrug-resistant bacteria.