Discussion
In total, we examined 108 rectal swabs originating from a pool of six
species of free-living animals and noted positive growth of E.
coli on the selective MacConkey agar medium in over 50% of the
samples. This is a lower percentage than that in a group of wildlife
predators analysed previously, in which we observed resistance to at
least one drug in approx. 70% of the tested animals (Osińska et al.,
2020). However, the lower number of resistant isolates in herbivores and
omnivores compared to carnivores is consistent with studies in mammals
(Nhung et al., 2015) and birds (Smith, Wang, Fanning, & McMahon, 2014).
These differences are related to the type of food consumed by these
groups of animals. Carnivores and predators are at the end of the food
chain and have more diverse food than herbivores, cumulating all
potential resistance determinants from prey. In the case of free-living
herbivores, the source of resistant bacteria is mainly assigned to the
polluted environment, including plants as a source of food, water, and
the ecological niche shared with other animal species (direct and
indirect contact).
Using several types of plates supplemented with various antimicrobial
substances, we were able to obtain up to 80 bacterial isolates from 55
single animals and, finally, using a two-step method, limit this number
to 70 E. coli strains that are distinct in terms of their
phenotype, genotype, or origin. This means that every third individual
of the resistant E. coli -positive animals carries more than one
strain, sometimes even three completely separate MDR clones, which may
indicate significantly reduced resistance results when analysed
according to the strategy “one randomly selected strain isolated from
one animal”. We also isolated a large number of multidrug-resistant
strains, estimated at over 70%, which confirms that regardless the
species of host, type of diet, and behaviour, free-living animals are
involved in the spread of resistant E. coli strains in Poland
despite the lack of direct exposure to antimicrobials (Osińska et al.,
2020; Nowakiewicz et al., 2020).
This fact is additionally confirmed by the finding of identical strain
clones in two different pairs of the red deer. Unfortunately, we do not
have information whether these animals were related to each other or
occupied the same area, but this does not change the fact that the same
strain was isolated from two different animals even in two cases. Red
deer and roe deer live in herds; therefore, close contact may promote a
horizontal transfer of resistance genes among animals sharing the same
ecological niche (Dolejska & Literak, 2019). It is also probable that
the statistically highest resistance to as many as four different
antimicrobials and the multidrug resistance of all the roe deer strains
may result from the herd lifestyle (swarm behaviour).
The high resistance to ampicillin, tetracycline, and sulfamethoxazole is
also consistent with our previous studies on predatory animals (Osińska
et al., 2020). This is also a typical result comparable to those
reported in studies of livestock animals in the world (Nhung et al.,
2015; de Alcântara Rodrigues, Ferrari, Panzenhagen, Mano, &
Conte-Junior, 2020) and in the same wildlife animal or species groups:
ungulates (Wasyl et al., 2018), squirrels (Jalal et al., 2019), and rats
(Nkogwe, Raletobana, Stewart-Johnson, Suepaul, & Adesiyun, 2011;
Himsworth et al., 2016).
We also recorded a high level of strain resistance to streptomycin and
chloramphenicol, almost identical to that in other wildlife animals in
Poland (Nowakiewicz et al., 2020; Osińska et al., 2020). As in the
previous study, we found only one gentamycin-resistant strain, which
confirms that resistance to gentamicin is rather sporadic among E.
coli isolates from wild animals.
In contrast to our previous studies, lower kanamycin resistance was
detected despite the use of this antibiotic for selective isolation of
the strains.
The phenotypic resistance of the strains was reflected in most cases in
the presence of at least one gene determining resistance to a given
antimicrobial. As with E. coli strains isolated from other
wildlife or domestic animals and strains isolated from nosocomial
infections, the gene profile was very similar, with the predominance of
the tetA (tetracycline-resistant strains) and sul 2 or bothsul 1 and sul 2 (sulfamethoxazole-resistant strains) (de
Alcântara Rodrigues, Ferrari, Panzenhagen, Mano, & Conte-Junior, 2020;
Hassan et al., 2020).
The nearly 40% level of chloramphenicol resistance was slightly higher
than that recorded previously in wildlife animal studies in Poland
(Osińska et al., 2020; Nowakiewicz et al., 2020) and as many as 59% of
the chloramphenicol-resistant strains exhibited the presence of theflo R gene responsible for both chloramphenicol and florfenicol
resistance. The latter antimicrobial is authorized for use in animals
also in Poland; therefore, such a high level of resistance to
chloramphenicol, although the drug is not approved for use in animals,
is likely to be associated with cross-resistance following exposure of
animals to florfenicol, residues of which were detected in environmental
samples, mainly in water (Hanna et al., 2018). Moreover, florfenicol was
considered as persistent against some water-treatment processes
(Charuaud et al., 2019). The ESBL phenotype strain had thebla CTX-M -27 gene. This variant has become one of
the most common contributors to the spread of blaCTX-M in humans in recent years (Cormier et al., 2019).
Currently, the producer of the MDR, CTX-M-27, ST 131 C1 cluster is
considered as a new epidemic clone (Fernandes et al., 2020). Although we
did not analyse the strains with the MLST method, it is worth noting
that the only strain with the ESBL phenotype isolated from the squirrel
was also resistant to the largest number of drug groups (up to seven)
among all strains tested similarly as the aforementioned cluster.
Virulence factors are an inherent attribute of pathogenic E. colistrains, unlike commensal strains, which naturally colonize the
digestive tract of humans and animals without causing clinical symptoms.
As many as 11.4% of the 70 strains with resistance to at least one
antimicrobial exhibited the presence of specific virulence factors,
qualifying them to specific pathotypes. However, the percentage of
simultaneously pathogenic and drug-resistant strains seems to be lower
than in other groups of free-living animals or other food-producing
animals from which strains were isolated randomly and not selected for
resistance (Nowak et al., 2017; Sarowska et al., 2019). Thus, a high
level of resistance is not necessarily accompanied by increased
virulence as in our study. We found mainly strains with the ExPEC
pathotype. These strains were isolated from the squirrels, rat, and
hedgehog samples. Due to the diverse and rich array of virulence
factors, it is difficult to determine clearly the origin of ExPEC
causing infections in humans. However, all the ExPEC strains in our
study were characterized by the presence of genes encoding fimbrial
adhesins (P, S, and F1C fimbriae) involved in adhesion to epithelial
cells in the intestine, kidney, bladder, and lower urinary tract
(Kuhnert, Boerlin, & Frey, 2000; Sarowska et al., 2019) as well as the
synthesis of capsular polysaccharide with antiphagocytic activity and
ferric aerobactin receptor involved in iron uptake transport (Chapman et
al., 2006; Kuhnert, Boerlin, & Frey, 2000). All these factors were most
often detected in UPEC or NMEC strains causing human infections
(Sarowska et al., 2019). However, a similar panel of virulence factors
was shown in strains from livestock (Bélanger et al., 2011; Wasiński,
2019) and ExPEC strains were isolated from pigs and cattle, where they
were responsible for urinary tract infections (Chapman et al., 2006),
pneumonia, mastitis, and meningitis (Tan et al., 2011). This pathotype
was also found in faecal microbiota of cats and dogs. It was shown that
dogs and cats in the same household can be carriers of the same UPEC
strains (Johnson, Miller, Johnston, Clabots, & DebRoy, 2009). This
indicates that the strains can overcome the species barrier and spread
between animals and humans (Bélanger et al., 2011) (Johnson et al.,
2008) (Johnson, Miller, Johnston, Clabots, & DebRoy, 2009).
Among other wild animal species, wild birds are the most widely studied
group in terms of virulence (Borges et al., 2017). Borges et al. (2017)
discovered the same virulence genes characteristic of APEC pathotype
strains that were also found in poultry, wild mammals (Chapman et al.,
2006), and humans (Maluta et al., 2014). This phenomenon suggests that
wild birds may also transfer virulence genes to ExPEC strains in other
hosts (Velhner, Suvajdžić, Todorović, Milanov, & Kozoderović, 2018)
therefore, the virulence factors do not appear to be in any way
correlated with the specific source of the strain.
We also found two isolates with the ETEC pathotype among samples
collected from the squirrels. Enterotoxigenic E. coli strains are
commonly isolated from farm animals and are responsible for diarrhoea,
especially in young animals. Since ETEC strains have a narrow host
species range due to specific adhesins, they are regarded as strains
posing a lower risk to public health. Moreover, enterotoxins are encoded
on plasmids, and ETEC strains are characterized by a large genotypic
diversity making it difficult to determine their original source (Nagy
& Fekete, 2005). In addition to specific adhesins, ETEC strains can
produce two main classes of toxins: heat-stabile (ST) and heat-labile
(LT) (Dubreuil, Isaacson, & Schifferli, 2016). Our strains contained
one or two genes responsible for producing ST enterotoxins that are
characteristic for ETEC of human, porcine, and calf hosts. Among
livestock, ETECs are often isolated from pigs. The pig environment is
one of the transmission pathways for ETEC strains. It has been confirmed
that ETECs can persist for at least six months when they are protected
by manure (Dubreuil, Isaacson, & Schifferli, 2016). In cattle, typical
ETEC strains only produce the STa toxin (Nagy & Fekete, 2005). This is
similar in dogs, but a small proportion of the strains were found to
produce the STb toxin as well (Dubreuil, Isaacson, & Schifferli, 2016).
The ETEC pathotype is very rarely isolated from wildlife animals and,
due to the low percentage of isolation and insufficient research, the
role of these strains in the aetiology of diarrhoea in this group of
animals is unknown (Milton et al., 2019).
We also revealed two E. coli strains with the EHEC pathotype
present in the squirrel and red deer samples; both of them produced
Shigatoxin 2. Shiga-toxin-producing strains (STEC) are a large class ofE. coli pathogenic strains and are commonly found in farm and
wild animals as well as humans. In humans, infection is mainly caused by
the consumption of bacterium-contaminated food or contact with
contaminated faeces (Milton et al., 2019). Cattle are the main reservoir
of EHEC strains in animals. Unlike humans, EHECs found in adult cattle
do not cause disease symptoms but may trigger life-threatening
conditions in young animals (Rice, Sheng, Wynia, & Hovde, 2003). In our
research, we only detected the presence of the stx2 gene, but thestx1 gene or both are detected equally often in animal-origin
strains (Milton et al., 2019). Stx1 or both genes dominated in
cattle (Tavakoli & Pourtaghi, 2017). Among wild animals, thestx2 gene is detected more frequently in birds (Sanches et al.,
2017) and ungulates (Milton et al., 2019). In contrast, in iguanas,
strains producing Shiga-toxins accounted for approx. 40% of pathotypes
(Bautista-Trujillo et al., 2020).