International Journal of

Environmental Research
and Public Health

Article
Comparison of Commensal Escherichia coli Isolates
from Adults and Young Children in Lubuskie
Province, Poland: Virulence Potential, Phylogeny and
Antimicrobial Resistance
Ewa Bok 1, *, Justyna Mazurek 1 , Andrzej Myc 1,2,3 , Michał Stosik 1 , Magdalena Wojciech 4
and Katarzyna Baldy-Chudzik 1
1 Department of Microbiology and Genetics, Faculty of Biological Sciences, University of Zielona Góra,
65-561 Zielona Góra, Poland; [email protected] (J.M.); [email protected] (A.M.);
[email protected] (M.S.); [email protected] (K.B.-C.)
2 Laboratory of Virology, Department of Immunology of Infectious Diseases, Ludwik Hirszfeld Institute of
Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland
3 Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan Medical
School, Ann Arbor, MI 48109-5648, USA
4 Department of Mathematical Statistics and Econometrics, Faculty of Mathematics, Computer Science and
Econometrics, University of Zielona Góra, 65-516 Zielona Góra, Poland; [email protected]
* Correspondence: [email protected]; Tel.: +48-68-328-73-33




Received: 6 March 2018; Accepted: 23 March 2018; Published: 28 March 2018


Abstract: Commensal Escherichia coli population is a dynamic structure which may be important in
the pathogenesis of extraintestinal infections. The aim of this study was the comparison of genetic
diversity of commensal E. coli isolates from two age group—adults and young children. E. coli strains
were isolated on MacConkey agar and identified by biochemical tests. Determination of four major
phylogenetic groups, identification of virulence genes and antimicrobial resistance determinants
were performed by using multiplex or simplex PCR. Phenotypic analysis of resistance was based on
disc-diffusion method. The prevalence of virulence genes was significantly higher among isolates from
adults than from young children. Phylogroup B2 predominated among E. coli from adults, whereas
phylogroup A was the most common in isolates from young children. The analyses of antimicrobial
resistance revealed that resistance to at least one antimicrobial agent and multidrug-resistance were
detected significantly more frequent in the isolates from adults than from young children. This study
documented that the commensal E. coli isolates from adults showed greater genetic diversity than from
young children and constitutes a substantial reservoir of the virulence genes typical for extraintestinal
pathogenic E. coli.

Keywords: commensal Escherichia coli; adults; young children; virulence genes; phylogenetic grouping;
antimicrobial resistance

1. Introduction
Escherichia coli bacteria is one of the most explored model organisms, partially due to its versatile
nature. These bacteria constitute a component of the natural microbiota of warm-blooded animals
including humans [1]. Some strains of the species are pathogens associated with intestinal or extraintestinal
diseases in some cases leading to morbidity and mortality [2–5]. The vast diversity of this species results
from the high recombination frequency and acquisition or loss of genetic information by horizontal gene
transfer [1]. Such great plasticity of the genome speeds up the adaptation to new niches [6]. The origin of
pathogenicity of E. coli remains unclear and it is unknown whether transition will go from commensal to

Int. J. Environ. Res. Public Health 2018, 15, 617; doi:10.3390/ijerph15040617 www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2018, 15, 617 2 of 19

pathogen or the other way around. Intestinal pathogenic E. coli strains (IPEC) are obligatory pathogens
and can be easily distinguished from commensal or extraintestinal pathogenic E. coli (ExPEC) based on the
presence of the selected virulence genes and phenotypic traits. ExPEC strains are facultative pathogens.
They are part of healthy intestinal microflora, however they may become pathogenic in immune-deficient
patients or during adaptation to other niches [3,7–9]. Various environmental factors may influence the
emergence of virulence genes and antimicrobial resistance among commensal E. coli strains, and such
strains constitute a reservoir of virulence and antimicrobial resistance [10–12].
E. coli strains colonize the infant’s intestine within a few hours after birth, likely deriving from
the mother’s fecal microflora but also from maternity ward staff contact [13–15]. It was reported
that each individual carries a single predominant strain. It is well known that the extraintestinal
pathogenic strains that cause urinary tract infection, neonatal septicemia or meningitis belong mainly
to phylogenetic group B2 and, to a lesser extent, group D [16,17]. Moreover, the strains of these
phylogroups harbor more virulence factors than the strains of the A and B1 groups [17,18].
E. coli strains inhabiting the intestine of an individual can be divided into two groups: the resident
strains that are present for months or years and the transient strains that appear and then disappear
within a few weeks [19]. The resident E. coli strains, persisting in the intestinal microflora, more
frequently carry the virulence genes encoding P fimbriae, K1 and K5 capsules, hemolysin, and other
compounds responsible for iron acquisition such as aerobactin, compared with colonic transient strains
in the same hosts [15,20]. These virulence genes are considered as encoding fitness factors to maintain
E. coli in the human intestinal tract, not always associated with virulence [21].
The use of antibiotics in medicine and in the environment has caused intestinal microorganisms
to become a reservoir of antibiotic resistance factors. Many recent studies revealed high, or increasing,
incidence of antibiotic resistance in commensal E. coli from healthy children and adults coming from
many countries [12,22,23]. Naturally, without the pressure of antibiotics in the gut, susceptible E. coli
strains should have an advantage over the resistant ones. However it has been reported that the
resistant E. coli are able to persist among intestinal microflora as well as susceptible strains [24].
Previous studies have reported the characteristics of commensal E. coli strains derived from
adults [25,26], schoolgirls aged 7–16 [20,21] and from infants [15,24,27], whereas there is limited data in
the literature regarding E. coli derived from young children aged 0.5–3. Moreover, it was also reported
that the diversity and composition of microorganisms found in the intestine changes dramatically
between birth and the age of 3 years [28]. It is important to ask if the pattern with dominance of
phylogroup B2 in E. coli from infants, will still persists among young children. The age of 0.5–3 years is
an interesting time period because the diet of young children is rather simple and consists of organic,
less processed food as compared to adult diet. Besides, young children aged 0.5–3 are very active
and interested in the surrounding world but not interested in hygiene practices, therefore they can
acquire many E. coli strains from the environment. This is the first report on the comparison of genetic
diversity of commensal E. coli isolates from two age group—adults and young children.
The aim of the present study was to characterize and compare commensal E. coli isolates derived
from healthy adults and young children. The investigation encompassed genotypic identification of the
virulence genes typically found in intestinal and extraintestinal pathogens, and the determination of
the main phylogenetic structure of E. coli. Additionally, the prevalence of the antimicrobial resistance
and resistance genes were determined. This study give the better insight into understanding of the
relations between virulence genes, phylogenetic structure and the resistance genes of E. coli from adults
and young children.

2. Materials and Methods

2.1. Sample Collection and E. coli Identification

A total of 382 fecal samples were collected from two groups of healthy humans living in Lubuskie
Province (Western Poland). The first group consisted of 296 unrelated adult volunteers (from separate
Int. J. Environ. Res. Public Health 2018, 15, 617 3 of 19

households) of both sexes (234 females and 62 males) aged 18–56 years (Figure S1), recruited mainly
from students and university staff. The second group consisted of 86 unrelated young children (aged
between 6 months and 3 years) of university staff and students attending the nursery. Freshly voided
fecal samples were collected at home by the participants or by the participants’ parents, in sterile
polystyrene containers. Samples from young children were collected in 2009–2010 and samples from
adults were collected in 2012–2015. The fecal sampling procedure followed laboratory standards
used in sampling for inoculation. The procedure ensured proper hygiene and posed no threat to
volunteers. The volunteers themselves transported the samples to the laboratory. The samples were
refrigerated upon arrival at the laboratory and processed the same day. After processing, samples were
properly discarded. Every volunteer was adequately informed of the research aims. This study was
approved by the bioethics committee at the District Medical Council in Zielona Góra (No. 03/85/2018).
The volunteers completed a short questionnaire concerning: health, age, sex, diet and antibiotics usage
within the period prior to sampling. The adults and young children used a regular diet appropriate to
their age. The volunteers had not been treated with any antimicrobial agents within the three-month
period prior to sampling. The stool samples were streaked on MacConkey agar and incubated at
37 ◦ C for 24 h. The bacterial colonies showing a typical E. coli morphology were randomly selected
and identified by biochemical IMVC tests (indole production, methyl red reaction, Voges-Proskauer
test and citrate utilization). A single isolate representing each sample was randomly picked for
further analysis. A single isolate was selected because it has been reported that individuals usually
carry a predominant E. coli strain that constitutes more than half of the colonies isolated from stool
sample [1,29]. It was also reported that the number of isolates sampled per individual (1, 5, or 10)
did not affect the general pattern of phylogenetic distribution among the analysed populations [25].
Our pilot studies (data not shown) also confirm that most of the strains isolated from one stool samples
were identical. All 382 E. coli isolates were stored frozen as a glycerol stock, at −80 ◦ C, until the
investigation. The DNA extraction was carried out using the thermal cell lysis method; 1.5–3 µL of the
boiled bacterial supernatant was used as a template in all the PCR reactions.

2.2. Virulence Genotyping

The isolates were examined for the presence of 30 different intestinal and extraintestinal virulence
associated genes, representing five functional categories presented in Table S1: List of virulence genes
tested in this study. Multiplex PCR-based genotyping was performed with primers and conditions
previously described [16,30–36]. The PCR amplification mixture in a volume of 25 µL contained:
buffer solution (Thermo Scientific, Waltham, MA, USA); 2.5 mM MgCl2 (Promega, Madison, WI, USA);
0.5 mM of each dNTP (Promega); 0.2 µM of each primer (IDT, Coralville, IA, USA); 1 U of DyNAzyme II
polymerase (Thermo Scientific) and 3 µL of DNA template. All PCR reactions included a negative
control containing no DNA template and a positive control, containing DNA template from an E. coli
isolates known to carry the identified gene (validated by sequencing). The PCR products were separated
in 1.5% or 2% agarose gel electrophoresis and stained with ethidium bromide.

2.3. Phylogenetic Typing

The E. coli isolates were assigned to four major phylogenetic groups—A, B1, B2 or D based on the
triplex PCR amplification method described by Clermont et al. [37].

2.4. Antimicrobial Susceptibility Testing

All the E. coli isolates were examined for their antimicrobial susceptibilities to 17 antimicrobial
agents using the disc-diffusion method on Mueller Hinton agar (Merck, Darmstadt, Germany). The tests
were performed following the European Committee on Antimicrobial Susceptibility Testing (EUCAST)
standard methods [38]. The growth inhibition zone diameters were measured and interpreted
according to the recommendations of the EUCAST [39] (for most antimicrobials) and the Clinical
and Laboratory Standards Institute (CLSI) [40] (for cephalothin, cefotaxime, streptomycin, tetracycline,
Int. J. Environ. Res. Public Health 2018, 15, 617 4 of 19

doxycycline, nalidixic acid and nitrofurantoin). The following antimicrobial agents were tested in
this study: ampicillin (AMP, 10 µg), amoxicillin-clavulanic acid (AMC, 30 µg), piperacillin (PIP, 30
µg), cephalothin (CF, 30 µg), cefuroxime (CXM, 30 µg), cefotaxime (CTX, 30 µg), streptomycin (S,
10 µg), gentamicin (GM, 10 µg), amikacin (AN, 30 µg), tetracycline (TE, 30 µg), doxycycline (D, 30 µg),
trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 µg), chloramphenicol (C, 30 µg), nalidixic acid (NA,
30 µg), norfloxacin (NOR, 10 µg), ciprofloxacin (CIP, 5 µg), and nitrofurantoin (FM, 300 µg). The E. coli
strains ATCC 25922 and ATCC 35218—a β-lactamase-producing strain were used as a negative and
positive quality controls, respectively. The susceptible or intermediately susceptible isolates were
considered to be susceptible. Multi-drug resistance (MDR) was defined as the non-susceptible profile to
≥1 agent in ≥3 antimicrobial categories. The antibiotic disks were purchased from Becton Dickinson
(Franklin Lakes, NJ, USA).

2.5. Identification of Resistance Genes

The E. coli isolates that showed resistance to ampicillin, cefotaxime, streptomycin, tetracycline,
trimethoprim/sulfamethoxazole and nalidixic acid were screened for the presence of genes encoding
resistance to these antimicrobials. The following genes associated with resistance to beta lactams
(blaTEM , blaSHV , blaCTX-M ), streptomycin (strA/strB, aadA1), tetracycline (tetA, tetB, tetC), trimethoprim
(dfrA1, dfrA7), sulfonamides (sul1, sul2, sul3), or quinolones (qnrA, qnrB, qnrS) were identified by
PCR. The PCR reactions were carried out using previously published primers and conditions [41–45].
The PCR amplification mixture was prepared in a volume 25 µL as described above.

2.6. Statistical Methods

The presence of the virulence genes and antimicrobial resistance were categorized as 1 = present
and 0 = absent. In order to determine whether there was a significant association between the prevalence
of the virulence genes (as well as the antimicrobial agent) and the host (adults vs. young children)
Pearson’s chi-squared test for independence or Fisher’s exact test were used. The Fisher’s exact test
was used if more than 20% of the cells in the contingency table have expected frequencies below five.
The evaluations of the frequency of the virulence genes among E. coli isolates from young children
and adults within each phylogenetic group were tested using the chi-squared test for proportions or
Fisher’s exact test for proportions, as appropriate. The null hypothesis assumes that the proportions in
isolates from adults and young children are equal. The alternative hypothesis is one-sided and assumes
that the proportion in one group (adults or young children) was lower or higher than in the other, as
appropriate. For all the statistical tests the level of statistical significance was defined as 0.05.
The relations between the presence of a virulence gene and the age of individual persons (adults)
were described using univariate logistic regression models. Given each model, the odds ratios (OR) and
their 95% confidence intervals for the population were estimated. This relation is deemed statistically
significant at the 0.05 level, if the confidence interval does not contain the value of 1.
In order to measure the strength of the associations for the cross tabulation of virulence genes
and virulence genes with antimicrobial resistance of the E. coli isolates from both adults and young
children, the Goodman and Kruskal tau coefficient was calculated.
Multiple correspondence analyses (MCA) were conducted to determine the overall relationship
between the group of the host (adults, young children) and the characteristic of the isolates
(the phylogenetic group and different profiles of the iron acquisition or protectins genes). The MCA
were performed on the Burt table using the host’s age as the supplementary categorical variable.
The statistical analyses were performed using the program R (R Core Team, R Foundation for Statistical
Computing, Vienna, Austria).
Int. J. Environ. Res. Public Health 2018, 15, 617 5 of 19

3. Results

3.1. Frequency of Virulence Associated Genes

Two virulence determinants typical for IPEC pathotypes escV (EPEC, EHEC) and east1 (EAEC),
and 15 typical for ExPEC—fimH, papA, sfaS, fyuA, iutA, iroN, ireA, kpsMT II, kpsMT III, ompT, traT, iss,
cnf1, hlyA, agn43—were identified among the analyzed isolates. The genes characteristic for the IPEC
pathotypes—bfpB (EPEC), ehxA (EPEC, EHEC), stx1, stx2 (EHEC), eltA, estI, estII (ETEC)—were not
found in any of the tested isolates. Table 1 presents the distribution of the virulence genes in two groups
of isolates, derived from adults and young children. Three of the detected virulence genes—sfaS,
escV, kpsMT III—occurred only among the E. coli isolates from adults. The most common detected
genes among tested functional category for the isolates from adults were: fimH (96.6%)—adhesins;
fyuA (76%)—iron acquisition; kpsMT II (67.9%)—protectins, with the most frequent variant K2 (64.9%);
and cnf1 (21.6%)—toxins. In the biofilm formation category allele K12 of the agn43 gene showed the
highest frequency of 59.5%. For the isolates from young children the following genes prevailed: fimH
(88.4%)—adhesins; fyuA (41.9%)—iron acquisition; traT (43%)—protectins; and east1 (25.6%)—toxins.

Table 1. Prevalence of virulence genes by functional categories among E. coli isolates from healthy
adults and young children.

Number (%) of E. coli Isolates with Virulence Genes

Functional Category Test of Independence
E. coli Pathotype Adults Young Children
Virulence Gene p-Value
n = 296 n = 86
Adhesins
fimH ExPEC 286 (96.6) 76 (88.4) 0.005 *
papA ExPEC 54 (18.2) 10 (11.6) 0.1482
sfaS ExPEC 4 (1.4) 0 0.5787
escV EPEC, EHEC 1 (0.3) 0 1
bfpB EPEC 0 0 -
Iron acquisition
fyuA ExPEC 225 (76) 36 (41.9) <0.0001 *
iutA ExPEC 184 (62.2) 17 (19.8) <0.0001 *
iroN ExPEC 110 (37.2) 4 (4.7) <0.0001 *
ireA ExPEC 87 (29.4) 6 (7) <0.0001 *
Protectins
kpsMT II ExPEC 201 (67.9) 16 (18.6) <0.0001 *
-K1 ExPEC 158 (53.4) 10 (11.6) <0.0001 *
-K2 ExPEC 192 (64.9) 15 (17.4) <0.0001 *
-K5 ExPEC 125 (42.2) 10 (11.6) <0.0001 *
kpsMT III ExPEC 18 (6.1) 0 0.02 *
ompT ExPEC 62 (21) 6 (7) 0.003 *
traT ExPEC 190 (64.2) 37 (43) 0.0004 *
iss ExPEC 70 (23.6) 6 (7) 0.0007 *
Toxins
cnf1 ExPEC, NTEC 64 (21.6) 6 (7) 0.002 *
hlyA ExPEC 50 (16.2) 4 (2.3) 0.004 *
east1 EAEC 28 (9.5) 23 (25.6) <0.0001 *
ehxA EPEC, EHEC 0 0 -
stx1 EHEC 0 0 -
stx2 EHEC 0 0 -
eltA ETEC 0 0 -
estI ETEC 0 0 -
estII ETEC 0 0 -
Biofilm formation
agn43 ExPEC 240 (81.1) 58 (67.4) 0.007 *
-a ExPEC 106 (35.8) 12 (14) 0.0001 *
-b ExPEC 73 (24.7) 4 (4.7) <0.0001 *
-K12 ExPEC 176 (59.5) 53 (61.6) 0.7179
* Statistically significant.
Int. J. Environ. Res. Public Health 2018, 15, 617 6 of 19

The most frequent variant in the group of agn43 genes was K12, involving 61.6% of young
children’s isolates. The comparison of the frequency of the isolates positive for particular virulence
genes between the two tested groups showed statistically significant differences (p < 0.02) for most
of the analyzed genes. The E. coli isolates derived from adults carried the virulence genes more
frequently than isolates from young children, with the exception of east1 and agn43 K12 genes. There are
no statistically significant differences between the two tested groups of E. coli for the papA, sfaS, escV
and agn43 K12 genes (Table 1).

3.2. Distribution of Virulence Genes among E. coli Isolates According to the Age of Adults
An interesting question was whether there were any associations between the age of individuals
and prevalence of virulence genes. The age range in the group of adults was high (from 18 to 56 years).
Figure 1 presents the influence of adults’ age on the distribution of virulence genes in the E. coli isolates.
The age of adults had a statistically significant effect on the prevalence of some genes (Figure 1).
The chance of the presence of ireA, variants K1 and K5 of the kpsMT II, cnf1 and hlyA genes increased
with age and was 2.8% (95% CI: 1.004–1.052), 2.9% (95% CI: 1.005–1.054), 2.7% (95% CI: 1.004–1.05),
5% (95% CI: 1.024–1.076) and 5.6% (95% CI: 1.029–1.084) respectively, while for the traT gene the
chance decreased with age and reached 5% (95% CI: 0.931–0.976). There was no statistically significant
influence of the age of young children (the age ranged from 0.5 to 3 years) on the prevalence of
virulence genes (data not shown).

Figure 1. Odds ratios (the dots) and their 95% confidence intervals for the association of the occurrence
of particular virulence genes in the E. coli isolates with the age of adults (18–56 years). The relations
between the virulence gene and age of adults were described by logistic models. If the 95% CI is above
the value of 1, this means that the ratio of the odds of the virulence gene’s occurrence increases with
age; otherwise this ratio decreases with age. A narrow confidence interval indicates higher precision of
the odds ratio.

3.3. Association between Virulence Genes

The statistical analysis of the association between the virulence genes of the E. coli isolates is shown
in Figure 2. A strong association occurred between the genes cnf1 and hlyA, (association coefficient of
Int. J. Environ. Res. Public Health 2018, 15, 617 7 of 19

0.74), among the isolates from adults (Figure 2, the part above the diagonal). In the group of isolates
from young children, very strong positive associations were found between the gene ireA and both
genes iss and ompT, with association coefficients of 1. Strong associations were observed between the
gene iroN and genes iss, ompT, ireA, with a coefficient of 0.65. The genes iutA and kpsMT II were also
positively associated, with a coefficient of 0.54. There were moderate association between the gene
kpsMT II and genes cnf1, fyuA, papA, with association coefficients of 0.33, 0.32 and 0.33, respectively,
in the isolates from young children (Figure 2, the part under the diagonal). There is good evidence of
a strong association between the genes ompT and iss among E. coli isolates from both adults and from
young children, with coefficients of 0.86 and 1 respectively. Weak association, with coefficients ≤0.3,
in the isolates from both young children and adults occurred between the remaining virulence genes.

Figure 2. Statistical association between virulence genes of the E. coli isolates derived from adults (the
part above the diagonal) and young children (the part under the diagonal). No values were introduced
in the case of undetected genes.

3.4. Phylogenetic Structure of Commensal E. coli

The E. coli isolates derived from adults and young children differed significantly in their
assignments to the phylogenetic groups (p < 0.0001) (Figure 3). The phylogroups B2 and D significantly
prevailed among the isolates from adults compared to young children, p < 0.0001 and p = 0.0005,
respectively. Conversely, phylogenetic group A was significantly more frequent in the isolates from
young children as compared to adults (p < 0.0001).
Int. J. Environ. Res. Public Health 2018, 15, 617 8 of 19

Figure 3. Phylogenetic structure of the E. coli isolates derived from two groups: adults and young children.

3.5. Distribution of Virulence Genes in Relation to Phylogenetic Groups

Figure 4 shows that the majority of the tested virulence genes were not randomly distributed
among the four main phylogenetic groups of isolates. Generally, among the isolates derived from both
adults and young children the frequencies of the particular genes were significantly lower (p < 0.05)
in the isolates of group A, whereas the isolates of groups B2 harbored the virulence determinants
significantly more frequently (p < 0.05).

Figure 4. The frequency distribution of virulence genes among E. coli phylogroups was compared by
Fisher’s exact test to its prevalence in a group consisted of the isolates of all the other phylogenetic
groups. The values significantly higher than among the other groups are indicated as follows:

p < 0.001, p < 0.01, p < 0.05. The values significantly lower than among the other groups

are indicated as follows: p < 0.001, p < 0.01, p < 0.05.

Int. J. Environ. Res. Public Health 2018, 15, 617 9 of 19

The further analysis encompassed comparing the prevalence of different virulence genes between
the isolates from adults and young children within each phylogenetic group. The results indicated that
group A and B2 isolates and to a lesser extent group B1 from adults carried frequently virulence factors
(p < 0.05) than the isolates from young children. The reverse situation was characteristic for the isolates
of group D; several virulence genes were detected less frequently (p < 0.05) in the isolates from adults
than from young children (Table 2). The fyuA, iutA, ireA, kpsMT II, ompT, traT and iss genes occurred
more frequently in the isolates of group A from adults than from young children, while the east1 gene
was more frequent detected among the isolates from young children. The three genes cnf1, hlyA and
agn43 occurred more frequently in the isolates of group B1 from adults. The genes for iroN, ireA, ompT,
iss and hlyA were found more frequently among the isolates of group B2 from adults than from young
children; the one exception was the papA gene detected more often in the E. coli from young children.
The papA, iroN, ompT and iss genes occurred less frequently in the isolates of group D from adults than
from young children (Table 2).

3.6. Multiple Correspondence Analysis

The virulence genes within two categories—iron acquisition and protectins—were particularly
prevalent among the tested E. coli isolates. To determine the overall relationships between two groups of
the host (adults, young children) and the characteristics of the isolates, multiple correspondence analysis
(MCA) was performed. The first analysis concerned the iron acquisition category. The projection of
the variables on the dimension1–dimension2 plane accounted for 27.47% of the total variance and
distinguished two major associations (Figure 5). The first association was positioned on the negative
values of the first dimension and the positive and negative values of the second dimension and
encompassed the isolates from adults, phylogenetic groups B1 and B2 variables and five complex
iron acquisition profiles, consisting of two, three or four genes. The isolates from young children,
the phylogenetic group A variables and three simple iron acquisition profiles consisting of one or
two genes formed the second association and were located on the positive values of both dimensions.
This analysis clearly distinguished adults, phylogroups B1 and B2 and the complex iron acquisition
profile variables on the negative values of the first dimension from the young children, phylogroups A
and D and the simple iron acquisition profile variables on the positive values of the first dimension.
The second analysis concerned the protectin category. The projection of the variables on the
dimension1–dimension2 plane accounted for 25.98% of the total variance (Figure 6). The first association
encompassed the isolates from adults, phylogroup B2 and D variables and eight protectin profiles
consisting of one to five genes, located on the negative values of the first dimension. The second association
was formed by the isolates from young children, phylogroup A and B1 variables and five profiles
consisting of one to three protectin genes, positioned on the positive values of the first dimension.
The most complex protectin profiles were located close to phylogroup B2 and the isolates from adults’
variables. The analysis clearly distinguished adults, phylogroups B2 and D and the complex protectin
profile variables from the young children, phylogroups A and B1 and the simple protectin profile variables.
Int. J. Environ. Res. Public Health 2018, 15, 617 10 of 19

Table 2. Comparison of the frequency of virulence genes among the E. coli isolates from adults and young children within each phylogenetic group using Pearson’s
chi-squared test or Fisher’s exact test for proportions as appropriate.

Number (%) of E. coli Isolates with Virulence Genes within Phylogenetic Groups
A B1 B2 D
Functional Category Virulence Genes
Adults Young Children Adults Young Children Adults Young Children Adults Young Children
n = 68 n = 60 n = 26 n = 10 n = 138 n = 12 n = 64 n=4
fimH 62 (91.2) 50 (83.3) 26 (100) 10 (100) 134 (97.1) 12 (100) 64 (100) 4 (100)
papA 0 0 4 (15.4) 0 32 (23.2) 6 (50) * 18 (28.1) 4 (100) ***
Adhesins
sfaS 0 0 0 0 4 (2.9) 0 0 0
escV 0 0 1 (0.3) 0 0 0 0 0
fyuA 32 (47.1) ** 12 (20) 20 (76.9) 10 (100) 131 (94.9) 10 (83.3) 42 (65.6) 4 (100)
iutA 30 (44.1) *** 2 (3.3) 14 (53.8) 4 (40) 96 (69.6) 7 (58.3) 44 (68.8) 4 (100)
Iron acquisition
iroN 4 (5.9) 0 14 (53.8) 2 (20) 88 (63.8) *** 0 4 (6.2) 2 (50) *
ireA 24 (35.3) *** 0 6 (23.1) 4 (40) 40 (29) * 0 17 (26.6) 2 (50)
kpsMT II 22 (32.4) *** 0 14 (53.8) 4 (40) 117 (84.8) 10 (83.3) 48 (75) 2 (50)
kpsMT III 2 (2.9) 0 2 (7.7) 0 12 (8.7) 0 2 (3.1) 0
Protectins ompT 8 (11.8) ** 0 4 (15.4) 4 (40) 46 (33.3) ** 0 4 (6.2) 2 (50) *
traT 34 (50) * 18 (30) 10 (38.5) 4 (40) 98 (71) 11 (91.7) 48 (75) 4 (100)
iss 12 (17.6) *** 0 4 (15.4) 4 (40) 50 (36.2) ** 0 4 (6.2) 2 (50) *
cnf1 2 (2.9) 0 10 (38.5) * 0 50 (36.2) 6 (50) 2 (3.1) 0
Toxins hlyA 2 (2.9) 4 (6.7) 10 (38.5) * 0 38 (27.5) * 0 0 0
east1 8 (11.8) 21 (35) ** 2 (7.7) 0 6 (4.3) 2 (16.7) 12 (18.8) 0
Biofilm formation agn43 36 (52.9) 40 (66.7) 20 (76.9) * 4 (40) 124 (89.9) 10 (83.3) 60 (93.8) 4 (100)
p-value: *** 0–0.001, ** 0.001–0.01, * 0.01–0.05.
Int. J. Environ. Res. Public Health 2018, 15, 617 11 of 19

Figure 5. Multiple correspondence analysis (MCA) of the characteristics of the isolates (the phylogenetic
group and different profiles of the iron acquisition genes) regarding two groups of the host (adults,
young children) among 382 E. coli isolates from humans. The projection of the variables on the
dimension1–dimension2 plane: the age of the host (adults, young children) is denoted by triangles,
the phylogenetic group (A, B1, B2, D) by squares, the profiles (combinations of the iron acquisition
genes fyuA, iutA, iroN, ireA) by circles. The number of isolates positive for a particular profile is
distinguished by color.

Figure 6. Multiple correspondence analysis (MCA) of the characteristics of the isolates (the phylogenetic
group and different profiles of the protectin genes) regarding two groups of the host (adults,
young children) among 382 E. coli isolates from humans. The projection of the variables on the
dimension1–dimension2 plane: the age of the host (adults, young children) is denoted by triangles,
the phylogenetic group (A, B1, B2, D) by squares, the profiles (combinations of the protectin genes:
kpsMT II kpsMT III, ompT, traT, iss) by circles. The number of isolates positive for a particular profile is
distinguished by color.
Int. J. Environ. Res. Public Health 2018, 15, 617 12 of 19

3.7. Prevalence of Antimicrobial Resistance

Resistance rates to 17 antimicrobial agents were compared between the E. coli isolates from adults
and young children and presented in Table 3. The most frequent resistance was detected for ampicillin
(38.2% vs. 31.4%), cephalothin (29.1% vs. 27.9%), streptomycin (46.3% vs. 22.1%) and tetracycline
(21.3% vs. 19.8%) in the isolates from adults and young children respectively. The resistance rates for
norfloxacin and ciprofloxacin (2.7% vs. 2.3%) and cefotaxime (1.4% vs. 2.7%) were the lowest among
the isolates from adults and young children respectively. Significantly higher frequency (p < 0.01)
in resistance to amoxicillin/clavulanic acid, piperacillin and streptomycin were observed in isolates
from adults as compared with young children. Moreover, resistance to at least one antimicrobial agent
(75.7% vs. 54.7%) and multidrug-resistance (30.4% vs. 14%) were detected significantly more frequent
(p = 0.0002 and p = 0.0024 respectively) in the isolates from adults than from young children (Table 3).

Table 3. Prevalence of resistance among the E. coli isolates from groups of adults and young children to
17 antimicrobials.

Number (%) of E. coli Isolates

Test of Independence
Antimicrobial Agent Adults Young Children
p-Value *
n = 296 n = 86
Ampicillin 113 (38.2) 27 (31.4) 0.2506
Amoxicillin/Clavulanic acid 40 (13.5) 3 (3.5) 0.0096 *
Piperacillin 72 (24.3) 5 (5.8) 0.0002 *
Cephalothin 86 (29.1) 24 (27.9) 0.8362
Cefuroxime 20 (6.8) 3 (3.5) 0.3152
Cefotaxime 4 (1.4) 2 (2.7) 0.6206
Streptomycin 137 (46.3) 19 (22.1) <0.0001 *
Gentamicin 32 (10.8) 9 (10.5) 0.9273
Amikacin 30 (10.1) 7 (8.1) 0.5818
Tetracycline 63 (21.3) 17 (19.8) 0.7610
Doxycycline 46 (15.5) 11 (12.8) 0.5287
Trimethoprim/sulfamethoxazole 33 (11.1) 12 (14) 0.4776
Chloramphenicol 18 (6.1) 0 0.0171
Nalidixic acid 39 (13.2) 8 (9.3) 0.3358
Norfloxacin 8 (2.7) 2 (2.3) 1
Ciprofloxacin 8 (2.7) 2 (2.3) 1
Nitrofurantoin 12 (4.1) 3 (3.5) 1
Antimicrobial susceptibility characteristic
R 224 (75.7) 47 (54.7) 0.0002 *
MDR 90 (30.4) 12 (14) 0.0024 *
R—resistant to at least one agent; MDR—multidrug-resistant. * Statistically significant.

3.8. Detection of Resistance Determinants

The isolates were screened according to their resistance phenotypes for the carriage of a set of
commonly encountered resistance genes. Genetic determinants associated with resistance to ampicillin,
cefotaxime, streptomycin, tetracycline, sulfamethoxazole, trimethoprim and nalidixic acid were tested
in this study. There were no statistically significant differences between the frequencies of the resistance
determinants among the resistant E. coli isolates from adults and young children (Table S2). The most
frequently observed gene encoding resistance to ampicillin was blaTEM with 47.8% vs. 48.1% for adults’
and young children’s isolates, respectively. The gene encoding the extended spectrum β-lactamase
blaCTX-M was detected in all the isolates resistant to cefotaxime. Regarding streptomycin resistant
E. coli the aadA1 gene occurred most often (8.8% vs. 21.1%, in the isolates from adults and young
children, respectively). The predominant gene among the tetracycline resistant isolates was tetB with
the frequencies 50.8% vs. 41.2% for adults’ and young children’s isolates, respectively. The genes dfrA7
(54.5% vs. 33.3%) and sul2 (60.6% vs. 25%) prevailed among the trimethoprim/sulfamethoxazole
resistant isolates from adults and young children, respectively. The sul3 gene was not detected in
Int. J. Environ. Res. Public Health 2018, 15, 617 13 of 19

any isolates. The qnrS gene was predominant among the E. coli resistant to nalidixic acid (17.9% vs.
25%, in the isolates from adults and young children, respectively). The qnrA gene was not detected in
any isolates.

3.9. Correlations between Virulence Genes and Antimicrobial Resistance

The results revealed that the associations between the virulence genes and antimicrobial resistance
were weak (association coefficients < 0.2) for E. coli isolates from adults and young children.

4. Discussion
To the best of our knowledge, this is the first study to compare the genetic diversity of commensal
E. coli isolates collected from two different age groups (adults and young children). E. coli isolates
from fecal samples were characterized in terms of the presence of virulence genes, phylogenetic
structure, and antimicrobial resistance. Our results indicate that E. coli isolated from adults differ in
their phylogenetic structure, harbour a greater variety of virulence genes, and multidrug-resistance
phenotypes, compared to E. coli from young children.
Our results have shown that commensal E. coli isolates from healthy humans constitute
a substantial reservoir of genes related to the extraintestinal pathotypes. All of the 15 tested virulence
genes typical for ExPEC were detected and, what is important, the prevalence of these genes was
significantly higher among the isolates from adults than from young children. In contrast, only two
of the nine tested virulence genes associated with the intestinal pathotypes were found: escV and
east1. The escV gene was identified only in one isolate from adults, while the east1 gene was more
common and was detected more frequent among the E. coli isolates from young children than adults.
The previous studies also reported that the presence of the intestinal virulence markers actually was
associated with pathogenesis [2,3,46,47]. The extraintestinal virulence genes such as those encoding
the fimbrial adhesins, iron acquisition systems, protectins or toxins occur not only in pathogens,
but also in commensal microflora of healthy people [3,9,21,47,48]. Previous reports indicated that
virulence genes associated with extraintestinal pathogenesis in fact help the E. coli strains to colonize
the human gut; therefore they may be considered as a fitness factor and the virulence is a coincidental
side effect [8,21,48].
The question arises whether the adult’s age has also an influence on the prevalence of virulence
genes. The analysis revealed that there are no statistically significant differences in the distribution
of virulence genes in the E. coli isolates in relation to the age of the adult host. The only exception
was some genes from the categories of iron acquisition (ireA), protectins (variants K1 and K5 of the
kpsMT II) and toxins (cnf1, hlyA), which tend to accumulate in E. coli over the life time of the host.
The opposite situation occurred only for the traT gene; the chance of accumulation decreases with
age. Overall, the results suggest that the difference in prevalence of virulence genes is not correlated
with age of adults, however, there is difference in prevalence of virulence genes between adults and
young children.
The phylogenetic analysis revealed that the distribution of four main E. coli phylogroups differ
significantly (p < 0.0001) between the isolates derived from adults and from young children. E. coli
assigned to phylogroup B2 dominated among adults, whereas phylogenetic group A isolates prevailed
among young children. The distribution of the phylogroups among the human population represents
two patterns, the first with predominance of group A typical for European population (in 1980s), Africa,
South America and Asia [1,25,26,49], and the second pattern that the isolates of group B2 prevailed
is characteristic for Europe in the 2000s, North America, Japan and Australia [1,25]. The changes in
the phylogenetic structure of the E. coli isolates from Europe during the period of 20 years may be the
result of the increasing of food processing and change in dietary habits [1,25]. The results of the present
study with the dominance of phylogroup B2 among E. coli isolates from adults are consistent with the
previous reports presenting the second pattern of phylogenetic structure, whereas the predominance
of isolates of phylogroup A among young children is in accordance with the first pattern [1,25,26,49].
Int. J. Environ. Res. Public Health 2018, 15, 617 14 of 19

The diet of young children aged 0.5–3 years is rather simple and in the most part consists of organic,
less processed food as compared to adult diet. The host diet may be a key factor which had an influence
on the phylogenetic structure of the E. coli isolates.
The analysis of the distribution of the virulence genes in relation to the phylogenetic groups
revealed that the E. coli group B2 isolates were an extensive reservoir of virulence genes while group A
was not.
Our results suggest that the higher frequencies of the virulence genes in the E. coli isolates from
adults is due to predomination of phylogroup B2, therefore the comparison between the E. coli isolates
from adults and young children with respect to the frequency of the virulence genes in each phylogroup
was performed. The results showed that the E. coli isolates from adults assigned to either phylogroup
A or B2 carried statistically significant (p < 0.05) more virulence determinants, particularly associated
with iron acquisition and protection, than the isolates from young children assigned to phylogroup
A and B2. Three genes from the toxin and biofilm formation categories occurred more frequently
among phylogroup B1 isolates from adults than from young children. Our results documented that
the genetic diversity of the isolates of phylogroups A, B2 and to a lesser extent B1 differs between
adults and young children. The isolates may acquire genetic diversity through horizontal gene transfer.
Possibly the resident strains that persist longer time in the intestinal tract have a greater chance to
undergo numerous horizontal transfer events resulting in accumulation of the virulence determinants.
Our studies may suggest that the strains of phylogroup D from adults are less diverse regarding the
virulent determinants than E. coli strains from young children, but these results should be confirmed
on a larger number of subjects.
Virulence genes are often clustered in the chromosomal genomic regions acquired by horizontal
gene transfer called pathogenicity islands [10,50], or in large plasmidic regions [51,52]. One such
region typical for the ExPEC plasmids has been recently described and called the conserved virulence
plasmidic (CVP) region. This region harbors eight virulence genes, including iroN, ompT and iss [52,53].
The results regarding the associations between the virulence genes revealed that there were fewer
statistically significant associations among the E. coli from adults than from young children. Strong
associations were found between the iroN, ompT and iss genes among the isolates from young children.
In the E. coli isolates from adults there remained only associations between the ompT and iss genes.
The prevalence of these genes was much higher among the E. coli isolates from adults than among
the isolates from young children. These results suggested that there was a tendency for accumulation
of these genes in different combinations in the isolates from adults. This observation indicated that
in commensal E. coli strains from adults there is low probability of presence of a specific pathogenic
plasmidic CVP region. Two genes from the toxin category—cnf1 and hlyA—were strongly associated
among the isolates from adults. What it was previously reported, these genes may be concealed
together on pathogenicity island PAI IIJ96 in pathogenic strains [10]. The existence of PAI IIJ96 in our
set of E. coli isolates from adults will be confirmed in near future.
Previous studies revealed that the ability of some E. coli strains to persist in the colonic microbiota
may be related to the carriage of the virulence genes for P fimbriae, aerobactin (iutA), yersiniabactin
(fyuA) and capsular antigen (kpsMT II variants K1 and K5) and that these genes may play a key role
as fitness factors [15,20,21,48]. In our studies the results of MCA analyses showed that the isolates
from adults correspond primarily to phylogroup B2 and these variables were positioned close to the
isolates positive for the more complex and also more numerous profiles both the iron acquisition and
protectin categories. The E. coli isolates from young children correspond strongly to phylogroup A
and to the isolates with simpler profiles. These results confirmed that the E. coli isolates derived from
adults exhibited more genetic diversity than isolates from young children. There were isolates carrying
three, four and even five genes of the same functional category. This indicated that the E. coli isolates
derived from adults, particularly of phylogroup B2, constitute a significant reservoir of extraintestinal
virulence genes and have a tendency to collect them.
Int. J. Environ. Res. Public Health 2018, 15, 617 15 of 19

The comparison of the resistance rates to 17 antimicrobial agents revealed that the E. coli isolates
showed significantly higher (p < 0.01) resistance to three antibiotics: amoxicillin/clavulanic acid,
piperacillin and streptomycin in adults as compared to young children. Likewise, significantly more
isolates from the adults than from the young children were classified as strains resistant to at least one
antimicrobial agent (p = 0.0002) and to multidrug-resistant strains (p = 0.0024). These results suggested
that the older the host, the higher accumulation of resistance determinants in E. coli strains occurs but
to a lesser degree as in the case of the virulence genes.
Our results indicated that the prevalence of resistance to ampicillin, cephalothin, streptomycin
and tetracycline among the E. coli isolates from both adults and young children was rather moderate
in the region of Western Poland, prevalence of resistance to trimethoprim/sulfamethoxazole was
low/moderate, whereas the rates of resistance to the third-generation cephalosporin cefotaxime
and fluoroquinolones were low. Depending on the country and different regions of the world,
higher [22,23,49,54–56], similar [23,54,55] or lower [24,49,54,57] rates of resistance to the antibiotics
mentioned above were observed among E. coli isolates from adults and children. The fact that the E. coli
isolates from young children exhibited moderate prevalence of resistance to tetracycline, a drug which
should not be administered to children, may be a consequence of the previous exposure of food animals
to growth-promoting antibiotics and the dissemination of these agents in the environment [58–60].
Some previous studies have revealed that resistant bacteria can be found even in conventional retail
foods and ready-to-consume items, which suggests that humans are often exposed to such bacteria
through daily food intake [61–63].
All the resistant E. coli isolates were screened for the carriage of a set of commonly encountered
resistance determinants. The results revealed no statistically significant differences between the
frequencies of the tested resistance genes in the resistant E. coli isolates from adults and young children.
It was probably due to the similar rates of resistance to most of the tested antibiotics in these two groups
of isolates. The blaTEM , aadA1, tetB, dfrA7, sul2 and qnrS genes related to ampicillin, streptomycin,
tetracycline, trimethoprim/sulfamethoxazole and quinolone resistance respectively were the most
common. The resistant isolates negative for the tested resistance determinants may have carried one of
the other resistance genes.
Previous studies have reported that strains carrying the papC or iutA gene were more often
resistant to at least one of the tested antibiotics than were strains without these traits [24,64]. Virulence
genes and resistance genes may occur together on mobile genetic elements and be co-selected.
The results of these studies revealed that the association between the virulence genes and antimicrobial
resistance were weak in both groups of E. coli isolates.

5. Conclusions
This study analyzed the relationships between the distribution of virulence genes, phylogenetic
structure and prevalence of antimicrobial resistance among E. coli isolated from adults and young
children in Lubuskie province, Poland. This is the first such comparison regarding only commensal
strains. The results revealed significant difference in the distribution of the virulence genes, phylogenetic
groups as well as the multidrug-resistance among E. coli from adults and young children. This study
confirmed that the commensal E. coli isolates, particularly that derived from adults, constitutes
a considerable reservoir of the virulence genes typical for ExPEC. The comparison of the frequency
of the virulence genes between the E. coli isolates from adults and young children in relation to each
phylogroup showed that greater genetic diversity exists in isolates from adults. This may suggest
accumulation of the virulence determinants among commensal E. coli strains through horizontal
gene transfer over the years. Further research will be needed to better understand the relationships
between environmental factors and the genetic diversity of E. coli commensal strains. Plasticity of E. coli
genome provides the ability to adapt to different nutrient conditions and niches in the host’s body [1,6].
An interesting question remains how the environmental conditions (diet, travel, cultural background,
etc.) effect the shift of E. coli from commensal to pathogen strain responsible for extraintestinal infection.
Int. J. Environ. Res. Public Health 2018, 15, 617 16 of 19

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/15/4/617/s1,

Figure S1: Age distribution among adults. Table S1: List of virulence genes tested in this study. Table S2: Prevalence
of the resistance genes among the antimicrobial-resistant E. coli isolates from healthy adults and young children.
Acknowledgments: We would like to thank Wanda Tomala, Katarzyna Janecka and Paweł Pusz for skillful
technical assistance in our work. This study was supported by Ministry of Science and Higher Education grant N
N304 176 340.
Author Contributions: E.B. and K.B.-C. conceived and designed the experiments. E.B. and J.M. performed the
experiments. E.B., K.B.-C., J.M., A.M., M.S. analyzed the data. M.W. performed all statistical analysis. E.B. wrote
the paper. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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