3. Results

The most common uropathogen in children with UTI, who were treated at the multispecialty regional children's hospital, is Escherichia coli, for which specific gravity equals from 33.92 1.7 to 62.96 1.2%. Most frequently, it was pointed out in outpatients (in 62.96 1.2% cases); less frequently, it was found at departments for newborns (33.92 1.7%). The second significant

etiological factor of UTI in children was E. faecalis, for which specific gravity ranged from 16.14 to 32.5% [22]. At the same time, E. faecalis was the most important factor in development of UTIs at departments for newborns: it was found in 57.2% of cases (Figure 1). Frequency of identifying E. faecalis in newborns diagnosed with UTI ranged from 30.8 to 74.5% of cases over 9 years.

р2–3˂0.001). In this group, lipolytic activity against Tween 60 was less pronounced, but the

Phenotypic and Genetic Diversity of Uropathogenic *Enterococcus faecalis* Strains Isolated…

E. faecalis isolated from patients older than 1 year (group 3) showed more lipolytic activity against Tween 80, compared to other age groups (p1–3˂0.001; p2–3˂0.01), gelatin's fermentation was determined more often than in enterococci of the group 2 (p˂0.001). The hemolytic activity of enterococci in the group 3 differed little compared to the group of newborn children. The other biochemical properties associated with pathogenicity factors of E. faecalis strains of this age group were similar to enterococci isolated from children under 1 year of age (Table 2).

Assessment of antibiotics resistance revealed that all studied cultures of uropathogenic enterococci (n = 71) are sensitive to vancomycin and nitrofurantoin. We found that enterococci are

1 year (n = 18)

19 19 (100) 14 14 (100) 21 21 (100)

21 12 (57.1 10.8) 17 6 (35.3 11.6) 19 8 (42.1 11.3)

examined cultures, n

Reduction of TTC \*■ 19 18 (94.7 5.1) 14 11 (78.6 11.0) 21 20 (95.2 4.7)

Mannitol 20 20 (100) 13 13 (100) 19 19 (100) Glucose ▲ 20 19 (95.0 4.9) 13 12 (92.3 7.4) 19 19 (100) Lactose ▲ 20 19 (95.0 4.9) 13 12 (92.3 7.4) 19 16 (84.2 8.4)

Sucrose 20 17 (85.0 8.0) 13 11 (84.6 10.0) 19 16 (84.2 8.4)

Milk \*▲ 21 18 (85.7 7.6) 18 9 (50.0 11.8) 21 10 (47.6 10.9) Gelatin \*▲■ 20 9 (45.0 11.1) 17 2 (11.8 7.8) 21 7 (33.3 10.3) Lecithinase activity 19 5 (26.3 10.1) 15 4 (26.7 11.4) 21 6 (28.6 9.9)

Tween 20 17 14 (82.4 9.2) 12 11 (91.7 7.9) 18 17 (94.4 5.4) Tween 60 \*▲ <sup>8</sup> 4 (50.0 17.2) 6 1 (16.7 15.2) 11 3 (27.3 13.4) Tween 80 ▲■ 21 14 (66.7 10.3) 17 12 (70.6 11.0) 19 16 (84.2 8.4) Note: \*, p < 0.05 between 1 and 2 groups; ▲, p < 0.05 between 1 and 3 groups; ■, p < 0.05 between groups 2 and 3; Abs.,

Table 2. Peculiarities of phenotypic manifestations biological properties of E. faecalis depending on the patient's age.

Rhamnose 21 0 13 0 19 0

Children over 1 year old

Abs. (Р mр, %)

89

(n = 21)

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examined cultures, n

Abs. (Р mр, %) Number of

Newborns (n = 21) Children from 29 days to

Abs. (Р mр, %) Number of

differences were significant only between groups 1 and 2 (p˂0.01).

Biological properties Enzymatic activity of E. faecalis

Number of examined cultures, n

Reduction of methylene blue

Presence of a capsule

Proteolytic activity in relation to:

Lipolytic activity in relation to:

\*▲

absolute.

Biochemical activity in relation to:

The present study analyzed the features of phenotypic manifestations of biological properties, including antibiotic resistance of E. faecalis (n = 71) isolated from urine of children with UTI. All analyzed uropathogenic E. faecalis had typical properties—morphology (cocci or oval Grampositive bacteria), biochemical activity against mannitol, methylene blue, its absence in rhamnose fermentation, variability in glucose, lactose, sucrose and 2,3,5-triphenyltetrazolium chloride (TTC), and lack of mobility and catalase (Table 2).

Most E. faecalis isolated from the urine of children with UTI had in vitro enzymatic activity associated with pathogenicity: hemolytic, proteolytic, lipolytic, and lecithinase. A capsule was found in 45.6 6.6% of uropathogenic enterococci. An inverse relationship was established between the presence of a capsule in E. faecalis with α-type hemolysis (r = 0.3, p = 0.0001), fermentation of milk (r = 0.31, p = 0.00), and a positive correlation with the microorganism titer in the urine (r = 0.33, p = 0.0332).

A direct correlation was established among the lecithinase and DNAase activity of E. faecalis (r = 0.31, p = 0.0438), the manifestation of hemolytic activity (α- or β-type) in uropathogenic enterococci, and the presence of the gene gelE (r = 0.49; p˂0.05).

The capsule (р1–2˂0.001; р1–3˂0.01), milk fermentation (р1–2˂0.001; р1–3˂0.01), gelatinous (р1– <sup>2</sup>˂0.001, р1–3˂0.05), and lipolytic activity with respect to the tween 60 (р1–2˂0.01, р1–3˂0.01) was determined more often in E. faecalis, isolated from the urine of newborn children (group 1) with UTI, than in other age groups.

E. faecalis, isolated from children under 1 year (group 2), have hemolytic activity more often than other age groups (р2–1˂0.05, р2–3˂0.05), but expressed less gelatinous activity (р1–2˂0.001;

Figure 1. The etiological structure of UTI in newborn.

р2–3˂0.001). In this group, lipolytic activity against Tween 60 was less pronounced, but the differences were significant only between groups 1 and 2 (p˂0.01).

etiological factor of UTI in children was E. faecalis, for which specific gravity ranged from 16.14 to 32.5% [22]. At the same time, E. faecalis was the most important factor in development of UTIs at departments for newborns: it was found in 57.2% of cases (Figure 1). Frequency of identifying E. faecalis in newborns diagnosed with UTI ranged from 30.8 to 74.5% of cases over 9 years.

The present study analyzed the features of phenotypic manifestations of biological properties, including antibiotic resistance of E. faecalis (n = 71) isolated from urine of children with UTI. All analyzed uropathogenic E. faecalis had typical properties—morphology (cocci or oval Grampositive bacteria), biochemical activity against mannitol, methylene blue, its absence in rhamnose fermentation, variability in glucose, lactose, sucrose and 2,3,5-triphenyltetrazolium chloride

Most E. faecalis isolated from the urine of children with UTI had in vitro enzymatic activity associated with pathogenicity: hemolytic, proteolytic, lipolytic, and lecithinase. A capsule was found in 45.6 6.6% of uropathogenic enterococci. An inverse relationship was established between the presence of a capsule in E. faecalis with α-type hemolysis (r = 0.3, p = 0.0001), fermentation of milk (r = 0.31, p = 0.00), and a positive correlation with the microorganism titer

A direct correlation was established among the lecithinase and DNAase activity of E. faecalis (r = 0.31, p = 0.0438), the manifestation of hemolytic activity (α- or β-type) in uropathogenic

The capsule (р1–2˂0.001; р1–3˂0.01), milk fermentation (р1–2˂0.001; р1–3˂0.01), gelatinous (р1– <sup>2</sup>˂0.001, р1–3˂0.05), and lipolytic activity with respect to the tween 60 (р1–2˂0.01, р1–3˂0.01) was determined more often in E. faecalis, isolated from the urine of newborn children (group 1)

E. faecalis, isolated from children under 1 year (group 2), have hemolytic activity more often than other age groups (р2–1˂0.05, р2–3˂0.05), but expressed less gelatinous activity (р1–2˂0.001;

(TTC), and lack of mobility and catalase (Table 2).

88 Microbiology of Urinary Tract Infections - Microbial Agents and Predisposing Factors

enterococci, and the presence of the gene gelE (r = 0.49; p˂0.05).

in the urine (r = 0.33, p = 0.0332).

with UTI, than in other age groups.

Figure 1. The etiological structure of UTI in newborn.

E. faecalis isolated from patients older than 1 year (group 3) showed more lipolytic activity against Tween 80, compared to other age groups (p1–3˂0.001; p2–3˂0.01), gelatin's fermentation was determined more often than in enterococci of the group 2 (p˂0.001). The hemolytic activity of enterococci in the group 3 differed little compared to the group of newborn children. The other biochemical properties associated with pathogenicity factors of E. faecalis strains of this age group were similar to enterococci isolated from children under 1 year of age (Table 2).

Assessment of antibiotics resistance revealed that all studied cultures of uropathogenic enterococci (n = 71) are sensitive to vancomycin and nitrofurantoin. We found that enterococci are


Note: \*, p < 0.05 between 1 and 2 groups; ▲, p < 0.05 between 1 and 3 groups; ■, p < 0.05 between groups 2 and 3; Abs., absolute.

Table 2. Peculiarities of phenotypic manifestations biological properties of E. faecalis depending on the patient's age.


highly resistant to erythromycin (77.1 5.02%), tetracycline (73.2 5.3%), fluoroquinolones of the II and the III generations (ciprofloxacin (55.1 5.9%), norfloxacin (48.6 9.9%), and levofloxacin (46.5 5.9%)). Were identified E. faecalis cultures, which are resistant (20.1 4.9%) and mid-resistant (8.9 3.5%) to the reserve drug linezolid. More than half (59.2 5.8%) of the studied cultures of uropathogenic enterococci were resistant to several

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91

It was found that enterococci, which are sensitive to penicillins, were characterized with lipolytic, lecithinase, and hemolytic (β-type) activity in vitro. Enterococci cultures which are resistant to fluoroquinolones, fermented sucrose, had proteolytic activity, and did not break down lactose. E. faecalis, which are resistant to gentamicin and erythromycin, had a capsule. Furthermore, E. faecalis, which are resistant to linezolid and chloramphenicol, had gelatinase, lecithinase, and

In that way, analysis of biological properties of E. faecalis, isolated from urine of children with UTI in Primorsky region, showed that overwhelming majority of cultures had typical properties.

lipolytic activities, as compared to other cultures (these data were not published).

Id Isolate ST Phenotype of antibiotic resistance Genotype 1785 PRA038 40 ТЕТ-ERT-AmG-CHL-LZD esp-efaA-eep-gelE

1780 PRV054 6 ТЕТ-ERT-AmG-CHL-FLQ aggA-efaA-eep-gelE

1788 PRU047 179 ТЕТ-ERT-AmG-LZD aggA-cylA-efaA-eep-gelE 1789 PRV052 ТЕТ-ERT-AmG aggA-esp-cylA-efaA-eep-gelE

1781 PR050 16 ТЕТ-ERT aggA-esp-cylA-efaA-eep 1782 PRV092 ТЕТ-ERT-AmG aggA-cylA-efaA-eep 1794 PR040 774 AmG-FX aggA-esp-efaA-eep 1793 PRAs081 ТЕТ-ERT-PEN-AmG-FLQ aggA-esp-efaA-eep-gelE 1795 PRA029 ТЕТ-ERT-PEN-FLQ aggA-efaA-eep-gelE

1786 PRV049 41 ТЕТ aggA-esp-efaA-eep-gelE

Note: TET, tetracycline; ERT, erythromycin; AmG, aminoglycosides; FLQ, fluoroquinolones; CHL, chloramphenicol; LZD,

1783 PRV082 21 FLQ efaA-eep

Table 4. Antibiotic resistance of uropathogenic E. faecalis depending on the sequence type.

antibiotics (four or more antimicrobial agents).

1784 PR055 ТЕТ-ERT-AmG-CHL

1791 PRV100 ТЕТ-ERT-AmG-CHL 1790 PRV105 ERT-AmG-CHL-FLQ 1787 PR042 116 ТЕТ-ERT-AmG-CHL-LZD

1792 PR051 ТЕТ-ERT-AmG-FLQ

linezolid; PEN, penicillins.

1779 PRN030 ТЕТ-ERT-AmG-CHL-FLQ

Note: nd, not determined.

Table 3. Genotypes of uropathogenic E. faecalis in Primorsky region.

highly resistant to erythromycin (77.1 5.02%), tetracycline (73.2 5.3%), fluoroquinolones of the II and the III generations (ciprofloxacin (55.1 5.9%), norfloxacin (48.6 9.9%), and levofloxacin (46.5 5.9%)). Were identified E. faecalis cultures, which are resistant (20.1 4.9%) and mid-resistant (8.9 3.5%) to the reserve drug linezolid. More than half (59.2 5.8%) of the studied cultures of uropathogenic enterococci were resistant to several antibiotics (four or more antimicrobial agents).

Id ST Isolate Genotype Total number of genes

1787 ST116 PR042 + + + + + + 6 1789 ST179 PRV 052 + + + + + + 6 1791 ST179 PRV100 + + + + + + 6 1790 ST179 PRV105 + + + + + + 6

90 Microbiology of Urinary Tract Infections - Microbial Agents and Predisposing Factors

1788 ST179 PRU047 + — + + ++ 5

1781 ST16 PR050 + + + + + — 5 1786 ST41 PRV049 + + — + ++ 5

1793 ST774 PRAs81 + + — + ++ 5

1780 ST6 PRV054 + — — + ++ 4 1779 ST6 PRN030 + — — + ++ 4 1795 ST774 PRA029 + — — + ++ 4 1792 ST774 PR51 + — — + ++ 4 1784 ST40 PR055 — + — + ++ 4 1785 ST40 PRA038 — + — + ++ 4

nd PRV086 + + + + + + 6 nd PR 230 + + + + + + 6

nd PR 181 + + + + + — 5

nd PRL079 + + — + ++ 5 nd PRV080 + + — + ++ 5

nd PR 198 + — — + ++ 4 nd PR 158 + — — + ++ 4

nd PR 228 + — +++ — 4

nd PR 215 + + — + + — 4 nd PR 223 + + — + + — 4

nd NCTC 12697 — — + + ++ 4 nd PR 97 — — + + ++ 4 nd PR 161 — —— + ++ 3 nd PR 206 — —— + ++ 3

1782 ST16 PRV092 + — +++ — 4

1794 ST774 PR040 + + — + + — 4

1783 ST21 PRV082 — —— + + — 2

Table 3. Genotypes of uropathogenic E. faecalis in Primorsky region.

Note: nd, not determined.

aggA esp cylA efaA eep gelE

It was found that enterococci, which are sensitive to penicillins, were characterized with lipolytic, lecithinase, and hemolytic (β-type) activity in vitro. Enterococci cultures which are resistant to fluoroquinolones, fermented sucrose, had proteolytic activity, and did not break down lactose. E. faecalis, which are resistant to gentamicin and erythromycin, had a capsule. Furthermore, E. faecalis, which are resistant to linezolid and chloramphenicol, had gelatinase, lecithinase, and lipolytic activities, as compared to other cultures (these data were not published).

In that way, analysis of biological properties of E. faecalis, isolated from urine of children with UTI in Primorsky region, showed that overwhelming majority of cultures had typical properties.


Note: TET, tetracycline; ERT, erythromycin; AmG, aminoglycosides; FLQ, fluoroquinolones; CHL, chloramphenicol; LZD, linezolid; PEN, penicillins.

Table 4. Antibiotic resistance of uropathogenic E. faecalis depending on the sequence type.

The mentioned variability of biochemical and fermentation activities of pathogenicity factors and resistance to antibiotics suggested a certain phenotypic heterogeneity of E. faecalis.

All analyzed uropathogenic E. faecalis had typical properties—morphology, biochemical activity against mannitol, methylene blue, its absence in rhamnose fermentation, variability in glucose,

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At the same time, weak and delayed acid formation from lactose was in three cultures (only on the third day), and in 12 isolates during fermentation of sucrose (by day 10). The results obtained with respect to a number of carbohydrates differ from the literature data, as it is known that E. faecalis ferment lactose and sucrose, and in relation to other sugars can be variable (Berdzhi). Possibly, this is associated with the spread of certain E. faecalis biovars in

According to results of research conducted in recent years, it was determined that enterococci produce many virulence factors, which conduce to development of the infectious process (hemolysin, gelatinase, enterococcal surface protein, aggregation substance, serine protease, capsule, etc.). The greatest number of virulence factors was found in E. faecalis isolated from urine. High proteolytic activity of E. faecalis (hydrolysis of gelatinase, casein, and collagen)

Moreover, the change in the properties of the microorganisms, causing the urinary tract infection, such as the development of resistance factors to antimicrobial drugs and the biofilm formation, makes it difficult to manage patients, especially with chronic persistent and often

Our research confirmed high virulence properties of E. faecalis isolated from urine of patients with and their manifestations depending on the patient's age. For example, there is a negative correlation between the proteolytic activity of E. faecalis and the age of the children (r = 0.28, р = 0.002). Most E. faecalis isolated from the urine of children with UTI had in vitro enzymatic activity associated with pathogenicity: hemolytic, proteolytic, lipolytic, and lecithinase.

Currently, lipase is referred to understudied factors of enterococcal persistence, although it is known that lipase may be a potential virulence factor of E. faecalis [12]. Among the studied enterococci isolated from children with UTI, lipolytic activity was determined in 85.0 8.2% of the cultures (more often with respect to Tween 20 and Tween 80). Enterococcus cultures showed heterogeneity in proteolytic and hemolytic activity. A reliable direct correlation between the phenotypic manifestation of β-type hemolytic activity with hydrolysis of gelatin (r = 0.58, p = 0.0001) and lecithinase activity (r = 0.52, p = 0.0004) of this uropathogen has been established. This confirms the combined effect of these pathogenic factors at a certain stage of the inflammatory process. A relationship was established between the phenotypic manifesta-

The most common properties of E. faecalisisolated from urine of newborn children were a capsule, proteolytic, and lipolytic (in relation to Tween 60) activity. Enterococci isolated from 1-year-old children with UTI most frequently were characterized with hemolytic activity. Lipolytic (in relation to Tween 80) activity was most frequently found in cultures isolated from patients older than 1 year. The prevalence of these virulence factors suggests that they are associated with virulence of this species in UTI. Such features of the manifestation of biological properties in vitro indicate the

selection of etiologically significant E. faecalis at the level of the macroorganism.

causes toxic damage to tissues and conduce to cicatricial changes in kidney [6, 19, 43].

lactose, sucrose and 2,3,5-TTC, and lack of mobility and catalase.

recurrent infection.

tion of pathogenicity factors and the age of patients.

the territory of Primorsky Krai or within a single multispecialty hospital.
