4. Discussion

Urinary tract infection is an inflammatory process in the organs of the urinary system without specifying the level of damage or growth of microorganisms in the urinary tract with possible development of local inflammatory changes. UTI refers to the factors that initiate the development of chronic kidney disease and depends on the age of the children [27]. Etiological multifactority is peculiarity of these infections. For a long time, commonly recognized pathogens of uroinfections are Gram-negative enterobacteria, among which Escherichia coli is prevalent [25, 26, 29, 31, 40, 44].

Interest to studying enterococci as participants of infectious diseases has increased in recent years. Our research has shown that E. faecalis are a common pathogen, which causes UTI in children, most often in newborns (from 30.8 up to 75% of cases). Perhaps this is not accidental, as according to some authors, this microorganism is detected in children from the first days of life and its amount exceeds the content of E. coli in the newborn period [19]. The reason for this microbial composition of urine appears to be functional immunodeficiency in this category of patients.

However, until now, the true etiological significance of these microorganisms in the development of the infectious process and unfavorable outcomes remains uncertain due to the everchanging properties of E. faecalis. It is known that the ability of bacteria to affect the kidneys and urinary tract is determined not by one but by a complex of properties necessary for this process, that at the different stages of the infectious process in the urinary system organs from the microorganism, priority expression of certain pathogenetically significant traits and/or their combinations is required [6].

All analyzed uropathogenic E. faecalis had typical properties—morphology, biochemical activity against mannitol, methylene blue, its absence in rhamnose fermentation, variability in glucose, lactose, sucrose and 2,3,5-TTC, and lack of mobility and catalase.

The mentioned variability of biochemical and fermentation activities of pathogenicity factors

E. faecalis was tested for genes, coding various pathogenicity factors, using PCR method. It was found that clinical strains (n = 30) of enterococci isolated from urine of children with UTI contained two and more studied pathogenicity genes. In this context, 27 out of 30 uropa-

Eleven variants of genes combinations, which code pathogenicity factors of E. faecalis, were identified. The most common variants are (aggA-esp-cylA-efaA-eep-gelE) and (aggA,efaA,eep, gelE). Multilocus sequence typing (MLST) divided 17 E. faecalis strains into eight (ST6, ST16, ST21, ST40, ST41, ST116, ST179, and ST774) sequence types (The results have not been published.) It was noticed that uropathogenic E. faecalis strains attributed to ST6, ST40, ST179, ST774, and

The results demonstrate broad variability of the range of genes, which code pathogenicity factors and reveal sequence types with multiple resistances to antimicrobial agents among

Urinary tract infection is an inflammatory process in the organs of the urinary system without specifying the level of damage or growth of microorganisms in the urinary tract with possible development of local inflammatory changes. UTI refers to the factors that initiate the development of chronic kidney disease and depends on the age of the children [27]. Etiological multifactority is peculiarity of these infections. For a long time, commonly recognized pathogens of uroinfections are Gram-negative enterobacteria, among which Escherichia coli is prevalent [25, 26, 29, 31, 40, 44]. Interest to studying enterococci as participants of infectious diseases has increased in recent years. Our research has shown that E. faecalis are a common pathogen, which causes UTI in children, most often in newborns (from 30.8 up to 75% of cases). Perhaps this is not accidental, as according to some authors, this microorganism is detected in children from the first days of life and its amount exceeds the content of E. coli in the newborn period [19]. The reason for this microbial composition of urine appears to be functional immunodeficiency in this category of patients.

However, until now, the true etiological significance of these microorganisms in the development of the infectious process and unfavorable outcomes remains uncertain due to the everchanging properties of E. faecalis. It is known that the ability of bacteria to affect the kidneys and urinary tract is determined not by one but by a complex of properties necessary for this process, that at the different stages of the infectious process in the urinary system organs from the microorganism, priority expression of certain pathogenetically significant traits and/or

and resistance to antibiotics suggested a certain phenotypic heterogeneity of E. faecalis.

thogenic E. faecalis had four and more of the studied genes (Table 3).

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

ST116 are resistant to four and more groups of antimicrobial agents (Table 4).

uropathogenic E. faecalis isolated from children with UTI in Primorsky region.

3.1. Molecular genetics typing of E. faecalis

4. Discussion

their combinations is required [6].

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

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

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 recurrent infection.

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 manifestation of pathogenicity factors and the age of patients.

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.

Furthermore, connection between sensitivity to antimicrobial drugs of E. faecalis and its biological properties was identified. It was found that uropathogenic enterococci characterized with proteolytic activity are resistant to antibacterial agents with different action mechanisms. In the work, it was found that E. faecalis, resistant to linezolid and chloramphenicol drugs that suppress protein synthesis at the level of the 50S subunit of the bacterial ribosome, possess a high pathogenic potential. These results require further research in this direction.

5. Conclusions

Acknowledgements

Conflict of interest

Author details

Russia

AAAA-A18-118031390014-9).

Zaitseva Elena Aleksandrovna<sup>1</sup>

Luchaninova Valentina Nikolaevna<sup>5</sup>

1. Important role in the etiology of UTI is played not only by Gram-negative bacteria of the Enterobacteriaceae family, but also by gram-positive E. faecalis, which are of paramount

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

http://dx.doi.org/10.5772/intechopen.80485

95

2. E. faecalis, isolated from the urine of children with UTI, have a complex of pathogenicity factors necessary for the development of the inflammatory process and their prolonged persistence in the urinary tract. The relationship between E. faecalis pathogenicity factors

3. Uropathogenic E. faecalis possess a polyantibiotic resistance, which is associated with its

4. A set of phenotypic manifestations of the biological properties of E. faecalis (the presence/ absence of hemolytic, gelatinase, lecithinase, lipolytic activities) established in the study may determine its clinical significance and serve as an in vitro diagnostic marker of

The research was supported with an internal grant from the university (registration No.

\*, Komenkova Tatiana Sergeevna<sup>1</sup>

,

resistance of the studied uropathogen to certain groups of antibacterial drugs.

biological properties and belonging to a particular sequence type.

importance in the development of UTI in newborns.

and the age of patients was determined.

The authors declare that there is no conflict of interests.

\*Address all correspondence to: elza200707@mail.ru

1 Pacific State Medical University, Vladivostok, Russia

2 Regional Children's Clinical Hospital № 1, Vladivostok, Russia

Russian Academy of Sciences, Pushchino, Moscow Region, Russia

Melnikova Elena Aleksandrovna1,2, Shadrin Andrey Mikhailovich3,4 and

3 G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms,

4 Pushchino State Institute of Life Sciences, Pushchino, Moscow Region, Russia

5 North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg,

Interesting data were obtained with regard to the sensitivity of uropathogenic E. faecalis to the reserve drug linezolid, recommended for treatment of infections caused by strains, which are resistant to vancomycin, aminoglycosides, and betalactams. In Primorsky Krai, E. faecalis cultures were found resistant and intermediate sensitivity to linezolid. However, in Russia in the period from 2005 to 2013, there were isolated single enterococcal strains with reduced sensitivity to linezolid.

This way, the mentioned variability of biochemical and fermentation activity of factors related to pathogenicity demonstrated phenotypic heterogeneity of enterococci and might have a certain diagnostic significance.

Pathogenicity factors of bacteria are genetically determined by properties which are localized in genome of microorganisms in the form of "pathogenicity islands" [17]. These genetic elements can contain various sets of virulence genes, which are important for the development of the enterococcal infectious process, including genes of antibiotics resistance [17, 24]. At the present stage, the association of antibiotic resistance of E. faecalis with pathogenic factors is actively studied. It is known that strains of enterococci resistant to ampicillin, ciprofloxacin, and gentamicin, but sensitive to vancomycin and nitrofurantoin have more pathogenicity factors (hemolysin, gelatinase, hyaluronidase, form biofilms) than vancomycin resistant [2, 28]. E. faecalis having the asa1 gene are more resistant to fluoroquinolones (norfloxacin, ciprofloxacin, and levofloxacin) than isolates lacking this gene. Resistance to ciprofloxacin is significantly higher in E. faecalis having the genes cylL and cylS than in strains with their absence [23, 28]. esp gene-positive E. faecalis are more resistant to doxycycline than esp gene-negative cultures [3]. Among the strains with multidrug resistance, a high prevalence of genes asa1 and esp was observed [23, 36].

The research implemented using PCR method enabled to characterize in greater detail the structure of E. faecalis population isolated from children with UTI from Primorsky region. In our research, significant variability in occurrence frequency of the studied genes was found. Two of them—efaA (coding the surface antigen A (EfaA), which initiates the infectious process) and eep (coding Eep protein, which conduces to formation of a biofilm, making it resistant to various biological stress factors) were found in all studied uropathogenic E. faecalis, which proves their involvement in certain stages of the infectious process.

MLST analysis conducted earlier revealed eight sequence types, five of which were characterized by multidrug resistant.

This way, clinically significant E. faecalis strains have a complex of virulent properties, which allow the bacteria to materialize their pathogenic potential on all stages of the inflammation process in urinary system. This makes further research of the listed factors in clinical E. faecalis necessary to estimate objectively the contribution of these properties of the agent into pathophysiologic mechanism of infectious and inflammatory diseases.
