Davydenko, Mishyna, Myasoedov, Pashchenko, Shtyker, Davydenko, and Pashchenko: A New Concept Of Enhancing The Efficacy Of Antimicrobial Therapy Of Pyo-Inflammatory Diseases In Children



Problem statement and analysis of the recent research

Pyo-inflammatory processes (PIP), as complications after surgical interventions, constitute a significant portion of all surgical diseases in children. Purulent diseases such as acute peritonitis, destructive pneumonia, acute hematogenous osteomyelitis have severe, often complicated course and can lead to adverse consequences [7].

One of the main factors of such phenomena is a high drug resistance of contemporary pathogenic microorganisms to antimicrobial agents, which significantly reduces the effectiveness of therapy [8, 12].

Consequently, despite the development of modern antimicrobials and introduction of new technologies to confront pathogens, the issues of treatment of purulent-inflammatory diseases remain unsettled [1, 2, 11].

Experimental data of recent decades indicate a complex organization of microbial communities in their natural habitat. The agents form a protective biofilm around and successfully protect themselves from disinfectants and antimicrobial agents [3, 5, 7, 10].

The formation of biofilms by pathogenic microorganisms complicates the treatment of pyo-inflammatory processes caused by them [3, 4, 5]. It is associated with the significant increase in the resistance of microorganisms to antimicrobials, as in the form of a biofilm pathogens implement processes which practically do not occur in free plankton forms of existence [9].

The big problem is that the biofilms are formed on the surface of medical devices – catheters, endoprostheses, endotracheal tubes, implants, surgical sutures, etc. These become the threat of serious complications of pyo-inflammatory processes, the basis of adverse course of the postoperative period and formation of chronic foci of inflammation [10].

At the present stage of the development of microbiology interesting studies concerning an identification of fluctuations in biological properties of microorganisms during the day have appeared. Researchers acknowledge that bacteria possess biological rhythms [4]. The study of such type of properties of microorganisms opens up new possibilities for the solution of both range of general biological and medical problems, namely, the determination of adaptation mechanisms, drug resistance of microorganisms, principal issues of diagnosis, treatment and prevention of pyo-inflammatory diseases in children.

In this context, the study of diurnal (twenty-four-hour) production of virulence factors of clinical strains on the example of S. aureus as the major pathogen of nosocomial infections, will allow us to take a new approach to the assessment of biological properties of the pathogen contributing to the development of a rational strategy of the microbiological diagnostics of pyo-inflammatory hospital-acquired infections as well as to creation of preconditions for further research pertaining to the implementation of effective antimicrobial means, and more consummate characterization of mechanisms that contribute to the development of infectious pathology.

Therefore, from a scientific and practical point of view it would be interesting to study the basic virulence factors of S. aureus during the day, as a result of response to the action of antimicrobials.

The purpose of the study – to study characteristics of production of virulence factors by S. aureus clinical strains during the day as well as to determine on this basis determine the directions of improving the efficiency of antimicrobial therapy of pyo-inflammatory diseases in children.

Materials and methods

The subjects of microbiological studies were as follows: the reference strain of S. aureus (ATSS 25923=NCDC 25923=F-49) and clinical strains isolated from venous cannulae, dressings and suture material, drainage systems and catheters (n=10) as well as from clinical material (wound tissue, purulent exudate) of children with pyo-inflammatory processes (n=10) who were treated in Kharkiv Regional Children Clinical Hospital No 1. Enzymatic identification was performed using identification kits MICRO-LA-TEST. The sensitivity of isolates to antimicrobial agents having different mechanism of action was evaluated using the microtest-system “TNKtestGr+” with semi-quantitative registration of the results. The formation of biofilms was studied on the surface of polystyrene plates [6]. The optical density of a bacterial solution was measured on the spectrophotometer “Multiskan EX 355” with a wavelength of 540 nm. The result was expressed in standard units of optical density (absorbance units – AU).

Results and Discussion

Adhesion is an initial stage of any infectious process. Pathogenetic mechanisms of pyo-inflammatory processes caused by S. aureus are associated with the peculiarities of the pathogen, which applies several mechanisms to overcome the antimicrobial defense of a macroorganism. These mechanisms are adhesion, invasion, and production of aggression enzymes. The mechanism of adhesion is realized by S. aureus with the help of surface structures and factors of adhesion: polysaccharides, protein A, teichoic acids etc. When determining the adhesive activity of S. aureus, it was found that it depends on the time of the day. Thus, strains of S. aureus, which were obtained from patients with PIP, demonstrated the highest activity at 12 a.m. (7.19±0.19 AU) and at 9 p.m. (7.42±0.48 AU) as compared with the activity at 6 a.m. and 6 p.m. (2.5±0.18 and 2.31±0.23 AU, respectively), when a tendency to the decrease in the index of adhesiveness of S. aureus was observed. The most highly adhesive strains were registered in the isolation of the pathogen from venous cannulae and drainage systems at 12 a.m. (7.69±0.14 AU) and 9 p.m. (7.84±0.15 AU).

The study of the daily dynamics of the content of S. aureus teichoic acids (Fig. 1) allowed us to identify that the maximum value in clinical strains of S.aureus extracted from patients with PIP was registered at 12 a.m. (0.95±0.08 AU) and 9 p.m. (0.89±0.04 AU). Clinical strains of S. aureus, obtained from venous cannulae, were distinguished by alteration to a lesser content of teichoic acids at 6 a.m. (0.29±0.06 AU) and 6 p.m. (0.31±0.05 AU) and by maximum content at 12 a.m. (0.94±0.06 AU) and 9 p.m. (0.98±0.06 AU), which makes it possible to identify informative criteria to assess the earliest changes in the indicators of physiological activity of pathogens, as well as to determine the mechanisms of adaptive responses of microorganisms to the action of antimicrobials.

Fig. 1.

Daily dynamics of the content of S. aureus teichoic acids

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The study of the daily dynamics of formation of dense biofilms by clinical strains of S. aureus and production of planktonic cells with the ability to form new biofilms enabled us to determine (Fig. 2), that the maximum index of the formation of daily dense biofilms with the production of planktonic cells by clinical strains of S. aureus was registered at 12 a.m. and 9 p.m. There was observed a tendency to lower density in the formation of biofilms with reduced activity of production of plankton cells at 6 a.m. and 6 p.m. (Fig. 3).

Fig. 2.

Daily dynamics of the formation of biofilms by clinical strains of S. aureus

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Fig. 3.

Daily dynamics of plankton cell production by biofilms of clinical strains of S. aureus

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Therefore, the formation of biofilms of bacteria depends on adhesive properties of the pathogen and content of teichoic acids with the final stage of development that consists in the dispersion of bacteria: as a result of the reproduction of bacteria, individual cells periodically detach from biofilms, and then, they are able to attach to the surface of the mucous membrane, medical devices, catheters, etc. and form new colonies, and subsequently a dense biofilm. The daily dynamics of the formation of dense biofilms with subsequent production of planktonic cells by clinical strains of S. aureus was observed at 12 a.m. and 9 p.m. indicating an adaptive response of pathogens associated with phenotypic variability and the development of resistance to antimicrobial agents by clinical strains in the form of biofilms. The decrease in the activity of plankton cell production by the isolates was registered at 6 a.m. and 6 p.m. in comparison with the formation of biofilms by the reference strain of S. aureus, in which the fluctuations of optical density of the biofilm and plankton cell production was not significantly different during the day.

At present, in clinical practice the most commonly used antimicrobial agents are fluoroquinolones – levofloxacin, and β-lactam antibiotics – Amoxiclav and Ceftriaxone, which are broad-spectrum drugs, and to which a high sensitivity of many pathogenic microorganisms remains for a long time. The task of the next stage of this investigation was the isolation of S. aureus isolates with the definition of the daily dynamics of plankton cell production and formation of biofilms under the influence of antimicrobials.

The study found that at 12 a.m. and 9 p.m. there was a high resistance of clinical strains of S. aureus to all antimicrobial agents that were taken in the experiment either in planktonic form (Fig. 4), or in the form of biofilm (Fig. 5). It should be noted that at 6 a.m. and 6 p.m. there was an increase in sensitivity of clinical strains to antimicrobial agents, which can be explained by the fact that at this time of the day there was noted a decreased ability of isolates to form biofilms.

Fig. 4.

The influence of antimicrobial agents on planktonic cells of clinical strains of S. aureus depending on the time of the day

gmj-23-gmj.2016.3.6-g4.jpg
Fig. 5.

The impact of antimicrobials on the daily biofilms of clinical strains of S. aureus depending on the time of the day

gmj-23-gmj.2016.3.6-g5.jpg

Therefore, when determining the action of antimicrobial agents being often used in medical practice in treatment of pyo-inflammatory processes depending on the form of the existence of S. aureus and the cultivation time during the day, there was established, that drug resistance of S. aureus is associated with an increased adhesive activity, teichoic acids production and the formation of a dense biofilm at a certain time of the day.

Thus, the study of biorhythmic properties of pathogenic microorganisms on the example of clinical strains of S. aureus has determined that under the influence of antimicrobial therapy the pathogens synchronously with the time of maximal concentrations of antimicrobials in an organism, namely at 12 a.m., and 9 p.m. increased the production of virulence factors, thereby significantly enhancing their drug resistance, i.e. reducing the effectiveness of antimicrobial therapy.

At the same time, at 6 a.m. and 6 p.m., when the concentration of antimicrobial agents in the blood and tissues decreased, the peaks of reduced adhesive activity and reduction in the density of biofilms were recorded, which was reflected in increased sensitivity of clinical strains to antimicrobial agents.

Thus, the study showed that pathogens implement their protective mechanism against antimicrobial agents by means of modification variability with active formation of biofilms.

It was established that the level of production of virulence factors varies throughout the day and depends on the time of antimicrobial agent administration.

It was experimentally proved that by the example of clinical strains of S. aureus isolated in children with pyo-inflammatory diseases in a definite hospital with a certain rhythm of antimicrobial agent administration the lowest level of virulence factors production was recorded at 6 a.m. and 6 p.m.

Thus, the identified diurnal rhythm of virulence factors production by clinical strains of causative agents of pyo-inflammatory diseases in children indicates that along with the development of well-known methods of suppressing drug-resistant pathogens (such as periodic replacement of certain antibiotics, there is a need in the development of a new treatment strategy, namely – periodic change in the time for administration of antimicrobial agents during the day so that the maximum concentration of drugs in blood plasma would coincide in time with minimum production of virulence factors, as the latter is accompanied with the maximum sensitivity of microorganisms to antimicrobials; therefore, such regimen could significantly increase the effectiveness of treatment when using usual therapeutic doses of antimicrobial drugs.

Conclusions

  • Pathogenic microorganisms demonstrate drug resistance by means of virulence factors production and formation of dense biofilms.

  • The production of virulence factors was not uniform within 24 hours. The highest levels were registered at 12 a.m. and 9 p.m. The decrease in the activity of production of virulence factors was registered at 6 a.m. and 6 p.m.

  • The ascertained daily dynamics of virulence factors production and drug resistance of pathogens indicates a need to change the time for administration of antimicrobial agents so that the period of maximum action of medications (maximum concentration in an organism) would coincide with periods of minimal drug resistance of pathogens.

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Copyright (c) 2017 V. B. Davydenko, M. M. Mishyna, V. V. Myasoedov, Yu. V. Pashchenko, S. Yu. Shtyker, N. V. Davydenko, K. Yu. Pashchenko

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