ANTI-CYTOKINE ACTIVITY AND PRODUCTION OF CYTOKINE-LIKE SUBSTANCES BY GRAM-NEGATIVE BACTERIA ISOLATED IN POSTOPERATIVE COMPLICATIONS

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Introduction
Microorganisms, in particular gram-negative bacteria of different species (primarily enterobacteria and non-fermenting bacteria), which cause postoperative infectious and inflammatory complications in patients in surgical hospitals, have not only pathogenicity factors, but also persistent properties that protect pathogens from immune effectors of the macroorganism and contribute to the realization of their pathogenic potential in infection of organs and fabrics [1]. On the other hand, it is known that the course and outcome of any infectious and inflammatory process are influenced by cytokines, which regulate the immune response of the macroorganism during the introduction of the pathogen [2]. At the same time, it has been established that microorganisms of different species have anti-cytokine activity (ACA), that is, they are able to inactivate certain pro- and anti-inflammatory cytokines [3-5], and can also produce cytokine-like substances (CPV) [6]. These features of bacterial pathogens can affect the local cytokine balance in infected tissues of the macroorganism, increasing the risk of infectious and inflammatory complications in surgical patients.  
In this regard, the purpose of this study was to characterize the anti-cytokine activity and the ability to produce cytokine-like substances in gram-negative bacteria of various species isolated during postoperative complications.

Materials and methods 
In vitro experiments included clinical strains of gram-negative bacteria of the following species: Escherichia coli (n=6), Klebsiella pneumoniae (n=6), Citrobacter freundii (n=6), C. braakii (n=6), Pseudomonas aeruginosa (n=6), P. putida (n=6), Stenotrophamonas maltophilia (n=6) isolated from patients with postoperative complications.  
The isolation of pure bacterial cultures was carried out by the bacteriological method, and their species identification was carried out by direct protein profiling using the MALDI TOF MS Microflex LT series mass spectrometer (BrukerDaltonics, Germany) and the MaldiBioTyper 3.0 software. 
Bacterial ACA in relation to a number of cytokines (anti–inflammatory –IL1RA and IL4 and pro-inflammatory - IL8 and TNFa) was determined by enzyme immunoassay (ELISA) according to a well-known technique [7] using kits from Cytokine LLC (St. Petersburg). To do this, 150 µl of bacterial suspensions (0.5 units according to the McFarland turbidity standard) and solutions of the corresponding cytokines were introduced into centrifuge tubes in a 1:1 ratio, the samples were incubated for 2 hours at 37 ° C. After incubation, the reaction was stopped in cold (+4 ° C), centrifuged with cooling at 3000 rpm for 15 minutes at +4 °With and selected the supernatants. Cytokine solutions with the addition of NaCl saline solution were used as a control. The final concentration of cytokines in experimental and control samples was 416.6 pg/ml for IL1RA, 28.6 pg/ml for IL4, 62.5 pg/ml for IL8, and 41.6 pg/ml for TNFa. The results were taken into account on a StatFax 2100 photometer (USA) at a wavelength of 450 nm. The levels of ACA and CPV production were calculated taking into account the proportion of inactivation or production of the corresponding cytokines in the experiment relative to the control, expressed in pg/ml. The presence of these signs in bacteria was recorded if their severity exceeded 5 pg/ml.  
The data obtained were statistically processed using the Student's criterion (Statistica 6.0; StatSoft, Inc.).  The results are presented in the form of averages and errors of averages (M±m) obtained in at least three independent experiments. The differences were considered significant at p<0.05.

Results and discussion
The experimental data obtained indicated that the individual studied strains of microorganisms of two groups (enterobacteria and non–fermenting gram-negative bacteria - NGOB) isolated from patients with postoperative complications were able to inactivate cytokines – IL1RA, IL4, IL8 and TNFa (Table. 1), as well as produce cytokine-like substances (CPV) corresponding to these cytokines (Table 2). At the same time, intergenerational and interspecific, as well as intraspecific (interstamp) variability of the studied gram-negative bacteria was observed, both in frequency of occurrence and in the severity of these properties.
Table 1. Prevalence and severity of anticytokine activity (ACA) in gram-negative bacterial strains of various species isolated during postoperative complications 

Group and type of bacteria (n is the number of strains)	The frequency of occurrence (abs./%) and the severity of ACA (pg/ml) in gram-negative bacteria against certain cytokines*
	IL1RА	IL4	IL8	TNFα
Enterobacteria (n=24), including:	6/25,0. 356,2±7,9	16/66,7  25,9±2,2	4/16,7 61,3±2,6	8/33,3/ 31,7±3,1
Escherichia coli (n=6)	2/33,3. 303,3±10,4	2/33,3 25,5±2,4	2/33,3 62,0±1,4	4/66,7 25,4±3,4
Klebsiella pneumoniae (n=6)	4/66,7 409,1±5,5	4/66,7 26,6±1,8	н/в	н/в
Citrobacter freundii (n=6)	н/в	4/66,7 24,8±2,5	н/в	2/33,3 40,5±4,4
Citrobacter braakii (n=6)	н/в	6/100,0 26,7±2,2	2/33,3 60,7±3,9	2/33,3 25,6±2,6
MGOB (n=18), including:	2/11,1 329,5±6,8	16/88,9 27,2±2,1	7/38,9 45,3±3,1	10/55,6 39,3±3,3
Pseudomonas aeruginosa (n=6)	н/в	6/100,0 26,2±2,2	3/50,0 38,7±2,8	6/100,0 38,9±2,4
Pseudomonas putida (n=6)	2/33,3 329,5±6,8	6/100,0 28,0±2,4	н/в	2/33,3 40,9±3,4
Stenotrophamonas maltophilia (n=6)	н/в	4/66,7 25,5±1,7	4/66,7 51,9±3,4	2/33,3 38,2±4,2
TOTAL (n=42)	8/19,0 342,8±7,3	32/76,2 26,5±2,1	11/26,2 53,3±2,8	18/42,9 35,5±3,2

 

Note: * the numerator indicates the frequency of occurrence of strains with this trait (abs./%); in the denominator – t level of severity of this trait (pg/ml); n/a – the trait was not detected; ** NGOB - non-fermenting gram-negative bacteria.  

From the data presented in Table 1, it can be seen that individual clinical strains of microorganisms of the studied genera/species from both groups (Enterobacteria and NGOB) were able to inactivate certain cytokines. Most often, they showed ACA against the anti–inflammatory cytokine IL4 (76.2%), and the prevalence of this trait among non-fermenting gram-negative bacteria (P. aeruginosa, P. putida, S. maltophilia) was slightly higher than in enterobacteria - E. coli, K. pneumoniae, C. freundii and C. braakii (88.9 vs. 66.7%, p<0.05). The ability to inactivate other cytokines (IL1RA, IL8 and TNFa) in clinical isolates of gram-negative bacteria was much less common with a gradient of: 19,0-26,2-42,9 %, respectively. At the same time, bacterial strains of certain species/genera in the analyzed samples lacked ACA for some cytokines. For example, isolates of microorganisms of the genus Citrobacter, as well as species P. aeruginosa and S. maltophilia did not show ACA for IL1RA, strains K. pneumoniae, C. freundii and P. putida did not show the ability to inactivate IL8, and in the sample K. pneumoniae cultures with ACA in relation to TNFa were absent. 
The studied strains of gram-negative bacteria demonstrated no less diversity in terms of the severity of ACA. In microorganisms exhibiting the ability to inactivate cytokines, relatively wide ranges of variation in ACA levels were observed with respect to inactivation of IL1RA and IL8 (303.3-409.1 and 38.7-62.0 pg/ml, respectively), whereas with respect to inhibition of IL4 and TNFa, ACA levels were more homogeneous (24.8-28.0 and 25.4-40.9 pg/ml, respectively). 
The analysis of the severity of the ability to inactivate cytokines in gram-negative microorganisms of the studied groups revealed higher values of ACA in enterobacteria than in NGOB with respect to IL1RA (356.2±7.9 vs. 329.5±6.8 pg/ml, p<0.05) and IL8 (61.3±2.6 vs. 45.3±3.1 pg/ml, p<0.01); According to other immunomodulators, the intergroup differences in the ACA of these bacteria were not reliable. 
Among enterobacteria and NGOB, there were intergenerational and interspecific differences in individual ACA variants. Thus, K. pneumoniae, in comparison with E. coli, had significantly higher ACA for IL1RA (409.1±5.5 vs. 303.3±10.4 pg/ml, p<0.01), C. freundii had a higher ACA level for TNFa than C. braakii (40.5±4.4 vs. 25.6±2.6 pg/ml, p<0.05), and S. maltophilia showed a higher ACA for IL8 than P. aeruginosa (51.9±3.4 vs. 38.7±2.8 pg/ml, p<0.05).     
It should be emphasized that the absolute values of ACA presented in Table 1 indicated that gram-negative bacteria are able to inactivate from 61 to 99% of cytokines in a reaction mixture with microorganisms. In addition, it should be noted that 8.0% of enterobacteria strains and 22.2% of NGOB isolates showed combined ACA against several cytokines, while the "core" of such combinations was the ability of bacteria to inactivate IL4, and the most frequent combinations were ACA against two cytokines – IL4+TNFa (14%) and IL4+IL8 (14%). Associated ACA for 3 cytokines (IL4+IL8+TNFa) was found only in P. aeruginosa strains in 50% of cases. 
Collectively, these results indicate a significant intra- and interspecific variability of gram-negative microorganisms (Enterobacteria and NGOB) in frequency of occurrence and the level of severity of their ability to inactivate individual cytokines, which, apparently, may affect the nature of the course of infectious and inflammatory processes caused by them, including by changing the local cytokine balance in infected tissues.
At the same time, it cannot be excluded that vegetative pathogens capable of producing cytokine-like substances (CPV) into the medium can make a certain contribution to the local cytokine status. The data we obtained indicated the production of such substances by some clinical strains of Gram-negative bacteria isolated from patients with infectious and inflammatory complications (Table 2). 
In particular, it was found that 66.7% of isolates of enterobacteria of different species (except K. pneumoniae) and 88.9% of NGOB strains were producers of IL1RA-like substances, whereas in the same groups the proportion of bacterial cultures producing other CPV (corresponding IL4, IL8 and TNFa) was significantly less (11.1-22.2%) and depended on their specific/generic affiliation. Thus, in the group of enterobacteria, there were no producers of IL4- and IL8-like substances among E. coli strains, C. freundii isolates did not secrete IL8- and TNFa-like substances into the medium, and C. cultures braakii are CPVS corresponding to all three of these cytokines (IL4, IL8 and TNFa). As for NGOB, the ability to produce IL4-, IL8- and TNFa-like substances was not found in P. aeruginosa bacteria, whereas 66.7% of P. putida strains were producers of IL8- and TNFa-like substances, and 33.3% of S. maltophilia isolates secreted IL4-like substances into the medium.

Table 2. Prevalence and severity of the ability to produce CPV in gram-negative bacterial strains of various species isolated from infectious and inflammatory postoperative complications

Group and type of bacteria (n is the number of strains)	The frequency of occurrence (abs./%) and the level of severity (pg/ml) of the ability to produce CPV in gram-negative bacteria
	IL1RА	IL4	IL8	TNFα
Enterobacteria (n=24), including:	16/66,7 165,7±5,2	4/16,7 11,5±0,5	6/25,0 14,2±1,2	8/33,3 16,1±0,9
Escherichia coli (n=6)	4/66,7 364,5±11,5	н/в	н/в	2/33,3 26,3±1,5
Klebsiella pneumoniae (n=6)	н/в	2/33,3 8,3±0,6	6/100 14,2±1,2	6/100 5,9±0,3
Citrobacter freundii (n=6)	6/100,0 55,0±1,8	2/33,3 14,7±0,4	н/в	н/в
Citrobacter braakii (n=6)	6/100 77,5±2,4	н/в	н/в	н/в
MGOB (n=18), including:	16/88,9 62,6±4,3	2/11,1 7,9±0,5	4/22,2 28,7±1,3	4/22,2 11,9±0,7
Pseudomonas aeruginosa (n=6)	6/100,0 58,9±3,4	н/в	н/в	н/в
Pseudomonas putida (n=6)	4/66,7 67,1±5,2	н/в	4/66,7 28,7±1,3	4/66,7 11,9±0,7
Stenotrophamonas maltophilia (n=6)	6/100,0 61,7±4,2	2/33,3 7,9±0,5	н/в	н/в
TOTAL (n=42)	32/76,2 114,15±4,7	6/14,3 9,7±0,5	10/23,8 21,5±1,2	12/28,6 12,1±0,7

 

 

Note: * the numerator indicates the frequency of occurrence of strains with this trait (abs./%); in the denominator – the level of severity of this trait (pg/ml); n/a – the trait was not detected; ** NGOB - non-fermenting gram-negative bacteria.  

The severity of the ability to produce individual CPV by gram-negative bacteria varied significantly depending on the group and species of microorganisms (Table 2). Thus, the average level of production of IL1RA-like substances in enterobacteria was twice as high as that in NGOB (165.7±5.2 versus 62.6±4.3 pg/ml, respectively, p<0.05) and It was maximal in E. coli strains (364.5±11.5 pg/ml), while in C. freundii and C. isolates. braakii it was 55.0±1.8 and 77.5±2.4 pg/ml, respectively (p<0.05), and in NGOB strains of different species/genera it varied in a relatively narrow range – 58.9-67.1 pg/ml (p>0.05). 
These data indicate the intergenerational/interspecific and intraspecific (interstitial) variability of gram-negative microorganisms (enterobacteria, NGOB) in terms of the prevalence and severity of their ability to produce certain CPV corresponding to human cytokines such as IL1RA, IL4, IL8 and TNFa, which, along with the above-described variability of these bacteria in ACA, makes a significant contribution contribution to the formation of phenotypic diversity and, obviously, the pathogenic potential of pathogens of infectious and inflammatory complications in surgical patients. 
If we look at the totality of the presented data, we can find that the structure of clinical strains of enterobacteria and NGOB included isolates that exhibit ACA in relation to a certain cytokine or produce CPV corresponding to it, as well as cultures that did not possess these signs (Figure 1). Moreover, the specific weight of such variants of bacterial strains depended both on their group affiliation (enterobacteria/NGOB) and on the variant of the analyzed cytokine. 
In addition, it should be noted that the studied clinical strains of gram-negative bacteria were able, on the one hand, to inhibit both anti-inflammatory cytokines (IL1RA and IL4) and pro-inflammatory immunomodulators (IL8 and TNFa), on the other hand, to produce substances similar to these regulatory molecules in the medium. It can be assumed that such a variety of cytokine-associated characteristics of pathogens can significantly "interfere" with the cytokine-mediated regulation of the inflammatory process of infectious etiology.   

Figure 1. The structure of clinical strains of enterobacteria and NGOB, taking into account the presence of ACA in relation to individual cytokines and the ability to produce certain CPV. 
Figure 1. The structure of clinical strains of enterobacteria and NGOB, taking into account the presence of ACA in relation to individual cytokines and the ability to produce certain CPV.

Conclusion 
Gram-negative bacteria (primarily E. coli, K. pneumoniae, Citrobacter spp. and P. aeruginosa) belong to the group of priority pathogens of postoperative infectious and inflammatory complications [8, 9]. In this regard, a comprehensive study of their pathogenic bioprophiles at both the phenotypic and genotypic levels is an urgent task of medical microbiology. Of particular interest is the question of the interaction of bacteria with immunoregulatory factors and, in particular, pro- and anti-inflammatory cytokines, which are directly involved in the development of the pathological process in infected organs and tissues [2]. 
Currently, there is increasing evidence that clinical strains of microorganisms of different taxonomic affiliation are able to inactivate/neutralize various cytokines, as well as produce cytokine-like substances (CPV) into the extracellular space. Today it is known that clinical strains of gram-positive cocci (staphylococci [6] and enterococci [3]) and fungi of the genus Candida [4] have anti-cytokine activity (ACA) against a number of pro- and anti-inflammatory cytokines, and are also capable of producing various CPVS in the culture medium, that is, they have "procytokine activity". In addition, ACA was also detected in gram-negative bacteria, in particular in clinical E. coli uroinsulates isolated from the urine of patients with urolithiasis [5]. Thus, it was found that all urostamps of E. coli isolated from patients with postoperative infectious and inflammatory complications were characterized by a wide prevalence and pronounced expression of ACA against proinflammatory (IL6, IL8 and TNFa) and/or anti-inflammatory (IL2, IL10) cytokines. When analyzing the ability of intestinal microsymbionts to inactivate cytokines, it was found that fecal strains of K. pneumoniae showed high ACA for IL10 and less pronounced for TNFa and IFNy, whereas pseudomonas exometabolites showed a high ability to reduce levels of oppositional cytokines TNFa, IFNy and IL10 in the culture medium [10].   
The results of our studies allowed us to characterize the intergenerational, interspecific and intraspecific/interstitial differences in the prevalence and severity of ACA in relation to IL1RA, IL4, IL8 and TNFa, as well as the production of CPV corresponding cytokines in clinical isolates of gram-negative microorganisms (representatives of the Enterobacteriaceae family and a group of non-fermenting bacteria) isolated in infectious and inflammatory complications in patients with surgical profile. At the same time, it should be recognized that the pathogenetic significance of the presence and expression of these properties in pathogens of postoperative complications does not yet have a significant evidence base, which requires further research, including those aimed at identifying the interrelationships of anti- and procytokine characteristics of bacterial isolates with clinical manifestations of the pathological process, the nature of its course and outcome options.  
The results of such studies may be promising for the development of algorithms for diagnosing and predicting the risk of postoperative infectious and inflammatory complications during surgical interventions.

About the authors

Olga Pashinina

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: olga25mikro@mail.ru
ORCID iD: 0000-0001-9944-3095
SPIN-code: 9638-7336

Candidate of Biological Sciences, Senior Researcher at the Laboratory of Persistence and Symbiosis of Microorganisms

Russian Federation

Tatiana Mikhailovna Pashkova

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: pashkova070782@mail.ru
ORCID iD: 0000-0001-8075-8249
SPIN-code: 5146-6190

Doctor of Biological Sciences, Leading Researcher at the Laboratory of Persistence and Symbiosis of Microorganisms 

Russian Federation

Olga Lvovna Kartashova

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: labpersist@mail.ru
ORCID iD: 0000-0002-1487-7546
SPIN-code: 9315-1070

Doctor of Biological Sciences, Leading Researcher at the Laboratory of Persistence and Symbiosis of Microorganisms

Russian Federation

Lyudmila Fomina

Институт иммунологии и физиологии УрО РАН

Author for correspondence.
Email: av_zurochka@mail.ru
ORCID iD: 0000-0001-8130-8979
SPIN-code: 2678-7689

Лаборатория иммунологии воспаления

Russian Federation

Viktor Alexandrovich Gritsenko

Orenburg Federal Research Center of the Ural Branch of the Russian Academy of Sciences

Email: vag59@mail.ru
ORCID iD: 0000-0002-2086-5170
SPIN-code: 7107-1300

Doctor of Medical Sciences, Chief Researcher at the Laboratory of Persistence and Symbiosis of Microorganisms

Russian Federation

References

Supplementary files