Antibiotic resistance changes in strains of bacteria and yeast-like fungi following their growth in established cell lines of human and animal origin

The phenomenon of the susceptibility and reversion of resistance to antibiotics in strains of bacteria and yeast-like molds has been identified upon the interaction of bacteria and molds with transplantable human and animal cell lines depending on the cultivation conditions. The ability of susceptibility reversion of bacteria and yeast-like fungi to some antibiotics is not stable, it varies with the passages. This phenomenon may be a reason of the poor efficacy of antibiotic treatment when the susceptibility of bacteria to antibiotics is assayed by the usual technique. The authors propose to modify the antibiotics-susceptibility assay currently used in clinics by co-culture of the isolated bacteria and fungi with the transplantable cell lines or human leukocytes.

Introduction.Bacterial and yeast-like organisms are usually isolated using nutrient media; the same media are taken to verify the antibiotic resistance of these microorganisms.However, a lot of different strains of bacteria and yeast-like organisms are parasites in habiting different human and animal organs con sisting of different type cells (epithelium-like, fibroblast-like, etc.).There are some published data [1 ] describing the adhesion process properties during shigellae interactions with human embryo intestinal mucosa.
Bacteria colonize the biomaterial, thereby adop ting a sessile mode of growth that progresses to the establishment of an antibiotic-resistant biofilm by the accretion of prospective glycocalyx [2,3].Amino-strains and 22 different Enterococcus strains (gramnegative bacteria); 17 representatives of the Candida genus and 2 Cryptococcus neoformans strains.Estab lished cell lines taken for these studies were: L41 (human lymphoblastoid cell line), OH-1 (murine lymphoblastoid cell line), RGNR (cell line originated from rat Gasser node neurinoma), and Vero (green African monkey kidney cell line).Cell cultures were grown in the RPMI-1640 supplemented both with calf foetal serum (10 %) and antibiotics [7].
We also manipulated with human leukocyte sus pension cultures (blood group 0).The cultivation of bacterial strains was performed as follows: 100 /Л of bacterial suspension containing 1-Ю 8 cells were ad ded to a leukocyte culture (3-Ю 6 cells/ml).Following bacterial growth during 24 h, 100 /Л of infected leukocyte suspension of the same concentration were transferred into a new leukocyte culture to carry out the 2 nd passage.The 3 rd one was made similarly.Antibiotic sensitivity of bacterial cultures for each passage was studied and compared with its original sensitivity to antibiotics.In these experiments we used two interferon (IFN) preparations -IFN-a-2b (Intron, 3-Ю 6 I. U., Shering-Plau, USA), and IFN-y (10 ъ I. U., product of the Ukrainian Academy of Agriculture).

Cultivation of bacteria and fungi. Each microbial or fungal strain was grown in cultured 24 h-old monolayer cell lines or in suspended human leuko cytes.
Each microbial/fungal suspension (10 8 cells/ml) was put into cell cultures or human leukocyte sus pension and cultivated during 24 h in RPMI-1640 without calf foetal serum and antibiotics.The degree of susceptibility of microbial/fungal strains to anti biotics was determined before their growth in eukaryotic cells grown in the Mueller-Hinton medium (initial susceptibility, IS) and following their culti vation (post-cultivation susceptibility, PCS).
The antibiotic susceptibility was evaluated by the Bauer-Kirby approach (a variant of the disk-diffusion method) [8]; all bacterial/fungal strains were grown on the Mueller-Hinton medium; commercial disks used were produced by the (St.Petersburg, Russian Federation).The disk quality control with the Muel ler-Hinton medium and different antibiotics was per formed each time as well as with any new disk series of nutrient media series.The criteria of their ade quacy were verified according to the zone diameter of inhibited growth for etalon strains -Escherichia coli 25922, S. aureus ATCC 25923, and P. aeruginosa ATCC 27853 received from the Alive Culture Museum of our Institute.The manufacturer's criteria were used to evaluate the efficacy of antibiotics against microbial/fungal agents.Taking into consideration the zone diameter of inhibited growth for investigated microorganisms, the strains were divided into three groups -sus ceptible strains (SS), moderately resistant strains (MRS), and strains highly resistant to antibiotic(s) (HRS) [9].We determined antibiotic-susceptib le/resistant properties of strains with the following compounds widely used in up-to-date clinical practice:

Enterobacteriaceae
susceptibility to antibiotics are given in Table 1.
The analysis of the data presented in Table 1 shows that the Enterobacteriaceae cultivation in the L41 cell line causes the development of two phenotypes: some antibiotic-sensitive strains become re sistant ones and vice versa.This Table proves the enterobacterial strains have not changed their sus ceptibility to gentamicin, cefoperazone, and imipe- nem.Some changes of antibiotic sensitivity were found in 14 enterobacterial strains, resistant strains becoming sensitive ones and vice versa.The Enterobacteriaceae changes from resistance to susceptibility (R -* S) were found for the following antibiotics: amikacin (for one strain), cefazolin (for three strains), and tobramicin (for 1 strain).
The data described above demonstrate the ente robacterial transition from to antibiotics susceptibility to antibacterial resistance and vice versa in the L41 cell line.We aimed to verify whether such phe nomenon is ubiquitous enough and if it may be found also during Enterobacteriaceae cultivation in cell lines  1), Vero (Fig. 2), OH-1 (Fig. 3), and L41 (Fig. 4).Among two Cr.neoformans strains studied here, the (R -> S) transition for fluconazole was registered for a single strain.

Table 2 Determination of enterobacterial strain 1646 susceptibility to antibiotics and its resistance to antibiotics during its growth in the L41 established cell line (24 h)
Antibiotic (the zone diameter, mm, of inhibited growth for etalon strains) The changes of microorganism susceptibility to antibiotics are due to numerous factors including properties of bacteria/yeast-like agents, properties of their host cells, culture media with all their com ponents, and metabolic products.The unstable results obtained in our investigations and their changes during the passages carried out led us to a hypothesis concerning the metabolic products as the main varia bility factor.To verify this hypothesis, we carried out the following experiment in a model system using an enterobacterial strain, 1646, with multiple changes of susceptibility to antibiotics.The bacteria were grown in: 1. L41 cell culture washed carefully from its culture medium before bacterial infecting (cell cul ture, CC); 2. Medium following the L41 cell line cultivation (medium post cell cultivation, MCC); 3. RPMI-1640 (cell medium, original, CMO).The results obtained are presented in Table 2.They show the susceptibility changes to occur in the MCC containing cell metabolic products including also cyto kines, e. g.IFN.
Due to these data, a question arose about pos sible IFN participation in changes of antibiotic sus ceptibility for different microorganisms.To answer this question, we carried out some additional re searches.The L41 cells were pre-treated by recom binant IFN preparations, IFN-a-2b, or by an IFN-y preparation (see above; both preparations contained 10 5 I. U.) (10 8 bacterial cells/ml).The results obtai ned are given in Table 3.
The data presented in Table 3

Fig. 1 . 8 Fig. 2 .
Fig. 1.Sensitivity changes (%) to antibiotics (7-70) following Enterobacteriaceae passages in RGNR cells: 7 -stable; 2 -unstable; 3 -amk; 4 -amp; 5chl; 6 -cip; 7 -kf; 8 -caz; 9 -его; юctx different cell lines revealed the same phenomenon.The next step of our investigations was the susceptibility determination of a gram-positive bac terium, S. aureus у during its growth in the cell line L41.Analyzing these results, it should be noted that the antibiotic susceptibility profile changed only for a single S. aureus strain; the (S R) transition was found for such antibiotic compounds as oxacillin, clindamycin, and cefazolin.The data concerning the antibiotic susceptibility changes for yeast-like fungi (Candida genus repre sentatives and Cr.neoformans) are shown in Fig. 5.The data obtained show that the (S -> R) and (R -» -» S) transitions are found in seven Candida strains among 17 studied.The (R -> S) transition was de tected for anti-Candida drugs: fluconazole (in 4 strains) and clotrimazole (in 2 strains).The transition (S R) was found for amphotericin В (in 5 strains), fluconazole (in 1 strain), and clotrimazole (in 1 strain).

Fig. 3 .Fig. 4 .Fig. 5 .
Fig. 3. Sensitivity changes (%) to antibiotics (1-13) following Enterobacteriaceae passages in OH-1 cells: / -stable; 2 -unstable; 3 -amp; 4 -amk; 5amx; 6 -ctx; 7 -tet; 8 -caz; 9 -tob; 10 -cip; 11 -его; 12 -chl; 13 -kf show that during two strains (724, 396) strains cultivation in L41 cell line, they change their susceptibility to antibiotics, the susceptible bacteria becoming resistant to amo xicillin/clavulanic acid, cefotaxime, ciprofloxacin, cef triaxone, and ceftazidime.The IFN-a-2b exerts no influence on susceptibility changes.The results obtained in the experiments with the IFN-y preparation concerning its effect on the strains 724 and 396 susceptibility/resistance suggest this cytokine to promote the R -» S transition for ami kacin, cefotaxime, ciprofloxacin, ceftriaxone, and cefazolin.Similar results were obtained while cultivating Enterococcus faecalis in human neutrophils with gamma-IFN (1) and Listeria monocytogenes in mac rophages with the same cytokine (2).All these data allow the conclusion that the Bauer-Kirby test as well as other similar tests using bacterial nutrient media do not give information sufficient for therapeutic schema elaboration including antibiotic use in vivo.They need additional correction to determine real antibiotic susceptibility for different infectious agents.The cell-produced IFNs may exert their effect on bacteria ability to change their anti biotic susceptibility.