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Research Article
Investigation of the activity of new derivatives of 1,3-diazinone-4 and their acyclic precursors with respect to bacteria of the genus Proteus
expand article infoSvetlana Luzhnova, Andrey Voronkov§, Narmina Gabitova, Souda Billel§
‡ Research Institute for the Study of Leprosy, Astrakhan, Russia
§ Pyatigorsk Medical and Pharmaceutical Institute, a branch of Volgograd State Medical University, Pyatigorsk, Russia
Open Access

Abstract

Introduction: The present paper provides a study of the activity of the new 1,3-diazinon-4 derivatives and their acyclic precursors under the laboratory cipher PYaTd1, PYaTs2, PYaTs3 and PYaTs4 against microorganisms of the genus Proteus, which is of high importance at the moment as the growing resistance of the Proteus to previously highly active antibiotics dictates the need to search for effective antimicrobial agents that meet modern safety requirements.

Materials and Methods: The study of the activity of the compounds was carried out on collection and freshly isolated strains from patients with different pathologies. The strains were identified using the BIOMIC V3 apparatus (Giles Scientific, USA) to verify genus and species identity. The strains used in the study were previously examined for susceptibility to antibacterial drugs by the Disc Method to assess the presence or absence of resistance. The activity of the new compounds was studied by the serial dilution method.

Results: The results of the study showed that the compounds PYaTd1, PYaTs2, PYaTs3 and PYaTs4 show a different activity against bacteria of the genus Proteus. The substance PYaTs2 is ineffective. With respect to strains P. mirabilis and P. rettgeri, the minimum inhibitory concentration of the compounds PYaTs3, PYaTs4 and PYaTd1 ranges from 4 μg/ml to 16 μg/ml.

Conclusion: Thus, by the average aggregate indices, regardless of the species and strain of bacteria, the most effective compound is PYaTd1, the MIC50 of which is within 10 μg/ml, which proves it to be promising and makes further development worthwhile.

Keywords

compounds PYaTd1, PYaTs2, PYaTs3, PYaTs4, P. mirabilis, P. rettgeri, P. vulgaris, activity, MIC.

Introduction

Urinary tract infections are amongst the most common bacterial infections in humans caused by bacteria of the genus Proteus (P. vulgaris, P. mirabilis, P. rettgeri) (Pellegrino et al. 2013, Budnik 2015, Liu et al. 2015). In 70% of cases, there are protei in cases of urolithiasis (Schaffer et al. 2016). P. vulgaris along with Escherichia and Pseudomonas aeruginosa, staphylococci and streptococci, as well as with anaerobic clostridia, often complicates the course of purulent and anaerobic infections. P. mirabilis often insulates wounds, forming biofilms, which protects the pathogen from the effects of antimicrobial agents and makes treatment difficult (Leblebicioglu and Esen 2003, Schaffer et al. 2016). P. mirabilis is known as an etiological agent of the onset of nosocomial pneumonia (Pаlаgin et al. 2012). There is an increased interest towards the role of Proteus in the etiology of rheumatoid arthritis (RA) and towards the development of chemotherapeutic drugs which are active against protei that can reduce the risk of and/or worsening RA development (Ushakova et al. 2001, Disaanayake et al. 2014).

Recent studies have shown that the problems of treating Proteus infections are linked to a growing number of strains resistant to antibiotics. In various countries of the world, studies of the resistance of protei to antimicrobial agents prove the proliferation of Proteus – producers of extended-spectrum β-lactamases (Cremet et al. 2011, Schmiemann et al. 2012, Corvec et al. 2013, Chen et al. 2015).

Despite intensive activities to contain and control antibiotic resistance, the problem remains extremely urgent. In this regard, WHO has published a list of “priority pathogens”, which includes enterobacteria (Budnik 2015).

The growing resistance of the pathogen to the previously highly active antibiotics makes it a high priority to search for effective antimicrobial agents that meet modern safety requirements.

Currently, there are opportunities to significantly increase the effectiveness of research on the synthesis of new pharmacologically active substances by using modern computer technologies that allow molecular design of target structures with a predictable effect (Filimonov and Porojkov 2006, WHO 2017).

New derivatives of 1,3-diazinon-4 and their acyclic precursors were created and synthesised by the staff of the Department of Organic Chemistry of Pyatigorsk Pharmaceutical Institute (Kodonidi 2010, Oganesyan et al. 2013). A preliminary analysis of the potential biological properties of these compounds was carried out using the PASS programme, which made it possible to conclude that the occurrence of various types of biological activity was likely. According to the computer forecast, they should have antibacterial actions. The primary screening of fifteen new derivatives of diazinon showed that they exhibited activity against protei. Some promising leaders were chosen for further development (Luzhnova et al. 2017).

Goal

To conduct a study of the activity of the new 1,3-diazinon-4 derivatives and their non-cyclic precursors coded in the laboratory as PYaTd1, PYaTs2, PYaTs3 and PYaTs4 for microorganisms of the genus Proteus.

Materials and methods of research

Investigation of the activity of the compounds PYaTd1, PYaTs2, PYaTs3 and PYaTs4 with reference to Proteus was carried out on the collection strains: P. mirabilis NIIL tya1, NIIL tya2 (collection “NIIL”, Astrakhan), P. mirabilis VK 294, VK 299 (State-funded Hospital JSC “SM Kirov City Clinical Hospital No. 3”, Astrakhan); isolates - P. rettgeri SES 11/01, SES 11/02 (isolated from the intestines of the patients - Sanitary and Epidemiological Station, Astrakhan); P. mirabilis VK 190, VK 194 (isolated from trophic ulcers), P. vulgaris VK 01, VK 02 (isolated from the intestine) - State-funded Hospital JSC “SM Kirov City Clinical Hospital No. 3”, Astrakhan.

All the strains used in the work were identified by means of the BIOMIC V3 software system (Giles Scientific, USA) to determine their belonging to the genus and species by using the bio Merieux APL kit, which includes strips containing individual biochemical markers, to identify protei.

The activity of the compounds was studied by the serial dilution method (Navashin and Fomin 1974). In these studies, the concentration of the substances under study in the series of serial dilutions was decreased in a geometric progression by a factor of 2: 128 μg/ml, 64 μg/ml, 32 μg/ml, 16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml, 1 μg/ml, 0.5 μg/ml and 0.25 μg/ml. Control was the tubes containing meat-peptone broth (MPB) without adding the compound and microorganisms (control of the medium sterility), with adding microorganisms without adding the substances (positive control), inoculations with a solvent (DMSO in equivolumes) and serial dilution series of the reference substances: sulphodimethoxin and streptocid for PYaTs2, PYaTs3 and PYaTs4 containing a fragment of sulphanilamide and dapsone for PYaTd1 containing a dapsone fragment.

The quantity of the compound (drug) of 4 mg was dissolved in 0.5 ml of dimexide, then 4.5 ml of physiological solution was added thereto to prepare a working solution from which, by means of dilution, a number of tubes with preset concentrations were formed.

To prepare a suspension of bacteria, a one-day culture was used. The bacterial suspension (0.05 ml) of a certain density (0.5 McFarland standard) was added to each tube with MPB BCH of a series of dilutions of the tested substances, including positive control. This culture was incubated for 24 hours at a temperature of +37 °C. At the end of this period, the presence of microorganisms growth was visually assessed in each of the tubes; then the contents of the tubes were centrifuged at 1500 rpm for 10 minutes. The supernatant was removed. The deposit was washed twice with physiologic saline solution. From each tube, 0.02 ml of the deposit was plated on Ploskirev’s medium. The culture was incubated for 24 hours at a temperature of +37 °C; then the grown colonies were counted, using the BIOMIC V3 microbiology system (Giles Scientific, USA). Based on the results obtained, the minimum inhibitory concentrations of the compounds – MIC50 and MIC90-100 were calculated (LibUSSR.RU 2014, Semina et al. 2004).

The strains used in this work were previously tested for susceptibility to antibacterial drugs by the Disc Test to determine the presence or absence of resistance (Semina et al. 2004). A set of indicator discs DI-PLS-50-01 (CJSC Research Centre for Pharmacotherapy, St. Petersburg) was used. The results were read using the BIOMIC V3.

The results were statistically processed using computer software BIOSTAT 2009 (Analist Soft Inc., USA). The variation series was checked for normality by the Kolmogorov-Smirnov criterion. The index of statistical significance was determined by the Student’s t-test. Statistically significant were the variations at p≤ 0.05-0.01.

Results and discussion

The strains of Proteus used in the work displayed a wide range of antibiotic susceptibility: from high sensitivity to antibacterial drugs to multidrug resistance (Table 1).

Sensitivity of strains of the genus Proteus to antibiotics

Antibiotics P. mirabilis P. rettgeri P. vulgaris
NIIL tya1 NIIL tya2 BK 294 BK 299 BK 190 BK 194 SES 11/01 SES 11/02 BK 01 BK 02
Ampicillin R R S R S S S S R R
Amoxicillin-clavulanate I I S I S S S S R R
Ciprofloxacin R R S I S S S S R R
Gentamicin S S S S S S S S R R
Levofloxacin I I S S S S S S R R
Ceftazidime I I S S S S S S S R
Cefixime S S S S S S S S S S
Cefotaxime R I S S S S S S S S
Cefepime I I S S S S S S S I
Ampicillin-sulbactam I I S I S S S S R S
Ticarcillin-clavulanate I I S S S S S S S S
Imipenem S S S I S S S S S S
Ceftriaxone R R S I S I S S S S
Amikacin R I S I S I I I S S
Norfloxacin I I S S S I S S S S

Analysis of the cultured collection strains P. mirabilis showed that the compounds codified as PYaTs3 and PYaTs4 suppress their growth within the concentration range of 128-4 μg/ml, with a concentration of 8-4 μg/ml being the MIC50 and 128 μg/ml corresponding to the MIC85–MIC100, i.е. close to bactericidal. These strains also displayed sensitivity for the PYaTs2 compound in, but within, the concentration ranges of 128-16 μg/ml: the MPC50 corresponded in most cases to 32 μg/ml and, with 128 μg/ml, the growth rate decreased by 65–6% at the most.

The reference drugs for this group of compounds were less effective: the MIC50 of sulphadimethoxin was usually 32 μg/ml and its concentration of 128 μg/ml inhibited the growth of 55% of the population at most; MIC50 of streptocid was somewhat lower (Table 2) and the effect when exposed to 128 μg/ml was similar to that of sulphadimethoxin.

Activity of the compounds towards collection strains P. mirabilis (μg/ml)

Compound P. mirabilis NIIL tya1 P. mirabilis NIIL tya2
MIC50 МIC90-100 МIC50 MIC90-100
PYATs2 8 16
PYATs3 4 128 8
PYATs4 4 128 4
PYATd1 4 128 4
Sulphadimethoxin 16 32
Streptocide 8 16
Dapson 1 128 2 128
P. mirabilis ВК 294 P. mirabilis ВК 299
PYATs2 16 16
PYATs3 8 8
PYATs4 8 4
PYATd1 8 4
Sulphadimethoxin 32 32
Streptocide 16 16
Dapson 2 128 4 128

The compound PYaTd1 suppressed the growth of strains in the same ranges as PYaTs3 and PYaTs4. In comparison with dapsone, it was somewhat less active.

The analysis of cultured freshly isolated strains of P. mirabilis showed that the PYaTs3 and PYaTs4 compounds had an MIC50 of 8 μg/ml and, at a concentration of 128 μg/ml, the growth of 65% of the population at most was inhibited. The compound PYaTd1 also retained its activity within the concentration range of 4-8 μg/ml (MIC50) and the concentration of 128 μg/ml corresponded to IPC65-70 (Table 3).

Activity of compounds towards Proteus mirabilis strains (μg/ml) isolated from patients

Compound P. mirabilis BK194 P. mirabilis BK190
MIC50 MIC90–100 MIC50 MIC90–100
PYATs2 32 64
PYATs3 8 8
PYATs4 8 8
PYATd1 4 8
Sulphadimethoxin 64 128
Streptocide 16 16
Dapson 1 100 1 100

The effect of the reference drugs sulphanilamides was twice as weak as PYaTs3 and PYaTs4 (Table 3). High activity was shown by dapsone.

Activity of compounds towards Proteus rettgeri Strains (μg / ml) isolated from patients

Compound P. rettgeri SES 11/01 P. rettgeri SES 11/02
MIC50 MIC90–100 MIC50 MIC90–100
PYATs2 64 32
PYATs3 8 8
PYATs4 16 8
PYATd1 8 4
Sulphadimethoxin 128 64
Streptocide 1 2
Dapson 1 128 1 128

The analysis of the cultured strains of P. rettgeri isolated from patients showed that the activity of the compounds towards this type of protei is somewhat lower than that of P. mirabilis: the range of MIC50 in the PYaTs3 and PYaTs4 compounds was 8–16 μg /ml, the concentration of 128 μg/ml inhibited the growth of 65–75% of the population and the PYaTs2 functioned much more weakly (Table 4). The activity of the substance PYaTd1 towards this species remained at the same level. Sulphadimethoxin was ineffective. The activity of streptocid was higher: its MIC50 was 1-2 μg/ml, the maximum level of growth inhibition (128 μg/ml) not exceeding 65%. It was dapsone that actively inhibited the growth of the pathogen: at high concentrations, its effect was bactericidal and the MIC50 was 1 μg/ml (Table 4).

The results of the cultured P. vulgaris can be seen in Table 5, which shows that, for the given type of bacteria, the use of the tested compounds and reference drugs is less effective. In all the compounds of “s” group, MIC50 is 64 μg/ml and application of a dose of 128 μg/ml inhibits no more than 60-65% of the population of strains. The MIC50 range of PYaTd1 shifts to higher concentrations (Table 5) and, at the concentration of 128 μg/ml, 65-75% inhibition of the growth of the microorganism population can be seen.

Activity of compounds towards Proteus vulgaris strains (μg/ml) ssolated from patients

Compound P. vulgaris ВК 01 P. vulgaris ВК 02
MIC50 MIC90–100 MIC50 MIC90–100
PYATs2 64 64
PYATs3 64 64
PYATs4 64 64
PYATd1 32 16
Sulphadimethoxin 128 128
Streptocide 128 128
Dapson 32 128 8 128

The reference drugs sulphadimethoxin and streptocide were ineffective: at a concentration of 128 μg/ml, they inhibited the growth of no more than 50-65% of the strain. The activity of dapsone towards these strains was also reduced, but it acted efficiently in the zone of comparatively lower concentrations (Table 5).

Mean values of the activity of the compounds are shown in Table 6. It follows from the table that the PYaTs3 and PYaTs4 compounds containing a fragment of sulphonamides are much more active than the reference drugs. PYaTs2 was also more active than the reference drugs, but it was inferior to PYaTs3 and PYaTs4, since its MIC50 was twice as high.

The PYaTd1 compound, by activity, outperformed the compounds containing a fragment of sulphonamide: its MIC50 was 2-4 times less. The ability of PYaTd1 to suppress the growth of strains of the genus Proteus was somewhat inferior to that of the reference drug dapsone. The difference in the value of their MIC50 was not statistically significant, but the dapsone median value was statistically significantly lower.

Average statistical values of the activity of compounds towards strains of the genus Proteus

Compound MIC50
М±m, mkg/ml Ме±m, mkg/ml
PYATs2 37.6± 7.5*# 32.0±2.3*#
PYATs3 18.8±7.5**## 8.0±3.0**##
PYATs4 18.8±7.6**## 8.0±3.0**##
PYATd1 9.2±2.8 6.0±1.0^
Sulphadimethoxin 75.2±15.1 64.0±6.0
Streptocide 34.7±15.6 16.0±6.2
Dapson 5.3±3.0 1.5±1.2

Conclusion

Thus, the tested compounds display different activities towards bacteria of the genus Proteus. The PYaTs2 substance is ineffective. With respect to strains of P. mirabilis and P. rettgeri, the minimum inhibitory concentration of the compounds PYaTs3, PYaTs4 and PYaTd1 ranges from 4 μg/ml to 16 μg/ml, which falls within an allowable range of concentrations required for antibacterial drugs. With respect to strains of P. vulgaris, the activity of the compounds is much lower. The most effective compound, according to the mean values regardless of the species and strain of bacteria, is the compound PYaTd1, having an MIC50 within 10 μg/ml, which proves it to be promising and makes further development worthwhile.

References

  • Budnik TV (2015) Antibiotic resistance in the context of a urinary tract infection. Family Medicine [Semejnaya meditsina] 4(60): 77. [Russian]
  • Chen L, Al Laham N, Chavda KD, Mediavilla JR, Jacobs MR, Bonomo RA, Kreiswirth BN (2015) First report of an OXA-48-producing multidrug-resistant Proteus mirabilis strain from Gaza. Palestine Antimicrob. Antimicrobial Agents and Chemotherapy 59(7): 4305–4307. https://doi.org/10.1128/AAC.00565-15 [PubMed]
  • Corvec S, Beyrouthy R, Crémet L, Aubin,GG Robin F, Bonnet R, Reynauda A (2013) TEM-187 a new extended-spectrum β-lactamase with weak activity in a Proteus mirabilis clinical strain. Antimicrobial Agents and Chemotherapy 57(5): 2410–2412. https://doi.org/10.1128/AAC.01761-12 [PubMed] [PMC]
  • Cremet L, Bemer P, Rome J, Juvin ME, Navas D, Bourigault C, Guillouzouic A, Caroff N, Lepelletier D, Asseray N, Perrouin-Verbe B, Corvec S (2011) Outbreak caused by Proteus mirabilis isolates producing weakly expressed TEM-derived extended-spectrum β-lactamase in spinal cord injury patients with recurrent bacteriuria. Scandinavian Journal of Infectious Diseases 43(11–12): 957–961. https://doi.org/10.3109/00365548.2011.601756 [PubMed]
  • Disaanayake DM, Faoagali J, Laroo H, Hancock G, Whitehouse M (2014) Efficacy of some colloidal silver preparations and silver salts against Proteus bacteria one possible cause of rheumatoid arthritis. Inflammopharmacology 22(2): 73–77. https://doi.org/10.1007/s10787-013-0198-0 [PubMed]
  • Filimonov DА, Lagunin АА, Gloriozova TА, Rudik АV, Druzhilovskij DS, Pogodin PV, Porojkov VV (2014) Prediction of spectra of biological activity of organic compounds using the web resource PASSONLINE. Chemistry of heterocyclic compounds [Khimiya geterotsiklicheskikh soedinenij] 3: 483–499. [Russian] [Abstract]
  • Filimonov DА, Porojkov VV (2006) Prediction of biological activity spectrum for organic compounds. Russian Chemical Journal [Rossijskij khimicheskij zhurnal] L(2): 66–75. [Russian]
  • Fusco A, Coretti L, Savio V, Buommino E, Lembo F, Donnarumma G (2017) Biofilm Formation and Immunomodulatory Activity of Proteus mirabilis Clinically Isolated Strains. International Journal of Molecular Sciences 18(2): E414. https://doi.org/10.3390/ijms18020414 [PubMed] [PMC]
  • Kodonidi IP (2010) Pharmacy [Farmatsiya]. Volume 1, 36–40. [Russian]
  • Leblebicioglu H, Esen S (2003) Hospital-acquired urinary tract infections in Turkey: a nationwide multicenter point prevalence study. The Journal of Hospital Infection 53(3): 207–210. https://doi.org/10.1053/jhin.2002.1362 [PubMed]
  • Luzhnova SА, Voronkov АV, Kodonidi IP, Gabitova NM, KHrapova АV, Souda Billel’ (2017) The activity of the new derivatives of 1,3-diazinon-4 and their non-cyclic precursors with respect to proteas. IX Annual All-Russian Congress on Infectious Diseases with international participation; March 27–29, 2017, Scientific and Publishing Center “Open Knowledge”, Moscow, 159 pp. [Russian]
  • Navashin SM, Fomin IP (1974) Reference book on antibiotics. Medicine, Moscow 415 рp. [Russia]
  • Oganesyan EHT, Kodonidi IP, Zolotykh DS, Lysenko TА (2013) Qsar for quinazolin-4-on derivatives, which poses anti-inflammatory, analgesic and antipyretic activity. Questions of Biological, Medical and Pharmaceutical Chemistry [Voprosy biologicheskoj. meditsinskoj i farmatsevticheskoj khimii] 11(1): 24–29. [Russian]
  • Pаlаgin IS, Sukhorukovа MV, Dekhnich AV, Ehjdel’shtejn MV, Shevelev AN, Grinev AV, Perepаnovа TS, Kozlov RS, Kogаn MI (2012) Current State of Antibiotic Resistance of Pathogens Causing Community-Acquired Urinary Tract Infections in Russia: “DARMIS” Study (2010–2011). Clinical Microbiology and Antimicrobial Chemotherapy [Klinicheskaya mikrobiologiya i antimikrobnaya khimioterapiya] 14(4): 280–302. [Russian]
  • Pellegrino R, Scavone P, Umpiérrez A, Maskell DJ, Zunino P (2013) Proteus mirabilis uroepithelial cell adhesin (UCA) fimbria plays a role in the colonization of the urinary tract. Pathogens and Disease 67(2): 104–107. https://doi.org/10.1111/2049-632X.12027 [PubMed]
  • Schaffer JN, Norsworthy AN, Sun TT, Pearson MM (2016) Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation. Proceedings of the National Academy of Sciences of the United States of America 113(16): 4494–4499. https://doi.org/10.1073/pnas.1601720113 [PubMed] [PMC]
  • Schmiemann G, Gágyor I, Hummers-Pradier E, Bleidorn J (2012) Resistance profiles of urinary tract infections in general practice – an observational study. BMC Urology 12: 33 https://doi.org/10.1186/1471-2490-12-33 [PMC]
  • Semina NА, Sidorenko SV, Rezvan SP, Grudinina SА, Strachunskij LS, Stetsyuk OU, Kozlov RS, EHndel’shtejn MV, Ved’mina EА, Stolyarova LG, Vlasova IV, Sereda ZS (2004) Методические указания МУК 4.2. 1890-04 "Determination of the sensitivity of microorganisms to antibacterial drugs". Federal Center for Sanitary Epidemiology of the Ministry of Health of Russia, 91 pр. [Russia]
  • Ushakova MА, Mouraviev YuV, Lebedeva VV (2001) To the problem of the possibile role of Proteus mirabilis in the development of rheumatoid arthritis. Scientific and practical rheumatology [Nauchno-prakticheskaya revmatologiya] 5: 52–57. [Russian]

Author Contributors

Svetlana A. Luzhnova, PhD in Biology, Senior Researcher, Research Institute for the Study of Leprosy, Ministry of Health of The Russian Federation, 3 Ostrovsky Lane, Astrakhan 414057, Russia; tel: +79171971467; e-mail: s.luzhnova@yandex.ru. Analysis and interpretation of the results and statistical processing.

Andrey V. Voronkov, Associate Professor, Doctor of Medical Sciences, Head of the Department of Pharmacology with a Course in Clinical Pharmacology; Deputy Director for Academic Affairs and Personal Development, Pyatigorsk Medical and Pharmaceutical Institute - a branch of Volgograd State Medical University, 11 Kalinin Ave., Pyatigorsk 357532 Stavropol region, Russia; phone: +79624273555; e-mail: prohor.77@mail.ru. Planning the study, analysing the results and literature on the research topic.

Narmina M. Gabitova, Junior Researcher, Research Institute for the Study of Leprosy, Ministry of Health of The Russian Federation, 3 Ostrovsky Lane, Astrakhan 414057, Russia; tel:+79061777286; e-mail: narmina85@inbox.ru. Performing laboratory studies and collecting source data.

Souda Billel, PhD student, the Department of Pharmacology with a Course in Clinical Pharmacology; Deputy Director for Academic Affairs and Personal Development, Pyatigorsk Medical and Pharmaceutical Institute - a branch of Volgograd State Medical University, 11 Kalinin Ave., Pyatigorsk 357532 Stavropol region, Russia; phone: +79624273555; e-mail: billel.souda@gmail.com. Setting tasks and goals of the experiment.