The influence of cultivation temperature on some phenotypic traits of Yersinia pseudotuberculosis Текст научной статьи по специальности «Биологические науки»

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THE INFLUENCE OF CULTIVATION TEMPERATURE ON SOME PHENOTYPIC TRAITS OF YERSINIA PSEUDOTUBERCULOSIS

Valerii USHKALOV1, Aidyn SALMANOV2, Liliia KALACHNIUK1, Yuriy VISHOVAN1, Sergiy BOIANOVSKIY1, Artem USHKALOV3, Andry GRANATE1, Goma M. HUWIAGE4, Oleksandra KEPPLE1

1National University of Life and Environmental Sciences of Ukraine, Ukrainian Laboratory of Quality and Safety of Agricultural Products

2Shupyk National Medical Academy of Postgraduate Education, Ukrainian National Committee of Infection Control (UCIC) and Antimicrobial Resistance (UCAST)

3Main administration of state service of Ukraine on food safety and consumer protection in Kharkov region 4High Institute of Medical Science & Technology, Algarabolli, Libya

Corresponding author: Valerii Ushkalov, e-mail: ushkalov63@gmail.com

DOI: 10.38045/ohrm.2020.1.14

UDC: 616.98:578.834.1(478)

Keywords: Yersinia pseudotuberculosis, cultivation temperature, phenotypic traits, antibiotics, resistance, sensitivity, biofilms.

Cuvinte cheie: Yersinia pseudotuberculosis, temperatura de cultivare, träsäturi fenotipice, antibi-otice, rezistenpä, sensibilitate, biofilme.

Introduction. Some causative agents of sapro-zoonotic infections can multiply in the external environment (water reservoirs, soils, plants), as well as in animals, including microorganisms of the genus Yersinia.

Material and methods. Isolation and identification of Y. pseudotuberculosis was carried out in accordance with the instructions on "Epidemiology, laboratory diagnosis ofyer-siniosis, organization and conduct of preventive and anti-epidemiological measures". Antibiotic sensitivity was performed via the disc diffusion method in accordance with EUCAST and national guidelines. Biofilm formation was tested using the spectrophotomet-ric assay.

Results. It was established that the studied cultures showed a decrease in the level of saccharolytic activity during cultivation at a temperature of+37°C in comparison with the results obtained at 25°C, changes in sensitivity to antibiotics depending on the temperature of cultivation were revealed. It was established that Y. pseudotuberculosis cultures were able to form denser (A 570) biofilms when cultured at 25°C, in comparison with biofilms formed at 37°C.

Conclusions. Biological characteristics of the studied Y. pseudotuberculosis isolates (changes in the saccharolytic activity, the level of sensitivity to antibiotics and the formation of biofilms were revealed) depends on the cultivation conditions.

INFLUENTA TEMPERATURII DE CULTIVARE ASUPRA UNOR PROPRIETATI FENOTIPICE ALE YERSINIA PSEUDOTUBERCULOSIS

Introducere. Unii agenti cauzali ai sapronozelor se pot multiplica atat in mediul extern (rezervoare de apa, sol, plante), cat si in organismul animalelor, la astfel de agenti pato-geni atribuindu-se si bacteriile din genul Yesinia.

Material si metode. Izolarea si identificarea Y. pseudotuberculosis a fost efectuata in conformitate cu instructiunea „Epidemiologia, diagnosticul de laborator al yersiniozei, organizarea si desfasurarea masurilor preventive si antiepidemice". Sensibilitatea la antibiotice a fost efectuata prin metoda difuzimetrica, in conformitate cu EUCAST si gidul national. Capacitatea de a forma biofilme, cat si densitatea lor, au fost determinate prin metoda spectrofotometrica dupa densitatea optica.

Rezultate. Culturile studiate au demonstrat o scadere a nivelului activitapii zaharolitice, in timpul cultivarii la temperatura de +37°C, in comparatie cu rezultatele obtinute la 25°C, totodata inregistrandu-se pi diferenpe ale sensibilitatii la antibiotice in functie de temperatura de cultivare. S-a mai stabilit ca culturile de Y. pseudotuberculosis au fost capabile sa formeze biofilme mai dense (A 570) la temperatura de 25°C, decat cele formate la 37°C. Concluzii. S-a stabilit ca, manifestarea proprietatilor biologice ale izolatelor studiate de Y. pseudotuberculosis (modificari ale activitapii zaharolitice, nivelul de sensibilitate la antibiotice si formarea de biofilme) depinde de conditiile de cultivare.

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INTRODUCTION

Some causative agents of sapro-zoonotic infections are capable of reproduction in the external environment (water reservoirs, soils, plants), as well as in the body of animals, including microorganisms of the genus Yersinia. The disease-causing pathogens occurring in humans and animals Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are quite common. Yersiniosis is a foodborne gastrointestinal tract zoonotic disease that can be transmitted via contaminated food or water. In Ukraine, there are active natural reservoirs of Y. pseudotuberculosis. Bacteriocarrier is recorded among synanthropic and agricultural animals, which determines the source of the pathogen and the factors of its transmission. Contamination of livestock products and/or food causes human diseases. There are evidences, suggesting that Y. pseudotuberculosis serotype O1b has been isolated due to in-tragenomic rearrangements and deletions of Y. pestis. The pathogenicity of Y. pseudotuberculosis is determined by genes that dictate invasive and cytotoxic properties of the pathogen (1). Therefore, the isolation of pure culture, as well as the identification of Y. pseudotuberculosis for a particular serovar, creates the basis for diagnosing pseudotuberculosis.

Pseudotuberculosis is an infectious disease of various animal species, accompanied by intoxication, the formation of caseous nodules and gran-ularnecrotic lesions like tuberculosis in various organs. The disease is characterized by fever, intoxication, damage to the small intestine and liver, scarlet fever-like rash.

The main route of infection is the alimentary one.

Yersinia spp. are bipolar-staining, gram-negative facultative anaerobic bacteria. The optimum cultivation temperature is 28-29°C, and can vary from 2 to 40°C. They retain mobility at 22-28°C (except Y. pestis) and become motionless at 37°C. It should also be noted that Y. enterocolitica has a higher motility compared to other Yersinias. At low temperature conditions (4-8°C), bacteria form smooth (S) colonies with slow accumulation.

At temperatures above 28°C, Y. Pseudotuberculosis dissociates into a rough (R) form. When isolated from the body of the patient or from animals, Y. pseudotuberculosis and Y. enterocolitica

exhibit an S-form. However, sometimes, the cultures of Y. pseudotuberculosis are isolated in R, SR-, R-S-forms (1, 2).

Demidova GV, Zyuzina VP, et al. (2009) examined the pathogenic strains of Y. pseudotuberculosis and found that, due to their sensitivity to polymyxin B, the studied strains, belonging to different serotypes can be divided into groups, particularly in cultures of serotypes I and IV, which were sensitive to polymyxin B at 28°C and resistant to 37°C (3).

Titareva GM, Fursova NK, Balakhonov SV. (2003) examined strains of Y. pestis and reported that the strain resistance, cultured at 25°C is higher than that of strains cultured at 37°C. Therefore, the study of the biological properties of cultures under incubation temperature conditions of 23-28°C and 37°C is an important feature of the differential diagnosis of Yersinia spp. (4). The ability to form biofilms is considered as a factor providing selective advantages of microorganisms in biological niches (according to CDC data, nearly 80% of human bacterial infections are caused by polymicrobial biofilms) (5, 6). The aim of the study was to study the biological properties of Y. pseudotuberculosis strains.

MATERIAL AND METHODS

The study carried out the Y. pseudotuberculosis isolation and identification in accordance with "Epidemiology, laboratory diagnosis of yer-siniosis, the organization and conduct of preventive and anti-epidemiological measures" instructions (7). Five cultures of Y. pseudotuberculosis were investigated, which have been observed in synanthropic rodents.

The morphological features and mobility of Yersinia cultures were determined according to the instructions (7). The morphological features were determined via microscope of Gram-stained smears. To determine the motility of bacteria, preparations such as "crushed drop" were prepared from daily agar cultures grown at 23°C and 37°C.

The enzymatic properties such as the ability of microorganisms to form acetyl methyl carbinol (acetoin) during glucose fermentation in the Foges-Proskauer reaction were determined; phenylalanine deaminase, the capacity for escu-lin hydrolysis, indole/urea, urease and orni-thine de carboxylase were detected; the saccha-

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rolytic properties on the Hiss medium with sugars were also revealed.

Antimicrobial susceptibility testing was performed using the EUCAST disk diffusion method. (version 10) and MU on "Determination of microorganisms susceptibility to antibacterial drugs" (MOH, 2007) (8, 9).

The ability of isolates to form biofilm (by method C. C. Heilmann E, 1996) and the biofilm optical density indexes (by spectrophotometrically) in three cultures of Y. pseudotuberculosis were evaluated, as well as in Pasteurella multocida, Salmonella Typhimurium, and Salmonella Enter-itidis, E. coli, S. aureus (10, 11) under different cultivation regimes (25°C and 37°C).

The nutrient media, commercial tests, and discs with antimicrobial drugs manufactured by HiMedia were used within the study.

RESULTS

The morphological features of the obtained cultures, as well as the motility in a "crushed drop" type drug have been determined via the microscope of the Gram-stained smears. To determine the bacterial motility, the one-day agar cultures have been prepared under two cultivation regimes (23°C and 37°C).

Over 24 hours, reddish transparent convex (sometimes greyish-yellow oily colonies with 0.5-1mm diameter), and granular or tubular colonies of S-, O- and R-forms are formed on meat-and-peptone agar, Endo agar, blood agar. The S-forms were represented by smooth, convex colonies. The R-forms had granular colonies with darkened center and thin lace-type periphery; this pattern was also found in the pathogen of zoonotic plague (camel plague).

The gram-negative organisms of ovoid (coccoid-like) form (0.8-5.0^m length, 0.4-0.8^m width), as well as the organisms with rounded tails (1.5 -6 |j.m length, 0.4-0.8^m width) were observed in the smears. Some bacterial cultures exhibited bipolarity properties. The study of motility in cultures showed that they were stationary at 37°C and mobile at 23°C.

Studies of bacterial enzyme properties showed that the studied cultures did not exhibit the ability to form acetyl methyl carbinol in the Foges-Proskauerre action at +23°C and +37°C; they hydrolyzed the esculin and did not form phe-nylpyruvic acid (determination of phenylalanine deaminase) and ornithine decarboxylase (tab. 1). A decrease in saccharolytic activity of the cultures was recorded when cultivated at +37°C (doubtful reaction).

Table 1. Enzymatic properties of Y. pseudotuberculosis.

Enzymatic properties

uu 3 « g £ S S g

g Mobility c ° s 8 I I i

okS ^ < ^ a s s

o £ tu ^ 3 re

i« _ 22 £ re re u

«

.5 — —

'g ^ w ö

« S S 2 s

— —

O- CU

^ w w S

S <V <V S

<d M M e

-C o os-

ex IX IX p

u u U u u u u u u u u u

o o o o o o o o o o o o

ro ro ro ro ro ro

CN ro CN ro CN ro CN ro CN ro CN ro

+ ± + ± + ± + ± + ± + ±

Y. p 1

+

+

Y. p 2 + - + -- -- + ± + ± + ± + ± + ± + -

Y. p 3 + - + - - - - + ± + ± + ± + ± + ± + ±

Y. p 4 + - + - - - - + ± + ± + ± + ± + ± + -

Y. p 5 + - + - - - - + ± + ± + ± + ± + ± + -

Antibiotic resistance was determined in 5 obtained isolates via the disc diffusion method (tab. 2), and the results were interpreted according to

EUCAST (version 10) and MU on "Determination of microorganism susceptibility to antibacterial drugs" (MOH, 2007).

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Table 2. Susceptibility to antibacterial drugs Y. pseudotuberculosis.

The name of the drug Y. pseudotuberculosis, diameter of crop growth inhibition, mm, (n = 5)

Y. p 1 Y. p 2 Y. p 3 Y. p 4 Y. p 5

23°C 37°C 23°C 37°C 23°C 37°C 23°C 37°C 23°C 37°C

Piperacillin <17; 20<; 35 26 34 25 34 28 28 20 37 28

Ampicillin <14; 14<; 31 17 30 16 20 11 13 6 20 11

Cefalexin <14; 14<; 31 23 30 22 36 24 28 20 28 20

Cefuroxime <19; 19<; 31 24 30 23 28 21 30 22 30 23

Cefotaxime <17; 20<; 37 23 36 22 32 23 28 20 36 22

Ceftriaxone <22; 25<; 40 30 40 30 34 25 34 25 34 25

Cefepime <24; 27<; 22 22 21 21 34 34 26 26 28 28

Imipenem <17; 22<; 24 24 24 24 36 36 30 30 32 32

Meropenem <16; 22<; 22 36 23 37 24 36 28 40 24 37

Gentamicin <17; 17<; 22 22 23 23 24 24 26 26 28 28

Nalidixic acid 30 30 31 31 36 36 42 42 30 30

Ciprofloxacin <22; 25<; 30 30 31 31 26 40 34 34 34 34

Norfloxacin <22; 22<; 26 34 27 35 32 40 20 28 30 38

Ofloloxacin <22; 24<; 34 43 36 38 26 43 30 38 26 36

Levofloxacin <19; 23<; 30 26 32 36 22 26 30 40 40 35

Chloramphenicol <17; 17<; 32 23 31 20 23 20 30 21 30 20

Polymyxin B 6 16 6 18 6 16 6 12 6 6

The examined cultures were predominantly sensitive to penicillins, cephalosporins, carba-penems, quinolones, gentamicin, and chloramphenicol, both at +23°C and at +37°C. Individual cultures exhibited resistance (Y. p 4 to Ampicillin; Y. p 1 and Y. p 2 to Cefepime) and moderate resistance (Y. p 4 to Cefepime) under given conditions.

The cultures of Y. p 3 and Y. p 5 exhibited sensitivity to Ampicillin at +23°C and resistance at +37°C. At +23°C, a culture Y. p 4 showed resistance to Norfloxacin. The Y. p 3 culture showed moderate level of resistance to Levofloxacin at +23 °C but remains uniformly susceptible to it at +37°C.

The culture sensitivity to Cefepime, Imipenem, Gentamicin, Nalidixic acid was the same under the given temperature conditions. In general, most bacterial cultures demonstrated changes in drugs susceptibility depending on temperature conditions. Thus, the lower level of bacterial susceptibility was recorded at +37°C - to Piperacillin, Ampicillin, Cefalexin, Cefuroxime, Cefo-taxime, Ceftriaxone, Chloramphenicol and at 23°C - to Meropenem, Norfloxacin, Ofloxacin, Levofloxacin, Polymyxin B.

The ability to form biofilms and their density (tab. 3) were determined in Y. pseudotuberculosis cultures (Y. p 1, Y. p 2, Y. p 3) at 25°C (S-form) and 37°C (R-form); the obtained results were compared with similar indicators of Pasteurella

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multocida culture (cultivation on heart-brain broth (HBB)), as well as Salmonella Typhimuri-um and Salmonella Enteritidis (cultivation on meat-peptone broth (MPB), E. coli (STX2) and

associated E. coli culture (STX2) + Proteus mirabilis (cultivation on MPB), S. aureus (cultivation on tryptone-soy broth (TSB)).

Table 3. Indicators of biofilm optical density produced by cultures under different cultivation conditions.

i n s

Culture ne s ¡1 S3 o +3 « > +3 o LO << o r t tn o u actual value

■> c 5 S a) u £ S i- s u u o H © LO << (À 570 - À 570 control)

Y. p 1

Y. p2

Y. p 3

25°C

2.8781

MPB

2.7358

37°C 1.6416 1.4993

25°C 2.9901 A -I A10 0.1423 2.8478

37°C 1.6734 1.5311

25°C 3.6737 3.5314

37°C 1.4557 1.3134

25°C 1.4218 1.1974

37°C 2.5968 0.2244 2.3734

37°C 0.9048 0.6804

P. multocida

S. Typhimurium S. Enteritidis

SMB

37°C

1.1209

0.8965

E. coli

37°C

0.3798

0.1423

E. coli+P. mirabilis

MPB

37°C

1.3265

0.1423

0.2375

1.1842

S. aureus

TSB

37°C

4.6778

0.3719

4.3049

Y. pseudotuberculosis were reported to form biofilms. All studied bacterial cultures (S colonies) formed high density bacterial associations at 25°C (Y. p1 - A 2.8781, Y. p2 - A 2.9901, Y. p3 -A 3.6737). When cultured at 37°C (in R-form), the biofilm optical density was significantly lower (Y. pi - A 1.6416, Y. p2 - A 1.6734, Y. p3 - A 1.4557), which is respectively 45.2%, 46.2%, 62.8% of the optical density of the cultures at 25°C.

The evaluation of the optical density in other cultures (S colonies) showed significantly lower optical densities of biofilms compared to cultures of Y. pseudotuberculosis, namely in S. Typhimurium - A 0,6804, S. Enteritidis - A 0.8965, E. coli A - 0.2375, and in the associated culture of E. coli O723 (STX2) + Proteus mirabilis A - 1.1842, which was 23.89%, 31.48%, 8.34%, and 41.58% respectively, from the optical density index of the S-shaped culture (at 25°C) Y. pseudotuberculosis 2 (A 2.8478).

The coagulase-positive culture of S. aureus formed a much higher density biofilm than Y.

pseudotuberculosis cultures. The optical density of S. aureus was A 4.3049, which made up 151.17% of optical density (at 25°C) Y. pseudotuberculosis 2 (A 2,8478).

The ability to form biofilms in P. multocida cultures was slightly lower than in Y. pseudotuberculosis cultures. At 25°C and 37°C, the optical density of P. multocida cultures were A 1.1974 and A 2.3734, respectively, that is, at 25°C the P. multocida optical density was 50.5% of that of P. multocida at 37°C.

DISCUSSIONS

The obtained results proved that the studied cultures of Y. pseudotuberculosis didn't show the ability to form acetyl methyl carbinol at +23°C and +37°C; they hydrolyzed the esculin and did not form phenyl pyruvic acid and ornithine decarboxylase; a constant decrease in the level of saccharolytic activity of cultures was observed during cultivation at +37°C.

An analysis of the results showed that, Y. pseudotuberculosis antibiotic susceptibility changes to

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most drugs depending on the cultivation temperature, decrease of sensitivity level to Piperacillin, Ampicillin, Cefalexin, Cefuroxime, Cefotaxime, Ceftriaxone, Chloramphenicolwasrecorded, and at 23°C - to Meropenem, Norfloxacin, Of-loloxacin, Levofloxacin, Polymyxin B at +37°C.

Biofilm formation is regarded as a pathogenicity factor. This study investigated the ability to form biofilmsin Y. pseudotuberculosis cultures compared with Pasteurella multocida, S. Typhimuri-um, S. Enteritidis, E. coli, S. aureus.

The obtained isolates of Y. Pseudotuberculosis were capable to form denser bacterial biofilms at cultivation temperature of 25°C. At 25°C and 37°C, the Pasteurella multocida culture (S colonies), whereas at 37°C, it formed a denser biofilm. The biofilm-forming capacity of Y. Pseudotuberculosis cultures (S and R coloniesj to form biofilms is higher than S-form in other studied cultures of enterobacteria (S. Typhimurium, S. Enteritidis, E. Coli) and Pasteurella multocida, compared to the culture of coagulase-positive S. aureus.

CONCLUSIONS

1. It was established that the manifestation of the biological properties of the studied Y. pseudotuber-culosis isolates (in particular, changes in the saccharolytic activity, antibiotic susceptibility and biofilm formation capacity were revealed) depends on the cultivation conditions.

2. The obtained isolates of Y. pseudotuberculosis were able to form denser bacterial biofilms at a culture temperature of 25°C, compared with the formation of biofilms at cultivation temperature +37°C.

3. The biofilm-forming capacity Y. pseudotuberculosis cultures (S and R colonies) is higher than S-form in other studied cultures of enterobacteria (S. Typhimurium, S. Enteritidis, E. Coli) and Pasteurella multocida, compared to the culture of coagulase-positive S. aureus.

CONFLICT OF INTERESTS

All authors declare no competing interests.

ACKNOWLEDGMENT

Research was carried out with the financial support of the Ministry of Education and Science of

Ukraine.

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Date of receipt of the manuscript: 03/04/2020 Date of acceptance for publication: 30/05/2020

Valerii USHKALOV, ORCID: 0000-0001-5694-632X, SCOPUS Author ID: 36130483300, Web of Science ID researcher AAS-4217-2020

Aidyn SALMANOV, ORCID: 0000-0002-4673-1154, SCOPUS Author ID: 56964145900

Liliia KALACHNIUK, ORCID: 0000-0002-5545-8495, SCOPUS Author ID: 6602997554, Web of Science Researcher ID: M-5019-2018

Yuriy VISHOVAN, ORCID: 0000-0003-1128-593X Sergiy BOIANOVSKIY, ORCID: 0000-000204621-5192 Artem USHKALOV, ORCID: 0000-0001-8317-7909 Andry GRANATE, ORCID: 0000-0003-3273-903X Goma M. HUWIAGE, ORCID: 0000-0002-2727-148X Oleksandra KEPPLE, ORCID: 0000-0002-8123-3310