Isolation of bacteriophages with lytic activity against a newly identified pantoea agglomerans

Pantoea agglomerans (known also as Erwinia herbicola) is a gram-negative aerobic bacteria from the family Enterobacteriaceae. Species from the genus Pantoea are mostly plant pathogens, they can be isolated from biological waste, plants, and soil (1). There they can act as pathogens as well as commensals (15). P. agglom- erans is one of the genus species that mostly isolated from human feces, urine and blood. Moreover, it is one of common causes of nosocomial infections (3, 4). Wound infections with P. agglomerans usually caused by follow piercing of skin with a plant infected material (11, 21). In hospital conditions, exogenous infection with P. agglomerans mostly results in septic arthritis or synovitis, but there are also reports about endophthalmitis, periostitis, endocarditis and osteomyelitis (19). Immunodeficient individuals are the most vulnerable to those infections, especially if contaminated medical equipment is used (18). Fatal cases of nosocomial septicemia have been described in several countries, both in adults and children (2, 6, 14). Even though P.agglomerans has a quite good susceptibility to a list of antimicrobials (13), it has a potential to develop resistance to those medicines in near future like other common human pathogens from Enterobacteriaceae .

Antimicrobial resistance became a big threat in recent decades. Members of family Enterobacteriaceae, Staphylococcus sp. and Streptococcus sp. became unsusceptible to many antibiotics we used to (9, 10). To defeat these multiresistant pathogens, physicians use chemicals and medicines with numerous side effects that also have influence on human organism and especially microflora (22). As a number of resistant microorganisms increases every year, more scientists start to work with bacteriophages. Bacteriophages (viruses of bacteria) are the only alternative to antimicrobials as they are biologically safe and highly specific. In contrast with antimicrobials, phages persist in human organism until the last bacterial cell's death. Furthermore, phages don't interact with other bacteria in the gut and have not side effects that makes them more safe than antibiotics (12). Some phages are so-called polyvalent phages that are able to infect bacterial strains from different species and genera Many enterobacteria phages are polyvalent that is very important for developing phage- based drugs (7, 20). Taking into account that bacteria can develop resistance to phages as well as to drugs, phage polyvalence helps to solve this problem.

For P. agglomerans, phage therapy can be used in different aspects, such as reduction of bacterial residues on fruits or vegetables, sanitation of appliances used in agriculture, or use as an alternative for antibiotics in human disease control. Aim of our work was to isolate and identify an Enterobacteriaceae member from onions with bacterial rot as well as to find bacteriophages active against this bacteria.

Materials and methods.The content of brown rot from onions was placed on LB growth medium (gms/l: tryptone - 10, yeast extract - 5, NaCl - 10, agar - 10, pH=7,5). Bacterial cells were stained according to Gram and examined on magnification x100 (MICROmed XS-4130). Type of respiration was established after tests on cytochromoxidase with Hugh-Leifson medium (main solution: peptone - 2, NaCl - 5, KH2PO4 - 0,3, agar - 3, bromothymol blue 0,1% solution - 3 ml, pH=7,1, glucose solution: 10 g glucose, 60 ml dH2O). Biochemical diagnostic features of bacteria were investigated at the clinical diagnostic laboratory of Public national institution "Scientific and practical center of preventive and clinical medicine" of State Directorate for Affairs (PNI "SPC PCM" SDA) with a number of selective medium: Kligler's agar (enzymatic digest of casein - 10, enzymatic digest of animal tissue - 10, lactose - 10, dextrose - 1, ferric ammonium citrate - 0.5, NaCl - 5, Na2S2O3 - 0.5, phenol red - 0.025, agar - 15, pH = 7.2), urease (urea - 20, Na2HPO4 - 9.5, KH2PO4 - 9.1, yeast extract - 0.1, phenol red - 0.01, pH=6.8), TTC (Pancreatic digest of gelatin - 10, NaCl - 5, beef extract - 3, agar - 4, triphenyltetrazolium chloride - 0.5, pH=7.3), Simmons citrate agar (MgSO4 - 0.2, NH4H2PO4 - 1, K2HPO4 - 1, sodium citrate - 2, NaCl - 5, bromothymol blue - 0.8, agar - 15, pH=6.8) and lysine decarboxylase (Glucose - 1, L-lysine - 5, Bromocresol purple - 0.016, pH=6.1).

The sensitivity to antibiotics was determined by sensitivity discs "HiMedia". Bacteria was streaked onto a Petri dish with Mueller-Hinton Agar to form a bacterial lawn and then disks with antimicrobials (max.5 per plate) were plated on. Results was determined by diameter of diffusion of antibiotics around disks and were verified according to the standard of sensitivity of EUCAST.

The morphological features of bacterial colonies were studied using stereoscopic microscope (Biomed MS-1 ZOOM) at the PNI "SPC PCM" SDA. Phytopathogenic properties of bacterial isolate was investigated on potato in vitro. Cut off potato cubes (3-4 cm) were inoculated with night culture of bacteria and left in plates with sterile saline at 27°C.

In order to isolate bacteriophages the samples of waste waters were used. To amplify bacteriophages in samples enrichment method was applied. The content of brown rot lesions was transferred to liquid LB broth and incubated for 48 hours at 27°C. After incubation LB broth was centrifuged (5000 r/m, 25 min), supernatant was mixed with chloroform to remove bacteria. The samples were plated on a bacterial lawn by agar overlay method. Separate phage plagues were than picked and transferred to sterile saline (1 ml). Isolated bacteriophages were purified by serial propagation of single plaques.

To obtain high titer phage lysates were prepared from confluent lysis plates by adding 10 ml of saline to 10 plates. The soft-agar layers were scrapped off after 30 min. Lysates were clarified by low speed centrifugation at 15.000g for 15 min.

For bacteriophage staining, phage solution was deposited on formvar coated copper grid for two minutes and stained by 2% (w/v) uranyl acetate, pH 4 - 4.5. The solution was drained through filter paper and phage particles were observed through transmission electron microscopy (JEOL 1400, instrumental magnification of 40.000-90.000).

Results and discussion. A total of 5 onion bulbs with symptoms of bacterial rot were taken from different markets in Kyiv, Ukraine. Samples were plated on LB media that is common for many enterobacteria. Three samples out of five resulted in bacterial growth on LB medium. One of the isolates formed yellow, domed, shining and mucoid colonies (fig.1).

Figure 1. A - Colonies of P.agglomerans on plate with LB agar; B - Simmons citrate agar

agricultural phytopathogenic bacteria

The bacterial colonies were stained according to Gram and studied using light microscope. Isolate was Gramnegative, rod-shaped bacteria, 1-4 pm in length.

In Hugh-Leifson medium bacteria changed color of medium in aerobic and anaerobic conditions that indicates its ability to ferment glucose. This feature is common for facultative anaerobic bacteria.

Bacterial biochemical characterization was conducted using selective medium for Gram-negative bacteria: Kligler's agar, urease, TTC, Simmons citrate agar and lysine decarboxylase tests. The results of bacterial identification are presented in tab 1. Kligler's test indicated the ability of bacteria to ferment glucose, without H2S release. The urease test was negative, so that the bacteria were not capable to secrete an urease enzyme. TTC test was more positive than negative6 bacteria showed a spread throughout the medium but after 48h of incubation. Furthermore, the bacteria was able to utilize a citrate in the Simmons citrate agar. The results of lysine decarboxylase test was negative, therefore it indicated the inability of the bacteria to use a lysine as a source of carbon and energy for growth. This isolate was classified as Pantoea agglomerans (formerly Erwinia herbicola) according to the results obtained and to the Bergey's Manual of Systematic Bacteriology (tab. 1).

The results of bacteral susceptibility to antibiotics showed that isolated P.agglomerans was resitant to some of them. The resistance to cefalexin, cefotaxime, ceftazidime, ciprofloxacin, chloramphenicol, cefuroxime and norfloxacin was observed that indicates a general resistance to Я-lactams. Also, some antibiotics showed boundary values between resistance and sensitivity that indicates development bacterial resistance to them. The results of bacterial susceptibility to antibiotics are presented in the table 2.

Table 1

Table 2

According to data obtained our isolate of P.agglomerans can be the source of antibiotic resistance factors. Taking into account that this bacteria is crosskingdom pathogen, horizontal gene transfer with plant and human bacteria in the environment can cause an evolving of new multiresistant bacteria.

Then detection of pathogenic properties was conducted on potato cubes. This model system was chosen in order to test bacteria on other typical vegetable as the preliminary phytopathogenicity test on onions yielded the expected positive result. Bacteria caused tissue maceration resulting in rotting and formation of bacterial plaque on slices. P.agglomerans isolate showed to be capable of developing bacterial infection in plants and biochemical tests indicate its potential of human pathogen.

According to the obtained results, we attempted isolation of specific bacteriophages from waste waters that is the source of phages of human pathogens due to constant intake of human feces . Waste waters were taken from treatment facilities, then filtrated and plated with P.agglomerans by agar overlay method. As a result, specific bacteriophage was isolated (fig.2).

Figure 2. Phage plaques on bacterial lawn of P.agglomerans

Bacteriophage, given work name Eh1, produced small plaques, d<1 mm. After serial propagation, bacteriophages formed negative colonies on bacterial test cultures with no decrease in phage titres, which indicates the lytic cycle of viral reproduction and, moreover, is an indispensable condition for developing phage-based products.

Phage isolate Eh1 was investigated with electron microscopy and it was indicated that this virus was member of family Myoviridae (fig 3). Isolate had icosahedral head 95X95±4 nm in diameter and tail 120±5 nm in length. Basal plate of the virus was also observed.

Figure 3. Electron microscopy of isolate Eh1 (family Myoviridae) R - resistance, S - sensitivity.

To investigate whether the isolate Eh1 is able to suppress bacterial infection in vitro method of potato slices was used again. As a result, bacterial growth on potato cubes was limited by bacteriophage successfully (fig.4).

Slices inoculated only by bacteria displayed the signs of rot while cubes inoculated with bacteria and phage were clear without any symptoms.

Figure 4. A - slices inoculated with bacteria and phage; B - only bacteria

In recent years researchers from different parts of the world report more often about isolation of enterobacteria from agricultural plants (5, 8, 17). Plant rhizosphere is high in nutrients and it is enough for human pathogens to live and survive. Violation of crop rotation could be the reason that led organisms such as P.agglomerans into new ecological niche. Introduced in agrocenoses with organic wastes, these bacteria adapted and started to invade new organisms - plants. Therefore, using of untreated human excreta contaminated with enteric pathogens to grow vegetables should be strictly controlled (16). Although, successful infection by these bacteria often occurs in co-infection with other phytopathogenic bacteria, this fact do not reduce the risk for human, for example those who eat fresh fruits and vegetables or drink juices. That is why it is important to revise classical strategies of bacterial control and to introduce alternative methods such as bacteriophages.

Conclusions. As the result bacteria was isolated from onion with bacterial rot symptom. Bacteria was identified as Pantoea agglomerans due to morphological and biochemical tests. Antimicrobial sensitivity profile of P.agglomerans was investigated, isolate showed resistance to some antibiotics. A specific bacteriophage was isolated from waste waters. Phage isolate Eh1 was examined by electron microscopy and identified as a member of family Myoviridae.

Ability of the phage to inhibit bacterial infection of P.agglomerans in vitro was demonstrated on potato slices.

References

1. Dust-borne bacteria in animal sheds, schools and children's day care centres / A.M. Andersson, N. Weiss, F. Rainey, M.S. Salkinoja-Salonen // J. of applied microbiology, 1999. - Vol. 86, N 4. - P. 622. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10212408.

2. Nosocomial outbreak of Pantoea agglomerans in a pediatric urgent care center / E.L. Bicudo, V. O. Macedo, M.A. Carrara et al. // The Brazilian journal of infectious diseases: an official publication of the Brazilian Society of Infectious Diseases, 2007. - Vol. 11, N.2. - P. 281. Available at :http://www.ncbi.nlm.nih.gov/pubmed/17625778.

3. Isolation of Pantoea agglomerans in two cases of septic monoarthritis after plant thorn and wood sliver injuries / C. De Champs, Le Seaux,

J.J. Dubost et al. // J. of clinical microbiology, 2000. - Vol. 38, N.2. - p. 4601. Available at :http://www.ncbi.nlm.nih.gov/pubmed/10618144.

4. Flatauer F.E. Septic arthritis caused by Enterobacter agglomerans / E.Flatauer, M.A. Khan // Archives of internal medicine, 1978. - Vol. 138, N.5. - P. 788. Available at: http://www.ncbi.nlm.nih.gov/pubmed/646543.

5. Pantoea agglomerans as a New Etiological Agent of a Bacterial Necrotic Disease of Mango Trees / J.A. Gutiйrrez-Barranquero, F.M. Cazorla, J.A. Torйs, A. de Vicente // Phytopathology, 2019. - Vol. 109, N.1. - P. 1726. doi: 10.1094/PHYTO-06-18-0186-R.

6. Habsah H. An outbreak of Pantoea spp. in a neonatal intensive care unit secondary to contaminated parenteral nutrition / H. Habsah // J. of Hospital Infection, 2005. - Vol. 61, N.3. - P. 213-218. doi: 10.1016/j .jhin .2005.01.004.

7. Characterization of two polyvalent phages infecting Enterobacteri- aceae / S. Hamdi, G.M. Rousseau, S.J. Labrie et al. // Sci. reports. Nature Publishing Group, 2017. - P. 40349. doi: 10.1038/srep40349.

8. First report of Serratia marcescens causing yellow wilt disease on sunflower in Russia / A.N. Ignatov, M.V. Khodykina, V.A. Polityko, M.V. Sukhacheva // New Disease Reports. British Society for Plant Pathology, 2016. - Vol. 33. - P. 8. doi: 10.5197/j.2044-0588.2016.033.008.

9. Iredell J. Antibiotic resistance in Enterobacteriaceae: mechanisms and clinical implications / J. Iredell, J. Brown, K. Tagg // BMJ, 2016. - P. h6420. doi: 10.1136/bmj.h6420.

10. Jeljaszewicz J. Antibiotic resistance in Gram-positive cocci / J. Jeljaszewicz, G. Mlynarczyk, A. Mlynarczyk // Internat. j. of antimicrobial agents, 2000. - Vol. 16, N. 4. - P. 473. Available at: http://www.ncbi.nlm.nih.goV/pubmed/11118861.

11. Pantoea agglomerans as a cause of septic arthritis after palm tree thorn injury; case report and literature review / A. Kratz, D. Greenberg, Y. Barki et al. // Archives of disease in childhood, 2003. - Vol. 88, N. 6. - P. 542. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12765929)..

12. Bacteriophage therapy against Enterobacteriaceae / Xu Y, Liu Y, Liu Y et al. // Virologica Sinica, 2015. - Vol. 30, N. 1. - P. 11-18. doi: 10.1007/s12250-014-3543-6.

13. Mardaneh J. Isolation, identification and antimicrobial susceptibility of Pantoea (Enterobacter) agglomerans isolated from consumed powdered infant formula milk (PIF) in NICu ward: First report from Iran / J. Mardaneh, M.M.S. Dallal // Iranian J. of Microbiol. Tehran Univ. of Med. Sci., 2013. - Vol. 5, N. 3. - P. 263. Available at: https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC3895565.

14. Enterobacter sepsis in infants and children due to contaminated intravenous fluids / N.S. Matsaniotis, V.P. Syriopoulou, M.C. Theodoridou et al. // Infection control : IC. - 1984. - Vol. 5, N. 10. - P. 471-477. Available at: http://www.ncbi.nlm.nih.gov/pubmed/6567611.

15. Monier J.-M. Aggregates of Resident Bacteria Facilitate Survival of Immigrant Bacteria on Leaf Surfaces / J.-M. Monier, S.E. Lindow // Microbial Ecology, 2005. - Vol. 49, N.3. - P. 343-352. doi: 10.1007/s00248-004-9.

16. Internalization of enteropathogenic human bacteria in lettuce and coriander plant tissue / G. Nduhiu, M.M. Gicheru, P.B. Gathura et al. // ISABB Jour. of Health and Environmental Sci., 2018. - Vol. 5, N. 3. - P. 28 doi: 10.5897/ISAAB-JHE2018.0043.

17. Nithya A. Prevalence of plant beneficial and human pathogenic bacteria isolated from salad vegetables in India / A. Nithya, S. Babu // BMC Microbiology. BioMed Central, 2017. - Vol. 17, N. 1. - P. 64. doi: 10.1186/s12866-017-0974-x.

18. Popoca E. O. F. Pantoea agglomerans in Immunodeficient Patients with Different Respiratory Symptoms / E.O.F. Popoca // The Sci. World Jour. Hindawi Limited, 2012. doi: 10.1100/2012/156827.

19. Raphael E. Infections Caused by Antimicrobial Drug-Resistant Saprophytic Gram-Negative Bacteria in the Environment / E. Raphael, L.W. Riley // Frontiers in medicine. Frontiers Media SA, 2017. - Vol. 4. - P. 183. doi: 10.3389/fmed.2017.00183.

20. Souza K. A. Isolation of a polyvalent bacteriophage for Escherichia coli, Klebsiella pneumoniae, and Aerobacter aerogenes / K.A. Souza, H.S. Ginoza, R.D. Haight // J. of Virology. American Society for Microbiology (ASM), 1972. - Vol. 9, N. 5. - P. 851-856. Available at: http://www.ncbi.nlm.nih.gov/pubmed/4554466.

21. Ulloa-Gutierrez R. Pantoea agglomerans and thorn-associated suppurative arthritis / R. Ulloa-Gutierrez, T. Moya, M.L. Avila-Aguero // The Pediatric infectious disease jour., 2004. - Vol. 23, N. 7. - P. 690. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15247618.

22. Side effects associated with the treatment of multidrug-resistant tuberculosis at a tuberculosis referral hospital in South Korea: A retrospective study / T.W. Yang, H.O. Park, H.N. Jang et al. // Medicine. Wolters Kluwer Health, 2017. - Vol. 96, N. 28. - P. e7482. doi: 10.1097/MD.0000000000007482.

23. Andersson AM et al. Dust-borne bacteria in animal sheds, schools and children's day care centres. J.appl.microbiol.1999; 86(4); 622-34. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10212408.

24. Bicudo EL et al. Nosocomial outbreak of Pantoea agglomerans in a pediatric urgent care center. Braz J Infect Dis. 2007; 11(2); 281-4. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17625778.

25. De Champs C. et al. Isolation of Pantoea agglomerans in two cases of septic monoarthritis after plant thorn and wood sliver injuries. J Clin microbiol. 2000; 38(1); 460-1. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/10618144..

26. Flatauer FE, Khan MA. Septic arthritis caused by Enterobacter agglomerans. JAMA Intern.Med.1978; 138(5); 788. Available at:

27. http://www.ncbi.nlm.nih.gov/pubmed/646543.

28. Gutiйrrez-Barranquero, J. A. et al. Pantoea agglomerans as a New Etiological Agent of a Bacterial Necrotic Disease of Mango Trees. Phyto- pathol. 2019; 109(1); 17-26. doi: 10.1094/PHYTO-06-18-0186-R.

29. Habsah H. et al. An outbreak of Pantoea spp. in a neonatal intensive care unit secondary to contaminated parenteral nutrition. J. Hosp. Infect. 2005; 61(3); 213-218. doi: 10.1016/j.jhin.2005.01.004.

30. Hamdi S. et al. Characterization of two polyvalent phages infecting Enterobacteriaceae. Sci. Rep.2017; 7; 40349. doi: 10.1038/srep40349.

31. Ignatov, A. N. et al. First report of Serratia marcescens causing yellow wilt disease on sunflower in Russia', New Dis. Rep. Brit. Soc. For Plant Pathol. 2016; 33; 8. doi: 10.5197/j.2044-0588.2016.033.008.

32. Iredell J, Brown J, Tagg K. Antibiotic resistance in Enterobacteri- aceae: mechanisms and clinical implications. BMJ.2016; h6420. doi: 10.1136/bmj.h6420.

33. Jeljaszewicz J, Mlynarczyk G, Mlynarczyk A. Antibiotic resistance in Gram-positive cocci. Int J Antimicrob Agents. 2000; 16(4); 473-8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11118861.

34. Kratz A et al. Pantoea agglomerans as a cause of septic arthritis after palm tree thorn injury; case report and literature review. Arch. Dis. Child.2003; 88(6); 542-4. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12765929.

35. Liu Y et al. Bacteriophage therapy against Enterobacteriaceae. Vir Sinica. 2015; 30(1); 11-18. doi: 10.1007/s12250-014-3543-6.

36. Mardaneh J, Dallal MMS. Isolation, identification and antimicrobial susceptibility of Pantoea (Enterobacter) agglomerans isolated from consumed powdered infant formula milk (PIF) in NICU ward: First report from Iran. I JM. 2013; 5(3); 263. Available at: https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC3895565.

37. Matsaniotis NS et al. Enterobacter sepsis in infants and children due to contaminated intravenous fluids. Infection control : IC.1984; 5 (10); 471-7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/6567611.

38. Monier J-M, Lindow SE. Aggregates of Resident Bacteria Facilitate Survival of Immigrant Bacteria on Leaf Surfaces. Microb Ecol. 2005; 49 (3); 343-352. doi: 10.1007/s00248-004-0007-9.

39. Nduhiu, G. et al. Internalization of enteropathogenic human bacteria in lettuce and coriander plant tissue. ISABB J Health Environ.l Sci. 2018; 5(3); 28-32. doi: 10.5897/ISAAB-JHE2018.0043.

40. Nithya, A. and Babu, S. Prevalence of plant beneficial and human pathogenic bacteria isolated from salad vegetables in India. BMC Microbiol. BioMed Centr. 2017; 17(1); 64. doi: 10.1186/s12866-017-0974-x.

41. Popoca EOF. et al. Pantoea agglomerans in Immunodeficient Patients with Different Respiratory Symptoms. Sci. World J. 2012; doi: 10.1100/2012/156827.

42. Raphael E, Riley LW. Infections Caused by Antimicrobial Drug- Resistant Saprophytic Gram-Negative Bacteria in the Environment. Front. Med. 2017; 4; 183. doi: 10.3389/fmed.2017.00183.

43. Souza KA, Ginoza HS, Haight RD. Isolation of a polyvalent bacteriophage for Escherichia coli, Klebsiella pneumoniae, and Aerobacter aero- genes.J. Virol.1972; 9(5); 851-6. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/4554466.

44. Ulloa-Gutierrez R, Moya T. Avila-Aguero ML. Pantoea agglomerans and thorn-associated suppurative arthritis. Pediatr. Infect. Dis. J. 2004; 23(7); 690. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15247618.

45. Yang TW et al. Side effects associated with the treatment of multidrug-resistant tuberculosis at a tuberculosis referral hospital in South Korea: A retrospective study. Medicine (Baltimore). 2017; 96(28); e7482. doi: 10.1097/MD.0000000000007482.

Размещено на Allbest.ru