Isolation and characterisation of bacteriophages against multi-drug-resistant bacteria of Proteus mirabilis from Sewage water sample

Shreeranjani U

Sri Bhuvanendra College, Karkala, Udupi dist., Karnataka 574104

Dr. Gopal Nath

Professor, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005

Abstract

​Proteus mirabilis is a member of Enterobacteriaceae family. It is a facultative anaerobic gram-negative bacilli, which is best known for its swarming motility on agar plates. This bacteria is widely distributed in environment like soil, water, fecally contaminated material and in humans as a part of normal flora of the gut. Proteus mirabilis species is an opportunistic pathogen and is known to cause septicemia, meningitis, wound infections, burn infections and urinary tract infections. This bacteria is associated with development of renal stones in patients with urinary tract infections. An alkaline urine sample is a possible sign of Proteus mirabilis due to its ability to produce high levels of urease. The bacteria has a distinct fishy odour and characteristic swarming motility which is usually observed in non-inhibitory agar as a wavelike spreading of organism across the entire surface of the agar. Current practice of treatment of bacterial infections is the use of antibiotics. But many of the bacterial pathogens are turning resistant to antibiotic regimes which is a serious problem not only in medical sector but also in food and industrial sector as well. An alternative potential technique in the age of multi-drug resistance is the use of Phage therapy. Unlike antibiotics, phages are host specific and usually less toxic as compared to antibiotics. The aim of the study is to isolate bacteriophages specific to Proteus mirabilis from water samples of different water bodies by standard techniques. The isolated phages will be harvested, purified and stored which can later be used for therapeutic purposes.

Keywords: bacilli, swarming motility, phages

Abbreviations

INTRODUCTION

Proteus mirabilis is a Gram negative bacilli belonging to Enterobacteriaceae family which is most commonly found in soil, water and human gastrointestinal tract. Normally the bacterium is non-pathogenic in nature however, if it gets into urinary tract can be pathogenic. Hence it is an opportunistic pathogen known to cause septecemia, meningitis, bacteremia, burn infections, wound infections and UTI. The bacterium exhibits swarming motility which is reported to be the important factor behind pathogenesis of UTI.

Currently the bacterial infections are being treated by the use of antibiotics all over the world. But due to the excessive use of antibiotics, almost all the pathogenic bacterial strains are becoming resistant towards all the antibiotics. This is unexceptional even in case of Proteus mirabilis. Most of the strains of the species are now resistant to antibiotics like ampicillin and benzylpenicillium. Hence there is a need for an alternative and effective technique replacing antibiotic treatment. Bacteriophages have been reported as a potential means to eliminate the bacterial pathogens.

Phage therapy is one of the potential applications for the treatment of bacterial infections in human as well as cattle. Bacteriophages are host specific in nature, effective in small numbers and have no effect on humans. Phage therapy is less expensive and involves simlpe techniques. Hence there is a need to implement this technique instead of the antibiotic treatment.

Objectives

• Isolation of bacterial pathogen from patient wound.
• Isolation of bacteriophage against specific bacterial pathogen.

LITERATURE REVIEW

Proteus mirabilis

Proteus belongs to the family of Enterobacteriaceae. The genus Proteus currently consists of 5 named species: P. mirabilis, P. vulgaris, P. penneri, P. myxofaciens and P. hauseri. Proteus was first discovered by Gustav Hauser, a German pathologist in 1885. The derivation of the word "Proteus" seems to be interesting as the origin of the word is mythological and historical. According to ancient Greek mythology, Proteus means an early prophetic sea god or god of oceans and rivers who changed shape to avoid capture. It is also believed that Proteus comes from the word "Protean" with the meaning of versatile, mutable and capable of assuming several forms. Species of Proteus are highly motile due to their swarming motility which is the striking and unique characteristic of the genus.

Classification

Kingdom : Bacteria

Phylum : Proteobacteria

Class : Gamma Proteobacteria

Order : Enterobacteriales

Family : Enterobacteriaceae

Genus : Proteus

Species : mirabilis

Environmental distribution

Proteus mirabilis is found as a part of normal flora of the gastrointestinal tract along with Escherichia coli and species of Klebsiella where E.coli seems to be prominant resident. Proteus mirablis is also found to be free living in soil and water in natural environment, which is often regarded as indicator of fecal contamination. However when it enters urinary tract, wounds or lungs can be infectious.

Laboratory characteristics

Proteus mirabilis is a gram-negative, rod shaped, facultative anaerobic bacterium. The bacteria is highly motile due to its swarming motility because of the presence of peritrichous flagella. The swarming motility is clearly visible as spreaded colonies on MHA, Blood agar and MacConkey agar plates with 1.5% agar concentration. The bacterium is known for it's swarming ability which is the unique characteristic of the genus Proteus among the family Enterobacteriaceae. Biochemically the bacterium is indole negative, H₂S producing, motile, fermentor of glucose, non-fermentor of lactose, sucrose and mannitol, urea positive, citrate positive, catalase positive, oxidase negative. Proteus mirabilis being non-lactose fermentor, doesn't give pink coloured colonies on MacConkey agar plates.

Morphological featues

Proteus mirabilis is a gram-negative bacilli. There is a presence of lipopolysaccharide membrane in the cell wall, hence are resistant to many of the antibodies. The bacteria has peritrichous flagella because of which it is highly motile. Optimum temperature for the growth Proteus mirabilis is 34 to 38°C which makes human the ideal host for its survival. Studies shows that, the bacterium needs a acidic pH for the growth.

Virulance factors

Among the species of Proteus, Proteus mirabilis is known to cause 90% of the Proteus infections. Proteus is the primary infectious agent in case of urinary tract infections. Many of the virulance factors may be resposible for the pathogenecity of P. mirabilis. A few of them are,

• Flagella, necessary for motility involved in causing infections (Harmaon et al., 1989)
• Urease, responsible for the formation of kidney stones at later stages of infections (Mobley & Hausinger, 1989)
• Haemolysin, cytotoxic for cultured urinary tract epithelial cells (Mobley et al., 1991). This has been shown to be correlated with the ability of bacteria for invading cells (Peerbooms et al., 1984)
• Swarming motility, correlated with all the virulance properties mentioned above (Allison & Hughes 1991a; Allison et al.,1992b; Liaw et al., 2000, 2001, 2004)

Infections assosiated with P. mirabilis

Proteus is most frequently a pathogen of UTI, specifically in elderly, patients with type 2 diabetes and in patients undergoing long term catheterization. It is known to be associated with the development of renal stones in patients with urinary tract infections. It causes infections in surgical wounds and burnt tissues. Relatively uncommon is the blood stream infection or septecemia due to Proteus mirabilis. In addition to this, P. mirabilis also causes bacteremia and meningitis.

Bacteriophages

Bacteriophages were discovered by English microbiologist Frederick William Twort in 1915. Bacteriophages are the viruses that infect bacteria. A typical bacteriophage consists of nucleic acid molecule surrounded by membranous protein units. Bacteriophage attach themselves to the bacterial surface and inject their genitic material into the host. The bacteriophage take control over the host machinary that induces the host to synthesize viral components and prevent the production of bacterial components. Eventually, newly synthesised bacteriophages assemble to pressurise the bacterial cell wall leading to the lysis of bacterial cell. The bacteriophages come out to infect other bacteria. This life cycle of a bacteriophages is called as lytic cycle and phages following lytic cycle are referred to as virulant phages. Occasionally after the injection of phage DNA into the host cell, the phage DNA integrates itself into host genome called prophage. This multiplies inside the bacterial cell and does not kill the bacteria. This life cycle of phages is lysogenic cycle and phages following this life cycle are referred to as temperate or non-virulant phages.

Recently bacteriophages are recognised to be the natural predators of bacteria. This has been effeciently used in modern biotechnology. The bacteriophages being host specific are abundant in nature. It is believed that for every bacteria there exist a bacteriophage. Hence antibiotics are able to be replaced by bacteriophages in treating infections of MDR strains of bacteria. This potential technique of therapeutic use of bacteriophages to treat bacterial infections is referred to as Phage therapy.

METHODOLOGY

The studies were conducted at Molecular Research Laboratory, Department of microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi (India) during 24^th May to 20^th July, 2019 under Indian Academy of Sciences.

MATERIALS AND METHODS

Materials Required

Methods

According to aims and objectives of project, methods are described as follows:

Isolation of bacterial strain from clinical sample

Clinical strains for the experiments were collected from wound pus on thigh of a patient suffering from severe injury and inflammation using swabs. The collected swab was cultured on Blood agar, MacConkey agar and Muller Hinton Agar (MHA). The Culture plates were incubated overnight at 37°C in incubator. And these cultures were processed further for confirmation by Gram's staining, biochemical tests and antibiotic susceptibility test.

Fig 1 Infected thigh of patient

Morphological and microscopic observation

The morphological characterisation was done by observing shape, size, colour and pattern of bacterial colonies on culture plates. The microscopic study was done by Gram's staining technique which is a preliminary method to distinguish between Gram's positive and Gram's negative bacteria. It has four basic steps:

• The heat-fixed thin bacterial smear on a glass slide is treated with Crystal violet for one minute and then rinsed with tap water.
• Gram's iodine is added for one minute and then rinsed with tap water. Iodine forms a complex with crystal violet.
• Rapid decolourisation is achieved by the addition of alcohol (ethanol) for 20-30 seconds and rinsed with tap water.
• Counter staining is done by the use of Safranin for 30 seconds and rinsed with tap water. The slide should be air dried.

After following these steps, the slide was observed under 10X, 40X and 100X (oil immersion) of microscope.

Biochemical tests

Biochemical tests are carried out for the identification of bacteria based on their differences in enzymatic and metabolic activities for different species. The bacteria was tested for metabolism of different sugars, H₂S gas production, indole production, motility etc. using a set of biochemicals such as TSI, SIM, Glucose, Lactose, Sucrose, Mannitol, Urea and Citrate.

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Fig 2 Biochemical tests

Antibiotic Drug Sensitivity Test (AST)

The clinical strain of Proteus mirabilis was lawned on MHA plate for antibiotic drug sensitivity pattern. Lawn culture was made using a bacterial suspension of 0.5 OD on MHA plate. Antibiotic discs of known concentrations were put on the marked place of media using a forcep.

Collection of sewage water sample

Sewage water samples were collected from different sites. The main sites of collection were:

• Sunderlal Hospital Sewage
• Umang pharmacy
• Lahartara Sewage
• Bhagvanpur Nala
• Assi Nala

Fig 3 Collected Sewage Water Samples

Isolation of Bacteriophages from Sewage Water

2 ml of water sample from each of the bottles were transferred to micro-centrifuge tubes using micropipette. All the tubes were treated with 1% (v/v) chloroform and were mixed thoroughly by shaking for 10 minutes. These tubes were centrifuged at 10,000 rpm at 4°C. After centrifugation, supernatant was collected for further processes.

• Bacterial Lawn Culture and Dropping

Bacterial Suspension of 2.0 OD was prepared in NS (Normal Saline). Lawn culture was done on MHA plate using sterile cotton swabs. The plates were then incubated at 37°C for 3 to 4 hours. After incubation, the collected supernatant was dropped on plates having lawn culture of Proteus mirabilis and were incubated overnight at 37°C.

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Fig 4 Making Lawn Culture

• Harvesting of plates and Centrifugation

After overnight incubation, dropped plates were harvested in TMG buffer using cotton swabs and were collected in micro-centrifuge tubes. All the tubes were treated with 1% (v/v) chloroform and were shaken for 10 minutes. Then the tubes were centrifuged thrice at 10,000 rpm for 10 minutes at 4°C. The supernatants containing phage lysates were collected for futher processes.

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Fig 5 Harvesting of Phages

• Overlay by Double Layer Agar Method

The collected phage lysates were diluted in TMG (TMG :Phage=450 µl :50 µl) by serial dilution. 100 µl of the diluted phages of different diliutions were added to different micro-centrifuge tubes containing mixture of 890 µl TMG and 10 µl bacterial suspension (>2.0 OD). The mixture was added to a test tube containing 3-4 ml of soft agar maintained at 45°C and mixed well by vortex. The soft agar mixture was poured and spreaded on labeled plates of MHA. After solidification the plates were incubated overnight at 37°C. Plaques of different morphology were observed on next day.

• Increasing the concentration of Phages

This was achieved by repeated phage dropping and harvesting on MHA plates. Another technique used to increase bacteriophage concentration was dropping of phages on roux bottle which has a large surface area. Phage concentration was also increased by inoculating the bacteria and phage in 2X LB broth. Bacterial suspension of 2.0 OD in normal saline was prepared and was inoculated in 2X LB broth for a minimum of 6 hours. After incubation, phages were added and the mixture was incubated again at 37°C overnight. After following any of these methods to increase the phage concentration, the activity was checked by dropping them on lawn MHA plate.

Fig 6 Dropped Phages on Roux bottles

• Purification of Phages by Membrane Dialysis

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Fig 7 Membrane Dialysis of Phage lysates

The aim of membrane dialysis was to remove all the endotoxins and exotoxins present in phage lysate. Membrane Dialysis was done as follows:

A dialysis membrane of 15cm length and pore size 0.22 µm was taken and its one end was tied tightly. The phage lysate was transferred inside the dialysis membrane through the untied end of the membrane and was then tied. The membrane containing the phage lysate was kept in a container containg PEG (Poly Ethyline Glycol). This was kept in refrigerator at 4°C overnight.

On next day PEG was changed with fresh PEG and phages were washed with PBS (Phosphate Buffer Saline) and again membrane end was tied and kept in container at 4°C in refrigerator, process was repeated three times.

After three day of dialysis phages were washed and collected by addition of 1 ml PBS which is free of Endotoxin and Endotoxins from phages.

• Storage of Bacteriophages

Bacteriophages were stored in Peptone stock media. Peptone stock media can be used to store bacteriophages both at room temperature and 4°C in which phages will remain intact for several years.

Fig 8 Stored Bacteriophages in Peptone stock media

Laboratory Characteristics

Isolation of bacterial strain from clinical sample

The cultured bacterial isolates were identified as Proteus mirabilis based on their morphology, microscopic observations and biochemical tests.

• Morphological Characteristics

Culture of Proteus mirabilis on MHA plates wih 1% agar showed irregular shaped spreaded colonies because of its swarming motility which makes it distinct from other members of the family Enterobacteriaceae.

a) MHA

b) MacConkey Agar

c) Blood Agar

Fig 9 Proteus mirabilis cultured on different media

Culture of Proteus mirabilis on MacConkey agar showed no pink coloured colonies which confirmed that it's a non-lactose fermentor and when cultured on Blood agar it showed grey coloured, spreaded colonies confirming the swarming motility, α-Haemolysis of Proteus mirabilis.

• Microscopic Observations

Proteus mirabilis under microscope was observed to be pink coloured, rod shaped bacilli. This confirmed that the bacteria is a Gram negative bacilli.

Fig 10 Gram's staining 

• Biochemical Tests

Results from the biochemical tests were as follows:

TSI test confirmed that the bacteria is a fermentor of Glucose, and non-fermentor of Lactose, Sucrose. Black colour in TSI test tube confirmed the production of H₂S gas by the bacteria. SIM test confirmed that the bacteria is Indole negative, motile and H₂S producing. Test for fermentation of sugars confirmed that the bacteria ferments Glucose but not Lactose, Sucrose and Manittol. Bacteria was positive for urea and citrate, confirming the capability of bacteria of urea hydrolysis and citrate utilizer.

Thus on the basis of biochemical tests, bacteria was confirmed to be Proteus mirabilis.

• Antibiotic Drug Sensitivity Test

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a) First Line Antibiotic Drug Sensitivity

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b) Second Line Antibiotic Drug Sensitivity

Fig 11 Antibiotic Drug Sensitivity Test

Proteus mirabilis was found to be sensitive to Gentamicin, Meropenem and resistant to all the other antibiotics tested. Thus confirming that the clinical strain was Multi Drug Resistant in nature.

Isolation of Bacteriophages

The sewage water collected was treated with chloroform and centrifuged. When centrifuged water samples were dropped on bacterial lawn culture of Proteus mirabilis for the first time, clearance was very less on MHA plate. After the repeated process of dropping and harvesting for several times, significant clearance was observed.

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Fig 12 Clearance on MHA plate

• Overlay by Double Layer Agar Method

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Fig 13 Overlay

After overlaying plaques were observed on MHA plates. Plaques were differentiated on the basis of their morphology in size. Then the plaques of different size were cut, added in tube containing 2X LB and incubated at 37° C overnight in incubator.

Next day, incubated phages stored in 2X LB were centrifuged at 10,000 rpm for 10 minutes at 4°C three times. Supernatants were collected.

• Membrane Dialysis

Membrane Dialysis was done to remove all the exotoxin and endotoxin present in Phage. The phages free of toxins were stored in Peptone stock media at room temperature.

CONCLUSIONS

MDR strains of bacteria is alarmingly serious problem reported in health sector across the world. None of the antibiotics are effective against these strains of bacteria. Hence there is a need to look for some effective alternative technique to cure bacterial infections. Bacteriophages are proved to be the potential means to fight against these MDR strains of bacteria

Bacteriophages are omnipresent and hence easily available. They being host specific have no interaction with human cells, hence harmless to humans. The number of bacteriophages increases with the number of bacteria. They are effective in small number and the process of isolation costs less. It is believed that for every bacteria, there is a bacteriophage present in nature. Hence it is possible to cure almost all the bacterial infections using Phage therapy.

Currently phages are being used for therapeutic purposes in Russia and Georgia to treat bacterial infections which do not respond to conventional antibiotics. India is one among those countries making highest use of antibiotics. Hence there is a need to focus on making people familiar with advantages and importance of Phage therapy in India.

REFERENCES

• Armbruster CE, Mobley HL. Merging mythology and morphology; the multifaceted lifestyle of Proteus mirabilis. Nat Rev Microbiol. 2012; 10(11): 743-54
• Armbruster CE, Hodges SA, Mobley HL. Initiation of swarming motility by Proteus mirabilis occurs in response to specific cues present in urine and requires excess L-Glutamine. Journal of bacteriology. 2013; 195(6): 1305-19
• Jones BV, Young R, Mahenthiralingam E, Stickler DJ. Ultrastructure of Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infections; Infect immun. 2004; 72(7): 3941-50
• Siegmund-Schultze N, Kroll HP, Martin HH, Nixdorff K. Composition of outer membrane of Proteus mirabilis progressive stages of cell form defectiveness; J Gen Microbiol.1991;137(12):2753-9
• Burke JP, Ingall MD, Klein JO, Gezon HM, Finland M. Proteus mirabilis infections in hospital nursery traced to a human carrier; N Engl J Med. 1971; 284: 115-121
• Schaffer JN, Pearson MM. Proteus mirabilis and urinary tract infections; Microbiol spectr. 2015; 3(5): 1110-28
• Haq IU, Chaudhry WN, Akhtar MN, Andleeb S, Qadri I. Bacteriophages and their implications on future biotechnology: a review- Virology journal 9. 2012; 9: 145-94
• Cisek AA, Dabrowska I, Gregorczyk KP, Wyzewski Z. Phage therapy in bacterial infections treatment: One hundred years after the discovery of bacteriophages; Current microbiology. 2017; 74(2): 277-283

ACKNOWLEDGEMENTS

First and foremost, I would like to express my deep sense of gratitude to Indian Academy of Sciences for providing me a golden opportunity to carry out this project. I owe my heartliest gratitude to respected Dr. Gopal Nath, M.D., Ph.D., MNAMS, Professor, Department of Microbiology, Institute of Medical Sciences, BHU, for his critical supervision, support and guidance.

I sincerely record my thanks to Mr. Virendra Bahadur Yadav and Ms. Pooja Gupta for their guidance during my project.

It is proud privilege for me to express my profound regards and deep sense of gratitude to Mr. Alakh Narayan Singh, Ms. Aprajita Singh, Ms. Pooja Patel, Mr. Anand Kumar, Ms. Ritu Kumari, Ms. Ragini Yadav, Ms. Kanika Bhargava who were always with me with their support and critical reviews despite their busy schedule during my project work.

I am equally grateful to Mr. Rajesh Kumar, Mr. Dilip Kumar Prajapati, Mr. Nilesh Gupta for their technical assistance during my project work.

I would like to sincerely thank Dr. Manjunath A Kotian, Principal and Ms. Deepthi, HOD, Department of Biotechnology, Sri Bhuvanendra College, Manglore University, Karkala for their permission and encouragement to work on this project.

I would also thank my lab mates and friends Ms. Irene Johnson, Mr. Asheq Shaik, Mr. Rahul Patel and Ms. Aleena Fatma who were always with me with their moral support and encouragement.

Last but never the least, I would like to express my deepest sense of gratitude towards my beloved parents, family who have always supported me with their valuable suggestions and guidance. I would like to express my special thanks to God and almighty for blessing me with all the knowledge and strength to complete this project successfully.

APPENDICES

• Muller Hinton Agar

• Luria Bertani broth

• Soft Agar (Luria Bertabi agar)

• Normal Saline

• TMG buffer

• Polyethylene Glycol (PEG)

• Phosphate Buffer Saline