Health Problems Caused By Antibiotic Resistant Bacterial Infection Biology Essay

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Nowadays, health problems caused by antibiotic resistance bacterial infection are getting more and more serious in the world. Inappropriate use of antibiotic and slow development of new antibiotics are the main factors to cause this problem. Therefore it is necessary to accelerate the development of new antibiotics.

The aim of this research is to discover and characterise antibiotic-producing soil microorganisms, with the aim of finding new antibiotics from the soil. Soil samples were collected from different areas of Bath and Keynsham in United Kingdom. Antibiotic producing microorganisms were isolated by crowded plate method. Classical streak plate method was carried out for analysing their antimicrobial activities against different strains of pathogenic bacteria. Bacteria producing compounds good antimicrobial activity were isolated for biochemical and morphological characterisations and further evaluation of antimicrobial activities. Finally they were identified by 16S RNA sequencing method.

Many antibiotic-producing microorganisms were found in the soil, some of which were found to have significant antimicrobial activity. A number of them have broad spectrum of activity against a range of bacteria while others had narrow spectrum of activity against a specific pathogenic bacteria. 16S RNA sequencing was carried out on 3 of them to identify their indentities. Results showed that two of the antibiotic-producing bacteria belong to different strains of Bacillus pumilus and one of them belongs to Streptomyces narbonensis.

INTRODUCTION

Antibiotics are chemical compounds which fight against bacterial infections in human or other animals. They exert their actions by targeting various cellular targets including cell wall, cell membrane, DNA, RNA and protein synthesis. Clinically, antibiotics are used for prophylaxis and treating various infections in human or other animals. Antibiotics can lower the mortality rate of infectious diseases [1]. However, if they are not used appropriately, this will lead to the development of antibiotic resistant bacteria [2]. Health problems caused by antibiotic resistance infections are getting more and more significant in the world [1][2].

Unnecessary use and slow development of new antibiotics are the main factors for the emergence of antibiotic resistant bacterial infections. When an antibiotic is used frequently or unnecessarily, bacteria will develop resistance mechanisms against that antibiotic. The resistant bacterial population can be developed by genetic mutation or plasmid which carry resistance gene [3]. Nowadays, antibiotics are sometimes used unnecessarily for mild infections or viral infections. Antibiotic associated diarrhoea is a common side effect of broad spectrum antibiotics. As they kill a large amount of normal healthy flora in the gut, the resistant Clostridium difficile predominates and excrets enterotoxin which causes diarrhoea [5]. Slow development of new antibiotics is also an important factor for resistant bacterial infections [2]. Many complex procedures are involved in developing a new drug include finding a suitable compound, structural modification, preclinical testing, animal testing, research and development, clinical testing and getting market authorisation [4]. In early 2010, the outbreak of multidrug resistance Enterobacteriaceae infection occurred in Pakistan, India and the United Kingdom. The antibiotic resistance mechanisms of Enterobacteriaceae in this infection were acquired by the plasmids which encode enzyme New Delhi metallo-β-lactamase 1 (NDM-1) [8]. John Conly, who is a professor at the Department of Medicine in the University of Calgary in Canada, believed that this resistance infection outbreak was mainly caused by inappropriate use of antibiotics. This outbreak also showed the urgency of developing new antibiotics. However, not many companies are willing to develop new a new antibiotic as it costs a lot of money and takes a long period of time with low return [4].

Most antibiotics currently used were discovered naturally from fungi and bacteria. In the 21th century, many drug companies ignore the natural resources and prefer using high throughput screening and combinatorial chemistry to design a new drug for a specific target. However, many antibiotics have the complex structures which prevents them from being synthesised artificially [4]. Moreover, exploring a new chemical takes a long period of time, many companies prefer modifying the structure of existing antibiotics to modify the pharmacokinetic properties rather than designing a new antibiotic, which also slows down the development of new antibiotics.

Antibiotic resistance in agricultural sector is also a great problem in the world. Pesticides are commonly used to prevent the agricultural products from being damaged by pests. Prolonged use of pesticides or other antimicrobial agents can impart pressure for bacterial population to develop antibiotic resistances in order to survive in the presence of antimicrobial agents. Besides, many farmers use antibiotics to promote the growth of their animals. The theory behind this is not known exactly. However, this practice will develop resistance population inside the animals. Consequently, antimicrobial agents and resistance bacteria can be passed on by food chain and finally reach the human [1].

Soil is a rich source of microorganisms [1,9] and many of them are capable of producing antibiotics [4,6]. Streptomyces species are one of the main sources of antibiotics [7]. Many antibiotics currently used are produced from Streptomyces species, like streptomycin, tetracycline and erythromycin [1,6,9]. There are many species in the Streptomyces family, each of them can produce different antibiotics. Many antibiotic-producing soil microorganisms have yet to be identified or discovered, so there is a great potential in finding new antibiotics from soil resources.

The aim of this research was to investigate the potentialities of discovering new antibiotics from soil by isolating and characterising antibiotic-producing soil microorganisms and their products. In this research, soil samples were collected from four different places in Bath and Keynsham in United Kingdom. They were collected at different places as different geographical locations have different pH, temperature and moisture content which can affect the growth of different microorganisms [9] Microorganisms which were capable to produce antimicrobial agents were isolated and tested for their activities against different pathogenic bacteria. Then they were characterised by a series of physical and biochemical assays. Finally, 16S RNA sequencing was carried out to find out their identities.

METHODS AND MATERIALS

Bacterial strains and growth conditions

The pathogenic bacterial strains used in this research are E. coli DH5α (New England Biolab), E. coli ER2420(pACYC184) (New England Biolabs), B. subtilis 168 [10], B. subtilis total-tat2 [11], E. faecium E1162 [12], Staphylococcus aureus MRSA252 [13]. E. faecium E1162 was maintained in TSB broth while the other strains were maintained in LB broth.

Collection of soil sample

Soil samples were collected from four different places in Bath and Keynsham by sterile tubes on 11th October, 2010, which was a sunny day. Sample 1, 2, 3 and 4 were collected at grass field near Pillow in Keynsham, a recently planted winter cornfield at the valley bottom below Pillow near Keynsham, a garden nearby London Road in Bath and the Lake at the University of Bath, Claverton Down respectively. After collection, they were stored at 5oC in refrigerator.

Crowded plate method and evaluation of antimicrobial activities

Isolation of antibiotic producing bacteria was performed in two phases. First, crowded plate method was carried out to isolate soil bacteria which were able to produce antimicrobial agents to antagonise the growth of other microorganisms. Soil samples were diluted by serial dilution and plated onto Luria Broth (LB) (Sigma), Tryptone Soya Bean (TSB) (OXOID), Nutrient (OXOID) and Vegitone (Fluka Analytical) agar plates [9,14]. Inhibition zones were observed and isolation of antibiotic producing soil microorganisms was carried out after incubating overnight at 37oC and three days at 25 oC.

Overlay method was carried out to isolate more antibiotic-producing bacteria. Serial dilutions of soil solution were spread onto the LB, TSB, Nutrient and Vegitone agar plates with 20mg/ml Nystatin solution (Sigma) in DMSO (Fisher) to inhibit the growth of fungi [15]. After overnight incubation, bacterial overlay was carried out as described [16] by covering the colonies with 0.8% soft agar containing 106 E. coli DH5α or B. subtilis 168. Inhibition zones were observed and isolation of antibiotic producing soil microorganisms was carried out after overnight incubation at 25oC and 37oC. This method was repeated to evaluate the antimicrobial activities of these soil bacteria. The ones which showed activities against these two pathogenic bacteria were selected for further analysis of their antimicrobial activities.

Classical streak method

Classical streak method was carried out to further analyse the antimicrobial activities of the selected bacteria. E. coli DH5α, E. coli ER2420(pACYC184), B. subtilis 168, B. subtilis total-tat2, E. faecium E1162 and MRSA 252 were streaked against antibiotic producing bacteria as described by Nanjwade et al. [17]. Inhibition of growth was recorded after incubating at 25oC for three days.

Enzyme detection

Selected antibiotic producing bacteria were analysed for their amylase and protease secretions. For amylase activity assay, the test was carried out as described by Lee [18] using Mueller Hinton agar (Oxoid) as testing medium. After overnight incubation, the agar plates were flooded with iodine solution containing 2% KI (Fisher) and 0.2% I2 (BDH) [19]. For protease activity assay, the test was carried out as described by Walker et al. [20] using nutrient agar containing 1% skimmed milk powder (MARVEL). Clear zone was recorded after overnight incubation.

Motility assay

Motility assay was performed to analyse the motility of microorganisms. The study was carried out as described by Yoon et al. [21] using LB soft agar (0.3% and 0.5%) as testing medium. After overnight incubation at 37oC, bacterial motility was determined by the size of bacterial zone.

Morphology characterisation

Gram staining and spore detections were carried out to characterise the selected soil microorganisms. Gram stain was performed as described by Preston et al. [22] to characterise the bacteria based on their cell wall properties. After staining procedures, they were observed under microscope (Axiostar plus Zeiss) with A-plan 100x objective.

They were examined for the ability to form endospores. Sporulation medium was prepared by as described by Schaeffer et al. [23]. After culturing the testing strains in sporulation medium for 2 days, 100μL chloroform was added to 900μL culture as described by Veening et al. [24]. Then 100μL suspension was plated out onto LB agar plates and the results were recorded after overnight incubation.

Carbon source utilization

The microorganisms were analysed for the catabolism of different carbon sources by assessing their growth on M9 minimal media agar with different carbohydrates. M9 minimal medium was prepared as described by Fluckingers et al. [25]. After that, 1ml of 1M MgSO4 (Sigma), 10ml of carbon sources (15% D-Xylose SLR (FISONS), L-(+)-Arabinose (Sigma), 18% β-D-(-)-Fructose (Sigma), D-Mannitol (Sigma), D-glucose (FISONS) and 34% Surcose(Fisher)) and 0.05ml 1M CaCl2 were added to each 500ml M9 minimal agar solution [25]. Finally, the bacterial cultures were streaked onto the sugar agar plates and were recorded for growth after incubating for 10 days [26].

Anaerobic growth test

This procedure was carried out to assess the ability of microorganisms to grow in the absence of oxygen. 100μL bacterial suspension (106cfu/ml) was spread over LB agar and incubated inside an anaerobic chamber (BBLTM GasPak PlusTM) at 25oC. Growth was recorded after 10 days.

Antibiotic susceptibility test

Antibiotic susceptibility test was carried out as described by Andrews [27] to compare the similarities between the antibiotics produced by soil microorganisms and the currently used antibiotics. 105 soil microorganisms on LB agar plates were tested against tetracycline 30μg (Oxoid), kanamycin 30μg (Oxoid), erythromycin 10μg (Oxoid), ampicillin 10μg (Oxoid), ciprofloxacin 1μg (Oxoid), chloramphenicol 10μg (Oxoid) and spectinomycin discs (Fluka Analytical). Finally diameter of zone of inhibitions was recorded after overnight incubation.

Further evaluation of antimicrobial activities

Agar well diffusion assay was carried out as described by Perez et al. [28] to further evaluate their antimicrobial activities against E. coli DH5α, E. coli ER2420(pACYC184), B. subtilis 168, B. subtilis total-tat2, E. faecium E1162 and MRSA 252. Five wells with 5mm diameter were punched on each LB agar plate after spreading with 100μL indicator bacteria suspension (106cfu/ml). After centrifugating the bacterial cultures at 11000g for 10 minutes, 350μL supernatant of each testing strain was pipetted into each well. Finally clear zone around the well was recorded after incubating at 25oC for 3 days.

Plasmid isolation and purification

Antibiotic-producing bacteria were analysed for the presence of plasmid DNA. All steps of plasmid isolation and purification were performed by Macherey Nagel Nucleospin Plasmid kit according to their instructions. Finally the samples were loaded onto 0.8% agarose TAE gel and visualised under UV light.

16S RNA sequencing and plasmid isolation

16s RNA sequencing was performed to analyse the sequences of interested antibiotic producing bacteria. Bacterial lysis and chromosomal DNA extraction were performed by using lysozyme(Sigma) and phenol-chloroform-isoamyl alcohol method as described by Leenhouts et al. [32]. Then 50μL PCR reaction was carried out to amplify their 16S RNA sequences. All steps of PCR were performed by using KAPA2G Robust PCR kit (include DNA polymerase, buffers and dNTP) and Eppendorf Mastercycle gradient PCR machine according to the instructions of KAPA Biosystems. 3 sets of primers (Sigma-Aldrich) were used in this analysis, which included broad range primers (forward primer 63f and reverse primer 1387r) [29], primers for gram positive rods (forward primer BAK11w and reverse primer BAK2) [30] and primers for Streptomyces sp. US24 strain [31]. After PCR, the products were cleaned by Macherey Nagel Nucleospin Extract II kit. Finally the PCR products were loaded onto 0.8% agarose TAE (Tris Acetate EDTA) gel and visualised under UV light. PCR products were compared according to their DNA length. Finally, the 16S RNA sequences were sent to Eurofins MWG for sequencing.

RESULTS

Isolation of antibiotic producing microorganisms and evaluation of antimicrobial activities

The crowded plate method was used to isolate antibiotic producing bacteria from 4 different soil samples.. 47 strains of antibiotic producing bacteria were isolated in this experiment. After that, bacterial overlay method was carried out to identify more antibiotic producing strains. 27 more antibiotic producing strains were isolated in this experiment. Finally bacterial overlay method was repeated on these 74 strains to test their activities against E. coli and B. subtilis. 10 strains were isolated for further analysis due to their good antimicrobial activities. Most of them were able to inhibit B. subtilis. However, only two of them were able to inhibit E. coli. Table 1 summarises the result of this study. Diameter of inhibition zone was recorded in millimetres.

Table 1: Diameters of zone of inhibitions (mm) by antibiotic producing bacteria

Antibiotic producing strain

E. coli

B. subtilis

S01/3

0

15

S01/5

10

0

S04/1

14

18

S07/1

0

13

S08/1

0

17

S09/1

0

16

S10/1

0

20

S14/2

0

15

S15/4

0

12

S17/1

0

50

Figure 1: Crowded plate method performed on TSB agar for soil sample 4. Inhibition zone was shown as indicated in this figure.

Figure 2: Colonies of antibiotic producing bacteria were covered with 0.8% consisting of Bacillus subtilis 168 (non-resistant strain) on LB agar plate. Clear zones were observed around the colonies of S01/4, S07/1 and S17/1.

Further characterisation and evaluation of antimicrobial activities

Classical streak method was performed to test the antimicrobial activities of these 10 testing strains. Result showed that six of them were able to produce compounds which inhibit two or more indicator bacterial strains. Table 2 summarises the result of this study. Length of inhibition was recorded in millimetres.

Figure 3: Classical streak test performed for S14/2 on LB agar. EC(c), E. coli DH5α; EC(r), E. coli ER2420(pACYC184); Bac(r), B. subtilis total-tat2; Bac(c), B. subtilis 168; Ent, E. faecium E1162; MRSA, MRSA 252.

Table 2: Mean length of inhibition (mm) of pathogenic bacteria by different strains of antibiotic producing bacteria

Strain

E. coli DH5α

E. coli ER2420(pACYC184)

B. subtilis 168

B. subtilis total-tat2

E. faecium E1162

MRSA 252

S01/3

0

0

0

0

0

0

S01/5

0

0

0

0

0

20

S04/1

16

0

0

0

0

6.5

S07/1

0

0

5

7

0

0

S08/1

0

0

0

0

0

0

S09/1

10.5

0

12.5

0

14.5

20

S10/1

0

0

0

0

0

9.5

S14/2

Completely inhibited

Completely inhibited

0

0

Completely inhibited

Completely inhibited

S15/4

Completely inhibited

19

Completely inhibited

Completely inhibited

Completely inhibited

Completely inhibited

S17/1

0

0

Completely inhibited

Completely inhibited

Completely inhibited

0

According to the result in table 2, S07/1, S09/1, S14/2, S15/4 and S17/1 were chosen as key strains as they were able to produce compounds with good antimicrobial activity. Further characterisations including enzyme assays, motility test, morphology assays, carbon utilisation tests, anaerobic growth assay and antibiotic susceptibility test were performed on these five strains. All of them were maintained in LB broth with the exception of S17/1 in TSB broth.

Enzyme detection

Enzyme detection tests were carried to characterise the antibiotic-producing bacteria according to their enzyme secretions. They were tested for amylase and protease secretions. For protease assay, positive result was indicated by clear zone around the colony. For amylase assay, result was recorded after flooding the agar plates with iodine solution. Positive result was indicated by clear zone around the colony. All of them were able to secrete protease and only two of them were able to secrete amylase. Table 3 summarises the results of these assays.

Table 3: Results of protease and amylase assays

Strain

Protease assay

(Diameter of clear zone measured in mm)

Amylase assay

S07/1

7

-

S09/1

15

-

S14/2

16

+

S15/4

16

-

S17/1

8

+

For protease, all testing strains were able to secrete protease. Diameter of clear zone (diameter of colony was also included) was recorded in millimetres. For amylase assay, + means amylase was present while - means no amylase was present.

Motility assay

This assay was performed to assess the motility of selected strains. First, 0.3% soft agar was used as testing medium. After overnight incubation, all of them except S17/1 were found to spread over the whole agar plate. So the experiment was performed again by increasing the agar hardness to 0.5%. However the result remained the same. So it can be concluded that S17/1 was non-motile while the others were highly motile.

Morphology characterisation

Gram staining was performed to assess the cell wall properties of the candidates. Result showed that all of them were Gram positive bacteria with rod structure. For endospore germination and detection, the testing strains were cultured in sporulation medium for spore formation. Then chloroform treatment was carried out as only bacteria with endospore formation were able to survive in the presence of chloroform. Finally the suspensions were plated out onto LB agar and the results were taken after overnight incubation. Results show that S09/1, S14/2 and S15/4 were able to germinate spores while S07/1 and S17/1 were unable to germinate spores.

Carbon utilisation

Selected strains were tested for their growth on M9 minimal media with different sugars in order to examine their ability to utilise different carbon sources. Result was recorded after 10 days. All of them were able to grow on the M9 carbohydrate plates. Table 4 summarises the results of this study.

Table 4: Level of growth of testing strains on different M9 minimal carbohydrate plates

Strain/Medium

Glucose

Sucrose

Fructose

Mannitol

Xylose

Arabinose

S07/1

+/-

++

+/-

+

+/-

++

S09/1

++

++

++

+

+/-

+/-

S14/2

+/-

+/-

+/-

+/-

+/-

+/-

S15/4

+/-

+/-

+/-

+/-

+/-

N/D*

S17/1

++

+

+

+/-

+/-

++

++ represents good growth; + represents moderate growth; +/- represents little growth.

*Arabinose plate of S15/4 was contaminated.

Anaerobic growth test

Anaerobic growth test was tested to investigate oxygen requirements of the candidates. They were incubated in anaerobic chamber and the results were taken after 10 days. As none of testing bacteria were able to grow in anaerobic condition, so it can be concluded that all of them were aerobic bacteria.

Antibiotic susceptibility test

Antibiotic susceptibility test was carried out to analyse the susceptibility of antibiotic-producing bacteria to different antibiotics. The rationale is, if the soil microorganism produces an antibiotic which is the same or similar to that antibiotic, then the soil microorganism would be resistant to that antibiotic. S14/2 and S15/4 were susceptible to all antibiotic disks while the other three were resistant to two or three antibiotic disks. Table 5 summarises the result of this study.

Table 5: Susceptibility of testing strains to different antibiotic disks

Strain/Antibiotic

Tetracycline

Kanamycin

Erythromycin

Ampicillin

Ciprofloxacin

Chloramphenicol

Spectinamycin

S07/1

+

+

-

-

+

-

+

S09/1

-

+

-

+

+

+

+

S14/2

+

+

+

+

+

+

+

S15/4

+

+

+

+

+

+

+

S17/1

+

+

-

-

-

+

+

+ represents Susceptible while - represents non-susceptible.

Further evaluation of antimicrobial activities

Agar well diffusion method was carried out to further analyse the antimicrobial actions of testing strains. Supernatants of soil microorganism suspensions were extracted by centrifugation and were tested against several strains of pathogenic bacteria. Their activities were weaker than that in classical streak method, only S17/1 was found to have good antimicrobial activity in this assay. Table 6 summarises the result of this study. Diameter of clear zone around the well was recorded in millimetres (Diameter of well was included).

Figure 4: Agar well diffusion assay with wells (3mm in diameter) performed for control B. subtilis 168

Table 6: Mean zone of inhibition (mm) of indicator bacteria by the testing antibiotic producing strains in wells diffusion assay

Strain

E. coli DH5α

E. coli ER2420(pACYC184)

B. subtilis 168

B. subtilis total-tat2

E. faecium

MRSA 252

S07/1

0

0

0

0

0

35

S09/1

0

0

0

0

0

32.5

S14/2

0

0

0

0

27.5

35

S15/4

0

0

0

0

0

0

S17/1

0

0

22

25

25

22.5

Plasmid isolation

As the genes responsible for producing antibiotics can be encoded on plasmid or chromosomal DNAs, this experiment was performed to analyse the presence of plasmid DNA in each testing antibiotic producing strain. Result showed that only S09/1 contains small amount of plasmid DNA while the others don't contain any plasmid.

16S RNA sequencing

16S RNA sequencing was performed to analyse their 16S RNA sequences and hence figure out their genus and species. This procedure was performed on S09/1, S15/4 and S17/1 as they have better antimicrobial activity than the other two. Broad range primers (forward primer 63f and reverse primer 1387r), primers for gram positive rods (forward primer BAK11w and reverse primer BAK2) and primers for Streptomyces species US24 strain were used in generating the PCR products. Then their products were analysed by gel electrophoresis. Broad range primers were unable to generate any DNA fragments, primers for gram positive rods generated 1.0 kilo base pairs (kb) DNA fragments and primers for Streptomyces species US24 strain generated 1.5 kb DNA fragments. Then PCR was performed again using primers for Streptomyces species US24 strain and the amplified sequences were sent to Eurofins MWG for sequencing. Sequencing results show that S09/1 and S15/4 belong to two different strains of Bacillus pumilus while S17/1 belongs to Streptomyces narbonensis.

Identity of newly discovered antibiotic producing bacteria

Series of morphological and biochemical assays were carried out to analyse the identities of S07/1, S09/1, S14/2, S15/4 and S17/1. Studies showed that all of them were aerobic gram positive rods which were able to survive in nutrient poor medium by utilising different carbon sources. Motility test was carried out and found that only S17/1 was non-motile. According to Bergey's Manual of Determinative Bacteriology 9th [33], they should belong to either Streptomyces species or Bacillus species. 16S RNA sequencing was performed on S09/1, S15/4 and S17/1 and found that S09/1 and S15/4 were two different strains of Bacillus pumilus while S17/1 was Streptomyces narbonensis. These results complied with the information of Bergey's Manual of Determinative Bacteriology as Bacillus bacteria were motile while Streptomyces bacteria were non-motile.

Analysis of antimicrobial activity

Antimicrobial activity of each testing strains (S07/1, S09/1, S14/2, S15/4 and S17/1) were tested by both classical streak method and agar well diffusion. Inhibitory effects observed in classical streak plates may not be caused by the antimicrobial products alone. Nutrient depletion may happen around the colonies of antibiotic producing bacteria, which prevent pathogenic bacteria from growing near to that area. So agar well diffusion method was performed to further analyse the activities of antimicrobial products. Their activities in agar well diffusion assays were lower than that in classical streak plates. This was probably caused by the low concentration of antibiotics in supernatant.

S09/1, S15/4 and S17/1 were shown to have better antimicrobial activities than S07/1 and S14/2. For S09/1, it showed significant activity against MRSA in both assays and weak activity against a range of bacteria in classical streak plate. For S15/4, it showed broad spectrum of activity against all of the testing bacteria. However, as the concentration of antimicrobial products were low in supernatant, it showed no activity in agar well diffusion assay. For S17/1, it showed narrow spectrum of activity with strong inhibition activities against both strains of B. subtilis and E. faecium E1162.

Two abnormalities occurred in the tests of S07/1 and S17/1. In classical streak plate, they were unable to inhibit MRSA, but in agar well diffusion assay, they were found to have inhibition actions against MRSA. The reason was probably due to the uneven spreading of MRSA on agar plate in the agar well diffusion and the slow growth rate of MRSA, which gave a false image of inhibition.

DISCUSSION

There are numerous amount of microorganisms in soil. Some microorganisms are able to compete for growth by producing antimicrobial agents to inhibit the growth of other soil microorganisms. Some of their antimicrobial products can be used clinically to treat bacterial infections of human or other animals. Streptomyces and Bacillus species are the most common microorganisms employed by pharmaceutical companies to produce clinically useful antibiotics. In this project, antibiotic-producing soil microorganisms were isolated from soil and characterised by different assays.

Relationship between antimicrobial products produced by selected antibiotic-producing bacteria and currently used antibiotics

Antibiotic susceptibility test was carried out to compare the similarities between the newly discovered antibiotics and the currently used antibiotics. It is assumed that the bacteria should be resistant to the antibiotic disk if it produces a product which is the same or related to that antibiotic. For S07/1, result suggests that it may produce some antimicrobial products similar to erythromycin and chloramphenicol. For S09/1, result suggests that it may produce some antimicrobial products similar to tetracycline and erythromycin. For S17/1, result suggests that it may produce some antimicrobial products similar to erythromycin and ciprofloxacin. For S14/2 and S15/4, it is possible that they produce some antimicrobial products which are totally not related to the currently used antibiotics they are susceptible to all of these currently used antibiotics. Although S07/1 and S17/1 are resistant to ampicillin, they are not likely to produce penicillin related antibiotics as no bacteria are found to produce pencillin related compounds. All of the currently used penicillin related antibiotics are synthesised from fungi [1].

16S RNA sequencing was performed on S09/1, S15/4 and S17/1 and found that they belong to two strains of B. pumilus and S. narbonensis respectively. According to the published literature, B. pumilus can produce a range of antibiotics include tetain, micrococcin P, pumilin [34] or some antifungal compounds [35] and S. narbonensis can produce josamycin [36] and narbomycin [37]. For B. pumilus, none of the published antibiotic relates to the ones used in antibiotic susceptibility test. For S. narbonensis, the result of antibiotic susceptibility test complies with the published journals as josamycin, narbomycin and erythromycin belong to marolide antibiotics.

Future directions

This research was performed successfully. Several strains were found to have good antimicrobial activities. However, this research is just in the early stage of identifying new antibiotics. Although the identities of the antibiotic-producing bacteria were identified, it is possible that they produce antibiotics other than the ones before. Therefore more studies need to be done in order to analyse the microbial products and improve antibiotics production.

More work should be performed to characterise the interested strains and their products. For bacterial identification, although 16S RNA was successfully performed to identify their genus and species, extensive characterisations should be done to analyse their growth behaviours in order to maximise their growth and antibiotic productions. For antimicrobial products, their structures and properties should be investigated in order to improve their pharmacokinetic properties. Thin layer chromatography (TLC) and fractional distillation should be performed to separate the compounds in the supernatants and find out the active antimicrobial products. Then spectroscopy should be performed to figure out the structures of newly discovered antibiotics and compare the similarities and differences with the currently used antibiotics. Stability, toxicity profile and bioavailability are also important in the development of new antibiotics. Molecular engineering should be performed to improve these properties. Glycosylation is commonly used to increase drug half life and reduce frequency of administration. William et al. [38] successfully broaden the activities of glycosyltransferase by making substitution of three amino acids on the glycosyltransferase encoding gene to glycosylate more macrolide antibiotics.

Genetic mechanisms of antibiotic production should also be investigated. Once the gene responsible for antibiotic production was identified, genetic engineering should be performed to modify the genes and generate a range of related antibiotics. Nguyen et al. [39] used this technique to generate a range of Daptomycin related compounds. In order to maximise the antibiotic production, more work should be done to find out the most suitable bacteria to carry the antibiotic encoding genes. The responsible genes can be plasmid or chromosomal encoded. If they are plasmid encoded, antimicrobial activity may be transferred to the other bacteria as plasmid DNA is mobile. In this research plasmid isolation and purification was performed on these antibiotic producing bacteria and found that plasmid DNA was found in S09/1 only. In the future, isolated plasmids from antibiotic producing bacteria should be transformed into different bacteria to analyse their roles in antibiotic production, and identify which bacteria is the most suitable to act as a host to carry the plasmids encoding for antibiotics in order to optimise the antibiotic production in pharmaceutical industry.

CONCLUSION

It is necessary to accelerate the development of new antibiotic as health problems caused by antibiotic resistant bacterial infection are getting more and more serious in the world. This research was performed successfully as several strains of antibiotic-producing bacteria were isolated in this study. However more work should be performed to accurately identify these antibiotic-producing bacteria and their products in order to optimise their antibiotic production.

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