This case study is the ideal example of a nurse’s failure to build a therapeutic relationship with their client. Empathy is pivotal when dealing with patients diagnosed with incurable diseases such as HIV. Nurses should be sensitive to patient’s pain and discomfort so that the patient feels understood and is more inclined to adhere to their medications.
A nursing assessment is part of the nursing process that allows the nurse to assess and collect relevant data to ensure that the patient’s needs are met. Throughout the process subjective and objective data are collected from the patient and the patient’s family. Subjective data refers to information from a patient’s point of view, such as pain levels, whereas objective data refers to information that is measured through physical examination and tests. Accurate data is vital for correct diagnosis and patient rehabilitation plans. In this process the nurse must engage in conversation and ask questions to get as much information as possible as well as determining if a patient has any communication barriers such as language and sensory deficits that may influence a patient’s ability to successfully communicate.Medicine and Law Essay Paper
To conclude, the main points that have been discussed in this essay are the importance of different forms of communication in nursing and how they can be utilized for a therapeutic nurse-patient relationship. Poor communication skills are still present and nurses must aim to be more empathetic to provide quality holistic care.
Over the years, the rate of discovery of new drugs has declined significantly, leading to an urge to explore alternative environmental sources apart from the soil, for novel antibiotics. In this study, water samples were collected from different places around Bath and they were screened for antibacterial activity against six test bacteria. A total of 106 isolates were isolated and tested against methicillin-sensitive Staphylococcus aureus (MSSA) to confirm their antagonistic activity. 34 isolates showed promising inhibition and were further tested against bacteria which included MSSA, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium (E. faecium), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) through perpendicular streak test. The supernatant of the isolates was also extracted by centrifugation and assayed for its antibacterial activity. For E. coli-inhibiting isolates, further tests against strains of E. coli with different antibiotic resistance were performed to identify the types of antibiotics produced. 11 active isolates were effective against both Gram-positive and Gram-negative bacteria in the perpendicular streak test. The supernatant of the isolates exhibited minimal antibacterial activity. Following the results of 16S rRNA sequencing, two active isolates belonged to Pseudomonas species while the other three isolates were classified as Bacillus species. Isolate 107 was identified as Bacillus pumilus and it demonstrated the strongest inhibition against MSSA, MRSA, E. faecium and E. coli with a zone of inhibition of 20.8mm, 24.3mm, 16.2mm and 13.6mm respectively. Moreover, isolates 18 and 107 both had strong activity against all strains of E. coli tested, while isolate 71 was only active against four strains of E. coli tested, suggesting that one of the antibiotics produced may be kanamycin. Our findings indicate that aquatic environment is a potent source for the isolation of bioactive microorganism potential for the production of antibacterial compounds. Medicine and Law Essay Paper
Microbes play an essential role in the production of antibiotics, antifungal as well as antiviral infections and this role is expanding each day (1). Owing to their ability to produce useful secondary metabolites, microbes have contributed greatly in the development of pharmaceutical industry and the control of many medical conditions as they are now widely used as antitumour drugs, immunosuppressants, enzyme inhibitors, and in many other applications (1). Back in 1928, Alexander Fleming found a compound produced by a mould, which was later identified as Penicillium notatum, had the capability of killing the bacterium Staphylococcus aureus in his laboratory. The active compound was known as penicillin, a beta-lactam antibiotic, and it was used massively as a potent antimicrobial drug during World War II (1). This discovery has marked the beginning of the microbial drug era as many useful antibiotics have since been isolated from soil bacteria, for instance, streptomycin, chloramphenicol, and tetracycline (2). These antibiotics produced have had remarkable biological activities to human beings, to illustrate, streptomycin was the first active drug against tuberculosis whereas chloramphenicol was the drug of choice for typhoid fever (3-5). Medicine and Law Essay Paper
The soil is incontestably a rich reservoir that allows the screening of drug compounds as it can harbour an enormous number of soil inhabitants, such as bacteria, fungi, algae, protozoa, insects, and other more complex living organisms (6). Many soil organisms have the ability to produce secondary metabolites which enable them to inhibit the growth of other microorganisms in the same niche in order to compete for survival (7). Over the past decades, a significant amount of bioactive compounds have been discovered from the terrestrial environment (3). The majority of the useful soil microorganisms belong to the genera Penicillium, Streptomyces, Cephalosporium, Micromonospora and Bacillus, and they are still being studied continuously (8). Bacillus is found abundantly in the soil environment and it is known to produce antibiotic like bacitracin, pumulin and gramicidin which are active against Gram-positive bacteria, and polymyxin, colistin and circulin which are effective against Gram-negative bacteria, demonstrating a vast range of antimicrobial activity (9; 10). Furthermore, more than 60% of antimicrobial agents used in human and animals were originated from the genus Streptomyces, some of which are chloramphenicol, erythromycin, and tetracycline, which have a broad spectrum of activity (5; 9). Medicine and Law Essay Paper
In the recent years, the search for antibiotics has plateaued whereby limited new antibacterial drugs are being introduced to the market, posing a challenge to the healthcare sector. The rate of discovery of new compounds has declined and the scientists are facing a bottleneck where the same known antimicrobial compounds have been isolated over the past few years (11). Nowadays, antibiotics are widely used therapeutically and prophylactically in the healthcare setting for human, animals and agricultural purposes. They are commercially exploited and the overconsumption of these compounds has brought about a rapid evolution in microorganisms where resistance to the antibiotics is spreading dramatically. Furthermore, the crisis of multidrug resistance is expanding uncontrollably, leading to a consistent demand for more effective and useful antibacterial medicines. Staphylococcus aureus, Pseudomonas sp., Klebsiella sp., and Enterococcus sp. are examples of common nosocomial bacteria and they often cause a serious problem to hospitalised patients (1). The development of resistance of these pathogens to currently available antibiotics, both natural and synthetic classes of antibiotics, has rendered many of the standard drugs ineffective (12). Medicine and Law Essay Paper
In view of the scarcity of new antimicrobial medications being found from terrestrial environment, there is an urge to explore different environmental sources. The marine environment consists of a wide diversity of microorganisms, and thus the discovery of many medically useful compounds. The antivirals acyclovir and cytarabine used for herpes virus and non-Hodgkin’s lymphoma respectively were originally isolated from marine sponges, showing the potential of marine life as a novel source of medicines (1). As reported in a study conducted in South East coast of India, organisms originated from the genera Vibrio, Pseudomonas, Marinobacter and Bacillus have been isolated and proven to exhibit antimicrobial activity, where the isolate belonged to Bacillus species had the highest activity against Bacillus subtilis, E. coli and C. albicans (13). Besides that, aquatic actinomycetes have also been reported to produce bioactive compounds with potential clinical uses such as salinosporamide-A (from Salinispora sp.), marinopyrroles (from Streptomyces sp.) and marinobactin (from Marinobacter sp.) (14-16). Antibacterial actinomycetes have been successfully isolated from Lake Tana, Ethiopia, of which 13 isolates showed antibacterial ability against at least one of the tested bacteria, such as K. pneumoniae, S. aureus, P. aeruginosa and E. coli (17). Similarly, a study conducted in Ghana has isolated 27 antibiotic-producing microorganisms from marine and freshwater sources and it has been found that one of the active isolates produced metabolites with a broad antibacterial activity against both tested Gram-positive and Gram-negative bacteria (18). Medicine and Law Essay Paper
Although it is apparent that the ocean is a boundless source for novel antibacterial compounds, the water sources remain underexplored and unexploited. The aim of this study was to isolate antibiotic-producing microorganisms from the aquatic environment around Bath area and to determine their antibacterial activity against six test bacteria, three of which belonged to Gram-positive bacteria and the other three were Gram-negative bacteria.
2. Methods and Materials
2.1 Sample collection
Water samples with sediments were collected from River Avon, Bath City Farm, Rainbow Wood Farm and pond in the University of Bath.
2.2 Isolation of microorganisms
The water samples were serially diluted up to 10-4 in phosphate buffered saline (PBS) (Oxoid). Each diluted sample was inoculated into media by spread plate technique and incubated at 28°C. Three different media were used, namely minimal salt agar (Sigma), nutrient agar (Oxoid) and tryptic soy agar (TSA) (Oxoid). Medicine and Law Essay Paper
2.3 Screening of isolates for antibacterial activity
Crowded plate technique was used to screen microorganisms with antagonistic activity. Inhibition activity can be demonstrated by the formation of clear zones surrounding the colonies, after being incubated for three days. Colonies of interest were selected and repeated streaking technique was used to purify the isolated colonies.
An overlay of soft agar with a concentration of 0.75% was performed using double-layer agar technique to confirm the antibacterial activity of the isolates. 10ml of soft agar maintained in a water bath at 42°C was mixed with 100l of methicillin-sensitive Staphylococcus aureus (MSSA) Newman (19) before pouring it onto the solid agar. Clear zones produced indicated the synthesis of compounds active against the test bacterium. Medicine and Law Essay Paper
Perpendicular streak test was carried out where isolates were streaked as a single line along the diameter of TSA and incubated at 37°C and 28°C for one day each or 28°C for three days. The test organisms were then cross streaked at right angles to the original streak of isolates. The test organisms used included MSSA Newman, methicillin-resistant Staphylococcus aureus (MRSA) 252 (20), Enterococcus faecium (E. faecium) E1162 (21), Escherichia coli (E.coli) BW25113 (22), Pseudomonas aeruginosa (P. aeruginosa) PA01 (23), and Klebsiella pneumoniae (K. pneumoniae) (departmental strain collection). The plates were incubated at 37°C for 24 hours and the length of zones of inhibition was measured. Control plates without isolates of interest were simultaneously streaked with test organisms to study their normal growth.
For isolates that showed activity against E. coli BW25113, they were further tested with four other E. coli strains: E. coli ER2420 (pACYC184) (24), E. coli ER2420 (pACYC177) (24), E. coli SURE (pET-Amy) (25), and E. coli XL1Blue (pSG1164) (26), which displayed different antibiotics resistance. Perpendicular streak test was carried out to identify the antibiotic(s) produced. Medicine and Law Essay Paper
2.4 Antibacterial activity of supernatant
Five isolates were selected and grown in the nutrient broth (NB) (Oxoid) for three days. The supernatant was obtained by centrifugation at 4 °C, 5000 g for 20 minutes and it was filter sterilised before use. The antibacterial activity of the supernatant of each isolate was investigated as described:
(i) 100l of the supernatant of each isolate was mixed with 100l of each test organism culture (106 cfu/ml) and plated in 96-well plates. Eight replicates were done for each isolate. 200l of NB served as a negative control, while a mixture of 100l of each test organism culture and 100l of NB served as a positive control. The plates were incubated at 37°C for 24 hours and the OD of the mixture was measured.
(ii) Agar well diffusion test was carried out where 100l of each test organism culture (106 cfu/ml) were plated on TSA and left for 30 minutes. Five wells of 9mm were punched in the TSA with a sterile cork borer and 100l of each supernatant sample was filled into the wells of each plate inoculated with different test organisms. The plates were incubated at 37°C for 24 hours and observed for growth inhibition zones. Medicine and Law Essay Paper
2.5 Characterisation of colonies of interest
The strains of interest were characterised using gram staining to identify their classes, shapes and sizes under a light microscope.
2.6 Genomic DNA extraction
Genomic DNA of each isolate was extracted by alkaline lysis as described in (27; 28) with some modifications. In this experiment, a loopful of cells was emulsified in 20l lysis buffer (0.25% sodium dodecyl sulfate (SDS) (SIGMA), 0.05N NaOH (Fisherbrand)) and heated at 95°C for 15 minutes (28). The cells were then centrifuged at 13,000rpm for 30 seconds and the supernatant was diluted with 180l sterile water and stored at -20°C for future use.
2.7 PCR primers and conditions
Five isolates of interest were chosen for 16S rRNA sequencing. The 16S rRNA gene was amplified by PCR using forward primer 27F (5’- AGAGTTTGATCMTGGCTCAG-3’) (Sigma) and reverse primer 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) (Sigma). PCR was conducted with One Taq @ Standard Reaction Buffer (5x) (New England Biolabs Inc.) using manufacturer’s instruction.
PCR protocol was as follow: at 94°C for 2 min followed by 30 cycles at 94°C for 30 s, 55°C for 60 s, and 68°C for 90 s, then 68°C for 5 min in a single step. The samples were then held at 4°C. The PCR products were washed using Monarch PCR & DNA Cleanup Kit (5g) (New England BioLabs Inc.) before performing agarose gel electrophoresis to evaluate their purity and quality. Medicine and Law Essay Paper
2.8 Agarose gel electrophoresis
Visualisation of the PCR products was carried out on 1% agarose-TAE gel (Invitrogen, ThermoScientific) as described in (27) and Bioline Hyperladder I 100lanes was used as the ladder. Following the result of electrophoresis, Mix2Seq protocol was followed to prepare the samples for 16S rRNA sequencing.
2.9 Statistical analysis
Student t-test was used to calculate the significance of the difference between the experimental and the control samples. Differences were considered significant at p<0.05.
3.1 Isolation and screening of microbial isolates
A total of 106 colonies were found to show inhibition of the growth of the surrounding microorganisms. The majority of the active isolates were collected from Bath City Farm while the least was collected from River Avon. It was also shown that most of the isolates of interest grew better on TSA compared to NA. Medicine and Law Essay Paper
Following primary isolation, the isolates were overlaid with MSSA to confirm their antibacterial activity as shown in Figure 1. Out of the 106 colonies, only 34 of them inhibited the growth of MSSA and they were further investigated through perpendicular streak test with six bacteria. The zone of inhibition from perpendicular streaking of the isolates indicated antibiotic-producing activity as depicted in Figure 2. From the 34 isolates, only 11 isolates showed good outcomes, five independent repeats were done and the results are shown in Table 1. The average length of the inhibition zone produced by the isolates varied greatly, depending on the susceptibility of the test organisms to the antibiotics produced. In the perpendicular streak test, MSSA, which served as a control, was inhibited by all of the tested isolates. It was found that none of the isolates was active against P. aeruginosa, while MRSA was sensitive to every antibiotic-producing isolate. The number of isolates that inhibited the growth of E. coli and E. faecium was six and five respectively, and only. Medicine and Law Essay Paper
Figure 1. An overlay of MSSA was performed on TSA to determine the antibacterial activity of the isolates. A prominent zone of inhibition was shown by isolate 44, where the growth of MSSA was inhibited.
three of the isolates antagonised the growth of K. pneumoniae. Out of the eleven isolates, five isolates were active against all three Gram-positive test organisms, three of which were active only against Gram-positive bacteria. Nine isolates demonstrated inhibition on at least three of the test organisms, of which five isolates inhibited four test organisms in total, and only two isolates inhibited two test organisms. The isolate 107 showed the most potent antagonistic activity against the test organisms as the lengths of inhibition observed were the longest among all (20.8 6.4 mm, 24.3 5.6 mm, 16.2 7.6 mm and 13.6 4.7 mm against MSSA, MRSA, E. faecium and E. coli respectively), while the isolate 103 exhibited the least potent
Isolates Degree of inhibition. Medicine and Law Essay Paper
MSSA MRSA E. faecium E. coli P. aeruginosa K. pneumoniae
18 ++ ++ – + – ++
33 + + – + – +
36 + ++ – – – –
40 + + – – – –
71 ++ ++ – ++ – ++
99 ++ ++ ++ + – –
101 + + ++ – – –
102 + + + – – –
103 + + – + – –
104 + + ++ – – –
107 +++ +++ ++ ++ – –
Figure 2. Perpendicular streak method was used to study the activity of the isolates against six bacteria. Colony 104 demonstrated clear inhibition of the growth of MSSA, MRSA and E. faecium.
antagonistic ability with the weakest inhibition against the test organisms MSSA, MRSA and E. coli (4.4 2.2 mm, 4.0 1.2 mm and 6.0 1.4 mm). MSSA and MRSA were both the Gram-positive bacteria most commonly inhibited by the isolates while E. coli was the most commonly inhibited Gram-negative bacterium among all.Medicine and Law Essay Paper
3.2 Antibacterial activity of the supernatant
Two tests were carried out to study the antibacterial activity of the supernatant of each isolate:
(i) In the agar well diffusion test, the diameter of the zone of inhibition was measured and recorded. A fairly weak zone of inhibition was shown by all
isolates, ranging from 9.5mm to 12.5mm. Isolate 18
was active against all bacteria, also demonstrating the strongest inhibition among all, with a maximum zone of inhibition of 12.5 0.7 mm shown against E. faecium. Isolate 71 had a fairly low activity against the test organisms E. coli (10.2 0.7mm), P. aeruginosa (10 0mm) and K. pneumoniae (11.5 2.1mm), and showed no effect against any of the Gram-positive bacteria. The supernatant of isolates 102, 104 and 107 exhibited some antibacterial activity against both Gram-positive and Gram-negative bacteria, although the activity was weak (<2mm zone of inhibition). Medicine and Law Essay Paper
(ii) In the 96-well assays, the OD of the isolates in test organisms’ broth was compared to the OD of the positive controls as shown in Figure 3. The reduction in OD indicated that there was antibacterial activity, whereas a similar reading of OD showed that there was no inhibition. It was shown that isolates 18, 71 and 102 demonstrated slight antagonistic activity against MSSA (only 102 showed significance with p=0.038), isolate 104 did not seem to have a great effect on MSSA while 107 had a mild promoting effect on the growth of MSSA (p=0.111).
All tested isolates antagonised MRSA and E. coli. For MRSA, the differences in the OD were significant, showing a significant inhibition effect from each isolate. The strongest inhibition against MRSA was shown by isolate 71 with a difference of 0.207 (90%) in the OD while isolate 18 showed the strongest activity against E. coli, giving a difference of 0.441 (97%) in the OD. Isolate 107 was less. Medicine and Law Essay Paper
active against both MRSA and E. coli, giving the minimum zone of inhibition in both tests.
There was no big difference in the OD shown among the isolates tested with E. faecium, except that isolate 18 was shown to have promoted the growth of the test organism, with a difference of 0.031 in the OD but it was not significant (p= 0.122). Isolates 18, 104 and 107 demonstrated slight enhancing effect while the other two isolates had an inhibitory effect on P. aeruginosa. All p values except for 104 are <0.05. A prominent inhibitory effect has been shown by isolate 18 against K. pneumoniae (p=0.0009), and isolate 71 gave a reduction in the OD (p=0.086). Three other isolates demonstrated a slight promoting effect on the growth of K. pneumoniae (p values >0.05).
As a whole, the supernatant of isolate 71 displayed the strongest activity against all test bacteria, while isolate 18 gave the greatest degree of zone inhibition, especially against MSSA, E. coli and K. pneumoniae. Medicine and Law Essay Paper