Identification Of Microorganisms And Antibiotic Treatment In The Essay.

1:  Explain how you arrived at the final identity of each of the organisms using the results obtained.

2: The patients profiles are listed below. For each patient decide whether the organism isolated is significant or not highlighting the reasons, with references, for you decision.

3: With respect to just organisms A and D and taking into account the clinical data given in Appendix 1, use your results to decide upon the most suitable antibiotic treatment for these patients. Justify your choice including a brief explanation of why you have discounted each of the other antibiotics you tested.

4:  Describe the appearance of the three types of haemolysis produced by Streptococcus species, the designation given to these three types and how Streptococci are classed according their haemolytic ability.

5: Explain in detail the principle of the Lancefield group test. Your answer should not be merely a description of the process. What are the limitations of the test?

6: Name three kinds of differential agar used in this practical series with a brief description of how each works. 

7:  Explain the principle of the Germ Tube Test and detail the main limitation associated with it.

8:  Give a brief description on how the RapID TM NF Plus test strip system works. Include two disadvantages of using this test versus other diagnostic methods.

9:  Compare and contrast the tube coagulase and staph latex tests as a means of identifying Staphylococcus aureus. 

Patient 1 (Organism A): This organism was isolated from an otherwise healthy 35 year old man with a painful boil on his face. His temperature was found to be normal.

Patient 2 (Organism B):  This organism was isolated from a 13 year old boy presenting with a fever, sore throat, difficulty swallowing and foul breath.

Patient 3: (Organism C): This organism was isolated from a 23 year old female presenting with white vaginal discharge and extreme itching of the vaginal area.

 Patient 4:  (Organism D): This organism was isolated from a 34 year old pregnant female (second trimester) presenting with a five day history of urinary urgency unpleasant smelling urine and a general sense of feeling unwell. 

Patient 5: (Organism E): This organism was isolated from a sputum sample from a 17 year old male admitted to hospital with symptoms of pneumonia.  This patient has previously been diagnosed with cystic fibrosis.

1. Explanation of the identification of each organism

Organism A: The organism has shown positive reaction on gram staining and appeared round shaped, which indicates identification of gram positive cocci. On catalase test positive result has been found which ensures enzyme catalytic nature. Strong clumps during staph latex test and positive result on tube coagulase test has been found. Further, grey colonies have occurred on blood agar which has indicated presence of alpha haemolytic staphylococci.

Organism B: The organism has appeared as round shaped and purple in colour, hence indicated presence of gram positive cocci. Catalase and staph latex test have provided negative result, which has ensured the absence of staphylococci. Further, positive test on lancefield grouping, beta hemolysis on blood agar and creamy white colonies on Colombia agar has confirmed the identification of Streptococci pyogenes.

Organism C: This organism has appeared purple colour and round shaped on gram staining, hence indicated the presence of gram positive cocci. The germ tube test specific for Candida albicans has shown positive result and chlamydospore have been found on corn meal agar plate. In addition, no hemolysis has been found on blood agar and bread yeast smell has been observed which has indicated the presence of Candida Albicans.

Organism D: Pink and rod shaped appearance on gram staining has indicated presence of gram negative bacteria and negative result on oxidase has confirmed anaerobic nature of the test organism. Furthermore, positive result on CLED agar plate and CUTI agar plate has indicated presence of UTI causing organism. Therefore, Escherichia coli has been identified as the gram negative, anaerobic, rod shaped, UTI causing bacteria.

Organism E: Pink coloured, rod shaped appearance has been detected upon gram staining and positive result has been found on oxidase test, which has indicated the presence of aerobic, gram negative bacteria. Further, white colonies have been observed on CLED agar and RapID test has confirmed the presence of Pseudomonas aeruginosa with code 714616.

Patient 1: The organism has been isolated from the painful boil on the face of the patient. According to Spellerberg and Brandt (2016), such boil is formed due to infection caused by Staphylococci which results in swollen area on skin through the accumulation of pus and dead tissue. Further, the tests performed for this organism have confirmed the presence of Staphylococci.

Patient 2: The organism has been isolated from a 13 year old boy suffering from sore throat, fever, foul breath and problem in swallowing. El?Boghdadly, Bailey and Wiles (2016), have informed that such consequence may occur due to infection caused by Streptococcus. Furthermore, the beta hemolysis and positive test on lancefield grouping has confirmed the presence of Streptococcus pyogenes.

Patient 3: The organism has been isolated from white vaginal discharge which usually caused due to candidiasis, trichomoniasis, and bacterial vaginosis (Venugopal et al. 2017). Further, the germ tube test and appearance of chlamydospore on corn meal agar has confirmed the identification of Candida albicans.

Patient 4: The organism has been isolated from urine of a pregnant women suffering from urinary urgency and unpleasant smelling of urine. As per the guidance of Conover et al. (2016) it has been assumed that the consequence has been developed due to UTI. Additionally, the tests done for this organism has confirmed the presence of gram negative, anaerobic, rod shaped, UTI causing bacteria, Escherichis coli.

2. Significance of isolated organisms for each patient

Patient 5: The organism has been isolated from the sputum of the patient developing the symptoms of pneumonia. The patient was diagnosed with cystic fibrosis earlier. Pseudomonas aeruginosa has been identified through the tests. Micek et al. (2015) have reported that this organism causes nosocomial infection and nosocomial infection may lead to suffering from pneumonia. Hence, it can be said that, the identification is appropriate for this patient.

As per the result, for patient 1, organism A, that is staphylococci, has been identified. Six antibiotics have been tested for examining the antibiotic resistance of the particular strain of staphylococci that has infected patient 1. As per the identification the organism has shown resistance to Erythromycin and Penicillin, hence, these antibiotics are not effective for patient 1. Further, sensitivity to four antibiotics has been detected such as, Tetracyclin, Gentamycin, Fusidic acid and Clindamycin. Among these antibiotics, the highest zone of inhibition has been identified for fusidic acid which inhibits the protein synthesis of the organism (Clark, Loeffler and Bond 2015). Therefore, Fusidic acid would be the best choice for curing staphylococci infection for this particular patient.

On the other hand, organism D, that is Escherichia coli, has been identified for patient 4. Six different antibiotics such as Trimethoprim, Nitrofurantoin, Ciprofloxacin, Augmentin, ampicillin and Cephalexin. Resistance for Trimethoprim and ampicillin has been detected in the test, hence these antibiotics are not effective for this particular patient. In addition, it has been found that the strain of Escherichia coli that has infected the patient 4 is sensitive to Nitrofurantoin, Ciprofloxacin, Augmentin and Cephalexin. Among these antibiotics, Ciprofloxacin has shown highest area of inhibition. Therefore, Ciprofloxacin would be the best choice for this particular patient as would act fast by inhibiting cell division (Ma et al. 2017).

Hemolysis is defined as the breakdown of red blood cells (Tille 2015). Roetzer et al. (2016) has reported that, according to the reaction observed in the blood agar, streptococci are classified into three hemolytic groups such as alpha haemolytic, beta hemolytic and gamma hemolytic, by Brown in 1919. These three types of hemolysis indicate complete degradation of red blood cell, partial degradation if red blood cell and no degradation of red blood cells on the blood agar.

Alpha haemolytic species: Alpha hemolysis is associated with partial degradation of red blood cells on the blood agar and green colonies occurred in the zone of hemolysis. This is mainly formed by the streptococci that produce hydrogen peroxide, therefore converts the oxidizing haemoglobin to the green methomoglobin (Roetzer et al. 2016). Example- Streptococcus pneumoniae.

Beta haemolytic species:  Beta hemolysis is associated with complete hemolysis or degradation of red blood cells on the blood agar. This type of hemolysis occur through two kinds of hemolysin such as hemolysin O and S which enhances the hemolysis reaction. Due to complete lysis of the red blood cell, clorless colonies occue in the blood agar plate.  Generally the Streptococci causing opportunistic infection are found to be beta haemolytic organism, for example, Streptococcus pyogenes (Roetzer et al. 2016).

Gamma haemolytic species: Gamma hemolysis is considered as no hemolysis. However, slight discoloration may occur in the blood agar plate. Generally gamma haemolytic streptococcus are non-pathogenic in nature, for example, Enterococcus faecalis (Roetzer et al. 2016).

3. Selection of suitable antibiotics for patients with organisms A and D

The process of lancefield group test is utilise in order to identify the coagulase negative and catalase negative bacteria on the basis of the carbohydrate composition of their antigen present on the cell wall. The test is developed by Rebecca Lancefield in order to identify the members of Streptococcaceae (van Sorge et al. 2014). Currently this test is used for the classification of haemolytic streptococci into different groups from A to O on the basis of their serologic action. This classification occurs according to the formation of precipitation when the organism is examined with group specific antisera. In order to extract the sero group from the cell wall, acid extraction is done with hydrochloric acid. Then it is treated with group specific antisera. The acid precipitable and heat coagulase proteins are removed through acid extraction and the antisera inhibits the formation of protein antibody, hence, the precipitation occurs on the basis of carbohydrate present in the antigen present on the cell wall (Rush et al. 2017).

The test has several limitations, for example, it is not a confirmatory test and the disc precipitates are too slight that they may escape detection as well. Minimal numbers of cross-reaction develops with this complex antigenic mosaic. Hence, it is difficult to identify haemolytic streptococci with lancefield group test and further confirmatory test is required for definite identification of streptococci (van Sorge et al. 2014).

Three types of differential agar that have been used in this practical series include-

• Blood agar
• Corn Meal agar
• CLED agar

Blood agar: Blood agar is considered as the enriched growth medium of bacteria, especially the fastidious organisms like streptococci, which fail to grow properly in other growth medium. The base of blood agar is mainly consists of protein source such as tryptone, sodium chloride, soybean protein digest and 5% blood of sheep. The composition of blood agar include, papaic digest of soy meal, pancreatic digest of casein, sodium chloride, agar and distilled water. Extra cellular enzymes are produced by some bacteria which act upon the red blood cells in blood agar and cause cell lysis. Such hemolysin leads to radial duffusion of the outwards of colony, which causes partial or complete destruction of RBC in the medium and forms colorless products due to denaturation of haemoglobin of the cells (Lim et al. 2016).

Corn meal agar: This agar is consists of infusion form of corn meal, agar, distilled water and polysorbate 80. Corn meal agar is mostly use as the mycological growth medium, especially for production of chlamydospore of Candida albicans. Corn meal infusion contains huge amount of nutrients and addition of polysorbate 80 which is oleic ester, influence the growth of chlamydospores. In addition, corn meal agar with 1% dextrose is used to identify some Trichophyton species as it produces red pigments when cultivate on this conventional media (Kim et al. 2016).

CLED agar: Cystine-lactose-electrolyte-deficient agar or CLED agar is used for the isolation of urinary microbes as it allows the growth of urinary pathogens. Due to such significance, it is used in the suspected cases of UTI. The composition of this agar include, lactose, pancreatic digest casein, pancreatic digest gelatin, beef extract, L-Cystine, bromothymol blue, agar and distilled water. It mainly works by discriminating the gram negative bacteria according to their ability of lactose fermentation. Lactose in the medium helps the bacteria to use the fermentative mechanism and bromothymol blue act as the pH indicator and lactose fermenting bacteria indicates lower pH and change the colour of the media from green to yellow (Mitra et al. 2015).

4. Different types of Streptococcus haemolysis and classification

Germ tube test is performed for the screening of Candida albicans, especially to differentiate Candida albicans from other yeast. The test is discovered by Reynold and Braude in the year 1956. It has been found that Candida albicans forms germs tube when allow to grow in sheep or human serum for 3 hours at 37⁰ C (Martín-Mazuelos et al., 2015). Martínez-Jiménez et al. (2014), have reported that due to excessive protein synthesis and production of ribonucleic acid germ tube is formed by this organism. The germ tube solution used in the test contains foetal bovine serum and tryptic soy broth which are considered as essential and effective nutrients for the synthesis of protein. Hence, the solution allows the organism to produce germ tube and then it is lyophilized for stability. In this way germ test helps to screen for the germ tube of Candida albicans which is defined as the virulence factor of this organism. Besides such effectiveness, one of the main limitation of this test is false interpretation of pseudohyphae of Candida tropicalis as germ tube. Hence, it is not a confirmation test, further test is required for definite identification of Candida albicans (Martín-Mazuelos et al., 2015).

The RapID TM NF Plus test strip system is used to identify the glucose non-fermenting, gram negative bacteria and some glucose fermenting, gram negative bacteria that do not belong to Enterobacteriaceae family. The system consists of RapID NF Plus Panels and RapID NF Plus reagent. Several reaction cavities are present in each panel. These cavities molded into circumference of plastic disposable tray. In addition the reaction cavities contain several dehydrated reactants. Simultaneous inoculation of the reaction cavities with pre-set amount of inoculum is allowed by the tray. The test organism is used as the inoculum in order to rehydrate and start the test. Then the system is allowed for incubation and after this colour development is examined for the reactivity of each reaction cavity. As the basis of identification positive and negative test scores are used and compared them with the reactivity pattern mentioned in the database of Electronic RapID Compendium or RapID NF Plus Differential Chart is also used in some cases. This colour change is manly occurred due to degradation of various substances by the test organisms (McPherson and Pincus 2017).

In addition to such advantages there are some disadvantages of this system as compare to other diagnosis system such as API 20E system. For example, the API 20E system is more convenient and provide an easier way to detect gram negative bacteria as compare to the RapID TM NF Plus test strip system. Further, Tille (2015), has reported that API 20E provides result within 4 hours of incubation at 37⁰ C, whereas RapID TM NF Plus test strip system takes 24 to 48 hours for the similar test. Hence, API 20E is faster than RapID TM NF Plus test strip system.

Both the coagulase tube test and staph latex test is effective and useful for the identification of Staphylococcus aureus, however, there are several difference in these two process which helps to compare between these tests. The tube coagulase test is performed to detect staphylocoagulase that are able to react with the coagulase reacting factor (CRF) which is a thrombin like reagent. The staphylocoaguase and CRF interact to convert fibrinogen to fibrin. Formation of clot indicates positive test and presence of Staphylococcos aureus (Osman et al. 2015). On the other hand, staph latex test helps to identify staphylococci that contain protein A or coagulase, especially staphylococcus aureus. In this test white latex molecules which are coated with human fibrinogen are used to identify the clumping factor and IgG to confirm the detection of protein A. When the test is performed with Staphylococcus aureus, the clumping factor produces strong agglutination of the latex molecules (Tille 2015). Both the process provides definite result and confirms the presence of Staphylococcus aureus, however, staph latex test allows to do rapid test and control latex is not required in this process. Whereas, tube coagulase is time consuming as compare to staph latex test and the negative tubes need to be kept for overnight for re-examination (McPherson and Pincus 2017). Therefore, it can be said that, both the tests are reliable for the identification of Staphylococcus aureus, nevertheless, staph latex test is comparatively advance due to the ability of allowing rapid test in less time.

5. Principle of Lancefield group test and its limitations

References:

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