Case Study 1
A 17 year old female with a one month history of worsening perineal and buttock lesions. Two weeks prior to admission, she presented to her family doctor for evaluation. At that time, the lesions were excoriated, inflamed and indurated. The lesions were multiple and scattered over the perineum and buttocks. The largest one measured approximately two centimetres in maximum dimension. The lesions became more painful and began to drain a purulent discharge. The patient was admitted for incision and drainage of the lesions. Fluid from the lesions was sent for investigation to the Microbiology Lab.
Two blood agar plates were inoculated and incubated – one aerobically and one anaerobically at 37˚C.
The following are the results obtained for the organism growing on the blood plate that was incubated aerobically and for the two organisms- A and B, that were growing on the blood agar plate that was incubated anaerobically. Both plates were incubated at 37˚C.The organism growing on the aerobic blood agar plate and also on the anaerobic plate as Organism A was identified as a coagulase negative Staphylococcus. Organism B that was growing on the anaerobic blood plate and that was the causative pathogen was identified as Bacteroides fragilis.
Table 1: Basic characterisation and further identification test results carried out on the two blood agar plates, incubated both aerobically and anaerobically at 37˚C for the patient in Case Study 1.
|Blood agar incubated aerobically||Blood agar incubated anaerobically||Controls:
|Test||Organism A||Organism B|
|Colonial morphology||White, 0.5mm, convex, entire, circular, gamma haemolysis, no odour.||White, 0.5mm, circular, entire, gamma haemolysis, no odour.||Grey, 1mm, mucoid, circular, entire, gamma haemolysis, ++ odour.||/||/|
|Gram stain||Gram positive cocci in clusters||Gram positive cocci in clusters||Gram negative bacilli, pleomorphic||/||/|
|Further Identification Tests:
|An – ident||/||/||+ for Bacteroides Fragilis||/||/|
For the blood agar that was incubated anaerobically, there were two different colonies growing on the agar. Organism A appears to be the same colony that was growing on the aerobic blood agar plate and is also growing on the anaerobic plate.
The catalase and oxidase test was not carried out for Organism A as there were no colonies left on the agar plate to carry out the testing.
Catalase: + = positive for the enzyme catalase- bubbles produced.
– = negative for the enzyme catalase -no bubbles produced
Oxidase: + = positive for the enzyme oxidase – purple colour formed
– = negative for the enzyme oxidase – no colour formed
Smell: ++ = strong, slightly pungent odour
Coagulase test: + = positive for the coagulase enzyme. Agglutination observed.
– = negative for the coagulase enzyme. No agglutination observed.
MTZ test: Positive = A zone of clearance (inhibition) of growth around the MTZ disc
Negative = no zone of clearance around the disc – the bacteria grows up to and around the MTZ disc.
Table 2: Antibiogram profile obtained for Organism B that grew on the anaerobic blood agar, for the An-ident test.
Legend: E60 = Erythromycin 60µg
RD15 = Rifampcin 15µg
CT10 = Colistin 10µg
P2 = Penicillin 2 units
K1000 = Kanamycin 1000µg
VA5 = Vancomycin 5µg
S = Sensitive
R = Resistant
An-ident test: + = the bacteria is sensitive (zone of inhibition – no growth around these antibiotic discs) to Erythromycin and Rifampicin and is resistant (no zone of inhibition – growth up to and around the antibiotic discs) to Colistin, Penicillin, Kanamycin and Vancomycin. This bacteria was identified as Bacteroides fragilis.
It is more than likely apparent that Organism A is a coagulase negative Staphylococcus. This organism was facultative as it grew on both the aerobic and anaerobic blood agar plates. This organism appeared as white, convex, circular, non-odorous, gamma haemolytic colonies on both agars. In the Gram stain this organism appeared as Gram positive cocci in a clustered formation. Organism A obtained a positive catalase test and a negative oxidase test. The coagulase test was then carried out to further identify this organism. It tested negative for the coagulase enzyme, hence leading to the identification of this organism as a coagulase negative Staphylococcus.
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From observing table 1, it is evident that Organism B is Bacteroides fragilis. This organism only grew on the anaerobic blood agar plate. It had a pungent smell, displayed gamma haemolysis, was circular, convex, grey in colour and 1mm in size on the anaerobic blood agar plate. In the Gram stain, this organism appeared as Gram negative bacilli that were pleomorphic in shape. In Case Study 1, the female patient possesses lesions that are draining a purulent discharge. A foul smell is associated with Bacteroides fragilis. This smell is due to the production of gases by the bacteria. Organism B obtained a positive catalase test and a negative oxidase test. An MTZ test was carried out on this organism and again, this organism obtained a positive result, emphasising that this organism is indeed anaerobic, as all anaerobic bacteria are sensitive to Metronidazole. In the An-ident disc antibiogram, the organism was shown to be Bacteroides fragilis. This anaerobic organism is infecting the patient. The site where the lesions are located – the perineum and buttocks are locations that this bacteria are prone to infect. The lesions are inflamed, excoriated and indurated as well as producing a purulent discharge are all indicative of tissue necrosis at the site of infection. Bacteroides fragilis is prevalently found on mucosal surfaces in indigenous flora. It initiates infection when tissue trauma or surgery occurs or when tumours are present on the mucosal surface. This organism can also become displaced from its normal site and hence, infect a site that is free from organisms. Bacteroides fragilis then penetrates host tissues and establishes infection. A reduced blood supply and necrosis of tissue facilitate the growth of this anaerobe due to them reducing the potential of oxidation-reduction.
Bacteroides fragilis possesses a wide array of virulence factors that aid in its pathogenesis. These include the production of various enzymes including neuraminidase that targets glycoproteins in the plasma of humans that contain neuraminic acid by altering this acid. Another enzyme produced by this bacteria includes heparinase. This facilitates clotting intravascularly. Hyaluronidase breaks down the hyaluronic acid between cells, facilitating the spread of this pathogen. Bacteroides fragilis also produces beta-lactamase that causes the destruction of the beta-lactam ring in beta-lactam antibiotics, initiating the resistance to the organism. DNase enzyme degrades host DNA or RNA, by catalysing the break-down of the phosphodiester bonds in the DNA backbone. This again is produced by this organism. Phosphatase is another enzyme produced by this organism. It is involved in signalling and the regulation of cells. The capsule is also a highly important virulent factor in Bacteroides fragilis. Succinate and butyrate, which are responsible for respiration in cells are produced by this organism and they possess a cytotoxic effect. This bacteria also possesses fimbriae, enabling it to adhere to the host’s epithelial cells. As this organism is Gram negative, it contains a Lipopolysaccharide layer (LPS) that activates coagulation via the intrinsic pathway (Wexler, H., 2007).
Bacteroides fragilis is one of the most resistant anaerobic bacteria to antimicrobials. This is due to the production of beta-lactamase, initiating resistance to the beta-lactam antibiotic family. This organism is resistant to many antibiotics including amikacin and gentamicin, members of the aminoglycoside family of antibiotics. Resistance is also shown to penicillin, vancomycin, colistin and kanamycin. Metronidazole (MTZ) inhibits the nucleic acid synthesis by Bacteroides fragilis, chloramphenicol, imipenem and antibiotics containing a mixture of both beta-lactams and an inhibitor of beta-lactamase are most effective against this bacterial strain. Rifampcin and erythromycin are also effective in treating infections by this organism (Brook et al., 2013).
This organism is responsible for a variety of infections throughout the human body. Intra-abdominal, soft tissue, bacteraemia, dental, oral, bone and female genital tract infections are among the most common infections caused by Bacteroides fragilis. Abscesses, lesions and infections below the waist are also associated with this bacteria. This organism can also play a virulent role in the pathogenesis of colon cancer and periodontal disease due to the organism’s production of enzymes such as neuraminidase and heparinase. These enzymes allow it to penetrate host tissues and cause infection following surgery or trauma to the tissue (Wexler, H., 2007).
In all, the two blood agar plates, one that was incubated aerobically and the other that was incubated aerobically were examined. Gram stains, colonial morphology, basic characterisation tests and further identification tests were all carried out to decipher the organisms that were present. These organisms were identified as Coagulase negative Staphylococcus – Organism A that was growing on both plates and Organism B – Bacteroides fragilis that was growing on just the anaerobic plate. Bacteroides fragilis is the principal organism that is causing the infection in the female patient. The clinical details of the patient were discussed in conjunction to the infecting pathogen. The virulence factors and pathogenesis of this organism were described, along with the antimicrobials used to treat and the infections caused by Bacteroides fragilis.
- Brook, I., Wexler, H. and Goldstein, E. (2013) Antianaerobic Antimicrobials: Spectrum and Susceptibility Testing. Clinical Microbiology Reviews [Online] 26(3), pp.526-546. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719496 [Accessed on: 5th October 2018]
- Wexler, H. (2007) Bacteroides: the Good, the Bad, and the Nitty-Gritty. Clinical Microbiology Reviews [Online] 20(4), pp.593-621. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2176045 [Accessed on: 5th October 2018]
Practical Risk Assessment Form
Practical title: Anaerobic Case Study 1
Practical description: Give a brief description of work to be undertaken and the nature of the materials and techniques to be used.
Working with two organisms – Bacteroides fragilis and coagulase negative Staphylococcus.
Colonial morphology, Gram stains, catalase, oxidase, MTZ test, An-ident and Coagulase test will be carried out on the organisms.
Blood agar plates will be streaked/lawned with the organisms and incubated anaerobically at 37˚C.
|Hazard||High||Medium||Low||Current control measures for this hazard||Options for improved controls|
Oxidase positive and negative controls
Catalase positive and negative controls
|Good aseptic techniques to control the exposure of this infectious agent.
The use of gloves and laboratory coat to avoid contamination of skin and clothes with these controls and bacteria.
Cleaning surface with disinfectant after working with these controls and the bacteria.
Disposing of gloves, slides and agar plates when they are finished with.
|Not touching face or mouth with gloved hands to prevent contamination with the bacteria and controls.|
Gram stains – Crystal Violet, Gram’s iodine, acetone and Carbol Fuschin
|Wearing gloves and laboratory coat to protect hands, clothes and skin from being stained with these chemical agents.
Wearing closed footwear and not sandals or open-toed shoes.
Not inhaling or ingesting the chemicals.
|Buttoning up laboratory coat properly.|
|Correct handling of the microscope.
Not tangling the lead or have it hanging down on the floor.
Turning it off when it is not in use.
|Not leaving it at the edge of the benchtop.|
|X||Proper handling of slides.
Disposing of them in the sharps bin when they are finished with.
|Not dropping them or disposing of them in the biohazard bins.|