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Meningitis is an inflammatory condition that occurs in the central nervous system affecting the membranes or meninges that cover the brain and spinal cord. Complications that can occur with meningitis patients include headache, fever, confusion, neck stiffness, vomiting, photophobia (inability to tolerate light) and phonophobia (inability to tolerate loud noises). A characteristic purple or red rash may also occur depending on the type of bacterial meningitis. The infection is usually spread by carriers, which are individuals who have the bacteria present in their throat, but they themselves do not develop an infection of the meninges. Bacterial means are the main cause of meningitis that can be due to the spread of contiguous infections from areas nearby the brain to the meningeal membranes such as sinusitis and mastoiditis, however most cases are caused by bloodborne pathogens. (Join-Lambert et al, 2010)
Normally, the CNS is a sterile area within the body and several mechanisms within the body protect this area from external bacteria, viruses or fungi. The blood-brain barrier (BBB) is one of the few mechanisms that protect the CNS from most bacteria which may have been transported in the bloodstream, however few extracellular pathogens pass this barrier that leads to bacterial meningitis. Such bacteria that cause meningitis include Escherichia coli and Streptococcus agalactiae (Group B Streptococcus) that occur mainly in newborns, while Neisseria meningitidis, Haemophilus influenzae type b and Strepto-coccus pneumoniae occur in children and adults. (Huang et al, 2000; Van de Beek et al, 2006).
Many of these bacterial organisms originate and enter the body in different areas. N. meningitidis, S.pneumoniae and H. Influenza are commensals of the nasopharynx, whereas E. coli and S.agalactiae enter via the digestive tract. There are many steps in how these organisms enter the CNS as they must have the ability to cross the oropharyngeal or digestive mucosal barrier, survive and replicate in the bloodstream, and to eventually cross the blood-cerebro- spinal fluid (CSF) barrier or blood-brain barrier (BBB). (Nassif et al, 2002).
In this experiment, we investigated and analysed a case study sample related to an unknown bacterial meningitis pathogen. We had to narrow down the possible pathogens and isolate the cause of the bacterial meningitis. The given case study involved a 2150g female neonate born at 32 weeks gestation. Her APGAR scores were 2 and 5, and she was intubated due to poor respiratory function. Her mother was febrile at the time of delivery and was given ampicillin. Blood cultures were taken from the newborn at birth and grew gram positive coccobacilli. Two lumbar punctures were attempted, both traumatic. (UTS, 2010).
From this case study, it can be hypothesised that the possible organism causing meningitis in the newborn could be Streptcoccus agalactiae, which is also known as group B streptococcus (GBS). This organism is one of the leading causes of neonatal meningitis, but it may also affect the elderly and those with underlying diseases. The gram positive coccobacilli may also give a clue in its possible identification, which is characteristic of Streptcoccus agalactiae. (Baker, 2000).
MATERIALS & METHODS
Case 7 - Streptcoccus agalactiae
A case isolate was provided and given on blood agar prepared from a CSF sample. The following screening tests (UTS, 2010) were carried out to help identify this organism:
The plate appearance including haemolysis type and colony morphology was observed and recorded.
A gram stain of the organism was then carried on a glass slide and observed under a microscope with oil at x100 magnification. The organism morphology and gram stain was recorded.
A catalase and oxidaise test was then done on the organism. A toothpick was used to pick up a small amount organisms from the same colony on the isolate plate and either placed in a small plastic test tube with catalase reagent or on a small piece of filter paper on a glass slide with oxidase reagent.
Aesculin agar. It was provided in a small glass jar and was inoculated with the organism using a sterile loop.
The Lancefield antigen latex kit found out if the organism belonged to a Lancefield group. (A, B, C, D, G). The kit was called the "Phadebact CSF Test" and was carried out as follows: The test reagents with the particular Lancefield group antibody were provided in the kit and ready to use. These reagents contained methylene blue for easy visualisation of results. Before testing, each specimen was heated at 37Â°C for 10 minutes with a heating block to eliminate nonspecific reactions. One drop of heated sample was then mixed with one drop of each test reagent on a disposable white card slide provided in the kit. The card slide was rocked manually for about a minute and observed for a co-agglutination reaction if the corresponding antigen is present. (Bactus, 2006).
An antibiotic sensitivity test (AST) on sensiblood agar using the CLSI (American) method. This was carried out by first inoculating a sterile saline solution with one or two well isolated colonies of the organism to produce a bacterial suspension. The turbidity of the suspension should be just visible to the eye (0.5 McFarlane). The sensiblood agar plate was then inoculated by a cotton wool swab dipped in the bacterial suspension, gently streaking the whole plate evenly with the swab at 120â° angles, rotating three times. This plate was then left to dry for three to five minutes. Selected antibiotic disks (maximum 6) with the correct strength was evenly placed apart on the plate using flamed forceps and pushed down. It was then incubated aerobically at 37â°C for 24 hours. After incubation the antibiotic disk zone diameters were measured and compared according to the CLSI table. (Page 29. UTS, 2010). The diameter results are either assigned as sensitive, intermediate or resistant. The antibiotic disks used for this experiment included Penicillin-G, Erythromycin, Tetracycline, Vancomycin, Cefotaxime and Opticin.
Plate appearance: A narrow soft clear beta haemolytic zone occurred around the colonies growing on the plate.
Colony morphology: 1-3mm in diameter, smooth, round, convex, rough colony edges, grey-white in colour.
Gram stain: Gram positive (purple stained) coccobacilli (intermediate shape between coccus and bacillus) in chains. i.e. a bacillus that is short and oval in shape.
Catalase: no bubbles formed in the catalase solution, thus a negative result for catalase activity.
Oxidase: no purple colour resulted with the oxidase reagent, thus a negative result for oxidase activity.
Aesculin test: the aesculin agar did not change to a black colour, thus a negative result.
Lancefield antigen test: the co-agglutination reaction only occurred with the Lancefield group B reagent. No reaction occurred with the other reagents. Thus this organism belongs to the Lancefield group B.
AST using the CLSI method: According to table 1, the organism was sensitive to a degree to all of the antibiotic disk types chosen, except for opticin where it was resistant.
Zone Diameter (mm)
Table 1 - The zone diameters and results from the antibiotic sensitivity test on sensiblood by the CLSI method.
Lumbar punctures can be used to collect cerebrospinal fluid (CSF) to diagnose or exclude a meningitis infection. It is carried out by inserting a needle into the spinal canal and extracting a sample of CSF. CSF has many functions such as a shock absorber for the brain and surrounds the brain and spinal cord. Once a CSF sample is collected, it must be analysed as soon as possible by the laboratory as it can be easily contaminated by external sources which affect the diagnosis. Microscopically, the CSF sample mainly contains many polymorphonuclear leukocytes which are pus cells due to infection, as well as varying loads of the bacteria responsible for the infection depending on the type of meningitis. Red blood cells may also be present if the lumbar puncture was traumatic. (Tunkel et al. 2004).
Treatment of an infected individual is usually promptly given antibiotics suitable for the type of meningitis or antivirals if the meningitis was by viral means. If not treated promptly, meningitis can cause long-term complications such as epilepsy and deafness. Fortunately, some types of meningitis can be prevented by immunisation such as meningitis caused by Haemophilus influenzae type b and Strepto-coccus pneumonia. (Tunkel et al. 2004)
In this experiment, the CSF meningitis isolate on blood agar could be caused by a range of bacterial organisms such as Escherichia coli, Streptococcus agalactiae (Group B Streptococcus), Neisseria meningitidis, Haemophilus influenzae type b, Listeria monocytogenes and Strepto-coccus pneumoniae. However, this range was narrowed down using simple tests used in the clinical laboratory. These included cell count, plate appearance, colony morphology, gram stain, catalase/oxidase test, aesculin test, Lancefield antigen test and antibiotic sensitivity tests. Tests must be selected carefully for suitability as the amount CSF collected are usually minute, especially CSF from infants. In an adult, CSF samples taken are usually 10-15mL, whereas for a baby approximately 0.5mL is taken. (UTS, 2010).
Cell counts are useful as it many help show the presence of an infection in the CSF. Normally, the CSF is sterile and white blood cells or red blood cells are not present. In an infected individual with meningitis, the CSF contains elevated white blood cells due to infection of the meninges and sometimes red blood cells if the lumbar puncture was traumatic. The type of white blood cell (i.e. lymphocytes) and their numbers present in CSF may help narrow down the type of bacterial meningitis suspected. (Tunkel et al. 2004) In the experiment, a cell count was not carried out in the experiment due to the lack of CSF samples, only the CSF isolate on an agar plate was provided.
On the blood agar the colony appeared to have a narrow soft clear beta haemolytic zone occurred around the colonies. This is suspicious for organisms such as Streptococcus agalactiae (Group B Streptococcus) or Listeria monocytogenes, as both of these organisms exhibit weak beta haemolytic zones around their colonies. (Hawkey et al, 2004).
The colony morphology consisted of medium sized 1-3mm in diameter colonies that were grey-white in colour but were round with rough edges. This is characteristic of a bacillus as coccus colonies are mainly characterised with very small perfect circular colonies such as Staphylococcus aureus. Possible bacillus organisms that show the colony morphology observed in the experiment include Streptococcus agalactiae (Group B Streptococcus), Listeria monocytogenes and Escherichia coli. Although Haemophilus influenzae has similar colony morphology, it can be excluded as it is a highly demanding facultative organism that struggles to grow on blood agar and requires choc agar instead. It will only grow on blood agar as satellite colonies around other bacteria as they provide the required X (haemin) and V (NAD) factors for growth. (Baker, 2000).
A gram stain of the isolate was carried out and observed microscopically, resulting in gram positive coccobacilli in chains, which is an intermediate shape between coccus and bacillus (i.e. a bacillus that is short and oval in shape). This further confirms that this organism is a bacillus due to the prediction from the colony morphology. Both Streptococcus agalactiae (Group B Streptococcus) and Listeria monocytogenes are short bacilli organisms that are gram positive, however Listeria monocytogenes can be excluded as no tumbling motility was observed. Another organism that has a coccobacilli shape is Haemophilus influenzae, however it is excluded since it exhibits a gram negative stain. (Hawkey et al, 2004).
A catalase test differentiates organisms based on their production of catalase enzyme that catalyses the reaction that decomposes hydrogen peroxide in to water and oxygen. A positive test for catalase enzyme production is the formation of bubbles in the solution due to the oxygen gas produced. (Hawkey et al, 2004). The CSF isolate provided was catalase negative and this further excludes Listeria monocytogenes from Streptococcus agalactiae (Group B Streptococcus) since Listeria monocytogenes is a catalase positive organism.
The oxidase test determines whether an organism produces cytochrome oxidases, which means it utilises oxygen by an electron transfer chain. (Hawkey et al, 2004). In the experiment, no purple colour was produced with the oxidase reagent, thus it is oxidase negative. This test that was carried out did not have much use for this organism as most gram positive organisms are oxidase negative.
An aesculin test was also carried out to further confirm and exclude Listeria monocytogenes from Streptococcus agalactiae (Group B Streptococcus). Aesculin is a glucoside that occurs naturally in the horse chestnut. It is incorporated into agar with ferric citrate and bile salts. Organisms that can hydrolyse aesculin with a certain enzyme forms aesculetin and glucose. The aesculetin forms dark brown or black complexes with ferric citrate, allowing the test to be read as a positive. (Hawkey et al, 2004). Listeria monocytogenes is an organism that contains an enzyme that hydrolyses aesculin, while GBS does not carry this reaction out and the agar is unchanged.
The Lancefield antigen latex kit found out if the organism belonged to a Lancefield group. (A, B, C, D, G). The kit used was called the "Phadebact CSF Test" which contained agents with antibodies corresponding to a certain Lancefield group. A positive result for a Lancefield group is a co-agglutination reaction that makes the solution look grainy. This reaction occurs where the antibody in the kit reagent reacts with its matching capsular antigen of the organism in the sample. During the experiment, the co-agglutination reaction only occurred with the Lancefield group B reagent. No reaction occurred with the other reagents. Thus it can be said that this organism belongs to the Lancefield group B which is indicative of Streptococcus agalactiae (Group B Streptococcus). To check the reliability of the Lancefield reagents, positive controls can be used which are provided in the kit. However although this test is quick and easy to find out which organism is present in the sample, further testing must be carried out before the final diagnosis as a false result can occur. For instance, those organisms without a capsule are non-reactive in this test due to the lack of capsular antigens that react with the Lancefield agent antibodies, thus a false negative result could occur. (Bactus, 2006)
An antibiotic sensitivity test (AST) on sensiblood agar using the CLSI (American) method was carried out. This test is used to find out if an organism is susceptible or resistant to a certain antibiotic. It can also be used to find the minimum inhibitory concentration (MIC) of an antibiotic for the tested bacteria, where generally larger zones around the antibiotic disk correlate with smaller MIC. This information can be useful in choosing the suitable antibiotic to treat the particular infection. The antibiotic disks used for this experiment included Penicillin-G, Erythromycin, Tetracycline, Vancomycin, Cefotaxime and Opticin. It was observed that the organism was sensitive to a degree to all of the antibiotic disk types chosen, except for opticin where it was resistant (Table 1). Alpha streptococcus such as Strepto-coccus pneumonia is susceptible to opticin thus inhibitory zoning would occur around the antibiotic disk, whereas beta streptococcus such as Streptococcus agalactiae (Group B Streptococcus) is resistant to opticin thus no zoning occurs around the antibiotic disk. This AST further confirms the presence of Streptococcus agalactiae (Group B Streptococcus) in the CSF isolate provided. (Hawkey et al, 2004).
Streptococcus agalactiae (Group B Streptococcus) is found normally in the human body, commonly be found in the intestine, vagina, and/or rectal area. Typical symptoms are nonspecific to a certain type of meningitis and include fever, vomiting and irritability. It can affect neonates and their mother, as well as chronically ill patients. Most women who are carriers of the GBS will not have any symptoms present, however, under certain circumstances, both the mother and/or newborn can develop GBS meningitis vertically in utero or during natural delivery. This organism causes one of the highest mortality and morbidity rates in neonates if left untreated. About 5% of infected infants die, and if they survive, they often suffer from severe neurological complications, such as mental retardation and vision and/or auditory disabilities, but development of a successful Group B Streptococcus vaccine is still currently under investigation. (Johri, 2006).
The CSF isolate provided relating to the given case study was successfully diagnosed as Streptococcus agalactiae (Group B Streptococcus). This diagnosed organism was correlated with the case study as a neonate was involved and the CSF sample produced gram positive coccobacilli. It was achieved by limiting and narrowing down the possible pathogens using various clinical laboratory test used in microbiology. These included the use of plate appearance, colony morphology, gram stain, catalase/oxidase test, aesculin test, Lancefield antigen test and antibiotic sensitivity tests.