# What bacteria results in a Gram +ve cocci and catalase +ve? What test comes next?

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# Results so far.

We are trying to determine unknown microorganisms in intro to microbiology course. I first did gram stain and they were all cocci morphology, purple color and clumped together (actually I'm not so sure if clumped is right word, but they definitely were not in chains and I didn't see any isolated coccus, so if that's qualifies as clumped then they were clumped. So I have a gram+ here.

Next I did catalase test and got bubbles on slide; I put thick chunk of the unknown on slide and dropped 2-3 drops of $${H}_{2}{O}_2$$; instant bubbling.

# Next test.

So now I will test for coagulase but I'm uncertain what a positive or negative result will tell me about he organism and whether this is the terminal test (for ID-ing organism I mean)

My notes say that positive for coagulase indicates S. aureus. But does that mean that it is unnecessary to do the Mannitol test at that point then? Or is Mannitol used as a confirmation test?

Further, if unknown tests negative for coagulase, my notes say that if its also novobiocin sensitive (pos) then it is S. epidermidis.

But my same question then is, is it necessary to still do the mannitol test?

At this point you have staph, you can use this flow chart (from here) to figure out what staph exactly.

If the test comes back as coagulase positive be very careful as Staph aureus is pathogenic.

## DNase Test: Principle, Procedure, Results

DNA hydrolysis test or Deoxyribonuclease (DNase) test is used to determine the ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy for growth.

An agar medium DNase agar, a differential medium is used to test the ability of an organism to produce deoxyribonuclease or DNase.

This medium is pale green in color because of the DNA-methyl green (indicator) complex (Note: Methyl green is a cation that binds to the negatively-charged DNA). It also contains nutrients for the bacteria.

Figure -1: DNA Hydrolysis test A. Positive Staphylococcus aureus B. Positive Serratia marcescens C. Negative: Staphylococcus epidermidis

If the organism that grows in the medium produces Deoxyribonuclease, it breaks down DNA into smaller fragments. When the DNA is broken down, it no longer binds to the methyl green, and green color fades and the colony is surrounded by a colorless zone (See fig-1).

#### Requirements:

1. Media: DNase Agar or DNase agar with Methyl green indicator.
2. Reagent: Hydrochloric acid (1mol/L) only when DNase agar without an indicator is used
3. Others: Inoculating loop, Bunsen burner

### Procedure of DNase (DNA hydrolysis test)

1. Dry the surface of agar plates before use. Each plate may be divided into sections by drawing lines on the bottom of the plate.
2. Inoculate the test agar medium: There are two types of inoculation that can be done.
• Touch a colony of the organism under test with a loop and inoculate it onto a small area of the DNase test agar plate, in the middle of one of the marked sections to form a thick plaque of growth 5-10 mm in diameter after incubation.
• Incubate the plate at 37°C for 18-24hr.
• Use a heavy inoculum and draw a line 3-4 cm long from the rim to the center of the DNase test agar plate
• Incubate the plate at 37°C for 18-24hr.
1. When using DNase agar without the indicator,
• Flood the plate with 1N Hydrochloric Acid.
• Leave the plate to stand for a few minutes to allow the reagent to absorb into the plate. Decant excess hydrochloric acid and then examine the plate within 5 minutes against a dark background.

Fig:2: DNase Test: M. catarrhalis (+ve) and N.gonorrhoeae (-ve). When DNase is produced by organisms, an acidic end product is formed and the pH indicator changes from red (alkaline) to yellow (acid).

## Starch Hydrolysis Test: Principle, Procedure, Results

Starch hydrolysis test is used to determine if the organism is capable of breaking down starch into maltose through the activity of the extra-cellular α-amylase enzyme. Starch, the most important source of carbohydrate for humans, is a polysaccharide mixture of two polymers, amylose, and amylopectin, the latter being predominant.

Amylose is a linear polysaccharide of several thousand α-D-glucose linked by 1,4-α-glycosidic bonds. Amylopectin is a branched-chain polysaccharide composed of glucose units linked primarily by α-1,4-glycosidic bonds but with occasional α-1,6-glycosidic bonds, which are responsible for the branching.

### Principle

Starch molecules are too large to enter the bacterial cell, so only bacteria that secrete exoenzymes (α -amylase and oligo-1,6-glucosidase) are able to hydrolyze starch into subunits (dextrin, maltose, or glucose). These molecules are readily transported into the bacterial cell to be used in metabolism.

In starch hydrolysis test (also known as amylase test), we use starch agar, which is a differential nutritive medium. The test organisms are inoculated onto a starch plate and incubated at 30°C until growth is seen (i.e. up to 48 hours). The Petri plate is then flooded with an iodine solution.

If there is no enzyme present, and therefore no hydrolysis, the amylose and iodine react together to form a blue color. Depending on the concentration of the iodine used, iodine turns blue, purple, or black in the presence of starch.

When bacteria capable of producing α-amylase and oligo-1,6-glucosidase are grown on starch agar, they secrete enzymes into the surrounding areas and hydrolyze the starch. As no amylose is present in the medium surrounding the bacterial colony, clearing around the bacterial growth is seen (there is no color development).

## Diseases caused by S.pyogenes

Mnemonic: Diseases caused by Streptococcus pyogenes: NIPPLES:
Necrotising fasciitis and myositis
Impetigo
Pharyngitis
Pneumonia
Lymphangitis
Erysipelas and cellulitis
Scarlet fever/ Streptococcal TSS

1. Necrotizing fasciitis (NF)
2. Streptococcal toxic shock syndrome (STSS)
3. Cellulitis
4. Bacteremia
5. Pneumonia
6. Puerperal sepsis

### Key Tests that are used to identify S. pyogenes:

The sample for the isolation/identification of S. pyogenes is either pharyngeal exudates, pus, blood, tissue, or body fluids depending on the sites and nature of infection.

## Acinetobacter: Disease, Properties, Resistance

Acinetobacter is a group of bacteria commonly found in soil, water, and dry environments. Acinetobacter poses very little risk to immune-competent people and the infections are mainly confined in healthcare settings housing very ill patients. People with a weakened immune system are susceptible to infections with Acinetobacter. They acquire Acinetobacter infections by person-to-person contact or contact with contaminated surfaces.

Immunocompromised patients i.e. people who have weakened immune systems, chronic lung disease, or diabetes are susceptible to this infection. Very ill patients on a ventilator, those with a prolonged hospital stay, persons having invasive devices like urinary catheters are at greater risk of Acinetobacter infections. Outbreaks of Acinetobacter infections typically occur in intensive care units (ICU).

Acinetobacter can live on the skin and may survive in the environment/inanimate surfaces for several days. Acinetobacter is associated with skin colonization of hospital personnel and may also “colonize” or live in a patient without causing infection or symptoms, especially in tracheostomy sites or open wounds.

While there are many species of Acinetobacter and all can cause human disease, Acinetobacter baumannii for about 80% of reported infections. Acinetobacter causes a variety of diseases, ranging from pneumonia to serious blood or wound infections, and the symptoms vary depending on the disease. It is an important cause of ventilator-associated pneumonia and catheter-related bacteremia.

Biochemical Properties:

: Gram-negative cocci or coccobacilli
1. Oxygen requirement: Strictly aerobic
2. Growth requirements: Non-fastidious: Non fermentative: Negative Positive (+ve): Negative (-ve): Positive (+ve) some species may not give a positive citrate utilization test.: Negative (-ve): Negative except. A.haemolyticus
3. Chloramphenicol: Resistant: +ve

Drug Resistance and Antibiotics in use

Acinetobacter is often resistant to many commonly prescribed antibiotics. Multiple Drug Resistance (MDR) patterns observed in Acinetobacter baumannii (MDR-AB) currently pose significant challenges for the management and treatment of infections. CDC has categorized Multidrug-resistant Acinetobacter as a serious threats to public health.

There are few antimicrobial agents that are commonly used for the treatment of infections with Acinetobacter baumannii .

## Introduction

Actinomyces spp and Propionibacterium propionicus (previously Arachnia propionica) are members of a large group of pleomorphic Gram-positive bacteria, many of which fhave some tendency toward mycelial growth. Both are members of the oral flora of humans or animals. Actinomyces species, in particular, are major components of dental plaque. A israelii, A gerencseriae (previously A israelii serotype II), and P propionicus cause actinomycosis in humans and animals. Other species of Actinomyces can be involved in mixed anaerobic and other infections, where they may not always play an obviously pathogenic role. In addition, some coryneform bacteria (diphtheroids) isolated from clinical samples, which had been placed into the Centers for Disease Control Coryneform groups 1, 2 and E, have been identified as new species of Actinomyces.

## Contents

The genus Corynebacterium was created by Lehmann and Neumann in 1896 as a taxonomic group to contain the bacterial rods responsible for causing diphtheria. The genus was defined based on morphological characteristics. Based on studies of 16S-rRNA, they have been grouped into the subdivision of gram-positive eubacteria with high G:C content, with close phylogenetic relationship to Arthrobacter, Mycobacterium, Nocardia, and Streptomyces. [7]

The term comes from Greek κορύνη, korýnē 'club, mace, staff, knobby plant bud or shoot' [8] and βακτήριον, baktḗrion 'little rod'. [9] The term "diphtheroids" is used to represent corynebacteria that are non-pathogenic for example, C. diphtheriae would be excluded. [ citation needed ] The term diphtheroid comes from Greek διφθέρα, diphthérā 'prepared hide, leather'. [10] [11]

Comparative analysis of corynebacterial genomes has led to the identification of several conserved signature indels which are unique to the genus. Two examples of these conserved signature indels are a two-amino-acid insertion in a conserved region of the enzyme phosphoribose diphosphate:decaprenyl-phosphate phosphoribosyltransferase and a three-amino-acid insertion in acetate kinase, both of which are found only in Corynebacterium species. Both of these indels serve as molecular markers for species of the genus Corynebacterium. Additionally, 16 conserved signature proteins, which are uniquely found in Corynebacterium species, have been identified. Three of the conserved signature proteins have homologs found in the genus Dietzia, which is believed to be the closest related genus to Corynebacterium. In phylogenetic trees based on concatenated protein sequences or 16S rRNA, the genus Corynebacterium forms a distinct clade, within which is a distinct subclade, cluster I. The cluster is made up of the species C. diphtheriae, C. pseudotuberculosis, C. ulcerans, C. aurimucosum, C. glutamicum, and C. efficiens. This cluster is distinguished by several conserved signature indels, such as a two-amino-acid insertion in LepA and a seven- or eight-amino-acid insertions in RpoC. Also, 21 conserved signature proteins are found only in members of cluster I. Another cluster has been proposed, consisting of C. jeikeium and C. urealyticum, which is supported by the presence of 19 distinct conserved signature proteins which are unique to these two species. [12] Corynebateria have a high G+C content ranging from 46-74 mol%. [13]

The principal features of the genus Corynebacterium were described by Collins and Cummins in 1986. [14] They are gram-positive, catalase-positive, non-spore-forming, non-motile, rod-shaped bacteria that are straight or slightly curved. [15] Metachromatic granules are usually present representing stored phosphate regions. Their size falls between 2 and 6 μms in length and 0.5 μm in diameter. The bacteria group together in a characteristic way, which has been described as the form of a "V", "palisades", or "Chinese characters". They may also appear elliptical. They are aerobic or facultatively anaerobic, chemoorganotrophs. They are pleomorphic through their lifecycles, they occur in various lengths, and they frequently have thickenings at either end, depending on the surrounding conditions. [16]

### Cell wall Edit

The cell wall is distinctive, with a predominance of mesodiaminopimelic acid in the murein wall [4] [15] and many repetitions of arabinogalactan, as well as corynemycolic acid (a mycolic acid with 22 to 26 carbon atoms), bound by disaccharide bonds called L-Rhap-(1 → 4)--D-GlcNAc-phosphate. These form a complex commonly seen in Corynebacterium species: the mycolyl-AG–peptidoglican (mAGP). [17]

### Culture Edit

Corynebacteria grow slowly, even on enriched media. In terms of nutritional requirements, all need biotin to grow. Some strains also need thiamine and PABA. [14] Some of the Corynebacterium species with sequenced genomes have between 2.5 and 3.0 million base pairs. The bacteria grow in Loeffler's medium, blood agar, and trypticase soy agar (TSA). They form small, grayish colonies with a granular appearance, mostly translucent, but with opaque centers, convex, with continuous borders. [15] The color tends to be yellowish-white in Loeffler's medium. In TSA, they can form grey colonies with black centers and dentated borders that look similar to flowers (C. gravis), or continuous borders (C. mitis), or a mix between the two forms (C. intermedium).

Corynebacterium species occur commonly in nature in the soil, water, plants, and food products. [4] [15] The nondiphtheiroid Corynebacterium species can even be found in the mucosa and normal skin flora of humans and animals. [4] [15] Unusual habitats, such as the preen gland of birds have been recently reported for Corynebacterium uropygiale. [18] Some species are known for their pathogenic effects in humans and other animals. Perhaps the most notable one is C. diphtheriae, which acquires the capacity to produce diphtheria toxin only after interacting with a bacteriophage. [19] [20] Other pathogenic species in humans include: C. amycolatum, C. striatum, C. jeikeium, C. urealyticum, and C. xerosis [21] [22] [23] [24] [25] all of these are important as pathogens in immunosuppressed patients. Pathogenic species in other animals include C. bovis and C. renale. [26] This genus has been found to be part of the human salivary microbiome. [27]

The most notable human infection is diphtheria, caused by C. diphtheriae. It is an acute and contagious infection characterized by pseudomembranes of dead epithelial cells, white blood cells, red blood cells, and fibrin that form around the tonsils and back of the throat. [28] In developed countries, it is an uncommon illness that tends to occur in unvaccinated individuals, especially school-aged children, elderly, neutropenic or immunocompromised patients, and those with prosthetic devices such as prosthetic heart valves, shunts, or catheters. It is more common in developing countries [29] It can occasionally infect wounds, the vulva, the conjunctiva, and the middle ear. It can be spread within a hospital. [30] The virulent and toxigenic strains are lysogenic, and produce an exotoxin formed by two polypeptide chains, which is itself produced when a bacterium is transformed by a gene from the β prophage. [19] [20]

Several species cause disease in animals, most notably C. pseudotuberculosis, which causes the disease caseous lymphadenitis, and some are also pathogenic in humans. Some attack healthy hosts, while others tend to attack the immunocompromised. Effects of infection include granulomatous lymphadenopathy, pneumonitis, pharyngitis, skin infections, and endocarditis. Corynebacterial endocarditis is seen most frequently in patients with intravascular devices. [31] Several species of Corynebacterium can cause trichomycosis axillaris. [32] C. striatum may cause axillary odor. [33] C. minutissimum causes erythrasma.

Nonpathogenic species of Corynebacterium are used for very important industrial applications, such as the production of amino acids, [34] [35] nucleotides, and other nutritional factors (Martín, 1989) bioconversion of steroids [36] degradation of hydrocarbons [37] cheese aging [38] and production of enzymes. [39] Some species produce metabolites similar to antibiotics: bacteriocins of the corynecin-linocin type, [30] [40] [41] antitumor agents, [42] etc. One of the most studied species is C. glutamicum, whose name refers to its capacity to produce glutamic acid in aerobic conditions. [43] This is used in the food industry as monosodium glutamate in the production of soy sauce and yogurt. [ citation needed ]

Species of Corynebacterium have been used in the mass production of various amino acids including glutamic acid, a food additive that is made at a rate of 1.5 million tons/ year. The metabolic pathways of Corynebacterium have been further manipulated to produce lysine and threonine. [ citation needed ]

L-Lysine production is specific to C. glutamicum in which core metabolic enzymes are manipulated through genetic engineering to drive metabolic flux towards the production of NADPH from the pentose phosphate pathway, and L-4-aspartyl phosphate, the commitment step to the synthesis of L-lysine, lysC, dapA, dapC, and dapF. These enzymes are up-regulated in industry through genetic engineering to ensure adequate amounts of lysine precursors are produced to increase metabolic flux. Unwanted side reactions such as threonine and asparagine production can occur if a buildup of intermediates occurs, so scientists have developed mutant strains of C. glutamicum through PCR engineering and chemical knockouts to ensure production of side-reaction enzymes are limited. Many genetic manipulations conducted in industry are by traditional cross-over methods or inhibition of transcriptional activators. [44]

Expression of functionally active human epidermal growth factor has been brought about in C. glutamicum, [45] thus demonstrating a potential for industrial-scale production of human proteins. Expressed proteins can be targeted for secretion through either the general secretory pathway or the twin-arginine translocation pathway. [46]

Unlike gram-negative bacteria, the gram-positive Corynebacterium species lack lipopolysaccharides that function as antigenic endotoxins in humans. [ citation needed ]

Most species of corynebacteria are not lipophilic. [ citation needed ]

### Nonlipophilic Edit

The nonlipophilic bacteria may be classified as fermentative and nonfermentative:

• Patients presenting with an infection need accurate diagnosis of the infective organism they are infected with to begin targeted treatment.
• To achieve this, a number of investigations can be carried out which can be used to identify the exact organism, including what drugs can be used to treat the organism.
• A gram stain is the main investigation used to identify bacterial infections - it involves looking at the colour and shape of a stained bacterial sample from the patient down a microscope.
• A serology is a common investigation used to detect antigens/antibodies within the patient's blood. It can be used for diagnosing all micro-organism types.
• A PCR test can be used to detect DNA/RNA within the fluids of the human body and can be used to diagnose any infection - but it takes a long time and is expensive. It can be used for diagnosing all micro-organism types.
• A blood culture can be taken to grow any bacteria that may be present within the usually sterile blood of the patient. Whatever grows in the culture can then be tested for antibacterial susceptibility. However, this process takes a long time and is not suitable for hyper-acute situations.
• In emergency/acute situations such as sepsis or meningitis (although blood/CSF samples will be taken) treatment is usually given empirically – antibiotics against the most likely causative organism without definite diagnosis using the above techniques

When a patient presents with an infection, it can often be difficult to tell the exact organism they are infected with from clinical signs and history alone. Many symptoms are common among many micro-organisms and thus investigations need to be carried out to confirm a diagnosis and begin targeted treatment.

This article covers a handful of diagnostic investigations that can be requested by a doctor to help with specific diagnosis of an infection. Some of these tests can be used for diagnosing other diseases that are not infectious in cause, but this is not within the scope of this article. Details of the methods of carrying out these investigations are not required past this level for medical students, but accurate interpretation of their results is vital.

Gram staining

Used for: Bacterial infections

Sample collected via: Sputum sample, stool sample, blood sample, urine sample

A gram stain involves looking at the shape and staining of a bacterial species under a microscope after carrying out a gram stain (using crystal violet and safranin).

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls through detecting peptidoglycan, which is present in the cell wall of gram-positive bacteria.

To understand gram staining, an understanding of the different shapes and components of the cell walls of bacteria is required.

The gram stain is almost always the first step in the preliminary identification of a bacterial organism. While gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique.

How is it done?

There are 4 basic steps to performing a gram-stain:

1. A primary stain of Crystal Violet is applied to a heat-fixed smear of a bacterial culture. This is stain is absorbed by the peptidoglycan in the walls of gram-positive bacteria. Therefore, gram positive cells will appear violet/purple in colour.
2. Iodide is added to keep the stain inside the cells and stop it from being washed out.
3. Rapid decolorization with ethanol or acetone. This essentially ‘washes out’ any remaining stain from the slide or from any cells which haven’t taken up the stain.
4. The gram-negative cells which haven’t taken up the initial stain will now be transparent due to the decolourization and can not be visualised. The sample is now counter-stained with safranin to visualise these cells which were not stained in the first step. Safranin is a pink colour therefore, gram-negative cells are pink in colour.

Bacterial shapes

Bacterial shapes can be either:

• Coccus shaped (pl. cocci) – these bacteria look circular
• Bacillus shaped (pl. bacilli) – these bacteria look like long tubes and are sometimes referred to as “rod” bacteria
• Spirillus/Spiral – specific to a handful of gram-negative bacteria, this is a rare finding on a gram stain

Image: The 3 shapes of bacteria. Note that cocci can arrange into singular cocci, diplococci (pairs of cocci) and larger groups such as strips and bunches

Creative commons source by CKRobinson [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

The arrangement of the bacteria within space is also important. For example:

• Streptococcus species are commonly arranged in strips of cocci (Remember: STREPS are STRIPS)
• Staphylococcus species are usually arranged in bunches (similar to grapes)

This information can be very useful in distinguishing between different species that may appear similar on a gram stain.

Figure: Gram Stain of a Streptococcus species. Notice the long chains of cocci ("strips") and purple colouring as all streptococci are gram positive

Figure: Gram Stain of a Staphylococcus species. Notice the bunches of cocci.

Creative commons source by Y Tambe [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0/)]

Bacterial cell wall components

Bacterial cell wall components often define how they infect the host, as well as their virulence and potential to cause disease. The cell wall components are also key in how an organism gram stains.

There are two main categories of bacteria:

Bacterial cell wall compositions vary based upon the category of bacteria:

• Gram-positive cell walls contain a plasma membrane and a LARGE peptidoglycan cell layer
• Gram-negative cell walls contain a plasma membrane a SMALL amount of peptidoglycan and then are often further encapsulated in a lipopolysaccharide cell wall (LPS) – this is a virulence factor that often leads to gram negative infections being more severe.

Diagram: A Gram-positive bacterial cell wall with its main components

SimpleMed original by Marcus Judge

Diagram: A Gram-negative bacterial cell wall with its main components. Note the LPS outer capsule that causes serious disease and is a virulence factor

SimpleMed original by Marcus Judge

Interpreting a gram stain

To reiterate interpretation is carried out by analysing the colour and shape of the bacteria on the microscope slide.

Peptidoglycan absorbs the crystal violet stain used in gram stains. As such:

• A gram-positive bacterium is PURPLE/VIOLET on a gram stain as it has more peptidoglycan.
• A gram-negative bacterium is PINK on a gram stain.

As previously discussed, the shape of a bacterium can be either cocci, bacillus or spiral.

Figure: Gram Positive vs Gram Negative gram stain

Converting this information into a diagnosis

Upon knowing the colour and shape of a bacterium the current differentials can be supported or rejected. This is why knowledge of the shape and gram of common species of bacterium is important for a physician
Here are some useful examples:

Gram positive cocci – (Purple circles)

• Staphylococcus species e.g. Staphylococcus aureus (causes cellulitis and skin abscesses)
• Streptococcus species e.g. Streptococcus pneumoniae (causes URT infections and pneumonia, is usually a diplococcus i.e. in pairs)

Often treated with: Penicillins and Carbapenems

Gram positive bacilli – (Purple rods)

• Clostridium difficile (also referred to as C.diff)(a hospital acquired infection that causes diarrhoea and other digestive complications, notable for its’ antibiotic resistance and spore forming ability making it hard to eradicate)
• Bacillus anthracis (commonly known as anthrax, this is a notifiable disease i.e. it must be reported to public health England if suspected)
• Listeria monocytogenes (causes listeriosis which can be an extremely severe life threatening condition)

Often treated with: Penicillins, Carbapenems and Erythromycin (macrolides)

Gram negative cocci – (pink circles)

• Neisseria meningitidis (causes meningococcal meningitis)
• Neisseria gonorrhoeae (causes gonorrhoea)

Often treated with: Cephalosporins and fluoroquinolones

Gram negative bacilli – (pink rods)

Often treated with: Cephalosporins and fluoroquinolones

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Other investigative techniques:

Definition: Serology refers to the study of serum (blood plasma without clotting factors) but in this context is used specifically to refer to diagnostic techniques within this field of study.

Used for: All types of infections and other non-infective conditions e.g. rheumatological conditions.

Used to detect: Bacteraemia (bacteria present in the bloodstream)/Syphilis/Hepatitis/HIV

Sample collected via: Blood (usually), other bodily fluids

Serological tests rely on the formation of antibodies in response to an infection (against a given microorganism). Presence of these antibodies in the blood implies that the body has begun to mount an immune response (which may be ineffective) against the organism. This diagnostic test is not suitable in immunocompromised individuals as they may lack the ability to form antibodies.

There are several serology techniques that can be used depending on the antibodies being studied. These include: ELISA, agglutination, precipitation, complement-fixation, fluorescent antibodies and chemiluminescence to name but a few examples (detailed knowledge of these tests is not required for this topic).

These techniques often involve the use of complementary antibodies to detect disease marker antibodies taken from the patient’s serum. The complementary antibodies may have a fluorescent marker or cause an observable enzymatic reaction in response to signal a positive test.

Full Blood Count

Definition: A very common test carried out on the patient’s blood to detect the levels and characteristics of certain benchmark cells within the blood. Anomalies in the number of red blood cells, their size and the number of immune cells/inflammatory markers etc. can be indicative of a disease. This investigation is not inherently diagnostic but can often show if a patient is infected and what cells are being affected within the blood.

Used for: All types of infections

Used to detect:

• Sepsis (raised inflammatory markers and either extremely high or low neutrophil count depending on stage of progression)
• AIDS (T-cell count below 200 cells/μL)
• Epstein Barr Virus (EBV) (high lymphocytes early on in infection, low B-cell count late)
• Any other acute viral or bacterial infections (raised inflammatory markers)

Sample collected via: Blood

A full blood count is almost always requested when a patient comes in acutely ill. It provides information such as:

• White cell count
• Neutrophil count: Raised levels may indicate bacterial infection. They may also be raised in acute viral infections.
• Lymphocyte count: Higher with some viral infections such as Epstein-Barr virus (although B-cell count will drop later on in this disease’s progression when it begins to infect B-cells). Counts may be decreased by HIV infection, as this causes the destruction of T-cells.
• Raised Monocytes: May be raised in bacterial infection, tuberculosis and malaria.
• Eosinophils: Increased in parasitic infections.

See our dedicated article in the haematology section

Definition: Polymerase chain reaction (PCR) is a technique that is used to amplify trace amounts of DNA (and in some instances, RNA) located in or on almost any liquid or surface where DNA strands may be deposited. Thus, a sample of fluid or blood taken from an infected patient can be passed through a PCR machine to amplify the specific DNA/RNA strands of certain viruses/bacteria that the technician is looking for. The amplified genetic information can then be used to match the sample with a microorganism on record.

Used for: Every infection and organism – the gold standard of infection investigations

Used to detect: Everything

Sample collected via: Blood, faecal sample, urine sample, throat swab, sputum sample

A PCR test is diagnostically the most accurate form of investigation commonly carried out, as it can detect the exact DNA of a certain microorganism within a sample and can be automated (reducing human error). It can even detect specific mutations that could cause resistance to certain medications. This allows further personalisation of treatment strategy to increase treatment efficacy.

PCR tests are powerful tools however, they take considerable time to carry out and are far more expensive to run than a gram stain or basic serology. They can however, be used in viral infections to detect the specific infective organism.

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Blood culture

Definition: As the name implies this technique is simply culturing a blood sample (the growing any bacteria found within the blood on a plate). It is used to detect infections have haematogenous spread (through the blood stream). This is possible as the bloodstream is usually a sterile environment therefore any positive culture indicates pathology. The technique can be used to grow large enough amounts of the bacteria from within a patient’s bloodstream to carry out tests such as antibiotic susceptibility (to identify the best antibiotic to treat that strain of the bacteria).

Used for: Suspected Bacterial infections, particularly those with likely antibiotic resistance.

Used to detect: Bacteria

Sample collected via: Blood

The bacteria are given an ideal growth environment on agar jelly. This allows rapid growth and detection of any bacteria within the blood. MIC (minimum inhibitory concentration) and susceptibility testing can then be carried out on the bacteria. This test is often paired with a gram-stain test, allowing for an excellent and full diagnosis of the infection present within the patient.

It is worth remembering that all these techniques do take time, therefore in acute emergency situations such as Sepsis empirical treatment must be given. Therefore, it is also useful to understand the most likely causative bacteria for infection in specific patient groups.

## Streptococcus pneumoniae symptoms?

Streptococcus pneumonia paves the way to a lot of pneumococcal diseases in people that have very low immune systems, children and elderly people. These diseases are contagious and can spread from a person to another. Along with that, it could also be life-threatening. Therefore, it is recommended to watch out for the symptoms of pneumococcal disease. The infections of the pneumococcal disease mostly happen around the sinuses, bloodstream, lungs, middle ear and meninges which is the lining of the spinal cord and brain which ultimately results in meningitis.

Hence, to mention a few of the streptococcus pneumoniae symptoms are:

• Cough
• Chills and fever
• Difficulty in breathing
• Rapid breathing
• Pain in the chest
• Stiff neck
• Disorientation or confusion
• Sensitivity to light
• Increased heart rate
• A sensation of cold and/or shivering and shaking
• Discomfort and pain
• Sweaty skin
• Short breath
• Sleepiness
• Ear pain
• The swollen or red eardrum
• Bloodstained sputum
• Nausea and vomiting
• Drowsiness

## Abstract

Group B streptococcal infection (Streptococcus agalactiae) is one of the leading causes of life-threatening disease in the early neonatal period, resulting in sepsis, pneumonia, and meningitis. During invasive infections, an excessive release of pro-inflammatory cytokine, such as interleukin-6 (IL-6), thus IL-6 gene is significant, as a diagnostic marker of systemic infection of the newborns. The present study aimed to describe the epidemiology diagnostic of GBS disease in neonatal by phenotypic and genotypic methods. Nine hundred and ninety-six samples were taken at Maternity and Children Hospital, Jeddah, Saudi Arabia for a period of one year (2011–2012). Results indicated that out of 217 infected samples, twenty (9.23.0%) were positive for group B Streptococci bacteria. This study also shows that female infants are more susceptible than males. The level of IL-6 was higher in mothers above 30 years. Twenty positive Streptococci group B isolates showed bands with the cylE gene primers in the border between 228 bp, 267 bp and 50 bp. Molecular detection by Real time polymerase chain reaction was also done to detect the target (Sip gene) encoding the Sip surface immunogenic protein. Specific primers and TaqMan probe were chosen for this purpose. A Real-time PCR method targeting the sip gene of GBS in neonates after delivery has been evaluated.