Uk standards for Microbiology Investigations Acknowledgments



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UK Standards for Microbiology Investigations




Acknowledgments

UK Standards for Microbiology Investigations (SMIs) are developed under the auspices of Public Health England (PHE) working in partnership with the National Health Service (NHS), Public Health Wales and with the professional organisations whose logos are displayed below and listed on the website http://www.hpa.org.uk/SMI/Partnerships. SMIs are developed, reviewed and revised by various working groups which are overseen by a steering committee (see http://www.hpa.org.uk/SMI/WorkingGroups).

The contributions of many individuals in clinical, specialist and reference laboratories who have provided information and comments during the development of this document are acknowledged. We are grateful to the Medical Editors for editing the medical content.

For further information please contact us at:

Standards Unit

Microbiology Services

Public Health England

61 Colindale Avenue

London NW9 5EQ

E-mail: standards@phe.gov.uk

Website: http://www.hpa.org.uk/SMI

UK Standards for Microbiology Investigations are produced in association with:

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UK Standards for Microbiology Investigations: Status



Users of SMIs

Three groups of users have been identified for whom SMIs are especially relevant:



  • SMIs are primarily intended as a general resource for practising professionals in the field operating in the field of laboratory medicine in the UK. Specialist advice should be obtained where necessary.

  • SMIs provide clinicians with information about the standard of laboratory services they should expect for the investigation of infection in their patients and the documents provide information that aids the electronic ordering of appropriate tests from hospital wards.

  • SMIs also provide commissioners of healthcare services with the standard of microbiology investigations they should be seeking as part of the clinical and public health care package for their population.

Background to SMIs

SMIs comprise a collection of recommended algorithms and procedures covering all stages of the investigative process in microbiology from the pre-analytical (clinical syndrome) stage to the analytical (laboratory testing) and post analytical (result interpretation and reporting) stages.

Syndromic algorithms are supported by more detailed documents containing advice on the investigation of specific diseases and infections. Guidance notes cover the clinical background, differential diagnosis, and appropriate investigation of particular clinical conditions. Quality guidance notes describe essential laboratory methodologies which underpin quality, for example assay validation, quality assurance, and understanding uncertainty of measurement.

Standardisation of the diagnostic process through the application of SMIs helps to assure the equivalence of investigation strategies in different laboratories across the UK and is essential for public health interventions, surveillance, and research and development activities. SMIs align advice on testing strategies with the UK diagnostic and public health agendas.



Involvement of Professional Organisations

The development of SMIs is undertaken within PHE in partnership with the NHS, Public Health Wales and with professional organisations.

The list of participating organisations may be found at http://www.hpa.org.uk/SMI/Partnerships. Inclusion of an organisation’s logo in an SMI implies support for the objectives and process of preparing SMIs. Representatives of professional organisations are members of the steering committee and working groups which develop SMIs, although the views of participants are not necessarily those of the entire organisation they represent.

SMIs are developed, reviewed and updated through a wide consultation process. The resulting documents reflect the majority view of contributors. SMIs are freely available to view at http://www.hpa.org.uk/SMI as controlled documents in Adobe PDF format.



Quality Assurance

The process for the development of SMIs is certified to ISO 9001:2008.

NHS Evidence has accredited the process used by PHE to produce SMIs. Accreditation is valid for three years from July 2011. The accreditation is applicable to all guidance produced since October 2009 using the processes described in PHE’s Standard Operating Procedure SW3026 (2009) version 6.

SMIs represent a good standard of practice to which all clinical and public health microbiology laboratories in the UK are expected to work. SMIs are well referenced and represent neither minimum standards of practice nor the highest level of complex laboratory investigation possible. In using SMIs, laboratories should take account of local requirements and undertake additional investigations where appropriate. SMIs help laboratories to meet accreditation requirements by promoting high quality practices which are auditable. SMIs also provide a reference point for method development. SMIs should be used in conjunction with other SMIs.

UK microbiology laboratories that do not use SMIs should be able to demonstrate at least equivalence in their testing methodologies.

The performance of SMIs depends on well trained staff and the quality of reagents and equipment used. Laboratories should ensure that all commercial and in-house tests have been validated and shown to be fit for purpose. Laboratories should participate in external quality assessment schemes and undertake relevant internal quality control procedures.

Whilst every care has been taken in the preparation of SMIs, PHE, its successor organisation(s) and any supporting organisation, shall, to the greatest extent possible under any applicable law, exclude liability for all losses, costs, claims, damages or expenses arising out of or connected with the use of an SMI or any information contained therein. If alterations are made to an SMI, it must be made clear where and by whom such changes have been made.

SMIs are the copyright of PHE which should be acknowledged where appropriate.

Microbial taxonomy is up to date at the time of full review.

Equality and Information Governance

An Equality Impact Assessment on SMIs is available at http://www.hpa.org.uk/SMI.

PHE is a Caldicott compliant organisation. It seeks to take every possible precaution to prevent unauthorised disclosure of patient details and to ensure that patient-related records are kept under secure conditions.

Suggested Citation for this Document

Public Health England. (). . UK Standards for Microbiology Investigations. ID 12Error: Reference source not found Issue xxx. http://www.hpa.org.uk/SMI/pdf.



Contents

Acknowledgments 2

UK Standards for Microbiology Investigations: Status 4

Amendment Table 7

Scope of Document 8

Introduction 8

Technical Information/Limitations 17

1 Safety Considerations27-35 18

2 Target Organisms2-4,7,9-11,14,16-18,22,24-26,39-50 18

3 Identification 18

4a Identification of Haemophilus species 24

4b Identification of HACEK group 25

5 Reporting 26

6 Referrals 27

References 28




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Amendment Table

Each SMI method has an individual record of amendments. The current amendments are listed on this page. The amendment history is available from standards@phe.gov.uk.

New or revised documents should be controlled within the laboratory in accordance with the local quality management system.



Amendment No/Date.

5/ Error: Reference source not found

Issue no. discarded.



Insert Issue no.

xxx

Section(s) involved.

Amendment.















Amendment No/Date.

4/27.06.12

Issue no. discarded.



Insert Issue no.



Section(s) involved.

Amendment.

Whole document.

Minor formatting amendments.

References.

Some references updated.


Scope of Document

This SMI describes the identification of Haemophilus species and other members of the HACEK group (Haemophilus species, Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans), Aggregatibacter aphrophilus (formerly Haemophilus aphrophilus and Haemophilus paraphrophilus), Cardiobacterium hominis, Eikenella corrodens and Kingella species.

This SMI should be used in conjunction with other SMIs.

Introduction



Taxonomy

There are currently fourteen species of Haemophilus1. The Haemophilus species associated with humans are H. influenzae2, H. aegyptius, H. haemolyticus, H. parainfluenzae, H. pittmaniae, H. parahaemolyticus, H. paraphrohaemolyticus, H. ducreyi and H. sputorum3. Nucleic acid hybridisation studies and 16S rRNA sequence homologies suggest H. ducreyi does not belong in the genus Haemophilus, though it does seem to be a valid member of the family Pasteurellaceae. Haemophilus aphrophilus and H. paraphrophilus have been re-classified as a single species on the basis of multilocus sequence analysis4, Aggregatibacter aphrophilus, which includes V-factor dependent and V-factor independent isolates. H. segnis has been re-classified as Aggregatibacter segnis4.



H. influenzae is the type species.

Characteristics

Haemophilus are Gram negative spherical, oval or rod-shaped cells less than 1 µm in width, variable in length, with marked pleomorphism, and sometimes forming filaments. The optimum growth temperature is 35–37°C. They are facultatively anaerobic and non-motile.

Members of the Haemophilus genus are typically cultured on blood agar plates as all species require at least one of the following blood factors for growth: haemin (factor X) and/or nicotinamide adenine dinucleotide (factor V). Chocolate agar is an excellent Haemophilus growth medium as it allows for increased accessibility to these factors. Alternatively, Haemophilus is sometimes cultured using the "Staph streak" technique: both Staphylococcus and Haemophilus organisms are cultured together on a single blood agar plate. In this case, Haemophilus colonies will frequently grow in small "satellite" colonies around the larger Staphylococcus colonies because the metabolism of Staphylococcus produces the necessary blood factor by-products required for Haemophilus growth. All Haemophilus species grow more readily in an atmosphere enriched with CO2; H ducreyi and some nontypable H influenzae strains will not form visible colonies on culture plates unless grown in CO2-enriched atmosphere. Aggregatibacter aphrophilus and Haemophilus paraphrohaemolyticus require CO2 for primary isolation.

On chocolate blood agar, colonies are small and grey, round, convex, which may be iridescent, and these develop in 24 hours. Iridescence is seen with capsulated strains.

Carbohydrates are catabolised with the production of acid. A few species produce gas. Nitrates are reduced to nitrites.

The medically important Haemophilus species are described as follows;

Haemophilus influenzae5

They are small, non-motile Gram negative bacterium in the family Pasteurellaceae. They are facultatively anaerobic. On chocolate blood agar, colonies are small and grey, round, convex, which may be iridescent, and these develop in 24 hours. Iridescence is seen with capsulated strains. There is no growth on MacConkey agar and show no β-haemolysis on sheep red blood cells. They also require the X and V factors for growth.

They are positive for oxidase, catalase, nitrate reduction and phosphatase. 11-89% of strains are positive for indole production and 80-89% of strains are positive for urease and Ornithine decarboxylase tests. They are also negative for ONPG, H2S production and aesculin hydrolysis. Acid is produced from D- Glucose, D- Galactose, Maltose, D- Ribose and D-xylose and not from lactose, D-mannitol, D- Mannose, sucrose, Inulin, Trehalose, Raffinose, L- Rhamnose, L. Sorbose, D-sorbitol, Fructose and Melibiose5.

There are 8 biotypes of H. influenzae (I-VIII) and Pittman also described six antigenically distinct capsular types, designated a-f4.



Haemophilus parainfluenzae5

They are small, non-motile Gram negative bacterium in the family Pasteurellaceae. They are facultatively anaerobic. There is no growth on MacConkey agar and show no β-haemolysis on sheep cells. They also require V factor (NAD) for growth and not the X- factor.

They are positive for oxidase, nitrate reduction and H2S production. Acid is produced from Fructose, D- Galactose, D- Glucose, Maltose, Sucrose and D- Mannose. 11- 89% of strains are positive for catalase, ONPG, Ornithine decarboxylase and Urease. They are negative for indole production and aesculin hydrolysis. Acid is not produced from D- Adonitol, L- Arabinose, Cellobiose, Dulcitol, D- Sorbitol, L- Sorbose, Trehalose, D-Xylose, Glycerol, Inulin, Lactose, D- Mannitol, Melibiose, Raffinose, L-Rhamnose, D- Ribose and Salicin.

There are eight biovars of Haemophilus parainfluenzae (I-VIII)4.



Haemophilus haemolyticus5

They are Gram negative, non-motile and non-spore-forming short to medium length rods. There is no growth on MacConkey agar and show β-haemolysis on sheep cells. They also require the X and V factors for growth.

They are positive for oxidase, catalase, nitrate reduction, phosphatase, urease and H2S production. 11-89% of strains are positive for Indole production. Acid is produced from D- Galactose, D- Glucose, Maltose and D- Ribose. 11-89% of strains produce acid from D- Xylose. They are negative for ONPG, ornithine decarboxylase and aesculin hydrolysis. Acid is not produced from D- Adonitol, L- Arabinose, Cellobiose, Dulcitol, Glycerol, Inulin, Lactose, D- Mannitol, D- Mannose, Melibiose, Raffinose, L- Rhamnose, Salicin, D-Sorbitol, L-Sorbose, Sucrose and Trehalose.

Haemophilus parahaemolyticus6

These usually differ morphologically from other haemophilic bacteria in that they are larger, stain more heavily and unevenly, and occur in long tangled thread forms with much pleomorphism.

The colonies tend to be larger, less translucent, and on blood agar, they are surrounded by a large colourless zone of haemolysis. In broth, there is stringy floccular sediment with clear supernatant. The V factor but not X is required for growth.

They are positive for oxidase, nitrate reduction, H2S production and urease tests. 11-89% of the strains are positive for catalase, ONPG, Ornithine decarboxylase and acid production from D- Galactose. Acid is produced from fructose, D- Glucose, maltose and sucrose. They are negative for indole production and aesculin hydrolysis. Acid is not produced from D- Adonitol, L- Arabinose, Cellobiose, Dulcitol, Glycerol, Inulin, Lactose, D- Mannitol, D- Mannose, Melibiose, Raffinose, L- Rhamnose, Salicin, D-Sorbitol, L-Sorbose, D- Xylose and Trehalose5.

The bacteria are associated frequently with acute pharyngitis and occasionally cause sub-acute endocarditis.

Haemophilus paraphrohaemolyticus 7

They are Gram negative, non-motile and non-spore-forming short to medium length rods measuring 0.75- 2.5µm and 0.4-.0.5µm. They grow well at 37°C both in air and in air with added CO2.

On blood agar plate, the colonies are smooth, round and dome-shaped and they also produce large zones of clear haemolysis. Chocolate agar promotes larger colonies than blood agar, irrespective of the presence or absence of CO2. They require the V factor for growth and not X factor. No growth is observed on inspissated serum or on MacConkey agar.

They are positive for catalase, oxidase, nitrate reduction, H2S production and urease tests. Acid is produced from fructose, D-Glucose, Maltose and sucrose. 11-89% of strains are positive for ONPG and produces acid from D- Galactose. They are negative for ornithine decarboxylase, Indole production and aesculin hydrolysis. Acid is not produced from D- Adonitol, L- Arabinose, Cellobiose, Dulcitol, Glycerol, Inositol, inulin, lactose, D-Mannitol, D- Mannose, melibiose, Raffinose, L- Rhamnose, D- ribose, Salicin, D- Sorbitol, L- Sorbose, Trehalose and D- Xylose5.

It has been isolated from sputum, throat, pharynx, thumb print and urethral discharge in humans7.

Haemophilus aegyptius 8

They are Gram negative, non-motile, non-spore-forming, non-encapsulated bacillus, 0.25 to 0.5µm by 1.0 to 2.5µm, with rounded ends and sometimes with a bipolar body. It is a facultative aerobe. It requires both haemin and V factors for growth. The optimum temperature is 35 to 37 °C with a range of 25 to 40°C. The colonies on blood agar are small and dew-drop-like without haemolysis; on transparent agar, they have a bluish tinge in transmitted light; and in semifluid medium they are granular to fluffy. They are soluble in sodium desoxycholate, reduce nitrates to nitrites, and do not produce indole. Slight acidity is formed from glucose and galactose; reaction on levulose is variable and on xylose negative. It agglutinates human red cells.

They can be differentiated from Haemophilus influenzae by means of serological means and to a certain extent, by growth characteristics and biochemical reactions.

Haemophilus pittmaniae 9

They are non-motile, facultatively anaerobic, Gram negative, small, pleomorphic rods, with occasional long, filamentous forms. Colonies on chocolate agar are greyish white and reach a diameter of 1–2mm after 24hr at 35 °C. A distinct β-haemolytic zone is produced around the colonies on horse or sheep blood agar. They depend on V-factor for growth on brain heart infusion agar plates, but are capable of growth on blood plates due to release of V-factor from lysed blood cells. They are positive for porphyrin test, negative or weakly positive in catalase and oxidase tests. Acid is produced from D-glucose, D-fructose, sucrose, D-mannose, D-galactose and maltose. A small amount of gas is produced from glucose. They also produce β-galactosidase (ONPG), alkaline phosphatase, acid phosphatase and leucine arylamidase, but not β-glucosidase (NPG), α-glucosidase (PNPG), β-glucosaminidase (GNAC), β-glucuronidase (PGUA) or α-fucosidase (ONPF).

They are negative for the indole, urease, in lysine and ornithine decarboxylase and arginine dihydrolase tests. Acid is not produced from lactose, D-xylose, D-mannitol, D-sorbitol, sorbose, melibiose, inulin, aesculin or amygdalin.

It was originally isolated from human saliva and is part of the normal flora of the oral mucous membranes of man. It is an opportunistic pathogen and has been isolated from various sites of infection, including blood and bile.



Haemophilus ducreyi10

Cells are Gram negative coccobacilli in “railroad track” arrangement. They grow best in microaerophilic conditions at 33-35°C in a humid atmosphere containing 5% CO2. The Identification of H. ducreyi growing from cultured specimens is not easy because the organism often cannot grow in the media used for routine biochemical testing; H. ducreyi grows on Mueller-Hinton agar with 5% sheep blood in a CO2 enriched atmosphere. They produce characteristic tan-yellow colonies that are highly self-adherent and can be ‘nudged’ intact over the surface of the agar. Furthermore, identification is not easy because H. ducreyi is asaccharolytic.

They require X factor for growth and this can most easily be evaluated using the porphyrin test. They are positive for oxidase and negative for catalase test.

They have been isolated from ulcers – leg, foot, perianal and penis11.

Haemophilus sputorum3

Cells are non-motile, small regular rods, 0.3–0.5µm × 2.0–3.0µm, with occasional coccoid forms. Colonies on chocolate agar are convex, whitish, opaque, and reach a diameter of 0.5–1.5mm within 24hr. Zones of β-haemolysis are produced around colonies on horse or sheep blood agar; occasional strains are non-haemolytic and consequently fail to grow on blood agar. Cells are unable to synthesize nicotinamide adenine dinucleotide de novo, ie growth is dependent on V factor. Porphyrins are synthesized from δ -aminolevulinic acid, ie haemin (X factor) is not required.

They are positive for oxidase and give variable results on catalase tests. Cells produce β-galactosidase, urease, and leucine arylamidase; the species are negative for indole test, arginine di-hydrolase, lysine decarboxylase, ornithine decarboxylase, and phenylalanine arylamidase. H2S is not or only weakly emitted (lead acetate test), IgA1 protease is not produced.

Acid is produced from fermentation of d-glucose, fructose, D-maltose and maltotriose; acid is not produced from N-acetyl-β-D-glucosamine, D-xylose, D-ribose, D-mannose, lactose, and D-malate. Variable fermentation is observed with D-galactose.



H. sputorum was orginially isolated from a case of human tooth and is occasionally involved in human infections and has been isolated from blood, sputum of patients with cystic fibrosis, and tooth alveolitis.

Other HACEK group of organisms

For the identification of Haemophilus species in the HACEK group see above.

A systematic approach is used to differentiate the HACEK group of clinically encountered, morphologically similar, aerobic and facultatively anaerobic Gram negative rods mainly associated with endocarditis and infections from normally sterile sites. These organisms are oropharyngeal/respiratory tract commensals12. The identification is considered together with the clinical details and the isolates may be identified further if clinically indicated. Isolates of clinically significant HACEK organisms from cases of endocarditis and normally sterile sites should be referred to the Laboratory of Health Care Associated Infections, PHE, (Colindale) for confirmation of identification and to the Antibiotic Resistance Monitoring and Reference Laboratory, PHE, Colindale for MIC testing.

Aggregatibacter species4

This is a member of the family Pasteurellaceae. They are Gram negative, non-motile, facultatively anaerobic rods or coccobacilli. Growth is mesophilic. Several species of the genus are capnophilic and primary isolation may require the presence of 5–10% CO2. There is no dependence on X factor and the requirement for V factor is variable. Granular growth in broth is common. Colonies on sheep- and horse-blood agar are greyish white and non-haemolytic. Acid is produced from glucose, fructose and maltose, whereas arabinose, cellobiose, melibiose, melezitose, salicin and sorbitol are not attacked. The fermentation of galactose, lactose, mannitol, mannose, raffinose, sorbose, sucrose, trehalose and xylose is variable and may aid in identification to the species level. They are also positive for nitrate reduction and alkaline phosphatase production, but strains are negative in tests for indole, urease, ornithine and lysine decarboxylases and arginine dihydrolase. Oxidase reaction is negative or weak; catalase is variably present.

The species of the genus are intimately associated with man; they are part of the human oral flora and are occasionally recovered from other body sites, including blood and brain, as causes of endocarditis and abscesses.

The type species is Aggregatibacter actinomycetemcomitans, originally described as ‘Bacterium actinomycetem comitans’.



Aggregatibacter actinomycetemcomitans4

(Previously known as Actinobacillus actinomycetemcomitans and then as Haemophilus actinomycetemcomitans).

They are small rods, 0.3-0.5 x 0.5-1.5µm, which may exhibit irregular staining and may appear as cocci in broth or actinomycotic lesions. They may occur singly, in pairs or in small clumps. Small amounts of extracellular slime may be produced. Cells are non-motile.

It grows best under microaerophilic conditions with added CO2 and is facultatively anaerobic. The optimal growth temperature is 37°C after 24hr incubation. Colonies on chocolate agar are small, with a diameter of ≤0.5mm after 24hr, but may exceed 1–2mm after 48hr. On primary isolation, the colonies are rough, textured and adherent and have an internal, opaque pattern described as star-like or like ‘crossed cigars’. The rough phenotype is related to fimbriation and to the production of hexoseamine-containing exopolysaccharide. Cells from rough colonies grow in broth as granular, autoaggregated cells that adhere to the glass and leave a clear broth. X and V factors are not required. If extracellular slime is produced, cultures may be sticky on primary isolation. Surface cultures have low viability and may die within 5-7 days.

They are positive for catalase, oxidase and acid is produced from glucose, fructose, maltose and mannose, whereas arabinose, cellobiose, galactose, lactose, melibiose, melezitose, trehalose, raffinose, salicin, sorbitol and sucrose are not fermented. Variable fermentation is observed with mannitol and xylose. They are negative for urease and ONPG hydrolysis.

The key tests for discrimination between Aggregatibacter actinomycetemcomitans and V factor-independent strains of Aggregatibacter aphrophilus are catalase and ONPG, plus fermentation of lactose, sucrose and trehalose.

They are indigenous to man, with primary habitat on dental surfaces. Aggregatibacter actinomycetemcomitans has regularly been isolated together with Actinomyces species from human actinomycosis. It has been sometimes found in other pathological processes such as endocarditis, brain abscess and urinary tract infections.



Aggregatibacter aphrophilus4

The species Haemophilus aphrophilus and Haemophilus paraphrophilus have been reclassified as a single species Aggregatibacter aphrophilus.

These are Gram negative, short regular rods, 0.5 x 1.5-1.7µm with occasional filamentous forms. They require 5-10% CO2 for primary isolation. Growth may be enhanced by haemin, but porphyrins are synthesized from δ-aminolaevulinic acid and X factor is not required. Some isolates require V-factor (formerly H. paraphrophilus) whilst others are V-factor independent (formerly H. aphrophilus). The colonies on chocolate agar are high convex, opaque, granular and yellowish and reach a diameter of 1.0-1.5mm within 24hr.

Acid is produced from glucose, fructose, lactose, maltose, mannose, sucrose and trehalose, whereas arabinose, cellobiose, mannitol, melibiose, melezitose, salicin, sorbose, sorbitol and xylose are not fermented. Variable fermentation is observed with galactose and raffinose. H2O2 is not decomposed; ONPG is hydrolysed. They are also catalase and urease negative, and oxidase variable.

Key tests for discrimination between V factor-dependent isolates of Aggregatibacter aphrophilus and strains of H. parainfluenzae biotype V (negative for indole, urease and ornithine decarboxylase) are fermentation of lactose and trehalose.

Aggregatibacter aphrophilus is a member of the normal flora of the human oral cavity and pharynx. May cause brain abscess and infective endocarditis and has been isolated from various other body sites including peritoneum, pleura, wound and bone.

Aggregatibacter segnis4

(Formerly called Haemophilus segnis.)

Cells are small, pleomorphic rods, often showing a predominance of irregular, filamentous forms. Growth on chocolate agar is slow and the colonies are smooth or granular, convex, greyish-white or opaque and 0.5mm in diameter after 48hr incubation. Growth in broth and fermentation media is slow, and reactions are negative or weakly positive. The growth of some strains is enhanced by 5-10% CO2. V-factor but not X-factor is required.

Small amounts of acid result from the fermentation of glucose, fructose, galactose, sucrose and maltose. Fermentation of sucrose is usually stronger than fermentation of glucose. Arabinose, cellobiose, lactose, mannitol, mannose, melibiose, melezitose, raffinose, salicin, sorbose, sorbitol, trehalose and xylose are not fermented. Catalase and β-galactosidase (hydrolysis of ONPG) are variably present. They are negative for oxidase, indole, urease and ornithine decarboxylase tests.

Aggregatibacter segnis is a regular member of the human oral flora, particularly in dental plaque, and can be isolated from the pharynx. It has occasionally been isolated from human infections including infective endocarditis.

Cardiobacterium species13

The genus Cardiobacterium contains 2 species, Cardiobacterium hominis and Cardiobacterium valvarum14,15. Cells are pleomorphic or straight rods, 0.5–0.75µm in diameter and 1–3µm in length with rounded ends, and long filaments may occur. Cells are arranged singly, in pairs, in short chains and in rosette clusters. They are Gram negative, but parts of the cell may stain Gram positive.

Growth on blood agar is poor. They do not require X or V factors, but may show an apparent requirement for X factor on first isolation. Very small colonies are produced unless incubated in a humid aerobic or anaerobic atmosphere with 5% CO2. After incubation for 2 days, colonies are 1mm in diameter, smooth, opaque and butyrous and show slight α- haemolysis. Some strains may pit the agar. They are facultatively anaerobic, but CO2 may be required by some strains on primary isolation. The optimum growth temperature is 30-37°C.

They are positive for oxidase, H2S production, indole (weakly), and are negative for nitrate reduction, catalase, urea and aesculin hydrolysis. They utilize dextrose, fructose, maltose, mannitol, sucrose, sorbitol, and mannose but do not utilize galactose, lactose, raffinose and xylose.



Cardiobacterium hominis is the type species.

Cardiobacterium hominis13

They are Gram negative pleomorphic to short, non-motile rods. Growth on blood agar is poor. C. hominis does not require X or V factors, but may show an apparent requirement for X factor on first isolation. Very small colonies are produced unless incubated in a humid aerobic or anaerobic atmosphere with 5% CO2. After incubation for 2 days, colonies are 1 mm in diameter, circular, smooth, entire, moist, glistening, opaque and butyrous and show slight α- haemolysis. Some strains may pit the agar. C. hominis is facultatively anaerobic, but CO2 may be required by some strains on primary isolation. The optimum growth temperature is 30-37°C.

They are positive for oxidase, H2S production, indole (weakly), and are negative for nitrate reduction, catalase, urease and aesculin hydrolysis. They utilize dextrose, fructose, maltose, mannitol, sucrose, sorbitol, and mannose but do not utilize galactose, lactose, raffinose and xylose.

C. hominis could be distinguished from other members of the HACEK group and from Pasteurella, Brucella, Streptobacillus moniliformis and Bordetella parapertussis. The main characteristics of C. hominis, distinguishing it from other closely related organisms are absence of catalase activity, positive oxidase reaction, production of indole and absence of nitrate production16.

They have been isolated from cerebrospinal fluid, blood as well as from nose and throat in healthy individuals.



Cardiobacterium valvarum17

They are fastidious Gram negative regular, pleomorphic to short rods. All strains are facultatively anaerobic and non-motile. Some strains have an acidulous. Its preferred culture medium is sheep blood agar, and visible colonies appear after an incubation period of 3 days. The colonies are round, elevated, opaque, smooth, and glistening. However, the colonies hardly reach 1 mm after extended incubation. Therefore, C. valvarum is more fastidious than C. hominis, whose colonies appear after a two-day incubation and reach a diameter of 2.2mm after 4 days. Microscopically, C. valvarum appears readily decolorized by acetone alcohol, and the cellular morphology varies depending on culture medium. When grown on blood agar, it is a fairly large regular rod, measuring 1 by 2 to 4µm. On chocolate agar, it is smaller and pleomorphic.

They are positive for the production of indole, cytochrome oxidase, and H2S but negative for catalase production, urea hydrolysis, aesculin hydrolysis, and nitrate reduction. It utilizes dextrose, fructose, sorbitol, and mannose, like C. hominis, but unlike C. hominis, does not utilize maltose, sucrose, or mannitol.

It was first isolated in 2001 from the blood of a 37-year-old man with endocarditis. Cardiobacterium valvarum is present in subgingival pockets and dental plaques14, and all the reported cases of endocarditis have been in persons who had recently undergone a dental procedure or had oral infection.



Eikenella corrodens18

The genus Eikenella contains only one species, Eikenella corrodens. Cells are straight, un-branched, non-sporing, slender Gram negative rods, 0.3-0.4 x 1.5-4µm in length.

Colonies may be very small on blood agar after overnight incubation or may not be visible for several days. The colonies have moist, clear centres surrounded by flat, and sometimes spreading, growth. Pitting of the medium may occur and yellow colouration may be seen in older cultures due to cell density. There may be colonial variation and spreading growth may vary between colonies of the same isolate. E. corrodens is non-haemolytic but a slight greening may occur around the colonies. Haemin is usually required for aerobic growth and rare strains remain X-dependent after further subculture. The optimum growth temperature is 35-37°C. E. corrodens is non-motile, but ‘twitching’ motility may be produced on some media. Strains are facultatively anaerobic and capnophilic. It may be confused with Bacteroides ureolyticus, which also exhibits pitting or corroding, but unlike E. corrodens is an obligate anaerobe and urease positive.

They are positive for oxidase, ornithine dacrboxylase and nitrate reduction and are negative for acidification of carbohydrates, production of indole, aesculin hydrolysis, catalase and urease tests.



Eikenella corrodens exists in dental plaque of both healthy people and periodontitis patients and can cause infections. Other clinical sources include head and neck infections and respiratory tract infections.

Kingella species19

The genus Kingella comprises four species, Kingella kingae, Kingella denitrificans, Kingella potus and Kingella oralis20. Kingella indologenes has been transferred to a new genus and classified as Suttonella indologenes19.



Kingella species are straight rods, 1.0µm in length with rounded or square ends. They occur in pairs and sometimes short chains. Endospores are not formed. Cells are Gram negative, but tend to resist decolourization. Two types of colonies occur on blood agar; a spreading, corroding type and a smooth, convex type. It does not require X or V factors. Growth is aerobic or facultatively anaerobic. The optimum growth temperature is 33-37°C 21.

They are non-motile, oxidase positive, catalase negative and urease-negative. Glucose and other carbohydrates are fermented with the production of acid but not gas.



Kingella species may grow on Neisseria selective agar and therefore may be misidentified as pathogenic Neisseria species. They can be differentiated from Moraxella and Neisseria species by a catalase test. Most Kingella species are catalase negative; Moraxella and most Neisseria species (except Neisseria elongata) are catalase positive.

K. denitrificans22

Previously designated CDC group TM-1. They are Gram negative, non-motile, plump rods 1.0µm in width. Small, translucent non-haemolytic colonies are produced on blood agar after 48hr of incubation at 37°C. Colonies may show pitting of the medium. Growth occurs anaerobically on blood agar. They are positive for oxidase, growth at 30 and 37°C, fermentative result in the O/F test, acid production from glucose, nitrate reduction, nitrite reduction, and production of gas from nitrite.

They are also negative for catalase, growth at 5 and 45°C, growth in the presence of 4 and 6% NaCl, growth on β-hydroxybutyrate in mineral medium, acid production from maltose unless serum was present23, starch hydrolysis and urease production. Isolated in the respiratory tract of man.

K. kingae24

The cells are coccoid to medium-sized rods, very much like those of Moraxella but slightly smaller, have square ends, and occur in pairs and short chains. They are Gram negative, with some tendency to resist decolourisation. They are also non-motile, non- encapsulated and no endospores are produced. On blood agar, two types of colonies occur; colonies of freshly isolated strains isolated strains appear as small depressions, 0.1 to 0.5mm in diameter, with a small central papilla initially but after 2 or more days incubation, there is considerable spreading growth and thin granular zones of growth often surround the colonies. Colonies when scrapped shows corrosion marks on the agar surface. The second colony, which often arises in subcultures of the first type, is small, delicate, translucent or slightly opaque, 0.1 to 0.6mm in diameter after 20hr on blood agar, low hemispherical, and smooth. On further incubation, the colonies increase in size but there is no evidence of corrosion or spreading. Both types of colonies are surrounded by distinct zones of β-haemolysis; their consistencies are soft or coherent and are not pigmented.

They are aerobic and grow at room temperature but their optimal growth is at 33-37°C. They are relatively fastidious and growth on high quality nutrient agar is as good as that on blood agar.

They are negative for catalase and urease tests. No acid is produced from fructose, lactose, saccharose, arabinose, xylose, rhamnose, mannitol, dulcitol, sorbitol, or glycerol. Gelatin and serum are not liquefied. Nitrate are not reduced or slightly reduced.

They are parasitic on human mucous membranes. Strains have been isolated from throat, nose, blood, bone lesions and joints.

K. oralis 25

They are Gram- negative rods or coccobacilli approximately 0.6 to 0.7µm in diameter by 1 to 3µm long with rounded ends. Cells can form pairs or chains. Cells have monopolar fimbriae up to 10µm long. There is a tendency to resist Gram decolourisation. Not motile by means of flagella, but cells form spreading colonies. They are aerobic or facultatively anaerobic. Growth is supported by 5% sheep blood agar supplemented with 5 mg of haemin per litre and 0.5 µg of menadione per ml in both anaerobic and aerobic environments with C02. They do not grow on MacConkey agar. Colonies are round with slightly irregular borders and flat to umbonate, and each colony has a granular periphery. Colonies appear to corrode the agar surface. They are positive for oxidase test and negative for nitrate, nitrite, indole, urease and aesculin hydrolysis tests. Acid is not produced from lactose, maltose, mannitol, sucrose, and xylose.

The habitat of K oralis appears to be human dental plaque and has been isolated from a supragingival plaque sample from a patient with adult periodontitis.

K. potus26

Cells are gram negative, non-spore-forming, non-motile rods. They are aerobic, DNase positive, oxidase positive, and catalase negative. Colonies are circular, low convex, yellow- pigmented, smooth, entire, approximately 1.5 to 2mm in diameter, and friable on Columbia blood agar after 48 h of incubation at 37°C. Colonies are non-haemolytic. Non-diffusible yellow pigments are produced. Nitrate and nitrite are not reduced. Aesculin and urea are not hydrolysed. Indole is not produced. Acid is not produced from fructose, glucose, mannose, mannitol, maltose, lactose, or sucrose. No alkaline phosphatase, α-glycosidase, β-galactosidase, or β-glucuronidase activity is detected. This has been isolated from the human wound caused by a bite by a kinkajou.

Tests that are useful in distinguishing Kingella potus from other Kingella species and members of the genus Neisseria are DNase test and its ability to pigment.

Principles of Identification

Colonies on blood or chocolate agar may be presumptively identified by colonial morphology, Gram stain, haemolysis and requirement for X and V factors and CO2. The porphyrin synthesis test (see TP 29 - Porphyrin synthesis (ala) test) may be used to differentiate haem producing Haemophilus species. Identification is confirmed by commercial biochemical tests, serotyping with type-specific antisera and/or referral to a Reference Laboratory.

Isolates of H. influenzae from normally sterile sites should be sent to the Haemophilus Reference Unit, Respiratory and Systemic Infection Laboratory, Public Health England, Colindale, for confirmation and typing.

Technical Information/Limitations

N/A

1 Safety Considerations27-35



Haemophilus influenzae is a Hazard Group 2 organism, and, and in some cases the nature of the work may dictate full Containment Level 3 conditions. All laboratories should handle specimens as if potentially high risk.

H. influenzae can cause serious invasive disease, especially in young children. Invasive disease is usually caused by encapsulated strains of the organism.

Vaccination against influenza is available; guidance is given in the DH Green Book36. Influenza vaccination is recommended for healthcare workers directly involved in patient care, who should be offered influenza immunisation on an annual basis.

Laboratory acquired infections have been reported37. The organism infects primarily by the respiratory route (inhalation), autoinoculation or ingestion in laboratory workers 38.

Laboratory procedures that give rise to infectious aerosols must be conducted in a microbiological safety cabinet. For the urease test, a urea slope is considered safer than a liquid medium. The use of needles, syringes, or other sharp objects should be strictly limited and eye protection must be used where there is a known or potential risk of exposure to splashes.

Refer to current guidance on the safe handling of all organisms documented in this SMI.

The above guidance should be supplemented with local COSHH and risk assessments.

Compliance with postal and transport regulations is essential.

2 Target Organisms2-4,7,9-11,14,16-18,22,24-26,39-50

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