Classification and pathogenicity of microbes

Publisher Summary

This chapter discusses the classification and pathogenicity of microbes. The microbial causes of human disease include viruses, chlamydiae, rickettsiae, mycoplasmas, bacteria, fungi, and protozoa. Viruses differ greatly from all the other microbes as they consist essentially of only nucleic acid surrounded by a protein coat and contain only one instead of two types of nucleic acid. Once inside human cells, the viruses remove the normal nuclear control of the cells to take over cellular metabolism for the synthesis of new virions. Chlamydiae and rickettsiae are also obligate intracellular parasites, have both DNA and RNA, and multiply by binary fission. Mycoplasmas, bacteria, and fungi can be cultured in cell-free media. Bacterial causes of disease are mainly lower bacteria that are unicellular. Multiplication is predominantly by asexual binary fission, although biological variation is facilitated in some species by sex, especially with Gram-negative species such as Escherichia coli. Only a few higher bacteria cause disease in man, such as Actinomycetes israelit, which are filamentous Gram-positive bacilli.

So, naturalists observe, a flea

Hath smaller fleas that on him prey;

And these have smaller fleas to bite ‘em,

And so proceed ad infinitum.

Jonathan Swift, On Poetry

The microbial causes of human disease include viruses, chlamydiae, rickettsiae, mycoplasmas, bacteria, fungi and protozoa. Basic features of these are included in Table 1.1 . Arthropods and worms are discussed in later chapters.

Table 1.1.

Classification of microbes

Type of microbe	Nucleic acids	Multiplication		Approx. size, μm	Seen by light microscope	Cell wall	Cytoplasmic membrane	Sensitive to ‘antibiotics’	Other features
		Intracellular	Extracellular
Viruses	DNA or RNA	+ (Virus takes over control of cell to synthesize new virions)	–	0·01–0·3	No	No	No	No	Host cell may show inclusions
Chlamydiae e.g. C. trachomatis, C. psittact)	DNA + RNA	+ (Multiplication by binary fission)	–	0·3	No	No	Yes	Yes (e.g. tetracyclines)	Host cell shows characteristic inclusions
Rickettsiae Coxiella (e.g. R. prowazeki)	DNA + RNA	+ (Multiplication by binary fission)	(Occasional exceptions)	0·3	Sometimes just visible by special stains	Rudimentary cell wall	Yes	Yes (e.g. tetracyclines)	‘Typhus’ transmitted by arthropods
Mycoplasmas (e.g. M. pneumoniae, M. hominis)	DNA +RNA	+ (Multiplication involves ‘elementary bodies	+	0·12–0·3	Sometimes just visible by special stains	No	Yes	Yes (e.g. tetracyclines)	Pleomorphic cells
Bacteria	DNA + RNA	±	+ (Multiplication by binary fission)	0·5–0·8 long	Yes	Yes (Muramic acid usually present)	Yes	Yes	Rigid cell wall
Fungi	DNA + RNA	+	+	Larger than bacteria (>5 long, >0·5 wide)	Yes	Yes Thicker than bacterial wall + contains sterol	Yes	No sensitive to anti-fungal drugs	Members of plant kingdom but no chlorophyll
Protozoa	DNA + RNA	± (Depends on particular species)	±	Larger than fungi	Yes	Yes	Yes	Not usually

Viruses differ greatly from all the other microbes as they consist essentially of only nucleic acid surrounded by a protein coat (capsid) and contain only one instead of two types of nucleic acid. Once inside human cells, the viruses remove the normal nuclear control of the cells to take over cellular metabolism for the synthesis of new virions. Chlamydiae and rickettsiae are also obligate intracellular parasites, have both DNA and RNA, and multiply by binary fission. Mycoplasmas, bacteria and fungi can be cultured in cell-free media unlike the above intracellular microbes.

Bacterial causes of disease are mainly ‘lower’ bacteria which are unicellular. Multiplication is predominantly by asexual binary fission although biological variation is facilitated in some species by ‘sex’, especially with Gram-negative species such as Escherichia coli. Only a few ‘higher’ bacteria cause disease in man, such as Actinomycetes israelii which are filamentous Gram-positive bacilli.

Protozoa pathogenic to man are divided into three main groups:

• 1Sarcodina (amoebae), e.g. Entamoeba histolytica
• 2Sporozoa, e.g. Plasmodium falciparum, Toxoplasma gondii
• 3Mastigophora (flagellates), e.g. Trichomonas vaginalis, Giardia lamblia, Leishmania and Trypanosoma species

CLASSIFICATION OF BACTERIA

There are three main groups of bacteria:

• 1Bacteria that are readily Gram-stained
• 2Acid-fast bacilli
• 3Spirochaetes

Bacteria that are Readily Gram-stained

These are classified into Gram-positive (blue-purple) or Gram-negative (pink-red) cocci or bacilli (Table 1.2 ).

Table 1.2.

Simple classification of Gram-stainable bacterial pathogens

Practical details of the Gram-stain are given in the Appendix to Chapter 2, p. 45. After the application of the methyl violet dye, Gram-positive bacteria stain blue and this colour is retained in spite of decolourization with acetone (or alcohol). Gram-negative bacteria initially stain blue after the methyl violet is applied, but the colour is lost after the application of acetone (or alcohol). They then take up the pink counterstain (saffronin, methyl red or carbol fuchsin).

The reason for the difference in colour after Gram-staining is not fully understood, but it is probably related to the large amount of mucopeptide and teichoic acid in the cell walls of Gram-positive bacteria. The fact that Gram-positive bacteria are more acidic than Gram-negative bacteria may account for their greater affinity for a basic dye. Even more important may be the greater permeability of Gram-negative cell walls which allow the methyl violet-iodine dye complex to diffuse out after treatment with acetone more readily than the cell walls of Gram-positive bacteria.

Within each subgroup, there are aerobic or anaerobic examples. The majority of bacterial pathogens can grow either aerobically or anaerobically, i.e. they are ‘facultative anaerobes’ such as Staphylococcus aureus or Escherichia coli; in Table 1.2 these have been included as ‘aerobes’. There are a few bacterial species which are strict aerobes, such as Pseudomonas aeruginosa, which will not grow at all anaerobically. Some bacterial species are strict anaerobes, such as Clostridium tetani or Bacteroides fragilis, which will not grow at all aerobically.

Exceptional Gram-stainable bacteria include Legionella pneumophila and Borrelia vincenti. Legionella pneumophila requires prolonged staining with the counterstain to be seen in tissues, although it appears readily as Gram-negative bacilli in smears made from colonies on agar media. Borrelia vincenti is the only spirochaetal pathogen that is easily seen by a Gram-stain.

Acid-fast Bacilli

Mycobacterial species are not readily seen by a Gram-stain, although they are weakly Gram-positive bacilli. Ziehl-Neelsen or other acid-fast stains are required for staining these organisms which have cell walls containing abundant lipids. Examples include Mycobacterium tuberculosis and Mycobacterium leprae.

Spirochaetes

Spirochaetes are thin-walled spiralled flexible organisms which are motile by means of an axial filament. They are not seen in a Gram-stain (except B. vincenti), but may be seen either by dark-ground illumination microscopy, or in a silver stain under the light microscope. Borrelia spirochaetes in the blood may also be seen in a Giemsa stain.

The three groups of spirochaetes include:

1. Treponema

Spirochaetes with regular spirals, approximately 1 μm apart from each other, 5–15 μm long and about 0·2 μm wide, e.g. Treponema pallidum (cause of syphilis)

2. Leptospira

Spirochaetes which have tightly coiled spirals, 5–15 μm long and about 01 μm wide. Characteristically, there is often a ‘hooked’ end, e.g. Leptospira icterohaemorrhagiae (cause of Weil's disease).

3. Borrelia

Large spirochaetes, 10–30 μm long and about 0·3 μm wide, with irregular spirals 2–4 μm apart from each other, e.g. Borrelia recurrentis (a cause of relapsing fever).

CLASSIFICATION OF VIRUSES

The classification of viruses depends on several factors including the type of nucleic acid present, the arrangement of the capsids into a cubical (icosahedral), helical or complex symmetry, the number of capsomeres, the size of the virus particle and whether the virion is naked or enveloped (often indicated by ether resistance or sensitivity, respectively, as well as by electron microscopic appearance). The main viruses causing disease in man are classified in Table 1.3 .

Table 1.3.

Classification of viruses

Nucleic acid	Capsid arrangement	Naked or enveloped	Size of virus particle, nm	Number of capsomers	Virus family	Virus examples	Diseases
DNA	Cubical(icosahedral)	Enveloped	100–200	162	Herpes viruses	Herpes simplex, I and II Varicella-zoster Cytomegalovirus	Mucocutaneous herpetic lesions Chickenpox and ‘shingles’ Cytomegalovirus inclusion disease
						Epstein-Barr virus	Glandular fever and Burkitt's lymphoma
DNA	Cubical	Naked	70–90	252	Adenoviruses	Over 30 serological types of adenoviruses	Pharyngo-conjunctivitis Lower respiratory infections in infants
						Adenovirus type 8	Epidemic kerato-conjunctivitis (‘shipyards eye’)
DNA	Cubical	Naked	45–55	72	Papova-viruses	Papilloma virus SV 40 type viruses	Warts Progressive multifocal leucoencephalopathy
DNA	Complex	Complex coat	Approx. 200×400		Pox viruses	Variola Monkeypox	Smallpox (now extinct) Monkeypox (rarely affects man)
						Vaccinia	Vaccinial skin lesions after vaccination
						Orf	Vaccinial skin lesions after vaccination
						Molluscum contagiosum	Molluscum contagiosum
RNA	Cubical	Enveloped	30–90		Toga viruses	Alpha and flavi viruses	Arthropod-borne fevers, e.g. equine encephalitis, yellow fever
RNA	Cubical	Naked	20–30	32	Picorna-	Enteroviruses—polio	Poliomyelitis
					viruses	—echo	Respiratory and CNS infections
						—Coxsackie A/B	Respiratory, CNS and heart infections
						Rhinoviruses	Colds
RNA	Cubical	Naked	60–80		Reoviruses	Rotavirus (wheel-like shape)	Gastro-enteritis
RNA	Helical	Enveloped	80–120		Orthomyxoviruses	Influenza A/B viruses	Influenza
					Paramyxoviruses	Para-influenza viruses Respiratory syncytial virus	Para-influenza Bronchiolitis, ‘croup’ and colds
						Mumps Measles	Mumps Measles
RNA	Helical	Enveloped	Approx. 70 × 170		Rhabdoviruses (‘bullet’ shaped)	Rabies virus	Rabies
RNA	Helical	Enveloped	90–100		Bunya-viruses	California arborviruses	Arthropod-borne fevers
RNA	Unknown	Enveloped	50–300		Arenaviruses	Lassa fever virus Lymphocytic choriomeningitis	Lassa fever Aseptic meningitis
RNA	Complex	Enveloped	80–130		Coronaviruses	Coronaviruses	Upper respiratory infections

One way of memorizing which viruses contain DNA is to remember that ‘PHAD’ is for DNA viruses, with P for pox and papova, H for herpes, AD for adenoviruses. Virtually all the remaining pathogenic human viruses are RNA viruses including the self-explanatory picorna viruses (‘PicoRNA’ viruses).

Some DNA viruses may cause tumours in man. These include papilloma virus causing warts, Epstein-Barr virus causing Burkitt's lymphoma and associated with naso-pharyngeal carcinoma, and herpes simplex virus which is associated with carcinoma of the cervix (see Jawetz et al., 1980). Other viruses, including certain papilloma viruses, have also been implicated in the aetiology of carcinoma of the cervix.

Retroviruses are RNA viruses which may cause tumours directly or indirectly in man and animals. The most important retrovirus is human immunodeficiency virus (HIV) which is the cause of acquired immune deficiency syndrome (AIDS)—see Chapter 21.

CLASSIFICATION OF FUNGI

The fungi causing diseases in man belong to the class ‘fungi imperfecti’. There are four main groups of pathogenic fungi: moulds (filamentous fungi), true yeasts, yeast-like fungi and dimorphic fungi.

1. Filamentous fungi

These grow as long filaments called ‘hyphae’ and the branched hyphae intertwine to form a ‘mycelium’. Reproduction is by spores including sexual spores which are used for identification. Culture in vitro of these fungi on Sabouraud's medium often shows ‘powdery’ colonies due to the presence of abundant spores, e.g. Trichophyton mentagrophytes.

2. True yeasts

These are unicellular round or oval fungi. Reproduction is by budding from the parent cell. Cultures in vitro characteristically show ‘creamy’ colonies, e.g. Cryptococcus neoformans.

3. Yeast-like fungi

These are like yeasts since they may appear as round or oval cells and grow by budding. They may also form long non-branching filaments known as ‘pseudohyphae’, e.g. Candida albicans.

4. Dimorphic fungi

These grow as yeast forms in the body and at 37 °C on culture media. They also form mycelia in the environment and on culture media at 22 °C. Several examples of this group of fungi grow intracellularly in reticuloendothelial cells in infected patients, e.g. Histoplasma capsulatum.

Fungi can also be classified according to whether they cause superficial or deep mycoses in infected patients and some examples are included in Table 1.4 . The deep mycoses most frequently occur in immunocompromised patients. Disease might also arise from the ingestion of mycotoxins in food: aflatoxins may be produced by Aspergillus flavus in cereals in less developed countries and ingestion of these toxins may cause liver damage possibly also predisposing to the development of hepatoma.

Table 1.4.

Classification of fungi

Fungi	Type of fungus	Disease examples	Geographical distribution
Fungi causing superficial mycoses
Dermatophytes including Microsporum, Trichophyton and Epidermophyton species	Filamentous	Tinea (ringworm) of skin, nails or hair	Worldwide
Aspergillus niger	Filamentous	Otitis externa	Worldwide
Candida albicans	Yeast-like	Oral thrush, monilial vaginitis Intertrigo, nappy rash Paronychia, granulomas in chronic mutocutaneous candidiasis	Worldwide
Malassezia furfur	Yeast-like	Pityriasis versicolor	Worldwide
Fungi causing deep mycoses
Aspergillus fumigatus	Filamentous	Pulmonary or disseminated aspergillosis	Worldwide
Mucor	Filamentous	Mucor mycosis	Worldwide
Allescheria boydii Madurella species	Filamentous	Madura mycosis (‘Madura foot’)	Tropics and subtropics
Candida albicans	Yeast-like	Septicaemia, endocarditis, bronchial and renal infections	Worldwide
Cryptococcus neoformans	True yeast	Cryptococcal meningitis or pulmonary infection (torulosis)	Worldwide
Histoplasma capsulatum	Dimorphic	Pulmonary or disseminated histoplasmosis	USA mainly
Blastomyces dermatidis	Dimorphic	North American blastomycosis	North America
Sporotrichum schenkii	Dimorphic	Sporotrichosis	USA and France mainly
Coccidioides immitis	Dimorphic (closest)*	Coccidioidomycosis (San Joaquin Valley fever)	USA—south western

^*‘Sporangia’ in tissues, filamentous at 22 C.

PATHOGENESIS: FACTORS AFFECTING THE ‘VIRULENCE’ AND SPREAD OF MICROBES

Pathogenicity

Microbes can be classified into ‘pathogens’, ‘commensals’ which are found in the normal body flora and ‘saprophytes’ which are found in environmental sites such as soil or plants. However, such a classification is of limited value since there are many examples of ‘commensals’, such as Escherichia coli, Staph. saprophyticus or Streptococcus viridans or saprophytes, such as Mycobacterium kansasii or Legionella pneumophila which may cause disease in patients under certain circumstances. The ‘pathogenicity’ of a microbe depends on host as well as on microbial factors and microbes can be usefully classified into ‘conventional pathogens’, ‘conditional pathogens’ and ‘opportunist pathogens’ (see Chapter 25). Host factors include the age of the patient, genetic factors, general host defences and local host defences against infection (see Chapter 6 and Immunodeficiency, in Chapter 8).

‘Koch's postulates’ have sometimes been useful for establishing the pathogenic relationship between a microbe and a disease. These postulates include the following: (1) the particular microbe is always associated with a given disease (this microbe may be either the cause or an incidental result of the disease); (2) the microbe may be isolated in the laboratory from specimens from a patient with the disease; (3) it is possible to produce a similar disease in animals by inoculation of the microbe into animals. Mycobacterium tuberculosis causing tuberculosis may be taken as an example where these three postulates may be fulfilled, but there are many other examples where complete fulfilment of these postulates does not occur, as with Treponema pallidum and syphilis, Epstein-Barr virus and glandular fever, Chlamydia trachomatis and non-specific urethritis.

Factors affecting ‘Virulence’

There is a lot of variation between strains of the same microbial species or between different species, in the ‘virulence’ of the microbe when considering the likelihood of disease being produced in a given ‘host’. An experimental measure of the ‘virulence’ can sometimes be obtained by estimating the LD₅₀ (lethal dose) which is the dose of organisms required to kill 50% of the animal population inoculated with the particular microbe. The more virulent the strain the lower is the LD₅₀.

The main known factors that affect virulence are concerned with pathogenicity, such as toxins and capsules in bacteria, examples of which are included in Table 1.5 . In recent years, there has been an increased interest in bacterial adhesiveness factors, such as the pili of gonococci or of E. coli strains that cause urinary tract infections (see Chapter 19). It has also become apparent that the ‘virulence’ of bacterial strains may also depend on the presence of transmissible genes contained in plasmids or mediated by bacteriophage. The adhesiveness to the ileal mucosa of an E. coli strain that produces enteritis in pigs is dependent on the presence of the K88 capsular antigen, a factor which is plasmid mediated. Enterotoxin production by this E. coli strain is also dependent on the presence of the appropriate plasmid. In man, the toxins produced by Corynebacterium diphtheriae and the erythrogenic toxin produced by Strep. pyogenes strains in scarlet fever patients are dependent on genes mediated by temperate phages. The fact that particular microbes appear to be more or less virulent at different times, might be due in part to the presence or absence of these types of transmissible genes. Scarlet fever is much less frequent than 50 years ago although streptococcal sore throats are still common. Skin sepsis due to Staph. aureus strains in hospital maternity units appears to be much less serious than in the 1950s. The microbial and the other factors that affect the virulence, and the spread of pathogens are often unclear (see Williams, 1976).

Table 1.5.

Factors affecting ‘virulence’ of bacteria—some examples

Virulence factor	Bacterial examples	Comment
I. Toxins
i. ‘Classic exotoxins’	Gram-positive bacteria mainly, e.g. Clostridium tetani toxin Clostridium perfringens (welchii) toxin Clostridium botulinum toxin Corynebacterium diphtheriae toxin	i.Exotoxins are highly toxic poly-peptides excreted by living bacteria (micrograms kill animals) They act at specific target sites, e.g. CNS, heart ii.They are highly antigenic (exception is Cl. tetani toxin) iii.Converted to antigenic non-toxic toxoids by formalin iv.The toxin is neutralized by anti-toxin V.The toxins are often destroyed by heat
ii. Other exotoxins	Streptococcal erythrogenic toxin Staph. aureus enterotoxin Bacillus anthracis toxic complex Vibrio cholerae enterotoxin Escherichia coli enterotoxin Shigella dysenteriae entero-/neurotoxins	Produced by Strep. pyogenes strains causing scarlet fever Heat stable Consists of three factors which combined in a complex cause oedema, haemorrhage and collapse These enterotoxins may produce diarrhoea after stimulating epithelial adenylate cyclase
iii. ‘Classic endotoxins’	Gram-negative bacteria mainly, e.g. Salmonella typhi Neisseria meningitides Escherichia coli Pseudomonas aeruginosa	i.Endotoxins are lipopolysaccharide (LPS) molecules in the outer layer of Gram-negative cell walls (released when organisms disintegrate). Lipid A is the main toxic component (hundreds of micrograms kill animals). They act non-specifically on RES cells stimulating release of mediators affecting vascular permeability and release of prostaglandins that may cause fever ii.The sugar chains present in the polysaccharide core of LPS confer ‘O’ antigen specificity and also affect virulence (‘rough’ strains instead of ‘smooth’ strains when the projecting sugar chains are shortened and the Gram-negative bacteria become less virulent) iii.Not converted to toxoids by formalin
		iv.Toxins do not naturally stimulate neutralizing antitoxin although antibodies to polysaccharide ‘O’ antigens result
		vEndotoxins are relatively heat stable
		viMay be detected by the Limulus test
		viiSevere endotoxaemia in patients may cause disseminated intravascular coagulation and/or fatal cardiovascular collapse
iv. Enzymes	e.g. Staph. aureus coagulase	Role of enzymes in man often unclear Coagulase may contribute to ‘walling off’ of staphylococcal lesions
	Strep. pyogenes streptolysins	Streptolysins can induce lysosomal discharge and kill polymorphs and inhibit Chemotaxis
II. Capsules and other surface antiphagocytic factors	e.g. Capsule of Haemophilus influenzae Pittman type b (polysaccharide)	These factors may contribute to the ‘invasiveness’ of some virulent bacteria by rendering the bacteria relatively resistant to either phagocytosis
	Capsule of Strep. pneumoniae (polysaccharide)	or killing within polymorphs or macrophages. Certain capsulated bacteria may
	Capsule of Bacillus anthracis (polyglutamic acid)	multiply in macrophages and be disseminated throughout the body as a result, e.g.
	K antigen of Escherichia coli (polysaccharide)	Salm. typhi bacilli with Vi antigen If specific antibody (opsonins)
	Vi antigen of Salm. typhi	has developed to the capsule or surface component the anti-
	‘M protein’ of Strep. Pyogenes	phagocytic effect may be reduced
	Protein A of Staph. aureus	Blocks phagocytosis of opsonized pathogenic strains of Staph. aureus possibly by interfering with attachment of Fc portions of IgG opsonins to surface of polymorphs

Factors affecting Spread

Epidemiological factors affecting the ‘host’ are relevant to the spread of microbes including the numbers of susceptible individuals in a geographically defined area, the proximity of the individuals to each other and to the source of infection, and the presence of other factors necessary for the transmission of infection, such as the correct climate or season, the presence of an essential arthropod vector, etc. These and other factors are discussed where relevant in the subsequent chapters where sporadic, endemic or epidemic infections are described.

Microbial factors that affect the spread depend partly on the ‘virulence’ of the microbe and partly on the ability of the microbe to survive or multiply in a given inanimate environment (‘fomites’ such as bedclothes, ‘vehicles’ such as milk or water) or on the hands of patients or hospital staff or in animals/arthropods. Above all, the microbe must have the ability to initiate an infection in a patient in as low a dose as possible, have an effective portal of entry for establishing infection, as well as a method of exit from the body where it can be shed in large numbers for as long as possible. ‘Carrier’ states clearly aid the transmission of bacteria. Gram-positive bacteria survive reasonably well in ‘dry’ environments while Gram-negative bacteria and some spirochaetes survive best in moist situations.

Microbes are either transmitted horizontally, i.e. between individuals of the same generation (such as the plague bacillus) or vertically, i.e. between individuals of different generations (such as congenital rubella from mother to infant). Hepatitis B is one example of an infection that is vertically transmitted between many millions of people in the less developed world.

Infection is either endogenous, from the patient's own flora, or exogenous, from a source outside such as another patient or person, an animal, a ‘vehicle’ or ‘fomite’. Modes of transmission of microbes include: (1) direct contact, such as with Neisseria gonorrhoeae; (2) ingestion, such as with Vibrio cholerae; (3) inoculation, such as with a ‘sharps’ injury transmitting hepatitis B, mosquito bite transmitting malaria or dog bite transmitting rabies; (4) inhalation, such as with measles virus, rhinoviruses or Mycobacterium tuberculosis. Numerous diseases are transmitted by the airborne route either by sprays of infected droplets or secretions (by coughing, sneezing or spitting) which contaminate clothing, hands, handkerchiefs (such as with rhinoviruses causing common colds) or by respiratory droplet nuclei (such as with measles virus). The droplet nuclei (1–10 μm in diameter) result from the evaporation of large droplets and may travel long distances as they become suspended in the air.

Appendix: Basic characteristics of some important bacterial pathogens

Notes on some basic characteristics of bacteria that can be Gram-stained are included in this appendix (see also Table 25.1, in Chapter 25; typing methods are also referred to in Chapter 25).

Gram-positive Bacteria

Staphylococci

Microscopy:	Gram-positive cocci mainly in clusters
Culture:	white, cream or golden yellow 0·5–1·5 mm colonies on blood agar after overnight aerobic incubation

Differential test

COAGULASE TEST

This is the main differential test. Coagulase is an enzyme which converts fibrinogen in plasma to fibrin, thus producing clumping when coagulase-positive staphylococci are mixed with plasma in a slide coagulase test and clot formation in a tube coagulase test.

Coagulase-positive staphylococci	indicates Staph. aureus
Coagulase-negative staphylococci	indicates Staph. albus (Staph. epidermidis) or Staph. saprophyticus

taphylococcus aureus

Found in the nose of 10–30% of normal people, but only occasionally on healthy skin, it is a common cause of infection in the community and in hospital. Most infections are sporadic but occasional outbreaks occur.

DISEASES INCLUDE

• 1Skin infections including boils, carbuncles, breast abscess, surgical wound infection, neonatal skin sepsis and rare toxic complications such as toxic epidermal necrolysis and toxic shock syndrome.
• 2Deep tissue infections including pneumonia, osteomyelitis, septic arthritis, endocarditis.
• 3Septicaemia and complications of septicaemia including disseminated intravascular coagulation, endocarditis and metastatic abscesses.
• 4Food poisoning; staphylococcal enterocolitis.

PREDISPOSING HOST FACTORS FOR STAPHYLOCOCCAL INFECTIONS INCLUDE

Diabetes mellitus, neutropenia, hypogammaglobulinaemia, and rare phagocyte defects as in chronic granulomatous disease.

ANTIBIOTICS

Penicillin, cloxacillin, erythromycin, lincomycin, fusidic acid and vancomycin are examples of narrow spectrum anti-staphylococcal antibiotics.

Greater than 90% hospital strains and 60% community strains are resistant to penicillin because of penicillinase production and these strains would also be resistant to other penicillins, such as ampicillin, but are usually sensitive to cloxacillin (or flucloxacillin).

Multiple antibiotic resistance to two or more different antibiotics may occur, especially in hospital, e.g. Staph. aureus resistant to penicillin, tetracycline, erythromycin, lincomycin and fusidic acid. Antibiotic resistance is often plasmid mediated and the spread of plasmids between different strains of Staph. aureus is facilitated by transducing phages. Increasing problems with epidemic strains of Staph. aureus resistant to methicillin (cloxacillin) and other antibiotics (MRSA) have occurred in hospitals throughout the world. Serious infections due to MRSA are best treated with intravenous vancomycin.

Staphylococcus epidermidis (Staph. albus), and ‘Staph. saprophyticus’

Found normally in the nose or skin flora of healthy people.

DISEASES INCLUDE

Urinary tract infections, endocarditis after heart surgery or in the elderly, shunt infections in infants with hydrocephalus and infections of hip joint prostheses.

ANTIBIOTICS

Antibiotic sensitivity patterns of different strains of Staph. epidermidis vary greatly, many strains being resistant to several antibiotics often including methicillin (cloxacillin), and serious infections may be treated with vancomycin.

Streptococci

Microscopy:	Gram-positive cocci, either in chains as with β-haemolytic streptococci or viridans streptococci, or as diplococci, as with pneumococci.
	Capsules of Strep. pneumoniae may occasionally be seen in Gram films and in special stained smears for capsules.
Culture:	Most streptococcal colonies on blood agar are apparent after 24–48 hours incubation aerobically. Some micro-aerophilic or anaerobic streptococci require up to 5 days incubation anaerobically before colonies are seen.
	Some pneumococcal strains, Strep. milleri strains and some viridans streptococci, such as Strep. mutans, grow best when 5–10% carbon dioxide is added to the atmosphere for incubation.

Differential tests

CLASSIFICATION ACCORDING TO HAEMOLYSIS ON BLOOD AGAR

Alpha (α) haemolysis	green colour around each colony due to altered haemoglobin, e.g. Strep. viridans
Beta (β) haemolysis	complete lysis of red cells around each colony. This is often most obvious on the anaerobic plate, e.g. Strep. pyogenes
GAMMA (γ) haemolysis	non-haemolytic colonies, e.g. Strep. faecalis

1. ALPHA-HAEMOLYTIC STREPTOCOCCI

Strep. pneumoniae and viridans streptococci. However, some viridans streptococci may also appear as non-haemolytic colonies. Pneumococcal colonies often classically have a ‘draughtsman’ appearance.

Optochin (diethylhydrocuprein) and bile solubility tests

Optochin disc test	pneumococci, but not viridans streptococci, show a zone of inhibition around optochin
Bile solubility test	pneumococci, but not viridans streptococci, are soluble in a bile salt suspension

Biochemical tests for viridans streptococci

Viridans streptococci can be identified further by biochemical tests, such as the production of dextran from sucrose, into species including Strep. mitior, Strep. sanguis, Strep. mutans, Strep. salivarius and Strep. milleri.

2. BETA-HAEMOLYTIC STREPTOCOCCI

These are differentiated mainly by Lancefield grouping.

Lancefield grouping and the ‘bacitracin test’

Polysaccharide antigen is extracted from the streptococcal cell walls for Lancefield grouping and the specific group antigen is identified using known antisera, such as in a precipitin test or in a coagglutination commercially available latex slide test.

The Lancefield grouping test may be used to identify some other streptococci which are not necessarily beta-haemolytic. In practice this test is most frequently carried out with beta-haemolytic streptococci. Important examples of different streptococci that can be put into Lancefield groupings include:

a. Lancefield group A—synonymous with ‘Strep. pyogenes’

Greater than 90% Strep. pyogenes strains are sensitive to a bacitracin identification disc. This bacitracin test is often used to presumptively identify beta-haemolytic streptococci on blood agar as Strep. pyogenes, especially in cultures of throat swabs. This test is not entirely reliable as other streptococcal species may sometimes be sensitive to bacitracin. Also a few strains of Strep. pyogenes may appear with reduced sensitivity to bacitracin.

The group A Lancefield antigen is distinct (polysaccharide) from the other cell wall antigens in Strep. pyogenes which are used to type strains in outbreaks such as the M ‘virulence’ protein and T protein antigens (see Typing, p. 604).

b. Lancefield group B—'strep. agalactiae’

Some group B streptococcal strains are only slightly beta-haemolytic. (There are selective media available to assist the isolation of group B streptococci in specimens from a site with mixed flora such as a vaginal swab.)

c. Lancefield group C and Lancefield group G streptococci

These beta-haemolytic streptococci are frequently isolated from normal (or infected) throat swabs or infected skin sites.

d. Lancefield group D

The main examples include Strep. faecalis and Strep. bovis although these species usually appear as non-haemolytic colonies on blood agar.

3. NON-HAEMOLYTIC STREPTOCOCCI

These species can be differentiated according to the results obtained with Lancefield grouping, biochemical tests and cultural tests on bile-aesculin agar or MacConkey agar.

Strep. faecalis grows on MacConkey agar (magenta colonies) and on bile-aesculin agar (turning this black).

Examples of streptococcal species that frequently appear as non-haemolytic streptococci include Strep. faecalis, Strep. bovis and some species of viridans streptococci, such as Strep. mutans and Strep. milleri.

Normal flora and streptococcal diseases

The main streptococcal pathogens and their associated diseases are included in Table 1.6 .

Table 1.6.

Streptococci and disease

Streptococcus	Usual haemolysis	Normal site	Main associated diseases include
Strep. pneumoniae	x	Throat and nose (up to 70% population)	Otitis media, sinusitis, mastoiditis, pneumonia, meningitis, brain abscess
	Mouth	Bacterial endocarditis Dental caries
and			(Strep. mutans)
Strep. milleri		Intestine	Collections of pus in abdomen, e.g. liver abscess; or in chest Bacterial endocarditis
Strep. pyogenes (Lancefield group A)	β	Throat (up to about 5% population)	Sore throat, scarlet fever, otitis media Later complications—rheumatic fever, acute glomerulonephritis Skin infections including erysipelas, impetigo, infected traumatic or eczematous lesions Wound infections and puerperal sepsis Septicaemia (e.g. complicating cellulitis)
Lancefield group B streptococci	β	Perineal skin lower vagina (5–30% women)	Neonatal septicaemia and meningitis
Lancefield group C or G streptococci	β	Throat	Sore throat (very occasionally), skin infections, septicaemia
Strep. faecalis	Non	Intestine	Urinary tract infection, bacterial endocarditis
Strep. bovis	Non	Intestine	Bacterial endocarditis
Micro-aerophilic or anaerobic streptococci	Non	Skin, throat or lower vagina	Meleney's synergistic gangrene (together with Staph. aureus) Cellulitis, such as skin or female genital tract infection Cerebral abscess (often mixed with other organisms)

Antibiotics

All Strep. pyogenes strains are sensitive to penicillin. Nearly all Strep. pneumoniae strains are sensitive to penicillin. The majority of ‘Strep. viridans’ strains are sensitive to penicillin. Strep. faecalis and Lancefield group B streptococci are only moderately sensitive to penicillin.

The great majority of streptococci are sensitive to erythromycin which may be particularly relevant for penicillin-allergic patients.

Bacillus species

These include Bacillus anthracis, Bacillus cereus and Bacillus subtilis.

Microscopy:	Bacillus anthracis is typically seen as large square-ended Gram-positive bacilli, sometimes in long chains. (Other aerobic-spore-bearing bacilli including Bacillus subtilis may appear as Gram-variable or Gram-negative bacilli.)
	Capsules of B. anthracis are stained purple in McFadyean's reaction with a polychrome methylene blue stain; other Bacillus species do not show capsules.
	B. anthracis spores are not apparent in spore stains of clinical specimens from infected patients but may be present in environmental specimens.
Culture	B. anthraciscolonies are seen after overnight culture on blood or nutrient agar as rough opaque colonies with edges resembling loose curls of hairs. Other Bacillus species do not have colonies with this type of edge. (B. subtilis, B. cereus and other bacilli are common blood culture contaminants which may sometimes be confused with ‘coliforms’ when colonies appear on MacConkey's medium.)
	B. anthracis gives a characteristic inverted fir tree growth in gelatin.

Differentiation of Bacillus species by animal inoculation tests

B. anthracis, but not the other Bacillus species, is pathogenic to mice and guinea-pigs. This test is too dangerous to use in the average hospital animal house. Infected animals may disseminate anthrax spores which could survive for many years in the environment.

Sources and diseases

B. anthracis can infect many different animal species including sheep and the anthrax spores may remain viable in animal products or in the soil for a long period. B. cereus and B. subtilis are found in the soil, dust and air and B. cereus may contaminate food such as boiled rice.

B. anthracis is the cause of anthrax in man and animals (see Chapter 16). B. cereus is one cause of food poisoning (see Chapter 14). B. subtilis is nearly always only a contaminant when noticed in cultures. However, it may rarely cause bacteraemia in patients on haemodialysis when the dialysis machines are contaminated or when an intravenous infusion has become contaminated.

Antibiotics

B. anthracis is characteristically sensitive to penicillin.

Corynebacterium species

These include Corynebacterium diphtheriae, Corynebacterium ulcerans, and ‘diphtheroid species’ including Corynebacterium xerosis and Corynebacterium hoffmani.

Microscopy:	The Gram-positive bacilli of C. diphtheriae are slightly curved and characteristically appear like ‘Chinese characters’ whereas the diphtheroid bacilli are often seen as palisade rows of bacilli.
	Metachromatic granules (‘Volutin granules’) are sometimes seen in an Albert's stain of diphtheria bacilli. However, these granules do not necessarily indicate that the bacilli are C. diphtheriae, nor do they reliably indicate that a diphtheria strain is toxigenic.
Culture:	Many ‘diphtheroids’ can easily be distinguished from C. diphtheriae according to the colonial appearances on tellurite media (such as Hoyle's or Downie's medium) and on blood agar. C. diphtheriae appears characteristically as grey-black colonies on tellurite. However, a few ‘diphtheroid’ strains may be difficult to differentiate in this way and any suspicious colonies require further tests.
	Loeffler's serum agar slope is also used for the isolation of C. diphtheriae and is useful for providing a suitable culture for toxigenicity tests.

Differential tests

Hiss's serum water sugar fermentation tests are inoculated and the pattern of results helps to identify the Corynebacterium species. Characteristically C. diphtheriae ferments glucose and maltose, rarely sucrose. The C. diphtheriae species can be further differentiated into the subspecies gravis (‘daisy head’ classically and ferments starch), mitis or intermedius but, in practice, this is not important except for epidemiological purposes. C. ulcerans can give some biochemical reactions similar to C. diphtheriae gravis but the urea slope test reaction is different.

Toxinogenicity tests

These urgent tests on suspicious C. diphtheriae cultures are performed by an Elek plate or guinea-pig method (see p. 200).

Normal flora and diseases

‘Diphtheroids’ are commonly isolated skin or throat commensals. Rarely, urinary tract infection or bacterial endocarditis affecting a prosthetic heart valve may be caused by these organisms.

C. diphtheriae is rarely found in the normal throat flora except during convalescent carriage. Toxigenic strains may cause diphtheria in susceptible individuals.

C. ulcerans may cause a severe sore throat and some marked constitutional upset but is rarely associated with the classic toxic complications of diphtheria.

Antibiotics

C. diphtheriae strains are characteristically sensitive to penicillin and erythromycin.

Listeria and Erysipelothrix

Listeria monocytogenes and Erysipelothrix rhusiopathiae

Microscopy:	Short Gram-positive bacilli may occasionally be confused with ‘diphtheroids’.
	Light microscopy of a wet preparation of a peptone water culture of Listeria monocytogenes, that has been incubated at room temperature, characteristically shows ‘tumbling motility’.
Culture:	Small colonies appear on blood agar after overnight incubation at 35 °C. Listeria colonies usually show beta-haemolysis but erysipelothrix colonies are usually alpha- or non-haemolytic. Listeria, but not Erysipelothrix, grows at 4°C.

Differential tests

Biochemical tests, as well as cultural characteristics, differentiate Listeria from Erysipelothrix including tests for aesculin hydrolysis and catalase production.

Diseases

Listeria monocytogenes is a cause of meningitis and/or septicaemia in neonates and in immunocompromised patients. It is a possible but rare cause of still-birth.

Erysipelothrix rhusiopathiae causes erysipeloid (see p. 382 and p. 518).

Antibiotics

Listeria and Erysipelothrix are both characteristically sensitive to ampicillin (or penicillin).

Nocardia and Actinomycetes

These Gram-positive branching filamentous bacilli cause nocardiosis and actinomycosis, respectively (see Chapter 13).

Clostridial species

These include Clostridium perfringens (welchii), septicum, oedematiens, histolyticum, tetani, botulinum, difficile.

The main characteristics of Gram-positive spore-forming anaerobic bacilli are described in Chapter 9.

Gram-negative Bacteria

Neisseria species

These include Neisseria gonorrhoeae, meningitidis, catarrhalis, pharyngis and lactamis.

Microscopy:	Gram-negative oval diplococci; some are characteristically intracellular when seen in clinical specimens.
Culture:	Neisseria are fragile and suitable transport of specimens with prompt culture is important (seepp. 232, 462).
	Small colonies on blood agar after 24–48 hours’ incubation in a moist aerobic atmosphere with 5–10% carbon dioxide added. Larger colonies on chocolate agar.
	Selective media are used for the isolation of Neisseria gonorrhoeae from genital tract or rectal specimens (seep. 462). Non-pathogenic Neisseria sometimes grow on plain agar.

Differential tests

Neisseria species are oxidase positive. The species are differentiated according to the results obtained with biochemical and immunological tests.

• 1 Biochemical tests Serum sugar agar slopes are used usually for carrying out sugar fermentation tests (not horse serum which contains maltose). Hydrocele fluid can be used instead of serum as a growth factor. Glucose only is characteristically fermented by gonococci. Maltose and glucose are characteristically fermented by meningococci (although a few strains do not ferment maltose). Sucrose or lactose are sometimes fermented by Neisseria species which are neither Neisseria gonorrhoeae nor meningitidis. The media used for sugar fermentation tests needs to be carefully quality controlled.
• 2 Immunological tests An immunological test as well as a biochemical test is desirable for the identification of possible gonococcal strains, especially when the isolate is from a female patient or from an unusual site, such as the throat (because atypical strains of other Neisseria species, isolated in these circumstances, may occasionally give similar biochemical reactions to those of gonococci). When an isolate shows strong immunofluorescence or gives a positive latex co-agglutination test with a specific anti-gonococcal serum, there is good evidence that the isolate is a gonococcus (but the medicolegal differentiation traditionally depends on the results of biochemical tests).

  Immunological tests are also used in reference centres to serogroup meningococci into one of the three main groups, A, B or C. Most strains in Britain are serogroup B.

Normal flora and diseases

Neisseria meningitidis is carried in the nasopharynx of 5–30% of the general population, and is one of the ‘three primary pathogens’ causing bacterial meningitis.

Neisseria gonorrhoeae is the cause of gonorrhoea and ophthalmia neonatorum. It is not found in the normal flora.

Antibiotics

All meningococcal strains outside South Africa are sensitive to penicillin. There are some gonococcal strains highly resistant to penicillin (penicillinase producers), but sensitive to spectinomycin and penicillinase-stable cephalosporins, such as cefotaxime. However, well over 90% gonococcal strains in Britain are still relatively sensitive to penicillin.

Enterobacteria (Coliforms)

These include scores of different genera and many hundreds of different species. Escherichia coli, Klebsiella aerogenes, Proteus, Salmonella and Shigella species are examples (see also Table 1.2).

Microscopy:	Gram-negative bacilli—the species are not differentiated by their Gram-stain appearance.
Culture:	Good growth on blood agar, MacConkey or cysteine lactose electrolyte deficient (CLED) medium is characteristic after overnight aerobic incubation.
	Selective media such as deoxycholate citrate agar (DCA), which suppress the growth of many E. coli strains, are used for the isolation of both salmonellae and shigellae from faeces and require up to 48 hours’ incubation.
	Enrichment liquid media, such as selenite F, are used to increase the yield of salmonella isolations from the faeces. The organisms are subcultured from these media on to selective media, usually after overnight incubation.

Differential tests

• 1 Lactose fermentation Escherichia coli, Klebsiella or other coliforms which are characteristically lactose fermenters appear as pink colonies after overnight culture on MacConkey or CLED media. However, some late lactose fermenting strains of these species may appear as ‘non-lactose fermenting’ colonies on MacConkey agar. Salmonella and Shigella species characteristically appear as ‘non-lactose fermenters’ on MacConkey or DCA media (but Shigella sonnei is a late lactose fermenter and may appear slightly pink on MacConkey after 24–48 hours’ incubation).
• 2 Motility and other biochemical tests A ‘hanging drop’ of an overnight peptone water culture may be examined by wet microscopy to see if the organism is motile or non-motile. E. coli and salmonellae are characteristically motile (they are flagellated coliforms) whereas klebsiellae and shigellae are characteristically non-motile. Semisolid agar methods are available (e.g. ‘Craigie tube’) for testing for the motility of a possible salmonella or shigella isolate, which are safer than the hanging drop method.

  Biochemical tests, in addition to lactose fermentation, include urea, glucose, mannite, sucrose, indole, citrate, hydrogen sulphide production and decarboxylases for apparent ‘non-lactose fermenters’ and indole, citrate and inositol for ‘lactose fermenters’.

  Commercial kits are available to assist this identification process which must be carried out on pure cultures. For the identification of ‘difficult’ organisms, a computer analysis of the results may be useful at a reference centre.

  A few simple characteristic examples of the results of biochemical tests include:

  Proteus species	urease positive, lactose negative
  Salmonella species	glucose fermented, mannite fermented, lactose negative, urea negative, sucrose negative
  Shigella species
  	glucose fermented
  	mannite fermented—mannitol positive
  	shigellae (e.g. Sh. sonnei)
  	urea negative, sucrose usually negative
  	mannite negative—mannitol negative shigellae
  E. coli	indole positive, lactose positive, citrate negative
  Serratia marcescens	DNAase positive.

  There are also classic ‘IMVIC’ tests for lactose fermenters isolated from possibly faecally contaminated water supplies—indole, methyl red, Voges-Proskauer, inositol and citrate. These biochemical tests may be performed at 44°C to recognize E. coli type I from a possible human faecal source (e.g. from polluted water).

• 3 Immunological tests Suspensions of suspected pathogenic faecal coliforms can be tested against known specific antisera in slide or tube agglutination tests. Specific ‘O’ (somatic antigen) antisera are used for the identification of isolates of salmonella, shigellae and enteropathogenic strains of E. coli. Specific ‘H’ (flagellar antigen) antisera are mainly used to identify Salmonella species. A salmonella culture on a nutrient agar is often used for agglutination tests but it may be in a ‘non-specific H phase’. To convert the Salmonella to a ‘specific H phase’, a ‘phase switch’ may be necessary using a Craigie tube method.

Normal flora

E. coli is the most common and most numerous aerobic Gram-negative species in the normal faecal flora. Other coliforms are also often present mixed with the E. coli, including Proteus, Klebsiella or Citrobacter species to mention just a few possibilities.

Salmonella and Shigella species are not found in the normal flora although they may be found in the faeces of healthy convalescent carriers (or permanently in a biliary tract carrier of Salm. typhi).

Diseases

‘Endogenous’ infections are most common with lactose fermenting coliforms, such as E. coli and certain non-lactose fermenting coliforms, such as Proteus species, including urinary tract infections, wound infections, abdominal sepsis and Gram-negative septicaemia.

‘Exogenous’ infections may also occur with the same organisms as those endogenous infections. Cross-infection or environmental infection in hospital may occur (see Chapter 25). Infections of the gastro-intestinal tract are also exogenous, due to salmonellae, shigellae, enteropathogenic E. coli, etc. (see Chapter 14).

Antibiotics

There is such an enormous variation in the antibiotic susceptibilities of different coliform strains that they can only be predicted to a limited extent.

• 1 Outside hospital Many coliforms are sensitive to ampicillin (although Klebsiella is an exception), provided no recent antibiotics have been given to the patient. The coliforms are usually sensitive to trimethoprim.
• 2 In hospital Coliforms are frequently resistant to ampicillin and are often also resistant to other agents, including sulphonamides, tetracycline and streptomycin. The antibiotic resistance patterns vary between different hospitals and between different places in the same hospital, as well as at different times. The patterns of antibiotic resistance depend greatly on the amounts of particular antibiotics used in a given hospital area, the prevalence of particular R factors which carry genes for multiple antibiotic resistance and the frequency of cross-infection in the hospital. In most hospitals in Britain, the great majority of coliforms are still sensitive to gentamicin and to new cephalosporins such as cefuroxime. However, outbreaks of gentamicin-resistant coliform infections occasionally occur.

Pseudomonas species

These include Pseudomonas aeruginosa, Pseudomonas cepacia and other Pseudomonas species.

Microscopy:	Gram-negative bacilli, indistinguishable from the ‘coliforms’ above on Gram-stain.
Culture:	Pseudomonas species are strictly aerobic. Good growth only occurs after overnight incubation in an aerobic atmosphere on a blood or nutrient agar plate in contrast to ‘coliform’ species which can grow well either aerobically or anaerobically as they are facultative anaerobes.
	Pseudomonas aeruginosa also grows well on a selective agar containing the disinfectant ‘cetrimide’ and in many solutions.
Oxidase test:	Pseudomonas species are, characteristically, strongly oxidase positive in contrast to ‘coliform’ species which are oxidase negative.

Differential tests

Pseudomonas aeruginosa (pyocyanea) produces a green ‘pyocyanin’ pigment on magnesium ion containing media whereas other Pseudomonas species do not produce this pigment. It metabolizes glucose by oxidation rather than by fermentation and this can be shown by a ‘Hugh and Leifson’ test. The other Pseudomonas species can be differentiated according to the results of biochemical tests (using ammonium salt sugars).

Normal flora and sources

Pseudomonas aeruginosa occurs infrequently in the faecal flora of patients outside hospital. Hospital patients receiving broad-spectrum antibiotics, such as oral cephalosporins, are frequently colonized by Pseudomonas aeruginosa in the lower intestinal tract.

Most Pseudomonas species may be isolated from moist environmental sites in the hospital including contaminated suction apparatus, contaminated disinfectants, respiratory ventilators and humidifiers. Bottles containing sterile distilled water or other solutions may become quickly contaminated by Gram-negative bacilli, including Pseudomonas species, once the bottles are opened.

Diseases

Endogenous or exogenous pseudomonas infections (cross-infection or environmental infection) include chronic urinary tract infections, wound infections, chronic osteomyelitis, chronic otitis externa, eye infections (rare), and various serious opportunistic infections including pneumonia and septicaemia.

Antibiotics

Pseudomonas aeruginosa is resistant to many antibiotics including ampicillin, sulphonamides, trimethoprim, tetracycline, cephaloridine and many cephalosporins, streptomycin and kanamycin. Nearly all Pseudomonas aeruginosa strains are sensitive to polymyxin but this antibiotic is mainly suitable for treating only superficial infections topically. Most strains are sensitive to the aminoglycosides, gentamicin or netilmicin, which are often valuable for systemic treatment. Many strains are sensitive to carbenicillin, ticarcillin, piperacillin or azlocillin and these anti-pseudomonas penicillins are usually used systemically together with the above aminoglycosides. There is a lot of variation in the sensitivity of different strains to these penicillins depending on whether the strains produce particular penicillinases. Most strains are sensitive to ceftazidime, ciprofloxacin and imipenem.

Vibrios

These include Vibrio cholerae, Vibrio parahaemolyticus and Campylobacter species (previously known as Vibrio foetus), such as Campylobacter jejuni.

Microscopy:	Vibrios characteristically appear as curved Gram-negative bacilli, like ‘comma’ bacilli, but they may also be indistinguishable from ‘coliforms’ in a Gram-stain.
	Campylobacters are characteristically seen as spiral or small ‘S’-shaped Gram-negative bacilli in a Gram-stain.
	Vibrios are typically motile when suspensions are examined by ‘wet microscopy’.
Culture:	Thiosulphate–citrate–bile salt–sucrose agar (TCBS) medium is the selective medium used for the isolation of Vibrio cholerae and Vibrio parahaemolyticus from faeces. V. cholerae and V. parahaemolyticus usually produce large yellow or green colonies, respectively, on TCBS medium after 24 hours incubation in an aerobic atmosphere.
	An alkaline peptone water enrichment culture is used in addition to TCBS selective medium for the isolation of V. cholerae.
	Campylobacter species grow well after 24–48 hours incubation on a selective blood agar medium containing polymyxin, vancomycin and trimethoprim. The plates have to be incubated in a micro-aerophilic atmosphere with added carbon dioxide, preferably at 40–42 °C. When this medium is used to isolate Campylobacter species from the faeces of a patient, a presumptive diagnosis of Campylobacter is often possible by seeing ‘S’-shaped or slim curved Gram-negative bacilli in a Gram-stain of the characteristic moist-looking, oxidase-positive colonies.

Differential tests for V. cholerae

Suspicious yellow colonies on TCBS medium are further identified by Gram-stain, oxidase test, subculture on to nutrient or blood agar for definitive immunological tests and rapid slide agglutination tests with specific V. cholerae anti-serum. A presumptive identification is made by the laboratory which urgently sends the culture to a reference laboratory for confirmatory tests including phage typing (with the Mukerjee phage). The results of phage and polymyxin sensitivity tests, haemolysis and other tests in the reference laboratory can also differentiate between ‘E1 Tor’ and the ‘classic’ biotypes of V. cholerae. Nearly all the patients with cholera seen in Europe, the Middle East and Africa have been infected by the ‘E1 Tor’ V. cholerae.

Diseases

V. cholerae causes cholera and the strains may be carried during convalesence.

V. parahaemolyticus is an uncommon cause of food poisoning in Britain.

Campylobacter jejuni is a common cause of gastro-enteritis and food poisoning.

Campylobacter pylori is associated with some types of peptic ulceration(see Chapter 14).

Antibiotics

V. cholerae (E1 Tor) is usually sensitive to tetracycline but the incidence of tetracycline-resistant strains is increasing. Antibiotics are of secondary importanc toflid and electrolyte replacement (see p. 356).

Campylobacter jejuni is nearly always sensitive to erythromycin.

Parvobacteria

These include: Haemophilus influenzae and other Haemophilus species; Bordetella pertussis and parapertussis; Pasteurella multocida (septica); Yersinia pseudotuberculosis, Yersinia enterocolitica and other Yersinia species; Brucella abortus and other Brucella species; Francisella tularensis; Pseudomonas mallei.

Microscopy:	The parvobacteria are short Gram-negative bacilli (coccobacilli). Many such as Haemophilus species are pleomorphic. Some bacilli, such as Pasteurella multocida may show bipolar staining. Occasionally, the capsules of capsulated strains of Haemophilus influenzae may be seen, e.g. Pittman type b strain, in stained smears or by immunofluorescent techniques using specific anti-capsular antibody.
Culture:	Parvobacteria are relatively fragile and specimens with these organisms need to be promptly cultured and preferably inoculated directly on to media for the best culture results. If delays are inevitable, appropriate transport media may be used for certain species such as Bordetella (seep. 196).
	Most parvobacteria species appear as small colonies on fresh blood agar after 24–48 hours’ incubation in an aerobic moist atmosphere with 5–10% added carbon dioxide. A few species will not grow on blood agar, such as Bordetella pertussis. Special enriched and selective media such as Bordet-Gengou, Lacey's medium or pertussis charcoal agar are needed for culture of Bordetella. Dorset's egg medium is required for culture of Francisella tularensis.
	Haemophilus species grow better on chocolate agar than on blood agar; moderately large colonies are apparent on chocolate agar after overnight incubation.

Differential tests

• 1 Haemophilus species The Haemophilus species can be identified according to the results of ‘satellitism’ tests. Haemophilus influenzae will not grow around a disc containing factor X (haemin) or factor V (coenzyme NAD) alone, but will around a combined X plus V disc on plain agar. On blood agar, Haemophilus influenzae shows improved satellite growth around Staph. aureus colonies due to the release of factor V from the staphylococci. (Haemophilus parainfluenzae does not require factor X but does require factor V.)

  Haemolysis is another cultural factor that is used for identifying less pathogenic Haemophilus species, such as Haemophilus haemolyticus, which may be found in the normal throat flora.

  Capsulated strains of Haemophilus influenzae often grow well with a green sheen on Levinthal's agar. The capsules can be Pittman typed using specific anti-capsular sera (in a Quelling type reaction). The usual capsular type H. influenzae infecting infants is Pittman type b. This capsular antigen may sometimes also be detected using an immunoprecipitation test.

• 2 Differentiation from coliforms Most parvobacteria species will not grow well (if at all) on MacConkey's agar after overnight incubation, in contrast to ‘coliforms’ and most Pseudomonas species. Most of the parvobacteria will not grow in peptone water sugars unlike coliforms, an important exception being Pasteurella multocida. Some parvobacteria will not grow anaerobically on blood agar after overnight incubation unlike coliforms.
• 3 Yersinia, Pasteurella and Bordetella species A range of cultural tests on MacConkey agar, blood agar, enriched and selective media combined with biochemical tests, motility and haemolysis tests help to identify a particular Yersinia or Pasteurella species. Immunological tests using slide or tube agglutinations of suspensions of the organisms against known antisera are also required for Yersinia and Bordetella species.

Normal flora

Haemophilus species are frequently found in the normal throat flora and occasionally in the nose flora (especially of infants). Pasteurella multocida is also occasionally found in the normal upper respiratory tract flora, especially in individuals who may have contact with rodents (because they work in an animal house or have rodent pets).

Diseases

Parvobacteria causing diseases are included in Table 1.7 . The most common parvobacteria infections in Britain include those due to Haemophilus influenzae, Bordetella pertussis and Pasteurella multocida. Other parvobacteria infections are very uncommon.

Table 1.7.

Some examples of infections due to parvobacteria

Parvobacteria	Main injections
Haemophilus influenzae
i. Capsulated Putman type b strains	Infections in children mainly, 3 months to 5 years (up to 12 years may occur):
	aRespiratory tract infections—pharyngitis, otitis media, sinusitis, acute epiglottitis (rare), pneumonia (very rare) bSepticaemia cMeningitis dOsteomyelitis and septic arthritis ePericarditis or endocarditis (both rare)
ii. Other strains	Infections mainly in adults: aInfective exacerbations of chronic bronchitis bChronic sinusitis cConjunctivitis
Bordetella pertussis	Whooping cough
Pasteurella multocida (septica)	Wound infections following animal bites: meningitis (rare), septicaemia (rare)
Yersinia pestis	Plague
Yersinia enterocolitica	Gastro-enteritis (possible ‘rheumatic fever’-like illness and arthritis possible)
Yersinia pseudotuberculosis	Mesenteric adenitis (may clinically mimic acute appendicitis)
Brucella species	Brucellosis
Francisella tularensis	Tularaemia
Pseudomonas mallei	Glanders

Antibiotics

Some parvobacteria species are relatively sensitive to penicillin in contrast to coliforms which are resistant to penicillin, a good example being Pasteurella multocida. Many parvobacteria are also sensitive to erythromycin in vitro, such as Bordetella pertussis, Pasteurella multocida. Yersinia and Brucella species are characteristically sensitive to tetracyclines.

Haemophilus influenzae strains are usually sensitive to ampicillin but the incidence of ampicillin-resistant beta-lactamase-producing strains is increasing and is greater than 10% for Pittman type b capsulated strains in some areas in Britain. Chloramphenicol is nearly always active against H. influenzae, including ampicillin-resistant strains and is recommended for treating haemophilus infections such as meningitis or acute epiglottitis. Tetracycline may be used instead of ampicillin for treating infective exacerbations of chronic bronchitis due to Haemophilus influenzae.

Legionella

Legionella pneumophila and some other Legionella species may cause severe pneumonia—Legionnaires’ disease.

Microscopy:	Gram-negative bacilli in Gram-stains of colonies from culture media; in tissues and clinical specimens the bacilli may stain poorly with an ordinary Gram-stain but better when a prolonged counterstain with carbol fuchsin is used. Gram films made from cultures may show long filamentous forms of the organism. The bacilli are also apparent in silver stains in tissue although they are best seen in tissues by immunofluorescence using specific anti-legionella antisera or by electron microscopy using immunoferritin techniques.
Culture:	No growth on ordinary media.
	Requires 3–5 days’ incubation on special legionella media containing blood, added cysteine and iron salts such as ferric pyrophosphate, at pH6·9.
	Guinea-pig inoculation may be necessary for the isolation of Legionella from environmental samples such as water.

Sources

Environmental: contaminated water in air conditioning plants, shower mixers, etc., causes infection by inhalation of contaminated air.

Disease

Legionnaires’ disease ranges from a mild pyrexia of unknown origin (PUO), or mild respiratory symptoms, to severe pneumonia with multisystem complications. It is fatal in about 15% of Legionella-infected patients requiring hospital admission for severe pneumonia. Other similar diseases due to ALLO (atypical Legionella-like organisms) have been recently reported.

Antibiotics

Legionella is characteristically sensitive to erythromycin and tetracycline but usually resistant to penicillins and aminoglycosides.

Anaerobic Gram-negative Bacilli

Strict non-sporing anaerobic organisms, including Gram-negative bacilli such as Bacteroides species, are described in Chapter 9.