Chapter 5:Small Gram-Negative Rods and Coccobacilli

Chapter 5:Small Gram-Negative Rods and Coccobacilli

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria

Chapter 5:Small Gram-negative rods and coccobacilli

Authors: Drs. M.M. Henton & J.A. Picard

Licensed under aCreative Commons Attribution license.

TABLE OF CONTENTS

INTRODUCTION

Table 5.1: Phenotypic characteristics separating selected genera of veterinary importance in the family Pasteurellaceae

Table 5.2: Histophilus and Haemophilus species of veterinary importance in production animals, dogs and poultry

Table 5.3: Identification of Haemophilus and Taylorella species of veterinary importance

Table 5.4: Actinobacillus species of veterinary importance in production animals and dogs

Table 5.5: Differential characteristics of the species belonging to the genus Actinobacillus

Table 5.6: Important Pasteurella and Mannheimia species in animals

Table 5.7: Identification of bacteria belonging to the genus Pasteurella. (All are non-motile, oxidase positive, nitrate positive and ferment glucose)

Table 5.8: Differential characteristics of the species belonging to the genus Pasteurella and some related species (Lonepinella koalarum and Phocoenobacter uteri are catalase and xylose negative

Table 5.9: Differential characteristic of Mannheimia species

Table 5.10: Identification characteristics of Gallibacterium species

REFERENCES

INTRODUCTION

The genera Haemophilus, Actinobacillus, Mannheimia and Pasteurella are currently classified in the family Pasteurellaceae. These species are major causes of respiratory and systemic diseases in humans, other mammals and birds. They exist as mucosal parasites, but are capable of being primary or opportunistic pathogens depending on the carrier status and health of the host.

Recently there have been many changes in the taxonomy of this group that now contains seven genera of veterinary importance: Actinobacillus, Avibacterium, Haemophilus, Histophilus, Mannheimia, Pasteurella and Bibersteinia. Important differential characteristics of genera in this family are included inTable 5.1.

Table 5.1: Phenotypic characteristics separating selected genera of veterinary importance in the family Pasteurellaceae

Histophilus / Haemophilus / Actinobacillus / Pasteurella
Haemolysis / D / D / D / -
Capnophilia / + / - / - / -
Yellowish pigmentation / + / - / - / -
V-factor dependence / - / + / D / D
X-factor dependence / - / + / - / -
Catalase / - / - / + / +
Oxidase / + / + / D / D
Urease / - / D / + / D
Voges–Proskauer (37 °C) / - / - / - / -
Indole / +d / D / - / +b
Ornithine decarboxylase / - / D / - / D
L-Arabinose / - / D / -
D-Fructose / - / D / + / +
D-Galactose / - / D / D / +
Meso-Inositol / - / - / -
Maltose / - / + / + / D
Mannitol / - / D / D
D-Mannose / D / D / +
D-Melibiose / - / D / -
D-Sorbitol / - / -C / D
Sucrose / - / - / + / +
Trehalose / - / - / D / D
ONPF (-fucosidase) / - / - / -
ONPG / -
PNPG / - / - / -

+, only positive reactions, -, only negative reactions; D, positive or negative; NK, not known.

aDeviating strains occur.

bP. aviumis indole negative

cAesculin, amygdalin, arbutin, cellobiose, salicin and NPG (-glucosidase)

dBisgaard taxon 9 late positive

Haemophilus/ Histophilus

There have been a number of taxonomic changes to organisms within the genus Haemophilus in recent years. The species Histophilussomni now includes the bovine commensal and opportunistic pathogen Haemophilussomnusand the ovine organisms Histophilusovisand Haemophilusagni. A list of HaemophilusandHistophilusspecies causing disease in animals are included in Table 5.2.

Table 5.2: Histophilusand Haemophilusspecies of veterinary importance in production animals, dogs and poultry

Species / Hosts / Disease
H. somni / Cattle (previously Haemophilussomnus)
Sheep(previously Haemophilusagni andHistophilusovis) / Septicaemia
Infarcts in the brain (Thrombotic meningoencephalitis)
Respiratory infections
Genital tract infections
Epididymitis
Pneumonia
Mastitis
Polyarthritis
Meningitis
Septicaemia
H. parasuis / Pigs / Polyserositis, meningitis (Glasser’s disease)
A. paragallinarum / Poultry / Infectious coryza in chickens and respiratory infection in turkeys
H. haemoglobinophilus / Dogs / Balanoposthitis, vaginitis
H. paracuniculus / Rabbits / Mucoid enteritis

Speciation of Haemophilusin the clinical laboratory has been primarily based on the requirement for haemin (a porphyrin also known as X factor) or NAD (also known as V factor or coenzyme I). The methods currently used for the determination of these growth factor requirements rely on the supplements being added to defined agar media or impregnated in disks or strips placed on agar plates.

Unlike human pathogens in this genus, no animal isolate is dependent on both factors. They are all dependent on only one factor with the exception of Histophilussomni, which is X and V factor independent. The X factor is present in adequate amounts in blood agar. In chocolate agar both factors are present by virtue of being released into the medium following haemolysis during heating of the blood. The V factor is also produced by an organism such as Staphylococcus aureus. When the latter is grown together with Haemophilus spp. on blood agar, the resultant enhanced growth of Haemophilus is referred to as satellitism. In veterinary laboratories, routine isolation on chocolate agar in an atmosphere of 5-10% CO2 (or candle jar) at 35-37°C for 3-4 days is satisfactory. Selective media can be used when one specifically suspects the presence of Haemophilusspecies.

Histophilussomni (formerly Haemophilussomnus, H. agniandHistophilusovis)

H. somni is an opportunistic pathogen of cattle. It is a common component of the normal urogenital and respiratory flora and sporadically causes thromboembolic meningoencephalitis, abortion, infertility and pneumonia. It is often the only bacterial isolate from the many reported cases of necrotic laryngitis, necrotic tracheitis, fibrinous bronchopneumonia and fibrinouspleuritis in cattle. There are probably many subtypes of H. somni.

This organism is a nutritionally demanding, moderately slow growing, capnophilic, Gram-negative bacillus. Colonies on blood agar are unremarkable. H. somnidoes not require X or V factors for growth. A selective growth medium for H. somnihas been described. It incorporates vancomycin (5 g/ml), neomycin (5 g/ml), sodium azide (50 g/ml), nystatin (100 g/ml) and cycloheximide (100 g/ml) into 5% horse blood agar. Addition of thiamine monophosphate (1 g/ml) also enhances growth of the organism.

When attempting to isolate H. somni from cases of suspected thromboembolic meningoencephalitis, brain tissue that includes visible lesions should be homogenised in sterile saline at the ratio of 5 mg tissue in 20 ml saline. Spread aliquots of 0.1 ml over the surface of blood agar. H. somni is considered to be homologous to Haemophilusagniand Histophilusovis both found in sheep and goats. There are slight biochemical differences between them e.g. H. agniis usually urease positive and indole negative. Histophilussomni may be weakly indole positive and when scraped together the colonies are usually yellow. Actinobacillusseminis is also very similar to H. somni. Both are sucrose negative, which distinguishes them from the rest of the community found in the Haemophilus/Pasteurella/Actinobacillus (HPA) group, but A. seminis is grey when the colonies are scraped together. Differential characteristics are summarized in Table 5.3.

Table 5.3: Identification of Haemophilusand Taylorella species of veterinary importance

Test / H. somnus / H. agni / H. parasuis / A. pleuropneumoniae / Avibacterium. paragallinarum / H. haemoglobinophilus / H. paracuniculus / T. equigenitalis
Requirement for X factor / - / - / - / - / - / + / - / -
Requirement for V factor / - / - / + / + / + / - / -
Growth on MacConkey / - / - / - / - / - / - / - / -
Catalase / - / - / + / d / - / + / + / +
Oxidase / + / + / - / d / - / + / + / +
Capnophilic / + / + / + / + / - / +
Haemolysis / - / - / - / + / - / - / -
Indole / +w / - / - / - / - / + / + / -
Urease / - / (+) / - / + / - / - / + / -
Nitrate reduction / + / + / + / + / + / + / + / -
Glucose (acid) / + / + / + / + / + / + / + / -
Aesculin hydrolysis / - / - / -- / d / - / - / -
ONPG / d / d / + / + / d / +
Phosphatase / - / + / + / + / - / + / +
Ornithine decarboxylase / + / + / - / - / - / - / + / -
Gas from glucose / - / - / - / - / - / - / -
Acid production from:
L-arabinose / d / +/w / - / - / - / -
Cellobiose / - / - / - / - / - / - / -
Dulcitol / d / - / - / - / - / - / -
Galactose / d / +/w / + / +/w / - / - / -
m-Inositol / d / - / d / - / - / - / -
Lactose / - / - / d / d / - / - / -
Maltose / + / +/w / + / + / (+) / + / +
Mannitol / + / +/w / - / + / + / + / -
Mannose / + / - / + / + / + / +
Melibiose / - / - / - / - / - / -
Raffinose / - / - / - / d / - / - / -
Salicin / - / - / - / - / - / - / -
Sorbitol / + / - / - / - / + / - / -
Sucrose / - / - / + / + / + / + / +
Trehalose / + / - / - / - / - / - / -
D-Xylose / + / +/w / - / + / d / + / -

Haemophilusparasuis

H. parasuis is considered to be part of the normal flora of the respiratory passages of conventionally raised pigs. It causes Glässer’s disease, characterized by polyserositis and meningitis. The disease only occurs sporadically, mainly in pigs two to four weeks of age, subjected to stressful environmental conditions. A selective medium for H. parasuis consists of chocolate agar containing 1.5 g/ml lincomycin, 5 g/ml bacitracin and 0.1 g/ml crystal violet. The specimen material should be spread with a sterile swab to completely cover the agar surface. Haemophilusparasuis grows weakly and may take 2 to 3 days to become apparent. Actinobacilluspleuropneumoniae in comparison grows rapidly (1 day), is CAMP and urease positive.

TayorellaEquigenitalis

This organism was formerly known as Haemophilusequigenitalis. It is an important cause of genital tract infections in horses and is either venereally transmitted or via assisted reproduction (artificial insemination). Swabs of the cervix, clitoral fossa and urethra can be examined in mares, and the urethral fossa and penile sheath (lamina interna) in stallions

For primary isolation agar bases such as Oxoid blood agar base No. 2, or Columbia agar (BBL) can be used with the addition of sodium sulphate 200 mg/L and L-cysteine HCl 100 mg/L. Chocolated blood (preferably sheep blood) are added at 10% concentration. Some plates should be prepared with streptomycin at 200 g/ml media and some without. One of each should be used to examine swab specimens. Plates are incubated in air with 5-10% CO2 for up to 6 days before discarding them. Colonies are small pinpoint to pin head in size.

The minimum profile of tests to be carried out on suspicious colonies is:

Motility
Oxidase
Catalase
Urease
Phosphatase / non-motile in Robertson's cooked meat medium
positive (rapid and strong reaction)
positive
negative
positive
Actinobacillus

Actinobacillus spp. are commensals on the mucous membranes of their hosts. They do not penetrate intact skin or healthy mucosa, but are responsible for endogenous infections following trauma. These organisms are aerobic, Gram-negative coccobacilli. Members of this genus that are of veterinary importance are listed in Table 5.4. Note that A. murishas only been found in rodents and A. actinomycetum-comitans and A. hominis in humans.

Table 5.4: Actinobacillusspecies of veterinary importance in production animals and dogs

Species / Hosts / Disease
A. lignieresii / Cattle
Sheep
pigs / Wooden tongue
Pyogranulomas
Pyogranulomas
Granulomatous mastitis
A. equuli / foals
mares
Pigs
dogs / Neonatal foal septicaemia
Arthritis
Abortion
Septicaemia
Arthritis
Opportunistic infections
A. suis / pigs / Pneumonia
Septicaemia
A. pleuropneumoniae / pigs / Fibrinouspleuropneumonia
A. seminis / rams / Epididymitis

They can all be isolated on blood agar and incubated aerobically for 24 hours at 37°C. Growth on MacConkey agar may take longer than 24 hours. Members of the genus Actinobacillus typically produce sticky colonies, grow well on MacConkey agar, and hydrolyze urea. The differential characteristics of the species belonging to the genus Actinobacillus are included in Table5.5.

Table 5.5: Differential characteristics of the species belonging to the genus Actinobacillus

Test / A. acetomycetum-comitans / A. capsulatus / A. arthrittidis / A. equuli subsp.equuli / A. equuli subsp. haemolyticus / A. hominis / A. lignieresii / A. muris / A. pleuropneumoniae / A. rossii / A. seminis / A. suis
Catalase / +a / (+) / d / (+) / - / d / + / d / (+) / + / +
Oxidase / + / + / (+) / + / + / (+) / + / d / (+) / d / (+)
Nitrate reduction / + / + / + / + / + / + / + / + / + / + / +
ONPG reaction / - / + / + / d / + / + / d / - / + / (+) / -b / (+)
Phosphatase / + / + / + / + / + / - / + / - / - / +
Ornithine decarboxylase / - / - / - / - / - / - / - / - / d / -
Indole production / - / - / - / - / - / - / - / - / - / -
Urease / - / + / + / + / + / + / + / + / - / +
Aesculin hydrolysis / - / +, L / - / (-) / d / - / +, L / d / - / d / +
NAD requirement / - / - / - / - / - / - / + / - / - / -
Growth on MacConkeyagarc / d / + / - / (+) / - / - / (+) / - / - / (+)w / - / +
Beta-haemolysis (sheep cells) / - / - / - / + / - / - / - / + / d / - / +
Fermentation f / + / NF / + / + / + / + / + / + / + / W, L / +
Gas production from glucosed / (+) / - / - / - / - / - / - / d / - / -
Acid production from: / -
L-arabinose / - / + / d / (-) / (-) / - / (-) / - / - / + / d, L / (+)
Arbutin / - / + / - / NT / - / + / NT / - / - / +
Cellobiose / - / + / - / + / - / - / +,L / - / - / - / +
Dulcitol / - / - / - / - / - / - / - / - / - / -
Galactose / + / + / + / + / + / + / d,w / +,w / + / d,L / (+)
m-Inositol / - / - / - / - / - / d,w / - / (+) / d / -
Lactose / - / + / + / + / + / d,L / - / d / + / - / +
Maltose / + / + / (+) / + / + / + / + / + / (-) / d,L / +
Mannitol / (+) / + / + / (-) / + / + / + / + / (+) / d,L / -
Mannose / + / + / + / - / + / + / + / d / - / +
Melibiose / - / + / + / + / + / +,L / - / +,L / - / (-) / - / +
Raffinose / - / + / + / + / + / + / d / + / d / (-) / - / +
Salicin / - / + / - / (+) / d / - / + / - / - / - / +
Sorbitol / (-) / + / + / - / (-) / - / (-) / - / - / (+) / - / -
Sucrose / - / + / + / + / + / + / + / (-) / - / +
Trehalose / - / + / + / + / + / - / + / - / - / - / +
D-Xylose / d / + / + / + / + / - / + / (+) / - / +
G+C content (mol% range) / 42.7-47.1 / 42.4 / 40-42.9 / 40.9 / 41.8-43.2 / 46.9 / 42 / 41.9 / 43.7 / 40.5

a +, ≥90% of the strains are positive; (+), 80 – 89% of the strains are positive; d, 21 to 79% of the strains are positive; (-), 11-20% of the strains are positive; -, ≤10% of the strains are positive; w, weak reaction; L, late reaction; NT, not tested.

bPositive according to Piechulla et al, and negative according to Sneath and Stevens.

c Results for A. pleuropneumoniais on media supplemented with NAD

d In Hugh-Leifson medium containing glucose, with incubation for 48 h at 37°C.

Actinobacilluslignieresii

This organism is a normal Gram-negative inhabitant of the oral cavity, and is the recognized cause of “wooden tongue” and local infections of the pharyngeal region, nostrils and neck of cattle. In sheep and goats, skin or lung lesions may be found. Drainage from these lesions tends to be non-putrid and watery. Tissues and pus from lesions may contain firm granules 1-3mm in diameter. They represent the so-called “sulphur” granules. Pus diluted with water and swirled around in a Petri dish may show up the small granules. Staining of crushed granules usually reveals masses of Gram-negative bacteria. In histological sections of granulomatous tissues, the masses of organisms are surrounded by Gram-negative club-shaped bodies.

Actinobacilluslignieresii is often difficult to isolate and only a few colonies may be present. It dies rapidly in the laboratory.

Actinobacillusrossii

This organism has been isolated from pigs and is a small Gram-negative rod. It produces smooth, grey colonies on blood agar incubated aerobically and anaerobically at 37 °C, but not at 42°C or at 25°C. It does not grow on MacConkey or Simmon’s citrate agar.

Actinobacillussuis

It is always strongly beta-haemolytic and can be distinguished from A. rossii on biochemical tests.

Actinobacilluspleuropneumonia

Pneumonic lung specimens are usually cultured on blood agar with a S. aureus streak or on chocolate horse blood agar as rare strains are V factor dependent. The selectivity of the medium is enhanced with the addition of bacitracin (500 units/ml), and cloxacillin (5 l/ml). Plates should be incubated for 24 hours aerobically or in an atmosphere of 5-10% CO2 (or a candle jar) at 37 °C. Colonies on chocolate agar are opaque and reach a diameter of 1-2mm within 48 hours. Two types of colonies often are visible: a round, hard, waxy type and a flatter, soft, glistening type. The haemolytic activity of this organism is characteristic of this species. The intensity of the haemolysis varies not only according to the type of erythrocyte used but also with the different serotypes.

Mannitol fermentation and positive urease reaction distinguish A. pleuropneumoniae from other respiratory pathogens of swine such as Haemophilusparasuis

Actinobacillusseminis

Actinobacillusactinomycetum-comitans mentioned in older texts should be considered to be A. seminis.

This organism is a highly pleomorphic, Gram-negative rod that does not grow on MacConkey agar, is not haemolytic, and does not require CO2 for growth. Colonies on blood agar are small (less than 1 mm), clear and glistening.

Actinobacillusseminis should be distinguished from other Gram-negative pleomorphic rods that can be isolated from the semen of rams such as Histophilusovis, Histophilussomniand Haemophilusagni. These three organisms cannot be distinguished on the basis of DNA studies, and should therefore be regarded as the same. They differ from A. seminisby the production of a yellow pigment, enhancement of growth in air with 10% CO2, are catalase negative and indole positive.

Actinobacillusequuli

Bacterial septicaemia in foals usually occurs during the first week of life and incurs a high mortality rate. Various pathogens can cause bacterial septicaemia in foals, also referred to as sleepy foal disease. They include A. equuli, E. coli, Streptococcus equisubsp. zooepidemicus, Salmonella Typhimurium, Klebsiellapneumoniaeand S. aureus.

Mannheimia/ Pasteurella/Gallibacterium

The genus Pasteurella is widely parasitic on mammals and birds. Under conditions of stress, these organisms can become invasive and play a significant role in the pathogenesis of a variety of infections in animals, including pneumonia, sinusitis, abortion, mastitis and septicaemia. Table 5.6 lists the most important species that can be isolated from ruminants, horses, pigs, companion animals, rabbits and birds. Colonies from mammals are small, grey and glistening, or larger, shiny colonies with a brown centre. Poultry strains are tiny beta-haemolytic colonies similar to Streptococcus. Pasteurellaspecies are small, Gram-negative rods or cocco-bacilli. Clinical material should routinely be inoculated on blood agar and MacConkey agar. Colonies of all species are usually visible after 24 hours.

Table 5.6: Important Pasteurellaand Mannheimiaspecies in animals

Species / Hosts / Disease
Mannheimiahaemolytica / Cattle
Sheep
Pigs / Pneumonia
Pneumonia
Gangrenous mastitis “Bluebag”
Septicaemia in lambs under 3 months of age
Pneumonia
B. trehalosi / Sheep / Septicaemia in lamds 5-12 months old
P. multocida type A / Cattle
Sheep/goats
Pigs
Rabbits
Poultry / Pneumonia (primary or secondary)
Occasional but severe mastitis
Pneumonia
Mastitis
Pneumonia (usually secondary)
“Snuffles”, pleuropneumonia, abscesses, otitis media, conjunctivitis and genital infections
Fowl cholera (primary infection)
P. multocida types B and E / Cattle and Water Buffalo / Haemorrhagicsepticaemia (type B in SE Asia and type E in Africa)
Nasopharynx of carrier animals
P. multocida type D / Pigs / Atrophic rhinitis
P. multocida type F / Turkeys mainly / Role as a pathogen is unclear
P. pneumotropica / Dogs and cats
Rodents / Normal nasopharyngeal micro flora
Pneumonia and abscesses
Commensal in the nasopharynx
P. canis / Dogs (humans) / Commensal in oral cavity
From dog bite injuries in humans
P. dagmatis / Dogs and cats / Commensal in oral cavity
From dog bite injuries in humans
P. stomatis / Dogs and cats / Commensal in respiratory tract
P. caballi / Horses / Respiratory infections including pneumonia
P. aerogenes / Pigs / Commensa; in digestive tract
P. anatis / Ducks / Intestinal commensal
P. gallinarum / Poultry / Commensal in respiratory mucosa. Occasional mild respiratory infections.
P. avium, P. langaa & P. volantium / Chickens / Commensal in the respiratory tract
P. testudinis / Turtles, tortoises / Abscessation
P. granulomatis / Cattle / Fibrogranulomatous disease

The differential characteristics of the species belonging to the genus Pasteurella only are given in Table 5.7 and that of Pasteurella and related species in Table 5.8.

Mannheimiahaemolyticaand Pasteurellatrehalosi

Pasteurellahaemolytica biotype A has been divided into the pathogenic Mannheimiahaemolytica, Mannheimiagranulomatis and Mannheimiavariegataand the non-pathogenic Mannheimiaglucosida. The former Pasteurellahaemolytica biotype T has been allocated to a new species known as Pasteurellatrehalosi. Of these,M. haemolytica and P. trehalosihave the greatest veterinary significance. Poultry strains of P. haemolytica retained their name. They are small Gram-positive cocco-bacilli that grow both aerobically and as facultative anaerobes. Mannheimiahaemolytica produces acid in arabinose, and P. trehalosi acid in trehalose. The fermentation reaction should be allowed to continue for up to 10 days. On blood agar, M. haemolytica strains produce small, grey colonies with distinct zones of haemolysis. The larger slightly yellow/brown colonies of P. trehalosi are less uniformly haemolytic.

On the basis of specific antisera, 17 serotypes of M. haemolytica have been identified. Mannheimiahaemolytica includes serotypes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13, 14; 16 and 17 and P. trehalosi includes serotypes 3, 4, 10 & 15. Selected characteristics of M. haemolytica are compared with other respiratory pathogens of cattle in Table 5.9.

Pasteurellamultocida

P. multocidacolonies are non-haemolytic and may be mucoid or non-mucoid in appearance. They do not grow on MacConkey agar. On the basis of their capsule polysaccharides, they have been divided in types A, B, D, E & F. In Africa, only types A, D & E have been described. Haemorrhagicsepticaemia is caused by types B and E.

Pasteurellacaballi

Strains isolated from horses, but not fulfilling the characteristics of any established species within the genus were described in 1989. Subsequent biochemical and genetic studies led to the proposed name of P. caballi. These strains differed from other Pasteurellaspecies in that all were aerogenic and catalase-and oxidase negative, and some strains produced acid from myo-inositol and L-rhamnose. They seem to have a limited host range and have only been isolated from horses. They are sporadically isolated from horses where they are opportunistic pathogens. They also produce gas from glucose, are urease-negative and ferment raffinose (slow), mannitol, maltose, dextrin, glucose and xylose. They utilize ODC and don’t hydrolyze aesculin. Colonies are yellow and non-haemolytic.

P. canisand P. dagmatis

These organisms are part of the normal oral flora of dogs but are potentially pathogenic for humans following bite wounds from dogs. When trying to isolate these bacteria from the buccal cavities of dogs, contaminating bacteria such as Staphylococcus, Streptococcus and Corynebacterium can be inhibited by the addition of 20 mg/litrethiostrepton to the culture media.