Lecture #11: GRAM NEGATIVE CURVED AND FLAGELLATED BACTERIA

 

Gram–Negative Curved and Flagellated Bacteria

A.   With Sheathed Flagella

1.     Helicobacter

2.     Vibrio

3.     Spirillum

B.     Unsheathed Flagella

1.     Campylobacter

2.     Aeromonas

 

THE CAMPYLOBACTER

 

Characteristics:

1.     The genus Campylobacter represents a distinct phylogenetic group within the class Epsilonproteobacteria that includes Arcobacter, Sulfurospirillum, Helicobacter, and Wolinella. 

2.     In 1989, Campylobacter pylori and the other campylobacteria with sheathed flagella became HELICOBACTER and two years later the name ARCOBACTER was adopted for the aerotolerant species.

3.     Campylobacters are Gram-negative, non-spore forming that exist as either curved or spiral shaped rods. In morphological terms, campylobacters are usually S-shaped or spiral rods with tapering ends (0.2–0.8 μm-wide by 0.5–5 μm-long). They commonly possess an unsheathed polar flagellum at one or both ends of the cell and this, presumably aided by its spiral morphology, imparts a high degree of motility to the cell.

4.     This bacterium has quite stringent requirements for its growth. Campylobacter species are microaerophilic, requiring a reduced O₂ concentration of 5%–8% and an elevated CO₂ concentration of 3%–10%. Most relevant species are also thermophilic, growing best among 40–42°C.

5.     They are non-saccharolytic and its catabolic capability is highly restricted. They do not ferment or oxidize carbohydrates neither complex substances. Energy is obtained from amino acids or tricarboxylic acid cycle intermediates.

6.     Campylobacters that are of relevance to the water industry are ascribed to the class of campylobacters known as the “thermophilic group.” This group is composed of Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari. The principal symptom of infection by Campylobacter in humans is acute diarrhea. A blood–free medium (CAT medium) containing Cefoperazone (8 μg/ml), Amphotericin (10 μg/ml), and Teicoplanin (4 μg/ml) has been used in the isolation of thermophilic Campylobacter.

7.     The two species most frequently associated with human disease are Campylobacter jejuni and Campylobacter coli which account for the majority of Campylobacter infections in humans.

8.     Campylobacter survives better under refrigeration temperatures than at room temperature. Freezing causes a significant reduction in organism counts, although it does not necessarily eliminate Campylobacter in meat products, and the organism is rapidly inactivated at normal cooking temperatures.

9.     Campylobacteriosis (Campylobacter infection) is an important foodborne infection. Foods of animal origin, mostly poultry and meat, play an important role in the infection. Barbecues appear to present special hazards for infection, because they permit easy transfer of Campylobacter from raw meats to hands and other foods. Milk is can also contaminated with Campylobacter and the consumption of raw milk can cause campylobacteriosis. Inadequate cooking and the consumption of poorly chlorinated drinking water or unpasteurized milk are other infection sources of Campylobacter. Campylobacter can survive in fresh cheese for only a short period of time. Campylobacter can contaminate shellfish. Campylobacter is sensitive to high temperatures, dry environments, and oxygen.

Laboratory Isolation:

Campylobacter species can be isolated from fecal specimens using a variety of selective media including blood-containing and blood-free formulations.

1.     Blood Based Selective Medium:

a.     Skirrow's medium is effective for isolating Campylobacters from human faeces but is less suitable for animal and environmental specimens owing to the presence of contaminating species. The medium contains Vancomycin, Trimethoprim, Polymyxin and Laked Horse Blood.

b.     Preston Campylobacter Selective Agar that has been formulated to facilitate the isolation of thermophilic Campylobacter species from all types of specimens. The medium is composed of beef extract, peptone, sodium chloride, polymyxin B, rifampicin, trimethoprim lactate, cycloheximide, agar, distilled or deionized water, and lysed horse blood.

c.      Butzler's medium – for the isolation of Campylobacter jejuni from human faeces is described. This medium contains novobiocin, cephazolin, cycloheximide, bacitracin, colistin and defibrinated horse blood.

d.     Blaser medium (aka Campy–thio/Campy–BAP) – a thioglycolate broth with added Vancomycin, Trimethoprim, Polymyxin B and Amphotericin B.

e.     CAMPYAIR – solid medium by adding 1.5% of agar-agar, 1.0% of soluble starch, 0.1% of deoxycholate, 10 μg/L TTC, and 10% of sheep blood. Campylobacter jejuni and Campylobacter coli

2.     Charcoal–based Solid Media

a.     Karmali Campylobacter Agar (+/– Potassium clavulanate or Polymyxin B) – a selective medium used for the isolation and cultivation of Campylobacter jejuni and Campylobacter coli from clinical specimens and food. Contains hemin, sodium pyruvate, cefoperazone, vancomycin, cycloheximide. The hemin component replaces the sodium sulphate in CCDA.

b.     Charcoal Cefoperazone Deoxycholate Agar (CCDA) was developed to replace blood with charcoal, ferrous sulphate and sodium pyruvate which enhances the growth and aerotolerance of Campylobacter species. The medium also contains casein hydrolysate which promotes the growth of Campylobacter lari.

c.      Campy–Cefex – a selective–differential medium for the isolation of Campylobacter jejuni from chicken carcasses. It contains Brucella agar, ferrous sulphate, sodium bisulphite, sodium pyruvate, water, and lysed horse blood.

 

CAMPYLOBACTER FETUS

1.     Campylobacter fetus has been most often recognized as an infectious agent of animals. The primary reservoir of Campylobacter fetus subspecie fetus is the gastrointestinal tracts of cattle and sheep; however, this subspecies can also be isolated from the feces of other animal species. In contrast, the natural niche of Campylobacter fetus subspecie venerealis is the bovine genital tract, where it can cause infection in cows, resulting in infertility or abortion.

2.     Campylobacter fetus infections of pregnant women have been described from early stages in the pregnancy up to a full-term birth. The clinical signs in the mother are fever, sometimes accompanied by diarrhea, but spontaneous abortions, without other clinical signs, have also been reported. In those cases, in which living infants were born, many of those infants suffered from Campylobacter fetus sepsis, frequently leading to meningitis.

3.     Campylobacter fetus comprises two subspecies: Campylobacter fetus subspecies fetus (Cff) and Campylobacter fetus subspecies venerealis (Cfv), which includes the biovar intermedius. The subspecies are genetically very closely related but have different habitats. They are differentiated by the 1% Glycine Tolerance Test, wherein Campylobacter fetus subspecie fetus is glycine–tolerant.

a.     Campylobacter fetus subspecie fetus is a commensal and pathogen of domesticated animals that can be transmitted to humans via contaminated food. The clinical features of human infection can be severe, especially in impaired hosts.

b.     Campylobacter fetus subspecie venerealis is a sexually transmitted pathogen essentially restricted to cattle.

c.      Campylobacter fetus can be either serotypes A, B, or AB based on its different O-antigens (i.e., LPS). Campylobacter fetus subspecie venerealis is always type A, whereas Campylobacter fetus subspecie fetus might be type A, type B, or rarely type AB. The different serotypes correspond also to the different S-layer protein types.

CAMPYLOBACTER JEJUNI

1.     Campylobacter jejuni possesses single, unsheathed polar flagellum at one or both poles of the cell, thus it is a highly motile organism, and the flagella are essential for colonization both in humans and animals. Campylobacter jejuni motility is also required for invasion of epithelial cells.

2.     It is now recognized as a leading cause of acute bacterial gastroenteritis in humans and raw milk has been implicated as a vehicle responsible for transmitting campylobacters to susceptible individuals.

3.     It can also cause post-infective syndromes such as arthritis, liver, or kidney inflammation, and particularly Guillain-Barré syndrome which is characterised by temporary paralysis of the peripheral nervous system.

4.     Campylobacter jejuni strains can be differentiated by using two different serotyping schemes, one based on heat–labile flagellar protein, referred to as Lior serotypes (there are over 100 Lior serotypes), the other based on heat–stable flagellar proteins referred to as Penner serotypes (there are over 60 Penner serotypes).

CAMPYLOBACTER UPSALIENSIS

1.     Campylobacter upsaliensis is gram–negative, microaerophilic, thermotolerant, motile, and curved. The organism has a single polar or bipolar flagellum and exhibits the darting movements characteristic of Campylobacter specie under phase-contrast microscopy. It is common in the feces of cats and dogs, especially in younger animals.

2.     It forms smooth, pinpoint, greyish or translucent colonies on blood agar plates. Swarming may be observed when the organism is grown on moist media. Growth in broth requires supplementation with sheep blood or fetal calf serum. The organisms are 0.3 to 0.4 μm wide and 1.2 to 3 μm long.

3.     Campylobacter upsaliensis is associated with acute self–limiting diarrhea but has also been isolated in the setting of chronic and recurrent diarrhea. Moreover, Campylobacter upsaliensis can cause bacteremia in debilitated and immunocompromised patients and has been associated with extraintestinal infections, spontaneous human abortion, hemolytic–uremic syndrome, and Guillain-Barré syndrome.

4.     This bacterium is oxidase positive, nitrate positive, and hippurate negative. Campylobacter upsaliensis is sensitive to nalidixic acid and, usually, to cephalothin. The presence of these antibiotics in the selective media generally used for the isolation of Campylobacter species (e.g., Skirrow’s medium) may well account for the suboptimal identification of Campylobacter upsaliensis in clinical specimens at most centers.

5.     The most useful biochemical tests for the identification of Campylobacter upsaliensis in the clinical microbiology laboratory include those for catalase production, hippurate hydrolysis, nitrate reduction, oxidase activity, H₂S production on triple sugar iron agar, and sensitivity to nalidixic acid.

CAMPYLOBACTER LARI

1.     Campylobacter lari (previously Campylobacter laridis) is the third most common species obtained from humans with gastroenteritis. Reactive arthritis may be a complication following enteritis.

2.     Because of its resistance to Nalidixic acid antibiotic Campylobacter lari was originally referred to as Nalidixic Acid-Resistant Thermophilic Campylobacter (NARTC) group.

3.     Useful tests to distinguish Campylobacter lari from other Campylobacter species include resistance to nalidixic acid, demonstration of anaerobic growth in the presence of trimethylamine-N-oxide, susceptibility to triphenyl tetrazolium chloride, hydrolysis of indoxyl acetate, and the absence of hippurate hydrolysis.

CAMPYLOBACTER RECTUS

1.     Campylobacter rectus is one of the anaerobic bacteria in the mouth, which was previously known as Wolinella recta. It was identified as a common pathogen closely related to human periodontal disease in 1979. In 1984, the first case of Campylobacter rectus infection outside the oral was reported. Then, it was reassigned to the genus Campylobacter in 1991.

2.     Campylobacter rectus is gram–negative, with no spores and can be cultured in microaerobic or anaerobic state. Its colonies are translucent, rough, flat, and non–hemolytic. The morphology of Campylobacter rectus is straight rod-shaped, arcuate, or S shape. Urease and oxidase tests are both negative.

3.     It is a member of the human oral flora and has been found in areas such as the periodontal sulcus, tongue, cheek mucosa, and saliva. To date, five other Campylobacter species (Campylobacter concisus, Campylobacter curvus, Campylobacter gracilis, Campylobacter showae, and Campylobacter sputorum) have been isolated from the human oral cavity. Campylobacter rectus is associated with human periodontal disease, being present in higher numbers in diseased than in healthy subgingival sites.

4.     Membrane filtration, a passive filtration culture method (the motile campylobacters pass through the filter to blood agar), with incubation in hydrogen-enriched microaerophilic atmosphere, also known as the “Cape Town protocol,” proved successful in the isolation of several of the “emerging” species such as Campylobacter rectus as well as Campylobacter concisus, Campylobacter sputorum, and Campylobacter curvus, the prevalence of which is underestimated where traditional methods are used.

THE ARCOBACTER

1.     Arcobacter are gram–negative, motile, aerotolerant campylobacter like microbes that grow at 30°C. Of the 10 described species, Arcobacter butzleri causes human foodborne bacterial gastroenteritis, is acknowledged as a significant zoonotic pathogen, and is closely related to Campylobacter jejuni.

2.     Some Arcobacter species (particularly, Arcobacter butzleri, Arcobacter skirrowii, and Arcobacter cryaerophilus) are associated with various diseases in humans and animals, their exact epidemiological and pathological role is not completely understood, and few cases of human infection are reported.

3.     The key distinguishing features of the genus Arcobacter differentiating them from Campylobacter are the ability to grow at 15°C but not at 42°C; the ability to optimally grow aerobically at 30 °C; G + C content of 27-30 mol% and methyl-substituted menaquinone-6 not present as a major isoprenoid quinone.

 

THE HELICOBACTER

1.     They are gram–negative curved or spiral rod distinguished by multiple, sheathed flagella and abundant urease. The release of urease by the gastric Helicobacter species, such as Helicobacter pylori, generates ammonia to neutralize the gastric mucosal niche to pH of 6–7 and may be a survival mechanism. In addition, alteration of surface lipopolysaccharides and proteins as well as surface phase changes to evade the host immune response favor colonization in the host.

2.     Most Helicobacter specie has a sheathed flagellum except Helicobacter canadensis, Helicobacter mesocricetorum, Helicobacter pullorum, Helicobacter winghamensis, and Helicobacter rodentium.

3.     Helicobacter species can be separated into gastric and enterohepatic groups.

4.     Enterohepatic Helicobacters (EHH) other than Helicobacter pylori colonize the bowel, biliary tree and liver of animals and human beings with pathogenic potential. They are classified into two groups, the first comprising of flagellated bacteria which in turn have periplasmic cilia. The second subgroup lacks these and is structurally similar to the microorganisms of the Campylobacter genus.

HELICOBACTER IN HUMAN INFECTION
Specie	No. of flagella	Sheathed Flagella	Catalase	Urease	42oC	Periplasmic Cilia
Helicobacter pullorum	Unipolar	–	+	–	+	–
Helicobacter winghamensis	Bipolar	–	–	–	–	–
Helicobacter bilis	Bipolar	+	+	+	+	+
Helicobacter hepaticus	Bipolar	+	+	+	–	–
Helicobacter cinaedi	Unipolar	+	+	–	–	–
Helicobacter fennelliae	Bipolar	+	+	–	–	–
Helicobacter canis	Bipolar	+	–	–	+	–

 

HELICOBACTER PYLORI

1.     Helicobacter pylori is a 2- to 6-µm long, 0.5-µm wide, gram-negative organism that has a surface flagellar sheath; the sheath is thought to provide acid resistance in its typical acidic gastric environment, along with the ureases the organism produces.

2.     It is clearly identified as the strongest risk factor for gastric cancer, the fourth most common malignancy and the second leading cause of cancer-related mortality worldwide.

3.     Significant evidence has emerged implicating the bacteria in the etiology of some cancer types such as Helicobacter pylori in non-cardia gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphomas, Chlamydia trachomatis in cervical cancer and Salmonella typhi in gallbladder cancer.

4.     In the routine clinical diagnostics, the urease test, histological examination, urea breath test, serology, bacterial culture, and stool antigen test are valuable methods of detecting Helicobacter pylori infection.

HELICOBACTER BILIS

1.     Helicobacter bilis (previously identified as Flexispira rappini, Helicobacter rappini, and Helicobacter specie Flexispira-taxa 2, 5, and 8) is an opportunistic, nonsporulating, and fusiform bacterium that has a fusiform body with 3–14 bipolar flagella and periplasmic fibers wrapped around the cell. It is a potentially zoonotic, weakly gram-negative, microaerophilic, motile, bacterium that may colonize human, dog, cat, and rodent intestine.

2.     Helicobacter bilis and Helicobacter pylori have been isolated from human bile samples of more than 75% of patients with gallbladder cancer and more than 50% of patients with chronic cholecystitis. It also causes inflammatory bowel disease (IBD).

HELICOBACTER CANIS

1.     They are gram-negative, non-spore-forming, helically curved and slender rod-shaped cells; typically, 0.25 x 4 µm. Cells have one to two spiral turns and carry single bipolar sheathed flagella. Exhibits darting motility in hanging-drop preparations of broth cultures.

2.     The colonies are pinpoint, non-pigmented, translucent, and α-haemolytic after 48 h on blood agar. Microaerophilic. No growth under aerobic or anaerobic conditions. No growth at 25°C, but growth at 37°C and 42°C (thermotolerant).

3.     Tolerant to 1.5 % bile, but not to safranin ‘O’. Resistant to polymyxin B and sensitive to nalidixic acid. The G-t-C content of genomic DNA by thermal denaturation is 48mol%.

HELICOBACTER FENNELLIAE

1.     Helicobacter fennelliae is a gram-negative, spiral bacillus that appears as thin-spread colonies on sheep blood agar and is similar to Helicobacter cinaedi. This organism is fastidious and difficult to culture.

2.     They demonstrate some biochemical differences from other Helicobacter species, such as lacking urease activity and being catalase-positive, nitrate-negative, indoxyl acetate hydrolysis-positive, alkaline phosphatase-positive, and gamma-glutamyl transpeptidase-negative.

3.     Nitrate reduction and alkaline phosphatase hydrolysis tests are useful techniques for diagnosing Helicobacter fennelliae in general laboratories from Helicobacter cinaedi.

HELICOBACTER PULLORUM

1.     Helicobacter pullorum commonly colonizes the gastrointestinal tract of poultry causing gastroenteritis. The bacterium may be transmitted to humans through contaminated meat where it has been associated with colitis and hepatitis.

2.     It is the only Helicobacter specie with unsheathed single polar flagellum.

3.     It is a motile, non-spore forming, microaerophilic bacterium, which best grows at 37 to 42°C.

4.     It produces catalase, reduces nitrates, but lacks urease, indoxyl acetate esterase, or alkaline phosphatase activity.

HELICOBACTER WINGHAMENSIS

1.     It is a gram-negative, slightly curved to spiral, non-spore-forming bacillus. The organism is approximately 2 μm in length by 0.3 to 0.6 μm in width, and it is motile by one or two bipolar, unsheathed flagella.

2.     Cultures grow on solid agar media supplemented with 10% sheep blood and exhibit a seemingly diverse colonial morphology of non-spreading and spreading colonies. The organisms grow in a microaerobic atmosphere at 37°C but fail to grow at 42°C or in aerobic or anaerobic atmospheres.

3.     This Helicobacter is oxidase and indoxyl acetate positive, tolerates 1% bile, and is alkaline phosphatase, catalase, and urease negative. It does not reduce nitrate. All isolates induced similar symptoms of gastroenteritis in humans, and these included general stomach malaise, vomiting, diarrhea, cramping, and mild fever.

4.     The absence of a flagellar sheath, uncharacteristic of most helicobacters, is shared by four other Helicobacter species: Helicobacter canadensis, Helicobacter mesocricetorum, Helicobacter pullorum, and Helicobacter rodentium.

HELICOBACTER CINAEDI

1.     Helicobacter cinaedi was first isolated from rectal cultures from homosexual men in 1984. It is formerly known as Campylobacter cinaedi and has been associated with proctocolitis, cellulitis, and bacteremia.

2.     It is primarily recovered from immunocompromised individuals; the organism is also recovered from chronic alcoholics as well as immunocompetent men and women.

3.     They exhibit pinpoint growth after 2 days of incubation, with flat, spreading colonies evident after 4 days of incubation.

THE VIBRIO

1.     Vibrios are curved gram-negative γ-proteobacteria with a single polar flagellum. They are ubiquitous and often highly motile in aqueous environments. Vibrio swimming motility is driven by a polar flagellum covered with a membranous sheath.

2.     Vibriosis is the common infection associated with some Vibrio specie by consuming raw or undercooked shellfish and seafood.

3.     Vibrios use sodium ions as the energy source for flagellar rotation.

4.     The medically important Vibrio species are divided into two groups:

a.     Choleragenic – such as Vibrio cholerae

b.     Non–choleragenic – such as Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cincinnatiensis, Vibrio fluvialis, Vibrio harveyi, and Vibrio metschnikovii. Additionally, Photobacterium damsela, subspecies damselae, and Grimontia hollisae formerly were included in the Vibrio genus (Vibrio damselae and Vibrio hollisae, respectively) and occasionally have been associated with human foodborne illness.

5.     Several molecular methods have been developed for the typing of Vibrio species and these include Restriction Fragment Length Analysis (RFLP) for virulence or virulence associated genes, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC) PCR, Ribotyping and Pulsed-Field Gel Electrophoresis (PFGE).

PROTEOLYTIC ENZYMES OF VIBRIOS
Species	Metalloprotease		Serine Protease
	Vibriolysin	Collagenase	Chymotrypsine –like protease
V. cholerae	+	–	–
V. fluvialis	+	–	–
V. mimiscus	+	–	–
V. vulnificus	+	–	+
V. metschnikovii	–	–	+
V. parahaemolyticus	–	+	+
V. alginolyticus	–	+	+

VIBRIO CHOLERAE

1.     Cholera is an acute diarrheal illness caused by infection of the intestine. Patients with cholera present with severe, voluminous watery (“rice-water”) diarrhea, abdominal cramps, nausea, and vomiting leading to serious electrolyte depletion, dehydration, acidosis, shock, and, if left untreated, to death.

2.     The most common time for Vibrio infections occur in the warmer months from May through October.

3.     The O antigens of Vibrio cholerae are heat-stable, shows enormous serological diversity and have proven to be valuable markers in both ecological and epidemiological studies. Of approximately 200 currently known O-antigen serogroups just two serogroups, O1 and O139, are associated with cholera. All strains identified as Vibrio cholerae based on biochemical tests but do not agglutinate with O1 or O139 antisera are collectively referred to as the “non-O1 non-O139” or also referred to as non-epidemic serogroups.

4.     The pathogenicity of Vibrio cholerae O1 strains is due to two virulence factors: cholera toxin (CT), an enterotoxin, and toxin-coregulated pilus (TCP), an intestinal colonization factor. The non–pandemic Vibrio cholerae (non-O1, non-O139) strains cause disease through a type III secretion system, and these strains lack genes encoding CT and TCP.

Virulence factor of Vibrio cholerae O1 Strain

1.     Cholera toxin (CTX) is the main virulence factor in toxigenic strains of Vibrio cholerae. It is responsible for the secretory diarrhea characteristic of cholera. It is secreted through the type II secretion system (T2S) and as cargo within outer membrane vesicles (OMVs).

2.     Toxin–coregulated pilus (TCP) is a type IV pilus with structural similarities to the T2SS. Bacterial aggregation in the form of microcolonies through pilus-pilus interaction with TCP is required to colonize the human intestine.

 

Laboratory isolation:

1.     Thiosulfate Citrate Bile-Salts Sucrose (TCBS) agar – colonies appear as translucent, flat, yellow colonies with elevated center.

2.     Tellurite Taurocholate Gelatin Agar (TTGA) – also known as Monsur medium. Colonies appear as colorless with a characteristic dark center after two days growth, surrounded by a halo, which appears due to the hydrolysis of gelatin.

3.     CHROMagar Vibrio – colonies appear as turquoise.

4.     Polymyxin Mannose Tellurite (PMT) – a selective and differential agar medium to easily differentiate colonies of Vibrio cholerae O1 from those of Vibrio cholerae non-O1. The differentiation between colonies of the two vibrios is based on mannose–fermentation. Colonies of V. cholerae O1 on the agar are agglutinated with O1 antiserum of Vibrio cholerae.

5.     Alkaline peptone water (APW) remains the recommended enrichment medium for vibrios in parallel with either Salt Polymyxin Broth (SPB) or Glucose teepol (or sodium dodecylsulphate) salt broth (GTSB) when tests for Vibrio parahaemolyticus are required.

Serogroups of Cholera

The serogroup is determined by the cell wall of the bacteria; the test for confirming the serotype is agglutination with a specific antiserum.

1.     Type O1 (Epidemic Cholera)

a.     Serotype – differ from one another only with respect to antigenic determinants on the O side chain of the lipopolysaccharide (LPS) antigen.

(1)   Ogawa – has antigenic determinants identified as A and B. The terminal sugar in the perosamine residue is a 2-O-methyl group.

(2)   Inaba – has antigenic determinants identified as A and C. The terminal sugar in the perosamine residue is the hydroxyl group.

(3)   Hiroshima – has antigenic determinants identified as A, B and C.

b.     Biotype – have distinct phenotypes and differ with respect to the severity of the disease they can cause, the ability to survive outside the human host, and the seasonal pattern of infection.

DIFFERENTIATING CHARACTERISTICS	EL TOR Biotype	CLASSICAL Biotype
Chicken Cell Agglutination	+	–
Hemolysis of Sheep Erythrocytes	+	–
Voges–Proskauer Test	+	–
Phage IV susceptibility	–	+
Polymyxin B susceptibility	–	+

2.     Type O139 or Bengal Type (Cholera gravis)

The structure of the O139 capsule is composed of one residue each of N-acetylglucosamine, N-acetylquinovosamine, galacturonic acid and galactose and 2 molecules of 3,6-dideoxyxylohexose. LPS of O139 contains colitose, glucose, l-glycero-d-manno-heptose, fructose, glucosamine and quinovosamine in its polysaccharide. Absence of perosamine.

VIBRIO PARAHAEMOLYTICUS

1.     Vibrio parahaemolyticus is a halophilic Vibrio species. It usually causes a self-limited, 72-hour, cholera-like gastroenteritis but some strains can penetrate the lamina propria, resulting in dysentery that resembles shigellosis.

2.     The Thermostable Direct Hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity as well as cardiotoxicity, and enterotoxicity.

3.     Their colonies were round, translucent, and purplish red on CHROMagar plates measuring 2–3 mm in diameter. They were round, translucent, and smooth green-colored colonies on TCBS plates.

4.     Vibrio parahaemolyticus synthesizes three major surface antigens: heat-stable somatic O antigen, heat-labile capsular K antigens and H flagellar antigens.

5.     It has dual flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum propels the swimmer cell in liquid environments. Numerous unsheathed lateral flagella move the swarmer cell over surfaces. The polar flagellum is produced continuously, whereas the synthesis of lateral flagella is induced under conditions that impede the function of the polar flagellum, e.g., in viscous environments or on surfaces.

VIBRIO VULNIFICUS

1.     Vibrio vulnificus is a gram–negative, halophilic, highly invasive pathogen associated with the consumption of raw or undercooked oysters and shellfish. This opportunistic pathogen is known to cause septicemia which progresses rapidly after an incubation period of approximately 26 hours with reported lethality rate of 50%.

2.     The vibriolysin of Vibrio vulnificus is a major toxic factor eliciting the secondary skin damage characterized by formation of the hemorrhagic brae. The vibriolysin from intestinal pathogens may play indirect roles in pathogenicity because it can activate protein toxins and hemagglutinin by the limited proteolysis and can affect the bacterial attachment to or detachment from the intestinal surface by degradation of the mucus layer.

3.     The preferred habitat of Vibrio vulnificus has been reported to be water temperature more than 18°C with salinities between 15 and 25 parts per thousand (ppt).

4.     Its concentration in water and shellfish show a strong seasonality, with greatest abundances observed at temperatures >20°C. At temperatures below 13°C it enters a dormant state, in which the cells cannot be grown on media; this stage is called viable-but-not-culturable (VNBC).

5.     Three biotypes (or two biotypes and one distinct serovar) of Vibrio vulnificus are currently recognized. The majority of clinical and environmental Vibrio vulnificus isolates reported to date are in Biotype 1. Strains initially classified as biotype 2 are responsible for sepsis in eels; they do not cause human disease. Biotype 3 strains have been described in association with wound infections related to handling of live fish (tilapia) from fish farms in Israel.

6.     A species-specific flagellar (H) antigen, which groups members of Vibrio vulnificus into a single serotype, has been exploited in the rapid identification of this pathogen.

7.     Sodium Dodecyl Sulphate Polymyxin Sucrose Agar Base is used for enrichment, isolation, and enumeration of Vibrio vulnificus from seafood samples.

VIBRIO FLUVIALIS

1.     Vibrio fluvialis, a halophilic Vibrio species (grow well in the presence of 7% NaCl), is found to be associated with acute diarrheal illness in humans, food poisoning due to consumption of contaminated raw shellfish, and extraintestinal infection.

2.     It is easily confused with Aeromonas hydrophila but can be differentiated by growth on media containing 6% sodium chloride. Patients experience diarrhea, abdominal pain, fever, and dehydration. Low numbers of Vibrio fluvialis can be isolated from fish and shellfish, and from warm seawater. Strains of Vibrio fluvialis express El Tor haemolysin, and a vacuolating toxin acting on HeLa cells, but the role of these in pathogenesis has not been demonstrated.

THE SPIRILLUM

Spirillum is microbiologically characterized as a gram-negative, motile helical cell with tufts of whip like flagella at each end. The helix of the largest spirillum, Spirillum volutans, is 5–8 μm across and 60 μm long.

Spirillum organisms can be identified in blood or biopsies from a lesion or adjacent lymph nodes.

Both Spirillum and Spirochete are spiral bacteria with spirillum being rigid with external flagella, and spirochetes being flexible with internal flagella.

SPIRILLUM VOLUTANS

1.     Spirillum volutans is a large, helical bacterium that occurs in stagnant freshwater sources. They have tufts of polar flagella (around 50 flagella at each pole).

2.     Even during enrichment, it is vastly outnumbered by other bacteria. For this reason and because it fails to form colonies on solid media, Spirillum volutans has been isolated only by used of a capillary tube method which allows it to outswim contaminants.

3.     Spirillum volutans grows only under microaerobic conditions in a Peptone-Succinate-Salts broth with the addition of potassium metabisulfite, norepinephrine, catalase, or superoxide dismutase (SOD). A combination of catalase and SOD had a synergistic effect on hydrogen peroxide. Spirillum volutans lacked catalase and had only a low level of peroxidase activity but did possess SOD activity (12 to 14 U/mg of protein).

4.     They are obligate microaerophile but can be maintained through weekly transfer in semi-solid (0.15% agar) like Modified-Peptone-Succinate-Salts medium (MPSS) under aerobic conditions with incubation and storage at 30^oC.

SPIRILLUM MINUS

1.     Spirillum minus is a short, thick, gram-negative, tightly coiled spiral rod measuring 0.2 to 0.5 µm by 3 to 5 µm. The organism has two to six regular helical turns. Terminal polytrichous flagella confer darting motility, which can be demonstrated with darkfield examination. The flagella can be stained with silver impregnation methods (e.g., Fontana-Tribondeau). Spirillum minus has not been cultured on artificial media, and its name derives from its appearance alone. No attempt at sequence analysis of the organism in body fluids has been reported.

2.     Spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy.

3.     Spirillum minus rat-bite fever is also called sodoku: “so” means “rat” and “doku” means “poison” in Japanese. Infection results from inoculation during the bite of a rat. Up to one-fourth of rats are colonized with Spirillum minus in sputum, conjunctiva, blood, or nasopharynx.

4.     The disease is asymptomatic in rodents. Humans develop a distinctive rash of red to purple-colored plaques. Compared to Streptobacillus moniliformis, arthritis rarely develops due to infection with this organism.

THE AEROMONAS

1.     Members of the genus Aeromonas, informally called “aeromonads,” are common in ponds, lakes, and soils. They are coccoid or straight rods with rounded ends; most are motile by means of unsheathed polar flagella. When growing anaerobically, they reduce nitrate to nitrite.

2.     Aeromonads are gram–negative, oxidase–positive, catalase–positive, facultatively anaerobic bacteria, resistant to novobiocin, are mostly arginine–dihydrolase–positive and produce DNAase, amylase, lipase, gas from glucose and acid from maltose, fructose, and trehalose. Aeromonas is conventionally distinguished from vibrios because Aeromonas species are unable to grow in the presence of 6% NaCl and are resistant to the vibriostatic agent, 2,4-diamino-6,7-diisopropyl-pteridine (O129).

3.     The family Vibrionaceae proposed by Véron in 1965 was initially composed of three genera (Vibrio, Aeromonas, Plesiomonas) that had several features in common, including ecologic habitats (freshwater, marine), similar disease syndromes (gastroenteritis, wound infections), and phenotypic features (oxidase positivity, facultatively anaerobic). It was not until years later that the phylogenetic investigations clearly demonstrated that Aeromonads belonged in their own family.

4.     Among species isolated from humans, >90% of strains produce β-hemolysis on sheep blood agar, except for Aeromonas popoffii and Aeromonas trota. Rare strains of Aeromonas caviae hydrolyze urea, a characteristic presumed to be negative among aeromonads. Earlier studies have indicated that three Aeromonas genomospecies (Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii biovar sobria) are responsible for the vast majority (≥85%) of human infections and clinical isolations attributed to this genus.

5.     Aeromonas produces biofilms, which are regulated by quorum sensing. Once established in the gastrointestinal tract, aeromonads can apparently produce diarrhea by elaboration of enterotoxigenic molecules, causing enteritis, or by invasion of the gastrointestinal epithelium, producing dysentery or colitis.

6.     Aeromonas are divided into two groups namely the non-motile psychrophilic aeromonads and the motile mesophilic aeromonads.

7.     Studies demonstrate that aeromonads belonging to serogroups O:11, O:16, O:18, and O:34 (Sakazaki and Shimada scheme) are associated with most cases of bacteremia, implying that lipopolysaccharide (LPS) antigens and architecture are important in systemic disease pathogenesis.

8.     Aeromonas can also generate a range of extracellular toxins, such as aerolysin and endotoxins. Two types of hemolysins are defined in Aeromonas, α and β, with physiological and functional differences but which can form pores in the membrane of the target cell generating their osmotic lysis.

9.     Aeromonas, like many other pathogenic bacteria, secrete lipases to the medium that acts as hydrolases on membrane lipids. These can provide nutrients or constitute virulence factors when interacting with human leukocytes or by affecting various functions of the immune system. An important lipase in the genus Aeromonas is glycerolphospholipid: cholesterol acyltransferases (GCAT), which could digest the membranes of erythrocytes and produce their lysis.

10.  Aeromonas has been shown to be a significant cause of infections associated with natural disasters (hurricanes, tsunamis, and earthquakes) and has been linked to emerging or new illnesses, including near-drowning events, prostatitis, and hemolytic-uremic syndrome.

Classification of Aeromonas by Moeller decarboxylase and dihydolase reaction pattern:

1.     Group 1 – positive for Ornithine decarboxylase (ODC) only

a.     Aeromonas veronii biovar veronii

b.     Aeromonas allosaccharophila

2.     Group 2 – positive for Lysine Decarboxylase (LDC) but positive or variable to Arginine dihydrolase (ADH)

a.     Aeromonas hydrophila

b.     Aeromonas veronii biovar sobria

c.      Aeromonas jandaei

d.     Aeromonas schubertii

e.     Aeromonas trota

f.      Aeromonas allosaccharophila

3.     Group 3 – negative for Lysine Decarboxylase (LDC), Ornithine decarboxylase (ODC) and Arginine dihydrolase (ADH)

a.     Aeromonas caviae

b.     Aeromonas media

c.      Aeromonas eucrenophila

4.     Group 4 – positive for Arginine dihydrolase (ADH) only

a.     Aeromonas bestiarum

b.     Aeromonas caviae

c.      Aeromonas media

d.     Aeromonas eucrenophila

e.     Aeromonas encheleia

f.      Aeromonas popoffii

5.     Group 5 – variable to Lysine Decarboxylase (LDC) and Arginine dihydrolase (ADH)

a.     Aeromonas bestiarum

b.     Aeromonas salmonicida

Laboratory Isolation:

1.     Pril–Ampicillin–Dextrin–Ethanol (PADE) agar – these medium employs dextrin as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (a) inhibition of the swarming of Proteus, (b) good reduction of the background, (c) inhibition of the over growth of Klebsiella species, (d) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (e) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.

2.     Ampicillin Dextrin Agar (ADA) – a medium for the enumeration of Aeromonas species in water by membrane filtration

3.     Starch Glutamate Ampicillin Penicillin C–glucose agar (SGAP–10C) – the addition of 10 mg/l of C–glucose prevents the growth of Pseudomonas from the medium.

4.     Starch–Ampicillin Agar (SAA) – is often used as an isolation medium for recovery of aeromonads from foods. SAA is composed of beef extract, proteose peptone, sodium chloride, phenol red, agar, soluble starch, reagent grade, ampicillin, and distilled or deionized water. It is found that yellow to honey-colored colonies, 2–3 mm in diameter, and surrounded by a light halo are considered as Aeromonas specie.

5.     Cary–Blair Transport Medium at room temperature yields the greatest recovery of Aeromonads.

AEROMONAS HYDROPHILA

1.     Aeromonas hydrophila is a nonspore-forming, Gram-negative, pleomorphic bacillus with a monotrichous flagellum. It is a fermentative, oxidase-positive, facultative anaerobe frequently found in fresh water and sewage.

2.     Aeromonas hydrophila pathogenicity and virulence are dependent on its ability to produce components related to gastroenteritis. Endotoxins, exotoxins, siderophores, cytotoxins, adhesins, invasins, S-layers and flagella are examples of these properties.

3.     It can be found in virtually all meat chillers but is responsible for only a small number of food-poisoning incidents. The gastroenteritis that it causes in immunocompromised people, however, can be severe. The incubation period is between 12 and 48 hours and illness are caused by heat-labile enterotoxins. It grows at a wide range of temperatures and can grow at temperatures as low as 0°C. The infective dose is not yet fully known. The organism is heat– sensitive and proper pasteurization of meat products reaching 70°C safely eliminates it.

4.     It possesses a number of characteristics of concern in relation to MAP prepared vegetables. It is a psychrotroph that grows slowly at 0°C, but temperatures of 4–5°C will support growth in foods. It is also a facultative anaerobe, capable of growing in atmospheres containing low concentrations of oxygen.

Other selective media:

1.     Rippey Cabelli agar - for differential and selective isolation of Aeromonas hydrophila species from water samples using membrane filter technique.

2.     ASBA30 – a sheep blood agar with 30 mg of ampicillin per liter where they produce grayish hemolytic colonies.

3.     Aeromonas hydrophila (AH) medium has been used to identify aeromonads. This test uses the ability of most aeromonads to ferment mannitol but not to decarboxylate ornithine to produce a yellow-grey butt and a purple band near the agar surface. In addition, some isolates may produce hydrogen sulfide resulting in a blackening of the medium, motility can be observed, and an indole reaction can be performed on the same tube.