Lecture #15: GRAM-NEGATIVE OBLIGATE INTRACELLULAR BACTERIA

 

Gram–negative Obligate Intracellular

1.     Rickettsia

2.     Anaplasma

3.     Ehrlichia

RICKETTSIAE

 

General characteristics:

1.  They are small, non–motile, gram negative, pleomorphic coccobacilli.

2.  They are obligate intracellular parasites and can be cultured only on guinea pigs or mice, chick embryo or tissue culture except for Rochalimea quintana (now Bartonella quintana) which can be grown on media containing high concentration of hemin and serum.

3.  They multiply in the intestinal tract of the arthropod then excreted in the feces or in salivary gland.

4.  Rickettsial growths are enhanced by sulfonamides but para–aminobenzoic acid (PABA) inhibits their growth.

5.  They survive pasteurization at 60^oC for 30 minutes and preserved by means of lyophilization.

6.  All grow intracellular as colonies or clusters in the cytoplasm of tissue cells except spotted fever group which grow both in the cytoplasm and nucleus.

7.  Coxiella burnetii is unique among rickets due to its resistance to heat, drying and chemical agents.

8.  Classification is based on:

a.     Anthropod vector

b.     Specificity of the antibody formed

c.      Intracellular localization

d.     Clinical manifestations

e.     Disease produced in experimental animals

Classification of Rickettsiae:

1.  According to clinical manifestation

Rickettsial disease are characterized by fever and rash except for Q–fever (no rash) and enlargement of spleen and liver.

a.  Typhus group

(1)   Epidemic typhus – initial stage

(2)   Brill Zinnser disease – a recrudescence of past or old infection

(3)   Endemic typhus – a milder form of epidemic typhus

b.  Spotted fever group

(1)   Rickettsialpox – rash resembles Varicella and produces a black eschar known also as Russian vesicular.

(2)   Rocky Mountain Spotted Fever (RMSF)

(3)   Mediterranean Fever / Kenya Fever / Marseilles Fever

(4)   North Asian Tick typhus

(5)   Queensland Tick typhus 

c.  Scrub typhus – clinically resembles epidemic typhus and presence of eschar.

d. Q–fever – resembles influenza, non–bacterial pneumonia and hepatitis.

e.  Trench fever – presence of raseolar rash

f.   SENLAT (Scalp Eschar and Neck Lymphadenopathy) – the old name of this disease is tick–borne lymphadenitis (TIBOLA) or Dermacentor–borne necrosis erythema lymphadenopathy (DEBONEL). Recently, the spectrum of the causative agents of SENLAT was extended, and several reports demonstrated that this syndrome can be caused by Borrelia burgdorferi, Bartonella henselae, Coxiella burnetii, Rickettisia rioja, and Francisella tularensis, all transmitted by the same ticks.

2.     According to intracellular localization in man

a.     Intranuclear – spotted fever group

b.     Intracytoplasmic – typhus fever group

3.     According to anthropod vector

Anthropod	Disease	Causative Agent
Pediculus humanus	Epidemic typus	R. prowazekii
	Trench Fever	R. quintana
Xenopsylla cheopis	Endemic typhus	R. typhi
Leptotrombidium deliense	Scrub typhus	R.tsutsugamushi (O. tsutsugamushi)
Leptotrombidium akamushi
Liponyssoides sanguineus	Rickettsialpox	R. akari
Dermacentor variabilis	Rocky Mountain Spotted Fever (RMSF)	R. ricketsii
Dermacentor andersonii
Rhipicephalus sanguineus	South–African	R. conorii
Dermacentor spp	North Asian	R. sibirica
Ixodes holocyclus	Queensland	R. australis
Ixodes tasmani
Amblyomma variegatum	African Tick Bite Fever	R. africae
Dermacentor marginatus	Scalp Eschar and Neck Lymphadenopathy (SENLAT)	R. slovaca
Dermacentor reticulatus		R. raoultii

Rickettsemia – associated with all rickettsial infection so blood is the principal source of organism.

Laboratory diagnosis

1.  Stained smear (using Macchiavello or Giemsa or Gimenez stain)

a.  Rickettsiae in yolk sacs are not stained well by the Macchiavello technique because the citric acid destaining step was not effective and that most of the basic fuchsin was lost from the rickettsiae during the application of methylene blue, another basic dye. A staining technique was then evolved with carbol basic fuchsin in pH 7.45 phosphate buffer (0.4% dye, 0.4% phenol, 0.07 M buffer), followed directly by 0.8% aqueous malachite green oxalate. This technique worked well for R. mooseri, R. prowazeki, R. rickettsii, R. akari, and R. buretii, but for R. tsutsugumushi a modification was needed, whereby 4% aqueous Fe(NO₃)₃·9H₂O (Ferric nitrate nonahydrate) was used as destaining solution, and 0.5% aqueous fast green as the counter-stain.

2.  Indirect Immunofluorescence Antibody

3.  Indirect Immunoperoxidase assays

4.  Weil–Felix test – a diagnostic serological test to detect rickettsia infection using Proteus OX strains. Serum of the patient is collected for this test to determine if the patient has had inapparent infection. The Weil-Felix test, based on cross-reactive antigens (OXK, OX19 & OX2) of Proteus vulgaris, is nonspecific and insensitive and has generally been abandoned where better tests are available.

EHRLICHIA AND ANAPLASMA

Ehrlichia is a genus of Rickettsiales bacteria that are transmitted to vertebrates by a lone star tick, Ambylomma Americanum.

1.  Ehrlichia chaffeensis is a tick-borne Gram-negative obligatory intracellular bacterium transmitted to humans through the bite of tick. Infection causes severe flu-like febrile disease called Human Monocytic Ehrlichiosis (HME), which is often accompanied by hematologic abnormalities and signs similar to those of hepatitis. Ehrlichia chaffeensis replicates within monocytes and macrophages, which are primary immune cells that recognize pathogen-associated molecular patterns (PAMPs) to unleash potent innate antimicrobial defenses.

2.  Ehrlichia ewingii is the Causal Agent of Canine (CGE) and Human (HGE) Granulocytic Ehrlichiosis. CGE causes moderately severe disease and polyarthritis in chronically infected dogs.

3.  Ehrlichia sennetsu (now known as Neorickettsia sennetsu)- is the causal agent of Sennetsu Fever with symptoms that may include a sudden high fever, headache, and muscle aches (myalgia) within a few weeks after initial infection.

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophilum) is an obligate gram-negative, intracellular bacterium that causes an acute febrile illness known as anaplasmosis or Human Granulocytic Anaplasmosis (HGA). In 2001, the organism was reassigned to the genus Anaplasma. The disease is transmitted to humans by tick bites primarily from the eastern black-legged tick (Ixodes scapularis) or the western black-legged tick (Ixodes pacificus). The main reservoir is the white-footed mouse, Peromyscus leucopus. Anaplasmosis is more frequent in men over the age of 40 years. Men are at higher risk of getting infection compared to women.

CHLAMYDIA OR BEDSONIA

General characteristics:

1.  All Chlamydiae exhibit the same morphologic feature, share a common antigen and multiply in the cytoplasm of their host by a developmental cycle. The mucosal epithelial cells are the primary target cell type in vivo. For most Chlaymydia trachomatis strains, infection is limited to the mucous membrane, although lymphogranuloma venereum (LGV) strains attack deep lymphatic tissues.

2.  They are gram negative obligate intracellular parasites and because of this characteristic they were once considered as viruses. They differ from viruses in the following important characteristics:

a.  They possess both DNA and RNA

b.  They multiply by binary fission

c.   They have a rigid cell wall

d.  They possess ribosomes and have a variety of metabolically active enzyme

e.   Their growth can be inhibited by many antimicrobial drugs

3.  They have a distinct staining property that differ somewhat at different stages of development 

Developmental cycle:

All Chlamydiae has a unique biphasic developmental cycle, which consists of two alternating cellular forms:

(1) The infectious, non-dividing elementary body (EB) which is about 300 nm in diameter.

(2) The proliferative, non-infectious reticulate body (RB) which is about 300–400 nm in diameter.

The developmental cycle is initiated by the attachment of an EB, which can survive extracellular environments, to the host cell plasma membrane. The EB is taken by the host cell into a vacuole. Inside the vacuole, which is designated inclusion, the EB differentiates into the larger RB, which proliferates by binary fission, and differentiates back into EBs in an asynchronous manner. The cycle completes upon the release of intracellular chlamydiae. Whereas a typical developmental cycle takes about 2–3 days, it can be altered in response to host immune reactions, antibiotic treatment, or other adversarial factors, leading to persistent infection. A persistent state is characterized by the formation of aberrant RBs, which is characterized by expansion of cellular contents without division. Aberrant RBs may convert back to normal RBs and re-enter the regular developmental cycle to produce infectious EBs upon the return of conditions favoring cell division.

Classification of Chlamydiae:

Property	Subgroup A		Subgroup B
Species name	Chlamydia trachomatis		Chlamydia psitacci
Host	Man		Birds and mammals
Susceptibility to Sulfonamides & D–cycloserin	Susceptible		Resistant
Intracellular inclusion	Compact or rigid with glycogen		Diffuse without glycogen
	Halberstaedter– Prowazek  bodies		Levinthal–Cole–Lillies bodies

Diseases produced by Chlamydia trachomatis:

1.     Trachoma – blindness among poverty stricken family.

2.     Inclusion conjunctivitis or swimming pool conjunctivitis

Trachoma	Inclusion conjunctivitis
Not sexually transmitted	Sexually transmitted
Transmitted by close contact under unhygienic condition	Also transmissible by fomites, swimming pool water and towel
Only the eyes are involved, no systemic manifestations	Aside from the eye, genito–urinary tract is involved primarily
Follicular hypertrophy of the conjunctiva	Purulent conjunctiva
Cornmeal scarring leading to blindness	No blindness

3.  Reiter’s syndrome – exhibits a triad of recurring symptoms including conjunctivitis, polyarthritis, and genital inflammation.

4.  TWAR strain (Taiwan Acute Respiratory) – associated with acute lower respiratory disease similar to Mycoplasma pneumoniae infections.

5.  Lymphogranuloma venereum (LGV) or Frei’s disease or climactic bubo – a minor venereal disease characterized by bilateral bubo.

Laboratory diagnosis

A characteristic initial or elementary body in the lesion and bubo. Isolation of the organism using tissue culture and chick embryo.

Frei test is a skin test employing injection of heat–inactivated egg–grown LGV.

Diseases produced by Chlamydia psittacis

1.  Psittacosis or Ornithosis – a disease of birds that may be transferred to humans by dust in bird’s cages. It produces a spectrum of clinical manifestations ranging from severe pneumonia and sepsis with a high mortality rate to mild inapparent infection.

2.  Meningopneumonitis

3.  Feline pneumonitis

Symptoms of disease caused by Chlamydiae

1.  In man, Chlamydia varies in severity from slight morning itching of the penis to discharge, painful urination, swelling of the testicles, eye disease and arthritis.

2.  In woman, there is a painful urination, vaginal discharge, menstrual disturbances, abscess and ruptured tubes, hepatitis and painless blocking of the tubes leading to permanent sterility.

Laboratory diagnosis

1.  Chlamydia psittacis – cultivated in the yolk sac of embryonated eggs, by intraperitoneal inoculation of mice or by inoculation of mice or by inoculating into HeLa 229 or other cell cultures. Inclusions in cell culture are demonstrated with Giemsa, methyl green – neutral red stains but not iodine.

2.  Chlamydia trachomatis – cell cultures, yolk sac, in mice after intracerebral or intraperitoneal inoculation. Identified by cytopathogenic effect (CPE)

3.  Halberstaedter–Prowazek bodies – 5% Lugol’s iodine in 10% Potassium Iodide (KI).

4.  Levinthal–Cole–Lillie’s bodies – Michiavello or Giemsa stain

Specimen

a.  Biopsy sample

b.  Whole secretion or as dacron or rayon tipped swabs from affected sites (e.g. eye, respiratory tract, cervix, urethra) and placed in a sucrose– phosphate buffer (2SP) for transport.

c.  McCoy cells (a strain of mouse heteroploid cells) – cycloheximide treated. Most commonly used method for isolation in cell culture.

Note:        Conjunctival scraping or swab should be used, as eye discharge

                  is inadequate for detection of Chlamydia trachomatis.

SEROVAR OF CHLAMYDIA TRACHOMATIS
Pathotype	Serovars	Transmission	Acute Disease	Complications
Ocular	A – C	Contact, fomites	Conjunctivitis (Trachoma)	Trachomatous trichiasis, blindness
Genital	D – K (non–LGV)	Sexual	Man: urethritis, epididymitis, proctitis	Premature birth, miscarriage, PID, infertility, ectopic pregnancy, sexually acquired reactive arthritis
			Woman: urethritis, vaginitis, cervicitis, endometritis, salpingitis, proctitis
		Perinatal	Infant conjunctivitis, pneumonia	?
	L1, L2, L3 (LGV)	Genital ulcer, proctitis, lymphadenopathy	Lymphogranuloma venereum

MYCOPLASMA

General characteristics:

1.  They are also known as PPLO (pleuropneumonialike organisms).

2.  They are pleomorphic because they lack a rigid cell wall and because of this, they are resistant to penicillin and staining.

3.  They are the smallest reproductive unit.

4.  They can reproduce in cell–free media. The colony is characteristically embedded beneath the surface of the agar.

5.  They do not revert to, or originate from, bacterial parental forms.

6.  They have an affinity for mammalian cell membrane.

7.  They cannot be studied by the usual bacteriologic methods because of the small size of their colony, their lack of cell wall, and their poor staining with aniline dyes.

Diseases:

1.  Ureaplasma urealyticum (formerly called T strain mycoplasma) – found in the urethra of some men with non–gonococcal urethritis. Such infection may play a role in male sterility.

2.  Mycoplasma pneumoniae – principal cause of non–bacterial pneumonia ranging from inapparent infection to mild or respiratory disease, ear involvement and pneumonia. Sometimes called "walking pneumonia" because it is usually mild and rarely requires hospitalization.

3.  Mycoplasma hominis – a commensal of the genital tract, is a potential underestimated pathogen causing both genitourinary and extragenital infections including neonatal infections. Septic arthritis, prosthetic joint infection, central nervous system (CNS) infections, infective endocarditis and abscess formation are common extragenital infections associated mainly with immunocompromised patients.

Virulence Factor of Mycoplasma:

1.  P1 adhesin – is highly immunogenic and is often detected by sera from Mycoplasma pneumoniae-infected patients. P1 plays an important role in the pathogenesis of Mycoplasma pneumoniae infection by mediating the attachment of the pathogen to host cells.

2.  P30 adhesin – originally regarded as an accessory protein, plays a role in localizing P1 to the terminal organelle and is involved in cell development; the amino acid sequence of P30 holds substantial homology with some eukaryotic proteins in humans, indicating that P30 could be associated with the occurrence of certain autoimmune diseases.

3.  HapE – a virulence factor that can produce H₂S by the desulfurization of cysteine which can lead to erythrocyte lysis. This enzyme mediates inflammatory reactions via adenosine triphosphate (ATP)–sensitive K⁺ channels. Oxidation of glycerol by the pathogen produces toxic metabolites including hydrogen peroxide which injures cells by causing inflammation. In addition, the Ca²⁺–dependent cytotoxic nuclease (encoded by MPN133) produced by M. pneumoniae can lead to apoptotic–like programmed cell death in the host.

4.  Community–Acquired Respiratory Distress Syndrome (CARDS) Toxin – this toxin is cytotoxic to mammalian cells by activation of the NLRP3–associated inflammasome and further promotes the release of IL–1β and IL–18. CARDS toxin increases the expression of the proinflammatory cytokines IL–1β, IL–6 and TNF–α in a dose– and activity–dependent manner.

Laboratory Tests:

1.  Serologic test – ELISA, PCR

2.  Dienes’ stain – a supravital stain which is a combination of methylene blue (2.5g), Azure II (1.25g), maltose (10g), sodium carbonate (0.25 g) and distilled water (100 ml) used to stain Mycoplasma colonies. Other stains used are the Hoechst stain and the DAPI stain.

3.  Heart infusion peptone broth with human ascitic fluid – “fried egg” appearance of colony.

4.  Eaton’s Agar 

Specimen:            throat swab, sputum, inflammatory exudates, respiratory,

                        urethral or genital secretions