Fastidious bacteria2

Jan TkadlecDept. of medical microbiology

Winter 2020

Topics

Fastidious gram negative bacili• Francisella• Legionella

• Brucella Spirochaetes• Leptospira• Treponema• Borrelia Rickettsia and rickettsia-like organisms

• Rickettsia• Anaplasma• Ehrlichia

• Bartonella

• Coxiella

Common features• Intracellular parasites• Culture is not effective

for diagnosis• Systemic infections• Vector borne (mostly)• Zoonosis (mostly)

Diagnosis of rare fastidiousbacteria is challengingWhen infection caused by fastidius patogen occurs rarely outcome could be worsebecause:

1. Looking for non fastidious patogens delays detection of true cause• Patogen will be detected after rulling out more common causes

2. Non fastidious patogen is taken as true cause• Treating wrong patogen

3. Not using apropriated method• Patogen would not be detected at al

It is important to know fastidious patogens and their typical symptomsand risk factors – it could by life-saving knowledge

Logistical challenge

Be aware that not all microbiology departments perform detection offastidious agents

1. It is too rare to be economical to have apropriate method

2. It is too laborious/expensive…

3. It is dangerous (highly infectious agens)

Detection or confirmatin of detection of some fastidious agents is performed only by specialised/reference laboratories

Francisella tularensis

Biology:Aerobic gram-negative cocobacillifacultative intracellular bacteriumSubspecies:F. tularensis tularensis

North America – more virulentF. tularensis holarctica

Europe and Asia – less virulentPotential bioterrorism agent

EcologyZoonosisReservoir: wild animals (rabbits, rodents, deer, birds, etc.)Survive also in water, grass, haystacks, animal carcassesTransmission (entry)• Respiratory system• Blood• Skin contactLow infection dose = highly infectiousBSL-3

Francisella tularensis

Rx:aminoglycosides, tetracyclines, orfluoroquinolones

Dg:Culture requires cystein forgrowthCO2 and prolonged cultivation35–37 °CChocolate agarSymptoms and patient history(contact with dead wildanimals)

Clinical signifikanceTularemiaUlceroglandular – throughskin contactPulmonary

F. tularensis on chocolate agar

BSL 3 - 4

BSL 1

BSL 2

(A) buffered

charcoal yeast

extract;

(B) chocolate agar

medium;

(C)sheep’s blood

agar;

(D)cysteine heart

agar.

Pathogen profile

dictionary

ppdictionary.com

Francisella tularensis - culture

Brucella

Biology:Strictly aerobic gram-negative cocobacillifacultative intracellular bacteriumSpecies:

B. abortus (cattle)B. melitensis (sheep and goat)B. suis (pig)B. canis (dog)

survive in soil, manure and dustfor weeks or months, and remain viable in dead fetal material for even longer

EcologyZoonosisReservoir: animals (sheep, cattle, orpigs, etc.)Transmission• contaminated food (such as

unpasteurized milk products, meatproducts)

• direct contact with an infected animal

• inhalation of aerosols

Low infection dose - level BSL-3B. melitensis is still prevalent in Mediterranean countries, the Middle East, central and southern Asia, and parts of Africa and South America

BrucellaClinical signifikanceBrucellosissepticaemic illness, undulant fever. Most human disease is caused by Brucella melitensis,B. abortus or B. suis.

Brucellosis can present as an acute or subacute pyrexial illness that may persist for months or develop into a focal infection that can involve almost any organ system. The characteristicintermittent waves of increased temperature that gave the name undulant fever to the human disease are now usually seen only in long-standing untreated cases.Affects gastrointestinal tract including anorexia, abdominal pain, vomiting, diarrhea, constipation, hepatomegaly, and splenomegaly

Less frequently arthritis (hip, knee, and ankle), spondylitis, osteomyelitis, and sacroiliitisRarely endokarditis

Infection in animal has economical impact – in pregnant animals oftenleads to abortion,

Brucella

Dg:isolation of the organism from blood (bloodculture); alternatively serology or PCRCulture: some species require CO2, media with glukose and animal serum37°C 1 to 6 weeks

Rx: tetracycline, usuallyin combination with an aminoglycoside or rifampicinPrevention –pasteurization of milk

Brucella melitensis,

blood agar

Legionella

Biology:Gram-negative rodsL. pneumophila

Other species less frequente.g. L. longbeachae

Epidemiology:Frequent in water – intracelularly in amoebas (Acanthamoeba, Naegleria) and other protozoaWarm water sourcesSpread through aircondition and untreatedwater supplyInhaling aerosol or droplets containinglegionellaInhaled bacteria are engulfed by monocytes and can survive therein as intracellular parasites

Legionella

LegionellaClinical significance:Legionnaires disease (mostly L. pneumophila serogroup 1)

pneumonia, up to 10% mortality when not treatedhigh fever, respiratory distress, scanty sputumconfusion, hallucinationsRenal impairement could be presentPotential to cause outbreak (hospitals!!!) –contaminated water supply

Risk factors: immunosuression, higher age (over 40y)Stays in hotels in low income countries

Pontiac fever – non-pneumonic, non-fatal, influenza-likesymptoms, high attack rate – most of the affected peopledevelop disease

History1976 first outbreak of 182 cases of pneumonia, mainly affecting members of the American Legion,occurred at a convention inPhiladelphia = legionnaires diseaseSpread through air condition

LegionellaDg: Sample: sputum and other respiratory specimen, lung biopsy –PCR or culturerequire cystein and iron grow best on buffered charcoal yeastextract agar (BCYE) with antibioticsCulture about 1 week, increased CO2Heat stable – sample could be heated to 50°C for 30 min to diminiss growth of other bacteriaATB testing is not performed (too laborious)Antigen detection in urine (ELISA) – commonly used, Serology – IG could be detected 8-10 days from start of thesymptoms

Rx:Legionnaires disese –intravenous azitromycin, combined withfluoroquinolones and/orrifampicin in severe cases

PreventionWater treatment:• heat• disinfection with chlorine or other biocides,including chlorine dioxide• copper–silver ionization.

Legionella pneumophila, Gram stain

By courtesy of MUDr. Petra Kabelíková

Legionella

pneumophila

BCYE agar

Courtesy of

MUDr. Petra

Kabelíková

Spirochaetes

Spirochaetes

Biology:Helical corkscrew –shapedbacteriaClose to gram negatives – haveouter and inner membranebut differently organised

flagella in periplasmic space –corkscrew-like movement = tissue penetration

Too thin to be seen by lightmicroscope- Dark field microscopy orimmunofluorescence

Borrelia Treponema Leptospira

Different shapes of spiropchaetes

BorreliaBiology:Microaerophilic spirochaetesPoorly stained by GramB. burgdorferi sensu latocomplex of related species:

• B. burgdorferi sensu stricto– USA and West EU

• B. garinii, B. afzelii - CZB. recurrentis

Epidemiology:arthropod vector - Lice or ticksRelapsing fever (B. recurrentis)– ticks (Argasidae) or lice (Pediculushumanus)Lyme disease (B. burgdorferi)- Ixodes ticks - I. dammini and I. pacificus

(USA); I. ricinus (EU)

Acridine orange stain of peripheral blood demonstrates Borrelia spp.

larvae nymph adults

Lyme disease (Lyme borreliosis)

B. burgdorferi sensu lato

• Reservoir mice and otherrodents, deer, sheep, cattle, horses and dogs.

• Transmission throughtick bite (Ixodes sp.)

• Multisystem infection• Non specific symptoms at

the begining• Affects skin, joints, heart,

and CNS• Three stages

Erythema migransTypical symptom but only in about 30% cases

Nymphs are most common vector in human Lyme disease

Kumar D, Ristow LC, Shi M, Mukherjee P, Caine JA, Lee W-Y, et al. (2015) PLoS Pathog 11(12): e1005333. doi:10.1371/journal.ppat.1005333

Lyme disease - patogenesis

Within one monthDx: antigen in CSFor PCR from CSF orskin biopsy

1 to 4 monthsDx: IgG and IgM in serum and CSF

More than 4 monthsDx: IgG and IgM in serum and CSF

Rx: doxycykline, amoxicillin, ceftriaxoneDuration from 2 weekup to 1 month

Relapsing fever (typhus recurrentis)

• Borrelia recurrentis

• Human is only reservoir

• Transmission: human louse (Pediculus humanus)

• Associated with poor hygiene conditions (low incomecountries, homeless people,…)

• Symptoms: Fever returning each 5-10 days, hepatosplenomegaly, ikterus

• Dx: Serology

• Rx: doxycycline

Treponema pallidum

Biology:Anaerobic to microaerophilic spirochaetesIntracellular patogenPoorly stained by GramSensitive to temperature above 40°C, oxygen etc.Transmission only through direct contactOnly human to human

Sexualy transmitedcongenital

SubspeciesT. pallidum palidum – cause syphilisT. p. endemicum – bejel or endemic syphilisT. p. pertenue - yaws

T. pallidum darkfield microscopy

Treponema pallidumClinical significance:Syphilis – STD

Penetration through skin lessions or through mucousmembranesTisuse destruction due to immune response

Forms/stages1. Primary

Localised necrosis and ulceration(ulcus durum) lymphadenophaty, highly infectious detectable Ig

2. SecondarySkin, mucous epitelia and systemic symptoms, CNS (encephalitis), highy infectious, detectable Igs

3. Latent – non infectious live long stage4. Tertiary – after 10 to 25 y - cardiovascular and CNS

(neurosyphilis) symptoms progressive paralysis• Congenital

Treatment - Penicilin

Prevention

Mandatory screening newborns etc

Monitoring of STDs

Dg:SampleUrineSwab (urethra, cervix, vagina)Swab from skin lessionsSerum

MethodsMicroscopy dark field or fluorescent antibody staining.PCR – only early stageSerologyNontreponemal (reagin) antibodies

Venereal Disease Research Laboratory (VDRL) test – CSF, flockulation, agglutinationRapid plasma reagin (RPR) test

Treponemal antibodiesT. pallidum indirect hemagglutination (TPHA), agglutinationT. pallidum particle agglutination (TP-PA) test, agglutinationELISA

Treponema pallidum

LeptospiraBiology:Obligate aerobic pathogenic or saprophyticspirochaetesStable in water environmentLeptospira interrogans

Epidemiology:Zoonosis

Reservoir wild animals (mammals rodents, etc), lives in kidneys of animals (withoutcausing illnes), released with urine

Pathogenic Leptospira do not multiply in the environment, but could survive in humidenvironments like stagnant water or contaminated soil.

Entry through:Skin abrasion, wound , ingestion, mucousmembrane (e.g. Mouth, nose, eye)

Faisal S.M., McDonough S.P., Chang YF. (2012) Leptospira: Invasion, Pathogenesis and

Persistence. In: Embers M. (eds) The Pathogenic Spirochetes: strategies for evasion of host

immunity and persistence. Springer, Boston, MA. https://doi.org/10.1007/978-1-4614-5404-5_8

Leptospira – risk factorsRisk:

• farmers, fishermen, garbage collectors and sewage workers

• Outdoor sport

• floods

LeptospiraDg:Sample: serum, urine, CSF, autopsyCultivation is possible patogenic species growworse than saprophyticEMJH medium with rabbit serum, 30°C, 4 - 7 daysPCRSerology – IgM ELISAAntigen detection - Latex agglutination etcMicroscopic agglutination test (MAT) is the reference test for the diagnosis of leptospirosispatient sera are mixed with different serovars of Leptospira – agglutination with IG is observedunder microscopeRx: intavenous penicillin G for severe

cases, amoxicillin, doxycycline

Clinical significance:LeptospirosisBacteria penetrates bloodstrem and spread through bodyvary from flu-like to severe illnes=Weil´s disease (meningitidis, renalfailure, icterus, bleeding, could affectliver, shock)

Symptoms:

Rickettsia and related patogens

RickettsialesRickettsiaAnaplasmaEhrlichia

Bartonella Coxiella

Rickettsia in general

Biology:Gram-negative, highly pleomorphic bacteria, obligateintracellular patogensSmall genomePrimar human patogenSensitive to environmentalchanges

Spoted fever groupR. rickettsiiR. akari

Typhus groupR. prowazekiiR. typhi

EpidemiologyzoonosesAnimal or human host arthropod vector

Dg:Symptom based suspitionSerology: Weil–Felix testMixing patient serum with rickettsial antigenWhen antibodies present agglutination appearsPCR

Penetrrates through skin into blood stream, infects endothelium of bloodstream vessels in multiple organs

Rx: tetracyclines - doxycycline

Spoted fever group

Rickettsia rickettsii (Western Hemisphere)• Rocky Mountain spotted fever• Tick-borne Dermacentor sp = Dog tick• Reservoir dogs and rodents• Infection in children during spring (tick activity)• Suden fever, headache, myalgia• Typical rash spreading from limbs to trunk• Fatal if untreated

Rickettsia akari (USA, former Soviet Union)• Rickettsialpox• Mite-borne Liponyssoides sp• Reservoir mice• Mild disease, nonfatal, self-limiting• Vesicular rash, fever, lymphadenopatty

Petechial rash on the arm caused by Rocky Mountain spotted fever

Rickettsialpox rash

Epidemic/recrudescent typhus

• Rickettsia prowazekii

• Vector – human body louse (Pedicullus humanus)

• War and poverty

• Africa, South america

• Symptoms: Chills, fever, headache, pain, stuporand delirium

• Signs f severe meningoencephalitis begin with rash

• Rash spreads from trunk to limbs

• Untreated is fatal

• In some people reactivity after many years –recrudescent typhus (Brill-Zinsser disease) – mildersymptoms

Murine/endemic typhus

• Rickettsia typhi

• Vector flea (Xenopsylla sp)

• Reservoir: Rats

• Symptoms: gradual onset of fever, chills severe headache, generalized pain

• Macular rash spreading from trunk to limbs

Ehrlichia and Anaplasma

Biology:Obligate intracellular patogensPreference to WBC – mononuclear cells

EpidemiologyZoonosisTick borne, reservoir: wild animals and dogs

Dg:PCR (blood)Serology

Rx: doxycycline

Clin. significanceHuman monocytic ehrlichiosis (HME)

Ehrlichia chaffeenisUSA

Human granulocytic ehrlichiosisAnaplasma phagocytophilumUSA but also EU and Asia

Bartonella

Biology:Gram-negative small rods or cocobacilliFacultative intracellular parasites

B. henselae, B. quintana, B. bacilliformis

Zoonosis (B. henselae)Human as primary host (B. quintana, B. bacilliformis)

Source: cat, mouse etc.

Transmission: vector borne (ticks, fleas, sand flies, mosquitoes)

Body louse

Bartonella

Clinical signifikance• Cat-scratch disease – felinosis (B.

henselae) – local lymphadenopathy, mild disease (fever, headachetiredness), slow recovery – 4 – 12 months, speed up by ATB

Endocarditis (B. henselae, B. quintana)

Trench fever (B. quintana)Soldiers (WWI) or today homeless people

Bacillary angiomatosis (B. henselae, B. quintana) – skin lessions(papules ornodules) – vascularisedPresent in internal organs – potentiallyfatal if not treated

Rx:Long term terapy (6 weeks and more)Tetracyclines, aminoglycosides, macrolides

Dg.Culture – CO2, at least two weeks, enriched mediaSample has to be treated withdetergent to release intracelularbacteria

Serology – antibody cross reactivitywith other bacteria, antibodies are often not producedPCR

Bacilary angiomatosis

Trench fever symptoms

felinosis

Coxiella burneti

Biology:small Gram-negative, coccobacilliobligate intracellular bacterial pathogenSurviving in phagolysosome of macorphagesResistant to environmental conditionsHighly infectious, primary patogens for human

Epidemiology:Zoonosis (cats, dogs, cattle,...)source – inhalation of aerosols frominfected animals or consumption ofmilk, or tick-borne transmissionRisk groups: farmers, slaughter house workers, veterinarians

Clinical significanceQ- Fever (query – the causing agent was not known for a long time)Survive in macrophages, intersticial (atypical) pneumonia, liver and spleen – granuloma, flu-like illness, gastroenteritiscomplications–meningoencefalitis, endocarditis

Dg: direct –cultivation using tissue culture, PCR,Undirect – serology: Weil-Felix reaction

Rx: doxycykline+ rifampicin (fluoroquinolones)

Possible transmission paths and potential hosts of C. burnetii

Take home messageFastidious bacteria (Francisella, Brucella, Legionella, Spirochates, Ricketssia etc.)

• Intracellular

• Culture is not possible or usefull – poor sensitivity and long time to result

• Serology! (or PCR, but not always)

• Symptoms (rash, lymphadenopaty, etc) and risk factors!

• Multi-organ infection, long term diseases = long term treatment.

• Zoonosis, vector borne (with exceptions e.g.T. pallidum)

Biggest thread in dealing with rare fastidious pathogens is that they could be easily forgotten and not treated in right time.