Mycobacteria and free-living amoebae

Free-living amoebae and mycobacteria: a dangerous association

Vincent THOMAS, PhDR&D – Biomarker Project ManagerEnterome / www.enterome.com94-96, Avenue Ledru Rollin75011 Paris - FRANCEMobile phone: +33 6 14 24 01 28

Lausanne, 25 September 2013

1- FLA & NTM: Ecological aspects2- Relation with resistance to drugs and biocides3- Relation with virulence4- Genomic/gene exchange

Last sentenceLast sentence of the paper:of the paper:

“We believe that testing

encysted mycobacteria may be necessary to properly

evaluatedecontamination

procedures for water and endoscopes.”

Mycobacteria can survive and develop within Free-Living Amoebae

Iovieno, A., D. R. Ledee, D. Miller, and E. C. Alfonso. 2010. Detection of bacterial endosymbionts in clinical Acanthamoeba isolates. Ophthalmology 117:445-52

Yu HS, Jeong HJ, Hong YC, Seol SY, Chung DI & Kong HH. 2007. Natural occurrence of Mycobacterium as an endosymbiont of Acanthamoeba isolated from a contact lens storage case. Korean J Parasitol 45: 11-18.

White, C. I., R. J. Birtles, P. Wigley, and P. H. Jones. 2010. Mycobacterium avium subspecies paratuberculosis in free-living amoebae isolated from fields not used for grazing. Vet Rec 166:401-2.

Glaser, K. C., N. D. Hetrick, and R. E. Molestina. 2011. Evidence for a previously unrecognized mycobacterial endosymbiont in Acanthamoeba castellanii strain Ma (ATCC 50370 ). Journal of Eukaryotic Microbiology 58:75-6.


Lamrabet O, Mba Medie F, Drancourt M. 2012. Acanthamoeba polyphaga-Enhanced Growth of Mycobacterium smegmatis. PLoS ONE 7:e29833.


Distribution

Stockage

Water treatmentplant

Distribution

Storage

Water treatmentplant

Free-living amoebaeFree-living amoebae: the Trojan horse of pathogenic bacteria

Adapted from Center for Biofilm Engineering, MSU-Bozeman 1996

Clarification Rapid sand filtration Ozonation Activated carbon filtration

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lorin

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Seine River

Distribution network

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Clarification Rapid sand filtration Ozonation Activated carbon filtration

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Seine River

Distribution network

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FLA and mycobacteria in drinking water treatment plants

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River water Sand biofilm Sand filtered Ozonated GAC biofilm GAC filtered Chlorinated Downstream Distant point

Other bacterial species isolated

Isolated chlamydiae

Isolated legionella

Isolated mycobacteria

Isolated amoebae

Indigenousamoebae

Bacteria resisting digestion by A. castellanii ATCC30010

Biofilms samples & water samples

Free-living amoebae: they are Free-living amoebae: they are everywhere!!!everywhere!!!


Ziehl-Neelsen staining

Mycobacteria: mainly “non-pathogenic” species


200 swab and water samples from hospital water network (Lausanne, Switzerland)

Emergency/ICU, Surgery and Internal Medicine Dpts

Cultivation of indigenous amoebae Seeding on A.castellanii ATCC30010

7.5% of the samples + 125 Amoebae-Resisting Bacteria isolatesrecovered from 45% of the samples

Hartmannella vermiformis +++ 35 M.gordonae, 3 M.kansasii, 5 M. xenopi9 L. anisa, 2 L. pneumophilaVarious known α-proteobacteria species considered as potentially pathogenicFour new α-proteobacteria species, one was recovered from 23 samples

FLA and mycobacteria in water distribution networks

• 23 liquid and 65 swab samples were collected• Amoebae isolated from 13 samples (12S, 1L): 6 Acanthamoeba sp, 8 Hartmannella

sp and 2 species related to Amoebozoa and Heterolobosea supergroups• 16 mycobacteria were isolated by co-culture from 6 of the 13 samples also

presenting amoeabe, and from 8 other samples (total: 14). • Identification: M. gordonae (n=9), M.chelonae (n=3), M. kansasii/gastri (n=1),

M.avium (n=1), M.peregrinum (n=1), M.mucogenicum (n=1).

FLA and mycobacteria in water distribution networks

Appl Environ Microbiol 2013; 79:3185-3192.


• M. avium-infected A. castellanii monolayers treated with rifabutin, clarithromycin, or azithromycin from the day after infection, for 4 days, showed no significant reduction in CFU per milliliter

• In contrast, all three compounds were found to be significantly active against M. avium within U937 macrophages

• To determine whether antimicrobials could penetrate A. castellanii and not be degraded intracellularly, A. castellanii monolayers were infected with a clinical strain of S. aureus and treated with the same antibiotics efficacy OK

Decreased uptake of the antimicrobials into the amoebae in comparison with the uptake of macrophages, inactivation of the compound within amoebae, or even change in the phenotype of the bacterium?.

(Faller et al., 2004, Science)

Porins

• Structural models indicate that erythromycin, kanamycin, and vancomycin are too large to move through the MspA channel

• Hydrophilic fluoroquinolones and chloramphenicol diffuse through porins in mycobacteria• Mutations resulting in less efficient porins or lower porin expression levels are likely to represent

a mechanism for the opportunistic pathogens M. avium, M. chelonae, and M. fortuitum, which have Msp-like porins, to acquire resistance to fluoroquinolones

Porins

• Deletion of the mspA gene caused high-level resistance of M. smegmatis to ampicillin and eightfold increased resistance to cephaloridine

• MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the mspA mutant were also increased 2- to 10-fold, possibly due to slower rate of transport of hydrophobic compounds across the OM

Porins: resistance to drugs and biocides

Dr Mary Jackson

An epidemic of postsurgical infections caused by M. massiliense in Brazil

• Prior to 2004, sporadic cases and small outbreaks (< 20 cases) of RGM infections, particularly in Rio de Janeiro and Šao Paulo

• First significant outbreak (311 suspected cases) in the city of Bélem, state of Pára, in 2004-2005 following laparoscopic surgeries. M. massiliense & M. bolletii = main causative agents

• 2006-2007, more than 2,000 cases of wound infections by RGM (MM) related to video laparoscopy were notified in 15 Brazilian states

• State of Rio: All 74 MM isolates analyzed belonged to one single clone (BRA-100); the same clone was found in other states as well…

• BRA-100 is glutaraldehyde-resistant (> 10 hrs exposure to 2% GTA)

Dr Mary Jackson

Effect of expressing mspA on the drug susceptibility of M. chelonae 9917 1

Drug ATCC 35752 ATCC/pZS01 9917 9917/pZS01 MIC (g ml-1) AMP > 500 > 500 > 500 > 500 KAN 50 nd 10 nd STR 200 250 10-20 10 VAN 5 5-10 > 500 25 CLA 25-50 50 > 250 25 AZI 500 500 500 500 HYG > 500 nd > 500 nd ERY 20-25 0.25 500 1 RIF 25 5 > 500 100 EMB > 500 25 > 500 250 CHL > 500 50 > 500 50 TOB 25-50 nd 25 nd GEN 7.5 7.5 15 20 INH > 400 > 400 > 400 > 400 ETH > 400 50 > 400 50 PZA > 500 nd > 500 nd TET 50 5-10 > 500 25 LIN 25 5 100 1 CIP 1.5 2.5 5 1 NOR 5 nd 5 nd

Large and hydrophilic

Large and hydrophobic

Small

Fluoroquinolone

AMP, ampicillin; AZI, azithromycin; CHL, chloramphenicol; CIP, ciprofloxacin; CLA, clarithromycin; EMB, ethambutol; ERY, erythromycin; ETH, ethionamide; GEN, gentamicin; HYG, hygromycin; INH, isoniazid; KAN, kanamycin; LIN, linezolid; NOR, norfloxacin; PZA, pyrazinamide; RIF, rifampicin; STR, streptomycin; TET, tetracycline; TOB, tobramycin; VAN, vancomycin.

Used in the clinical treatment of RGM infections


Dr Mary Jackson

Svetlikova Z, Skovierova H, Niederweis M, Gaillard JL, McDonnell G, Jackson M. 2009. The role of porins in the susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to aldehyde-based disinfectants and drugs. Antimicrob Agents Chemother 53:4015-4018.

Porin mutants: MN01 mspAML10 mspAmspCSMR5, WT parent

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OPA 0.05 %

OPA 0.1 %

* OPA: Cidex® OPA (J&J)

GTA 2 %


Dr Mary Jackson

ML02: mspA mutant,ML10: the mspA mspC mutant,ML16: mspA mspC mspD mutant.


Porins: pathogenicity

Disrupting mspA and mspC in M. smegmatis enhances the intracellular persistence of the bacterium in murine macrophages… and amoebae!

Porins: pathogenicity

Drug resistance

Biocideresistance Virulence

Porins: the link between drug resistance, Porins: the link between drug resistance, biocide resistance and virulence…biocide resistance and virulence…

PORIN

PO

RIN

PO

RIN

1- FLA & NTM: Ecological aspects2- Relation with resistance to drugs and biocides3- Relation with virulence4- Genomic/ gene exchange

Amoebae: A training field for macrophage resistance?

Amoebae display similar bactericidal mechanisms to macrophages:

- phagosome/lysosome fusion- lysosomal enzymes- oxidative stress (ROI)- phagosome acidification- antimicrobial peptides

• 24 of 29 tested M. avium strains enter and replicate in A. castellanii ATCC 30234 (24°C-37°C)• M. smegmatis mc² 155 and ATCC 11727 are able to infect but are digested within 2 days• M. avium inhibits lysosomal fusion and replicates in vacuoles• Compared to growth of bacteria in broth, growth of M. avium in amoebae enhances both entry

and intracellular replication in amoebae, humand epithelial cells and macrophages• Amoeba-grown M. avium was also more virulent in the beige mouse model of infection



• Mycobacterium avium genomic library with GFP-promoter trap was created in Mycobacterium smegmatis

• 10 000 clones were screened for increased fluorescence emission during infection of A. castellanii

• 20 selected due to high gfp expression• Eight out of these 20 genes were found to be the same as those upregulated upon human

macrophage infection

Proc Natl Acad Sci U S A 2007; 104:11038-11043.

Identification of a 8 genes pathogenicity island that is unique to M. avium and promotes infection of A. castellanii and macrophages

• ORF7 is involved in inducing actin polymerization and is required for efficient M. avium entry into macrophages

• ORFs 5 – 8 function would be to transport actin-interacting protein ORF7• Function of ORFs 1 – 4 is unknown


Mycobacterium kansasii : NTM species that is frequently involved in human infections Several sub-types (hsp65) :

• sub-type 1 virulent,• sub-type 2 opportunistic (HIV, immunodeficiency),• sub-type 3 commensal• pathogenicity of other sub-types (4 to 7) not well defined

Pathogenicity of these sub-types to amoebae?




a melting pot of organisms favouring inter-kingdoms gene exchangesFLA:FLA:

Phylogenetic tree depicting relationships of Legionella drancourtii sterol delta-7 reductase with closest homologues retrieved by performing a BlastP analysis against NCBI protein database and Naegleria gruberi ongoing genome

a melting pot of organisms favouring inter-kingdoms gene exchanges

Lamrabet O, Merhej V, Pontarotti P, Raoult D, Drancourt M. 2012. The genealogic tree of mycobacteria reveals a long-standing sympatric life into free-living protozoa. PLoS One 7:e34754.

Schematic representation of two alternative explanations of the evolutionary history of the pyridine nucleotide disulfideoxidoreductase gene

FLA: a melting pot of organisms favouring inter-kingdoms gene exchanges

Additional gene exchanges to be described in the future…

a melting pot of organisms favouring inter-kingdoms gene exchanges

Paper Discussion

Appl Environ Microbiol 2013; 79:3185-3192.

Mostly a methodology paper

Species Strain Protozoa * Survival within Survival within Multiplication

trophozoïtes cysts in trophozoites

"M. habana" (M. simiae ) TMC 5135 A. castellanii + (30°C) ? ?

M. abscessus CIP 104536T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. aurum CIP 104465T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. avium 29 env. & clin. strains A. castellanii ATCC30234 + (24 to 37°C) ? + (24 to 37°C)

M. avium serotype 4 clinical str. A. polyphaga ATCC 30872 + (33°C) + (33°C) ?

M. avium subsp. avium ATCC 35719 A. castellanii + (30°C) ? ?

M. avium subsp. avium CIP 104244T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. avium subsp. paratuberculosis 2 animal & 1 clinical str. A. polyphaga CCAP 1501/18 + (room temp.) + (room temp.) + (room temp.)

M. avium subsp. paratuberculosis NCTC 8578 A. castellanii CCAP 1501/1B + (25°C) ? + (25°C)

M. avium subsp. paratuberculosis NCTC 8578 A. polyphaga CCAP 1501/3B + (25°C) ? + (25°C)

M. bohemicum CIP 105811T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. bovis NCTC 10772 A. castellanii CCAP 1501/1A + (15°C) + (15°C) -

M. bovis BCG ATCC 35734 A. castellanii CCAP 1501/1A - - -

M. chelonae CIP 104535T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. fortuitum CIP 104534T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. fortuitum clinical str. A. castellanii ATCC30234 + ? +

M. fortuitum subsp. fortuitum ATCC 6841 A. castellanii + (30°C) ? ?

M. gastri CIP 104530T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. goodii ATCC 700504T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. gordonae CIP 104529T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. immunogenum CIP 106684T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. intracellulare CIP 104243T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. kansasii 14 clinical strains A. castellanii ATCC30010 + (33°C) ? + (33°C)

M. kansasii CIP 104589T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. lentiflavum CIP 105465T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. leprae clinical str. A. culbertsoni ATCC 30171 + (?°C) ? not demonstrated

M. mageritense CIP 104973T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. malmoense CIP 105775T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. marinum clinical strain M A. castellanii ATCC30234 + at 32°C, - at 37°C ? + at 32°C, - at 37°C

Thomas V, McDonnell G. 2007. Relationship between mycobacteria and amoebae: ecological and epidemiological concerns. Lett Appl Microbiol 45:349-357.

Genotype T4, mostly A. polyphaga Linc-Ap1 (identical to CCAP 1501/18) and A. castellanii

M. marinum ATCC 927 A. castellanii + (30°C) ? ?

M. marinum CIP 104528T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. massiliense CIP 108297T A. polyphaga Linc-AP1 + ? ?

M. massiliense CIP 108297T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. mucogenicum ATCC 49650T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. peregrinum CIP 105382T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. phlei TMC 1523 A. castellanii + (30°C) ? ?

M. porcinum CIP 105392T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. septicum ATCC 700731T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. simiae CIP 104531T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. simiae TMC 5134 A. castellanii + (30°C) ? ?

M. smegmatis ATCC 14468 A. castellanii + (30°C) ? ?

M. smegmatis ATCC 19420T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. smegmatis ATCC 11727 A. castellanii ATCC30234 - ? -

M. szulgai CIP 104532T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. terrae CIP 104321T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. tusciae CIP 106367T A. polyphaga Linc-AP1 + (33°C) + (33°C) ?

M. ulcerans Tub/71 A. castellanii + (30°C) ? ?

M. xenopi environmental str. A. polyphaga Linc-AP1 + (33°C) + (33°C) +

M. avium subsp. avium ATCC 35713 Dictyostelium discoideum AX2 + (24°C) * + (24°C)

M. marinum clinical str. Dictyostelium discoideum AX2 + (25°C) * + (25°C)

M. avium 3 clin. & 1 env. str Tetrahymena pyriformis ATCC 30202 + (30°C) + (30°C) + (30°C)

M. intracellulare ATCC 13950 Tetrahymena pyriformis ATCC 30202 + (30°C) + (30°C) + (30°C)

M. scrofulaceum ATCC 19981 Tetrahymena pyriformis ATCC 30202 + (30°C) + (30°C) + (30°C)

Thomas V, McDonnell G. 2007. Relationship between mycobacteria and amoebae: ecological and epidemiological concerns. Lett Appl Microbiol 45:349-357.

Genotype T4, mostly A. polyphaga Linc-Ap1 (identical to CCAP 1501/18) and A. castellanii

PLoS One 2010, 5:e9823.

mCherry strains provided by Dr Mary Jackson

A. lenticulata ATCC 30841

Acanthamoeba H16S1(hospital strain)

M. avium H87 (hospital strain) M. avium 104

mCherry strains provided by Dr Mary Jackson

M. avium H87 (hospital strain) M. avium 104

H. vermiformis ATCC 50237

Naegleria clarki CCAP 1518/15

• Capacity of M. avium 104 and M. avium recovered from a hospital sample to proliferate within a large number of amoebal strains, including H. vermiformis, which is frequently isolated from water networks

• M. avium 104 and H87 can potentially survive but not proliferate in H. vermiformis and Naegleria spp.

• The two M. avium isolates tested proliferate more efficiently in A. lenticulata collection strain than any other Acanthamoeba strain tested, including both environmental and collection strains.

• Improved growth of M. avium in A. lenticulata may significantly impact the epidemiology of this pathogenic NTM in drinking water networks. Indeed, A. lenticulata, one and only representative species of genotype T5, is frequently isolated from environmental and clinical samples. In the large study by Fuerst et al, A. lenticulata was the second most frequent genotype recovered from environmental samples after genotype T4 organisms (34). Half of Acanthamoeba isolates recovered from rivers, canals and lakes in Arizona were A. lenticulata (35). A. lenticulata have also been isolated from biofilm samples collected from hospitals and swimming pool water in Brazil (36, 37), and soils and tap water samples in Florida (38).

34. Fuerst PA, Booton GC, Visvesvara GS, Byers TJ. Genotypic identification of non-keratitis infections caused by the opportunistically pathogenic ameba genus Acanthamoeba. J Eukaryot Microbiol. 2003;50 Suppl:512-3.35. De Jonckheere JF. Molecular identification of free-living amoebae of the Vahlkampfiidae and Acanthamoebidae isolated in Arizona (USA). European journal of Protistology. 2007 Jan;43(1):9-15.36. Carlesso AM, Artuso GL, Caumo K, Rott MB. Potentially pathogenic acanthamoeba isolated from a hospital in Brazil. Curr Microbiol. 2010;60(3):185-90.37. Caumo K, Rott MB. Acanthamoeba T3, T4 and T5 in swimming-pool waters from Southern Brazil. Acta Trop. 2011 Mar;117(3):233-5.38. Booton GC, Rogerson A, Bonilla TD, Seal DV, Kelly DJ, Beattie TK, et al. Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis. J Eukaryot Microbiol. 2004 Mar-Apr;51(2):192-200.

Not mentioned in the paper:

• Acanthamoeba spp do degrade antibiotics (amikacin, ampicillin) very quickly (proteases?)

• Mycobacteria grow very well in the presence of amoebal supernatant (see Steinert M, Birkness K, White E, Fields B, Quinn F. 1998. Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba

polyphaga and survive within cyst walls. Appl Environ Microbiol 64:2256-2261.)• It might consequently be very tricky to perform intra-

amoebal growth tests with rapidly growing NTM (difficult to differentiate between intracellular and saprozoical growth???)

Pathogènes avérés: Legionella pneumophila

Utilisation d’une souche GFP-mip afin de suivre &

quantifier la prédation et la

croissance intracellulaire

Amibe 1 Amibe 2

Amibe 3 Amibe 4

Not mentioned in the paper:

Thank You!