Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection

Amoebae are the natural hosts for Legionella pneumophila and play essential roles in bacterial ecology and infectivity to humans. When L. pneumophila colonizes an aquatic installation, it can persist for years despite repeated treatments with disinfectants. We hypothesized that freshwater amoebae play an important role in bacterial resistance to disinfectants, and in subsequent resuscitation of viable non-culturable (VNC) L. pneumophila that results in re-emergence of the disease-causing strain in the disinfected water source. Our work showed that in the absence of Acanthamoeba polyphaga, seven L. pneumophila strains became non-culturable after treatment by 256 p.p.m. of sodium hypochlorite (NaOCl). In contrast, intracellular L. pneumophila within A. polyphaga was resistant to 1024 p.p.m. of NaOCl. In addition, L. pneumophila-infected A. polyphaga exhibited increased resistance to NaOCl. When chlorine-sterilized water samples were co-cultured with A. polyphaga, the non-culturable L. pneumophila were resuscitated and proliferated robustly within A. polyphaga. Upon treatment by NaOCl, uninfected amoebae differentiated into cysts within 48 h. In contrast, L. pneumophila-infected A. polyphaga failed to differentiate into cysts, and L. pneumophila was never detected in cysts of A. polyphaga. We conclude that amoebic trophozoites protect intracellular L. pneumophila from eradication by NaOCl, and play an essential role in resuscitation of VNC L. pneumophila in NaOCl-disinfected water sources. Intracellular L. pneumophila within trophozoites of A. polyphaga block encystation of the amoebae, and the resistance of both organisms to NaOCl is enhanced. To ensure long-term eradication and complete loss of the VNC state of L. pneumophila, we recommend that Legionella-protozoa co-culture should be an important tool to ensure complete loss of the VNC state of L. pneumophila.     

Growth inhibitory and biocidal activity of some isothiazolone biocides

Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizosaccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1-0.5 micrograms/ml for CMIT, 15-20 micrograms/ml for BIT and 40-250 micrograms/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this compound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.     

Chemical reactivity of some isothiazolone biocides

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Growth inhibitory and biocidal activity of some isothiazolone biocides

C ollier , P.J., R amsey , A.J., A ustin , P. & G ilbert , P. 1990. Growth inhibitory and biocidal activity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 569–577.
Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizo-saccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1–0.5 μg/ml for CMIT, 15–20 μg/ml for BIT and 40–250 μg/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this com-pound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.     

The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila

The ability of Legionella pneumophila to cause legionnaires' disease is dependent on its capacity to replicate within cells in the alveolar spaces. The bacteria kill mammalian cells in two phases: induction of apoptosis during the early stages of infection, followed by an independent and rapid necrosis during later stages of the infection, mediated by a pore-forming activity. In the environment, L. pneumophila is a parasite of protozoa. The molecular mechanisms by which L. pneumophila kills the protozoan cells, after their exploitation for intracellular proliferation, are not known. In an effort to decipher these mechanisms, we have examined induction of both apoptosis and necrosis in the protozoan Acanthamoeba polyphaga upon infection by L. pneumophila. Our data show that, although A. polyphaga undergoes apoptosis following treatment with actinomycin D, L. pneumophila does not induce apoptosis in these cells. Instead, intracellular L. pneumophila induces necrotic death in A. polyphaga, which is mediated by the pore-forming activity. Mutants of L. pneumophila defective in expression of the pore-forming activity are indistinguishable from the parental strain in intracellular replication within A. polyphaga. The parental strain bacteria cause necrosis-mediated lysis of all the A. polyphaga cells within 48 h after infection, and all the intracellular bacteria are released into the tissue culture medium. In contrast, all cells infected by the mutants remain intact, and the intracellular bacteria are 'trapped' within A. polyphaga after the termination of intracellular replication. Failure to exit the host cell after termination of intracellular replication results in a gradual decline in the viability of the mutant strain bacteria within A. polyphaga starting 48h after infection. Our data show that the pore-forming activity of L. pneumophila is not required for intracellular bacterial replication within A. polyphaga but is required for killing and exiting the protozoan host upon termination of intracellular replication.     

Chemical reactivity of some isothiazolone biocides

C ollier , P.J., R amsey , A., W aigh , R.D., D ouglas , K.T., A ustin , P. & G ilbert , P. 1990. Chemical reactivity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 578–584.
Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7–10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Cocultivation of Legionella pneumophila and free-living amoebae.

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.     

Counting Legionella cells within single amoeba host cells

Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite(-1), respectively.     

Ultra-structure and localisation of formazan formed by human neutrophils and amoebae phagocytosing virulent and avirulent Legionella pneumophila

Legionella pneumophila (LP) strains of differing virulence were incubated with a solution of nitroblue-tetrazolium (NBT) at a concentration of 1 mg.ml-1 in the presence of Acanthamoeba polyphaga or human polymorphonuclear neutrophils (PMN). Reduction of NBT to formazan occurred at a faster rate in the presence of virulent strains. Reduction appeared to be temperature dependent; at 37 degrees C the reaction rate was higher than at 20 degrees C. On microscopic examination, deposits of formazan around Legionella cells were observed inside amoebae similar to those deposited in human neutrophils. Electron microscopy revealed electron-dense particles surrounding virulent legionellae, which appeared to be associated with formazan formation. Formazan formation inside amoebae may suggest the presence of a respiratory burst against LP, which is more intense with virulent strains.     

Decreased biocide susceptibility of adherent Legionella pneumophila

J.B. WRIGHT. I. RUSESKA AND J.W. COSTERTON. 1991. In a study of the in vitro effectiveness of biocides against Legionella pneumophila , some aspects of the cooling tower environment were replicated in the laboratory, paying particular attention to water hardness and pH. Pieces of Douglas fir and polyvinyl chloride were colonized in a recirculating system and the comparative efficacy of two biocides (Bronopol and Kathon) against the sessile and planktonic populations was examined. While the biocides were relatively effective against the planktonic L. pneumophila population over a short period of time (minimum 9-12 h), substantially longer periods of time (maximum > 48 h) were required to reducc the number of cultivable bacteria to below detectable levels in the adherent population. The results indicate that failure to monitor the sessile population of L. pneumophila in laboratory studies of biocides may result in the use of incorrect dosages and/or contact times in field trials with apparently reduced in situ efficacy.     

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.     

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.     

Decreased biocide susceptibility of adherent Legionella pneumophila.

In a study of the in vitro effectiveness of biocides against Legionella pneumophila, some aspects of the cooling tower environment were replicated in the laboratory, paying particular attention to water hardness and pH. Pieces of Douglas fir and polyvinyl chloride were colonized in a recirculating system and the comparative efficacy of two biocides (Bronopol and Kathon) against the sessile and planktonic populations was examined. While the biocides were relatively effective against the planktonic L. pneumophila population over a short period of time (minimum 9-12 h), substantially longer periods of time (maximum greater than 48 h) were required to reduce the number of cultivable bacteria to below detectable levels in the adherent population. The results indicate that failure to monitor the sessile population of L. pneumophila in laboratory studies of biocides may result in the use of incorrect dosages and/or contact times in field trials with apparently reduced in situ efficacy.     

Ultra-structure and localisation of formazan formed by human neutrophils and amoebae phagocytosing virulent and avirulent Legionella pneumophila

Abstract Legionella pneumophila (LP) strains of differing virulence were incubated with a solution of nitroblue-tetrazolium (NBT) at a concentration of 1 mg · ml⁻ in the presence of Acanthamoeba polyphaga or human polymorphonuclear neutrophils (PMN). Reduction of NBT to formazan occurred at a faster rate in the presence of virulent strains. Reduction appeared to be temperature dependent; at 37°C the reaction rate was higher than at 20°C. On microscopic examination, deposits of formazan around Legionella cells were observed inside amoebae similar to those deposited in human neutrophils. Electron microscopy revealed electron-dense particles surrounding virulent legionellae, which appeared to be associated with formazan foundation. Formazan formation inside amoebae may suggest the presence of a respiratory burst against LP, which is more intense with virulent strains.     

Protozoa and pathogenic bacteria: lessons learned from Legionella pneumophila

Bacterivorous protozoa and bacteria have been in co-existence since the origin of life. This co-existence has led unequivocally to the evolution of many different co-interactions. Most bacteria are ingested and digested, but many escape ingestion for various reasons. Others are ingested but evade digestion, and a few, notoriously Legionella pneumophila , even have the capacity of multiplying within the protozoan host. The aims of this study were to elucidate the interactions of various multi-drug-resistant Staphylococcus aureus (MRSA) strains, Listeria monocytogenes sv4b, and Escherichia coli K12 with the amoeba, Acanthamoeba polyphaga . To evaluate the interactions, we set up co-cultures in Neffs' amoebic saline, at a multiplicity of invasion (MOI) of 1:100 of amoeba to bacteria, and a temperature of 37°C, although the effects of MOI and temperature were also assessed. Survival of bacteria and amoeba was checked at regular intervals, coupled with microscopy. It was discovered under our test conditions, that E. coli was ingested and digested by A. polyphaga , but in contrast, L. monocytogenes , had the capacity to flourish in the presence of A. polyphaga . We also report, for the first time, that all six MRSA isolates tested, survived and replicated in association with A. polyphaga , in comparison to conditions where amoebae were absent. Indeed, we also have evidence suggesting that increases in MRSA, in the presence of A. polyphaga , may be attributable to intracellular survival and replication. These findings have profound implications for the hospital environment, where Acanthamoeba sp., are also commonly isolated. In conclusion, this study illustrates the significance of protozoa as vehicles augmenting the survival of MRSA and L. monocytogenes in the environment.     

Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii.

Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.     

The effect of in vitro growth conditions on the resistance of Acanthamoeba cysts

Despite increasing concerns of direct pathogenicity and/or their role as hosts for other microorganisms there are currently no standard methods for the inactivation of amoebae that belong to the genus Acanthamoeba. Methods used to grow amoebae and produce cysts for these tests may be important as they can dramatically modify cyst susceptibility. We compared resistance of cysts produced from trophozoites grown in peptone-yeast extract-glucose broth or by feeding on HEp-2 cells and then encysted in Neff's medium. We observed that trophozoites grown using HEp-2 cells as a nutrient source produce cysts that are significantly more resistant to SDS and to most biocides tested, including heat. Increased resistance is likely due to a higher proportion of mature cysts presenting thicker cell walls as demonstrated using transmission electron microscopy. This was confirmed by calcofluor white staining demonstrating higher cellulose content in cysts produced from trophozoites grown using HEp-2 cells as a feeding source. These results demonstrate that not only methods used to produce cysts from trophozoites are critical, but that methods used to grow trophozoites before encystment should also be chosen carefully. This should be taken into account for the development of protocols to evaluate biocides and antimicrobials against amoebal cysts.     

Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages

Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.     

Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella

Axenically and monoxenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were examined by polyacrylamide isoelectric focusing in the pH range 3–10. Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared. Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A. castellanii, A. polyphaga and H. vermiformis. Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns. Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM.\par Comparison of zymograms from axenically and monoxenically grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures. The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.