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.     

Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.

Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata . The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions.     

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis survival within amoebal cysts

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.     

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.     

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.     

Antitumor effects of a variety of non-viable bacteria against the murine EL-4 lymphoma

Summary Suppression of growth of the EL-4 murine lymphoma was achieved in syngeneic animals by the simultaneous injection of EL-4 cells and a variety of non-viable bacteria. Heat-killed Staphylococcus aureus, Mycobacterium bovis, Propionibacterium acnes, Streptococcus mutans, and Escherichia coli injected together with viable EL-4 cells resulted in significant tumor suppression. The antitumor effects observed were dependent upon the numbers of bacteria injected. Heat-killed Listeria monocytogenes caused no significant tumor suppression. Injections of S. aureus, M. bovis, P. acnes, and S. mutans into sites previously injected with EL-4 cells also produced significant antitumor effects, while L. monocytogenes and E. coli did not.     

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 μg ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 μg ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 μg ml(-1) (FA) and 5000 μg ml(-1) (GA), for L. monocytogenes it was >5000 μg ml(-1) (for both phenolic acids), and for S. aureus it was 5000 μg ml(-1) (FA) and >5000 μg ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.     

Amoebae promote persistence of epidemic strains of MRSA

The control of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) infection is of concern worldwide. Given the evidence that several pathogenic species replicate within amoebae and emerge more virulent and more resistant and the abundance of amoebae in healthcare settings, we investigated interactions of Acanthamoeba polyphaga with epidemic MRSA isolates. MRSA proliferated in the presence of amoebae, attributable partly to intracellular replication. Following 24 h of co-culture, confocal microscopy revealed that c. 50% amoebae had viable MRSA within phago-lysosomes and 2% of amoebae were heavily infected with viable cocci throughout the cytoplasm. Infection control strategies should recognize the contribution of protozoa.     

Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.     

Effect of milk proteins on adhesion of bacteria to stainless steel surfaces.

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins alpha-casein, beta-casein, kappa-casein, and alpha-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system.

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Relationship between the susceptibility of various bacteria to active oxygen species and to intracellular killing by macrophages

The susceptibilities of six micro-organisms to active oxygen species generated in the xanthine oxidase-mediated bactericidal system were as follows: Escherichia coli 81 greater than or equal to Listeria monocytogenes EGD greater than or equal to Salmonella typhimurium HKB-1 greater than or equal to Staphylococcus aureus Smith much greater than Mycobacterium tuberculosis H37Rv approximately equal to Candida albicans NIH A207 (the last two organisms were essentially resistant to this system). The H2O2-Fe-mediated halogenation system exhibited a higher microbicidal activity. When the micro-organisms were compared for their sensitivity to bactericidal activity of resident mouse peritoneal macrophages (M phi s), C. albicans, Staph. aureus and E. coli were killed rapidly, whereas M. tuberculosis, L. monocytogenes and S. typhimurium were more resistant. In tests for the ability to trigger an oxidative burst in mouse peritoneal M phi s (as measured by chemiluminescence), Staph. aureus showed the highest activity followed by the other organisms in the following order: C. albicans greater than E. coli greater than L. monocytogenes congruent to M. tuberculosis. S. typhimurium exhibited no triggering activity. The high susceptibility of Staph. aureus and E. coli to M phi bactericidal activity, and the partial resistance of L. monocytogenes and M. tuberculosis, correlated with their susceptibility to active oxygen and the H2O2-Fe-mediated halogenation reaction.     

Marine Amoebae from Waters of Northwest Spain, with Comments on a Potentially Pathogenic Euryhaline Species

Of 17 species of free-living amoebae identified in various samples of salt water, only 1. Acanthamoeba polyphaga , is known to be a potential pathogen. While no deaths occurred when laboratory animals were inoculated with A. polyphaga to test for pathogenicity, the protozoa were present in the brain, liver and lungs of some but not all of the animals.     

Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat

Ten strains of bacteriocin-producing lactic acid bacteria were isolated from retail cuts of meat. These 10 strains along with 11 other bacteriocin-producing lactic acid bacteria were tested for inhibitory activity against psychotrophic pathogens, including four strains of Listeria monocytogenes, two strains of Aeromonas hydrophila, and two strains of Staphylococcus aureus. Inhibition due to acid, hydrogen peroxide, and lytic bacteriophage were excluded. The proteinaceous nature of the inhibitory substance was confirmed by demonstration of its sensitivity to proteolytic enzymes. Eight of the meat isolates had inhibitory activity against all four L. monocytogenes strains. Bacteriocin activity against L. monocytogenes was found in all of the strains obtained from other sources. Activity against A. hydrophila and S. aureus was also common.     

Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat.

Ten strains of bacteriocin-producing lactic acid bacteria were isolated from retail cuts of meat. These 10 strains along with 11 other bacteriocin-producing lactic acid bacteria were tested for inhibitory activity against psychotrophic pathogens, including four strains of Listeria monocytogenes, two strains of Aeromonas hydrophila, and two strains of Staphylococcus aureus. Inhibition due to acid, hydrogen peroxide, and lytic bacteriophage were excluded. The proteinaceous nature of the inhibitory substance was confirmed by demonstration of its sensitivity to proteolytic enzymes. Eight of the meat isolates had inhibitory activity against all four L. monocytogenes strains. Bacteriocin activity against L. monocytogenes was found in all of the strains obtained from other sources. Activity against A. hydrophila and S. aureus was also common.     

Marine amoebae from waters of northwest Spain, with comments on a potentially pathogenic euryhaline species

Of 17 species of free-living amoebae identified in various samples of salt water, only 1, Acanthamoeba polyphaga, is known to be a potential pathogen. While no deaths occurred when laboratory animals were inoculated with A. polyphaga to test for pathogenicity, the protozoa were present in the brain, liver and lungs of some but not all of the animals.     

Effects of acidified sodium chlorite, cetylpyridinium chloride and hot water on populations of Listeria monocytogenes and Staphylococcus aureus on beef

AIMS: The present study was designed to determine the individual and combined effects of acidified sodium chlorite (ASC, 0.1%, 24 +/- 1 degrees C), cetylpyridinium chloride (CPC, 0.5%, 24 +/- 1 degrees C) and hot water (HW, 93 +/- 1 degrees C) treatments on the survival of Listeria monocytogenes and Staphylococcus aureus. METHODS AND RESULTS: Beef samples inoculated with L. monocytogenes and S. aureus were treated with nine different applications singly or in combination. Treatment groups comprised (i) untreated control; (ii) sterile tap water; (iii) 0.1% ASC; (iv) 0.5% CPC; (v) HW; (vi) HW followed by 0.1% ASC; (vii) HW followed by 0.5% CPC; (viii) 0.1% ASC followed by HW; (ix) 0.5% CPC followed by HW. Compared with the untreated control group, the reductions in L. monocytogenes populations were 1.14-2.31 log CFU g(-1), while the reductions in S. aureus populations were 0.83-2.74 log CFU g(-1) on day 0. CONCLUSION: The reduction effect that occurred after combined treatment with ASC followed by HW, HW followed by ASC, CPC followed by HW and HW followed by CPC was found to be significantly greater (P < 0.05) than after treatment with ASC and CPC alone on days 0, 2 and 4 of storage. SIGNIFICANCE AND IMPACT OF THE STUDY: ASC, CPC and HW treatments can be used to reduce L. monocytogenes and S. aureus, which would provide an additional measure of safety on the production line.     

Functional activity of mouse peritoneal macrophages contaminated with gram-positive bacteria

The analysis of the functional and enzymatic activity of mouse peritoneal macrophages contaminated with Staphylococcus aureus and Listeria monocytogenes virulent strains is presented. The low bactericidal and digestive activity of these cells with respect to the above-mentioned microorganisms was determined. In this study a decrease in the activity of plasmatic membrane enzymes (5'-nucleotidase and ATP-ase) of macrophages contaminated with S. aureus and L. monocytogenes was observed, which was indicative of the stimulation of phagocytes. A rise in the activity of the oxygen-dependent system of macrophages contaminated with S. aureus and L. monocytogenes was detected by means of the nitro blue tetrazolium test. At the same time a decrease in the intracellular content of nitrogen oxide end metabolites in macrophages was detected with a rise in content of nitrogen oxide in the supernatants.