A New Primer Pair for Detection of Chlamydia pneumoniae by Polymerase Chain Reaction

In order to improve the detection and identification of Chlamydia pneumoniae, new primers for polymerase chain reaction (PCR) were designed based on the DNA base sequence within the 53-kDa protein gene, which is specific for C. pneumoniae. The primers permitted the identification of 24 C. pneumoniae strains collected from different geographical locations, but no reaction was observed with C. trachomatis, C. psittaci nor C. pecorum. The primers were unable to amplify the DNA of bacteria commonly related to respiratory tract infections. The positive amplification was achieved with only 9 EBs/assay. Therefore, the new primers seem to be useful in the diagnosis of C. pneumoniae infections.     

Interaction of Pasteurella multocida with Free-Living Amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Two distinct sensing pathways allow recognition of Klebsiella pneumoniae by Dictyostelium amoebae

Recognition of bacteria by metazoans is mediated by receptors that recognize different types of microorganisms and elicit specific cellular responses. The soil amoebae Dictyostelium discoideum feeds upon a variable mixture of environmental bacteria, and it is expected to recognize and adapt to various food sources. To date, however, no bacteria‐sensing mechanisms have been described. In this study, we isolated a Dictyostelium mutant (fspA KO) unable to grow in the presence of non‐capsulated Klebsiella pneumoniae bacteria, but growing as efficiently as wild‐type cells in the presence of other bacteria, such as Bacillus subtilis. fspA KO cells were also unable to respond to K. pneumoniae and more specifically to bacterially secreted folate in a chemokinetic assay, while they responded readily to B. subtilis. Remarkably, both WT and fspA KO cells were able to grow in the presence of capsulated LM21 K. pneumoniae, and responded to purified capsule, indicating that capsule recognition may represent an alternative, FspA‐independent mechanism for K. pneumoniae sensing. When LM21 capsule synthesis genes were deleted, growth and chemokinetic response were lost for fspA KO cells, but not for WT cells. Altogether, these results indicate that Dictyostelium amoebae use specific recognition mechanisms to respond to different K. pneumoniae elements.     

Prevalence and characterization of Chlamydia DNA in zoo animals in Japan

Because many people visit zoos, prevention of zoonoses is important from the standpoint of public health. This study examined the prevalence of Chlamydia among zoo animals in Japan by PCR and characterized these bacteria by performing phylogenetic analyses of the sequences of the variable domain (VD) 2 and VD4 regions of the ompA gene, which encodes the Chlamydia major outer membrane protein. Fecal samples were collected from 1150 zoo animals in five zoos and examined for Chlamydia DNA. Chlamydia psittaci DNA was found in 3.9% of mammals, 7.2% of birds and 8.1% of reptiles. The prevalence of Chlamydia pneumoniae DNA was significantly higher in reptiles (5.8%) than in mammals (0.3%) and birds (0.3%). Phylogenetic analysis of the ompA VD2 region from 18 samples showed that nine were in three different clusters containing C. psittaci strains, six were in a cluster containing C. pneumoniae strains and three each formed a distinct branch. Furthermore, phylogenetic analysis of the ompA VD4 region showed that C. pneumoniae DNAs from reptiles were close to those from human patients. The C. pneumoniae DNAs from the European glass lizard, Emerald tree boa, and Panther chameleon were classified in clusters that were distinct from other strains, suggesting that these reptiles had each been infected with a specific C. pneumoniae genotype. This study showed that diverse Chlamydia strains have been prevalent among a variety of zoo animals.     

Infection of Acanthamoeba castellanii by Chlamydia pneumoniae.

Chlamydia pneumoniae is an intracellular respiratory pathogen, which, similar to Legionella, might have developed mechanisms to escape the intracellular bactericidal activity of both human host cells and amoeba. We therefore investigated the intracellular growth and survival of C. pneumoniae in Acanthamoeba castellanii by using cell culture, immunofluorescence microscopy, and electron microscopy. A castellanii was incubated with purified elementary bodies of C. pneumoniae TW 183 at a concentration of 10(6) inclusion-forming units (IFU)/ml to give a ratio of approximately 1 IFU of C. pneumoniae per amoeba. Quantitative determination of chlamydial growth within A. castellanii revealed viable and infective C. pneumoniae in the range of 10(4) to 10(5) IFU/ml between days 7 and 14 postinfection. Immunofluorescence analysis and transmission electron microscopy with subsequent immunogold staining confirmed evidence of infection of the amoebae by C. Pneumoniae and additionally revealed that C. pneumoniae entered the typical growth cycle. Our results show that amoebae allow the survival of C. pneumoniae, suggesting that amoebae may serve as an additional reservoir for Chlamydia or Chlamydia-related organisms.     

Interaction of Pasteurella multocida with free-living amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Seroepidemiological survey of Chlamydia pneumoniae infection among Singapore university undergraduates: comparison between microimmunofluorescence and neutralization tests

Chlamydia pneumoniae causes acute human respiratory tract infections, and has been implicated in the pathogenesis of atherosclerosis. A seroepidemiological study using the microimmunofluorescence (MIF) technique was conducted to determine the prevalence of C. pneumoniae IgG antibodies (at titres of at least 1:16) among 205 apparently healthy Singapore university undergraduates. The overall seroprevalence was 35.1%, with significantly higher seropositivity rates among males than females (48.2% vs. 18.7%, P < 0.001). Out of 78 samples subjected to further neutralization tests in vitro, complement-independent neutralizing antibodies were detected in 22.2% (14/63) of MIF-positive sera, but only in 6.7% (1/15) of MIF-negative sera. The percentages of MIF-positive sera with neutralizing activity increased with the grade of MIF positivity, i.e. 0% (1+), 7.1% (2+), 18.8% (3+), and 63.6% (4+), with the latter being comparatively significant (P < 0.05). The percentages of reduction in the inclusion-forming unit (IFU) count correlated well with the MIF data, as reflected by their mean percentages of IFU reduction of 0.6% for MIF-negative sera, 15.4, 26.5, 40.1 and 51.5% for MIF 1+, 2+, 3+ and 4+ sera, respectively, with these differences being statistically significant. The relatively high and gender-biased seroprevalence of antibodies to C. pneumoniae among young adults highlights the importance of this common yet under-recognized infection in the local community. Furthermore, high grade MIF positivity may represent a useful serological marker of predictive value for neutralizing activity.     

Detection of Ralstonia solanacearum in Soil and Weeds from Commercial Tomato Fields Using Immunocapture and the Polymerase Chain Reaction

A detection assay for Ralstonia solanacearum in soil and weeds was developed by combining immunocapture and the polymerase chain reaction (IC‐PCR). Anti‐R. solanacearum polyclonal antibodies were produced in a white female rabbit and Dynal^® super‐paramagnetic beads were coated with purified immunoglobulinG (IgG). Using IC‐PCR, the 718 bp target DNA was amplified at a detection threshold of approximately 10⁴ colony‐forming units (CFU) bacteria per millilitre of suspension. DNA was not amplified in soil suspensions derived from autoclaved and non‐autoclaved soils, which contained R. solanacearum at 1–10⁵ CFU/g soil. However, a positive PCR result was obtained when bacteria in the soil suspensions were first enriched in nutrient broth. IC‐PCR detected R. solanacearum in tomato stems 24 h after inoculation by stem puncture with a suspension containing approximately 10⁵ CFU/ml. IC‐PCR detected the bacterium in 28 of 55 (51%) weeds and 10 of 32 (31%) soil samples. Of the weeds, Physalis minima, Amaranthus spinosus and Euphorbia hirta had the highest incidence of infection. R. solanacearum was not detected in soil taken from fallow fields, but it was discovered in some weed species. Symptomless tomato and pepper plants collected from the fields in which tomato bacterial wilt had previously occurred were found to contain R. solanacearum. These discoveries suggest that weeds and latent hosts may play a role in the survival of R. solanacearum between cropping cycles.     

Minor contribution of mutations at iniA codon 501 and embC-embA intergenic region in ethambutol-resistant clinical Mycobacterium tuberculosis isolates in Kuwait

Background

Mycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI) assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples.

Objective

The aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay.

Methods

A total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands). We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea).

Results

Following discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq) per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool.

Conclusion

The ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.     

Survival of Botrytis cinerea in Soil in South-Eastern Spain

The survival of Botrytis cinerea in sterile and unsterile soil at different temperatures and relative air humidities was investigated in south‐eastern Spain. Conidia survived only 7 days at 40^°C but, depending on relative humidity, for 30–90 days at 22^°C. High air humidity (95%) was needed to maintain soil humidity (8%) at a level that favoured conidial survival. Conidia survived better in sterile soil than in unsterile soil, probably because of the presence in the latter of soil microorganisms antagonistic to B. cinerea. Survival of conidia in environmental conditions simulating those in a greenhouse was less than 28 days. Results showed that B. cinerea conidia cannot survive over summer in south‐eastern Spain, and other primary sources of inocula are discussed.     

The Effect of Different Environmental Conditions on the Viability of Naegleria fowleri Amoebae

Naegleria fowleri, a free‐living amoeba found in soil and freshwater environments, is the causative agent of Primary Amoebic Meningoencephalitis. Infection occurs when amoebae enter the nasal cavity, attach to the nasal mucosa and travel along olfactory neurons towards the olfactory bulb. Upon reaching the central nervous system, the amoebae replicate very rapidly and can cause death in 3–10 days. Little is known about the conditions in which the amoeba can survive in the environment. We have tested conditions beyond the known boundaries on the viability of amoebae by introducing them into moderate and extreme salinity, pH, and temperatures. Our data shows that although viability expectedly decreases towards each of these extreme conditions, their tolerance was much greater than anticipated, including viability in moderate salinity, a wide pH range, and temperatures higher than the previously reported 45 °C.     

Development of a TaqMan Real‐Time Polymerase Chain Reaction Assay for Detection and Quantification of Fusarium oxysporum f. sp. lycopersici in Soil

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is an important disease of tomato. Pathogenicity and vegetative compatibility tests, although reliable, are laborious for the identification of FOL isolates and cannot efficiently quantify population densities of FOL in the soil. The objective of this study was to develop a rapid, sensitive and quantitative real‐time polymerase chain reaction (PCR) assay for detecting and quantifying FOL in soil. An inexpensive and relatively simple method for soil DNA extraction and purification was developed based on bead‐beating and a silica‐based DNA‐binding method. A TaqMan probe and PCR primers were designed using the DNA sequence of the species‐specific virulence gene SIX1, which is only present in isolates of FOL, not in isolates of other formae speciales or non‐pathogenic isolates of F. oxysporum. The real‐time PCR assay successfully amplified isolates of three races of FOL used in this study and quantified FOL DNA in soils, with a detection limit of 0.44 pg of genomic DNA of FOL in 20 μl of the real‐time PCR. A spiking test performed by adding different concentrations of conidia to soil showed a significant linear relationship between the amount of genomic DNA of FOL detected by the real‐time PCR assay and the concentration of conidia added. In addition, the real‐time PCR assay revealed a significant quadratic regression for a glasshouse experiment between disease severity and DNA concentration of FOL. The soil DNA extraction method and real‐time PCR assay developed in this study could be used to determine population densities of FOL in soil, develop threshold models to predict Fusarium wilt severity, identify high‐risk fields and measure the impact of cultural practices on FOL populations in soils.     

Increased persistence of Salmonella enterica serovar Typhi in the presence of Acanthamoeba castellanii

Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of the systemic disease typhoid fever. Transmission occurs via ingestion of contaminated food or water. S. Typhi is specific to humans, and no animal or environmental reservoirs are known. As the free-living amoeba Acanthamoeba castellanii is an environmental host for many pathogenic bacteria, this study investigates interactions between S. Typhi and A. castellanii by using cocultures. Growth of both organisms was estimated by cell count, viable count, flow cytometry, and fluorescence microscopy. Results indicate that S. Typhi can survive at least 3 weeks when grown with A. castellanii, as opposed to less than 10 days when grown as singly cultured bacteria under the same conditions. Interestingly, growth rates of amoebae after 14 days were similar in cocultures or when amoebae were singly cultured, suggesting that S. Typhi is not cytotoxic to A. castellanii. Bacteria surviving in coculture were not intracellular and did not require a physical contact with amoebae for their survival. These results suggest that S. Typhi may have a selective advantage when it is associated with A. castellanii and that amoebae may contribute to S. Typhi persistence in the environment.     

Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.     

Trace element balance is changed in infected organs during acute <Emphasis Type="Italic">Chlamydophila pneumoniae</Emphasis> infection in mice

Most infectious diseases are accompanied by changed levels of several trace elements in the blood. However, sequential changes in trace elements in tissues harbouring bacterial infections have not been studied. In the present study the respiratory pathogen Chlamydophila pneumoniae (C. pneumoniae), adapted to C57BL/6J mice, was used to study whether the balance of trace elements is changed in infected organs. Bacteria were quantitatively measured by real-time PCR in the blood, lungs, liver, aorta, and heart on days 2, 5, and 8 of the infection. Concentrations of 13 trace elements were measured in the liver, heart, and serum by inductively coupled plasma mass-spectrometry (ICP-MS). Infected mice developed expected clinical signs of disease and bacteria were found in lungs, liver, and heart on all days. The number of bacteria peaked on day 2 in the heart and on day 5 in the liver. The copper/zinc (Cu/Zn) ratio in serum increased as a response to the infection. Cu increased in the liver but did not change in the heart. Iron (Fe) in serum decreased progressively, whereas in the heart it tended to increase, and in the liver it progressively increased. C. pneumoniae may thus cause a changed trace element balance in target tissues of infection that may be pivotal for bacterial growth.     

Starvation effects on the ultrastructure of amoeba mitochondria

Summary Mitochondria in non-starved giant amoebae, Pelomyxa carolinensis, contain tubules lying at random in the matrix. Many mitochondria in starved amoebae have enlarged tubules aligned in a zigzag pattern. Tubules within the zigzag region are separated by very little matrix material. Some of these altered mitochondria are found in 70% of amoebae starved for only 24 hours, and in nearly all P. carolinensis starved for 8 days or longer. The percentage of such altered mitochondria increases from zero in most well-fed amoebae, to about 60% after two weeks of continuous starvation. Most P. carolinensis starved at 25° C survive less than three weeks. Microfilament bundles are observed in the matrix of some mitochondria in amoebae starved for more than two days.Work supported by the U. S. Atomic Energy Commission.The authors acknowledge the assistance of Miss Doris Jean Buer and Miss Patricia Ann Sustarsic.     

Root exudates modify bacterial diversity of phenanthrene degraders in PAH-polluted soil but not phenanthrene degradation rates

To determine whether the diversity of phenanthrene‐degrading bacteria in an aged polycyclic aromatic hydrocarbon (PAH) contaminated soil is affected by the addition of plant root exudates, DNA stable isotope probing (SIP) was used. Microcosms of soil with and without addition of ryegrass exudates and with ¹³C‐labelled phenanthrene (PHE) were monitored over 12 days. PHE degradation was slightly delayed in the presence of added exudate after 4 days of incubation. After 12 days, 68% of added PHE disappeared both with and without exudate. Carbon balance using isotopic analyses indicated that a part of the ¹³C‐PHE was not totally mineralized as ¹³CO₂ but unidentified ¹³C‐compounds (i.e. ¹³C‐PHE or ¹³C‐labelled metabolites) were trapped into the soil matrix. Temporal thermal gradient gel electrophoresis (TTGE) analyses of 16S rRNA genes were performed on recovered ¹³C‐enriched DNA fractions. 16S rRNA gene banding showed the impact of root exudates on diversity of PHE‐degrading bacteria. With PHE as a fresh sole carbon source, Pseudoxanthomonas sp. and Microbacterium sp. were the major PHE degraders, while in the presence of exudates, Pseudomonas sp. and Arthrobacter sp. were favoured. These two different PHE‐degrading bacterial populations were also distinguished through detection of PAH‐ring hydroxylating dioxygenase (PAH‐RHD_α) genes by real‐time PCR. Root exudates favoured the development of a higher diversity of bacteria and increased the abundance of bacteria containing known PAH‐RHD_α genes.     

Development of a PCR-based Assay for the Detection of Plasmodiophora brassicae in Soil

A single-tube nested polymerase chain reaction (STN PCR) method was developed for detecting the causal agent of clubroot disease, Plasmodiophora brassicae. Outer primer PBTZS-2 (5′-CCGAATTCGCGTCAGCGTGA-3′) to amplify a 1457 bp-fragment from P. brassicae DNA and nested primers, PBTZS-3 (5′-CCACGTCGATCACGTTGCAAT-3′) and PBTZS-4 (5′-GCTGGCGTTGATGTACTGGAA-TT-3′), to amplify a 398 bp-fragment internal of the 1457 bp-fragment were used for the STN PCR. The 398 bp-fragment was amplified from as little as 1 fg of P. brassicae DNA with the STN PCR. A protocol for extracting P. brassicae DNA directly from soil was developed. By using the protocol, DNA was extracted from artificially infested soil containing various numbers of P. brassicae resting spores and the resulting DNA was used as template for the STN PCR. As little as one resting spore of P. brassicae per g of soil was detectable with the STN PCR. The STN PCR was applied to naturally infested soil from 3 fields and one canal bed. The 398 bp-fragment was amplified from soil of 2 fields and the canal bed. To improve the detection of P. brassicae, the STN PCR products were subjected to second PCR amplification (double PCR) using the nested primers PBTZS-3 and PBTZS-4. The double PCR amplification generated a single 398 bp-DNA band which was visualized clearly on the agarose gel for all the 4 soil samples tested. A combination of the STN PCR and the double PCR appears a useful assay method for detecting P. brassicae resting spores in field soil.     

Role of Bacterial Surface Structures on the Interaction of Klebsiella pneumoniae with Phagocytes

Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis.     

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.