Strains of the food-borne pathogen Listeria monocytogenes, showing either intermediate or high-level resistance to class IIa bacteriocins, were investigated to determine characteristics that correlated with their sensitivity levels. Two intermediate and one highly resistant spontaneous mutant of L. monocytogenes B73, a highly resistant mutant of L. monocytogenes 412, and a highly resistant, defined (mptA) mutant of L. monocytogenes EGDe were compared with their respective wild-type strains in order to investigate the contribution of different factors to resistance. Decreased mannose-specific phosphotransferase system gene expression (mptA, EIIAB(Man) component) was implicated in all levels of resistance, confirming previous studies by the authors' group. However, a clear correlation between d-alanine content in teichoic acid (TA), in particular the alanine : phosphorus ratio, and a more positive cell surface, as determined by cytochrome c binding, were found for the highly resistant strains. Furthermore, two of the three highly resistant strains showed a significant increase in sensitivity towards d-cycloserine (DCS). However, real-time PCR of the dltA (d-alanine esterification), and dal and ddlA genes (peptidoglycan biosynthesis) showed no change in transcriptional levels. The link between DCS sensitivity and increased d-alanine esterification of TA may be that DCS competes with alanine for transport via the alanine transporter. A possible tendency towards increased lysinylation of membrane phospholipid in the highly resistant strains was also found. A previous study reported that cell membranes of all the resistant strains, including the intermediate resistant strains, contained more unsaturated phosphatidylglycerol, which is an indication of a more fluid cell membrane. The results of that study correlate with the possible lysinylation, decreased mptA expression, d-alanine esterification of TA and more positive cell surface charge found in this study for resistant strains. The authors' findings strongly indicate that all these factors could contribute to class IIa bacteriocin resistance and that the combination and contribution of each of these factors determine the level of bacteriocin resistance.
Les amibes libres sont des micro-organismes eucaryotes omnipresents dans les reseaux d’eau naturels et anthropiques. Bien que les amibes libres soient rarement decrites comme directement pathogenes pour l’homme, elles sont considerees comme un reservoir permettant la persistance et la propagation de bacteries pathogenes et indesirables dans les reseaux d’eau. Ce constat justifie ainsi la necessite de mieux comprendre la dynamique des populations amibiennes dans le contexte de l’approvisionnement en eau potable. Alors que leur presence dans les reseaux d’eau potable est largement documentee, l’ecologie de ces micro-organismes dans ce type d’environnement reste a ce jour tres peu decrite. Sur la base d’une campagne de prelevement d’eau couvrant le reseau d’eau potable de la ville de Paris, sur une periode d’un an, la presence et la composition des communautes amibiennes, ainsi que l’ensemble des bacteries en association avec ces dernieres (designees comme leur microbiome) ont ete caracterises par pyrosequencage. La collection de divers parametres physico-chimiques tels que l’origine de l’eau, la temperature, le pH et la concentration en chlore, a permis de mettre en evidence le fort impact de ces facteurs abiotiques dans la composition et la dynamique des communautes amibiennes omnipresentes au sein du reseau d’eau potable etudie.
Vermamoeba vermiformis is a free-living amoeba (FLA) widely distributed in the environment, known to colonize hot water networks and to be the reservoir of pathogenic bacteria such as Legionella pneumophila. FLA are partly resistant to biocides, especially in their cyst form. The control of V. vermiformis in hot water networks represents an important health issue, but there are very few data on their resistance to disinfection treatments. The sensitivity of cysts of two strains of V. vermiformis to three disinfectants frequently used in hot water networks (chlorine, heat shock, peracetic acid (PAA) mixed with hydrogen peroxide (H2O2)) was investigated. In vitro, several concentrations of biocides, temperatures and exposure times according to the French regulation were tested. Cysts were fully inactivated by the following conditions: 15 mg/L of chlorine for 10 min; 60 °C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min. For the first time, the strong efficacy of subtilisin (0.625 U/mL for 24 h), a protease, to inactivate the V. vermiformis cysts has been demonstrated. It suggests that novel approaches may be efficient for disinfection processes. Finally, V. vermifomis cysts were sensitive to all the tested treatments and appeared to be more sensitive than Acanthamoeba cysts.
Abstract Nontuberculous mycobacteria (NTM) are environmental bacteria increasingly associated to public health problems. In water systems, free-living amoebae (FLA) feed on bacteria by phagocytosis, but several bacteria, including many NTM, are resistant to this predation. Thus, FLA can be seen as a training ground for pathogenic bacteria. Mycobacterium llatzerense was previously described as frequently associated with FLA in a drinking water network. The present study aimed to characterize the interactions between M. llatzerense and FLA. M. llatzerense was internalised by phagocytosis and featured lipid inclusions, suggesting a subversion of host resources. Moreover, M. llatzerense survived and even multiplied in presence of A. castellanii . Using a genomic-based comparative approach, twelve genes involved in phagocytosis interference, described in M. tuberculosis , were identified in the M. llatzerense genome sequenced in this study. Transcriptomic analyses showed that ten genes were significantly upregulated during the first hours of the infection, which could partly explain M. llatzerense resistance. Additionally, M. llatzerense was shown to actively inhibit phagosome acidification. In conclusion, M. llatzerense presents a high degree of resistance to phagocytosis, likely explaining its frequent occurrence within FLA in drinking water networks. It underscores that NTM should be carefully monitored in water networks to prevent human health concerns.
Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of Mycobacterium avium subsp. paratuberculosis is poorly understood and several studies suggest that free-living amoebae might be a potential environmental host. Free-living amoebae are protozoa found in water and soil that are described as reservoirs of pathogenic bacteria in the environment. Indeed, bacteria able to survive within these amoebae would survive phagocytosis from immune cells. In this study, we assessed the in vitro interactions between several strains of Mycobacterium avium subsp. paratuberculosis and Acanthamoeba castellanii. The results indicate that the bacteria were able to grow within the amoeba and that they can survive for several days within their host. To explore the presence of Mycobacterium avium subsp. paratuberculosis in environmental amoebae, we sampled water from farms positive for paratuberculosis. A Mycobacterium avium subsp. paratuberculosis strain was detected within an environmental amoeba identified as related to the poorly described Rosculus genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various Mycobacterium avium subsp. paratuberculosis strains were able to grow within amoebae and that these bacteria could be found on farm within amoebae isolated from the cattle environment. It validates that infected amoebae might be a reservoir and vector for the transmission of Mycobacterium avium subsp. paratuberculosis.
