Elsevier

Experimental Parasitology

Volume 115, Issue 2, February 2007, Pages 181-186
Experimental Parasitology

Acanthamoeba: Could it be an environmental host of Shigella?

https://doi.org/10.1016/j.exppara.2006.08.002Get rights and content

Abstract

Shigellosis is a serious public health problem in Korea, because large outbreaks of Shigella sonnei infections were recorded in many parts of the country during the period 1998–2000. However, the epidemiological features of shigellosis are not well known. In this study, we devised conditions suitable for the growth and replication of Shigella in an amoebic intracellular environment, and investigate whether medium conditions affect the survival and replication of Shigella within Acanthamoeba. We evaluated the uptake rates of invasive and non invasive S. sonnei strains by three Acanthamoeba species, namely, A. castellanii Neff, A. astronyxis Ray & Hayes, and A. healyi OC-3A. When A. castellanii Neff was infected with S. sonnei 99OBS1 or 80DH248, shigellae was maintained for a longer time in cytoplasms than in other Acanthamoeba species. S. sonnei 99OBS1 strain (a virulent strain) was recovered in higher numbers than the non-virulent S. sonnei 80DH248 strain in all experiments. Moreover, S. sonnei was more easily engulfed by Acanthamoeba at 18 °C. The shigellae uptake rates of Neff strain, which was cultured in free-media (less nutrition), were higher (>10-fold) than those observed in original amoeba culture media (PYG medium) in all time points. S. sonnei 99OBS1 was localized, with an intact membrane, to the vacuoles of Acanthamoeba. We conclude that free-living amoebae more likely act as environmental hosts for shigellae, and thus, may have contributed to outbreaks of shigellosis in Korea.

Introduction

Free-living amoebae and bacteria interact in complex ways. It is known that amoebae act as environmental hosts for several intracellular pathogens, such as, Legionella, Chlamydia, Mycobacterium, and Listeria spp. (Amann et al., 1997, Ly and Muller, 1990, Neumeister et al., 1997, Steinert et al., 1998). Interestingly, some of the gene functions required by Legionella spp. for infecting protozoans are also required for infecting mammalian cells (Hales and Shuman, 1999, Segal and Shuman, 1999). It was even suggested that growth in an amoebic intracellular environment might assist bacteria adaptation to the mammalian phagocytic cell environment (Harb and Kwaik, 2000, Harb et al., 2000). Moreover, the incorporation of bacteria into amoebic cysts has been shown to allow bacteria to survive under adverse environmental conditions, for example, exposure to biocidal agents (Barker and Brown, 1994).

Bacillary dysentery caused by Shigella species is an important cause of acute diarrheal disease in both developing and industrialized countries (Vila et al., 1994). Shigellosis is viewed as a serious public health problem in Korea, because large outbreaks of Shigella sonnei infections in several parts of the country occurred during the period 1998–2000. The annual incidence of shigellosis was estimated at about 10 cases before 1997, but increased explosively to about 1000–2500 cases during the period 1998–2000 (National Institute of Health in Korea, 2002). Nevertheless, comparatively little is known about the epidemiological features of shigellosis, or of vectors capable of transmitting Shigella, which can only infect primates.

Free-living amoebae, such as Acanthamoeba spp., are commonly found in natural aquatic systems and in soil (Martinez and Visvesvara, 1997), and even within the intestines of humans (Zaman et al., 1999) and reptiles (Sesma and Ramos, 1989), and hence, they are expected to encounter and ingest Shigella. In fact, King et al. (1988) reported on the survival of Salmonella enterica serovar Typhimurium within Acanthamoeba castellanii during chlorination, and suggested that the amoeba afforded a protective intracellular habitat for the bacteria. In addition, Escherichia coli O157 can survive in a soil protozoan (Barker et al., 1999). However, the role played by protozoa in the environmental survival of Shigella has not been studied, and there is growing concern about the survival of these pathogens in sewage sludge disposed on land now that green laws limit sea dumping. Soils contaminated with organic matter and sewage waste contain vast numbers of protozoans like Acanthamoebae (Rodriguez-Zaragoza, 1994). Thus, it is probable that Shigella in soil and slurry will be preyed on by free-living amoebae, which could be potential vectors for the spread of this pathogen. Here, using laboratory microcosms we studied the ability of the ubiquitous free-living protozoan, Acanthamoeba, to support the growth of Shigella.

In this study, we describe the establishment of conditions required to support the uptake of Shigella in an amoebic intracellular environment.

Section snippets

Bacterial strains

Two S. sonnei strains were used in this study, 80DH248 and 99OBS1, both were isolated from human infections in Korea. 80DH248 strain was isolated at 1980 and had become avirulent after being maintained in artificial medium and being allowed to reproduce many times. The other strain, 99OBS1, was isolated during a significant outbreak shigellosis and had retained its virulence.

Cell lines and culture conditions

Three amoebae isolates were obtained from the American Type Culture Collection (ATCC), namely, A. castellanii Neff strain

Plaque formation test for virulent and non-virulent Shigella sonnei

We initiated our experiments by HeLa cell plaque forming test by S. sonnei 99OBS1 and 80DH248 strains. HeLa cells co-incubated with S. sonnei 80DH248 strain maintained an intact morphology (Fig. 1A and B), but S. sonnei 99OBS1 strain infected HeLa cells and induced aggregation (initiated after about 18 h of co-culture, Fig. 1C and D). After 24 h of co-culture, most of aggregated cells were detached from cell culture plate and many plaques were formed (Fig. 1E and F). Moreover, plaque sizes and

Discussion

Free-living amoebae are attracting research attention because they influence our perceptions of human bacterial pathogens in the environment. For example, the facultative intracellular mammalian pathogens Legionella pneumophila and mycobacteria are also known to prosper within free-living amoebae, implying that such bacteria are adapted to an intracellular environment (Neumeister et al., 1997, Steinert et al., 1998). Furthermore, L. pneumophila is known to use the same sets of genes for

Acknowledgment

This work was supported by Pusan National University Research Grant, 2004.

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