Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Summary

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Abstract

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Introduction

Before the advent of molecular diagnosis, microorganisms present in environmental niches or in clinical samples were often detected by cultivating them on different selective media, mainly on agar in Petri dishes. The phenotype of the bacterial colonies and their metabolic activity then allowed bacterial classification at species level. Broth may also be used to increase the sensitivity of detection. However, both techniques do not allow the recovery of bacteria that grow slowly or not at all on these media. This is the reason why molecular approaches are so widely used nowadays. Nevertheless, detection of DNA provides no clue on the viability of the bacteria. Moreove....

Protocol

1. Amoebal Coculture

1.1 Sample preparation

1. Environmental sample
   1. Water samples
      Filter the water sample (500 ml to 1 L) through a 0.22 μm pore size membrane. Then, shake the membrane in Page's amoeba saline medium PAS (120 mg of NaCl, 4 mg of MgSO₄•7H₂O, 4 mg of CaCl₂•2H₂O, 142 mg of Na₂HPO_4, and 136 mg of KH₂PO₄ in 1 L of distilled water).
   2. Solid samples
      Resuspend solid samples like soil or sand samples and semi-solid samples such as act....

Results

Using amoebal coculture and amoebal enrichment, a whole range of environmental and/or pathogenic bacteria were discovered (Table 1).

Amoebal coculture was used by our group and others to analyze environmental samples, water treatment plants and water distribution systems. A broad range of microorganisms could be isolated with this technique. The most common bacteria isolated by amoebal coculture are members of the Mycobacterium genus that could be recovered from water.......

Discussion

Amoebal coculture and amoebal enrichment are efficient methods that allowed the isolation of many new bacterial and amoebal species. Results obtained with these methods confirm the ubiquitous presence of both amoebae and amoeba-resisting bacteria in the environment, and most interestingly in manmade water networks that are considered to be controlled by chemical treatments such as chlorination and ozonation. Amoebal coculture and amoebal enrichment are essential tools to isolate and cultivate these potentially pathogenic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glucose monohydrate	Merck, Darmstadt, Germany	108342
0.22 μm pore size membrane	Merck Millipore, Darmstadt, Germany	SCVPU11RE
proteose peptone	Becton-Dickinson, Franklin Lakes, NJ	211693
yeast extract	Becton-Dickinson, Franklin Lakes, NJ	212750
Cell culture flasks	Becton-Dickinson, Franklin Lakes, NJ	353135
Kova slide	Hycor, Indianapolis, IN	87144
cell culture microplates	Corning Inc, Corning, NY	3524
Diff-Quik staining kit	Siemens Healthcare diagn., Munich, Germany	130832
Ziehl fuchsin	Fluka, St-Louis, MI	21820
basic fuchsin	Sigma, St-Louis, MI	857843
Phenol	Sigma, St-Louis, MI	P1037	Corrosive and mutagenic
malachite green oxalate	Fluka, St-Louis, MI	63160
Paraformaldehyde 16% solution	Electron Microscopy Sciences, Hatfield, PA	15710
Saponin	Sigma, St-Louis, MI	84510