Development of a modified gentamicin protection assay to investigate the interaction between Campylobacter jejuni and Acanthamoeba castellanii ATCC 30010

The aim of this study was to evaluate Campylobacter jejuni NTCT 11168 adhesion to abiotic and biotic surfaces when grown in co-culture with Escherichia coli ATCC 11229 and/or Listeria monocytogenes 4b. Adhesion of C. jejuni to polystyrene and to Caco-2 cells and Acanthamoeba castellanii was lower for at least 3 log CFU/mL compared to E. coli and L. monocytogenes. Electron micrographs of ultrathin sections revealed interactions of C. jejuni with host cells. In co-culture with E. coli and L. monocytogenes, adhesion of C. jejuni to all tested surfaces was significantly increased for more than 1 log CFU/mL. There was 10% higher aggregation for C. jejuni than for other pathogens, and high co-aggregation of co-cultures of C. jejuni with E. coli and L. monocytogenes. These data show that C. jejuni in co-cultures with E. coli and L. monocytogenes present significantly higher risk than C. jejuni as mono-cultures, which need to be taken into account in risk evaluation. C. jejuni adhesion is a prerequisite for their colonization, biofilm formation, and further contamination of the environment. C. jejuni survival under adverse conditions as a factor in their pathogenicity and depends on their adhesion to different surfaces, not only as individual strains, but also in co-cultures with other bacteria like E. coli and L. monocytogenes.

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Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci

Infection and Immunity ◽

10.1128/iai.00119-19 ◽

2019 ◽

Vol 87 (5) ◽

Cited By ~ 7

Author(s):

Jin-Hahn Kim ◽

Akhilesh Kumar Chaurasia ◽

Nayab Batool ◽

Kwan Soo Ko ◽

Kyeong Kyu Kim

Keyword(s):

Host Cells ◽

Intracellular Bacteria ◽

Protection Assay ◽

Host Cell Membrane ◽

Bacterial Killing ◽

Content Type ◽

Gentamicin Protection Assay ◽

Enzyme Protection ◽

The Difference ◽

Kinetics Of

ABSTRACTPrecise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellularStaphylococcus aureus. We found that the difference between the two methods for the recovery of intracellular CFU ofS. aureuswas about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

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Effect of environmental stress factors on the uptake and survival of Campylobacter jejuni in Acanthamoeba castellanii

BMC Microbiology ◽

10.1186/1471-2180-12-232 ◽

2012 ◽

Vol 12 (1) ◽

pp. 232 ◽

Cited By ~ 12

Author(s):

Xuan Bui ◽

Klaus Qvortrup ◽

Anders Wolff ◽

Dang Bang ◽

Carole Creuzenet

Keyword(s):

Environmental Stress ◽

Campylobacter Jejuni ◽

Acanthamoeba Castellanii ◽

Stress Factors

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Survival of Campylobacter jejuni in Amoebae enhances subsequent invasion of mammalian cells

10.1101/2021.11.25.469992 ◽

2021 ◽

Author(s):

Fauzy Nasher ◽

Brendan W. Wren

Keyword(s):

Epithelial Cells ◽

Campylobacter Jejuni ◽

Mammalian Cells ◽

Acanthamoeba Castellanii ◽

Public Health Problem ◽

Fold Increase ◽

Unicellular Eukaryote ◽

Major Public Health Problem ◽

Survival Assay ◽

Insight Into

The ubiquitous unicellular eukaryote, Acanthamoeba, is known to play a role in the survival and dissemination of Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne gastroenteritis world-wide and is a major public health problem. The ability of C. jejuni to interact and potentially invade epithelial cells is thought to be key for disease development in humans. We examined C. jejuni grown under standard laboratory conditions,11168HCBA with that harvested from within Acanthamoeba castellanii (11168HAC/CBA) or Acanthamoeba polyphaga (11168HAP/CBA), and compared their ability to invade different cell lines. C. jejuni harvested from within amoebae had a ~3.7-fold increase in invasiveness into T84 human epithelial cells and a striking ~11-fold increase for re-entry into A. castellanii cells. We also investigated the invasiveness and survivability of six diverse representative C. jejuni strains within Acanthamoeba spp., our results confirm that invasion and survivability is likely host cell dependent. Our survival assay data led us to conclude that Acanthamoeba spp. are a transient host for C. jejuni and that survival within amoebae pre-adapts C. jejuni and enhances subsequent cell invasion. This study provides new insight into C. jejuni interactions with amoebae and its increased invasiveness potential in mammalian hosts.

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Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages

BIO-PROTOCOL ◽

10.21769/bioprotoc.1235 ◽

2014 ◽

Vol 4 (18) ◽

Author(s):

Sargurunathan Subashchandrabose ◽

Harry Mobley

Keyword(s):

Bacterial Survival ◽

Protection Assay ◽

Gentamicin Protection Assay

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Survival of Campylobacter jejuni in co-culture with Acanthamoeba castellanii: role of amoeba-mediated depletion of dissolved oxygen

Environmental Microbiology ◽

10.1111/j.1462-2920.2011.02655.x ◽

2011 ◽

Vol 14 (8) ◽

pp. 2034-2047 ◽

Cited By ~ 26

Author(s):

Xuan Thanh Bui ◽

Anne Winding ◽

Klaus Qvortrup ◽

Anders Wolff ◽

Dang Duong Bang ◽

...

Keyword(s):

Dissolved Oxygen ◽

Campylobacter Jejuni ◽

Acanthamoeba Castellanii

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Construction of Aminoglycoside-Sensitive Burkholderia cenocepacia Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay

Applied and Environmental Microbiology ◽

10.1128/aem.03024-09 ◽

2010 ◽

Vol 76 (10) ◽

pp. 3170-3176 ◽

Cited By ~ 51

Author(s):

Mohamad A. Hamad ◽

Alexander M. Skeldon ◽

Miguel A. Valvano

Keyword(s):

Efflux Pump ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Eukaryotic Cell ◽

Burkholderia Cenocepacia ◽

Intracellular Bacteria ◽

Raw 264.7 ◽

Protection Assay ◽

Intracellular Infection ◽

Gentamicin Protection Assay

ABSTRACT Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.

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