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An ex vivo porcine nasal mucosa explants model to study MRSA colonization.

ABSTRACT: Staphylococcus aureus is an opportunistic pathogen able to colonize the upper respiratory tract and skin surfaces in mammals. Methicillin-resistant S. aureus ST398 is prevalent in pigs in Europe and North America. However, the mechanism of successful pig colonization by MRSA ST398 is poorly understood. To study MRSA colonization in pigs, an ex vivo model consisting of porcine nasal mucosa explants cultured at an air-liquid interface was evaluated. In cultured mucosa explants from the surfaces of the ventral turbinates and septum of the pig nose no changes in cell morphology and viability were observed up to 72 h. MRSA colonization on the explants was evaluated followed for three MRSA ST398 isolates for 180 minutes. The explants were incubated with 3×10(8) CFU/ml in PBS for 2 h to allow bacteria to adhere to the explants surface. Next the explants were washed and in the first 30 minutes post adhering time, a decline in the number of CFU was observed for all MRSA. Subsequently, the isolates showed either: bacterial growth, no growth, or a further reduction in bacterial numbers. The MRSA were either localized as clusters between the cilia or as single bacteria on the cilia surface. No morphological changes in the epithelium layer were observed during the incubation with MRSA. We conclude that porcine nasal mucosa explants are a valuable ex vivo model to unravel the interaction of MRSA with nasal tissue.

SUBMITTER: Tulinski P

PROVIDER: S-EPMC3543263 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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An ex vivo porcine nasal mucosa explants model to study MRSA colonization.

Tulinski Pawel P Fluit Ad C AC van Putten Jos P M JP de Bruin Alain A Glorieux Sarah S Wagenaar Jaap A JA Duim Birgitta B

PloS one 20130111 1

Staphylococcus aureus is an opportunistic pathogen able to colonize the upper respiratory tract and skin surfaces in mammals. Methicillin-resistant S. aureus ST398 is prevalent in pigs in Europe and North America. However, the mechanism of successful pig colonization by MRSA ST398 is poorly understood. To study MRSA colonization in pigs, an ex vivo model consisting of porcine nasal mucosa explants cultured at an air-liquid interface was evaluated. In cultured mucosa explants from the surfaces of ...[more]

PMID: 23326505

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Project description:The nasal mucosa provides first line defense against inhaled pathogens while creating a unique microenvironment for bacterial communities. Studying the impact of microbiota in the nasal cavity has been difficult due to limitations with current models including explant cultures, primary cells, or neoplastic cell lines. Most notably, none have been shown to support reproducible colonization by bacterial communities from human donors. Therefore, to conduct controlled studies of the human nasal ecosystem, we have developed a novel ex vivo mucosal model that supports bacterial colonization of a cultured host mucosa created by immortalized human nasal epithelial cells (NEC). For this model, immortalized NEC established from 5 male and 5 female donors were cultured with an air-interfaced, apical surface on a porous transwell membrane. NEC were grown from nasal turbinate tissues harvested from willed bodies or from discarded tissue collected during sinonasal procedures. Immortalized cells were evaluated through molecular verification of cell type, histological confirmation of tissue differentiation including formation of tight junctions, NEC multilayer viability, metabolism, physiology and imaging of the luminal surface by scanning electron microscopy. Results showed proper differentiation and multilayer formation at seven to 10 days after air interface that was maintained for up to 3 weeks. The optimized mucosal cultures created an environment necessary to sustain colonization by nasal microbiomes (NMBs) that were collected from healthy volunteers, cryogenically preserved and characterized with customized quantitative polymerase chain reaction (qPCR) arrays. Polymicrobial communities of nasal bacteria associated with healthy and inflamed states were consistently reproduced in matured NEC co-cultures by transplant of NMBs from multiple community types. The cultured NMBs were stable after an initial period of bacterial replication and equilibration. This novel ex vivo culture system is the first model that supports controlled cultivation of NMBs, allowing for lab-based causation studies and further experimentation to explore the complexities of host-microbe and microbe-microbe interactions.

Project description:Methicillin-resistant Staphylococcus aureus (MRSA) infection is a leading cause of hospitalization and medical visits among individuals experiencing homelessness and also among persons who inject drugs (PWID), populations with significant overlap in urban centers in the USA. While injection drug use is a risk factor for MRSA skin infections, MRSA is also known to transmit easily in crowded, public locations in which individuals have reduced personal hygiene. Individuals in urban centers who experience homelessness or drug addiction may spend significant amounts of time in environments where MRSA can be easily transmitted, and may also experience reduced access to facilities to maintain personal hygiene. We assessed the relationship between homelessness, personal hygiene, and MRSA nasal colonization, a proxy for MRSA infection risk, in a study of PWID in Boston, MA (n?=?78). Sleeping in a homeless shelter for at least one night in the last 3 months was significantly associated with MRSA nasal colonization (OR 3.0; p?=?0.02; 95% CI 1.2, 7.6). Sleeping at more than one place during the last week (considered a metric of elevated housing instability) was also associated with a threefold increase in odds of MRSA nasal colonization (OR 3.1; p?=?0.01; 95% CI 1.3, 7.6). MRSA nasal colonization was strongly associated with use of public showers (OR 13.7; p?=?0.02; 95% CI 1.4, 132.8), although few people in this study (4 of 78) reported using these public facilities. Sharing bedding with other people was also associated with increased risk of MRSA colonization (OR 2.2; p?=?0.05; 95% CI 1.0-4.7). No associations between hand hygiene, frequency of bathing or clothes laundering, or street sleeping were observed. Use of public facilities supporting persons experiencing homelessness and housing instability, including shelters and public showers, is associated with an increased risk of MRSA nasal colonization in this study. Personal hygiene behaviors appear less associated with MRSA nasal colonization. Environmental assessments of MRSA contamination in homeless shelters and public sanitation facilities are warranted so as to inform appropriate intervention activities.

Dataset Information

An ex vivo porcine nasal mucosa explants model to study MRSA colonization.

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An ex vivo porcine nasal mucosa explants model to study MRSA colonization.

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