Dataset Information

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

ABSTRACT: Methicillin-resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well-characterized Staphylococcus aureus invasive strains belonging to the main ST-MRSA-SCCmec clones. To overcome the limitations of the cell culture method, we comparatively analyzed the ability of internalization within human MG-63 osteoblasts with imaging flow cytometry (IFC). After evaluation by cell culture assay, the MRSA clones in the study were all able to readily internalize at 3h postinfection, the persistence of intracellular bacteria was evaluated at 24h both by routine cell culture and IFC assay, after vancomycin-BODIPY staining. A statistical difference of persistence was found in ST5-SCCmecII (26.59%), ST228-SCCmecI (20.25%), ST8-SCCmecIV (19.52%), ST239-SCCmecIII (47.82%), and ST22-SCCmecIVh (50.55%) showing the same ability to internalize as ATCC12598 (51%), the invasive isolate used as control strain for invasion and persistence assays. We demonstrated that the intracellular persistence process depends on the total number of infected cells. Comparing our data obtained by IFC with those of the cell culture assay, we obtained greater reproducibility rates and a number of intracellular bacteria, with the advantage of analyzing live host cells. Moreover, with some limitations related to the lack of whole-genome sequencing analysis, we validated the different proclivities to persist in the main Italian HA-MRSA invasive isolates and our results highlighted the heterogeneity of the different clones to persist during cell infection.

SUBMITTER: Bongiorno D

PROVIDER: S-EPMC7221431 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

Bongiorno Dafne D Musso Nicolò N Lazzaro Lorenzo Mattia LM Mongelli Gino G Stefani Stefania S Campanile Floriana F

MicrobiologyOpen 20200401 5

Methicillin-resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well-characterized Staphylococcus aureus invasive strains belonging to the main ST-MRSA-SCCmec clones. To overcome the limitations of the cell culture method, we comparatively anal ...[more]

PMID: 32237200

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Project description:Rapid laboratory diagnosis is critical for treating, managing, and preventing methicillin-resistant Staphylococcus aureus (MRSA) infections. We evaluated and compared the potential for MRSA detection of five chromogenic media, Brilliance MRSA agar (Oxoid), ChromID (bioMérieux), MRSASelect (Bio-Rad), CHROMagar (CHROMagar Microbiology), and BBL-CHROMagar (BD Diagnostics). Media were tested with log serial dilutions (10(0) to 10(6) CFU) of pure isolates of MRSA (n = 60), non-MRSA (n = 27), and defined mixtures thereof simulating clinical samples (n = 84). Further evaluations were done on pre-enriched nasal and groin screening swabs (n = 213) from 165 hospitalized patients. Randomized samples were spiral plated on each medium and independently scored by five investigators for characteristic colonies at 24 and 48 h of incubation. Confirmatory testing of up to five putative MRSA colonies recovered from each medium was done. The cumulative average sensitivity with isolates, mixtures, and clinical samples was the highest for Brilliance MRSA agar (97%) and similar for the other four media (>or=92%). The cumulative average specificity was the highest for BBL-CHROMagar (99%), followed by MRSASelect (98%), CHROMagar (97%), ChromID (89%), and Brilliance MRSA agar (86%). All of the media detected MRSA at 10 and 1 CFU, although at these low loads, few MRSA samples harboring SCCmec type III or IV were misinterpreted as non-MRSA by investigators. False-positive results were mainly due to methicillin-resistant S. epidermidis. For an arbitrary MRSA prevalence of 5% and based on patient sample evaluations, the positive predictive values for BBL-CHROMagar and CHROMagar ( approximately 84%) were the highest. The negative predictive values of all of the media were >or=92% for MRSA prevalences ranging from 5% to 30%. In conclusion, BBL-CHROMagar and CHROMagar gave the best overall results for detection of MRSA, irrespective of the sample concentration, investigator, or incubation period.

Project description:Methicillin resistant Staphylococcus aureus (MRSA) infection is becoming refractory to existing antibiotic therapy owing to the inherent ability of S. aureus to develop rapid resistance and is considered a major threat to public health. We found that a natural isolate of Bacillus pumilus from the Columbia River Estuary produces a strong anti-MRSA compound, amicoumacin A. As amicoumacin A has been reported to exhibit anti-microbial, anti-inflammatory, and anti-ulcer activities, we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in the expression of genes involved in several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which has been shown to be induced in response to a collapse of membrane potential. In order to gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by serial passage method was carried out. Single point mutations resulting in codon substitutions were uncovered in several distinct genes: ksgA, RNA dimethyltranferase; fusA, elongation factor G; dnaG, primase, ; lacD, tagatose 1,6-bisphosphate aldolase, ; and SACOL0611, encoding a putative glycosyl transferase gene. Based on these results, a candidate approach was undertaken to recreate the same amino acid substitution individually in FusA and KsgA, each of which resulted in two-fold resistance towards amicoumacin A. The fusA gene is known as the site for fusidic acid- resistant mutations; however the codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance occur in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.

Dataset Information

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

Publications

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

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