Evaluation of the MC-Media Pad® Rapid Aerobic Count Device for the Enumeration of Total Aerobic Counts in a Variety of Foods: Collaborative Study, First Action Official MethodSM 2019.02.

BACKGROUND: The MC-Media Pad® Rapid Aerobic Count (RAC) is a ready-to-use culture device combining a test pad coated with medium and water absorption polymers that are designed for the rapid quantification of total aerobic bacteria in food products. OBJECTIVE: The MC-Media Pad RAC was compared to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, Chapter 3.02: Quantitative Analysis of Bacteria in Foods as Sanitary Indicators for raw ground pork and the Standard Methods for the Examination of Dairy Products, Chapter 6: Microbial Count Methods for yogurt drink. METHOD: The candidate method was evaluated against the reference methods using a paired study design in a multi-collaborator study, following the current AOAC INTERNATIONAL Official Methods of AnalysisSM Appendix J guidelines. Three target contamination levels (low, medium, and high) were evaluated. MC-Media Pad RAC devices were enumerated after 24 and 48 h of incubation. RESULTS: Plate counts obtained by both methods were log10-transformed and the difference of means (including 95% confidence intervals), repeatability SD, and reproducibility SD were determined for each contamination level. All 95% confidence intervals for mean difference fell easily within ±0.10, the performance requirement being ±0.5. CONCLUSION: The MC-Media Pad RAC (for both 24 and 48 h) and both reference methods for each contamination level were therefore shown to be equivalent, with 97.5% confidence. HIGHLIGHTS: The new method offers a convenient alternative to the reference methods for detection of aerobic plate count in food products, yielding reliable and comparable results in 24 or 48 h compared to 48 h for the reference methods.

Evaluation of the MC-Media PadTM Yeast and Mold Device for the Enumeration of Yeast and Mold: Collaborative Study, First Action 2018.02.

Background: The MC-Media Pad™ Yeast and Mold (YM) is a ready-to use culture device that combines a test pad coated with medium and water-absorption polymers that is designed for the rapid quantification of yeast and mold in food products. Objective: The MC-Media Pad YM was compared with the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the enumeration of yeast and mold in frozen orange juice concentrate. Methods: The candidate method was evaluated using a paired study design in a multilaboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels (low, 10-100 CFU/g; medium, 100-1000 CFU/g; and high 1000-10 000 CFU/g) and an uninoculated control level (0 CFU/g) were evaluated. MC-Media Pad YM devices were enumerated after 48 and 72 h of incubation. Results: Plate count obtained by both methods were log-transformed and the difference of means (including 95% confidence intervals), repeatability SD, and SD were determined for each contamination level. Conclusions: No statistical difference was observed between the MC-Media Pad YM (for both 48 and 72 h) and the FDA BAM for each contamination level. Highlights: The new method offers a convenient alternative to the reference method (FDA BAM) for detection of yeast and mold contamination in food products, yielding reliable and comparable results in 48 h compared to 5 days for the reference method.

Development of a micromanipulation method for single cell isolation of prokaryotes and its application in food safety.

For nearly a century, conventional microbiological methods have been standard practice for detecting and identifying pathogens in food. Nevertheless, the microbiological safety of food has improved and various rapid methods have been developed to overcome the limitations of conventional methods. Alternative methods are expected to detect low cell numbers, since the presence in food of even a single cell of a pathogenic organism may be infectious. With respect to low population levels, the performance of a detection method is assessed by producing serial dilutions of a pure bacterial suspension to inoculate representative food matrices with highly diluted bacterial cells (fewer than 10 CFU/ml). The accuracy of data obtained by multiple dilution techniques is not certain and does not exclude some colonies arising from clumps of cells. Micromanipulation techniques to capture and isolate single cells from environmental samples were introduced more than 40 years ago. The main limitation of the current micromanipulation technique is still the low recovery rate for the growth of a single cell in culture medium. In this study, we describe a new single cell isolation method and demonstrate that it can be used successfully to grow various types of microorganism from picked individual cells. Tests with Gram-positive and Gram-negative organisms, including cocci, rods, aerobes, anaerobes, yeasts and molds showed growth recovery rates from 60% to 100% after micromanipulation. We also highlight the use of our method to evaluate and challenge the detection limits of standard detection methods in food samples contaminated by a single cell of Salmonella enterica.

A new ultrasonic high-throughput instrument for rapid DNA release from microorganisms.

Rapid detection methods for microbiological contamination are requested by many industries that respond to public health concerns. The control laboratories are replacing traditional culturing methods with faster assays based on nucleic acid amplification technologies, such as real-time PCR. However, an optimal nucleic acid sample preparation method is critical for the sensitivity and specificity of such tools. A high-throughput automated external ultrasonic device was developed for rapid lysing of microorganisms. Based on Ct values obtained from real-time PCR, there was efficient DNA release from the 16 microorganisms tested, including Gram positive and negative bacteria, bacterial spores, yeasts and spores of molds. Linearity of the lysis method was also demonstrated for Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Candida albicans and the spores of Aspergillus brasiliensis, with correlation coefficients (r(2)) between 0.90 and 0.98. After sonication, PCR analysis of the lysates revealed earlier Ct values (0.83 to 7.49) for S. aureus, P. aeruginosa and S. enterica compared to the bead-beating method of lysis. These results demonstrated more efficient DNA release from bacteria with the ultrasonication system. Nevertheless, for fungi, the Ct values were 0.94 to 1.61 later for sonication than for bead beating. This study demonstrates that 4min of sonication with this new automated high-throughput instrument allows for the efficient lysis of a large range of microorganisms.

Fluorescence-based rapid detection of microbiological contaminants in water samples.

Microbiological contamination of process waters is a current issue for pharmaceutical industries. Traditional methods require several days to obtain results; therefore, rapid microbiological methods are widely requested to shorten time-to-result. Milliflex Quantum was developed for the rapid detection and enumeration of microorganisms in filterable samples. It combines membrane filtration to universal fluorescent staining of viable microorganisms. This new alternative method was validated using European and United States Pharmacopeia definitions, with sterile water and/or sterile water artificially contaminated with microorganisms. The Milliflex Quantum method was demonstrated to be reliable, robust, specific, accurate, and linear over the whole range of assays following these guidelines. The Milliflex Quantum system was challenged to detect natural contaminants in different types of pharmaceutical purified process waters. Milliflex Quantum was demonstrated to detect accurately contaminants 3- to 7-fold faster than traditional membrane filtration method. The staining procedure is nondestructive allowing downstream identification following a positive result. The Milliflex Quantum offers a fast, sensitive, and robust alternative to the compendial membrane filtration method.

The AcrAB-TolC pump is involved in macrolide resistance but not in telithromycin efflux in Enterobacter aerogenes and Escherichia coli.

The role of the AcrAB-TolC pump in macrolide and ketolide susceptibility in Escherichia coli and Enterobacter aerogenes was studied. Efflux pump inhibitor restored erythromycin, clarithromycin, and telithromycin susceptibilities to multidrug-resistant isolates. No modification of telithromycin accumulation was detected in E. aerogenes acrAB or tolC derivatives compared to that in the parental strain. Two independent efflux pumps, inhibited by phenylalanine arginine beta-naphthylamide, expel macrolides and telithromycin in E. aerogenes.

Enterobacter aerogenes OmpX, a cation-selective channel mar- and osmo-regulated.

The ompX gene of Enterobacter aerogenes was cloned. Its overexpression induced a decrease in the major porin Omp36 production and consequently a beta-lactam resistance was noted. Purified outer membrane protein X (OmpX) was reconstituted into artificial membranes and formed ion channels with a conductance of 20 pS in 1 M NaCl and a cationic selectivity. Both MarA expression and high osmolarity induced a noticeable increase of the OmpX synthesis in the E. aerogenes ATCC 13048 strain. In addition, OmpX synthesis increased under conditions in which the expression of the E. aerogenes major non-specific porins, Omp36 and Omp35, decreased.

RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes.

Multidrug resistance (MDR) in Enterobacter aerogenes can be mediated by induction of MarA, which is triggered by certain antibiotics and phenolic compounds. In this study, we identified the gene encoding RamA, a 113-amino-acid regulatory protein belonging to the AraC-XylS transcriptional activator family, in the Enterobacter aerogenes ATCC 13048 type strain and in a clinical multiresistant isolate. Overexpression of RamA induced an MDR phenotype in drug-susceptible Escherichia coli JM109 and E. aerogenes ATCC 13048, as demonstrated by 2- to 16-fold-increased resistance to beta-lactams, tetracycline, chloramphenicol, and quinolones, a decrease in porin production, and increased production of AcrA, a component of the AcrAB-TolC drug efflux pump. We show that RamA enhances the transcription of the marRAB operon but is also able to induce an MDR phenotype in a mar-deleted strain. We demonstrate here that RamA is a transcriptional activator of the Mar regulon and is also a self-governing activator of the MDR cascade.

Modification of outer membrane protein profile and evidence suggesting an active drug pump in Enterobacter aerogenes clinical strains.

Two clinical strains of Enterobacter aerogenes that exhibited phenotypes of multiresistance to beta-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two known outer membrane proteins, OmpX and LamB. In addition, the full-length O-polysaccharide phenotype was replaced by a semirough Ra phenotype. Moreover, in one isolate the intracellular accumulation of chloramphenicol was increased in the presence of the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone, suggesting an energy-dependent efflux of chloramphenicol in this strain. The resistance strategies used by these isolates appear to be similar to that induced by stress in Escherichia coli cells.

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes.

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.

mar Operon involved in multidrug resistance of Enterobacter aerogenes.

We determined the sequence of the entire marRAB operon in Enterobacter aerogenes. It is functionally and structurally analogous to the Escherichia coli operon. The overexpression of E. aerogenes MarA induces a multidrug resistance phenotype in a susceptible strain, demonstrated by a noticeable resistance to various antibiotics, a decrease in immunodetected porins, and active efflux of norfloxacin.

Molecular characterization of amoxicillin-clavulanate resistance in a clinical isolate of Escherichia coli.

The resistance phenotype of the clinical isolate of Escherichia coli 1941 was characterized by high-level resistance to penicillins and to combinations amoxicillin-ticarcillin/clavulanate and ampicillin/sulbactam. This resistance was carried by the conjugative plasmid pEC1941 that encoded a beta-lactamase activity. The purified enzyme focused at pI 5.4 and was strongly inhibited in vitro by clavulanic acid (IC50 = 0.09 microM). Nucleotide sequence analysis revealed identity between the plasmid borne blaTEM gene of E. coli 1941 and the blaTEM-1B gene, except for a single C-to-T substitution at position 32 in the promoter region leading to the overlapping promoters Pa and Pb. No alterations in the expression of outer membrane porins OmpC and OmpF have been detected. These findings show that the resistance of E. coli 1941 to the combinations of beta-lactams with beta-lactamase inhibitors is related to high-level production of TEM-1 enzyme expressed from the strong promoters Pa and Pb.