Microbiological Control of Cellular Products: The Relevance of the Cellular Matrix, Incubation Temperature, and Atmosphere for the Detection Performance of Automated Culture Systems.

BACKGROUND: The microbiological control of cellular products sometimes causes significant procedural issues for quality control laboratories. According to the European Pharmacopoeia (EP), the microbiological control of cellular products requires a 7- to 14-day incubation period at two different incubation temperatures using aerobic and anaerobic growth media. However, the suitability of these test conditions for efficient quality control can be influenced by many conditions, such as the expected microbial spectrum of contamination or the texture and composition of the cellular product. Because of interference, direct inoculation and membrane filtration as reference methods of pharmacopoeia are largely unsuitable for the microbiological control of cellular products; therefore, alternative and, above all, automated methods are the focus of interest. OBJECTIVE: The aim of our study was to evaluate the method suitability and possible effects of cell matrix, incubation temperature, and oxygen pressure on the detection performance of automated culture systems. METHODS: The BacT/ALERT® 3DTM Dual T system (bioMérieux, Nürtingen, Germany) was used to evaluate the factors influencing automated microbiological control of cellular products. The tests were performed using microbial strains recommended by the EP for microbiological method suitability testing and additional relevant possible contaminants of human-derived stem-cell products under varying culture and cell matrix conditions. RESULTS: All contaminants were detected by the system in the required period of 2-5 days. Low incubation temperatures (22°C) had overall negative effects on the detection kinetics of each type of microbial contamination. The adverse effects of the accompanying cell matrix on the detection properties of the system could be compensated in our study by incubation at 32°C in both the aerobic and the anaerobic culture conditions. CONCLUSION: Automated culture techniques represent a sufficient approach for the microbiological control of cellular products. The negative effects of the cell matrix and microbial contamination on the detection performance can be compensated by the application of variable culture conditions in the automated culture system.

Distribution of carbapenem resistance mechanisms in clinical isolates of XDR Pseudomonas aeruginosa.

Our study aims to define the epidemiology of carbapenem resistance mechanisms in clinical isolates of Pseudomonas aeruginosa (PA). We evaluated 11,457 clinical PA strains isolated between 2009 and 2015 at the tertiary care University Hospital in Heidelberg, Germany. Thirty-four percent of the isolates (3867/11,457) were MDR (multidrug-resistant), 16% (1816/11,457) were XDR (extensively drug resistant), and less than 1% (82/11,457) had a PDR (pandrug-resistant) profile. Of those, 23% carried a carbapenemase gene (CPM positive) with 12% VIM-2, 10% VIM-1, and less than 1% IMP-1. Comparing MIC (minimal inhibitory concentration) distributions, the mean rank for meropenem, imipenem, gentamicin, and fosfomycin was significantly higher in the CPM-positive group than in the CPM-negative XDR group (p ≤ 0.004). oprD (outer membrane protein) mutations were found in 19/19 tested strains; 12/19 carried a CPM and had a higher mutation rate. Meropenem resistance was mostly associated with the presence of CPM. Only 1/19 strains was meropenem resistant in the absence of CPM genes; nevertheless, it carried an oprD mutation in a strategic site (loop 2). Of 19 CPM-negative strains tested, 7 (36%) showed EP (efflux pumps) hyperexpression versus 12 in the CPM-positive strains. In our study, nearly 50% of the PA isolates exhibited resistance to the tested first-line antibiotics. Our study also demonstrates that carbapenemase genes can be isolated in approximately 23% of XDR PA strains in our population. This finding supports the clinical relevance of PA driven by the possible presence of multiple resistance mechanisms acquired under exposure to antibiotics or by horizontal transfer of resistance genes.

Natural isothiocyanates express antimicrobial activity against developing and mature biofilms of Pseudomonas aeruginosa.

BACKGROUND: The antimicrobial properties of natural isothiocyanates (ITCs) found in plants such as nasturtium (Tropaeolum majus) and horseradish (Armoracia rusticana), and the need of new chemotherapeutic options for treatment of infections caused by multidrug-resistant and biofilm-forming Gram-negative bacteria such as Pseudomonas aeruginosa (Pa), led us to evaluate the effects of three major ITCs, allylisothiocyanate (AITC), benzylisothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC), and a mixture (ITCM) adapted to the ITC composition after release of active components out of natural sources. MATERIAL/METHODS: Out of 105Pa isolates 27 isolates with increased biofilm formation were selected for testing. The effects of ITCs on Pa were evaluated regarding (1) planktonic bacterial proliferation, (2) biofilm formation, (3) metabolic activity in mature biofilms, and (4) synergism of ITCs and antibiotics. RESULTS: (1) Each ITC had anti-Pa activity. Mean minimum inhibitory concentrations (MICs) were (µg/ml, mean±standard deviation): AITC 103±6.9; BITC, 2145±249; PEITC 29,423±1652; and ITCM, 140±5. (2) Treating bacteria with PEITC and ITCM in concentrations below the MIC significantly inhibited biofilm formation. Particularly, ITCM reduced biofilm mass and bacterial proliferation. (3) ITCs significantly inhibited metabolic activity in mature biofilms. (4) Combining ITCs with meropenem synergistically increased antimicrobial efficacy on Pa biofilms. CONCLUSIONS: ITCs represent a promising group of natural anti-infective compounds with activity against Pa biofilms.

Sterility Testing of Injectable Products: Evaluation of the Growth-based BacT/ALERT® 3D™ Dual T Culture System.

Sterility testing as described in the European Pharmacopoeia Chapter 2.6.1 as well as the United States Pharmacopeia Chapter 71 requires a 14 day incubation period of the test product in two different media and at two different temperatures. Because of extensive personnel requirements for test performance and quality assurance, alternative and partially automated methods for product sterility testing are of interest. The study objective was to evaluate the applicability of the BacT/ALERT® 3D™ Dual T system (Biomérieux, Nürtingen, Germany) for detection of microbial contaminants according to current pharmacopoeia standards. In addition, we compared the BacT/ALERT® 3D™ Dual T system to conventional pharmacopoeia sterility testing using the direct inoculation method. The results showed no significant disadvantages of sterility testing by BacT/ALERT® 3D™ Dual T compared to the direct inoculation method regarding the ability to detect microbial contamination. Furthermore, product testing using the BacT/ALERT® 3D™ Dual T system met the compendia requirements for method qualification. Altogether, our data provide evidence that the BacT/ALERT® 3D™ Dual T system is a promising alternative for sterility testing of injectable products of sample volume below 10 mL and without antimicrobial activity. LAY ABSTRACT: Sterility is defined as the freedom from the presence of viable microorganisms. Injectable pharmaceutical products that are sometimes used for treatment of patients need to be free of microorganisms and therefore tested in the laboratory to confirm sterility. The testing procedures are described in detail in the European Pharmacopoeia. To test for sterility, the products need to be incubated for 14 days at two different temperatures and using two different growth media to ensure growth of various microorganisms. These tests, however, are cost- and labor-intensive. Therefore, we sought to use an automated system to reduce costs and hands-on time, as well as to improve quality assurance. The study objective was to evaluate the applicability of the automated BacT/ALERT® 3D™ Dual T system (Biomérieux, Nürtingen, Germany) for detection of microorganisms according to current pharmacopeia standards. We compared the BacT/ALERT® 3D™ Dual T system to standard sterility testing using the so-called direct inoculation method. The results showed no major disadvantages of sterility testing by the automated system compared to the standard method with regard to the detection level of microorganisms. Furthermore, product testing using the BacT/ALERT® 3D™ Dual T system met the compendia requirements for method qualification. Additionally, the new automated method provided reliable results and promises a higher robustness to human errors than do standard manual methods, which might reduce the potential for errors and improves quality assurance. Altogether, our data provide evidence that the BacT/ALERT® 3D™ Dual T system is a promising alternative for sterility testing of injectable products.