Fungal culture of musculoskeletal tissue: what’s the point?

There have not been any studies that review the prevalence of fungal isolates using selective media from samples of banked musculoskeletal tissue retrieved from living and cadaveric donors. A total of 2,036 swab and 2,621 biopsy samples of musculoskeletal tissue from tissue banks were received from the 1st August 2008 till 31st December 2010. Routine culture for fungi using selective media with a prolonged incubation period failed to demonstrate a greater prevalence of fungal isolates than by using non-selective culture media alone. Using selective culture fungi were recovered from only two Sabouraud agar plates (0.1%) but not from non-selective media. During the same period fungi were isolated from three graft samples cultured in non-selective broth media only (0.1%). There was no correlation of fungal isolates from selective or non-selective media inoculated at the same time nor from multiple graft samples collected from the same donor supporting the possibility of an exogenous source for fungal isolates rather than an endogenous source.     

Roadmap to approval: use of an automated sterility test method as a lot release test for Carticel, autologous cultured chondrocytes

BACKGROUND: In February 2004, FDA approved a supplement to our biologics license for Carticel, autologous cultured chondrocytes, to use the BacT/ALERT microbial detection system as an alternative to the compendial sterility test for lot release. This article provides a roadmap to our approval process. The approval represents more than 4 years of development and validation studies comparing the Steritest compact system to the BacT/ALERT microbial detection system. METHODS: For this study, freshly cultured chondrocytes were prepared from a characterized cell bank. Microbial isolates were prepared from either American Type Culture Collection (ATCC) strains or from in-house contaminants. For each test condition, a suspension of chondrocyte cells and test organisms was inoculated into both aerobic media (SA standard adult culture bottles, FA FAN, tryptic soy broth) and anaerobic media (SN standard adult culture bottles, FN FAN, fluid thioglycollate media) and tested for sterility using the Steritest compact system (Millipore, Bedford, MA, USA) and the BacT/ALERT microbial detection system (bioMerieux, Durham, NC, USA). Negative control bottles were inoculated with chondrocytes and no microorganisms. All bottles were incubated for 14 days and read daily. Bacterial growth was determined by either visual examination of Steritest canisters or detection of a positive by the BacT/ALERT system. A gram stain and streak plate were used to confirm positive bottles and negative bottles after 14 days. RESULTS: The detection of a positive by either the Steritest compact system or the BacT/ALERT system was summarized for each organism in each validation study. Data generated from studies reducing the incubation temperature from 35 degrees C to 32 degrees C improved detection times in the automated method compared with the compendial method. Other improvements included the use of FAN aerobic and anaerobic media to absorb the gentamicin contained in the culture media of prepared chondrocyte samples. Chondrocytes alone did not generate positive results in either the compendial method or the automated method. DISCUSSION: Data from validation studies support the use of the BacT/ALERT microbial detection system as an alternative sterility test for Carticel.     

Residual Antibiotics in Decontaminated Human Cardiovascular Tissues Intended for Transplantation and Risk of Falsely Negative Microbiological Analyses

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.     

Comparison of rapid NFT and API 20E with conventional methods for identification of gram-negative nonfermentative bacilli from pharmaceuticals and cosmetics

The accuracy of the Rapid NFT and the API 20E identification systems was evaluated by comparing them with conventional biochemical methods for the identification of gram-negative, nonfermentative bacilli. The organisms were recovered from preserved, nonsterile pharmaceutical and cosmetic products. A total of 123 test isolates that are commonly encountered in these products were used. By using the criteria of accurate and reliable identification without employing additional tests, Rapid NFT was found to be more accurate after 48 h of incubation than API 20E for characterizing isolates to the species level. Therefore, close agreement between NFT and conventional methods for identification of industrial gram-negative isolates provides evidence that the Rapid NFT system is an improved and rapid method for identifying these organisms to the species level with minimal use of supplementary tests.     

Comparison of rapid NFT and API 20E with conventional methods for identification of gram-negative nonfermentative bacilli from pharmaceuticals and cosmetics.

The accuracy of the Rapid NFT and the API 20E identification systems was evaluated by comparing them with conventional biochemical methods for the identification of gram-negative, nonfermentative bacilli. The organisms were recovered from preserved, nonsterile pharmaceutical and cosmetic products. A total of 123 test isolates that are commonly encountered in these products were used. By using the criteria of accurate and reliable identification without employing additional tests, Rapid NFT was found to be more accurate after 48 h of incubation than API 20E for characterizing isolates to the species level. Therefore, close agreement between NFT and conventional methods for identification of industrial gram-negative isolates provides evidence that the Rapid NFT system is an improved and rapid method for identifying these organisms to the species level with minimal use of supplementary tests.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery.

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Rapid, Direct Tissue Culture Test for Toxigenicity of Corynebacterium Diphtheriae

A method for testing toxigenicity of Corynebacterium diphtheriae in tissue culture is described. The technique, called the colony overlay test (COT), involves inoculating material from an isolated colony of C. diphtheriae to a small area on the surface of an agar medium which overlays a monolayer of toxin-susceptible HeLa cells. If toxin is produced during incubation at 37 C, it diffuses to the tissue monolayer and destroys the cells below the inoculation site. Twenty-four hours after inoculation, organisms are killed and tissue cells are fixed with formaldehyde. The agar overlay is then removed, and the monolayer is stained with crystal violet. Toxin-affected areas fail to stain or stain poorly. A second plate with antitoxin incorporated in the overlay serves as a control for specificity. Forty-eight strains of C. diphtheriae were tested by the COT, guinea pig, and in vitro, gel diffusion tests. The COT is as specific as the other two tests, is easy to read, and can be used to test large numbers of isolates for toxin action more conveniently than by animal inoculation.     

Characterization of some anaerobic bacteria from the liquid phase of a mesophilic anaerobic digester fed with a prefermented cheese whey substrate

Bacterial counts on the contents of an anaerobic fixed-bed digester receiving a whey substrate were conducted using the modified roll tube technique. Average anaerobic counts after 48 hours incubation on lactate containing media were 3.12 × 10⁹ and 3.7 × 10⁹ ml^–1, respectively. These counts were between 140 and 190 times higher than aerobic counts on the same media. Seventy-four strains from both media were isolated and characterized, and the relationship between the organisms was calculated according to the similarity coefficient of Sokal and Michener [20]. The organisms were clustered using the unweighted pair group method and the results were presented in the form of a simplified dendrogram. The isolates clustered in three major groups (A, B, and C) at a similarity level of 76%. A small diffuse group of five organisms was also found. The organisms in two (A and B) of the major clusters were obligate anaerobes. Cluster A represented 34% of the isolates, cluster B represented 50%, and the facultative streptococci in cluster C, 11%. The isolates in clusters A and B could only tentatively be identified as members of the generaBacteroides andPeptostreptococcus, respectively. The isolates could not be positively identified.     

Validation of an alternative microbiological method for tissue products

According to the European Pharmacopoeia sterility testing of products includes an incubation time of 14 days in thioglycollate medium and soya-bean casein medium. In this case a large period of time is needed for product testing. So we designed a study to evaluate an alternative method for sterility testing. The aim of this study was to reduce the incubation time for the routinely produced products in our tissue bank (cornea and amnion grafts) by obtaining the same detection limit, accurateness and recovery rates as the reference method described in the European Pharmacopoeia. The study included two steps of validation. Primary validation compared the reference method with the alternative method. Therefore eight bacterial and two fungi test strains were tested at their preferred milieu. A geometric dilution series from 10 to 0.625 colony forming unit per 10 ml culture media was used. Subsequent to the evaluation the second part of the study started including the validation of the fertility of the culture media and the parallel testing of the two methods by investigating products. For this purpose two product batches were tested in three independent runs. Concerning the validation we could not find any aberration between the alternative and the reference method. In addition, the recovery rate of each microorganism was between 83.33 and 100 %. The alternative method showed non-inferiority regarding accuracy to the reference method. Due to this study we reduced the sterility testing for cornea and amniotic grafts to 9 days.     

Usefulness of Electrophoretic Pattern of Cell Envelope Protein as a Taxonomic Tool for Fishhold Slime Moraxella Species

Nine independent Moraxella cultures were isolated from the accumulated slime in fishholds of fishery trawlers. It is significant that none of these isolates was viable above 30°C, a temperature well below the usual incubation temperature for plate counts of food samples. The traditional taxonomic parameters showed no significant dissimilarities among these closely related marine organisms or between them and conventional moraxellas. However, cell envelope protein profiles examined on sodium dodecyl sulfate-polyacrylamide gels revealed that the organisms fell into several distinct groups. The cell envelope protein profile could be a simple and quick test to determine the fine relationships between individual isolates.     

Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination.

Randomly amplified polymorphic DNA (RAPD) analysis using two or more primers has been reported to provide additional discriminatory ability over one primer used individually. This may be of particular application in epidemiological typing of clonal organisms, such as Shiga toxin-producing E. coli O157, where strain differentiation can be difficult. Using four arbitrary primers individually, and in all possible permutations, E. coli O157 isolates and other arbitrarily chosen E. coli strains were typed using RAPD analysis. For most nonclonal strains, the use of two primers resulted in increased differentiation between isolates; however, more than two primers did not increase further the discriminatory capacity. E. coli O157 isolates that produced virtually identical profiles using one primer did not show increased differentiation when using two or more primers, demonstrating that in some cases, where strains of an organism are highly related, there is limited advantage to using more than one primer in RAPD analysis.     

Intense association of non-culturable endophytic bacteria with antibiotic-cleansed in vitro watermelon and their activation in degenerating cultures

The study was undertaken with a view to unravel the source of bacterial colony growth observed in a section of micropropagated triploid watermelon cultures that were supposedly cleansed of the associated endophytic bacteria through antibiotic treatment, and thereafter maintained under stringent sterility checks to prevent lateral intrusion of contaminants. Five different bacteria were retrieved from colony growth-displaying watermelon cultures that were previously treated with gentamycin and five isolates from cefazolin-treated stocks with the organisms showing tolerance to the respective antibiotic. These watermelon cultures were in degeneration phase (over 6 months after the previous sub-culturing), while the actively maintained counterpart stocks appeared healthy with no colony growth on different bacteriological media during tissue-screenings. The latter cultures, however, revealed abundant motile, tetrazolium-stained bacterial cells in microscopy, suggesting tissue colonization by non-culturable endophytes. PCR screening on healthy cultures endorsed tissue colonization by different bacterial phylogenic groups. A few organisms could be activated to cultivation from healthy watermelon stocks through host tissue extract supplementation, which also enhanced the growth of all the organisms. The study indicated that a fraction of antibiotic-tolerant bacteria survived intra-tissue in non-culturable form during the preceding cleansing activity, multiplied to substantial numbers thereafter, and turned cultivable in degenerating cultures contributed by tissue breakdown products. This study brings out the existence of a deep endophyte association in tissue cultures which is not easily dissociable. It also signifies the utility of in vitro system for investigations into plant–endophyte association and to bring normally non-culturable novel organisms to cultivation facilitating their future exploitation.     

USE OF PCR ANALYSIS FOR STERILITY TESTING IN PHARMACEUTICAL ENVIRONMENTS

To determine the sterility of pharmaceutical samples, highly conserved bacterial ribosomal DNA sequences were used in a PCR-based assay. Finished products, raw materials, growth media, and diluents were artificially contaminated with different types of microorganisms. Samples were incubated for 24 h. After incubation, microbial DNA was extracted from enrichment broths using a Tris-EDTA-Tween 20 buffer containing proteinase K. Extracted DNA was added to Ready-To-Go PCR beads and eubacterial primers. Contaminated samples were found to contain the conserved 1.5 kilobase (kb) DNA fragment of the bacterial genome by using the PCR assay. None of the uninoculated samples was found to show the presence of the 1.5 kb fragment. PCR test results were compared with standard conventional methods. There was a 100% correlation between standard conventional methods and the PCR assay. However, the PCR-based assay was completed within 27 h while conventional methods required 4–5 days. Rapid PCR analysis using a simple sample preparation reduced the time for sterility testing of pharmaceutical samples allowing optimization of risk assessment and implementation of corrective actions.     

Characterization of Canine Mycoplasmas by Polyacrylamide Gel Electrophoresis and Immunodiffusion

Canine mycoplasmas which had been characterized by biological and serological methods were further studied by using polyacrylamide gel electrophoresis (PGE) and double diffusion in agar gel. The three dog mycoplasmas previously characterized, Mycoplasma canis, M. maculosum, and M. spumans showed distinctive patterns by PGE. Five additional representative isolates from dogs had been characterized serologically and biologically into three new groups, A, C, and D. An additional mycoplasma (group B) was indistinguishable from M. canis by growth inhibition and PGE but was more broadly reactive with field isolates serologically. The group A organisms were distinctive in pattern and similar to those studied by Razin and Rottem, tentatively designated M. edwardii. The group C organisms were represented by two isolates which were similar by fluorescent-antibody studies but different by growth inhibition tests. These two isolates were also different from each other by PGE. The group D serotypes were also distinctive by PGE from all other dog mycoplasmas tested. It was found, during these studies, that two different mycoplasmas showed different PGE patterns at different intervals during incubation. Immunodiffusion studies showed a relationship among all the canine mycoplasmas, and bands of nonidentity between the two group C mycoplasmas were demonstrated.     

Pathogenesis of Cryptococcus neoformans is Associated with Quantitative Differences in Multiple Virulence Factors

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG)under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU).Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strain's combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.This revised version was published online in October 2005 with corrections to the Cover Date.     

Rapid tests for esculin hydrolysis by anaerobic bacteria

Esculin hydrolysis is one of the biochemical tests used in the identification of anaerobic microorganisms. The conventional method by use of growing microbial cells requires 24–48 hours of incubation. On the other hand, growth independent methods like the buffered esculin test, the spot test, and the PathoTec strip test utilize the presence of constitutive enzymes and, therefore, yield results in 1–4 hours. A total of 817 anaerobic organisms were used in this study to determine the sensitivity and specificity of the three rapid methods. All three rapid methods gave excellent correlation with the standard conventional method. Over 99% of the organisms gave comparable results with the spot test and the buffered esculin test within one hour; the PathoTec strip test required up to 4 hours. The former two were not only more rapid but also more economical than the PathoTec strip test.     

Factors influencing microbiological assay of cell-culture mycoplasma.

A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%). M. orale and A. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aeorbic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains of M. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas.     

Microbiological cultivation ofMycoplasma hyorhinis from cell cultures

The failure of many cell culture isolates ofMycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly prepared yeast extract in mycoplasmal media together with incubation in 5% CO₂/air successfully detectedM. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures. This study was supported by grant AI 15748 from NIH. We thank Ronnie Vanaman and Judi Sarama for their technical assistance.     

A QUANTITATIVE METHOD FOR ASSESSING THE EFFECT OF DISINFECTANTS AND CLEANING AGENTS ON BACTERIA DEPOSITED ON SURFACES

SUMMARY: A test has been developed for evaluating disinfectant action on surfaces, which relies on the pH response of Strep. faecalis , since with this organism the pH of a buffered broth at the end of incubation depends on the weight of the inoculum, i.e. the numbers of organisms which survive.
The organisms are deposited as a wet film on the surface of small strips of tinned mild steel, which are dried and treated with the disinfectant. The strips are finally placed in tubes of media and, after incubation, the pH is measured.
The standardization of the different variables of the test are described. The condition of the test organism is particularly important: at the time the films are dried it must be in its stationary phase of growth, when the cells are homogeneous and more resistant to disinfectant action than during the logarithmic phase.
Incubation is terminated when the control tubes containing strips which had no disinfectant treatment have pH values averaging 5.50. Under these conditions, with hypochlorite a linear response over the range of 0–6 p/m available chlorine is generally obtained. Chlorine may therefore be used as a standard for comparison with other disinfectants.
As an example, the testing of a quaternary ammonium compound is described.