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.     

Evaluation of enrichment broths for their ability to recover heat-injured Listeria monocytogenes

J.R. PATEL AND L.R. BEUCHAT. 1995. Listeria selective enrichment broth (LEB), University of Vermont (UVM) broth, modified UVM (MUVM) broth and Fraser broth (FB) were compared for their ability to recover cells of L. monocytogenes from heated tryptose phosphate broth. Three strains of L. monocytogenes were heated at 54C for 30 min, inoculated into enrichment broths supplemented with 400 µg catalase ml⁻¹, and incubated for 8 h at 30°C. After incubation for 4 h, the total viable cell populations either decreased or did not change, whereas the number of healthy (non-injured) cells of all strains increased significantly in all broths except FB inoculated with the LCDC strain. With an increase in incubation time to 8 h, the number of healthy cells of all strains increased in all broths. At 8 h, the difference between populations of total (injured plus healthy cells) and healthy cells detected in LEB inoculated with two strains was not significant. Overall, recovery of heat-treated cells was significantly higher in LEB, followed by MUVM broth, UVM broth and FB. The addition of catalase to enrichment broths significantly enhanced recovery of heat-injured cells. A slight reduction of catalase activity of heated cells of all test strains in all enrichment broths except FB was observed by extending the incubation period from 4 to 8 h. A test strain that produces relatively higher catalase activity compared to the other strains exhibited the greatest resistance to exogenous hydrogen peroxide. Enumeration of viable L. monocytogenes cells in heated foods should be done using LEB supplemented with 400 µg catalase ml⁻¹ to maximize the recovery of injured cells.     

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.     

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.     

A Comparison of Methods for the Enrichment of Salmonellae from Sheep Faeces

S ummary : A 10-tube MPN technique was used to test the efficiency of nutrient, tetrathionate, mannitol-selenite and mannitol-selenite-cystine broths as enrichment media for detecting salmonellae. Small numbers of broth grown salmonellae could be detected in all 4 media in the presence of 5% of sheep faeces. In naturally infected sheep faeces small numbers of salmonellae were not detected with either nutrient or tetrathionate broths. With mannitol-selenite the sensitivity of salmonella detection increased with both incubation temperature (37–43°) and the addition of faeces. The most sensitive and reliable medium for detecting salmonellae in naturally infected sheep faeces was mannitol-selenite-cystine broth. Neither incubation temperature (37–43°) nor the addition of faeces had a statistically significant effect on its sensitivity.     

Pre-Enrichment and Enrichment Methods for Isolating Small Number of Campylobacteria from Contaminating Flora

A method was developed to detect fewer than 100 CFU of campylobacteria from SIFF transport medium to which thawing drip from deep frozen broiler carcasses was added as a source of contamination and which was then stored at room temperature for 20 h. The method was made possible by using pre–enrichment in 1 % buffered peptone water under a microaerophilic atmosphere for 5 h at 43°C before selective enrichment either in brucella enrichment broth and on brucella blood selective agar supplemented with Skirrow antibiotics or in CCD enrichment broth and on blood free CCD selective agar. The other pre–enrichment broth studied was alkaline peptone water with reducing agents (RAPW) and the other enrichment broths and selective agars were Preston broth and agar, THAL broth and alkaline tryptose broth (ATB) and brucella agar with ATB antibiotics. Contaminating flora can be a problem when using enrichment broths and selective agars with limited antibiotic supplementation.     

The detection of Salmonella in skimmed milk powder enrichments using conventional methods and immunomagnetic separation

Skimmed milk powders were spiked with one of three Salmonella serovars and incubated in buffered peptone water for 24 h. No false-negative results were obtained by immunomagnetic separation (IMS), compared to seven for selenite cysteine, one for Müller-Kauffmann tetrathionate and two for Rappaport-Vassiliadis enrichment broths. Salmonella virchow was detected and enumerated during the pre-enrichment incubation by IMS and indirect conductance techniques. The Salm. virchow cell number did not increase after 12 h incubation and remained at 3 times 10⁶ cfu ml^-1. IMS was able to capture Salm. virchow cells at cell numbers ca 50 ml^-1 in the presence of a 1000 greater number of non-salmonella cells.     

FLUORESCENCE CONCENTRATION IMMUNOASSAY FOR RAPID DETECTION OF CAMPYLOBACTER SPP. IN CHICKEN RINSE WATER

A total of 215 freshly processed post-chill whole chicken carcasses were assessed for Campylobacter spp. contamination by a fluorescence concentration immunoassay (FCIA) procedure. Whole chicken carcasses were sampled with low volume water rinses from which 5 ml portions were enriched with brucella enrichment broth with or without oxyrase supplement in a test tube enrichment system. After a 24h stationary incubation at 42C, each sample was assayed using a FCIA procedure for the presence or absence of campylobacters. The FCIA procedure indicated Campylobacter spp. contamination in 84% of carcasses using oxyrase supplemented enrichment, while only 47% of the chicken carcasses were positive from nonsupplemented enrichment. The corresponding incidence rates detected by culture method were 92% and 87% for oxyrase supplemented and unsupplemented samples, respectively. The FCIA procedure can be completed in less than 1 h with 48 samples including a positive and a negative control assayed on one plate. In summary, the test tube oxyrase-supplemented stationary enrichment system followed by the use of the FCIA procedure was found to be an effective, rapid method for the detection of Campylobacter spp. in chicken rinse water.     

Determination of the optimal culture conditions for detecting thermophilic campylobacters in environmental water

This study evaluated alternative protocols for culturing thermophilic campylobacters in environmental water. All samples were filtered through a sterile 0.45 μm pore-size membrane, which was then incubated in Preston enrichment broth. Four variables were compared: water sample volume (2000 mL vs. 500 mL), enrichment broth volume (25 mL vs. 100 mL), enrichment incubation duration (24 h vs. 48 h), and number of enrichment passages (one vs. two). In addition, DNA extracts were prepared from all final broths and analyzed using three rRNA PCR assays. River water was collected at 3 sampling sites weekly for 9 weeks. Among these 27 collections, 25 (93%) yielded Campylobacter spp. under at least one of the 16 culture conditions. By univariate analysis, yields were significantly better for the 2000 mL sample volume (68.5% vs. 43.0%, p < 0.0001) and the 25 mL enrichment broth volume (64.5% vs. 47.0%, p < 0.0004). Neither of the enrichment period had a significant effect, although there was a trend in favor of 48 h incubation (59.5% vs. 52.0%, p = 0.13). The three PCR methods gave concordant results for 66 (33%) of the culture-negative samples and 103 (50%) of the culture-positive samples. Compared with culture results, Lubeck's 16S PCR assay had the best performance characteristics, with a sensitivity of 82% and a specificity of 94%. Of the 12 culture-negative samples positive by Lubeck's PCR assay, 11 (92%) samples were also positive by Denis' 16S PCR assay, suggesting that in these cases the culture might have been falsely negative. Based on our results, we conclude that the optimal conditions for detecting Campylobacter spp. in natural waters include 2000 mL sample volume and a single enrichment broth of 25 mL PB incubated for 48 h.     

Isolation of E. coli O157:H7 and non-O157 STEC in different matrices: review of the most commonly used enrichment protocols

AIMS: To review and characterize the enrichment protocols used for detecting all Shiga-Toxin producing Escherichia coli (STEC) from different matrices. METHODS AND RESULTS: Firstly, the frequency distribution of the factors characterizing the enrichment protocols is described; secondly, a multiple correspondence analysis is performed to display profiles of association of these factors, and thirdly, published results concerning the relative performances of the protocols are summarized. Trypticase Soy Broth (TSB) is reported as the most frequently used enrichment broth. More often, one antibiotic is added in enrichment broths and these broths are incubated for a duration of 16-24 h at 35-37 degrees C. It also appears that the incubation temperature does not seem to be related to the type of serogroup looked for and that antibiotics are used regardless of the matrix analysed. Finally, results relating to the enrichment protocol efficacy are rare and differ from one study to another. CONCLUSIONS: Statistical studies must be conducted so as to assess the efficacy of the main enrichment protocols investigated in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reviews the most commonly used enrichment protocols and highlights the lack of results as to their relative efficacy.     

Evaluation of methods for enumeration of Vibrio parahaemolyticus from seafood

The efficiency of several enrichment broths in recovering Vibrio parahaemolyticus inoculated into fish homogenates was studied. Recovery by the most probable number technique was very low in all the broths, while direct plating on thiosulfate citrate bile salt sucrose agar yielded better recovery. A decrease in the enrichment time to 8 from 18 h did not improve recovery. At concentrations exceeding 2.5 micrograms/ml, polymyxin was inhibitory to V. parahaemolyticus.     

Factors affecting the recovery of Campylobacter spp. from retail packs of raw, fresh chicken using ISO 10272-1:2006

Aims:  This study sought to determine the most effective protocol for the detection of Campylobacter spp. in retail packs of fresh, raw chicken based on ISO 10272-1:2006.
Methods and Results:  Three sample preparation protocols were studied; two based on excision and one combining excision with a rinse of the remaining sample. Enrichment cultures were incubated both in closed bottles and microaerobically, and sub-cultured at 24 and 48 h. Packs of chicken (110) were analysed and only two yielded no Campylobacter spp. Subculturing enrichment broths at 24 h gave the same prevalence as at 48 h, P  >   0·4 but microaerobic incubation yielded approximately 50% more positive samples than did incubation in closed bottles. Sampling based on excision plus rinsing gave the highest Campylobacter prevalence (92·7%).
Conclusions:  To isolate Campylobacter spp. from retail packs of chicken, enrichment cultures must be incubated in a microaerobic atmosphere and sub-cultured at 24 h and, possibly, 48 h. Sampling packs by excision plus rinsing maximized recoveries.
Significance and Impact of the Study:  ISO 10272-1:2006 permits the use of inefficient protocols which markedly underestimate the true prevalence of Campylobacter spp. in retail, fresh chicken. Equivalent results could be obtained 24 h earlier, with consequent savings. Its revision is essential.     

Evaluation of methods for enumeration of Vibrio parahaemolyticus from seafood.

The efficiency of several enrichment broths in recovering Vibrio parahaemolyticus inoculated into fish homogenates was studied. Recovery by the most probable number technique was very low in all the broths, while direct plating on thiosulfate citrate bile salt sucrose agar yielded better recovery. A decrease in the enrichment time to 8 from 18 h did not improve recovery. At concentrations exceeding 2.5 micrograms/ml, polymyxin was inhibitory to V. parahaemolyticus.     

Effects of aluminium on ATP pools and utilization in the cyanobacterium Anabaena cylindrica: a model for the in vivo toxicity

ATP pools extracted from the cyanobacterium Anabaena cylindrica , grown in the absence or presence of AlCl₃, were measured using the luciferin-luciferase assay. Addition of low concentrations of AlCl₃ (3.6–36 μ M ) increased the ATP pool 20–40% within 24 h, the effect being more marked with time. When using the Tris-EDTA boiling technique for extraction of cellular ATP, the ATP from aluminium-exposed cells appeared more stable during the extraction than the ATP from untreated cells. The higher ATP pools in aluminium-exposed cells were also evident after dark treatment and addition of the phosphorylating inhibitors carbonylcyanide m -chloro-phenylhydrazone (CCCP) and N,N-dicyclohexylcarbodiimide (DCCD). The formation of elevated ATP pools in cells exposed to aluminium was curtailed by high concentrations of cellular phosphate and postincubation at high pH (>8). These results favour the hypothesis that intracellular aluminium binds to ATP by competing with Mg²⁺ and, as a consequence, the stable Al³⁺-ATP complex formed is no longer available for cellular metabolism. The cyanobacterium is assumed to compensate by increasing the total pool of ATP. At high AlCl₃-concentrations, and in particular at low phosphate: aluminium molar ratios (<1), aluminium apparently also interferes with the membranes in A. cylindrica as indicated by inhibited O₂ production, reduced ATP production and cell lysis.     

Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility

The effects of tributyltin (TBT) on the energy metabolism and motility of fish spermatozoa were investigated in vitro in African catfish and common carp. A significant (P<0.05) decrease of the duration and the intensity of motility was observed in catfish spermatozoa exposed to 0.27 microg/l TBT for 24 h. Exposure of catfish spermatozoa to 2.7-27 microg/l TBT caused an instant decrease in ATP content. In the presence of 27 microg/l TBT approximately 55% of the initial ATP concentration in catfish semen was lost after 60 min incubation while AMP concentrations increased and the total adenine nucleotide (TAN) pool remained unchanged. The reduction in sperm ATP levels could not be attributed to cell death since viability decreased only slightly over the period of exposure. In carp by contrast, none of the adenylates concentrations studied (ATP, ADP and AMP) were affected by TBT exposure at any experimental condition. However, carp sperm motility was significantly reduced by exposure to 2.7 microg/l TBT. Among the enzymes investigated only lactate dehydrogenase (LDH) in catfish sperm was significantly (P<0.01) affected by 27 microg/l TBT treatment with a reduction in activity of approximately 75%. Compared with carp sperm before TBT exposure, that of catfish had lower adenylate contents and overall lower enzymatic activities; this explains its slower sperm velocity and shorter duration of movement as measured by computer assisted sperm analysis (CASA). The present in vitro study shows that catfish spermatozoa are more sensitive to TBT exposure (and probably to other toxicants) than those of carp.     

Metabolic and Energetic Changes During Apoptosis in Neural Cells

Abstract: Changes in cellular energetic and metabolic parameters were analyzed at several time points during apoptosis of differentiated PC12 cells following removal of nerve growth factor (NGF). As approximately 60% of the population died during the period of study (24 h), most of the measured metabolic indicators declined over time. However, this decline paralleled the overall decrease in cellular viability, suggesting that, in individual cells, a compromised metabolic state occurred suddenly and very late in the death process. For example, when expressed as a function of viable cells, protein and RNA synthesis did not decrease until 24 h. Glucose utilization in live cells was never significantly reduced relative to control levels; lactate production decreased slightly within 4–8 h after NGF removal, but eventually rebounded to 122% of control levels by 24 h. ATP levels dropped 27% in an early predeath period, but then returned to near control levels (on a per-live-cell basis) once the population actively began to die. The ATP/ADP ratio remained at least 84% of control throughout. UTP/UDP and GTP/GDP ratios did not change significantly at any time point.     

Salmonella-TEK, a rapid screening method for Salmonella species in food.

A micro-enzyme-linked immunosorbent assay (micro-ELISA) using the Salmonella-TEK screen kit was tested for the detection of Salmonella spp. in pure cultures as well as in 30 artificially contaminated food samples and in 45 naturally contaminated food samples. Different raw, fleshy foods and processed foods were used as test products. The artificially contaminated minced meat samples were preenriched in buffered peptone water, and after incubation, different selective enrichment broths were tested. The micro-ELISA optical density values after enrichment and isolation of the different broths were very analogous. The quickest method to detect Salmonella spp. in different foods is to enrich them with Salmosyst broth, which reduces the total analysis time to 31 h. The Salmonella-TEK kit for Salmonella spp. provides a promising test for the detection of Salmonella antigens in food even when they are present at a low concentration (1 to 5 CFU/25 g). The cross-reaction of the anti-Salmonella antibodies, especially to other gram-negative bacteria, is nil.     

Salmonella-TEK, a rapid screening method for Salmonella species in food

A micro-enzyme-linked immunosorbent assay (micro-ELISA) using the Salmonella-TEK screen kit was tested for the detection of Salmonella spp. in pure cultures as well as in 30 artificially contaminated food samples and in 45 naturally contaminated food samples. Different raw, fleshy foods and processed foods were used as test products. The artificially contaminated minced meat samples were preenriched in buffered peptone water, and after incubation, different selective enrichment broths were tested. The micro-ELISA optical density values after enrichment and isolation of the different broths were very analogous. The quickest method to detect Salmonella spp. in different foods is to enrich them with Salmosyst broth, which reduces the total analysis time to 31 h. The Salmonella-TEK kit for Salmonella spp. provides a promising test for the detection of Salmonella antigens in food even when they are present at a low concentration (1 to 5 CFU/25 g). The cross-reaction of the anti-Salmonella antibodies, especially to other gram-negative bacteria, is nil.     

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.     

Preslaughter Holding Environment in Pork Plants Is Highly Contaminated with Salmonella enterica

The objective of this study was to determine whether abattoir pens can provide a Salmonella enterica infection source during the 2 to 4 h of preharvest holding. Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens. For 24 groups of pigs studied (~150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded. Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period (~2.5 h). After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group. All samples were cultured for the isolation and identification of S. enterica by primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification. The study pens were highly contaminated with S. enterica; all holding pens sampled had at least one positive sample. Additionally, 33% (8 of 24) of drinking water samples were positive for S. enterica. All 24 groups of pigs had S. enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples. From pigs, trailers, and pens, 586 isolates representing 36 different Salmonella serovars were isolated. Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers. Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source. This study demonstrates highly contaminated abattoir holding pens and watering sources. It also demonstrates that holding pens can serve as an infection source. This study identifies the abattoir holding pens as a significant hazard and a potential control point for Salmonella contamination in the preharvest pork production chain.