A comparison of methods for detecting bacteriophage contamination of tissue culture sera

Summary Detection of bacteriophage contamination of tissue culture sera by direct plating has been compared with detection methods based on batch enrichment and on the Poisson distribution (PD plating). Batch enrichment is extremely sensitive for detecting the presence of phage contamination. PD plating combines sensitive, withy isolation of each contaminating phage in pure culture. Both batch enrichment and PD plating are more sensitive than direct plating. Neither method requires highly trained personnel or specialized equipment.     

A rapid and sensitive method for the detection of Yersinia enterocolitica strains from clinical samples

AIMS: To compare three culture methods to detect Yersinia enterocolitica from oral or rectal swabs from experimentally infected pigs. METHODS AND RESULTS: The three methods used were: direct plating on Cefsulodin-Irgasan-Novobiocin (CIN) agar, cold enrichment in phosphate buffered saline (PBS) followed by plating on CIN agar and selective enrichment with Luria-Bertani-Bile Salts Irgasan (LB-BSI) followed by plating on CIN agar. Selective enrichment with LB-BSI produced the highest recovery rate (63%), when compared with cold enrichment (52%) and plating on CIN agar alone (43%). Selective enrichment with LB-BSI was significantly (P < 0.02) more sensitive than direct plating on CIN agar and more sensitive than cold enrichment (P < 0.1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Selective enrichment with LB-BSI was more sensitive than the widely accepted method of cold enrichment and it reduced the time required for detection of Y. enterocolitica by three weeks. Selective enrichment with LB-BSI was also compatible with a multiplex PCR technique.     

A Bayesian modelling framework to estimate Campylobacter prevalence and culture methods sensitivity: application to a chicken meat survey in Belgium

Aims: To estimate the true prevalence of Campylobacter and the diagnostic sensitivity of routine detection methods by applying a Bayesian modelling approach. Methods and Results: Results from a Belgium‐wide survey of Campylobacter contamination in chicken meat preparations (n = 656 samples) showed that Campylobacter was detected in 24·2% of the samples by enrichment, compared with 41% detected by direct plating. Combining positive results from both methods increased the apparent prevalence to 48·02%. Bayesian model was set up in WinBUGS software, the model estimates Campylobacter prevalence as 60% (95% Credibility interval (CI): 47–82%), and the sensitivity of enrichment culture and direct plating as 41% (95% CI: 31–52%) and 69% (95% CI: 50–85%), respectively. Conclusions: The parallel use of direct plating and enrichment culture adds value for Campylobacter detection from chicken meat preparations, but the false‐negative results from each culture method must be taken into account. Significance and Impact of the Study: Monitoring data could be strongly biased by the microbiological techniques used to generate it. To circumvent this bias, we describe an applied Bayesian framework for better interpretation of Campylobacter survey data in view of the imperfect test characteristics of routine culture methods.     

Genetic Diversity through the Looking Glass: Effect of Enrichment Bias

The effect of enrichment bias on the diversity of 2,4-dichlorophenoxyacetate (2,4-D)-degrading (2,4-D(sup+)) bacteria recovered from soil was evaluated by comparing the diversity of isolates obtained by direct plating to the diversity of isolates obtained from 85 liquid batch cultures. By the two methods, a total of 159 isolates were purified from 1 g of soil and divided into populations based on repeated extragenic palindromic sequence PCR (rep-PCR) genomic fingerprints. Approximately 42% of the direct-plating isolates hybridized with the tfdA and tfdB genes from Alcaligenes eutrophus JMP134(pJP4), 27% hybridized with the tfdA and tfdB genes from Burkholderia sp. strain RASC, and 30% hybridized with none of the probes. In contrast, the enrichment isolates not only represented fewer populations than the isolates obtained by direct plating but also exhibited, almost exclusively, a single hybridization pattern with 2,4-D catabolic gene probes. Approximately 98% of the enrichment isolates possessed pJP4-type tfdA and tfdB genes, whereas isolates containing RASC-type tfdA and tfdB genes were obtained from only 2 of the 85 enrichment cultures. The skewed occurrence of the pJP4-type genes among the isolates obtained by enrichment suggests that the competitive fitness of 2,4-D(sup+) populations during growth with 2,4-D may be influenced either by specific tfd alleles or by genetic factors linked to these alleles. Moreover, the results indicate that evaluation of the diversity and distribution of catabolic pathways in nature can be highly distorted by the use of enrichment culture techniques.     

Evaluation of four protocols for the detection and isolation of thermophilic Campylobacter from different matrices

Aims: To identify the optimal method for detection of thermophilic Campylobacter at various stages in the food chain, three culture‐dependent (direct plating, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38). Methods and Results: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006‐1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest number of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real‐time qPCR yielded the highest number of positive samples. Conclusion: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less‐contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensitive and powerful evaluation tool. Significance and impact of the study: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distributed, which suggests this is still a significant risk for consumers.     

Metabolic limitation of DNA-polymerase I synthesis by Escherichia coli strain CM5199

The work was aimed at studying DNA polymerase I synthesis after induction of the vector prophage lambda polA (NM 964) in lysogenic Escherichia coli CM 5199 in the course of batch cultivation in different media and at various growth phases as well as upon "nutrient shifts" caused by adding organic compounds to the minimal medium. The enzyme activity was highest when the phage was induced at the exponential phase of growth and in media richer in their composition. The enzyme synthesis, the dynamics of protein and RNA content were studied after induction of the phage and enrichment of the medium; the studies have shown that synthesis of DNA polymerase I is influenced by limitation at two levels: (1) biosynthesis of amino acids and (2) biosynthesis of components of the protein-synthesizing apparatus. There is a direct correlation between DNA polymerase I biosynthesis under the control of vector DNA and the growth rate of the culture.     

Baseline Data from a Belgium-Wide Survey of Campylobacter Species Contamination in Chicken Meat Preparations and Considerations for a Reliable Monitoring Program

From February to November 2007, chicken meat preparations (n = 656) were sampled at 11 processing companies across Belgium. All samples were tested for Campylobacter by enrichment culture and by direct plating according to standard methods. Almost half (48.02%) of the samples were positive for Campylobacter spp. The mean Campylobacter count was 1.68 log₁₀ CFU/g with a standard deviation of ± 0.64 log₁₀ CFU/g. The study revealed a statistically significant variation in Campylobacter contamination levels between companies; processors with a wider frequency distribution range of Campylobacter counts provided chicken meat preparations with higher Campylobacter incidences and concentrations. There was no significant difference between the counts of Campylobacter spp. in various preparation types. However, the Campylobacter counts and incidences in chicken wings were the highest and portioned-form products (legs, wings, and breasts) showed a higher probability of being Campylobacter positive compared to minced-form products (sausages, burgers, and minced meat). The proportion of Campylobacter-positive samples was significantly higher in July than in other months. Recovery of Campylobacter spp. recovery by direct plating was higher (41.0%) compared to detection after enrichment (24.2%). Statistical modeling of the survey data showed that the likelihood of obtaining a positive result by enrichment culture increases with an increase in the Campylobacter concentration in the sample. In the present study, we provide the first enumeration data on Campylobacter contamination in Belgian chicken meat preparations and address proposals for improving Campylobacter monitoring programs.     

Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods.

A511::luxAB is a recombinant derivative of a broad-host-range bacteriophage specific for the genus Listeria, transducing bacterial bioluminescence into infected cells. In this study, we have evaluated its use for rapid and easy testing of contaminated foods and environmental samples for the presence of viable Listeria cells, in comparison to the standard plating procedure. With a short preenrichment step of 20 h, the system was capable of detecting very low initial contamination rates in several foods artificially contaminated with Listeria monocytogenes Scott A cells. In ricotta cheese, chocolate pudding, and cabbage, less than one cell per g of food could be clearly identified by comparing the light emission of phage-infected samples to that of controls without lux phage. In foods having a large and complex microbial background flora, such as minced meat and soft cheese, at least 10 cells per g were necessary to produce a positive bioluminescence signal. Of 348 potentially contaminated natural food and environmental samples, 55 were found to be Listeria positive by the lux phage method. The standard plating procedure detected 57 positive samples. Some differences were observed with respect to the individual samples, i.e., the lux phage procedure detected more positive samples among the dairy products and environmental samples, whereas the plating procedure revealed more contaminated meat and poultry samples. Overall, both methods performed similarly, i.e., were equally sensitive. However, the minimum time required for detection of Listeria with the luciferase phage assay was 24 h, which is much shorter than the 4 days needed by the standard plating method. Furthermore, a most probable number technique with three parallels, based on the use of A511::luxAB for differentiation of positive and negative tubes, is described. The method enables rapid enumeration of low levels of Listeria cells in several foods tested, against the background of a competing microflora.     

1株产L-天冬氨酸酶大肠埃希菌噬菌体分离和生理特性初步研究

对产L-天冬氨酸酶大肠埃希菌噬菌体进行分离和生理特性研究,有助于为生产过程中噬菌体污染的防治提供指导。采用双层平板法对噬菌体进行分离纯化。利用透射电镜观察噬菌体形态。进行噬菌体全基因组测序和比对。通过测定不同处理条件下噬菌体活性,研究温度、pH、有机溶剂氯仿、去垢剂SDS对噬菌体的影响。从噬菌体污染的L 天冬氨酸酶生产菌种大肠埃希菌HY-05C发酵培养液中分离出1株噬菌体。电镜结果表明,该噬菌体由呈多面体对称的头部和极短的尾部构成。基因组测序和比对结果表明,噬菌体与T7样噬菌体的相似性最高。生理特性研究表明,噬菌体对高温和去垢剂SDS敏感,对有机溶剂氯仿不敏感;最适pH为7.0,碱性条件下活力较为稳定,酸性条件容易失活。噬菌体保藏编号为CICC 80001。     

Procedure for Isolation and Enumeration of Vibrio parahaemolyticus,

An evaluation of criteria used in the identification of Vibrio parahaemolyticus showed that cultural responses varied with respect to growth in broth with 10% NaCl, type of hemolysis, reactions in triple sugar-iron-agar, and serological reactions. With few or no exceptions, cultures were positive for cytochrome oxidase, utilized glucose fermentatively, were sensitive to pteridine (0/129) and novobiocin, and failed to grow in Trypticase soy broth (TSB) without NaCl. A procedure employing a direct plating technique, with or without prior enrichment, was designed for the isolation and enumeration of V. parahaemolyticus. The plating medium consisted of 2.0% peptone, 0.2% yeast extract, 1.0% corn starch, 7% NaCl, and 1.5% agar, with the pH adjusted to 8.0. The enrichment broth was TSB with 7% NaCl. Dilutions of food homogenates were either spread directly on the plates or inoculated into enrichment broth. TSB enrichments were incubated at 42 C for 18 hr. A loopful of the TSB tubes then was streaked onto the direct plating medium. Incubation of plates was at 42 C for 24 to 48 hr. Smooth, white to creamy, circular, amylase-positive colonies were then picked as suspect V. parahaemolyticus. Confirmation of gram-negative, fermentative, oxidase-positive, pleomorphic rods sensitive to pteridine 0/129 was made by a fluorescent-antibody technique. With this procedure, a satisfactory quantitative recovery of known V. parahaemolyticus from inoculated seafoods was made possible. V. parahaemolyticus was nto isolated from other salted foods.     

Determination of the microbial contamination of tablets of tetracycline series antibiotics]

The experiments showed that the method of direct plating of tetracycline tablet suspensions on solid nutrient media containing magnesium sulfate may be used for determination of microbial contamination. The method provided determination of both the antibiotic resistant and the antibiotic sensitive organisms which may be present in the drug. Dilution of the basic suspensions increased the probability of the microbe detection in the tablets.     

Rifamycin: XXXIII. Isolation of Actinophages Active on Streptomyces mediterranei and Characteristics of Phage-Resistant Strains

Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.     

Enumeration of Listeria monocytogenes in raw milk

Enumeration of Listeria monocytogenes in raw milk was compared by direct plating and Most Probable Number (MPN) techniques involving both direct and two-stage enrichments. Direct plating was found to lack sensitivity for the enumeration of low numbers of listerias found in milk but an MPN technique with direct selective enrichment was found to be more suitable than one with two-stage enrichment.     

Rifamycin: XXXIV. Physicochemical Characterization of Actinophages Active on Streptomyces mediterranei

Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.     

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

AIMS: To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity. METHODS AND RESULTS: Water samples from 40 dental units were analysed. Three different techniques for detecting Legionella spp. were compared: (i) culture approach, (ii) direct FISH and (iii) FISH with a previous R2A medium enrichment (R2A/FISH). The FISH detection was confirmed by PCR. The use of the direct FISH does not improve significantly the detection of legionellae when compared with the culture. On the contrary, when R2A/FISH was performed, sensitivity was, respectively, two- and threefold higher than that with the direct FISH and culture approach. Using R2A/FISH, 63% of water samples analysed showed a contamination by legionellae. CONCLUSIONS: Legionellae detection by direct FISH and R2A/FISH in dental unit water is possible but is more rapid and more sensitive (R2A/FISH) than the culture approach. SIGNIFICANCE AND IMPACT OF THE STUDY: R2A/FISH showed that several pathogens present in DUWL are viable but may not be culturable. Unlike PCR, R2A/FISH is designed to detect only metabolically active cells and therefore provides more pertinent information on infectious risk.     

Enrichment for Estuarine Petroleum-Degrading Bacteria using Liquid and Solid Media

Bacteria in oil-contaminated and oil-free estuarine water and sediment were isolated on solid and in liquid petroleum media. A greater variety of bacteria was noted when direct plating techniques were employed, compared with liquid enrichment. In addition, strains isolated by liquid enrichment were found more frequently to be capable of utilizing model petroleum in vitro , compared with isolates obtained by direct plating.     

Prevalence of Broad-Host-Range Lytic Bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa

Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with Sphaerotilus natans ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.     

Nature of the carrier state of bacteriophage SP-10 in Bacillus subtilis

Although the association of phage SP-10 with Bacillus subtilis W-23-S(r) persists in heat- and antiserum-resistant form through the spore stage, it is unstable in vegetative cells and frequently terminates in loss of the carried phage or in lysis. On low-tonicity media, the plating efficiency of carrier cells is low. However, high concentrations of succinate or sucrose or a slowed growth rate preserve viability: on 0.48 m succinate-agar, the viable count per optical density unit is the same as that of a noncarrier control culture. Carrier clones retain phage on 0.48 m succinate-agar. At higher succinate levels, many colonies emerge free of phage; at 1 m succinate, all are cured, probably because high succinate inhibits reinfection. Growth of carrier cells in liquid medium with antiphage serum results in rapid curing; events in such cultures with and without succinate were studied quantitatively by tracing the emergence of sensitive cells, the multiplication and induction of carrier cells, and the sensitivity of carrier cells to superinfection with virulent phage. During log phase, 40 to 70% of the carrier cells became sensitive to virulent phage, although the same cells were insensitive during lag and stationary phase. Apparently, fluctuations in repressor levels are responsible. Spontaneous induction of carrier cells followed a qualitatively similar pattern, perhaps in response to changes in level of the same repressor. Production of sensitive segregants by carrier followed a different course, presumably because the repressor does not affect segregation. Many sensitive cells were found two to three divisions after inoculation in antiserum medium. This suggests that each inoculum cell contained one or only a few phage replicons. The data are compatible with the idea that the carrier state in media without antisera is maintained entirely by reinfection and without replication of phage in the latent state. Alternative models which involve replication of latent phage are not ruled out, however.     

The effect of suspended particulate material on cyanobacteria—cyanophage interactions in liquid culture

B arnet Y vonne M., D aft , M.J. S tewart , W.D.P. 1984. The effect of suspended particulate material on cyanobacteria-cyanophage interactions in liquid culture. Journal of Applied Bacteriology 56 , 109–115.
The effect of the lytic phage LPP-DUN 1 on the cyanobacterium Plectonema boryanum has been investigated in batch and in continuous cultures in the presence and absence of silt. In batch culture Plectonema without added phage grew normally; the presence of phage caused rapid lysis of the cyanobacterium and the addition of silt prevented lysis by the phage. In continuous culture the numbers of cyanobacterial cells and phage particles oscillated in a reciprocal manner, but the addition of silt damped down the oscillations in Plectonema biomass without decreasing the numbers of phage particles isolated from the cultures. The presence of silt thus appears to protect the cyanobacterium from lysis by phage, although the total numbers of phage particles are relatively unaffected by the silt, at least in the short-term.     

Characterization of a temperate actinophage,MPphiWR-1, capable of infectingMicromonospora purpurea ATCC 15835

Summary A temperate actinophage was isolated from soil using the gentamicin-producing microorganism,Micromonospora purpurea ATCC 15835 as host. The characterization of the phage represents the initial step in its development as a cloning vector. The phage isolated, MPphiWR-1, formed red- to purple-pigmented turbid plaques. Cells isolated from these plaques were resistant to superinfection with lytic mutants of MPphiWR-1. Southern blots of genomic DNA from a resistant culture showed that MPphiWR-1 integrated into the host genome. The phage was UV- or Mitomycin C-inducible. The integration, resistance to superinfection and inducibility indicated a lysogenic relationship with the host.Using MPphiE-RCPM, a lytic derivative, the phage host range was demonstrated to include members of three genera: one species each ofAmpullariella andCatellatospora, and 12 species ofMicromonospora. The phage belonged to Ackerman's B1 morphotype having an isometric head and a flexible noncontractile tail. The density of the phage was 1.525 g/cc. Restriction site mapping demonstrated that the phage DNA was 57.9 kb long and had cohesive ends. Using EDTA enrichment, viable mutants with deletions of at least 3.5 kb were isolated and mapped. Phage adsorption, sensitivities and plating efficiency were investigated. Non-liposome PEG-mediated transfection was demonstrated.