Replica Plating Method Research Articles

A replica plating method for efficient, high-throughput screening of antibiotic gene clusters in bacteria uncovers a holomycin-like cluster in the clinical isolate, Pantoea agglomerans 20KB447973

Bacterial natural products remain a major untapped source for novel antimicrobial scaffolds. Many of these products are encoded by biosynthetic gene clusters (BGCs), which can be identified using functional genomics. We developed a replica-plating approach to quickly screen for antibiotic production mutants from transposon mutant libraries and identify candidate antibiotic BGCs. In this technique, filter paper is used to transfer up to 200 mutants simultaneously onto a soft agar overlay or spread plate containing a target microbe to identify antibiotic-production mutants. These mutants can then be analyzed to identify disrupted genes and antibiotic BGCs. We first tested and optimized this technique by screening for previously characterized BGCs in Pantoea. We then applied the technique to uncover the gene cluster responsible for the production of an unknown broad-spectrum antibiotic from P. agglomerans 20KB447973, which we call Pantoea Natural Product 5 (PNP-5). Analysis of the predicted gene cluster for PNP-5 showed similarity to previously identified gene clusters for the broad-spectrum dithiolopyrrolone antibiotic, holomycin. Analysis of the spectrum of activity of PNP-5 showed activity against members of the Enterobacteriaceae, Erwiniaceae, and Streptococcaceae, including clinically relevant pathogens such as Klebsiella sp. and Escherichia coli. We also identified the production of a second antibiotic, pantocin A. Our findings demonstrate the utility of our replica-plating mutant transfer method in exploring unknown antibiotic BGCs. Adoption of this technique may accelerate the identification of potentially novel antimicrobial BGCs within strain collections, advancing the search for novel antimicrobials that can be used to treat multi-drug resistant infections.

The metagenomics of soil bacteria and fungi and the release of mechanical dormancy in hard seeds.

Persistence in the soil is a function of seed physiology, particularly non-germination and inherent lifespan. However, for seeds with mechanical dormancy, non-germination is also a function of the composition and activity of the soil microbiota. We attempted to screen out microorganisms in the soil that can specifically and rapidly decompose the hard fruit pericarps of Tilia miqueliana Maxim., a unique native tree species in China. Using the classical replica plating method, more than 100 different culturable microorganisms that could rapidly erode the pericarp were collected from the surface of pericarps under different culture conditions. At the same time, we successfully extended the concept of metagenomics and applied it to the identification of mixed artificial cultures. The decomposition process of the pericarps in soil was also simulated artificially. The physical and chemical data suggested a potential mechanism of microbial scarification and cracking in pericarp, whilst the embryos inside the eroded fruits retained good viability. Our discoveries could pave the way for the removal of physical and mechanical obstacles that prevent hard coat seeds from germinating. We anticipate that the use of this technology will improve the germination of other hard coat seeds. More research is needed to investigate the impacts on other seeds. The findings of this research can inform the design of experiments on the seed ecology of persistence.

A Genome-Wide Screen for Genes Affecting Spontaneous Direct-Repeat Recombination in Saccharomyces cerevisiae.

Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionarily conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan

We here developed a novel loop-mediated isothermal amplification (LAMP) method to detect Streptococcus suis in raw pork meat. This method, designated LAMPSS, targeted the recombination/repair protein (recN) gene of S. suis and detected all serotypes of S. suis, except those taxonomically removed from authentic S. suis, i.e., serotypes 20, 22, 26, 32, 33, and 34. The specificity of LAMPSS was confirmed and its detection limit was 5.4cfu/reaction. Among the 966 raw pork meat samples examined, including sliced pork, minced pork, and the liver, tongue, heart, and small intestine, 255 samples tested positive with LAMPSS. The rate of contamination was higher in the organs than in pork. No significant difference was observed in the total bacterial count between LAMPSS-positive and -negative samples. The number of shops that provided LAMPSS-positive pork was slightly higher in those that sold swine organs and pork than in those that sold only pork, suggesting that cross contamination occurred from the organs to pork. Among the 255 which tested positive for LAMPSS, only 47 samples tested positive for the previously described LAMP specific for S. suis serotype 2. Two isolates of S. suis serotype 2, belonging to sequence type 28, which is potentially hazardous to humans, as well as those of some other serotypes were obtained from 19 out of 47 samples by combining LAMP with a replica plating method. These results suggest that LAMPSS will be a useful tool for the surveillance of raw pork meat in the retail market.

Isolation and Identification of Bacteria Micro Flora of White Shrimp, Litopenaeus vannamei, with Antagonistic Properties Against Vibrio Species

The emergence of antibiotic-resistant bacterial pathogen has led aquaculture attentions to the use of probiotics as an alternative to antibiotics. This study was conducted to isolate bacterial micro flora from digestive tract of healthy juvenile white shrimp, Litopenaeus vannamei based on antagonistic activity against shrimp pathogen, Vibrio parahaemolyticus. In this study, potential probiotic strains were isolated using replica plating method to screen the bacteria with antagonism properties. Six isolates were identified as Pseudomonas sp. using conventional biochemical tests and Biolog GN microplates. In addition three Vibrio species including V. parahaemolyticus, Vibrio alginolyticus and Vibrio cholerae were isolated from moribund shrimp and used as pathogen in this study. The pathogenicity of three Vibrio species was tested on shrimp L. vannamei by injecting 0.1 mL of 107 CFU mL-1 of each pathogen into the third abdominal segment. Among three Vibrio species only V. parahaemolyticus found to be highly virulent to shrimp with 43% cumulated mortality after 10 days. Subsequently, all Pseudomonas sp. isolates were tested for antibacterial activity against three Vibrio species using cross streak assay. Strong antibacterial activity was recorded for Pseudomonas sp. isolates number 5, 7, 15 and 30 against three pathogens. In addition a reasonable antibacterial activity was observed for isolates number 9 and 12. On the basis of great antibacterial activity of Pseudomonas sp. isolates these species may be considered for future challenge experiments in shrimp as a very promising alternative to the use of antibiotics.

PREVALENCE OF ANTIBIOTIC RESISTANT STRAINS OF ESCHERICHIA COLI AND ENTEROCOCCUS SPP. IN ROE DEER (CAPREOLUS CAPREOLUS) AND RED DEER (CERVUS ELAPHUS) AT THE PARCO NAZIONALE DEI MONTI SIBILLINI, ITALY

A case control study was performed in the Parco Nazionale dei Monti Sibillini, Italy, to find out whether roe deer (Capreolus capreolus) and red deer (Cervus elaphus) were more likely to harbour antibiotic resistant Escherichia coli in their faeces, compared to Enterococcus spp. Ten areas were selected and samples were collected during a fourmonths (May to August, 2008) sampling period. Samples of water (n=12) and feces (n=59), collected at 10 different sites, were cultured for E. coli and Enterococcus spp. The resulting colonies were screened for tetracycline, ampicillin and kanamycin resistance using the Lederberg Replica Plating method (breakpoint 4 μg/ml). All resistant isolates were then selected, and subjected to the CLSI antimicrobial plate susceptibility test (7). Among the water specimens contaminated by E. coli, 80% were found to be resistant to ampicillin, 80% to tetracycline and 40% to kanamycin. Among the water specimens contaminated by Enterococcus spp., 14.29% were found to be resistant to ampicillin, 14.29% to tetracycline and 71.3% to kanamycin. Among the 39 strains of E. coli isolated from red deer feces, 12 were resistant to ampicillin (30.77%), 5 to tetracycline (12,82%) and 3 to kanamycin (7.69%). Among the 19 strains of Enterococcus spp. isolated from red deer feces, 0 were resistant to ampicillin (0%), 1 to tetracycline (5.26%) and 19 to kanamycin (100). These are significant findings, indicating that antibiotic resistance can be found in naive animal populations and that red deer and fallow deer could act as sentinels for antimicrobial resistance. Key words Antibiotic-resistance, red deer, fallow deer, Escherichia

PREVALENCE OF ANTIBIOTIC RESISTANT STRAINS OF ESCHERICHIA COLI AND ENTEROCOCCUS SPP. IN ROE DEER (CAPREOLUS CAPREOLUS) AND RED DEER (CERVUS ELAPHUS) AT THE PARCO NAZIONALE DEI MONTI SIBILLINI, ITALY

A case control study was performed in the Parco Nazionale dei Monti Sibillini, Italy, to find out whether roe deer (Capreolus capreolus) and red deer (Cervus elaphus) were more likely to harbour antibiotic resistant Escherichia coli in their faeces, compared to Enterococcus spp. Ten areas were selected and samples were collected during a fourmonths (May to August, 2008) sampling period. Samples of water (n=12) and feces (n=59), collected at 10 different sites, were cultured for E. coli and Enterococcus spp. The resulting colonies were screened for tetracycline, ampicillin and kanamycin resistance using the Lederberg Replica Plating method (breakpoint 4 μg/ml). All resistant isolates were then selected, and subjected to the CLSI antimicrobial plate susceptibility test (7). Among the water specimens contaminated by E. coli, 80% were found to be resistant to ampicillin, 80% to tetracycline and 40% to kanamycin. Among the water specimens contaminated by Enterococcus spp., 14.29% were found to be resistant to ampicillin, 14.29% to tetracycline and 71.3% to kanamycin. Among the 39 strains of E. coli isolated from red deer feces, 12 were resistant to ampicillin (30.77%), 5 to tetracycline (12,82%) and 3 to kanamycin (7.69%). Among the 19 strains of Enterococcus spp. isolated from red deer feces, 0 were resistant to ampicillin (0%), 1 to tetracycline (5.26%) and 19 to kanamycin (100). These are significant findings, indicating that antibiotic resistance can be found in naïve animal populations and that red deer and fallow deer could act as sentinels for antimicrobial resistance. Key words Antibiotic-resistance, red deer, fallow deer, Escherichia

Case-Control Study To Determine whether River Water Can Spread Tetracycline Resistance to Unexposed Impala ( Aepyceros melampus ) in Kruger National Park (South Africa)

A case-control study was performed in the Kruger National Park (KNP), South Africa, to find out whether impala (Aepyceros melampus) were more likely to harbor tetracycline-resistant Escherichia coli (TREC) in their feces when they drank from rivers that contained these bacteria than when they drank from rivers that were uncontaminated with TREC. The following five perennial rivers were selected: the Crocodile, the Letaba, the Olifants, the Sabie, and the Sand. Samples of river water (n = 33) and feces (n = 209), collected at 11 different sites, were cultured for E. coli. The resulting colonies were screened for tetracycline resistance by use of the Lederberg replica plating method (breakpoint, 4 mg/liter). A resistant and/or a susceptible isolate was then selected from each sample and subjected to the CLSI MIC broth microdilution test for tetracyclines. Among the 21 water specimens contaminated by E. coli, 19.05% (n = 4) were found to be resistant by the MIC method (breakpoint, >/=8 mg/liter). This led to the Crocodile, Olifants, and Letaba rivers being classified as TREC positive. Among the 209 impala feces sampled, 191 were positive for the presence of E. coli (91.38%). Within these (n = 191), 9.95% (n = 19) of the isolates were shown to be TREC by the MIC method. It was found that 1.11% (n = 1) of the E. coli isolates cultured from the feces of the control group (n = 90) were TREC, in comparison with 17.82% (n = 18) of those in feces from the exposed group (n = 101). The calculation of the odds ratio showed that impala drinking from TREC-contaminated rivers were 19.3 (2.63 to 141.69) times more likely to be infected with TREC than were unexposed impala. This is a significant finding, indicating that surface water could be a possible source of antimicrobial resistance in naïve animal populations and that impala could act as sentinels for antimicrobial resistance.

Effects of three dosages of oral oxolinic acid treatment on the selection of antibiotic-resistant Aeromonas: Experimental approach in farmed trout

To determine the effect of oxolinic acid (OA) dosage treatment on the selection of antibiotic-resistant bacteria in farmed trout, three different dosages of OA were administered over seven consecutive days, via medicated feed, to healthy rainbow trout reared in experimental tanks supplied with river water. OA dosage licensed in France (12 mg/kg/day), over-dosage (24 mg/kg/day) and under-dosage (6 mg/kg/day) were tested. The importance of trout gut and rearing waters as potential sites of selection for antibiotic-resistant bacteria in fish farming were investigated (i) by quantification of heterotrophic microbiota on GSP selective medium and of motile Aeromonas on TSA medium, and (ii) by characterisation of Aeromonas OA susceptibility levels before, during and until two weeks after treatment. In the intestines of treated and control fish, bacterial counts were severely reduced during the entire experiment, and Aeromonas populations remained susceptible to OA. In contrast, bacterial counts in output water of both medicated and control tanks were significantly higher than in input water. OA minimum inhibitory concentration distributions of Aeromonas estimated with a replica plating method (GSP plates supplemented with OA concentrations from 0.03 mg/l to 2 mg/l) indicated that the treatment, whatever the dosage, had induced the emergence of OA-resistant Aeromonas in output water. OA MIC of resistant Aeromonas isolated on GSP agar (estimated MIC ≥ 4 mg/l) determined using an agar dilution method on Mueller–Hinton agar confirmed the presence in output water of highly OA-resistant Aeromonas (MIC ≥ 128 mg/l). Between one and two weeks after the end of treatment, those highly OA-resistant Aeromonas were all the more prevalent among total Aeromonas populations that treatment dosage was higher. Results suggested that selection and emergence of OA-resistant Aeromonas had probably occurred in faecal matters after excretion, rather than in the fish intestines.

Emergence of Clarithromycin‐Resistant Helicobacter pylori (CRHP) with a High Prevalence in Children Compared with Their Parents

Clarithromycin-resistant Helicobacter pylori (CRHP) is increasing worldwide. Clarithromycin resistance in H. pylori from familial members has not been investigated. Biopsy specimens were taken from 13 families living in Tokyo, Yokohama, and Niigata between 1998 and 2001. Drug resistance was tested with the replica plating method. The minimum inhibitory concentrations of antimicrobial agents for H. pylori strains were determined by the agar dilution method. Molecular analyses of H. pylori strains were performed by ribosomal RNA gene restriction pattern analysis. The DNA region, associated with clarithromycin resistance, was analyzed by PCR and sequencing. Helicobacter pylori strains isolated from a 5-year-old-son displayed clarithromycin resistance with a mutation (A --> G at position 2143) in the 23S ribosomal RNA, whereas H. pylori strains from his parents did not. DNA analyses revealed that the boy was infected with his father's strain. The boy had repeatedly developed otitis media and received clarithromycin since the age of 2 years. Studies on an additional 12 families demonstrated that clarithromycin resistance in the children's strains reached 42.9% and was significantly higher than those of H. pylori strains from their parents (0%) or from adult patients (11.1%) (p <.05). The rate of clarithromycin resistance in H. pylori strains from Japanese children was extremely high, in contrast to those from their parents or adult patients. Prior history of clarithromycin usage in a child suggested development of clarithromycin resistance in resident H. pylori, which was originated from a parent.

Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios.

We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.

Isolation of High-Malate-Producing Sake Yeasts from Low-Maltose-Assimilating Mutants.

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.

Isolation of high-malate-producing sake yeasts from low-maltose-assimilating mutants

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.

Reversion of argE3 ochre strain Escherichia coli AB1157 as a tool for studying the stationary-phase (adaptive) mutations.

Adaptive (starvation-associated) mutations occur in non-dividing cells and allow growth under the selective conditions imposed. We developed a new method for the determination of adaptive mutations in Escherichia coli. The system involves reversion to prototrophy of the argE3OC mutation and was tested on AB1157 strains mutated in the mutT and/or mutY genes. The bacteria that mutated adaptively grow into colonies on minimal medium plates devoid of arginine (starvation conditions) when incubated longer than 4 days. Using the replica plating method we solved the problem of discrimination between growth-dependent and adaptive argE3-->Arg+ revertants. Phenotype analysis and susceptibility of the Arg+ revertants to a set of T4 phage mutants create an additional possibility to draw a distinction between these two types of Arg+ revertants.

A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance.

The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics. Described here is the use of 5-fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae. Counterselection using 5-fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5-fluoroanthranilic acid. Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica-plating method to rapidly assess genetic linkage in self-replicating shuttle vectors, can now be carried out using the TRP1 marker gene. In addition, novel tryptophan auxotrophs can be selected using 5-fluoroanthranilic acid.

Isolation and enzyme determination of Candida tropicalis mutants for DCA production.

Techniques, named two-step enrichment and double-time replica-plating method (TEDR), are described that allow a mutated population of Candida tropicalis to be enriched efficiently for mutants deficient in the alkane degradation pathway (Alk(-)) and to be selected easily for mutants increasing in the DCA (dicarboxylic acids) excretion pathway. After C. tropicalis was mutated with ethyl methane sulphonate and ultraviolet, the Alk(-) mutants were enriched (the first step enrichment, up to eightfold in one round of enrichment) by treatment with nystatin in medium SEL1-1. The mutagen-treated cells were then cultured in medium YPD containing chlorpromazine for further enriching (the second-step enrichment, up to threefold in one round) the mutants with an increasing capacity of alpha- and omega-oxidation. On the other hand, the Alk(-) mutants were readily isolated by the SEL1 replica-plating method by using alkane or glucose as the sole carbon source. A total of 43 Alk(-) mutants were isolated from 2x10(8) mutagen-treated cells. In the following steps, by using SEL2 replica plating, the screening studies showed that of the 43 Alk(-) mutants, 11 strains could accumulate DCA greatly from alkane, and strains 1-12 and 1-3, especially, could produce nearly three times as much DCA as the wild-type organism could. The results showed that the strains had more cytochrome P450 activity and a higher converting capacity of alkane.

Isolation of mutagen-sensitive Chinese hamster cell lines by replica plating.

Mutant rodent cell lines hypersensitive to DNA-damaging agents have provided a useful tool for the characterization of DNA repair pathways and have contributed to a better understanding of the mechanisms involved in the cellular responses to mutagenic treatment. Here we present a detailed description of how to isolate mutagen-sensitive mutants from hamster "wild-type" cell lines. First, cells are treated with ethyl nitrosourea, and then the mutagenized cell populations are screened for cells with an increased sensitivity to various mutagens using a replica-plating method. Mutagen-sensitive clones are identified and then characterized by assessing their stability, degree of sensitivity to various mutagens, and by genetic complementation analysis.

Antimicrobial and mercury resistance in aerobic gram-negative bacilli in fecal flora among persons with and without dental amalgam fillings.

Antimicrobial resistance is more widespread than can be accounted for as being a consequence of the selection pressure caused by the use of antibiotics alone. In this study, we tested the hypothesis that a high mercury content in feces might select for mercury-resistant bacteria and thus for antimicrobial resistance linked to mercury resistance. Three subject groups with different exposures to dental amalgam fillings were compared. None of the subjects had taken antimicrobial agents during the three preceding months or longer. The group exposed to dental amalgam (n = 92) had 13 times more mercury in feces than the group that had never been exposed to amalgam (n = 43) and the group whose amalgam fillings had been removed (n = 56). No significant differences in either mercury resistance or antibiotic resistance in the fecal aerobic gram-negative flora of these subject groups were seen. The following antimicrobial resistance frequencies were detected with a replica plating method: > or = 1% resistance was seen in 40% of the subjects for ampicillin, 14% of the subjects for cefuroxime, 6% of the subjects for nalidixic acid, 14% of the subjects for trimethoprim, 19% of the subjects for sulfamethoxazole, and 25% of the subjects for tetracycline. The amount of mercury in feces derived from amalgam was not selective for any resistance factors in aerobic gram-negative bacteria, but antimicrobial resistance was widespread even among healthy subjects with no recent exposure to antibiotics.

Analysis of plasmid stability by the difference in colony size

Due to the reduced growth rate, plasmid-haboring cells give smaller colonies or agar plates than their plasmid-free counterparts. Based on the difference in colony size, a new method to measure plasmid stability is proposed. The new method is flexible, fast, and agrees well with the replica plating method.

Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.