Rolf D. Joerger

16S rRNA-Based Analysis of Microbiota from the Cecum of Broiler Chickens

ABSTRACT The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.

Evaluation of a polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruits and vegetables.

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administrations (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.

Pressure inactivation kinetics of phage lambda cI 857.

Inactivation curves of phage lambda cI 857 inactivated by high hydrostatic pressure were obtained at three pressure levels (300, 350, and 400 MPa) in buffered media and ultrahigh-temperature 2% reduced fat milk. Pressurization of phage lambda in buffered media at 300 MPa for 300 min, 350 MPa for 36 min, and 400 MPa for 8 min reduced the titer of phage lambda by 7.5, 6.7, and 7.7 log, respectively. Pressurization of phage lambda in milk at 300 MPa for 400 min, 350 MPa for 80 min, and 400 MPa for 20 min reduced the titer of phage lambda by 5.4, 6.4, and 7.1 log, respectively. Tailing was observed in all inactivation curves, indicating that the linear model was not adequate for describing these curves. Among the three nonlinear models studied, the Weibull and log-logistic models consistently produced best fits to all inactivation curves, and the modified Gompertz model the poorest. Because there were no significant differences in the values of shape factor (n) for suspension medium buffer, we reduced the number of parameters in the Weibull model from two to one by setting n at the mean value. The simplified Weibull model produced a fit comparable to the full model. Additionally, the simplified Weibull model allowed predictions to be made at pressures different from the experimental pressures. Menstruum was found to significantly affect the pressure resistance of phage lambda. Comparison of pressure inactivation of hepatitis A virus and phage lambda indicated that phage lambda is more sensitive to pressure than hepatitis A virus in Dulbeccos modified Eagle medium with 10% fetal bovine sera.

Study of the Chemical Composition and Antimicrobial Activities of Ethanolic Extracts from Roots of Scutellaria baicalensis Georgi

Scutellaria baicalensis Georgi (SBG), commonly named Huangqin, showed strong in vitro antimicrobial effects. However, limited research is available to systematically evaluate the effects of extraction methods on the phytochemical composition of SBG and its associated antimicrobial effects. In addition, limited studies have tested SBG as a natural antimicrobial agent on fresh produce such as tomatoes. In the current study, powered roots of SBG were extracted with 60, 80, and 100% ethanol, and their antiviral and antibacterial activities were evaluated. SBG ethanol extracts (SBGEEs) at 6.25 mg/mL showed limited antiviral activities against coliphage MS2 and hepatitis A virus. The SBG 80% ethanol extract (SBG80%EE) exhibited the lowest minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) against six foodborne pathogens. SBG80%EE had the highest contents of flavonoids and phenolic acids determined by high-performance liquid chromatography (HPLC). Among these bioactive compounds, ferulic acid had the lowest MIC and MBC values, 0.4 and 1.0 mg/mL, respectively, followed by baicalein and baicalin. Washing with SBG80%EE (12.5 mg/mL) resulted in >1 log reduction of Salmonella enterica serovars Typhimurium, Kentucky, Senftenberg, and Enteritidis on surface-inoculated grape tomatoes. None of SBGEE solutions changed the total phenolic content, color, or pH values of grape tomatoes. The quantification of these antimicrobial flavonoids and phenolic acids is important to maintain the quality and antimicrobial efficacy of SBG extracts. In addition, the application of SBG on tomatoes has provided valuable insights on the potential usage of this antimicrobial extract.

Comparison of genetic and physiological properties of Salmonella enterica isolates from chickens reveals one major difference between serovar Kentucky and other serovars: response to acid.

For unknown reasons, Salmonella enterica Kentucky has become the serovar most frequently isolated from chickens and chicken carcasses in the United States. In an attempt to identify traits that may underlie this phenomenon, genetic and physiological features of 30 serovar Kentucky chicken isolates were compared with those of chicken isolates belonging to a range of other S. enterica serovars. Most of the well-known Salmonella virulence genes were detected in the serovar Kentucky isolates by PCR, but the cdtB, spvB, spvC, and pefA genes were not found. The serovar Kentucky isolates were as invasive as the non-Kentucky isolates in in vitro assays involving chicken embryo hepatocytes, but were less invasive than the Enteritidis, Mbandaki, and Typhimurium isolates when incubated with human HCT-8 cells. Statistical comparison of growth, biofilm formation, and stress survival data from the serovar Kentucky and those from the serovar Enteritidis, Hadar, Mbandaka, Senftenberg, Typhimurium, and Worthington isolates demonstrated either no differences or differences with only a few of the serovars; however, three data sets were different. These data sets included the OD(600) values of cultures grown in tryptic soy broth (TSB) adjusted to pH 5.5 with acetic acid and survival counts of cells grown in either TSB pH 7 or TSB adjusted to pH 5.5 with acetic acid and then transferred into TSB adjusted to pH 2.5 with HCl. Most notable was the log(10) reduction for acetic acid pre-exposed Kentucky isolates of 3.1 versus <1 log(10) for the other isolates upon transfer to pH 2.5. The connection, if any, between this acid response phenotype and the prevalence of the serovar Kentucky in poultry remains to be elucidated, but it is possible that slightly better growth in the presence of acetic acid in conjunction with not mounting a strong, energy-consuming acetic acid-induced adaptive acid response provides a small competitive advantage to this serovar in low acid environments such as the cecum where the pH is around 5.5.

Survey of retail alfalfa sprouts and mushrooms for the presence of Escherichia coli O157:H7, Salmonella, and Listeria with BAX, and evaluation of this polymerase chain reaction-based system with experimentally contaminated samples

BAX, a polymerase chain reaction (PCR)-based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocylogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.

Differences in pressure tolerance of Listeria monocytogenes strains are not correlated with other stress tolerances and are not based on differences in CtsR.

Thirty strains of Listeria monocytogenes were screened for their pressure tolerance phenotype at 400 MPa for 2 min at 21 degrees C. The strains exhibited reductions ranging from 1.9 to 7.1 log(10)CFU/ml in tryptic soy broth with 6% yeast extract (TSBYE). The 3 most and the 3 least pressure-tolerant strains were further tested for their thermal resistance (based on their ability to survive at 55 degrees C), acid tolerance (based on their ability to survive in acidified TSBYE; pH 2.0) and for their nisin sensitivity. No correlation between pressure tolerance and heat, acid or nisin resistances was found. Nucleotide sequence analysis of the ctsR region in these 6 strains demonstrated that this gene codes for a CtsR protein with identical predicted amino acid sequences. The sequences of the 200-bp region located immediately upstream of the ctsR start codon of the different strains were virtually identical and it is therefore likely that differences in pressure tolerance are based on factors other than the stress gene regulator CtsR. The pressure sensitivity of a cocktail of the 2 most pressure-resistant strains and a cocktail of the 2 most-sensitive strains was investigated when the cocktails were inoculated into a real food system consisting of ground chicken meat. We demonstrated that the nature of the suspending substrate or the temperature did not change the expected pressure tolerance of the cocktails.

Gene expression profiling of a pressure-tolerant Listeria monocytogenes Scott A ctsR deletion mutant

Listeria monocytogenes is a food-borne pathogen of significant threat to public health. High hydrostatic pressure (HHP) treatment can be used to control Listeria monocytogenes in food. The CtsR (class three stress gene repressor) protein negatively regulates the expression of class III heat shock genes. A spontaneous pressure-tolerant ctsR mutant 2-1 that was able to survive under HHP treatment has been identified previously. So far, there is only limited information about the mechanisms of survival and adaptation of this mutant to high pressure. Microarray technology was used to monitor the gene expression profiles of the ctsR mutant 2-1 under HHP treatment. Compared to pressure-treated L. monocytogenes Scott A wild type, 17 genes were up-regulated (>2-fold increase) in the ctsR mutant 2-1, whereas 58 genes were down-regulated (<−2-fold decrease). The entire clpC operon was up-regulated in the ctsR mutant 2-1, indicating that the mutant CtsR protein was not a functional repressor. The increased levels of expression of stress-related genes in ctsR mutant 2-1 may contribute to its survival under high pressure. The reduced expression levels of the genes related to virulence, flagella synthesis, and cell division in the ctsR mutant 2-1 correlate with its characteristics (elongated cells, reduced virulence, and absence of flagella). The gene expression changes determined by microarray assays were confirmed by real-time reverse transcriptase PCR analyses. This study enhances our understanding of how Listeria monocytogenes survives under HHP and may contribute to the design of effective and economically feasible HHP treatment in food processing.

Effect of high hydrostatic pressure on four genotypes of F‐specific RNA bacteriophages

Aims:  The pressure responses of four genotypes of F‐specific RNA bacteriophages, f2, GA, Qβ and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium.

Gene Expression Analysis of Salmonella enterica Enteritidis NalR and Salmonella enterica Kentucky 3795 Exposed to HCl and Acetic Acid in Rich Medium

In the United States, serovar Kentucky has become one of the most frequently isolated Salmonella enterica serovars from chickens. The reasons for this prevalence are not well understood. Phenotypic comparisons of poultry Salmonella isolates belonging to various serovars demonstrated that serovar Kentucky isolates differed from those of most other serovars in their response to acid. Microarray and qPCR analyses were performed with aerated exponentially growing poultry isolates, Salmonella enterica serovar Kentucky 3795 and Enteritidis Nal(R), exposed for 10 min to tryptic soy broth (TSB) adjusted to pH 4.5 with HCl and to pH 5.5 with HCl or acetic acid. Data obtained by microarray analysis indicated that more genes were up- or down-regulated in strain Kentucky 3795 than in Enteritidis Nal(R) under acidic conditions. Acid exposure in general caused up-regulation of energy metabolism genes and down-regulation of protein synthesis genes, particularly of ribosomal protein genes. Both strains appear to similarly utilize the lysine-based pH homeostasis system, as up-regulation of cadB was observed under the acidic conditions. Expression of regulatory genes (rpoS, fur, phoPQ) known to be involved in the acid response showed similar trends in both isolates. Differences between Kentucky 3795 and Enteritidis Nal(R) were observed with respect to the expression of the hdeB-like locus SEN1493 (potentially encoding a chaperone important to acid response), and some differences in the expression of other genes such as those involved in citrate utilization and motility were noted. It appears that the early stages of the transcriptional response to acid by isolates Kentucky 3795 and Enteritidis Nal(R) are similar, but differences exist in the scope and in some facets of the response. Possibly, the quantitative differences observed might lead to differences in protein levels that could explain the observed differences in the acid phenotype of serovar Kentucky and other Salmonella serovars.