Chin-Yi Chen

A 6×6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli

A protocol was developed using 96-well plates and multichannel pipettes for serial dilutions, followed by drop plating on agar in a 6 x 6 format. This protocol permits simultaneous plating of six dilutions which greatly decreases the number of plates utilized thereby saving incubator space for organisms such as Campylobacter which require unique environmental conditions.

Quorum Sensing and Production of Autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica Serovar Typhimurium in Foods

ABSTRACT Autoinducer molecules are utilized by gram-negative and gram-positive bacteria to regulate density-dependent gene expression by a mechanism known as quorum sensing. PCR and DNA sequencing results showed that Campylobacter jejuni and Campylobacter coli possessed luxS, which is responsible for autoinducer-2 (AI-2) production. Using a Vibrio harveyi luminescence assay, the production of AI-2 was observed in milk, chicken broth, and brucella broth by C. coli, C. jejuni, Salmonella enterica serovar Typhimurium, and Escherichia coli O157:H7 under different conditions.

Sequence of the Escherichia coli O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic E. coli O121 by PCR Amplification of the wzx and wzy Genes

ABSTRACT The DNA sequence of the 15,155-bp O-antigen gene cluster of Escherichia coli O121 was determined, and 14 open reading frames were identified (all had the same transcriptional direction). Analyses of results indicated that the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes were E. coli O121 specific, so regions in these two genes were chosen for development of PCR assays. The PCR assays using DNA from 99 E. coli O121 strains, strains representative of non-O121 E. coli serogroups, and strains of other bacterial genera and PCR assays using DNA from seven enrichments of swine fecal samples naturally contaminated with E. coli O121 showed specificity for E. coli O121. Thus, the PCR assay can be employed to reliably identify E. coli O121 and to potentially detect the organism in food, fecal, and environmental samples.

Analysis of AI-2/LuxS-Dependent Transcription in Campylobacter jejuni Strain 81-176

Autoinducer-2 (AI-2) is a quorum-sensing signal molecule that controls a variety of cellular activities in response to cell density in both gram-negative and gram-positive bacteria. The production of AI-2 is dependent upon LuxS, the last enzyme in the AI-2 biosynthesis pathway. For this study, we constructed a luxS null mutation (Delta luxS) in Campylobacter jejuni strain 81-176, and showed that it abolished AI-2 production. The Delta luxS mutant had a longer doubling time in Mueller-Hinton (MH) broth and reduced swarming on MH soft agar at 37 degrees C compared to the wild type (wt), whereas growth rate or swarming at 42 degrees C was not affected. The Delta luxS mutant was also more sensitive to hydrogen peroxide (H(2)O(2)) and cumene hydroperoxide than the wt by disc inhibition assays at 42 degrees C, though minimum inhibitory concentration comparisons were inconclusive. Differences in genome-wide gene expression between wt and Delta luxS mutant with and without H(2)O(2) treatments were compared using DNA microarrays. The genes that showed differential expressions (wt/Delta luxS) include operons/pathways involved in AI-2 synthesis and S-adenosylmethionine (SAM) metabolism (metE, metF, and pfs), flagellar assembly/regulation, stress response (ahpC, tpx, and groES), ABC transporters/efflux systems, and two genes of unknown function located downstream of luxS (Cj1199 and Cj1200). The wt/Delta luxS expression ratios of ahpC (encoding alkyl hydroperoxide reductase) and tpx (encoding thiol peroxidase) were increased only with H(2)O(2) treatment, consistent with our finding that the Delta luxS mutant exhibits higher sensitivity to oxidative stress than wt. Our microarray results agreed with the Delta luxS mutant phenotypes, and suggested that LuxS plays a role in central metabolism involving SAM metabolism, but it is uncertain whether AI-2 functions as a true quorum-sensing signal in C. jejuni.

Antimicrobial activity of spherical silver nanoparticles prepared using a biocompatible macromolecular capping agent: evidence for induction of a greatly prolonged bacterial lag phase

BackgroundWe have evaluated the antimicrobial properties of Ag-based nanoparticles (Np s) using two solid phase bioassays and found that 10-20 μL of 0.3-3 μM keratin-stabilized Np s (depending on the starting bacterial concentration = CI) completely inhibited the growth of an equivalent volume of ca. 103 to 104 colony forming units per mL (CFU mL-1) Staphylococcus aureus, Salmonella Typhimurium, or Escherichia coli O157:H7 on solid surfaces. Even after one week at 37°C on solid media, no growth was observed. At lower Np concentrations (= [Np]s), visible colonies were observed but they eventually ceased growing.ResultsTo further study the physiology of this growth inhibition, we repeated these experiments in liquid phase by observing microbial growth via optical density at 590 nm (OD) at 37°C in the presence of a [Np] = 0 to 10-6 M. To extract various growth parameters we fit all OD[t] data to a common sigmoidal function which provides measures of the beginning and final OD values, a first-order rate constant (k), as well as the time to calculated 1/2-maximal OD (tm) which is a function of CI, k, as well as the microbiological lag time (T).Performing such experiments using a 96-well microtitre plate reader, we found that growth always occurred in solution but tm varied between 7 (controls; CI = 8 × 103 CFU mL-1) and > 20 hrs using either the citrate-([Np] ~ 3 × 10-7 M) or keratin-based ([Np] ~ 10-6 M) Np s and observed that {∂tm/∂ [Np]}citrate ~ 5 × 107 and {∂tm/∂ [Np]}keratin ~ 107 hr·L mol-1. We also found that there was little effect of Np s on S. aureus growth rates which varied only between k = 1.0 and 1.2 hr-1 (1.1 ± 0.075 hr-1). To test the idea that the Np s were changing the initial concentration (CI) of bacteria (i.e., cell death), we performed probabilistic calculations assuming that the perturbations in tm were due to CI alone. We found that such large perturbations in tm could only come about at a CI where the probability of any growth at all was small. This result indicates that much of the Np-induced change in tm was due to a greatly increased T (e.g., from ca. 1 to 15-20 hrs). For the solid phase assays we hypothesize that the bacteria eventually became non-culturable since they were inhibited from undergoing further cell division (T > many days).ConclusionWe propose that the difference between the solid and liquid system relates to the obvious difference in the exposure, or residence, time of the Np s with respect to the bacterial cell membrane inasmuch as when small, Np-inhibited colonies were selected and streaked on fresh (i.e., no Np s present) media, growth proceeded normally: e.g., a small, growth-inhibited colony resulted in a plateful of typical S. aureus colonies when streaked on fresh, solid media.

Phenotypic and Genotypic Characterization of Biofilm Forming Capabilities in Non-O157 Shiga Toxin-Producing Escherichia coli Strains

The biofilm life style helps bacteria resist oxidative stress, desiccation, antibiotic treatment, and starvation. Biofilm formation involves a complex regulatory gene network controlled by various environmental signals. It was previously shown that prophage insertions in mlrA and heterogeneous mutations in rpoS constituted major obstacles limiting biofilm formation and the expression of extracellular curli fibers in strains of Escherichia coli serotype O157:H7. The purpose of this study was to test strains from other important serotypes of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O113, O121, and O145) for similar regulatory restrictions. In a small but diverse collection of biofilm-forming and non-forming strains, mlrA prophage insertions were identified in only 4 of the 19 strains (serotypes O103, O113, and O145). Only the STEC O103 and O113 strains could be complemented by a trans-copy of mlrA to restore curli production and Congo red (CR) dye affinity. RpoS mutations were found in 5 strains (4 serotypes), each with low CR affinity, and the defects were moderately restored by a wild-type copy of rpoS in 2 of the 3 strains attempted. Fourteen strains in this study showed no or weak biofilm formation, of which 9 could be explained by prophage insertions or rpoS mutations. However, each of the remaining five biofilm-deficient strains, as well as the two O145 strains that could not be complemented by mlrA, showed complete or nearly complete lack of motility. This study indicates that mlrA prophage insertions and rpoS mutations do limit biofilm and curli expression in the non-serotype O157:H7 STEC but prophage insertions may not be as common as in serotype O157:H7 strains. The results also suggest that lack of motility provides a third major factor limiting biofilm formation in the non-O157:H7 STEC. Understanding biofilm regulatory mechanisms will prove beneficial in reducing pathogen survival and enhancing food safety.

Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serovars.

ABSTRACT Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kanr) phenotypes, 102 KanrSalmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the KanrSalmonella isolates were amplified by PCR, sequenced, and showed four different aph(3′)-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.

Multiple mechanisms responsible for strong Congo-red-binding variants of Escherichia coli O157:H7 strains.

High variability in the expression of csgD-dependent, biofilm-forming and adhesive properties is common among Shiga toxin-producing Escherichia coli. Although many strains of serotype O157:H7 form little biofilm, conversion to stronger biofilm phenotypes has been observed. In this study, we screened different strains of serotype O157:H7 for the emergence of strong Congo-red (CR) affinity/biofilm-forming properties and investigated the underlying genetic mechanisms. Two major mechanisms which conferred stronger biofilm phenotypes were identified: mutations (insertion, deletion, single nucleotide change) in rcsB region and stx-prophage excision from the mlrA site. Restoration of the native mlrA gene (due to prophage excision) resulted in strong biofilm properties to all variants. Whereas RcsB mutants showed weaker CR affinity and biofilm properties, it provided more possibilities for phenotypic presentations through heterogenic sequence mutations.

Binding of nontarget microorganisms from food washes to anti-Salmonella and anti-E. coli O157 immunomagnetic beads: most probable composition of background Eubacteria

We present herein the composition of bacterial communities occurring in ground chicken and the changes which arise in these populations based upon nonselective partitioning by commercially-available Dynal anti-Salmonella and anti-E. coli O157 immunomagnetic beads (IMB). Our enumeration and colony selection protocol was based upon a 6 × 6 drop plate method (n = 18 for each 25-g sub-sampling) using a dilution which resulted in ca. 4–8 colonies per drop. An average of 82 ± 13 colonies were selected from three 25-g ground chicken subsamplings per batch, each of which was repeated seasonally for one year. DNA was extracted from each colony and the composition of Eubacteria in each of these harvests was determined by sequence-based identification of 16S rDNA amplicons. The Gram-positive bacteria Brochothrix thermosphacta and Carnobacterium maltaromticum were the most commonly found organisms in both the total chicken wash (PBS) and in the IMB-bound (PBS-washed) fractions. The remaining background organisms which also adhered to varying degrees to commercial IMBs were: Pseudomonas oleovorans, Acinetobacter lwoffi, Serratia spp., and one Rahnella spp. A large number of the organisms were also cladistically evaluated based on rDNA basepair disparities: all Brochothrices were monophyletic; twelve different Pseudomonads were found along with eight Carnobacteria, seven Acinetobacteres, four Serratiae, and two Rahnellae. Carnobacterium alone showed an IMB-based concentration enhancement (ca. two to sixfold).

Peroxide resistance in Escherichia coli serotype O157:H7 biofilms is regulated by both RpoS- dependent and -independent mechanisms

In many Escherichia coli serotype O157 : H7 strains, defences against peroxide damage include the peroxiredoxin AhpCF and three catalases: KatG (catalase/peroxidase), KatE (catalase) and the plasmid-encoded KatP (catalase/peroxidase). AhpC and KatG basal expression is maintained by RpoS, and AhpC, KatG and KatP are all induced by OxyR/σ(70) in exponential phase. KatE is regulated by RpoS during stationary growth and is independent of OxyR. In a previous study we used mutant strains of ATCC 43895 (EDL933) with deletions of katG, ahpC, katE and katP in all possible combinations to characterize peroxide resistance during both exponential and 18-24 h growth in Luria-Bertani broth at 37 °C. In this study, we used triple deletion strains that isolated each catalase/peroxidase gene to investigate their role in the peroxide resistance of biofilm-forming variant 43895OR in 48 and 72 h biofilms. We also used quantitative real-time reverse transcriptase PCR and translational lacZ fusions to study gene expression. Peroxide resistance was greater (P<0.05) in biofilm cells than in planktonic cells, and full resistance required rpoS but not oxyR. In 72 h biofilms, katG and katE were the major protective genes. katG, ahpC and katE peroxide protection had both rpoS-dependent and rpoS-independent components, but katP protection was independent of rpoS. H(2)O(2) challenge induced (P<0.05) katG, ahpC and katP expression in biofilm cells, suggesting that peroxide induction of the OxyR-dependent resistance genes may contribute to the RpoS-independent protection in Shiga toxin-producing E. coli biofilms.