The complex multicellular morphology of the food spoilage bacteria Brochothrix thermosphacta strains isolated from ground chicken.

Herein we describe a highly structured, filamentous growth phenotype displayed by an isolate of the food spoilage microorganism Brochothrix thermosphacta. The growth morphology of this B. thermosphacta strain (strain BII) was dependent on environmental factors such as the growth media, incubation temperatures, and the inoculum concentration. Inoculation of cultures in highly dilute suspensions resulted in the formation of isolated, tight aggregates resembling fungal growth in liquid media. This same strain also formed stable, mesh-like structures in 6-well tissue culture plates under specific growth conditions. The complex growth phenotype does not appear to be unique to strain BII but was common among B. thermosphacta strains isolated from chicken. Light and electron micrographs showed that the filaments of multiple BII cells can organize into complex, tertiary structures resembling multistranded cables. Time-lapse microscopy was employed to monitor the development of such aggregates over 18 h and revealed growth originating from short filaments into compact ball-like clusters that appeared fuzzy due to protruding filaments or cables. This report is the first to document this complex filamentous growth phenotype in a wild-type bacterial isolate of B. thermosphacta.

A method for correcting standard-based real-time PCR DNA quantitation when the standard's polymerase reaction efficiency is significantly different from that of the unknown's.

Standard-based real-time or quantitative polymerase chain reaction quantitation of an unknown sample's DNA concentration (i.e., [DNA](unk)) assumes that the concentration dependence of the standard and unknown reactions (related to reaction efficiency, E) are equivalent. In our work with background food-borne organisms which can interfere with pathogen detection, we have found that it is generally possible to achieve an acceptable E (1 ± 0.05) for standard solutions by optimizing the PCR conditions, template purity, primer sequence, and amplicon lengths. However, this is frequently not true for the solutions containing unknown amounts of target DNA inasmuch as cell extracts are more chemically complex than the standards which have been amplified (2(30)-fold) as well as undergone a purification process. When significant differences in E occur, it is not possible to accurately estimate unknown target DNA concentration from the standard solution's slope and intercept (from threshold cycle number, or C(T), versus Log[DNA] data). What is needed is a standard-mediated intercept which can be specifically coupled with an unknown solution's PCR concentration dependence. In this work, we develop a simple mathematical procedure to generate a new standard curve with a slope (∂C(T)/∂Log[Dilution](unk)) derived from at least three dilutions of the unknown target DNA solution ([DNA](unk)) and an intercept calculated from the unknown's C(T)s, DNA concentrations interpolated from the standard curve (i.e., the traditional estimate of [DNA](unk)), and ∂C(T)/∂Log[Dilution](unk). We were able to achieve this due to our discovery of the predictable way in which the observed and ideal C(T) versus Log[DNA] slopes and intercepts deviate from one another. This "correction" in the standard-based [DNA](unk) determination is typically 20-60% when the difference in the standard and unknown E is >0.1.

Sequence analysis and plasmid mobilization of a 6.6-kb kanamycin resistance plasmid, pSNC3-Kan, from a Salmonella enterica serotype Newport isolate.

Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. A series of diverse ColE-like kanamycin resistance plasmids ("KanR plasmids") from Salmonella enterica were characterized previously. In this study, the 6.6-kb pSNC3-Kan from a Salmonella enterica serotype Newport isolate was investigated. It possessed highly conserved RNA I/II and Tn602 (IS903-aph-IS903) regions to two other KanR plasmids pSe-Kan and pSBardo-Kan, but carried a mobC-mobA/BD operon. The mobilization proteins encoded by the mob operon of pSNC3-Kan showed high sequence identity (~95%) to those of an E. coli plasmid pEC34B, except that MobE was not present; and were much less conserved to those of another KanR plasmid pSN11/00Kan (43% - 86% identity). Four structurally different KanR plasmids were investigated for their ability to be mobilized by the conjugal transfer (tra) genes from F and IncP plasmids. Transfer genes derived from IncP plasmids can efficiently mobilize KanR plasmids possessing the mob operons (mobC-mobA/BD), such as pSNC3-Kan and pSN11/00Kan, in bi-parental mating experiments. On the other hand, F tra genes were able to mobilize pU302S, pSNC3-Kan and pSe-Kan, but not pSN11/00Kan. A plasmid-borne mob operon was not required for mobilization of the oriT(F)-bearing pSe-Kan by the F tra genes. This study underscores the complexity of plasmid interaction and the importance of how small mobilizable plasmids may contribute to the spread of antibiotic resistance genes.

Genomic Comparison of Conjugative Plasmids from Salmonella enterica and Escherichia coli Encoding Beta-Lactamases and Capable of Mobilizing Kanamycin Resistance Col-like Plasmids.

Salmonella enterica and Escherichia coli are important human pathogens that frequently contain plasmids, both large and small, carrying antibiotic resistance genes. Large conjugative plasmids are known to mobilize small Col plasmids, but less is known about the specificity of mobilization. In the current study, six S. enterica and four E. coli strains containing large plasmids were tested for their ability to mobilize three different kanamycin resistance Col plasmids (KanR plasmids). Large conjugative plasmids from five isolates, four S. enterica and one E. coli, were able to mobilize KanR plasmids of various types. Plasmids capable of mobilizing the KanR plasmids were either IncI1 or IncX, while IncI1 and IncX plasmids with no evidence of conjugation had disrupted transfer regions. Conjugative plasmids of similar types mobilized similar KanR plasmids, but not all conjugative plasmid types were capable of mobilizing all of the KanR plasmids. These data describe some of the complexities and specificities of individual small plasmid mobilization.

Evidence for a bimodal distribution of Escherichia coli doubling times below a threshold initial cell concentration.

BACKGROUND: In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (tau) only up to a certain threshold cell density. Below this cell concentration (100 CFU mL-1), the tau values were distributed unimodally (mutau = 18 +/- 0.71 min; n = 174). Inclusion of a small amount of ethyl acetate to the LB caused a collapse of the bimodal to a unimodal form. Comparable bimodal tau distribution results were also observed using E. coli cells diluted from mid-log phase cultures. Similar results were also obtained when using either an E. coli O157:H7 or a Citrobacter strain. When sterile-filtered LB supernatants, which formerly contained relatively low concentrations of bacteria(1,000-10,000 CFU mL-1), were employed as a diluent, there was an evident shift of the two populations towards each other but the bimodal effect was still apparent using either stationary or log phase cells. CONCLUSION: These data argue that there is a dependence of growth rate on starting cell density.

The relationship between purely stochastic sampling error and the number of technical replicates used to estimate concentration at an extreme dilution.

For any analytical system the population mean (µ) number of entities (e.g., cells or molecules) per tested volume, surface area, or mass also defines the population standard deviation (σ = square root(µ)). For a preponderance of analytical methods, σ is very small relative to µ due to their large limit of detection (>10(2) per volume). However, in theory at least, DNA-based detection methods (real-time, quantitative or qPCR) can detect ≈ 1 DNA molecule per tested volume (i.e., µ ≈ 1) whereupon errors of random sampling can cause sample means (mean) to substantially deviate from µ if the number of samplings (n), or "technical replicates", per observation is too small. In this work the behaviors of two measures of sampling error (each replicated fivefold) are examined under the influence of n. For all data (µ = 1.25, 2.5, 5, 7.5, 10, and 20) a large sample of individual analytical counts (x) were created and randomly assigned into N integral-valued sub-samples each containing between 2 and 50 repeats (n) whereupon N × n = 322 to 361. From these data the average µ-normalized deviation of σ from each sub-sample's standard deviation estimate (s(j), j = 1 to N; N = 7 [n = 50] to 180 [n = 2]) was calculated (Δ). Alternatively, the average µ-normalized deviation of µ from each sub-sample's mean estimate (mean(j)) was also evaluated (Δ'). It was found that both of these empirical measures of sampling error were proportional to ⁻²âˆšn . µ. Derivative (∂/∂n · Δ or Δ') analyses of our results indicate that a large number of samplings (n ≈ 33 +/- 3.1) are requisite to achieve a nominal sampling error for samples with a µ ≈ 1. This result argues that pathogen detection is most economically performed, even using highly sensitive techniques such as qPCR, when some form of organism cultural enrichment is utilized and which results in a binomial response. Thus, using a specific gene PCR-based (+ or -) most probable number (MPN) assay one could detect anywhere from 0.2 to 10(5) CFU mL(-1) using 6 to 48 reactions (i.e., 8 dilutions × 6 replicates per dilution) depending on the initial concentration of the pathogen and volume sampled.

Isolation and characterization of two novel groups of kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals.

While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.

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.