Electrical Counting and Sizing of Mammalian Cells in Suspension

A recently developed method of determining the number and size of particles suspended in a conducting solution is to pump the suspension through a small orifice having an immersed electrode on each side to supply electrical current. The current changes due to the passage of particles of resistivity different from that of the solution. Theoretical expressions are developed which relate the current change caused by such particles to their volume and shape. It is found that most biological cells may be treated as dielectric particles whose capacitive effects are negligible. Electrolytic tank measurements on models confirm the theoretical development, and electric field plots of model orifices are used to predict the observed pulse shapes. An equivalent circuit of the orifice-electrode system is analyzed and shows that the current pulse may be made conductivity-independent when observed with a zero input impedance amplifier

Fructose-Asparagine Is a Primary Nutrient during Growth of <i>Salmonella</i> in the Inflamed Intestine

<div><p><i>Salmonella enterica</i> serovar Typhimurium (<i>Salmonella</i>) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that <i>Salmonella</i> encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for <i>Salmonella</i> fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10^−/− mice). The locus encoding F-Asn utilization, <i>fra</i>, provides an advantage only if <i>Salmonella</i> can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the <i>fra</i> phenotype is lost in <i>Salmonella</i> SPI1⁻ SPI2⁻ or <i>ttrA</i> mutants, respectively). The severe fitness defect of a <i>Salmonella fra</i> mutant suggests that F-Asn is the primary nutrient utilized by <i>Salmonella</i> in the inflamed intestine and that this system provides a valuable target for novel therapies.</p></div

Protection of mice against <i>Salmonella</i> serovar Typhimurium strain 14028 by <i>Enterobacter cloacae</i> strain JLD400.

<p>Germ-free C57BL/6 mice were divided into two groups. One group was colonized with 10⁷ cfu of <i>Enterobacter cloacae</i> via the intragastric route (i.g.) and one group was not. One day later both groups were challenged i.g. with 10⁷ cfu of <i>Salmonella</i>. After 24 hours, the cecum and spleen were homogenized and plated to enumerate <i>Salmonella</i>. Each point represents the CFU/g recovered from one mouse with the geometric mean shown by a horizontal line. Statistical significance between select groups was determined by using an unpaired two-tailed Student <i>t</i> test. ** = P value<0.01, *** = P value<0.001.</p

Quantitation of <i>Salmonella</i> in feces on days 1 through 4, and cecum on day 4, post-infection.

<p>Groups of five C57BL/6 mice heterozygous for <i>Nramp1</i> were orally inoculated with 10⁷ CFU of IR715 (wild-type), MA59 (<i>fraB1</i>::kan mutant), or ASD6000 (<i>fraB1</i>::kan mutant with complementation plasmid pASD5006). The geometric mean+SE is shown. Statistical significance between select groups was determined by using an unpaired two-tailed Student <i>t</i> test. * = P value<0.05, ** = P value<0.01.</p

Fitness defect of a <i>fraB1</i>::kan mutant as measured by competitive index (CI) in various genetic backgrounds and mouse models.

<p>A) 10⁷ wild-type MA43 and <i>fraB1</i>::kan mutant MA59 in germ-free (GF) C57BL/6 mice, via the intragastric route (i.g.) and recovered from the cecum after 24 hours. B) 10⁷ wild-type MA43 and <i>fraB1</i>::kan mutant MA59 in germ-free C57BL/6 mice mono-associated with <i>Enterobacter cloacae</i>, via the i.g. route and recovered from the cecum after 24 hours. C) 10⁹ wild-type MA43 and <i>fraB1</i>::kan mutant MA59 in C57BL/6 conventional mice, via the i.g. route and recovered from the cecum after 24 hours. D) 10⁷ wild-type IR715 and <i>fraB1</i>::kan mutant MA59 in streptomycin-treated (ST) C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. E) 10⁷ wild-type IR715 and <i>fraB1</i>::kan mutant MA59 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. F) Complementation of the <i>fraB1</i>::kan mutation with a plasmid encoding the entire <i>fra</i> island, pASD5006. 10⁷ ASD6090 and ASD6000 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. G) 10⁷ wild-type IR715 and <i>fraB4</i>::kan mutant CS1032 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. H) 10⁷ wild-type IR715 and <i>fraB4</i>::kan mutant CS1032 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. I) Complementation of the <i>fraB4</i>::kan mutation with a plasmid encoding the entire <i>fra</i> island, pASD5006.10⁷ wild-type ASD6090 and <i>fraB4</i>::kan mutant ASD6040 in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. J) 10⁷<i>fra</i>⁺ MA4301 and <i>fraB1</i>::cam mutant MA5900, both strains are a SPI1⁻ SPI2⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. K) 10⁷<i>fra</i>⁺ MA4301 and <i>fraB1</i>::cam mutant MA5900, both strains are a SPI1⁻ SPI2⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. L) 10⁷<i>fra</i>⁺ MA4301 vs <i>fraB1</i>::cam mutant MA5900, both strains in a SPI1⁻ SPI2⁻ background, in germ-free C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. M) 10⁷<i>fra</i>⁺ MA4310 vs <i>fraB1</i>::kan mutant MA5910, both strains are a <i>ttrA</i>⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. N) 10⁷<i>fra</i>⁺ MA4310 vs <i>fraB1</i>::kan mutant MA5910, both strains are a <i>ttrA</i>⁻ background, in streptomycin-treated C57BL/6 mice, via the i.g. route and recovered from the cecum after 4 days. O) 10⁷<i>fra</i>⁺ MA4310 vs <i>fraB1</i>::kan mutant MA5910, both strains are a <i>ttrA</i>⁻ background, in germ-free C57BL/6 mice, via the i.g. route and recovered from the cecum after 24 hours. P) 10⁴ wild-type MA43 and <i>fraB1</i>::kan mutant MA59 in conventional C57BL/6 mice, via the intraperitoneal route (i.p.) and recovered from the spleen after 24 hours. Q) 10⁴ wild-type MA43 and <i>fraB1</i>::kan mutant MA59 in streptomycin-treated C57BL/6 mice, via the i.p. route and recovered from the spleen after 24 hours. Each data point represents the CI from one mouse with the median shown by a horizontal line. Statistical significance of each group being different than 1 was determined by using a one sample Student's <i>t</i> test. Statistical significance between select groups was determined using a Mann-Whitney test. * = P value<0.05, ** = P value<0.01, *** = P value<0.001.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Competitive index (CI) measurements of a <i>sirA</i> mutant in mouse models.

<p>A) 10⁷ wild-type MA43 and <i>sirA</i> mutant MA45 in germ-free mice, via the intragastric route (i.g.) and recovered from the cecum after 24 hours. B) 10⁷ wild-type MA43 vs <i>sirA</i> mutant MA45 in germ-free mice mono-associated with <i>Enterobacter cloacae</i>, via the i.g. route and recovered from the cecum after 24 hours. Each point represents the CI from one mouse with the median shown by a horizontal line. Statistical significance of each group being different than 1 was determined by using a one sample Student's <i>t</i> test. Statistical significance between groups was determined using a Mann-Whitney test. * = P value<0.05, *** = P value<0.001.</p

Growth of <i>Salmonella</i> on F-Asn in the presence or absence of tetrathionate or oxygen.

<p>Growth of wild-type MA43 and <i>fraB1</i>::kan mutant MA59 on 5 mM F-Asn or 5 mM glucose anaerobically (A and B) or aerobically (C and D) in the presence (A and C) or absence (B and D) of 40 mM tetrathionate (S₄0₆²⁻). Bacteria were grown overnight in LB at 37°C shaking, centrifuged, resuspended in water, and subcultured 1∶1000 into NCE medium containing the indicated carbon source. The optical density at 600 nm was then read at time points during growth at 37°C with shaking. Each point represents the mean of four cultures with error bars indicating standard deviation.</p

Oligonucleotides used.

<p>Oligonucleotides used.</p