Regional differences in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of aspergillus flavus along a transect within the United States

Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B1 and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 micrometer in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 micrometer in diameter. The S-strain isolates generally produced high levels of aflatoxin B1, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B1 production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B1 and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 microgram of aflatoxin B1 per ml. The percentages of isolates producing >10 microgram of aflatoxin B1 per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B1 or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.     

A Colorimetric Technique for Detecting Trichothecenes and Assessing Relative Potencies

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B₁, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B₁ and M₁ be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.     

Occurrence of Beauvericin and Enniatins in Wheat Affected by Fusarium avenaceum Head Blight

We evaluated Fusarium contamination and the levels of hexadepsipeptide mycotoxins in 13 wheat samples affected by head blight in Finland. Fusarium avenaceum was the dominant species (91%) isolated from all samples, but isolates of F. culmorum (4%), F. tricinctum (3%), and F. poae (2%) also were recovered. Beauvericin (0.64 to 3.5 μg/g) was detected in all 13 samples. Enniatin B (trace to 4.8 μg/g) was detected in 12 samples, enniatin B₁ (trace to 1.9 μg/g) was detected in 8 samples, and enniatin A₁ (trace to 6.9 μg/g) was detected in 10 samples. Ten of 13 strains of F. avenaceum and 2 strains of F. poae and F. tricinctum produced beauvericin in culture on rice (trace to 70, 9.4, and 33 μg/g, respectively). All strains also produced enniatins (trace to 2,700 μg/g). This is the first report on the natural cooccurence of beauvericin and enniatins in wheat infected predominantly by F. avenaceum.     

Divergence of West African and North American Communities of Aspergillus Section Flavi

West African Aspergillus flavus S isolates differed from North American isolates. Both produced aflatoxin B₁. However, 40 and 100% of West African isolates also produced aflatoxin G₁ in NH₄ medium and urea medium, respectively. No North American S strain isolate produced aflatoxin G₁. This geographical and physiological divergence may influence aflatoxin management.     

Potential for aflatoxin B1 and B2 production by Aspergillus flavus strains isolated from rice samples

In this study, we investigated the potential for aflatoxin B₁ (AFB₁) and B₂ (AFB₂) production in rice grain by 127 strains of Aspergillus flavus isolated from rice grains collected from China. These strains were inoculated onto rice grains and incubated at 28 °C for 21 days. AFB₁ and AFB₂ were extracted and quantified by high-performance liquid chromatography coupled with fluorescence detection. Among the tested strains, 37% produced AFB₁ and AFB₂ with levels ranging from 175 to 124 101 μg kg⁻¹ for AFB₁ and from not detected to 10 329 μg kg⁻¹ for AFB₂. The mean yields of these isolates were 5884 μg kg⁻¹ for AFB₁ and 1968 μg kg⁻¹ for AFB₂. Overall, most of the aflatoxigenic strains produced higher levels of AFB₁ than AFB₂ in rice. The obtained information is useful for assessing the risk of aflatoxin contamination in rice samples.     

Effects of Aflatoxin on Seeding Growth and Ultrastructure in Plants

Nineteen plants belonging to 11 species of the cruciferae were studied to determine the effects of aflatoxin B₁ on seed germination and seedling development. Germination was not inhibited in any test organism at a concentration of 100 μg of aflatoxin per ml of agar substrate. Inhibition of elongation of the hypocotyls and roots in the species studied varied from 29 to 93% and from 22 to 91% in the respective tissues. Lepidium sativum was the most susceptible plant studied and exhibited the maximal inhibitory response noted above at concentrations of 8 μg of aflatoxin per ml. The ultrastructure of Lepidium root cells treated with crystalline aflatoxin B₁ exhibited morphological changes characteristic of those found in aflatoxin-treated animal cells. In addition to changes in the cytoplasmic organelles, numerous ring-shaped nucleoli with prominent nucleolar caps were produced. The effect of aflatoxin on plant cells is compared with similar effects induced by actinomycin D. Seed germination and seedling development is discussed in relation to the effects of both compounds on deoxyribonucleic acid-dependent ribonucleic acid biosynthesis.     

Carbohydrate and Amino Acid Fermentation in the Free-Living Primitive Protozoon Hexamita sp.

Hexamita sp. is an amitochondriate free-living diplomonad which inhabits O₂-limited environments, such as the deep waters and sediments of lakes and marine basins. ¹³C nuclear magnetic resonance spectroscopy reveals ethanol, lactate, acetate, and alanine as products of glucose fermentation under microaerobic conditions (23 to 34 μM O₂). Propionic acid and butyric acid were also detected and are believed to be the result of fermentation of alternative substrates. Production of organic acids was greatest under microaerobic conditions (15 μM O₂) and decreased under anaerobic (<0.25 μM O₂) and aerobic (200 to 250 μM O₂) conditions. Microaerobic incubation resulted in the production of high levels of oxidized end products (70% acetate) compared to that produced under anoxic conditions (20% acetate). In addition, data suggest that Hexamita cells contain the arginine dihydrolase pathway, generating energy from the catabolism of arginine to citrulline, ornithine, NH₄⁺, and CO₂. The rate of arginine catabolism was higher under anoxic conditions than under microaerobic conditions. Hexamita cells were able to grow in the absence of a carbohydrate source, albeit with a lower growth rate and yield.     

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B₁ complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B₁ remained bound. Nonviable bacteria retained the highest amount of aflatoxin B₁. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B₁ from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B₁ to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B₁. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B₁ released. Binding of aflatoxin B₁ appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.     

Production of Beauvericin, Moniliformin, Fusaproliferin, and Fumonisins B1, B2, and B3 by Fifteen Ex-Type Strains of Fusarium Species

Fifteen Fusarium species were analyzed by high-performance liquid chromatography for the production of six mycotoxins in corn grits cultures. Production of mycotoxins ranged from 66 to 2,500 μg/kg for fumonisin B₁, 0.6 to 1,500 μg/g for moniliformin, 2.2 to 720 μg/g for beauvericin, and 12 to 130 μg/g for fusaproliferin. Fumonisin B₂ (360 μg/kg) was produced by two species, fumonisin B₃ was not detected in any of the 15 species examined, and Fusarium bulbicola produced none of the six mycotoxins that we analyzed.     

Maternal dietary intake of folate,vitamin B12 and MTHFR 677C>T genotype: their impact on newborn’s anthropometric parameters

In this study, we evaluated the effects of dietary intake of vitamin B₁₂ and folate during pregnancy and their interactions with maternal polymorphism of MTHFR (677C>T; 1298A>C) on intrauterine development. Anthropometric parameters were obtained from 231 newborns that belong to a prospective birth cohort in Morelos, Mexico. Maternal dietary intake of vitamin B₁₂ and folate was assessed using a semi-quantitative questionnaire administered during the first and third trimesters of the pregnancy. Maternal MTHFR 677C>T and 1298 A>C genotypes were determined by PCR–RFLP. The associations between deficient dietary intake of vitamin B₁₂ (<2.0 μg/d) and folate (<400 μg/d) in the first and third trimesters and maternal polymorphisms of MTHFR on anthropometric parameters at birth were estimated using a multivariate linear regression model. During pregnancy, the deficient dietary intake was roughly 60 % for folate and 19 % for vitamin B₁₂. Allelic frequencies of 677T and 1298C were 59 and 10 %, respectively. After adjusting for confounders, deficiency in maternal dietary intake of vitamin B₁₂ (<2.0 μg/d) was associated with a significant reduction in length (β ~ −2.4; 95 % CI −4.3; −0.6) and length-for-age at birth (β ~ −1.2; 95 % CI −2.3; −0.1) among infants whose mothers were carriers of the 677TT genotype (p for interaction = 0.02). In contrast, no association was observed between deficiency in maternal dietary intake of folate (<400 μg/d) and any anthropometric parameter of newborns. These results suggest that supplementation with vitamin B₁₂ during pregnancy could have a favorable impact on intrauterine fetal development mainly in populations that are genetically susceptible.     

Co-occurrence of aflatoxin B<Subscript>1</Subscript> and cyclopiazonic acid in sour lime (<Emphasis Type="Italic">Citrus aurantifolia Swingle</Emphasis>) during post-harvest pathogenesis by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

During hot and humid seasons, extensive rot of sour lime was observed to be caused by Aspergillus flavus. In view of this, investigations were undertaken to obtain data on the production of various toxins by A. flavus during post harvest pathogenesis of sour lime. Sixty percent of the pathogenic A. flavus isolates were detected to be aflatoxin B₁ producers in sour lime tissue. It was also noted that thirty three percent of aflatoxigenic A. flavus isolates had the potential to coproduce cyclopiazonic acid (CPA). Such aflatoxigenic isolates produced quantitatively more CPA (ranging from 250.0 to 2501.3 g/kg) than aflatoxin B₁ (ranging from 141.3 to 811.7 g/kg) in the affected sour lime. This study demonstrates for the first time that sour lime are a favourable substrate for aflatoxin B₁ and cyclopiazonic acid production by A. flavus isolates. This is of great concern to the health of consumers.     

In Vivo Availability of Pro-Resolving Lipid Mediators in Oxazolone Induced Dermal Inflammation in the Mouse

The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA₄ on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B₄ and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA₄ (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B₄ was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA₄. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

β-Lactam Effects on Mixed Cultures of Common Respiratory Isolates as an Approach to Treatment Effects on Nasopharyngeal Bacterial Population Dynamics

Background

Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae are bacteria present in the nasopharynx as part of normal flora. The ecological equilibrium in the nasopharynx can be disrupted by the presence of antibiotics.

Methodology/Principal Findings

A computerized two-compartment pharmacodynamic model was used to explore β-lactam effects on the evolution over time of a bacterial load containing common pharyngeal isolates by simulating free serum concentrations obtained with amoxicillin (AMX) 875 mg tid, amoxicillin/clavulanic acid (AMC) 875/125 mg tid and cefditoren (CDN) 400 mg bid regimens over 24 h. Strains and MICs (µg/ml) of AMX, AMC and CDN were: S. pyogenes (0.03, 0.03 and 0.015), S. pneumoniae (2, 2 and 0.25), a β-lactamase positive H. influenzae (BL⁺; >16, 2 and 0.06) and a β-lactamase positive AMC-resistant H. influenzae (BLPACR, >16, 8 and 0.06). Mixture of identical 1∶1∶1∶1 volumes of each bacterial suspension were prepared yielding an inocula of ≈4×10⁶ cfu/ml. Antibiotic concentrations were measured both in bacterial and in bacteria-free antibiotic simulations. β-lactamase production decreased AMX concentrations and fT_>MIC against S. pneumoniae (from 43.2% to 17.7%) or S. pyogenes (from 99.9% to 24.9%), and eradication was precluded. The presence of clavulanic acid countered this effect of co-pathogenicity, and S. pyogenes (but not BL⁺ and S. pneumoniae) was eradicated. Resistance of CDN to TEM β-lactamase avoided this co-pathogenicity effect, and CDN eradicated S. pyogenes and H. influenzae strains (fT_>MIC >58%), and reduced in 94% S. pneumoniae counts (fT_>MIC ≈25%).

Conclusions/Significance

Co-pathogenicity seems to be gradual since clavulanic acid countered this effect for strains very susceptible to AMX as S. pyogenes but not for strains with AMX MIC values in the limit of susceptibility as S. pneumoniae. There is a potential therapeutic advantage for β-lactamase resistant cephalosporins with high activity against streptococci.     

Prevalence and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx₁ and/or stx₂, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx₂, eae, katP, etpD, and enterohemorrhagic E. coli hly (hly_EHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx₁. Only 7.0% (n = 5) of the isolates were positive for hly_EHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf_O113, saa, lpfA_O157/01-141, and lpfA_O157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.     

Microcystin Production by Microcystis aeruginosa in a Phosphorus-Limited Chemostat

The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C₁₈ cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C₁₈ reverse-phase column. The specific growth rate (μ) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing μ. The MC-LR and MC-RR contents on a dry weight basis were highest at μ of 0.1/day at 339 and 774 μg g⁻¹, respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower μ, whereas the MC-producing rate was linearly proportional to μ. The C fixation rate at an ambient irradiance (160 microeinsteins m⁻² s⁻¹) increased with μ. The ratios of the MC-producing rate to the C fixation rate were higher at a lower μ. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.     

Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis

Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 μg of β-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 μg/ml. However, these amounts of β-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of β-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of β-exotoxin I.     

Antimicrobial Resistance in Salmonella enterica Serovar Heidelberg Isolates from Retail Meats, Including Poultry, from 2002 to 2006

Salmonella enterica serovar Heidelberg frequently causes food-borne illness in humans. There are few data on the prevalence, antimicrobial susceptibility, and genetic diversity of Salmonella serovar Heidelberg isolates in retail meats. We compared the prevalences of Salmonella serovar Heidelberg in a sampling of 20,295 meats, including chicken breast (n = 5,075), ground turkey (n = 5,044), ground beef (n = 5,100), and pork chops (n = 5,076), collected during 2002 to 2006. Isolates were analyzed for antimicrobial susceptibility and compared genetically using pulsed-field gel electrophoresis (PFGE) and PCR for the bla_CMY gene. A total of 298 Salmonella serovar Heidelberg isolates were recovered, representing 21.6% of all Salmonella serovars from retail meats. One hundred seventy-eight (59.7%) were from ground turkey, 110 (36.9%) were from chicken breast, and 10 (3.4%) were from pork chops; none was found in ground beef. One hundred ninety-eight isolates (66.4%) were resistant to at least one compound, and 49 (16.4%) were resistant to at least five compounds. Six isolates (2.0%), all from ground turkey, were resistant to at least nine antimicrobials. The highest resistance in poultry isolates was to tetracycline (39.9%), followed by streptomycin (37.8%), sulfamethoxazole (27.7%), gentamicin (25.7%), kanamycin (21.5%), ampicillin (19.8%), amoxicillin-clavulanic acid (10.4%), and ceftiofur (9.0%). All isolates were susceptible to ceftriaxone and ciprofloxacin. All ceftiofur-resistant strains carried bla_CMY. PFGE using XbaI and BlnI showed that certain clones were widely dispersed in different types of meats and meat brands from different store chains in all five sampling years. These data indicate that Salmonella serovar Heidelberg is a common serovar in retail poultry meats and includes widespread clones of multidrug-resistant strains.     

Role of periplasmic trehalase in uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: K_m = 156 ± 11 μM and V_max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (K_m = 0.11 ± 0.03 μM and V_max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (K_m = 46 ± 3 μM and V_max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.     

Effects of Orally Administered Tetracycline on the Intestinal Community Structure of Chickens and on tet Determinant Carriage by Commensal Bacteria and Campylobacter jejuni

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 μg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at <2 μg/ml for Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 μg/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.