Intestinal microbiota profiles in a genetic model of colon tumorigenesis correlates with colon cancer biomarkers.

Faecal (FM) and colon mucosal associated microbiota (MAM) were studied in a model of colorectal cancer (CRC), the Apc-mutated Pirc rats, and in age-paired wt F344 rats. Principal Coordinates Analysis indicated that samples' distribution was driven by age, with samples of young rats (1 month old; without tumours) separated from older ones (11-month-old; bearing tumours). Diversity analysis showed significant differences between FM and MAM in older Pirc rats, and between MAM of both Pirc and wt rats and the tumour microbiota, enriched in Enterococcus, Escherichia/Shigella, Proteus and Bifidobacteriaceae. In young animals, Pirc FM was enriched in the genus Delftia, while wt FM was enriched in Lactobacillus and Streptococcus. Some CRC biomarkers and faecal short chain fatty acids (SCFAs) were also measured. Colon proliferation and DClK1 expression, a pro-survival mucosal marker, were higher in Pirc than in wt rats, while the mucin MUC2, was lower in Pirc rats. Branched SCFAs were higher in Pirc than in wt animals. By Spearman analysis CRC biomarkers correlated with FM (in both young and old rats) and with MAM (in young rats), suggesting a specific relationship between the gut microbiota profile and these functional mucosal parameters deserving further investigation.

Morphological features and mechanical properties of nanofibers scaffolds of polylactic acid modified with hydroxyapatite/CdSe for wound healing applications.

Ternary nanocomposites, including graphene oxide (GO), hydroxyapatite (HAP), and cadmium selenite (CdSe) have been encapsulated into nanofibrous scaffolds of polylactic acid. These compositions were indexed as HAP@PLA (C1), CdSe@PLA (C2), HAP/CdSe@PLA (C3), HAP/GO@PLA (C4), and HAP/CdSe/GO@PLA (C5). Structural confirmation is executed by XRD and XPS techniques, while FESEM performs morphological characteristics. CdSe and GO dopants cause a significant increase in nanofiber diameter, HAP/GO@PLA (C4), showing thin surface fibers with fiber diameter up to 3.1 µm, followed by HAP/CdSe/GO@PLA (C4) composite that belongs to filament size up to 2.1 µm. On the other hand, the mechanical properties reveal that the dual dopant composites HAP/CdSe@PLA (C3) and HAP/GO@PLA (C4) hit the maximum tensile fracture values with 1.49 ± 0.3 and 0.99 ± 0.2 MPa. Further, the ternary C5 composite represents the lowest contact angle of 86.1 ± 3.7°. The antibacterial activity increased from 32.4 ± 9.7 and 28.4 ± 6.5% to be 85.3 ± 4.6 and 88.1 ± 5.6% for C1 and C5, respectively, against both E. coli and S. aureus in dark conditions. Moreover, the antibacterial potency enhanced from 75.4 ± 7.6 to be 83.5 ± 6.5 from dark to light conditions against E. coli for the composition of PLA containing the binary composition of HAP/CdSe.

Development of XRM-MacConkey agar selective medium for the isolation of Escherichia albertii.

Escherichia albertii has increasingly been recognized as an emerging pathogen. However, lack of selective medium for E. albertii is the bottleneck for clinical and epidemiological investigations. In this study, a selective medium for E. albertii named XRM-MacConkey agar, which is modified MacConkey agar supplemented with xylose (X), rhamnose (R), and melibiose (M) instead of lactose, was developed and evaluated. All 49 E. albertii and 6 different species out of 23 grew as colorless colonies, whereas 17 remaining species grew as red colonies. Detection limit of E. albertii by this medium was 105 CFU/g stool when examined with spiked healthy human stool. Furthermore, colorless colonies on XRM-MacConkey agar obtained from 7 E. albertii-positive diarrheal stools were consistently E. albertii. In contrast, 57%, 18%, and 36% colorless colonies on MacConkey, DHL, and mEA agars, respectively, were non-E. albertii. We concluded that XRM-MacConkey agar could specifically be used for isolation of E. albertii.

Altered mucosa-associated microbiota in the ileum and colon of neonatal calves in response to delayed first colostrum feeding.

The present study investigated whether delaying the first feeding of colostrum affected ileum and colon mucosa-associated microbiota in calves. Twenty-seven male Holstein calves were randomly assigned to 1 of 3 groups, fed colostrum at 45 min, 6 h, and 12 h after birth, respectively. Ileum and colon mucosa were collected at 51 h after birth, and their associated microbial profiles were assessed using amplicon sequencing. Both ileum and colon mucosa-associated microbiota were predominated by genus Escherichia-Shigella. The negative correlation between the molar proportion of short-chain fatty acids (SCFA) and ileum mucosa-associated opportunistic pathogens, and the positive correlation between the molar proportion of SCFA and colon mucosa-associated beneficial bacteria, suggest that SCFA might play an important role in maintaining the gut health of 2-d-old calves. A higher relative abundance of ileum mucosa-associated Enterococcus and Streptococcus was detected when the first colostrum feeding was delayed for 12 h. The relative abundance of colon mucosa-associated Lactobacillus tended to be lower in calves fed colostrum 12 h than those under the other 2 treatments, whereas that of Faecalibacterium tended to be lower in calves fed colostrum immediately after birth than those fed colostrum 6 and 12 h after birth, respectively. Our findings suggest that delayed first colostrum feeding affects the establishment of ileum and colon mucosa-associated bacteria, which may have long-term effects on gut health of calves.

The Evasive Enemy: Insights into the Virulence and Epidemiology of the Emerging Attaching and Effacing Pathogen Escherichia albertii.

The diarrheic attaching and effacing (A/E) pathogen Escherichia albertii was first isolated from infants in Bangladesh in 1991, although the bacterium was initially classified as Hafnia alvei Subsequent genetic and biochemical interrogation of these isolates raised concerns about their initial taxonomic placement. It was not until 2003 that these isolates were reassigned to the novel taxon Escherichia albertii because they were genetically more closely related to E. coli, although they had diverged sufficiently to warrant a novel species name. Unfortunately, new isolates continue to be mistyped as enteropathogenic E. coli (EPEC) or enterohemorrhagic E. coli (EHEC) owing to shared traits, most notably the ability to form A/E lesions. Consequently, E. albertii remains an underappreciated A/E pathogen, despite multiple reports demonstrating that many provisional EPEC and EHEC isolates incriminated in disease outbreaks are actually E. albertii Metagenomic studies on dozens of E. albertii isolates reveal a genetic architecture that boasts an arsenal of candidate virulence factors to rival that of its better-characterized cousins, EPEC and EHEC. Beyond these computational comparisons, studies addressing the regulation, structure, function, and mechanism of action of its repertoire of virulence factors are lacking. Thus, the paucity of knowledge about the epidemiology, virulence, and antibiotic resistance of E. albertii, coupled with its misclassification and its ability to develop multidrug resistance in a single step, highlights the challenges in combating this emerging pathogen. This review seeks to synthesize our current but incomplete understanding of the biology of E. albertii.

Rapid change of fecal microbiome and disappearance of Clostridium difficile in a colonized infant after transition from breast milk to cow milk.

BACKGROUND: Clostridium difficile is the most common known cause of antibiotic-associated diarrhea. Upon the disturbance of gut microbiota by antibiotics, C. difficile establishes growth and releases toxins A and B, which cause tissue damage in the host. The symptoms of C. difficile infection disease range from mild diarrhea to pseudomembranous colitis and toxic megacolon. Interestingly, 10-50 % of infants are asymptomatic carriers of C. difficile. This longitudinal study of the C. difficile colonization in an infant revealed the dynamics of C. difficile presence in gut microbiota. METHODS: Fifty fecal samples, collected weekly between 5.5 and 17 months of age from a female infant who was an asymptomatic carrier of C. difficile, were analyzed by 16S rRNA gene sequencing. RESULTS: Colonization switching between toxigenic and non-toxigenic C. difficile strains as well as more than 100,000-fold fluctuations of C. difficile counts were observed. C. difficile toxins were detected during the testing period in some infant stool samples, but the infant never had diarrhea. Although fecal microbiota was stable during breast feeding, a dramatic and permanent change of microbiota composition was observed within 5 days of the transition from human milk to cow milk. A rapid decline and eventual disappearance of C. difficile coincided with weaning at 12.5 months. An increase in the relative abundance of Bacteroides spp., Blautia spp., Parabacteroides spp., Coprococcus spp., Ruminococcus spp., and Oscillospira spp. and a decrease of Bifidobacterium spp., Lactobacillus spp., Escherichia spp., and Clostridium spp. were observed during weaning. The change in microbiome composition was accompanied by a gradual increase of fecal pH from 5.5 to 7. CONCLUSIONS: The bacterial groups that are less abundant in early infancy, and that increase in relative abundance after weaning, likely are responsible for the expulsion of C. difficile.

Changes in microbial diversity of brined green asparagus upon treatment with high hydrostatic pressure.

The application of high hydrostatic pressure (HHP, 600MPa, 8 min) on brined green asparagus and the changes in bacterial diversity after treatments and during storage at 4 °C (30 days) or 22 °C (10 days) were studied. HHP treatments reduced viable cell counts by 3.6 log cycles. The residual surviving population did not increase during storage at 4 °C. However, bacterial counts significantly increased at 22 °C by day 3, leading to rapid spoilage. The microbiota of green asparagus was composed mainly by Proteobacteria (mainly Pantoea and Pseudomonas), followed by Firmicutes (mainly Lactococcus and Enterococcus) and to a less extent Bacteroidetes and Actinobacteria. During chill storage of untreated asparagus, the relative abundance of Proteobacteria as well as Enterococcus and Lactococcus decreased while Lactobacillus increased. During storage of untreated asparagus at 22 °C, the abundance of Bacteroidetes decreased while Proteobacteria increased during late storage. The HHP treatment determined a reduction of the Proteobacteria both early after treatment and during chill storage. In the HHP treated samples stored at 22 °C, the relative abundance of Pseudomonas rapidly decreased at day 1, with an increase of Bacteroidetes. This was followed by a marked increase in Enterobacteriaceae (Escherichia) simultaneously with increase in viable counts and spoilage. Results from the study indicate that the effect of HHP treatments on the viability ofmicrobial populations in foods also has an impact on the dynamics of microbial populations during the storage of the treated foods.

Bifidogenic effect of grain larvae extract on serum lipid, glucose and intestinal microflora in rats.

The main objective of this study was to investigate whether orally administered Korean grain larvae ethanol extract (GLE) had a bifidogenic effect in normal rats. Male Sprague-Dawley rats were divided into a negative control group (CO) and GLE orally administered (5.0, 7.0 and 9.0 mg/100 g body weight) groups. Thymus and spleen weights dosedependently increased by 128.58 percent and 128.58 percent, respectively, but abdominal fat decreased by 19.18 percent after GLE administration compared with that in the CO group (p less than 0.05). Serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, and glucose decreased by 30.26 percent, 7.33 percent, 27.20 percent, and 6.96 percent, respectively, whereas highdensity lipoprotein cholesterol increased by 129.93 percent in the GLE groups compared with those in the CO group (p less than 0.05). IgG, IgM, IgA in the GLE groups increased 203.68 percent, 181.41 percent, and 238.25 percent, respectively, compared to that in the CO group (p less than 0.05). Bifidobacteria and Lactobacillus increased by 115.74 percent and 144.28 percent, whereas Bacteroides, Clostridium, Escherichia, and Streptococcus decreased by 17.37 percent, 17.46 percent, 21.25 percent, and 19.16 percent, respectively, in the GLE groups compared with those in the CO group (p less than 0.05). Total organic acids, acetic acid, and propionic acid increased by 151.40 percent, 188.09 percent, and 150.17 percent, whereas butyric acid and valeric acid decreased by 40.65 percent and 49.24 percent, respectively, in the GLE groups as compared with those in the CO group (p less than 0.05). These results suggest that Korean GLE improves the bifidogenic effect by increasing cecal organic acids and modulating gut microflora via a selective increase in Bifidobacterium in normal rats.

Paneth cell α-defensin 6 (HD-6) is an antimicrobial peptide.

Defensins protect human barriers from commensal and pathogenic microorganisms. Human α-defensin 6 (HD-6) is produced exclusively by small intestinal Paneth cells but, in contrast to other antimicrobial peptides (AMPs) for HD-6, no direct antibacterial killing activity has been detected so far. Herein, we systematically tested how environmental factors, like pH and reducing conditions, affect antimicrobial activity of different defensins against anaerobic bacteria of the human intestinal microbiota. Remarkably, by mimicking the intestinal milieu we detected for the first time antibacterial activity of HD-6. Activity was observed against anaerobic gut commensals but not against some pathogenic strains. Antibiotic activity was attributable to the reduced peptide and independent of free cysteines or a conserved histidine residue. Furthermore, the oxidoreductase thioredoxin, which is also expressed in Paneth cells, is able to reduce a truncated physiological variant of HD-6. Ultrastructural analyses revealed that reduced HD-6 causes disintegration of cytoplasmic structures and alterations in the bacterial cell envelope, while maintaining extracellular net-like structures. We conclude that HD-6 is an antimicrobial peptide. Our data suggest two distinct antimicrobial mechanisms by one peptide: HD-6 kills specific microbes depending on the local environmental conditions, whereas known microbial trapping by extracellular net structures is independent of the reducing milieu.

Degradation of curcuminoids by in vitro pure culture fermentation.

Colonic bacteria may mediate the transformation of curcuminoids, but studies of this metabolism are limited. Here, the metabolism of curcuminoids by Escherichia fergusonii (ATCC 35469) and two Escherichia coli strains (ATCC 8739 and DH10B) was examined in modified medium for colon bacteria (mMCB) with or without pig cecal fluid. LC-MS analysis showed that 16-37% of curcumin, 6-16% of demethoxycurcumin (DMC) and 7-15% of bis-demethoxycurcumin (Bis-DMC), and 7-15% of bis-demethoxycurcumin (Bis-DMC) were converted following 36 h of fermentation, with the amount of curcuminoids degraded varying depending on the bacterial strain and medium used. Three metabolites (dihydrocurcumin (DHC), tetrahydrocurcumin (THC), and ferulic acid (FA)) were found in fermentation cultures with all strains used. In addition, a compound with m/z [M - H](-) 470 was found and identified to be a curcumin adduct (curcumin-l-cysteine), using accurate mass FT-ICR-MS. This study provides insights into the bacterial metabolism of curcuminoids.

Antibiotic resistance and growth of the emergent pathogen Escherichia albertii on raw ground beef stored under refrigeration, abuse, and physiological temperature.

Escherichia albertii is an emerging gram-negative facultative rod that has been implicated in multiple cases of human diarrheal disease, particularly in young children. When biochemical and other typing methods have been used, this organism has often been misidentified due to similarities with other members of the family Enterobacteriaceae. Isolates have been reported to be capable of producing attachment and effacement lesions via the synthesis of intimin, cytolethal distending toxin, and a variant form of Shiga toxin. The purposes of this study were to characterize the antibiotic resistance characteristics and the growth of individual strains of E. albertii on raw ground beef at different storage temperatures. Nalidixic acid-resistant strains of E. albertii were inoculated onto raw ground beef to a target of 4.0 log CFU/g, and samples were then aerobically incubated at 5, 22, or 35°C for various time periods prior to microbiological enumeration of the pathogen on lactose-free MacConkey agar containing 50 mg of nalidixic acid per liter and 0.5% L-rhamnose. Antibiotic resistance was determined using a broth microdilution assay. E. albertii did not grow at 5°C, with populations declining slowly over 14 days of refrigerated storage. Strains of the organism grew well under abusive storage, increasing by 2.5 to 3.1 log CFU/g and 4.1 to 4.3 log CFU/g after 24 h at 22 and 35°C, respectively. All strains were resistant to tetracycline but were sensitive to tested cephalosporins and chloramphenicol. Resistance to penicillin was observed, but susceptibility to other members of the b -lactam group, including ampicillin, amoxicillin, and clavulanic acid, was recorded. E. albertii represents an emerging pathogen with a probable foodborne transmission route. Future research should focus on verifying food process measures able to inactivate the pathogen.

Antibacterial activity of thyme and lavender essential oils.

Strong antiseptic activity of essential oils has been known for a long time. The antibacterial activity of oils was tested against clinical bacterial strains of Staphylococcus, Enterococcus, Escherichia and Pseudomonas genera. The agar diffusion method was used for microbial growth inhibition at various concentrations of the oils from T. vulgaris and L. angustifolia. Susceptibility testing to antibiotics and chemotherapeutics was carried out using disc-diffusion method. 120 strains of bacteria isolated from patients with infections of oral cavity, respiratory, genitourinary tracts and from hospital environment were investigated. The results of experiments showed that the oil from T. vulgaris exhibited extremely strong activity against all of the clinical strains. Thyme oil demonstrated a good efficacy against antibiotics resistant strains of the tested bacteria. Lavender oil has been less activity against clinical strains of Staphylococcus, Enterococcus and Escherichia genus. The worst results have been observed against all strains of Pseudomonas aeruginosa.

Remission of histiocytic ulcerative colitis in Boxer dogs correlates with eradication of invasive intramucosal Escherichia coli.

BACKGROUND: Historically, histiocytic ulcerative (HUC) (or granulomatous) colitis of Boxer dogs was considered an idiopathic immune-mediated disease with a poor prognosis. Recent reports of dramatic responses to enrofloxacin and the discovery of invasive Escherichia coli within the colonic mucosa of affected Boxer dogs support an infectious etiology. HYPOTHESIS: Invasive E. coli is associated with colonic inflammation in Boxer dogs with HUC, and eradication of intramucosal E. coli correlates with clinical and histologic remission. ANIMALS: Seven Boxer dogs with HUC. METHODS: Prospective case series. Colonic biopsies were obtained at initial evaluation in 7 dogs, and in 5 dogs after treatment with enrofloxacin. Biopsies were evaluated by standardized histopathology, and fluorescence in situ hybridization (FISH) with probes to eubacteria and E. coli. RESULTS: Intramucosal E. coli was present in colonic biopsies of 7/7 Boxers with HUC. Clinical response was noted in all dogs within 2 weeks of enrofloxacin (7 + or - 3.06 mg/kg q24 h, for 9.5 + or - 3.98 weeks) and was sustained in 6 dogs (median disease-free interval to date of 47 months, range 17-62). FISH was negative for E. coli in 4/5 dogs after enrofloxacin. E. coli resistant to enrofloxacin were present in the FISH-positive dog that relapsed. CONCLUSIONS AND CLINICAL RELEVANCE: The correlation between clinical remission and the eradication of mucosally invasive E. coli during treatment with enrofloxacin supports the causal involvement of E. coli in the development of HUC in susceptible Boxer dogs. A poor response to enrofloxacin treatment might be due to colonization with enrofloxacin-resistant E. coli.

The ybiN gene of Escherichia coli encodes adenine-N6 methyltransferase specific for modification of A1618 of 23 S ribosomal RNA, a methylated residue located close to the ribosomal exit tunnel.

N(6)-Methyladenosine 1618 of Escherichia coli 23 S rRNA is located in a cluster of modified nucleotides 12 A away from the nascent peptide tunnel of the ribosome. Here, we describe the identification of gene ybiN encoding an enzyme responsible for methylation of A1618. Knockout of the ybiN gene leads to loss of modification at A1618. The modification is restored if ybiN knock-out strain has been co-transformed with a plasmid expressing the ybiN gene. On the basis of these results we suggest that ybiN gene should be renamed to rlmF in accordance with the accepted nomenclature for rRNA methyltransferases. Recombinant YbiN protein is able to methylate partially deproteinized 50 S ribosomal subunit, so-called 3.5 M LiCl core particle in vitro, but neither the completely assembled 50 S subunits nor completely deproteinized 23 S rRNA. Both lack of the ybiN gene and it's over-expression leads to growth retardation and loss of cell fitness comparative to the parental strain. It might be suggested that A1618 modification could be necessary for the exit tunnel interaction with some unknown regulatory peptides.

[Secreted expression of the combinant antimicrobial peptide PL in Pichia pastoris and its antibacterial activity in vitro].

In order to obtain a high activity antibacterial peptide, An expression vector pPICZalphaA-pl is constructed with a tandem of four antimicrobial peptides in the same direction,which includes Protegrin-1 (PG-1), Scorpion Defensin (SD), Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide (SMAP-29) (serial number in GenBank are AAB27599, AAAB27538, P80409 and P49928 respectively). At the same time the expression vector pPICZalphaA-sd which express Scorpion Defensin was contructed. The expression vectors of pPICZalphaA-pl and pPICZalphaA-sd were linearized and transformed into the yeast host strain X-33 respectively. Under the control of the promoter AOX1 (alcohol oxidase1), the peptides PL and SD were secreted expressed. Their heat-stable property, acid-stable property and MIC were detected in vitro. The results suggest the peptides PL and SD have good heat-stable and acid-stable properties, and the combinant PL peptide showes higher antibacterial activity against several Gram-positive bacteria (G+) and Gram-negative bacteria (G-) than the peptide SD, especially against Escherichia coli. The antibacterial activity of combinant antimicrobial peptide PL shows its far exploiting perspective.

An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts.

F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions. A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception was traW. A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.

The Escherichia coli ynfEFGHI operon encodes polypeptides which are paralogues of dimethyl sulfoxide reductase (DmsABC).

The ynfEFGHI operon is a paralogue of the Escherichia coli dmsABC operon. ynfE and ynfF are paralogues of dmsA. ynfG and ynfH are paralogues of dmsB and dmsC, respectively. YnfI (dmsD) has no dms paralogue. YnfE/F and YnfG could be detected by immunoblotting with anti-DmsAB antibodies when expressed under the control of a tac or dms promoter. Cells harbouring ynfFGH on a multicopy plasmid supported anaerobic growth with dimethyl sulfoxide (DMSO) as respiratory oxidant in a dmsABC deletion, suggesting that YnfFGH forms a heterotimeric enzyme complex similar to DmsABC. Exchange of DmsC by YnfH (DmsAB-YnfH) resulted in membrane localization, anaerobic growth on DMSO, and binding of 2-n-heptyl 4-hydroxyquinoline-N-oxide, indicating that YnfH was a competent anchor. YnfG can also replace DmsB as the electron transfer subunit and assembled [Fe-S] clusters as judged by electron paramagnetic resonance spectroscopy. YnfE and/or YnfF could not form a functional complex with DmsBC and expression of YnfE prevented the accumulation of YnfFGH.

Effects of 810 nm laser irradiation on in vitro growth of bacteria: comparison of continuous wave and frequency modulated light.

BACKGROUND AND OBJECTIVES: Low intensity laser therapy may modify growth of wound bacteria, which could affect wound healing. This study compares the effects on bacteria of 810 nm laser using various delivery modes (continuous wave or frequency modulated light at 26, 292, 1000, or 3800 Hz). STUDY DESIGN/MATERIALS AND METHODS: Staphylococcus (S.) aureus, Escherichia (E.) coli, and Pseudomonas (P.) aeruginosa were plated on agar and then irradiated (0.015 W/cm(2); 1-50 J/cm(2)) or used as controls (sham irradiated); growth was examined after 20 hours of incubation post exposure. RESULTS: There were interactions of species and modulation frequency in the overall effects of irradiation (P = 0.0001), and in the radiant exposure mediated effects (P = 0.0001); thus individual frequencies and each bacterium were analysed separately. Bacteria increased following 3800 Hz (P = 0.0001) and 1000 Hz (P = 0.0001) pulsed irradiation; at particular radiant exposures P. aeruginosa proliferated significantly more than other bacteria. Pulsed laser at 292 and 26 Hz also produced species-dependent effects (P = 0.0001; P = 0.0005); however, the effects for different radiant exposures were not significant. Bacterial growth increased overall, independent of species, using continuous mode laser, significantly so at 1 J/cm(2) (P = 0.02). Analysis of individual species demonstrated that laser-mediated growth of S. aureus and E. coli was dependent on pulse frequency; for S. aureus, however, there was no effect for different radiant exposures. Further tests to examine the radiant exposure effects on E. coli showed that growth increased at a frequency of 1000 Hz (2 J/cm(2); P = 0.03). P. aeruginosa growth increased up to 192% using pulsed irradiation at 1000-3800 Hz; whereas 26-292 Hz laser produced only a growth trend. CONCLUSIONS: The findings of this study point to the need for wound cultures prior to laser irradiation of infected wounds. Similar investigations using other common therapeutic wavelengths are recommended.

Oligonucleotide probe for the visualization of Escherichia coli/Escherichia fergusonii cells by in situ hybridization: specificity and potential applications.

There are several occasions when enumeration of Escherichia coli cells is needed. These include examination of urine specimens and water or food samples. Present methods rely on growth in more or less selective media (colony-forming units on agar or the most probable number method using liquid media). Unfortunately, no really selective medium with 100% efficiency of plating is available for E. coli. A 24-mer oligonucleotide probe (Colinsitu), complementary to a piece of 16S ribosomal ribonucleic acid, has been tested for specifically visualizing E. coli cells by in situ hybridization and epifluorescence microscopy. The fluorescent dye-labeled probe was able to stain cells of E. coli, Shigella spp. and E. fergusonii. Shigella spp. are known to belong to the E. coli genomospecies and E. fergusonii is the nomenspecies closest to E. coli by DNA-DNA hybridization. The probe did not stain any strain of 169 other genomospecies of the family Enterobacteriaceae or of a few other species frequently encountered in the environment. Revivification without cell division allowed the visualization of E. coli cells in contaminated water. In situ hybridization using the Colinsitu probe is a potential tool for the confirmation of (atypical) E. coli in reference centers and the rapid (3-6 h) detection and enumeration of E. coli in urine specimens, contaminated water and food. More work is needed to include in situ hybridization in laboratory routine.

Escherichia hermanii--a new bacterial strain for chlorobenzene degradation.

A new bacterial strain capable of chlorobenzene degradation has been isolated from sludge of an industrial wastewater treatment plant. The micro-organism is short, rod-shaped, Gram-negative, yellow-pigmented and has been identified as Escherichia hermanii. It was observed that high chlorobenzene concentrations (up to 394 mg l-1) had low toxic effects towards this strain, which was able to degrade chlorobenzene without any previous adaptation.