Antimicrobial and anti-biofilm efficacy of different inorganic and organic zinc forms against multidrug-resistant Escherichia, Klebsiella, Staphylococcus and Pseudomonas.

In our study antibacterial and anti-biofilm efficacy of 2 inorganics (Zn(II) sulphate monohydrate; Zn(II) sulphate heptahydrate) and 3 organic Zn(II) substances (Zn(II) chelate of protein hydrolysate: Zn-Bio; Zn(II) chelate of amino acid hydrate: Zn-AMK; Zn(II) chelate of glycine hydrate: Zn-Gly) were explored and compared against multidrug resistant Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Klebsiella oxytoca (K. oxytoca) and Pseudomonas aeruginosa (P. aeruginosa) using by the 96- wells microtiter plate-based resazurin and/or crystal violet assay. Our finding confirmed that Zn(II)-sulphates and Zn(II)-amino acid complexes exhibit dose and genus-based antibacterial and anti-biofilm potential. Organic compounds (Zn-AMK and Zn-Gly) were more effective against bacterial growth, except P. aeruginosa. Besides Zn-AMK, others organic and inorganic forms of Zn(II) caused predominantly statistically significant decrease of biofilm production in all of tested bacteria. Current data highlights that Zn(II) in various forms has a great potential to be developed as antibacterial and anti-biofilm agents.

Virulence determinants and antibiotic resistance in staphylococci isolated from the skin of captive bred reptiles.

Knowledge of the composition and properties of skin microbiota in healthy reptiles is essential for preservation strategies and thus the prevention of skin dysbiosis leading to dermatological diseases. Despite the greatly increasing popularity of reptiles as pets, only a few studies have dealt with this topic. Therefore, the aim of this work was to analyse species composition of bacteria isolated from skin swabs of 40 reptiles (17 species) using MALDI-TOF spectrometry and to characterise the virulence properties of identified staphylococci (n = 51). The most common species were Staphylococcus xylosus and S. sciuri. Bacilli, enterococci, Escherichia coli, Salmonella sp. and Acinetobacter sp. were also common. The most frequent antimicrobial resistance in staphylococcal isolates was observed for ampicillin (100.0%) and cefoxitin (98.0%) with the blaZ gene being most prevalent (58.8%). In contrast, all staphylococci were susceptible to gentamicin, kanamycin and imipenem. Slime and biofilm production was observed in 86.3% and 76.5% of isolates, respectively. Gelatinase, DNase, protease and lipase activity was found more rarely (41.2%; 25.5%; 27.5% and 21.6%). Since reptiles are a reservoir of bacteria for their owners, common multi-drug resistance (84.3%, MAR index average 0.29 ± 0.09) and biofilm formation must be kept in mind, especially in the case of injury when handling reptiles.

Distribution of virulence-associated genes, antibiotic resistance and phylogenetic groups in Escherichia coli isolated from domestic and racing pigeons.

Despite a lot of information about virulence and resistance of Escherichia coli (E. coli) in poultry, very limited data are currently available on its occurrence in pigeon isolates, although this poses a threat to human and animal health. Therefore, this study was conducted to explore the phylogenetic classification, antibiotic sensitivity, and virulence factors in E. coli isolated from cloacal swabs of domestic pigeons bred for meat (n = 47) and racing pigeons (n = 44). The most frequent phylogroup in racing pigeons was E (36, 82.00%), unlike domestic pigeons (B2- 19, 40.00%). The most abundant iron uptake system in both groups of bird was feoB (racing = 40, 90.90%; domestic = 44, 93.61%). The presence of ibeA (52, 57.10%) and kpsMTII (46, 50.50%) genes was detected in more than half of all strains belonging exclusively to phylogroups B2, D, E, F, clade I. Antibiotic resistance was higher in racing pigeons. All racing pigeon isolates were resistant to tetracycline and trimethoprim + sulphonamide. Resistance to ciprofloxacin was determined in three isolates (6.38%) of domestic and 33 isolates (75%) of racing pigeons. Aminoglycosides and ß-lactamases resistance were also recorded. One of the important detected phenotypic mechanisms of resistance occurring in isolates from racing pigeons was AGL AAC(6´)I. Our study confirms that healthy pigeons are a reservoir of antibiotic-resistant E. coli containing an arsenal of virulence factors, thus capable of potentially causing infection. Pigeons with the option to fly to multiple places can transfer virulent and resistant bacteria. Direct contact with pigeons and their faeces and the contamination of water and food pose a threat of infection to humans and other animal species.

Antimicrobial activity of organic acids against canine skin bacteria.

Canine skin is often a source of bacterial strains that are characterized by the presence of important virulence factors and a high antimicrobial resistance. These bacteria are involved in the pathogenesis of infectious skin diseases, which are very frequent in dogs. Moreover, canine skin isolates are easily spread to other animals and humans. The aim of this study was to evaluate the inhibitory and bactericidal activity of eight organic acids (L-lactic, acetic, propionic, butyric, citric, succinic, glycolic, L-ascorbic acid) against 14 canine skin isolates (11 Gram-positive and three Gram-negative species). The advantages of the tested organic acids are their gentleness to the skin and their affordability. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution method. All tested acids showed a bactericidal effect against the selected bacteria, with the exception of their bacteriostatic effect against the Bacillus cereus strain. The lowest MIC showed acetic acid (MIC between 0.5 and 2.0 mg/mL) and propionic acid (MIC 0.8 - 3.3 mg/mL), whereas L-ascorbic acid (MIC 4.0 - 16.0 mg/mL) seems to be weaker among the tested acids. Two Staphylococcus aureus strains and a strain of Escherichia coli were observed to be more resistant compared to coagulase-negative staphylococci.

The effect of exposure to non-ionising radiofrequency field on Escherichia coli, Klebsiella oxytoca and Pseudomonas aeruginosa biofilms.

An environment with a higher accumulation of electromagnetic non-ionising radiofrequency (RF) emissions generated by various telecommunication, data transport and navigation devices (mobile phones, Wi-Fi, radar, etc.) may have a major impact on biological systems. This study aimed to evaluate the incidence of an electromagnetic field (EMF) on the development of bacterial biofilm. Quantification of biofilm production was done by using microtiter plate assay. Bacterial isolates of Escherichia coli, Klebsiella oxytoca and Pseudomonas aeruginosa were exposed with EMF of frequencies 1-5 and 2.4 GHz with an exposure time 3 or 24 h, respectively. Exposure of bacteria to EMF produced a statistically significant increase in biofilm production mainly at 1, 2 and 4 GHz, and in contrast, a significant inhibition of biofilm development occurred at frequencies 3 and 5 GHz, both with exception of K. oxytoca and P. aeruginosa. Wi-Fi operating at 2.4 GHz caused biofilm reduction. The results indicate that EMF exposure act on bacteria in both ways, depending on the frequency: as stressful by enhancing bacterial biofilm formation (under environmental stress, bacteria produce a polysaccharide matrix and aggregate to form biofilms to increase virulence and resistance), although some frequencies leading to biofilm damage could be caused by changes to the physicochemical properties of bacteria.

Antibiofilm Activity of Weissella spp. and Bacillus coagulans Isolated from Equine Skin against Staphylococcus aureus.

The aim of this study was to evaluate the antimicrobial and antibiofilm activity of Weissella cibaria, Weissella hellenica and Bacillus coagulans, isolated from equine skin, against biofilm-forming Staphylococcus aureus CCM 4223 and clinical isolate methicillin-resistant S. aureus (MRSA). Non-neutralized cell-free supernatants (nnCFS) of tested skin isolates completely inhibited the growth and biofilm formation of S. aureus strains and caused dispersion of the 24 h preformed biofilm in the range of 21-90%. The majority of the pH-neutralized cell-free supernatants (nCFS) of skin isolates inhibited the biofilm formation of both S. aureus strains in the range of 20-100%. The dispersion activity of B. coagulans nCFS ranged from 17 to 77% and was significantly lower than that of nnCFS, except for B. coagulans 3T27 against S. aureus CCM 4223. Changes in the growth of S. aureus CCM 4223 in the presence of catalase- or trypsin-treated W. hellenica 4/2D23 and W. cibaria 4/8D37 nCFS indicated the role of peroxides and/or bacteriocin in their antimicrobial activities. For the first time, the presence of the fenD gene, associated with biosurfactants production, was detected in B. coagulans. The results of this study showed that selected isolates may have the potential for the prevention and treatment of biofilm-forming S. aureus infections.

Virulence Factors and Antibiotic Resistance of Enterobacterales.

In the class Gammaproteobacteria, Enterobacterales are Gram-negative, facultatively anaerobic bacteria [...].

Distribution and Characterization of Staphylococci Isolated From Healthy Canine Skin.

Despite increasing interest to study skin microbiota with progressive methods, there are almost no data on staphylococcal species distribution on skin of healthy dogs available. Therefore we decide to characterize staphylococci isolated from 8 different body sites (inner pinna, chin, nasal skin, back, axilla, abdomen, interdigital area and perianal region) of healthy canine skin. A total of 91 staphylococci were isolated from 30 dogs living in East Slovakia. Swabs of each dog were cultivated and colonies analysed using MALDI-TOF spectrometry. The vast majority of isolated staphylococci belonged to S pseudintermedius species (48%) followed by S hominis (15%) and S aureus (10%). S haemolyticus, S warneri, S epidermidis, S capitis, S xylosus, S pasteuri, S intermedius and S succinus were also isolated (<10%). The most frequent resistance in staphylococcal isolates was observed for chloramphenicol (73%) and penicillin (67%) followed by erythromycin (42%), tetracycline (26%), and oxacillin (20%). Multi-drug resistance was found in 50% of isolates. All strains were gentamicin and vancomycin sensitive and were strong or moderate biofilm producers with high acid and alkaline phosphatase activities. Over half of strains were haemolytic (57%) and produced gelatinase (54%), DNAse (84%) and lipase (64%). It seems, multiresistant biofilm forming staphylococci could be commonly detected also in healthy dogs and could probably serve as reservoir for other dogs or owners because of constant exchange of their microbiota.

Antimicrobial Susceptibility and Detection of Virulence-Associated Genes in Escherichia coli Strains Isolated from Commercial Broilers.

The aim of this study was to investigate the presence of iron-uptake and virulence genes, antibiotic resistance profiles, and phylogenetic relatedness in 115 Escherichia coli (E. coli) strains isolated from broilers in Slovakia and to determine their potential threat to human health. The most frequent phylogroups were B1 (37%) and A (21%), and 33.9% strains were included in pathogenic groups. The commonly observed iron-uptake genes were feoB (94%), sitA (83%), and iutA (58%). Protectins (iss, kpsMTII) were identified in 30% of samples. Four percent of B2-associated broilers carried the papC (P fimbria) gene connected with upper urinary tract infection. The dominant resistance was to tetracycline (49%), ampicillin (66%), ampicillin + sulbactam (27%), ciprofloxacin (61%), and trimethoprim + sulfonamide (34%); moreover, sporadically occurring resistance to cephalosporins, aminoglycosides, fluoroquinolones, and polypeptide colistin was observed. Genotypic analysis of resistance revealed the presence of blaCTX-M-1 and blaCTX-M-2 in two isolates from broilers. Commercial broilers can be reservoirs of virulent and resistant genes as well as E. coli causing (extra-)intestinal infections, which can be a potential threat to humans via direct contact and food.

Escherichia coli Specific Virulence-Gene Markers Analysis for Quality Control of Ovine Cheese in Slovakia.

Shiga toxin-producing and extra-intestinal pathogenic Escherichia coli (E. coli) have the potential to spread through faecal waste, resulting in contamination of food and causing foodborne disease outbreaks. With the aim of characterizing unpasteurized ovine cheese in Slovakia, a total of 92 E. coli strains were examined for eleven representative virulence genes typical for (extra-)intestinal pathogenic E. coli and phylogenetic grouping. Phylogenetic groups B1 (36%) and A (32%) were the most dominant, followed by groups C (14%) and D (13%), while the lowest incidence was recorded for F (4%), and E (1%), and 43 (47%) samples carried at least one virulent gene, i.e., potential pathogens. Isolates present in groups E, F and D showed higher presence of virulence genes (100%, 75%, and 67%), versus 55%, 39%, and 28% in commensal B1, C, and A, respectively. Occurrence of papC and fyuA (both 24%) was highest, followed by tsh, iss, stx2, cnf1, kpsII, cvaC, stx1, iutA and eaeA. Nine E. coli strains (almost 10% of all tested and around 21% of our virulence-gene-associated isolates) harboured stx1, stx2 or eae. Ovine cheeses in Slovakia are highly contaminated with E. coli including potentially pathogenic strains capable of causing intestinal and/or extra-intestinal diseases, and thus may pose a threat to public health while unpasteurized.

Antibiotic Resistant and Biofilm-Associated Escherichia coli Isolates from Diarrheic and Healthy Dogs.

Bacteria isolated from companion animals are attracting concerns in a view of public health including antimicrobial resistance and biofilm development, both contributing to difficult-to-treat infections. The purpose of this study was to evaluate the minimum inhibitory concentrations (MIC) of 18 antibiotics in Escherichia coli isolated from two groups of dogs (healthy and diarrheic). Isolates were classified into phylogroups, examined for the presence of resistance genes and biofilm-formation capacity. In healthy dogs, phylogenetic analysis showed that 47.37% and 34.22% of E. coli isolates belonged to commensal groups (A; B1) in contrast to diarrheic dogs; 42.2% of isolates were identified as the B2 phylogroup, and these E. coli bacteria formed a stronger biofilm. The results of healthy dogs showed higher MIC levels for tetracycline (32 mg/L), ampicillin (64 mg/L), ciprofloxacin (8 mg/L) and trimethoprim-sulphonamide (8 mg/L) compared to clinical breakpoints. The most detected gene encoding plasmid-mediated resistance to quinolones in the healthy group was qnrB, and in dogs with diarrhea, qnrS. The resistance genes were more frequently detected in healthy dogs. The presence of the integron int1 and the transposon tn3 increases the possibility of transfer of many different cassette-associated antibiotic-resistance genes. These results suggest that dogs could be a potential reservoir of resistance genes.

Cell-Free Lactobacillus casei 21L10 Modulates Nitric Oxide Release and Cell Proliferation/Cell Death in Lipopolysaccharide-Challenged HT-29 Cells.

Lactobacillus casei (L. casei) is one of the probiotic strains that may influence intestinal injury and inflammation in nonspecific intestinal diseases. We aimed to evaluate the effect of cell-free Lactobacillus casei 21L10 supernatant (LC) on the cell line HT-29 challenged with lipopolysaccharide (LPS) in order to modulate production of NO, cell proliferation, and apoptosis. Cell line HT-29 was stimulated with LPS in the presence or absence of LC. Our results showed that LC from L. casei 21L10 did not affect the viability of unstimulated HT-29 cells line. HT-29 cell line treatment with LC caused significant decrease of LPS induced NO production after 3 h, and 24 h, but not after 48 h. Proliferation activity of LPS stimulated HT-29 cell line analysed with MTT assay significantly decreased after 24 h and 48 h, but not after 3 h. The majority of LPS stimulated HT-29 cell line treated with LC showed annexin V/PI positivity at 48 h survival, which corresponded to late apoptotic/necrotic cell features. The observed differences suggest that cell-free L. casei 21L10 supernatant could participate in attenuation of LPS-induced inflammation, and may exhibit anti-proliferative and pro-apoptotic/necrotic effects. This study provides pilot data for the further development of L. casei exoproducts as an anti-inflammatory or anti-proliferative agent for the treatment of inflammatory and cancer diseases in gut. However, more data is needed before final conclusions of L. casei cell-free supernatant's efficacy can be drawn.

Canine Bone Marrow Mesenchymal Stem Cell Conditioned Media Affect Bacterial Growth, Biofilm-Associated Staphylococcus aureus and AHL-Dependent Quorum Sensing.

The present study investigated the in vitro antibacterial, antibiofilm and anti-Quorum Sensing (anti-QS) activities of canine bone marrow mesenchymal stem cell-conditioned media (cBM MSC CM) containing all secreted factors <30 K, using a disc diffusion test (DDT), spectrophotometric Crystal Violet Assay (SCVA) and Bioluminescence Assay (BA) with QS-reporter Escherichia coli JM109 pSB1142. The results show a sample-specific bacterial growth inhibition (zones varied between 7-30 mm), statistically significant modulation of biofilm-associated Staphylococcus aureus and Escherichia coli bioluminescence (0.391 ± 0.062 in the positive control to the lowest 0.150 ± 0.096 in the experimental group, cf. 11,714 ± 1362 to 7753 ± 700, given as average values of absorbance A550 ± SD versus average values of relative light units to growth RLU/A550 ± SD). The proteomic analysis performed in our previous experiment revealed the presence of several substances with documented antibacterial, antibiofilm and immunomodulatory properties (namely, apolipoprotein B and D; amyloid-ß peptide; cathepsin B; protein S100-A4, galectin 3, CLEC3A, granulin, transferrin). This study highlights that cBM MSC CM may represent an important new approach to managing biofilm-associated and QS signal molecule-dependent bacterial infections. To the best of our knowledge, there is no previous documentation of canine BM MSC CM associated with in vitro antibiofilm and anti-QS activity.

In vitro evaluation of the safety and probiotic properties of Lactobacilli isolated from chicken and calves.

A total of 73 chicken and calves isolates were diagnosed using matrix-assisted laser desorption ionization-time-of flight mass spectrometry (Maldi-Tof MS). After a preliminary subtractive screening based on the high acid tolerance at pH 2.5 and bile resistance at 0.3% oxgall, twenty isolates belonging to the species Lactobacillus salivarius, Lactobacillus agilis, Lactobacillus reuteri, Lactobacillus murinus and Lactobacillus amylovorus were in vitro screened for the safety assessment and probiotic properties, including antibiotics susceptibility patterns, biochemical activity and potential for competitive exclusion of biofilm producing pathogens determined by crystal violet and/or quantitative Fluorescent in situ Hybridisation (FISH) assays utilizing 5'Cy 3 labelled probe Enter1432 for enteric group. Antibiotic susceptibility testing was performed according to the ISO norm 10932. The sixteen strains were susceptible to certain antimicrobial agents, except for two chicken (L. salivarius 12K, L. agilis 13K) and two calves (L. reuteri L10/1, L. murinus L9) isolates with the presence non wild-type ECOFFs (epidemiological cut-off) for gentamicin (≥256 µg ml(-1)), tetracycline (≥128 µg ml(-1)), kanamycin (≥256 µg ml(-1)) and streptomycin (≥96 µg ml(-1)). The two referenced chicken isolates gave positive aac(6')Ie-aph(2″)Ia and tet(L) PCR results. The wild-type ECOFFs isolates were subjected to the apiZYM analysis for enzyme profile evaluation and amino acid decarboxylase activities determined by qualitative plate method and multiplex PCR for the detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via eithers ornithine decarboxylase, odc, or agmatine deiminase, agdi). From examined strains only two chicken isolates (L. reuteri 14K; L. salivarius 15K) had no harmful ß-glucuronidase, ß-glucosidase activities connected with detrimental effects in the gastrointestinal tract and together no amino acid decarboxylase activities and no genes associated with biogenic amines production though only chicken L. salivarius 15K whole cells and acid supernatants shown strong suppressive potential against biofilm-forming Klebsiella and Escherichia coli. Our results highlight that above-mentioned isolate L. salivarius 15K fulfils the principle requirements of a qualified probiotic and may be seen as a reliable candidate for further validation studies in chicken.

Effect of antibiotic treatment on intestinal microbial and enzymatic development in postnatally overfed obese rats.

OBJECTIVE: To investigate the effect of the microbiota-induced changes and early overfeeding after amoxicillin administration (a) in suckling pups via their dams up to 15 days of lactation and (b) in weaned pups on intestinal microbial/functional adaptability and obesity development in male Sprague-Dawley rats. DESIGN AND METHODS: Postnatal nutrition was elicited by adjusting the number of pups in the nest to 4 (small litters [SLs]) and 10 (normal litters [NLs]), while from days 21 to 40, both groups were fed with a standard diet. The numbers of Bacteroides/Prevotella (BAC) and Lactobacillus/Enterococcus (LAB) in the jejunum and colon were determined by fluorescence in situ hybridization technique, and jejunal alkaline phosphatase (AP), α-glucosidase and aminopeptidase activity was assayed histochemically. RESULTS: On day 40, the SL in comparison with NL animals displayed excess weight/fat gain accompanied by higher LAB and lower numbers of BAC, and with permanently higher AP activity. Moreover, these acquired changes continued in SL vs. NL rats and were not influenced by antibiotic treatment, which induced significant decrease in the quantity of LAB and BAC. CONCLUSIONS: These findings highlight the role of early life overfeeding upon the gut microbial/functional ontogeny and allow to distinguish their potential involvement in later risk of obesity.

Broilers as a source of quinolone-resistant and extraintestinal pathogenic Escherichia coli in the Czech Republic.

Extraintestinal Escherichia coli infections are associated with extraintestinal pathogenic E. coli (ExPEC) strains. A total of 114 E. coli isolates were characterized regarding their antimicrobial resistance in a prospective study of 319 broilers from 12 slaughterhouses in the Czech Republic, a European Union member, during 2008. PCR-based assays to define ExPEC-associated traits were performed in resistant strains. Consumption of antimicrobial drugs by poultry in the Czech Republic was also analyzed. Antibiotic resistance was detected in 82% of isolates. Resistance to nalidixic acid and ciprofloxacin was predominant. Plasmid-mediated quinolone resistance genes, qnrB19 and qnrS1, were detected in 1 and 3 of 93 resistant isolates, respectively. Twenty-three percent of resistant isolates were considered as ExPEC. In total, 972 kg of flumequine, enrofloxacin, and difloxacin were used in poultry in the Czech Republic during 2008. High prevalence of broilers with ciprofloxacin-resistant E. coli isolates was linked to consumption of quinolones in poultry. Broilers may comprise an important vehicle for community-wide dissemination of fluoroquinolone-resistant E. coli and ExPEC. Withdrawal of fluoroquinolones from use in chicken production should be seriously considered in the Czech Republic and the European Union as well.

Functional properties of Lactobacillus strains isolated from dairy products.

Twenty-four acid- and bile-tolerant lactobacilli isolates from dairy products were identified and further in vitro characterized for the presence of functional traits potentially useful for probiotic applications, which included desirable and undesirable traits, such as biofilm formation, ability to inhibit intestinal pathogens, antibiotic susceptibility, and enzyme activity. The majority of examined strains were susceptible to certain antimicrobial agents (streptomycin, gentamicin, clindamycin, erythromycin, tetracycline, quinupristin-dalfopristin), except for three strains of Lactobacillus rhamnosus with minimal inhibitory concentration levels for streptomycin higher than the microbiological breakpoints (≥32 µg/mL), which are considered as resistant. Undesirable traits such as α-chymotrypsin or N-acetyl-ß-glucosaminidase activities were not detected, but low ß-glucuronidase, and moderate and high ß-glucosidase activities were recorded in nine strains, which were eliminated from further examination together with three isolates showing unsuitable antibiotic resistance. Of the remaining 12 isolates, 4 (Lactobacillus fermentum 202, Lactobacillus gallinarum 7001, L. rhamnosus 183, and Lactobacillus plantarum L2-1) manifested an outstanding potential to inhibit selected intestinal pathogens in an agar spot test, including Escherichia coli and Salmonella spp., and simultaneously demonstrated strong biofilm-forming capacity. In conclusion, the results of our in vitro experiments showed that the above four strains had a potential probiotic value and met the criteria to be identified as a possible probiotic microorganism, with the necessity of verification through well-designed in vivo experimental, clinical, and technological studies before the strains can be used as probiotics or as starter probiotic cultures.

Inhibitory potential of lactobacilli against Escherichia coli internalization by HT 29 cells.

A quantitative fluorescent in situ hybridization method was employed to evaluate the competitive inhibitory effect of three Lactobacillus strains (Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus plantarum) against Escherichia coli internalization in a model system of HT 29 cells. Furthermore, aggregation and adhesion abilities of the Lactobacillus strains were examined. All lactobacilli were able to attach to the HT 29 cells and aggregate with pathogens; however, the adhesion and aggregation degree was strain-dependent. L. reuteri possessed a high capacity of adhesion (6.80 ± 0.63; log CFU ± SEM per well), whereas lower capacities were expressed by L. gasseri (4.52 ± 0.55) and L. plantarum (4.90 ± 0.98). Additionally, L. reuteri showed the rapid or normal ability to aggregate with selected E. coli in comparison with remaining two lactobacilli, which showed only slow or negative aggregative reaction. Internalization of E. coli into the cell lines was markedly suppressed by L. reuteri, while L. gasseri and L. plantarum caused only a minimum anti-invasion effect. The fact that L. reuteri in our experiments showed an outstanding potential for adhering to the colon epithelial cell line, compared with the rest strains, suggested that one of the possible mechanisms of preventing pathogen adhesion and invasion is simple competitions at certain receptors and capability to block receptor binding sites, or that an avid interaction between L. reuteri and the host cell might be modulating intracellular events responsible for the E. coli internalization. Moreover, L. reuteri exhibited a strong ability to aggregate with E. coli, which could be another limiting factor of pathogen invasion.

Developmental changes of gut microflora and enzyme activity in rat pups exposed to fat-rich diet.

The aim of this study was to investigate the effect of a high-fat (HF)/energy diet on the intestinal microbiota, the alkaline phosphatase (AP) activity, and related parameters of growth and obesity during the suckling and weaning periods in male Sprague-Dawley rats. From birth, nutrition in suckling pups was manipulated by feeding rat dams either HF or a standard diet, and then after weaning, by exposure of experimental pups to the HF, and control rats to normal diet. On days 15, 20, 40 the numbers of 2 microbial groups, i.e., Bacteroides/Prevotella (BAC) and the Lactobacillus/Enterococcus (LAB) in the jejunum, were determined by fluorescent in situ hybridization technique, and the AP activity was assayed histochemically. During all investigated periods HF pups gained body fat more rapidly than control animals, but from weaning they displayed significantly stunted growth resulting in final body weight loss. Obesity in HF rats was also accompanied by higher LAB and lower numbers of BAC and with permanently higher AP activity. Correlation of these data showed significant negative correlation between LAB, AP, and weight gain and energy efficiency, and significant positive correlation of BAC and AP activity with body fat. These data support the concept that postnatal nutritional experience represents an important factor affecting the ontogeny of intestinal microbial communities and intestinal function. These acquired changes could be a component of regulatory mechanisms involved in adverse and/or positive consequences of HF diet for adiposity, body weight, and energy-balance control in later life.

Essential oils--their antimicrobial activity against Escherichia coli and effect on intestinal cell viability.

Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria. The main objective of this study was to evaluate possible harmful effects of four commonly used essential oils and their major components on intestinal cells. Antimicrobial activity of selected plant extracts against enteroinvasive Escherichia coli was dose dependent. However, doses of essential oils with the ability to completely inhibit bacterial growth (0.05%) showed also relatively high cytotoxicity to intestinal-like cells cultured in vitro. Lower doses of essential oils (0.01%) had only partial antimicrobial activity and their damaging effect on Caco-2 cells was only modest. Cell death assessment based on morphological and viability staining followed by fluorescence microscopy showed that essential oils of cinnamon and clove and their major component eugenol had almost no cytotoxic effect at lower doses. Although essential oil of oregano and its component carvacrol slightly increased the incidence of apoptotic cell death, they showed extensive antimicrobial activity even at lower concentrations. Relatively high cytotoxicity was demonstrated by thyme oil, which increased both apoptotic and necrotic cell death incidence. In contrast, its component thymol showed no cytotoxic effect as well as greatly-reduced ability to inhibit visible growth of the chosen pathogen in the doses used. On the other hand, the addition of all essential oils and their components at lower doses, with the exception of thyme oil, to bacterial suspension significantly reduced the cytotoxic effect of E. coli on Caco-2 cells after 1h culture. In conclusion, it is possible to find appropriate doses of essential oils showing both antimicrobial activity and very low detrimental effect on intestinal cells.