Intestinal Isolates Research Articles

Presence of Anti-Microbial Antibodies in Liver Cirrhosis – A Tell-Tale Sign of Compromised Immunity?

BackgroundBacterial translocation plays important role in the complications of liver cirrhosis. Antibody formation against various microbial antigens is common in Crohn's disease and considered to be caused by sustained exposure to gut microflora constituents. We hypothesized that anti-microbial antibodies are present in patients with liver cirrhosis and may be associated with the development of bacterial infections.Methodology/Principal FindingsSera of 676 patients with various chronic liver diseases (autoimmune diseases:266, viral hepatitis C:124, and liver cirrhosis of different etiology:286) and 100 controls were assayed for antibodies to Saccharomyces cerevisiae(ASCA) and to antigens derived from two intestinal bacterial isolates (one gram positive, one gram negative, neither is Escherichia coli). In patients with liver cirrhosis, we also prospectively recorded the development of severe episodes of bacterial infection. ASCA and anti-OMP Plus™ antibodies were present in 38.5% and 62.6% of patients with cirrhosis and in 16% and 20% of controls, respectively (p<0.001). Occurrence of these antibodies was more frequent in cases of advanced cirrhosis (according to Child-Pugh and MELD score; p<0.001) or in the presence of ascites (p<0.001). During the median follow-up of 425 days, 81 patients (28.3%) presented with severe bacterial infections. Anti-microbial antibody titers (p = 0.003), as well as multiple seroreactivity (p = 0.036), was associated with infectious events. In logistic regression analysis, the presence of ascites (OR:1.62, 95%CI:1.16–2.25), co-morbidities (OR:2.22, 95%CI:1.27–3.86), and ASCA positivity (OR:1.59, 95%CI:1.07–2.36) were independent risk factors for severe infections. A shorter time period until the first infection was associated with the presence of ASCA (p = 0.03) and multiple seropositivity (p = 0.037) by Kaplan-Meier analysis, and with Child-Pugh stage (p = 0.018, OR:1.85) and co-morbidities (p<0.001, OR:2.02) by Cox-regression analysis.Conclusions/SignificanceThe present study suggests that systemic reactivity to microbial components reflects compromised mucosal immunity in patients with liver cirrhosis, further supporting the possible role of bacterial translocation in the formation of anti-microbial antibodies.

In vitro adherence of two candidate probiotics from Atlantic cod and their interference with the adhesion of two pathogenic bacteria

The potential of two candidate probiotic bacteria (GP21 and GP12), isolated from the gut of Atlantic cod, to adhere to primary cultures of the epithelial cells from the different regions of the intestine and to interfere with the adhesion of two pathogens, Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida were investigated. The intestinal isolates showed clear preference in adhering to the cells from the different intestine segments. GP12 adhered strongly to the fore- and mid intestine cells. The adherence of GP21 was most to the cells from the hind intestine followed by those from the mid-segment. The adhesion of V. anguillarum was affected by both GP21 and GP12; GP12 interfered through competition, but a specific mode of action was not observed for GP21. In the case of A. salmonicida, competition was the principal mechanism by which GP21 interfered with their adhesion, while exclusion mechanism was favoured by GP12. In addition, GP21 was more auto-aggregative than GP12, but the latter was more co-aggregative with both the pathogens. The isolates were also capable of lowering lactate dehydrogenase activity compared to that by the pathogen and they reduced the caspase-3 activity in the epithelial cells from the hind intestine, to which the pathogens adhered the most. Thus it could be concluded that the adhesion of the candidate probiotics is segment-specific and their interference with the adhesion of pathogens is dependent on both source of the epithelial cells and the mechanism adopted by the isolates. This information is novel in the case of fish and the manner in which potential probiotic organisms interfere with the pathogen adhesion provides supportive information for disease control.

Effect of Thymus vulgaris essential oil on intestinal bacterial microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum) and bacterial isolates

The application of natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics. We evaluated the effect of diet supplementation with Thymus vulgaris essential oil (TVEO) on the allochthonous microbial composition of rainbow trout. DNA was extracted directly from the intestinal contents, and the V3-V4 regions of the 16S rRNA genes were amplified by PCR. The bacterial composition was analysed using temporal temperature gradient electrophoresis (TTGE). No significant changes (P>0.05) were detected in the TTGE profiles of TVEO-treated trout compared with the controls. The Dice similarity index revealed a high stability (Cs >70%) of the intestinal microbiota in both groups during the 5-week period. Sequence analyses of the TTGE bands revealed the same bacterial composition in both groups, with most bacteria belonging to the Proteobacteria and Firmicutes phyla. The in vitro antibacterial activity of TVEO was assessed using a range of normal intestinal isolates and fish pathogens. The inhibitory concentrations for all the tested bacteria were higher than the TVEO levels used in trout, which may explain the in vivo results.

Emergence of fluoroquinolone‐resistant clonal group A: clonal analysis of Norwegian and Russian E. coli isolates

We describe a study of urinary tract and intestinal isolates of Escherichia coli from Norway and Russia using automated ribotyping, single nucleotide polymorphism analysis for clonal group A (CgA) supplemented with phylogrouping, virulence gene profiling and resistance profiling. CgA comprised 19% of the Norwegian UTI isolates from 2001. Two highly multiresistant fluoroquinolone-resistant CgA isolates were found. Ribotypes clustered into four major and six minor groups (ribogroups). Fluoroquinolone-resistant isolates and phylogroups A and B1 were associated with ribogroup (R)A. Ribogroup (R)B predominated among Russian UTI isolates and was predominantly phylogroup A and depleted in P-fimbriae. Ribogroup (R)C predominated among Norwegian UTI isolates and was rich in virulence factors (S-fimbriae, haemagglutinin and haemolysin) and predominantly phylogroup B2 and D. Ribogroup (R)G was associated with CgA and predominantly phylogroup D. Ribogroups (R)D, (R)E and (R)F had too few members for statistical analysis. The correlation between ribotype and phylogenetic group was not as strong as reported in other studies.

Metatranscriptome Analysis of the Human Fecal Microbiota Reveals Subject-Specific Expression Profiles, with Genes Encoding Proteins Involved in Carbohydrate Metabolism Being Dominantly Expressed

The human gastrointestinal (GI) tract provides home to a complex microbial community, collectively termed microbiota. Although major efforts have been made to describe the diversity and stability of the microbiota, functional studies have been largely restricted to intestinal isolates and include few community studies. The aim of this study was to explore the in situ gene expression of the fecal microbiota and to evaluate the RNA fingerprinting method cDNA-AFLP (cDNA amplified fragment length polymorphism) for this purpose. To this end, cDNA-AFLP analysis of enriched mRNA revealed that two healthy subjects showed highly divergent expression profiles with considerable fluctuations in time. Subsequent excision and sequence determination of bands from the mRNA-enriched profiles resulted in 122 identifiable sequences (transcripts and rRNAs). The classification of retrieved transcripts into functional clusters based on COG (cluster of orthologous genes) annotation showed that most assigned transcripts belonged to the metabolism cluster (26% of all sequences), underlining that even at the very end of the intestinal tract the microbiota is still very active. This study furthermore revealed that cDNA-AFLP is a useful tool to compare gene expression profiles in time in complex microbial communities.

Effects of humic acids on the growth of bacteria

The influence of humic acids of different origins on the growth of bacterial cultures of different taxa isolated from the soil and the digestive tracts of earthworms (Aporrectodea caliginosa)—habitats with contrasting conditions—was studied. More than half of the soil and intestinal isolates from the 170 tested strains grew on the humic acid of brown coal as the only carbon source. The specific growth rate of the bacteria isolated from the intestines of the earthworms was higher than that of the soil bacteria. The use of humic acids by intestinal bacteria confirms the possibility of symbiotic digestion by earthworms with the participation of bacterial symbionts. Humic acids at a concentration of 0.1 g/l stimulated the growth of the soil and intestinal bacteria strains (66 strains out of 161) on Czapek’s medium with glucose (1 g/l), probably, acting as a regulator of the cell metabolism. On the medium with the humic acid, the intestinal bacteria grew faster than the soil isolates did. The most active growth of the intestinal isolates was observed by Paenibacillus sp., Pseudomonas putida, Delftia acidovorans, Microbacterium terregens, and Aeromonas sp.; among the soil ones were the representatives of the Pseudomonas genus. A response of the bacteria to the influence of humic acids was shown at the strain level using the example of Pseudomonas representatives. The Flexom humin preparation stimulated the growth of the hydrocarbon-oxidizing Acinetobacter sp. bacteria. This effect can be used for creating a new compound with the elevated activity of bacteria that are destroyers of oil and oil products.

Selection of candidate probionts by two different screening strategies from Atlantic cod ( Gadus morhua L.) larvae

Two primary selection criteria were used to collect a pool of nearly 500 candidate probiotic bacteria from Atlantic cod ( Gadus morhua L.) larvae, i.e. the dominant intestinal bacterial flora and isolates with antagonistic activity against Vibrio anguillarum. Bacteria were isolated from cod larvae from five rearing groups with variable rearing technologies. The bacteria were brought to pure culture and characterized phenotypically. Based on properties such as uniqueness, dominance and fermentative ability, a selection of ∼10% of the isolates were chosen from the initial pool of bacteria to reduce the number of candidates. These 55 isolates were characterized further in vitro regarding antagonism, adhesion to mucus, growth in mucus, production of extracellular enzymes, fish bile resistance and haemolytic properties. Based on the results of the in vitro tests, the number of probiotic candidates was further reduced to seven isolates. To evaluate the probiotic potential and to assure that the seven isolates were not harmful to the host, yolk sac larvae of cod were exposed to the isolates in a small-scale in vivo experiment. The in vivo experiment excluded two of the candidate bacteria due to increased mortality of cod larvae, whereas three isolates from the dominant ( Vibrio and two different strains of Microbacterium) and two from the antagonistic ( Ruegeria and Pseudoalteromonas) group improved the survival of larvae compared to the positive control. Thus, a combination of the two screening methods was suited for making multistrain probiotics with complementary modes of action.

Antimicrobial resistance of fecal bacteria in waters of the Seine river watershed (France)

We studied the prevalences of antimicrobial resistance (AR) and multiple antimicrobial resistance (MAR) among the fecal bacteria found in the rivers of a large watershed under strong anthropogenic pressures, the Seine river watershed (France). Two groups of fecal indicator bacteria, Escherichia coli and intestinal enterococci, were tested for their susceptibility to 16 and 10 antimicrobials respectively, using the disk diffusion method. We found that 42% of the 214 E. coli river isolates were AR (resistant to at least one antimicrobial) and 35% were MAR (resistant to at least two antimicrobials). Among the 148 intestinal enterococci isolates from rivers, 83% were AR and 49% were MAR. We also investigated the sources of AR fecal bacteria found in the rivers of the watershed. A total of 715 E. coli isolates and 476 intestinal enterococci isolates were collected in point sources (municipal and hospital wastewaters) and non-point sources (surface runoff and soil leaching waters from agricultural or forest areas). For E. coli, the prevalence of AR differed widely from source to source and ranked in this order: hospital wastewaters (71%) > municipal wastewaters (44%) > agricultural non-point sources (16%) > forest non-point sources (2%). The prevalence of MAR ranked similarly, and the same trend was observed for intestinal enterococci. The AR level of fecal bacteria in the sources was related to their expected exposure level to antimicrobials before their release into the environment. A MAR index was calculated for every source and a good discrimination between them was thus obtained. At the global scale of the Seine river watershed, domestic wastewaters seemed more likely to be the predominant source of the AR fecal bacteria found in the rivers. This was corroborated by the similarity of the MAR indices from river and municipal wastewater isolates for both fecal indicators.

Limited genetic diversity among genotypes of Enterocytozoon bieneusi strains isolated from HIV-infected patients from Sydney, Australia

Microsporidia are intracellular parasites, with over 1200 species belonging to 143 genera described to date. They are opportunistic pathogens in humans and can cause chronic diarrhoea in immunosuppressed patients. Both Enterocytozoon bieneusi and Encephalitozoon intestinalis cause intestinal disease, with Enterocytozoon bieneusi more commonly identified in patients with human immunodeficiency virus (HIV) infection. In this study, intestinal microsporidial clinical isolates from patients in Sydney, Australia, were genotyped. All specimens were from HIV-infected men with low CD4(+) T-cell counts (<100 cells mm(-3)). Genotyping of the internal transcribed spacer regions of the rRNA gene showed the presence of only one genotype, the anthroponotic Enterocytozoon bieneusi genotype B strain. This study thus highlighted the limited genetic diversity among Australian Enterocytozoon bieneusi isolates, and it is hypothesized that, due to the reduced incidence of microsporidia and the subsequent reduction in the human reservoir of the anthroponotic genotype B, locally acquired intestinal microsporidiosis will rarely be seen in HIV-infected persons undergoing highly active antiretroviral therapy in the future in Australia.

Produkcija hemolizina i hemaglutinacija kod urinarnih izolata Escherichia coli

Escherichia coli (E.coli) is the most frequent agent of urinary infections in the outpatients. The aim of the work was to determine the production of hemolyzin and hemaglutination as the virulence factors of uropatogenic E.coli (UPEC). Production of hemolyzin and mannose-sensitive (MSHA) and mannose-resistant (MRHA) hemaglutination were examined in 200 urinary and 50 control intestinal isolates of E.coli. Hemolysis on blood agar was shown by 80 (40%) urinary isolates and 5 (10%) control intestinal isolates. In the examined group, MSHA was shown by 39 isolates (19,5%), and MRHA was shown by 83 isolates (41,5%). In control group, MSHA was shown by 9 isolates (18%), and MRHA was shown by 4 isolates (8%). It was found a statistically highly significant difference between the examined and control group, both in production of hemolyzin (p<0,0001) and in hemaglutination (p<0,0001). Urinary isolates of E.coli which produce hemolizin and show MSHA and MRHA, may be considered UPEC.

Cross-transmission of clinicalEnterococcus faeciumin relation toespand antibiotic resistance

To investigate clonality among clinical Enterococcus faecium isolates and normal intestinal microflora isolates as well as cross-transmission between patients in relation to the presence of the esp gene and antibiotic resistance. Blood-culture isolates (n = 101) deriving from tertiary, secondary and primary hospitals were analysed. Antibiotic susceptibility was investigated. Polymerase chain reaction and pulsed-field gel electrophoresis were used for detection of esp and genotyping, respectively. Nearly half (43%) of the patients included were involved in a cross-transmission event with Ent. faecium. These strains disseminated both within and between all hospitals. The antibiotic resistance and presence of esp were highest in isolates from the tertiary hospital. Isolates harbouring esp showed less genetic diversity compared with esp negative ones. Cross-transmission with Ent. faecium between patients was readily detected, indicating that hospital-adapted clones circulate within and between hospitals. Acquired characteristics, such as antibiotic resistance and esp, seem to accumulate in the isolates disseminating in the tertiary hospital. It is important to characterize Ent. faecium isolates causing infections and to determine the extent of dissemination in order to prevent further spread of these pathogens.

Ulcerative enteritis in Homarus americanus: Case report and molecular characterization of intestinal aerobic bacteria of apparently healthy lobsters in live storage

An intermoult male American lobster, Homarus americanus, with severe intestinal lesions was encountered while collecting samples of aerobic intestinal bacteria from lobsters held in an artificial sea-water recirculation aquarium system. Grossly, the intestine was firm, thickened, and white. Histologic examination revealed a severe, diffuse, ulcerative enteritis which spared the chitin-lined colon, somewhat similar to hemocytic enteritis of shrimp. The bacterial isolates from this lobster were compared to 11 other lobsters lacking gross intestinal lesions. Two organisms, one identified as Vibrio sp. and another most similar to an uncultured proteobacterium (98.9%), clustering with Rhanella and Serratia species using 16S rDNA PCR, were isolated from the intestines of the 11, grossly normal, lobsters and the affected lobster. An additional two intestinal isolates were cultured only from the lobster with ulcerative enteritis. One, a Flavobacterium, similar to Lutibacter litoralis (99.3%), possibly represented a previously described commensal of the distal intestine. The second, a Vibrio sp., was unique to the affected animal. While the etiology of the ulcerative enteritis remains undetermined, this report represents the first description of gross and histologic findings in H. americanus of a condition which has morphologic similarities to hemocytic enteritis of shrimp. An additional observation was a decrease in the number of intestinal isolates recovered from the 11 apparently healthy lobsters compared to that previously reported for recently harvested lobster. More comprehensive studies of the relationship between the health of lobsters, gut microbial flora and the husbandry and environment maintained within holding units are warranted.

Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use

A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF 37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.

Antibiotic Resistance and Adherence Properties of Hafnia alvei Clinical Isolates: A 19-Month Study in the Hospital of Orléans, France

We studied the epidemiology of antibiotic resistance and adherence properties among all Hafnia alvei clinical isolates collected from August 2003 to February 2005 from patients hospitalized in the hospital of Orléans, France. The isolates were typed by random amplified polymorphic DNA (RAPD), screened for antibiotic resistance and bacterial adherence to A549 respiratory and T24 bladder cells. Six intestinal, 3 respiratory, and 8 isolates from different body sites were collected. A total of 12 RAPD profiles were found, demonstrating a high genetic diversity. All the isolates were resistant to amoxicillin + clavulanate and cephalothin and sensitive to aminoglycosides, fluoroquinolones, cefepime and imipenem. Six isolates had a high-level and constitutive cephalosporinase production phenotype. Three independent isolates were resistant or had intermediate sensitivity to nalidixic acid, sulfonamide and trimethoprim or chloramphenicol. All the isolates adhered efficiently to the two cell lines with a higher effectiveness of adherence to bladder cells. The respiratory isolates adhered more efficiently to epithelial cells than intestinal isolates. No relationship was found between antibiotic resistance phenotypes, adherence properties, and RAPD types. In conclusion, H. alvei is an unusual nosocomial pathogen with little acquired antibiotic resistance able to adhere to human epithelial cells from different human body compartments.

Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae

While the normal microbiota has been implicated as a critical defense against invading pathogens, the impact of enteropathogenic infection and host inflammation on intestinal microbial communities has not been elucidated. Using mouse models of Citrobacter rodentium, which closely mimics human diarrheal pathogens inducing host intestinal inflammation, and Campylobacter jejuni infection, as well as chemically and genetically induced models of intestinal inflammation, we demonstrate that host-mediated inflammation in response to an infecting agent, a chemical trigger, or genetic predisposition markedly alters the colonic microbial community. While eliminating a subset of indigenous microbiota, host-mediated inflammation supported the growth of either the resident or introduced aerobic bacteria, particularly of the Enterobacteriaceae family. Further, assault by an enteropathogen and host-mediated inflammation combined to significantly reduce the total numbers of resident colonic bacteria. These findings underscore the importance of intestinal microbial ecosystems in infectious colitis and noninfectious intestinal inflammatory conditions, such as inflammatory bowel disease.

Salivaricin P, One of a Family of Two-Component Antilisterial Bacteriocins Produced by Intestinal Isolates of Lactobacillus salivarius

Lactobacillus salivarius DPC6005, a porcine intestinal isolate, produces a two-component bacteriocin, salivaricin P, with homology to ABP-118 produced by a human probiotic L. salivarius strain. Indeed, molecular characterization revealed that while the peptides Sln1 and ABP-118alpha are identical, their companion peptides (Sln2 and ABP-118beta, respectively) differ by two amino acids. This observation suggests that two-component bacteriocins may be a common feature of intestinal L. salivarius strains.

A comparison of phylogenetic group, virulence factors and antibiotic resistance in Russian and Norwegian isolates of Escherichia coli from urinary tract infection

Isolates of Escherichia coli from 31 Norwegian and 31 Russian females with significant bacteruria who presented with clinical signs of urinary tract infection (UTI) were tested for antimicrobial sensitivity, the presence of virulence genes, phylogroup distribution and clonal affinity. Twenty isolates, representing the full clonal diversity of a collection of 138 intestinal isolates of E. coli from healthy Norwegian females, served as a reference group. Russian UTI isolates belonged more often to phylogroup A and possessed fewer virulence genes than did Norwegian isolates. UTI isolates of E. coli were genetically heterogeneous and had a high degree of antimicrobial sensitivity.

Increased conjugation frequencies in clinical Enterococcus faecium strains harbouring the enterococcal surface protein gene esp

This study compared the in-vitro ability of Enterococcus faecium isolates of different origin to acquire vanA by conjugation in relation to the occurrence of the esp gene. In total, 29 clinical isolates (15/29 esp+), 30 normal intestinal microflora isolates (2/30 esp+) and one probiotic strain (esp-) were studied with a filter-mating assay. Conjugation events were confirmed by PCR and pulsed-field gel electrophoresis. Among the infection-derived isolates, the esp+ isolates had higher conjugation frequencies compared with esp- isolates (p < 0.001), with a median value of 6.4 x 10(-6) transconjugants/donor. The probiotic strain was shown to acquire vanA vancomycin resistance in in-vitro filter mating experiments.

Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products

Lactobacillus strains from traditional African fermented milk products, as well as human intestinal isolates were identified and investigated in vitro for their technological and functional characteristics as potential new probiotic strains. To test survival under gastrointestinal conditions, first the protective effect of milk and the effects of medium composition, lysozyme, pepsin, and pH of the medium on bacterial viability were assessed in vitro using the Plackett–Burman statistical model and the commercially used L. johnsonii LA1 probiotic strain. The use of either an artificial gastric electrolyte solution or MRS did not play a significant role in the viability of the cultures, while lysozyme, acidic conditions (pH 2.5), pepsin and the presence of milk significantly influenced the survival of the strain. Therefore, these parameters were selected as important test variables in a model stomach passage survival trial. Five strains identified as L. plantarum and two identified as L. johnsonii showed good survival under simulated gastrointestinal conditions. These selected strains also showed antimicrobial activity, probably due to production of organic acids. All strains exhibited bile salt hydrolase activity, while only the L. plantarum strains showed β-galactosidase activity.

Resistance of Klebsiella Pneumoniae Isolated From Different Infections to Antibiotics

During the period (September, 1998-March, 1999), 95 isolates of Klebsiella from respiratory tract, urinary tract, middle ear, intestinal tract, wound and blood infections were collected.. Morphological studies and biochemical tests were used for the identification of this bacterium. (50) isolates of Klebsiella pneumoniae were selected and used in the study. It was found that all isolates were resistant to one or more antibiotic and that Amikacin was the most active antibiotic against this species as all isolates were susceptible to it. Also Ciprfloxacin and Norfloxacin were very active with resistance rates of (8 %) and (14 %) respectively. Middle ear isolates were found to be more susceptible to antibiotics whereas blood and intestinal tract isolates were highly resistant to them. The study indicated that this bacterium has a high ability to produce -lactamase enzymes with total production rate of (88 %). The production rate of extended spectrum -lactamases was (36 %) where blood and intestinal tract isolates gave rates of (100 %) and (66.7 %) respectively, whilst urinary tract isolates gave rate of (55.6 %), whereas the production rate of respiratory tract and wound isolates was (25 %) for each. Middle ear isolates didn’t produce these enzymes