Detection of Specific Strains and Variants of Streptococcus cremoris in Mixed Cultures by Immunofluorescence

Antisera against four different strains of Streptococcus cremoris were raised by injecting rabbits with washed suspensions of whole cells. These antisera interacted specifically with the corresponding strain in a mixture of up to nine different S. cremoris strains. The antisera could be used for analyzing the composition of mixed cultures containing these strains by immunofluorescence. Competition experiments were performed in batch and continuous cultures under amino acid limitation. A bacteriophage-sensitive variant of S. cremoris SK11 (SK1128) could be distinguished from a bacteriophage-resistant variant (SK1143) by the same immunofluorescence technique. The competition between the two variants and the stability of both variants in pure cultures were followed with the specific antibodies. Antibodies against the purified proteolytic system of S. cremoris Wg2 were used to determine the presence of proteases by immunofluorescence in several S. cremoris strains under different culture conditions. The described immunofluorescence methods can be used to analyze complex mixed starter cultures common in the dairy industry as the strains and variants present in these mixtures can be recognized microscopically.     

Antibiotic resistance genes in anaerobic bacteria isolated from primary dental root canal infections

Fourty-one bacterial strains isolated from infected dental root canals and identified by 16S rRNA gene sequence were screened for the presence of 14 genes encoding resistance to beta-lactams, tetracycline and macrolides. Thirteen isolates (32%) were positive for at least one of the target antibiotic resistance genes. These strains carrying at least one antibiotic resistance gene belonged to 11 of the 26 (42%) infected root canals sampled. Two of these positive cases had two strains carrying resistance genes. Six out of 7 Fusobacterium strains harbored at least one of the target resistance genes. One Dialister invisus strain was positive for 3 resistance genes, and 4 other strains carried two of the target genes. Of the 6 antibiotic resistance genes detected in root canal strains, the most prevalent were blaTEM (17% of the strains), tetW (10%), and ermC (10%). Some as-yet-uncharacterized Fusobacterium and Prevotella isolates were positive for blaTEM, cfxA and tetM. Findings demonstrated that an unexpectedly large proportion of dental root canal isolates, including as-yet-uncharacterized strains previously regarded as uncultivated phylotypes, can carry antibiotic resistance genes.     

Mycobacterium chimaera as an Underestimated Cause of NTM Lung Diseases in Patients Hospitalized in Pulmonary Wards

Mycobacterium chimaera is the newly described species belonging to Mycobacterium avium complex (MAC), with morphology and growth characteristics closely related to Mycobacterium intracellulare. The aim of this retrospective study was to analyze the frequency and clinical significance of M. chimaera identification in the population of patients with previous positive respiratory cultures for M. intracellulare or MAC. 200 strains of M. intracellulare or MAC, isolated from respiratory specimens of patients hospitalized in pulmonary wards, between 2011 and 2020, were retrospectively analyzed with GenoType NTM-DR test. 88 (44%) of strains were re-classified to M. chimaera species. Analysis of clinical data in 30 patients with positive M. chimaera isolates revealed that they were diagnosed with chronic obstructive pulmonary disease (COPD) – 27%, past tuberculosis – 20%, or interstitial lung diseases – 17%, respectively. Non-tuberculous mycobacterial lung disease (NTMLD) caused by M. chimaera has been recognized in 53% of patients, most often in those presenting with post-tuberculous lung lesions. M. chimaera was almost exclusively isolated from respiratory specimens of patients with underlying lung diseases, especially those with COPD and/or past tuberculosis. NTMLD due to M. chimaera was diagnosed predominantly in patients with past tuberculosis.     

Siderophore and chitinase producing isolates from the rhizosphere of Nicotiana glauca Graham enhance growth and induce systemic resistance in Solanum lycopersicum L.

A screening for Plant Growth Promoting Rhizobacteria (PGPR) was carried out in the rhizosphere of wild populations of Nicotiana glauca Graham in south-eastern Spain. Nine hundred and sixty strains were isolated and grouped in four parataxonomic groups: Gram positive endospore forming bacilli, Gram positive non-endospore forming bacilli, Gram negative bacilli and others. Two groups were selected to continue the study: Gram negative bacilli since it was the most abundant, and Gram positive sporulated bacilli, seeking their sporulating capacity as an advantage for inoculants formulation. The ability of these to release siderophores and chitinases in vitro was evaluated. Ninety six isolates were siderophore producers, and 56 of them were also able to produce chitinases. Fifty percent of these were tested for growth promotion in tomato. The best results were obtained with 5 Gram negative bacilli and one Gram positive sporulated bacilli; 5 strains increased all growth parameters while one of them, N21.4, severely compromised plant growth. The ability of these 6 strains to induce systemic resistance against the leaf pathogen Xanthomonas campestris in tomato was evaluated. Five of them effectively reduced disease symptoms (up to 50%). The six strains were identified by 16s rDNA sequencing resulting in 3 Pseudomonas, 1 Bacillus and 2 Stenotrophomonas; it’s striking that 2 Pseudomonas protected up to 50% while the other increased disease incidence. This indicates that systemic induction is strain specific and not necessarily related to production of siderophores and chitinases.     

Presence of pathogenic Vibrio Parahaemolyticus in waters and seafood from the Tunisian Sea

The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected from various exported seafood products comprising of fishes and shellfish (Mytilus edulis and Crassostrea gigas) or seawater, was studied. Eight strains were confirmed as V. parahaemolyticus by toxR -based polymerase chain reaction and only one strain out of these 8 strains was positive for tdh and trh genes. Toxigenic V. parahaemolyticus isolates are present in Tunisian coastal areas and they may also be present in Tunisian exported seafood products.     

Isolation of Phenylalanine-Negative Proteus vulgaris

Three phenylalanine-negative Proteus vulgaris strains were isolated from three different sources. The significance of these Proteus strains has not been fully recognized.     

Patterns of Reactivity between a Panel of Monoclonal Antibodies and Forage Rhizobium Strains

A panel of 11 monoclonal antibodies raised against vegetative cells of Rhizobium leguminosarum biovar trifolii or Rhizobium meliloti was tested by enzyme-linked immunosorbent assay for reactivity with 47 strains of R. leguminosarum biovar trifolii and 60 strains of R. meliloti. The goal of the study was to define the degree of specificity associated with each antibody and to gain an understanding of the amount of antigenic diversity found among the strains and between the species. Each antibody was tested against each Rhizobium strain in four forms: washed steamed cells, washed unsteamed cells, cell-free culture broth, and nodule squash material. Each antibody showed a different pattern of reactivity among the 107 strains. One of each of the antibodies developed against R. meliloti and R. leguminosarum biovar trifolii reacted in a highly specific manner with cells or antigen from the immunogenic strain only. Nine of the antibodies recognized secreted as well as cellular antigen from many of the strains. Analysis of patterns of reactivity between the 107 strains and the 11 antibodies separated the strains into 28 groups of which 12 were represented by one strain only.     

Selective Isolation and Distribution of Sporichthya Strains in Soil

A simplified enrichment method in which centrifugation is used for selective isolation of Sporichthya strains from soil is described. Gellan gum plus 2 mM CaCl₂ stimulated growth of Sporichthya polymorpha KCC A0089 so that this organism was readily recognized on an isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0) and then incubating the preparation at 27°C for 60 min and centrifuging it at 1,000 × g for 10 min. Dry heat treatment at 80°C for 60 min increased the ratio of Sporichthya colonies to nonfilamentous bacteria on a gellan gum plate. Since S. polymorpha was sensitive to 14 antibiotics, including nalidixic acid, addition of these antibiotics was not suitable for isolating Sporichthya strains. Our isolates were identified as Sporichthya strains on the basis of their morphological and chemotaxonomic characteristics. By combining the techniques described above, we isolated a number of Sporichthya strains from 21 soil samples, which were collected in Belgium, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States. Sporichthya strains are widely distributed in the world. To our knowledge, this is the first time that Sporichthya strains have been isolated from locations other than the United States or Japan.     

Detection of plasmid-mediated quinolone resistance in clinical isolates of Enterobacteriaceae strains in Hamadan,West of Iran

Plasmid mediated quinolone resistance (PMQR) determinants have arisen as a significant concern in recent years. The aim of this study was screening of resistant-clinical isolates to fluoroquinolone antibiotics and detection of qnr and aac(6′)-Ib-cr genes.For this purpose we collected 100 fluoroquinolone-resistant Enterobacteriaceae which were from 3 hospitals in Hamadan, west provinces of Iran, between October 2012 and June 2013. The all samples were identified by biochemical tests and confirmed by PCR method. Antimicrobial susceptibility to 14 antimicrobial agents including levofloxacin and ciprofloxacin were determined by disk diffusion methods and ciprofloxacin MIC was obtained by broth microdilution method as Clinical Laboratory Standards Institute (CLSI) recommendations. The isolates were screened for the presence of qnrA, qnrB, qnrS and aac(6′)-Ib-cr genes using PCR assay. Among the screened isolates, 64 strains (64%) of Escherichia coli, 23 strains (23%) of Klebsiella pneumoniae, 13 strains (13%) of Proteus mirabilis were collected as quinolone-resistant isolates. out of 100 isolates, two (2%) were positive for qnrS, seventeen (17%) isolates were positive for qnrB and we did not find qnrA gene in any of the isolates. There were also 32 positive isolates for aac(6′)-Ib-cr determinant. We described the prevalence of qnr and aac(6′)-Ib-cr genes in fluoroquinolone-resistant Enterobacteriaceae in Hamadan city. The carriage rate of multidrug-resistant Enterobacteriaceae in healthy people in Hamadan City is extremely high. Moreover, genes encoding transferable quinolones, in particular aac(6′)-Ib-cr, are highly prevalent in these strains.     

Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.     

Surveillance and characterisation of Cronobacter spp. in Czech retail food and environmental samples

Cronobacter spp. (formerly Enterobacter sakazakii) are emerging, opportunistic pathogens that are linked with food-borne infections in neonates and infants. In the present study, 291 samples of food, 36 samples from a dairy farm and 140 samples of dust from vacuum cleaners were examined for the presence of Cronobacter spp. using chromogenic media and biochemical tests. Altogether, 72 Cronobacter spp. strains were isolated in accordance with the reference standard ?SN P ISO/TS 22964 (2006). No Cronobacter spp. strains were detected in 10 samples of infant milk formula or in samples from a dairy farm. Twelve out of 20 positive food samples were dry products. The incidence of Cronobacter spp. in instant and powdered products and spices (12 positive isolates out of 82 samples) was significantly higher than that in other foods (P?=?0.002), but lower than that in samples of dust (52 isolates; P?<?0.001). The incidence of Cronobacter spp. in dust from restaurants, bars and hotels (13 positive isolates in 20 samples) was significantly higher than that in dust from households (P?=?0.010). The polymerase chain reaction assay for the species-specific detection of the rpoB gene was performed in 49 isolates. Thirty-four Cronobacter spp. isolates were identified as Cronobacter sakazakii, nine isolates as Cronobacter malonaticus and one isolate as Cronobacter turicensis.     

Isolation and Identification of Ice Nucleation Active Fusarium Strains from Rapid Apple Declined Trees in Korea

In biological particles such as Fusarium species, ice nucleation activity (INA) has been observed. Fusarium strains isolated from apple declined trees in Korea were identified with a multilocus sequence analysis using the tef1 and rpb1 genes. Droplet-freezing and tube-freezing assays were used to determine the INA of the strains, using Pseudomonas syringae pv. syringae KACC 21200 as a positive control and resulting in seven INA+ fungal strains that were identified as F. tricinctum (KNUF-21-F17, KNUF-21-F18, KNUF-21-F29, KNUF-21-F32, KNUF-21-F38, KNUF-21-F43, and KNUF-21-F44). The effect of Fusarium INA+ KNUF-21-F29 was compared to that of INA– strains on Chrysanthemum morifolium cv. Shinma explants. A higher callus formation and no-shoot formation were observed, suggesting that fungal INA could play a role in cold injuries and be a factor to consider in rapid apple decline. To the best of our knowledge, this is the first report of INA fungal strains isolated in Korea.     

Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.     

Serotyping, stx2 Subtyping, and Characterization of the Locus of Enterocyte Effacement Island of Shiga Toxin-Producing Escherichia coli and E. coli O157:H7 Strains Isolated from the Environment in France

Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx₁, and 24 were positive for stx₂ (10 were positive for stx_2vh-a or stx_2vh-b, 19 were positive for stx_2d, and 15 were positive for stx_2e). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.     

Molecular and Phenotypic Characterization of Escherichia coli O26:H8 among Diarrheagenic E. coli O26 Strains Isolated in Brazil

Escherichia coli strains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among the several genes related to type III secretion system-secreted effector proteins, espK was found to be highly specific for EHEC O26:H11 and its stx-negative derivative strains isolated in European countries. E. coli O26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lack stx genes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26 stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive for fliC_H11, and 11 were fliC_H8 positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive for espK and could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and its stx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragments ex vivo confirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content of stx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.     

Cloning and Nucleotide Sequence of the gyrB Gene of Vibrio parahaemolyticus and Its Application in Detection of This Pathogen in Shrimp

Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic probe. The gyrB genes of V. parahaemolyticus and closely related Vibrio alginolyticus were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within gyrB specific for V. parahaemolyticus. These primers recognized 117 of 117 reference and wild-type V. parahaemolyticus strains, whereas amplification did not occur when 90 strains of 37 other Vibrio species or 60 strains representing 34 different nonvibrio species were tested. In 100-μl PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of gyrB primers for the routine identification of V. parahaemolyticus in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. parahaemolyticus-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of V. parahaemolyticus cells per g of shrimp homogenate. By this approach, we were able to detect V. parahaemolyticus in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.     

Evidence for Coexistence of Distinct Escherichia coli Populations in Various Aquatic Environments and Their Survival in Estuary Water

Escherichia coli, a commensal bacterium from the intestinal tracts of humans and vertebrate animals, has been used as one of two bacterial indicators of fecal contamination, along with intestinal enterococci, to monitor the microbiological quality of water. However, water environments are now recognized as a secondary habitat where some strains can survive. We investigated the survival of E. coli isolates collected from bodies of water in France exhibiting distinct profiles of contamination, defined according to the following criteria: vicinity of the point sources of contamination, land use, hydrology, and physicochemical characteristics of the receiving water. We selected 88 E. coli strains among a collection of 352 strains to carry out a microcosm experiment in filtered estuarine water for 14 days at 10°C. The relationship between the survival of E. coli strains and genotypic and phenotypic characteristics was analyzed. This work showed that distinct E. coli survival types, able to survive from between 7 and 14 days to less than 2 days, coexisted in the water. E. coli isolates that rapidly lost their culturability were more frequently isolated in water recently contaminated by fecal bacteria of human origin, and most were multiresistant to antibiotics and harbored several virulence factors. In contrast, persistent strains able to survive from 4 to 14 days were more often found in water with low levels of fecal bacteria, belonged mainly to the B1 phylogroup, often harbored only one virulence factor, kspE or ompT, and were able to grow at 7°C.     

Characterization of Verotoxin-Encoding Phages from Escherichia coli O103:H2 Strains of Bovine and Human Origins

The objectives of this study were to induce and characterize verotoxin-encoding phages from a collection of 91 verotoxin-producing Escherichia coli (VTEC) O103:H2 strains of human and bovine origins. All the strains carried the vt1 gene, and two carried the vt2 gene as well. The phages were induced by UV irradiation and characterized by DNA restriction fragment length polymorphism (RFLP), genome size, morphology, and Q and P genes, characteristic of lambdoid phages. A total of 32 vt-positive phages were induced and isolated from 31 VTEC O103:H2 strains. Thirty phages were vt1 positive, and two were vt2 positive. Ten of the 30 vt1-positive phages (33.3%) were from cattle strains, and 20 (66.6%) were from human strains. The two vt2-positive phages were from human strains. Phages belonged to 21 RFLP profiles, of which 17 were single-phage profiles and 4 were multiple-phage profiles. The estimated genome size of the phages ranged from 34 to 84 kb. Two phages that were examined by electron microscopy possessed hexagonal heads with long tails, and one had an elongated head with a long tail. The Q and P genes were amplified in all 32 phages, and the Q-stxA₁ gene region yielded an amplicon in 19 phages (59.3%). It is concluded that the VTEC O103:H2 strains of human origin were more readily inducible than those of bovine origin and that the genotypic profiles of verotoxin-encoding phages were highly diverse, as revealed by their RFLP profiles.     

Mixed infection with cagA positive and cagA negative strains of Helicobacter pylori lowers disease burden in The Gambia

Background

The prevalence of Helicobacter pylori including strains with putatively virulent genotypes is high, whereas the H. pylori-associated disease burden is low, in Africa compared to developed countries. In this study, we investigated the prevalence of virulence-related H. pylori genotypes and their association with gastroduodenal diseases in The Gambia.

Methods and Findings

DNA extracted from biopsies and H. pylori cultures from 169 subjects with abdominal pain, dyspepsia or other gastroduodenal diseases were tested by PCR for H. pylori. The H. pylori positive samples were further tested for the cagA oncogene and vacA toxin gene.One hundred and twenty one subjects (71.6%) were H. pylori positive. The cagA gene and more toxigenic s1 and m1 alleles of the vacA gene were found in 61.2%, 76.9% and 45.5% respectively of Gambian patients harbouring H. pylori. There was a high prevalence of cagA positive strains in patients with overt gastric diseases than those with non-ulcerative dyspepsia (NUD) (p = 0.05); however, mixed infection by cagA positive and cagA negative strains was more common in patients with NUD compared to patients with gastric disease (24.5% versus 0%; p = 0.002).

Conclusion

This study shows that the prevalence of H. pylori is high in dyspeptic patients in The Gambia and that many strains are of the putatively more virulent cagA⁺, vacAs1 and vacAm1 genotypes. This study has also shown significantly lower disease burden in Gambians infected with a mixture of cag-positive and cag-negative strains, relative to those containing only cag-positive or only cag-negative strains, which suggests that harbouring both cag-positive and cag-negative strains is protective.     

PCR detection of nitrite reductase genes (nirK and nirS) and use of active consortia of constructed ternary adherent staphylococcal cultures via mixture design for a denitrification process

In this study, three adherent Staphylococcus strains able to use nitrate as electron acceptor were isolated from textile wastewater activated sludge. Based on the biochemical profiles, bacterial strains were identified as Staphylococcus lentus, Staphylococcus warneri and Staphylococcus epidermidis the PCR amplification of the nir genes reveal that S. lentus and S. epidermidis were nirK positive and that S. epidermidis was nirS positive. The three strains were also icaA/icaD positive. To obtain the optimal formulation of pure cultures of the staphylococci, the influence of the different mixtures of organisms was studied using mixture design. The regression model of microorganism composition and main metabolites was established. The most predictable reduction of nitrate was 87.2 and 12% of COD consumption. The results suggested that the predictable production of nitrite would reach a minimum of 6.1 mg/l. Based on this, the response values that satisfied all expectations were optimized using MINITAB^® 14 analysis software. The most optimal proportion combination was 49.75% of S. lentus (curve value), 36.85% of S. warneri (curve value) and 13.39% S. epidermidis (curve value).