Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.     

Detection of periodontopathogenic bacteria in pregnant women by traditional anaerobic culture method and by a commercial molecular genetic method

To culture facultative and strict anaerobic bacteria is a well-established method for analyzing subgingival plaque samples. Micro-IDent^® and micro-IDent^® Plus (HAIN Lifescience GmbH, Nehren, Germany) tests are two commercially available rapid PCR-based methods for the identification and quantification of putative periodontopathogen bacteria. In this study, we compared these commercial PCR-based hybridization methods with conventional anaerobic culture technique. A total of 36 subgingival plaque samples were collected from periodontal pockets of pregnant women with chronic localized periodontitis. Aliquots of these samples were evaluated with species-specific probes provided by micro-IDent^® and micro-IDent^® Plus tests simultaneously, and from the same samples anaerobic and capnophylic bacteria were cultured on selective media. The overall agreement between both methods was excellent for Eubacterium nodatum, Tannerella forsythia and Porphyromonas gingivalis (97–92%), fair for Capnocytophaga sp, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Prevotella intermedia (91–89%) and poor for Fusobacterium nucleatum, Parvimonas micra (Micromonas micros), and Campylobacter rectus (86–78%). Discrepancies in the results may be explained by inability of culture method to distinguish between closely related taxa (e.i P. intermedia/Prevotella. nigrescens), and problems of keeping periodontopathogen bacteria viable, which is required for successful detection by standard culture method. Nucleic acid-based methods may replace cultivation method as frequently used methods in microbiological diagnosis of progressive periodontitis, thus micro-IDent^® andmicro-IDent^® Plus tests can be recommended where culture of periodontopathogenic bacteria is not performed in routine microbiology laboratories to analyze subgingival plaque samples.     

Interactions between Eikenella corrodens and ‘Streptococcus milleri-group’ organisms: possible mechanisms of pathogenicity in mixed infections

Interactions between the ‘Streptococcus milleri-group’ organisms (SMG; S. intermedius, S. constellatus and S. anginosus) and Eikenella corrodens were investigated. Coaggregation reactions occurred frequently between S. anginosus (83% of strain combinations) or S. constellatus (87%) and E. corrodens isolates, but were infrequent between S. intermedius and E. corrodens (28%). No enhancement of enzyme activities against lipid, phosphate, peptide and saccharide substrates tested were detected with combinations of species in comparison to the species alone. Exponential growth of S. constellatus and S. intermedius, in mixed culture with E. corrodens, occurred within 6h post inoculation, in comparison to 25h without E. corrodens. No growth stimulation of S. anginosus was observed. It is concluded that both coaggregation and growth stimulation occur between E. corrodens and SMG isolates, and may be important mechanisms in the establishment of mixed infections involving these bacteria.     

Presence of Salmonella and Campylobacter spp. in Wild Small Mammals on Organic Farms

The presence of Salmonella and Campylobacter spp. in rodents and insectivores (n = 282) was investigated on organic farms. Infections were encountered in house mice (8 of 83 Campylobacter positive and 1 of 83 Salmonella sp. strain Livingstone positive) and brown rats (1 of 8 Campylobacter positive) but not in other species. No shared Campylobacter genotypes were found between rodent and pig manure isolates. Effective on-farm rodent management is recommended.     

Effect of Conventional and Organic Production Practices on the Prevalence and Antimicrobial Resistance of Campylobacter spp. in Poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

Epidemiological Typing of Campylobacter Isolates from Meat Processing Plants by Pulsed-Field Gel Electrophoresis, Fatty Acid Profile Typing, Serotyping, and Biotyping

Campylobacter spp. are a leading cause of bacterial gastroenteritis. Foods of animal origin, particularly undercooked poultry, are common sources of Campylobacter species associated with disease in humans. A collection of 110 Campylobacter jejuni and 31 C. coli human and environmental isolates from different Ontario, Canada, abattoirs were analyzed by pulsed-field gel electrophoresis, fatty acid profile typing, and biotyping. Previously collected serotyping data for the same isolates were also analyzed in this study. Pulsed-field gel electrophoresis was found to be the most discriminatory of the typing methods, followed by serotyping, fatty acid profile typing, and biotyping. A wide variety of typing profiles were observed within the isolates, suggesting that several different Campylobacter sp. strains were present within the abattoirs.     

Description ofCampylobacter laridis,a new species comprising the nalidixic acid resistant thermophilicCampylobacter (NARTC) group

Ten strains of the nalidixic acid-resistant thermophilicCampylobacter (NARTC) group, of which 2 were isolated from human feces, were compared with 12 reference strains representing various species ofCampylobacter. The NARTC strains were a homogeneous group with respect to their cell morphology and 28 physiological and biochemical characters. All were microaerophilic, motile (amphitrichate), gram-negative, curved, S-shaped or helical rods, and representative strains had mean DNA base compositions of 31 to 32 mol % G+C. Distinctive features of the 10 strains were resistance to nalidixic acid and anaerobic growth in the presence of trimethylamine N-oxide hydrochloride (TMAO). The latter feature may account for the common occurrence of NARTC strains in the fecal contents of seagulls. DNA-DNA hybridizations indicated high (≥76%) base sequence relatedness within the group and low (≤15%) relatedness to other species ofCampylobacter. The 10 strains were classified in the genusCampylobacter but they could not be assigned to any previously defined species. Therefore, a new species, with the nameCampylobacter laridis, is proposed for these 10 strains; the type strain is NCTC 11352.     

Neptuniibacter pectenicola sp. nov. and Neptuniibacter marinus sp. nov., two novel species isolated from a Great scallop (Pecten maximus) hatchery in Norway and emended description of the genus Neptuniibacter.

Nine isolates obtained from a great scallop hatchery in Norway were characterized using a polyphasic approach. Strains were Gram-negative, aerobic and motile rods with oxidative metabolism. Phylogenetic analysis based on the sequences of 16S rRNA and rpoB genes showed that these strains formed two different groups associated with members of the genus Neptuniibacter. DNA–DNA hybridization (DDH) and Average Nucleotide Identity (ANI) demonstrated that the isolates constituted two novel species of this genus, which can be phenotypically differentiated from their closest relatives. The names Neptuniibacter marinus sp. nov. and Neptuniibacter pectenicola sp. nov are proposed, with ATR 1.1^T (=CECT 8938^T = DSM 100783^T) and LFT 1.8^T (=CECT 8936^T = DSM 100781^T) as respective type strains.     

Investigation of the Effect of Type 2 Diabetes Mellitus on Subgingival Plaque Microbiota by High-Throughput 16S rDNA Pyrosequencing

Diabetes mellitus is a major risk factor for chronic periodontitis. We investigated the effects of type 2 diabetes on the subgingival plaque bacterial composition by applying culture-independent 16S rDNA sequencing to periodontal bacteria isolated from four groups of volunteers: non-diabetic subjects without periodontitis, non-diabetic subjects with periodontitis, type 2 diabetic patients without periodontitis, and type 2 diabetic patients with periodontitis. A total of 71,373 high-quality sequences were produced from the V1-V3 region of 16S rDNA genes by 454 pyrosequencing. Those 16S rDNA sequences were classified into 16 phyla, 27 classes, 48 orders, 85 families, 126 genera, and 1141 species-level OTUs. Comparing periodontally healthy samples with periodontitis samples identified 20 health-associated and 15 periodontitis-associated OTUs. In the subjects with healthy periodontium, the abundances of three genera (Prevotella, Pseudomonas, and Tannerella) and nine OTUs were significantly different between diabetic patients and their non-diabetic counterparts. In the subjects carrying periodontitis, the abundances of three phyla (Actinobacteria, Proteobacteria, and Bacteriodetes), two genera (Actinomyces and Aggregatibacter), and six OTUs were also significantly different between diabetics and non-diabetics. Our results show that type 2 diabetes mellitus could alter the bacterial composition in the subgingival plaque.     

Transfer and Expression of the Catabolic Plasmid pBRC60 in Wild Bacterial Recipients in a Freshwater Ecosystem

3-Chlorobenzoate (3Cba)-degrading bacteria were isolated from the waters and sediments of flowthrough mesocosms dosed with various concentrations of 3Cba and inoculated with a 3Cba-degrading Alcaligenes sp., strain BR60. Bacteria capable of 3Cba degradation which were distinct from BR60 were isolated. They carried pBRC60, a plasmid introduced with Alcaligenes sp. strain BR60 that carries a transposable element (Tn5271) encoding 3Cba degradation. The isolates expressed these genes in different ways. The majority of pBRC60 recipients were motile, yellow-pigmented, gram-negative rods related to the group III pseudomonads and to BR60 by substrate utilization pattern. They were capable of complete 3Cba degradation at both millimolar and micromolar concentrations. Two isolates, Pseudomonas fluorescens PR24B(pBRC60) and Pseudomonas sp. strain PR120(pBRC60), are more distantly related to BR60 and both produced chlorocatechol when exposed to 3Cba at millimolar concentrations in the presence of yeast extract. These species showed poor growth in liquid 3Cba minimal medium but could degrade 3Cba in continuous cultures dosed with micromolar levels of the chemical. Laboratory matings confirm that pBRC60 can transfer from BR60 to species in both the beta and gamma subgroups of the proteobacteria and that 3Cba gene expression is variable between species. Selection pressures acting on pBRC60 recipients are discussed.     

Infinite Serovar and Ribotype Heterogeneity Among Oral Fusobacterium nucleatum Strains?

Fusobacterium nucleatum is part of the residential human microbiota and is associated with various infections. It is characterised by broad genetic heterogeneity, but reliable phenotypic markers are lacking. The purpose of the present study was to generate antibodies for the detection of F. nucleatum , to characterise expression patterns of the detected surface antigens on oral isolates, to investigate the prevalence of distinguishable subtypes in clinical samples from the oral cavity, and to compare antigenic with ribotype heterogeneity. Thirty-seven monoclonal antibodies (mAbs) were generated and characterised using strains from 52 taxa. Antibody-binding bacteria were monitored in 35 samples of supra- and subgingival plaque from healthy sites and sites affected by gingivitis or periodontitis. Results indicated broad but structured antigenic heterogeneity. Detecting at least 28 different epitopes, the mAbs defined 19 serovars. Epitopes were expressed on periodate-sensitive polysaccharide chains. Ribotyping of 40 oral F. nucleatum strains (Pvu II digestion) resulted in the detection of similarly broad genetic heterogeneity, which rarely corresponded to the observed phenotypic diversity. Clinical samples were generally positive for multiple (up to eight) serovars of which some colonised supra- and subgingival plaques from both healthy and diseased sites, whereas others were restricted to inflamed sites. The majority of the studied isolates could not be grouped with reference strains of the five established subspecies of F. nucleatum , corroborating doubts about the usefulness of the current classification scheme. Although, as a whole the described monoclonal antibodies can only recognise a part of the overwhelming heterogeneity of this ‘species’, they should prove of value to investigations of the importance, the antigenic stability and the origin of positive subtypes of F. nucleatum from human infections     

Antimicrobial-Resistant Campylobacter Species from Retail Raw Meats

The antimicrobial susceptibilities of 378 Campylobacter isolates were determined. Resistance to tetracycline was the most common (82%), followed by resistance to doxycycline (77%), erythromycin (54%), nalidixic acid (41%), and ciprofloxacin (35%). Campylobacter coli displayed significantly higher rates of resistance to ciprofloxacin and erythromycin than Campylobacter jejuni, and Campylobacter isolates from turkey meat showed a greater resistance than those from chicken meat.     

Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels.     

Lack of Evidence for Vertical Transmission of Campylobacter spp. in Chickens

Campylobacter jejuni is a major cause of bacterial food-borne infection in the industrial world. There is evidence that C. jejuni is present in eggs and hatchery fluff, opening the possibility for vertical transmission from hens to progeny. Poultry operations in Iceland provide an excellent opportunity to study this possibility, since breeding flocks are established solely from eggs imported from grandparent flocks in Sweden. This leaves limited opportunity for grandparents and their progeny to share isolates through horizontal transmission. While Campylobacter was not detected in all grandparent flocks, 13 of the 16 egg import lots consisted of eggs gathered from one or more Campylobacter-positive grandparent flocks. No evidence of Campylobacter was found by PCR in any of the 10 relevant quarantine hatchery fluff samples examined, and no Campylobacter was isolated from the parent birds through 8 weeks, while they were still in quarantine rearing facilities. After the birds were moved to less biosecure rearing facilities, Campylobacter was isolated, and 29 alleles were observed among the 224 isolates studied. While three alleles were found in both Sweden and Iceland, in no case was the same allele found both in a particular grandparent flock and in its progeny. We could find no evidence for vertical transmission of Campylobacter to the approximately 60,000 progeny parent breeders that were hatched from eggs coming from Campylobacter-positive grandparent flocks. If vertical transmission is occurring, it is not a significant source for the contamination of chicken flocks with Campylobacter spp.     

Subgingival microbiota from Cebus apella (capuchin monkey) with different periodontal conditions

This present study evaluated the subgingival microbiota of the Cebus apella with different periodontal conditions kept by the Tufted Capuchin Monkey Procreation Center (São PauloState University – UNESP) or free-ranging monkeys. For this purpose, clinical specimens of subgingival biofilm were collected from 52 monkeys, of both genders, 40 kept in captivity and 12 free-ranging monkeys. The primates were submitted to periodontal evaluation and biofilm samples were transferred to VMGA III transport medium and ultrapure water. The microbiota was cultivated in selective and non-selective culture media and microbial DNA was extracted and the presence of periodontal pathogens was evaluated using PCR and real-time PCR. The actinomycetes, fusobacteria, Campylobacter rectus, Eikenella corrodens, black-pigmented Gram-negative anaerobic rods, Tannerella forsythia, staphylococci and streptococci represent the predominantly detected microorganisms. Aggregatibacter actinomycetemcomitans, Dialister pneumosintes and Prevotella nigrescens were rarely observed, whereas Treponema denticola was not found. Populations of C. rectus, E. corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, T. forsythia and the total microbial load were significantly higher in animals with bone loss and, in smaller extension, in animals with gingival bleeding.     

Evaluation of novel agars for the enumeration of Campylobacter spp. in poultry retail samples

The purpose of this study was to examine the performance of novel agars for the identification and enumeration of Campylobacter species. The analytical sensitivity and specificity of Campylobacter Selective agar (CASA), Brilliance CampyCount agar (BCCA) and CampyFoodIDagar (CFA) for 84 Campylobacter spp. isolates and 50 non-Campylobacter spp. isolates from 37 distinct genera were of 100% sensitivity, with a 98% specificity for BCCA and CFA, and a 100% specificity for CASA. The application of these selective agars for Campylobacter spp. enumeration in comparison to the conventional agars, modified charcoal cefoperazonedeoxycholate agar (mCCDA) and Campy-Cefex (CCA) was examined using Campylobacter jejuni and Campylobacter coli inoculated samples. From C. jejuni inoculated samples, recovery on BCCA was significantly greater than other media (p < 0.05). Recovery on CASA was not significantly different from mCCDA and CCA (p > 0.05). With C. coli inoculated samples, recovery was significantly greater on BCCA and CASA than with other media (p < 0.05). The recovery of both C. jejuni and C. coli from inoculated samples with CFA was significantly less than with other media (P < 0.05). CASA was able to effectively inhibit and differentiate Campylobacter spp. from background microflora while false positive organisms occurred with BCCA and CFA. An examination of 483 randomly selected suspect Campylobacter colonies from naturally contaminated samples demonstrated a colony confirmation rate for CCA, CFA, BCCA, mCCDA, and CASA, of 84%, 87%, 88%, 90%, and 100%, respectively. The media evaluated present an alternative to conventional selective agars for the identification and enumeration of thermotolerant Campylobacter spp. from samples of poultry origin through the farm to fork continuum.     

Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments.

Strict anaerobic culture techniques were used to quantitatively and qualitatively evaluate the anaerobic heterotrophic bacteria present at the sediment-water interface of hyperutrophic Wintergreen Lake (Augusta, Mich.). Anaerobic plate counts remained constant from March through December, 1973, ranging from 2.4 X 10(6) to 5.7 X 10(6) organisms/g (dry weight) of sediment. The isolatable bacteria represented a small percentage of the total microbial community, which was shown by direct microscopic counts to be 2.0 X 10' organisms/g (dry weight) of sediment during June and July. Bacteria of the genus Clostridium dominated the isolates obtained, accounting for 71.8% of the 960 isolates examined. A single species, Clostridium bifermentens, comprised 47.7% of the total. Additional bacterial groups and the percentage in which they were isolated included: Streptococcus sp. (10.8%), unidentified curved rods (9.5%y, gram-positive nonsporing rods (5.6%), and motile gram-negative rods (1.9%). Temperature growth studies demonstrated the ability of all the isolates to grow at in situ sediment temperatures. Gas-liqid radiochromatography was used to determine the soluble metabolic end products from [U-14C]glucose and a U-14C-labeled amino acid mixture by representative sedimentary clostridial isolates and by natural sediment microbial communities. At in situ temperatures the natural sediment microflora produced soluble fermentative end products characteristic of those elaborated by the clostridial isolates tested. These results are considered strong presumptive evidence that clostridia are actively metabolizing in the sediments of Wintergreen Lake.