The effects of primary and secondary bacterial exposure on the seahorse (Hippocampus erectus) immune response.

Evolutionary adaptations in the Syngnathidae teleost family (seahorses, pipefish and seadragons) culminated in an array of spectacular morphologies, key immune gene losses, and the enigmatic male pregnancy. In seahorses, genome modifications associated with immunoglobulins, complement, and major histocompatibility complex (MHC II) pathway components raise questions concerning their immunological efficiency and the evolution of compensatory measures that may act in their place. In this investigation heat-killed bacteria (Vibrio aestuarianus and Tenacibaculum maritimum) were used in a two-phased experiment to assess the immune response dynamics of Hippocampus erectus. Gill transcriptomes from double and single-exposed individuals were analysed in order to determine the differentially expressed genes contributing to immune system responses towards immune priming. Double-exposed individuals exhibited a greater adaptive immune response when compared with single-exposed individuals, while single-exposed individuals, particularly with V. aestuarianus replicates, associated more with the innate branch of the immune system. T. maritimum double-exposed replicates exhibited the strongest immune reaction, likely due to their immunological naivety towards the bacterium, while there are also potential signs of innate trained immunity. MHC II upregulated expression was identified in selected V. aestuarianus-exposed seahorses, in the absence of other pathway constituents suggesting a possible alternative or non-classical MHC II immune function in seahorses. Gene Ontology (GO) enrichment analysis highlighted prominent angiogenesis activity following secondary exposure, which could be linked to an adaptive immune process in seahorses. This investigation highlights the prominent role of T-cell mediated adaptive immune responses in seahorses when exposed to sequential foreign bacteria exposures. If classical MHC II pathway function has been lost, innate trained immunity in syngnathids could be a potential compensatory mechanism.

Vibrio syngnathi sp. nov., a fish pathogen, isolated from the Kiel Fjord.

A new Vibrio strain, K08M4T, was isolated from the broad-nosed pipefish Syngnathus typhle in the Kiel Fjord. Infection experiments revealed that K08M4T was highly virulent for juvenile pipefish. Cells of strain K08M4T were Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain grew aerobically at 9-40° C, at pH 4-10.5 and it tolerated up to 12 % (w/v) NaCl. The most prevalent (>10 %) cellular fatty acids of K08M4T were C16 : 1 ω7c and C16 : 0. Whole-genome comparisons revealed that K08M4T represents a separate evolutionary lineage that is distinct from other Vibrio species and falls within the Splendidus clade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 and 1, 587,964 bp) and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the name Vibrio syngnathi sp. nov. The type strain is K08M4T (=DSM 109818T=CECT 30086T).

Microbiological findings in early and late implant loss: an observational clinical case-controlled study.

BACKGROUND: Implants are a predictable and well-established treatment method in dentistry. Nevertheless, looking at possible failures of dental implants, early and late loss have to be distinguished. The intent of the study was to report microbiological findings on the surface of implants with severe peri-implantitis, which had to be explanted. METHODS: 53 specimens of implants from 48 patients without severe general illnesses have been examined. The groups investigated were implants that had to be removed in the period of osseointegration (early loss, 13 patients with 14 implants) or after the healing period (late loss, 14 patients with 17 implants). The implant losses were compared with two control groups (implants with no bone loss directly after completed osseointegration, two to four months after implant placement (17 patients with 17 implants) and implants with no bone loss and prosthetic restoration for more than three years (5 patients with 5 implants)). Data about the bacteria located in the peri-implant sulcus was collected using amplification and high throughput sequencing of the 16S rRNA gene. RESULTS: The biofilm composition differed substantially between individuals. Both in early and late implant loss, Fusobacterium nucleatum and Porphyromonas gingivalis were found to be abundant. Late lost implants showed higher bacterial diversity and in addition higher abundances of Treponema, Fretibacterium, Pseudoramibacter and Desulfobulbus, while microbial communities of early loss implants were very heterogeneous and showed no significantly more abundant bacterial taxa. CONCLUSIONS: Specific peri-implant pathogens were found around implants that were lost after a primarily uneventful osseointegration. P. gingivalis and F. nucleatum frequently colonized the implant in early and late losses and could therefore be characteristic for implant loss in general. In general, early lost implants showed also lower microbial diversity than late losses. However, the microbial results were not indicative of the causes of early and late losses.

Impact of dental cement on the peri-implant biofilm-microbial comparison of two different cements in an in vivo observational study.

BACKGROUND: The type of cement used in cemented fixed implant-supported restorations influences formation of undetected excess cement and composition of the peri-implant biofilm. Excess cement and dysbiosis of the biofilm involve the risk of peri-implant inflammation. PURPOSE: The aim of the study was to investigate the impact of two different cements on the peri-implant biofilm and inflammation. MATERIALS AND METHODS: In an observational study, the suprastructures of 34 patients with cemented fixed implant-supported restorations were revised. In 20 patients, a methacrylate cement (Premier Implant cement [PIC]) and in 14 patients, a zinc oxide eugenol cement (Temp Bond [TB]) were used. After revision, TB was used for recementation. During revision and follow-up after 1 year, microbial samples were obtained. RESULTS: Excess cement was found in 12 (60%) of the 20 patients with PIC. Suppuration was observed in two (25%) implants with PIC without excess cement (PIC-) and in all 12 (100%) implants with PIC and excess cement (PIC+). Implants cemented with TB had neither excess cement nor suppuration. The taxonomic analysis of the microbial samples revealed an accumulation of periodontal pathogens in the PIC patients independent of the presence of excess cement. Significantly, fewer oral pathogens occurred in patients with TB compared to patients with PIC. TB was used in all cases (PIC and TB) for recementation. In the follow-up check, suppuration was not found around any of the implants with PIC-, only around one implant with PIC+ and around one implant with TB. Bacterial species associated with severe periodontal infections that were abundant in PIC- and PIC+ samples before the revision were reduced after 1 year to levels found in the TB samples. CONCLUSIONS: The revision and recementation with TB had a positive effect on the peri-implant biofilm in cases with PIC. The cementation of suprastructures on implants with TB is an alternative method to be considered.

Effect of dental cements on peri-implant microbial community: comparison of the microbial communities inhabiting the peri-implant tissue when using different luting cements.

BACKGROUND: Cementing dental restorations on implants poses the risk of undetected excess cement. Such cement remnants may favor the development of inflammation in the peri-implant tissue. The effect of excess cement on the bacterial community is not yet known. The aim of this study was to analyze the effect of two different dental cements on the composition of the microbial peri-implant community. METHODS: In a cohort of 38 patients, samples of the peri-implant tissue were taken with paper points from one implant per patient. In 15 patients, the suprastructure had been cemented with a zinc oxide-eugenol cement (Temp Bond, TB) and in 23 patients with a methacrylate cement (Premier Implant Cement, PIC). The excess cement found as well as suppuration was documented. Subgingival samples of all patients were analyzed for taxonomic composition by means of 16S amplicon sequencing. RESULTS: None of the TB-cemented implants had excess cement or suppuration. In 14 (61%) of the PIC, excess cement was found. Suppuration was detected in 33% of the PIC implants without excess cement and in 100% of the PIC implants with excess cement. The taxonomic analysis of the microbial samples revealed an accumulation of oral pathogens in the PIC patients independent of the presence of excess cement. Significantly fewer oral pathogens occurred in patients with TB compared to patients with PIC. CONCLUSION: Compared with TB, PIC favors the development of suppuration and the growth of periodontal pathogens.

Microbial analysis of biofilms on cement surfaces: An investigation in cement-associated peri-implantitis.

The cementation of implant-supported restorations always poses the risk of excess cement retained in the peri-implant sulcus despite careful clinical control. Excess cement can become the basis of colonization by oral microorganisms. As a result of the biofilm formation peri-mucositis or peri-implantitis may develop. Complications were observed in the routine prosthetic restoration of implants when a methacrylate-based cement was used. These developed a few weeks after cementation of the suprastructure and caused bleeding on probing as well as suppuration from the peri-implant tissue. In the revision therapy, excess cement in the peri-implant sulcus was found in many cases. This excess cement was sampled from ten patients and investigated for biofilm formation. For this purpose, the cement samples were collected and analyzed for bacterial in situ colonization by 16S rDNA-based methods. In laboratory experiments, the methacrylate-based cement and two other dental cements were then investigated for their proneness to form biofilm. The results of the in situ and in vitro investigations revealed a strong tendency towards bacterial invasion of the methacrylate-based cement by opportunistic species and pathogens.

Diversity of patients microflora on orthopaedic and dental implants.

The aim of this study was to compare the diversity of microbial colonization on implant material from different individuals. Eubacterial DNA was extracted, separated and sequenced from orthopaedic metallic implant material, tissues or body fluids, and skin of 4 patients as well as from identical dental cement material from 10 individuals after revision and routine removal. Additionally, the composition of the bacterial population of the dental cement and the oral swab sample from one individual after direct extraction of bacterial DNA was compared to extraction after conventional microbiological enrichment. The latter investigation proved that the commonly used cultivation technique gave different results than direct extraction of DNA, especially as regards the detection of anaerobes. Comparing the bacterial colonization of implant materials from different patients showed significant individual diversity. The common focus on a constricted pathogen spectrum may have to be expanded toward a multispecies population. Moreover, the dependence of the bacterial population on the individual host has to be integrated in discussing implant colonization and infection.

Bacterial DNA from orthopedic implants after routine removal.

Bacterial 16S rDNA was monitored and identified from orthopedic metallic implants after routine or septic removal from patients in a German hospital. From March to June 2009, 28 metallic implants, 10 human biopsies, and 6 foam dressings from 28 patients were investigated. After analysis of this first collective, the methods were optimized to enhance sensitivity and to reduce interference with human DNA. Then a second collective consisting of 21 metallic implants from 21 patients was investigated from June 2009 to January 2010. In the first collective, 71% of the metallic implants were negative for eubacterial DNA. Pathogens such as Staphylococcus aureus and opportunists such as Lactobacillus rhamnosus were identified in 11% of the samples, whereas the residual 18% positive results were classified as from skin sources or could not be confirmed. Tissue, secretion, and bone samples as well as foam dressings from the same collective also contained pathogens and opportunists. After the optimization of the methods, a considerable increase of positive samples was seen: in the second collective 19 of the 21 metallic implants proved to be positive for eubacterial 16S rDNA. Bacterial DNA from environmental sources was detected in 13 samples, and in 20 specimens, predominantly mostly the skin. Opportunistic pathogens were detected in 19 samples. Interestingly, septic complications did not occur despite the presence of bacterial DNA. The results obtained up to now encourage us not only to continue a directed monitoring of bacterial DNA on orthopedic implants in practice but also to look intensely for possible sources of bacterial contamination during and after insertion or during removal of such implants.

MALDI-ToF mass spectrometry-multivariate data analysis as a tool for classification of reactivation and non-culturable states of bacteria.

Some bacterial life states are only difficult to describe and to detect because they are on the border of active metabolism. A prominent example is the so-called viable but non-culturable state, which is mainly characterized by the inability of bacteria to grow on synthetic media. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF/MS) in combination with multivariate data analysis represents a powerful tool for mass-spectrometric pattern recognition of biological samples. This method is already used for differentiation of bacterial strains. In this study we present a rapid readout method based on MALDI-ToF/MS in combination with principal component analysis to classify the bacterial non-culturable state using Enterococcus faecalis as a model organism. By applying this technique to samples of different physiological states, distinct clusters were calculated and all mass spectra were classified correctly into groups of similar type concerning their physiological state.

Use of quantitative real-time RT-PCR to analyse the expression of some quorum-sensing regulated genes in Pseudomonas aeruginosa.

P. aeruginosa living in biofilm populations sends out diffusive signalling molecules, called autoinducers, for example acylated homoserine lactone (AHL) or the P. aeruginosa quinolone signal (PQS). So far, two quorum-sensing systems, LasR and VsmR, have been identified in P. aeruginosa, both of which are required for all virulence determinants. The expression of specific genes involved in quorum-sensing regulatory mechanisms has been analysed with molecular biology methods. Real-time quantitative PCR is a highly sensitive and powerful technique for quantification of nucleic acids. Expression of the genes vsmR, lasI, and PA4296 was studied by use of reverse transcriptase and subsequent quantitative real-time PCR of the cDNAs. In parallel, expression of ribosomal 16S rRNA, used as a housekeeping gene that was constitutively expressed in all analyses, was also monitored. Biofilm was compared with planktonic bacteria, and in contrast to vsmR and Pa4296, the lasI gene was found to be down-regulated in biofilm. Extended experiments were run with synthetic signal molecules inducing regulated processes in bacterial populations. It was shown that the genes under investigation were up-regulated in mature biofilm in the presence of the signal molecule N-(3-oxododecanoyl)-L-homoserine lactone.

Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration.

Populations of bacteria in biofilms from different sites of a drinking water production system were analysed. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses revealed changing DNA band patterns, suggesting a population shift during bank filtration and processing at the waterworks. In addition, common DNA bands that were attributed to ubiquitous bacteria were found. Biofilms even developed directly after UV disinfection (1-2m distance). Their DNA band patterns only partly agreed with those of the biofilms from the downstream distribution system. Opportunistic pathogenic bacteria in biofilms were analysed using PCR and Southern blot hybridisation (SBH). Surface water appeared to have a direct influence on the composition of biofilms in the drinking water distribution system. In spite of preceding filtration and UV disinfection, opportunistic pathogens such as atypical mycobacteria and Legionella spp. were found in biofilms of drinking water, and Pseudomonas aeruginosa was detected sporadically. Enterococci were not found in any biofilm. Bacterial cell counts in the biofilms from surface water to drinking water dropped significantly, and esterase and alanine-aminopeptidase activity decreased. beta-glucosidase activity was not found in the biofilms. Contrary to the results for planktonic bacteria, inhibitory effects were not observed in biofilms. This suggested an increased tolerance of biofilm bacteria against toxic compounds.