The oral microbiota is a reservoir for antimicrobial resistance: resistome and phenotypic resistance characteristics of oral biofilm in health, caries, and periodontitis.

BACKGROUND: Antimicrobial resistance (AMR) is an ever-growing threat to modern medicine and, according to the latest reports, it causes nearly twice as many deaths globally as AIDS or malaria. Elucidating reservoirs and dissemination routes of antimicrobial resistance genes (ARGs) are essential in fighting AMR. Human commensals represent an important reservoir, which is underexplored for the oral microbiota. Here, we set out to investigate the resistome and phenotypic resistance of oral biofilm microbiota from 179 orally healthy (H), caries active (C), and periodontally diseased (P) individuals (TRN: DRKS00013119, Registration date: 22.10.2022). The samples were analysed using shotgun metagenomic sequencing combined, for the first time, with culture technique. A selection of 997 isolates was tested for resistance to relevant antibiotics. RESULTS: The shotgun metagenomics sequencing resulted in 2,069,295,923 reads classified into 4856 species-level OTUs. PERMANOVA analysis of beta-diversity revealed significant differences between the groups regarding their microbiota composition and their ARG profile. The samples were clustered into three ecotypes based on their microbial composition. The bacterial composition of H and C samples greatly overlapped and was based on ecotypes 1 and 2 whereas ecotype 3 was only detected in periodontitis. We found 64 ARGs conveying resistance to 36 antibiotics, particularly to tetracycline, macrolide-lincosamide-streptogramin, and beta-lactam antibiotics, and a correspondingly high prevalence of phenotypic resistance. Based on the microbiota composition, these ARGs cluster in different resistotypes, and a higher prevalence is found in healthy and caries active than in periodontally diseased individuals. There was a significant association between the resistotypes and the ecotypes. Although numerous associations were found between specific antibiotic resistance and bacterial taxa, only a few taxa showed matching associations with both genotypic and phenotypic analyses. CONCLUSIONS: Our findings show the importance of the oral microbiota from different niches within the oral cavity as a reservoir for antibiotic resistance. Additionally, the present study showed the need for using more than one method to reveal antibiotic resistance within the total oral biofilm, as a clear mismatch between the shotgun metagenomics method and the phenotypic resistance characterization was shown.

Prevalence of Candida albicans and Candida dubliniensis in caries-free and caries-active children in relation to the oral microbiota-a clinical study.

OBJECTIVES: The correlation between caries and the oral prevalence of Candida spp. in children is contradictory in literature. Thereby, authors focused on Candida albicans as the most isolated Candida species from the oral cavity. Therefore, the aim of the present study was to compare caries-free and caries-bearing children regarding their oral carriage of Candida spp. MATERIAL AND METHODS: Twenty-six caries-free (CF group) and 26 caries-active children (CA group) were included into this study. Three different types of specimens were assessed, saliva and plaque, and in the case of caries, infected dentine samples were microbiologically analyzed for aerobic and anaerobic microorganisms and their counts. Special attention was given to the differentiation between C. albicans and Candida dubliniensis. Additionally, different biochemical tests, VITEK 2 (VITEK®2, bioMérieux, Marcy-l'Etoile, France) and 16S and 18S ribosomal DNA (rDNA) sequencing, were applied for identification. RESULTS: The detection of C. albicans did not differ between the CF and CA groups. C. dubliniensis was never detected in any specimen of the CF group, but occurred in one quarter of the CA group (27 % in plaque, 23 % in saliva), thus leading to a statistically significant difference between the two groups (p < 0.05). In six of these cases, C. dubliniensis was detected concomitantly in saliva and plaque and once only in plaque. CA group harbored statistically more Streptococcus mutans than the control group revealing a correlation between S. mutans and C. dubliniensis regarding the caries group. CONCLUSIONS: This is the first study reporting a frequent detection of C. dubliniensis in caries-active children, which could have been underestimated so far due to difficulties in differentiation between this yeast species and C. albicans. CLINICAL RELEVANCE: Microbiological diagnostic-especially of oral Candida species-is an important determinant for identifying etiological factors of dental caries in children.

Bacterial colonization on different suture materials--a potential risk for intraoral dentoalveolar surgery.

In this in vivo and in vitro study on resorbable (Monocryl and nonresorbable (Deknalon) monofilament sutures used in intraoral dentoalveolar surgery the bacterial colonization was compared. For the in vivo study the sutures were applied in 11 patients during dental surgery. Eight days postoperative the sutures were removed and the adhered bacteria were isolated and identified by biochemistry, morphology, antibiotic susceptibility, and gas chromatography. The colonization was studied by scanning electron microscopy. Aerobic and anaerobic bacteria were isolated in nearly equal colony-forming units (cfu) on each suture. In comparison with Monocryl about 15% more aerobic and anaerobic strains were isolated on Deknalon. Regarding the pathogens only, about three times more anaerobic strains were isolated on both sutures in total. Additionally, more pathogens were found on Deknalon than on Monocryl (aerobic >40%, anaerobic >25%). The variety of bacteria correspond with purulent infections, not with normal oral flora. Intraindividual comparisons of cfu showed differences in dependence of the patient as described for subgingivale plaques. For the in vitro study the sutures were incubated with Streptococcus intermedius and Prevotella intermedia for 0.5 h. Scanning electron microscopy was performed to examine qualitatively the level of bacterial adherence. After 0.5 h the bacteria adhered very well. The colonization rate of Streptococcus intermedius on both sutures was similar. Coccoid bacteria within biofilms were seen. The growth of Prevotella intermedia was much better on Deknalon than on Monocryl. The risk of bacteremia at the time of suture removal is discussed.

Photic entrainment of circannual rhythms in golden-mantled ground squirrels: role of the pineal gland.

Entrainment of circannual rhythms of body mass and reproduction was monitored for 3 years in female golden-mantled ground squirrels maintained in a simulated natural photoperiod. Both pinealectomized and pineal-intact squirrels generated circannual rhythms of body mass and estrus, but only the intact animals entrained these rhythms to a period of 365 days. In the second and third years after treatment, the period of the body mass rhythm was significantly shorter than 365 days for pinealectomized squirrels, and variance in tau among these animals was significantly greater than for intact squirrels. A similar pattern was evident in the rhythm of reproduction, which was phase-disrupted in pinealectomized squirrels but entrained in intacts. Seasonal changes in duration of nocturnal melatonin secretion by the pineal appear to be necessary to produce phase-delays required to entrain the circannual clock to a period of 12 months.

Intraperitoneal and intravenous cefoperazone kinetics during continuous ambulatory peritoneal dialysis.

Serum cefoperazone (CFP) kinetics after a 1-gm dose added to the peritoneal dialysate were followed in seven patients undergoing continuous ambulatory peritoneal dialysis (CAPD). In a randomized order five of the seven patients received 1 gm IV CFP. Serum samples were collected over 10 hr during one dialysate exchange interval. CFP concentrations were determined by HPLC. After intravenous dosing CFP mean peak and 6-hr serum concentrations were 104.2 +/- 29.1 micrograms X ml-1 and 8.5 +/- 3.8 micrograms X ml-1, mean body clearance was 80 +/- 20 ml X min-1, and mean apparent volume of distribution was 14.6 +/- 3.2 l. The elimination rate constant (kel) varied from 0.29 to 0.38 hr-1 and was almost identical to kel derived from intraperitoneal application (range 0.29 to 0.42 hr-1). Instillation of CFP with the peritoneal dialysate resulted in a rapid rise of serum levels (Tmax = 1.9 +/- 0.7 hr; absorption rate constant ka = 0.68 +/- 0.11 hr-1), and sufficient CFP concentrations (Cmax = 33.2 +/- 5.3 micrograms X ml-1), were maintained over 6 hr (C6 hr = 17.3 +/- 5.8 micrograms X ml-1). Mean systemic availability of intraperitoneal CFP was 95% +/- 12%. Intraperitoneal administration of CFP in patients undergoing CAPD resulted in serum levels of CFP adequate for systemic treatment of bacterial infections.

Routine identification of Campylobacter jejuni and Campylobacter coli from human stool samples.

Correct identification of Campylobacter jejuni and Campylobacter coli isolates to the species or subspecies level is a cumbersome but nevertheless important task for a routine diagnostic laboratory. The widely used biochemical tests might be often misleading while more sophisticated phenotypic or genotypic methods are not generally available. This investigation was performed to assess the performance of common biochemical identification in comparison with species-specific PCR and gas liquid chromatography of whole cell fatty acid extracts (GLC). A total of 150 consecutive isolates from human stool samples were investigated (134 C. jejuni ssp. jejuni, 14 C. coli, two Helicobacter pullorum). From these 144, 145 and 149 isolates were correctly identified by biochemistry, GLC and PCR, respectively. Biochemical identification of all C. jejuni isolates was confirmed by PCR. GLC detected both H. pullorum strains but misidentified two C. coli strains as C. jejuni and one C. jejuni strain as C. coli. No single method can be defined as 'gold standard' for identification of C. jejuni and C. coli but a combination of techniques is needed. Therefore a stepwise identification scheme starting with biochemical reactions is suggested. All results other than C. jejuni should be confirmed by further methods. For indoxyl acetate-positive isolates species-specific PCR is recommended while GLC seems to be advantageous in indoxyl acetate-negative isolates.

Discrimination of Helicobacter pullorum and Campylobacter lari by analysis of whole cell fatty acid extracts.

Helicobacter pullorum and Campylobacter lari are rarely isolated from humans with acute enteritis. Hitherto the two species could only be identified by genotypic techniques. Gas liquid chromatography of whole cell fatty acid extracts is described as the first phenotypic method for discrimination of the two species. Cholesteryl glucoside, a characteristic feature of the genus Helicobacter, but seldom found in other bacteria, could not be detected in Helicobacter pullorum. Therefore, rapid determination of this glycolipid may serve as a discrimination marker for Helicobacter pullorum from most other Helicobacter species.

Surveillance of nosocomial infections in a neurology intensive care unit.

To identify overall and site-specific nosocomial infection (NI) rates in patients receiving neurological intensive care therapy, a prospective study was started in 1997 in the ten-bed neurological intensive-care unit (NICU) of the University Hospital of Freiburg, Germany. Case records and microbiology reports were reviewed twice a week, and ward staff were consulted. NI were defined according to the Center for Disease Control and Prevention (CDC) criteria and were categorised by specific infection site. Within 30 months, 505 patients with a total of 4,873 patient days were studied (mean length of stay: 9.6 days). 122 NI were identified in 96 patients (74 patients with one, 18 with two and 4 with three infections. An incidence of 24.2/100 patients and incidence density of 25.0/1,000 patient days of NI in the neurological ICU were documented. Site-specific incidence rates and incidence densities were: 1.4 bloodstream infections per 100 patients (1.9 central line-associated BSIs per 1,000 central line-days), 11.7 pneumonias per 100 patients (20.4 ventilator-associated pneumonias per 1,000 ventilator-days), 8.7 urinary tract infections per 100 patients (10.0 urinary catheter-associated urinary track infections (UTIs) per 1,000 urinary catheter-days). Additionally, 0.4 cases of meningitis, 0.8 ventriculitis, and 1.2 other infections (catheter-related local infection, diarrhea) were documented per 1,000 patient days. 15% of nosocomial pathogens were A. baumannii (due to a outbreak of an nosocomial pneumonia with A. baumannii), 13% S. aureus, 10% E. coli, 7% CNS,7% Bacteroides spp., 7% Enterobacter spp., 6,5% Klebsiella spp.,5.9% enterococci, 5.9% streptococci, and 4.7% Pseudomonas spp. In eight cases of NI no pathogen could be isolated. In future, data on NI in NICUs should be assessed in greater detail, both to improve the quality of care and serve as a basis for identification and implementation of the most effective measures by which to prevent these infections in patients receiving intensive neurological care.

Fluorescence in situ hybridization for direct visualization of gram-negative anaerobes in subgingival plaque samples.

Fluorescent oligonucleotide probes complementary to variable regions of Porphyromonas gingivalis and Bacteroides forsythus 16S ribosomal RNA were used to identify these organisms in smears of formaldehyde-fixed subgingival plaque samples from patients suffering from periodontitis. Fluorescence in situ hybridization represents a useful method for assessing the microbial ecology of the periodontal flora.

Is the reuse of needles for insulin injection systems associated with a higher risk of cutaneous complications?

Twenty diabetic patients participated in a study to assess if multiple use of needles for insulin injection systems (Pens) is safe under normal daily conditions. The previous mean duration of Pen therapy was 16.3 months. During this time, the 20 patients carried out altogether more than 33,000 injections without any sign of local infection despite needle reuse. Patients were told to use needles if possible for 1, 3, 6, 9, and 12 injections before bacteriological assessment. Bacteriological investigation of these needles showed no contamination, except with one needle used three times, which was colonized with coagulase negative Staphylococcus. In contrast, half of the needles' plastic ground points which touched the skin were contaminated. No signs of infection were observed at the injection sites throughout the study. We conclude that, based on the bacteriostatic effects of commercially formulated insulin and on the siliconisation of needles' surfaces, bacterial growth is sufficiently prevented. Therefore, we can recommend the reuse of pen needles as a simple, safe and cost-beneficial procedure.

Inflammatory consequences of the translocation of bacteria and endotoxin to mesenteric lymph nodes.

BACKGROUND: Translocation of intestinal bacteria to mesenteric lymph nodes (MLNs) has been documented in humans under a variety of circumstances, yet its clinical significance remains to be established. The aim of this study was to correlate detectable translocation to MLNs of bacteria and endotoxin with local and systemic signs of inflammation. METHODS: From each of 10 patients with carcinoma of the cecal region two MLNs were harvested prior to resection. The presence of bacteria and endotoxin in the lymphatic tissue and blood was determined by culture methods and DNA preparation (PCR) and by a Limulus assay, respectively. Inflammatory mediators were determined in plasma and in MLN homogenates. RESULTS: Viable bacteria were detected in MLNs of 7 patients and in 9 of 20 lymph nodes. PCR revealed traces of bacteria in 4 patients and in 6 of their MLNs. Combining both modalities, the translocation rate was 80% and 55% for patients and MLNs, respectively. There was no detectable bacteremia. Endotoxin was found in the plasma of 7 patients and in 9 MLNs from 5 patients. There was no correlation between culture findings and endotoxin concentrations. Moreover, bacteriological data did not correspond to local or systemic inflammation. The group of MLN with detectable endotoxin differed significantly from LPS-negative nodes with respect to interleukin-6, interleukin-10, and sCD14. Systemic concentrations of endotoxin and inflammatory parameters did not correspond to levels within MLNs. CONCLUSION: Translocation to MLNs occurs in patients with cecal carcinoma. This, however, seems not to be of major clinical significance if no additional physiologic insults are encountered. Irrespective of the presence of bacteria, there are variations in inflammatory reactions between lymph nodes from one and the same patient, probably reflecting fluctuating response mechanisms to low-grade translocation.

Antibacterial properties of electron beam-sterilized Gutta-Percha cones.

The purpose of the study was to determine the effect of electron beam sterilization on gutta-percha cones (GPCs) at different times after sterilization. An agar diffusion test was used with -one aerobic bacterium (Bacillus subtilis) and five oral anaerobic bacteria (Fusobacterium nucleatum, Peptostreptococcus micros, Porphyromonas gingivalis, Propionibacterium acnes, and Veillonella parvula). With each microorganism 30 agar plates were prepared, evenly distributed among three groups (group 1: unsterilized GPCs; groups 2 and 3: GPCs sterilized by electron beam irradiation 5 months and 5 yr before, respectively). One GPC of the selected group was placed in each plate. After incubation the area of inhibition was read on the agar plates. Inhibition of growth was significantly different for the tested microorganisms. However no significant difference was observed between the sterilized and unsterilized GPCs. Both the tested sterilized and unsterilized GPCs impair the growth of endodontic pathogens, with no influence of the time elapsed since sterilization.

Pharmacokinetics and bactericidal activity of a single daily dose of netilmicin in the treatment of CAPD-associated peritonitis.

Single daily dosage of netilmicin is generally accepted in systemic infections, due to biphasic bactericidal activity and prolonged postantibiotic effect of aminoglycosides. Since little is known about the efficacy of single daily intraperitoneal application of netilmicin in the treatment of CAPD-associated peritonitis, we conducted this prospective study. Seven patients with CAPD-associated peritonitis were treated with a single daily dose of netilmicin (loading dose 1.5 mg/kg, followed by 40 mg/21 bag/day). Serum and intraperitoneal levels as well as bactericidal activity of netilmicin against Acinetobacter baumanii, E. coli and Pseudomonas aeruginosa were measured for 48 hours. Serum and peritoneal levels widely varied among the patients due to different interindividual plasma clearance of netilmicin. The intraperitoneal antibacterial action of netilmicin was decreased, more over, substantial differences in the bactericidal activity were found among the patients. However, with high initial netilmicin levels sufficient bactericidal activity was found for Acinetobacter and E. coli, but not for Pseudomonas aeruginosa. Hence, a single daily dosage of netilmicin can be a suitable treatment of CAPD-associated peritonitis, only if the dose is adapted according to the first serum and peritoneal levels. In infections with Pseudomonas aeruginosa higher peritoneal levels of netilmicin and the combination with other antibiotics will be needed for a sufficient peritoneal bactericidal activity.

[Concentration of antibiotics in long term embolized kidneys (author's transl)]. / Experimentelle Untersuchungen zur Antibiotikakonzentration in embolisierten Nieren.

In 7 dogs renal arterial embolization was performed using Seldinger technique and inflatable Swan Ganz balloon catheter by injection of methyl methacrylate/butyl 2 cyano acrylat. Six months later tissue concentration of cefazolin was measured in the embolized and kontralateral kidneys and in serum two hours after a short infusion of 1 g of Cefazolin. The concentrations in serum and homogenized kidney tissue were determined by means of the agar well diffusion method. Angiography studies showed no vessels in the embolized kidneys. Histologically there was total atrophy of the tissue especially the tubular system. The mean tissue level of Cefazolin in the embolized kidney was 70% of serum. The tissue concentration was always above 10 microgram/g. This concentration is high enough to inhibit nearly all strains of E. coli, Klebsiella, Salmonella, Shigella and a large part of Proteus mirabilis, whereas nearly all gram-positive bacteria are inhibit at a lower concentration. Value and necessity of antibiotic therapy in embolized kidney cancer is discussed.

Effect of different crown contours on periodontal health in dogs. Microbiological results.

OBJECTIVES: In some clinical circumstances, i.e. in cases when the upper anterior region has to be restored by prosthetic means, it is necessary to place the margins of crowns and fixed partial dentures subgingivally. In addition, in periodontally compromised patients the restoration sometimes has to be overcontoured in order to replace the lost interdental papilla. The overcontoured crown margin may influence the subgingival bacterial composition. Therefore, the aim of the present investigation was to evaluate the effect of three different subgingival crown contours in dogs on the composition of the subgingival microbiota. METHODS: In four adult beagle dogs the second and third premolars were prepared in three quadrants and restored with single gold crowns. The unprepared second and third premolars in the last quadrant served as controls. The crowns had three different emergence profiles including a normal contour, a 30 degrees and a 50 degrees over-contour. During the entire study period, professional oral hygiene was performed seven times a week. Microbiological samples were harvested from four sites of test and control teeth (mesial, distal, buccal and lingual) at baseline, after 3 months, and after 5 months. RESULTS: The microbiological analysis (DNA-DNA hybridization technique) of the subgingival microbial flora revealed a dominance of P. intermedia, T. denticola and C. showae in all test and control groups at baseline. At three months, the total amount of bacteria increased and a broader variety of bacterial species could be detected. The detection frequency of most bacterial species increased from baseline to the 5-month evaluation. The frequency of detection of some species was higher in the 30 degrees and 50 degrees overcontoured test groups compared to the normal contour group and to the natural teeth. CONCLUSIONS: It can be concluded within the limits of this investigation that overcontoured gold crowns placed subgingivally have only slight effects on the microbiological composition in dogs when an intensive oral hygiene regimen was executed.

[Endophthalmitis: improvement of culture results]. / Endophthalmitis.Verbesserung des Keimnachweises.

BACKGROUND: Infectious endophthalmitis is a rare, but serious complication. Identification of the responsible microorganism is important for prognosis and therapy. In the literature positive cultures have been reported in 35-85% of cases. The purpose of our work was to improve the proportion of positive culture results. METHODS AND PATIENTS: All necessary materials (culture media: Columbia, Hämatin, ENDO and yeast-cystein-blood agar, fungus media) and instructions were always available in the operation theater. Thus, intraocularly derived samples could be cultured by less experienced residents right in the operation theater, especially off duty. At night the culture plates were incubated in the Department of Ophthalmology under optimal conditions and passed on to an experienced microbiologist later. Culture results of the first 21 patients suffering from endophthalmitis (acute or chronic postoperative, endogen) after introduction of this "endophthalmitis set" were compared to the results of the 21 patients before. RESULTS: As a result of the introduction of the "endophthalmitis set", the proportion of positive culture results improved from 11/21 to 18/21. The detected microorganisms were all representative of the pathogens commonly causing endophthalmitis (gram-positive bacteria, anaerobic bacteria). CONCLUSION: An appropriate culture set that is always available in the operation theater may increase the number of positive cultures dramatically and hence help to find the adequate antibiotic treatment in endophthalmitis cases.

Development of nosocomial and iatrogenic urinary tract infections (UTI) following urological interventions. A prospective clinical study.

Development of nosocomial and iatrogenic UTI was investigated over 3 months period of time. Included were 140 patients following 228 instrumentations or endoscopical or surgical interventions. Of these 84 were carried out under antimicrobial medication. 30 UTIs developed 1-8 days after the various procedures (= 13.2%): 10% after TUR-B, 14% after TUR-P and ureteral instrumentation, 16% following cystoscopy and catheterisation. No significant difference was observed between: Males and females, patients with or without antimicrobial medication, younger and older patients and after long or short procedures. Indwelling catheters remain sterile for 3 days. Though antimicrobial prophylaxis did not significantly influence the generally low rate of infection it appeared as a trend that the elderly patient who requires repeated interventions and who suffers from additional diseases as diabetes, consuming tumours and renal insufficiency may benefit from medication.

Airborne microbes in different dental environments in comparison to a public area.

OBJECTIVE: The aim of this study was to investigate quantitatively and qualitatively the airborne microbial load in a multi-chair dental clinic, a normal dental practice and a non-dental public area over a time period of four days and at different time points to estimate the risk of infections during dental surgery. METHODS: A multi-chair and a single chair treatment room each were examined in comparison to a non-medical public area over a period of four days. The colony forming units m(-3) (CFUs) were determined and isolated bacteria were characterised by morphological and biochemical analysis, gas chromatography and by 16S rRNA-gene sequencing. In the analyses enterococci were selectively searched for. RESULTS: The CFUs in the multi-chair treatment room were between 20 and 1050 CFU m(-3). During treatment the maxima reached were below 800 CFU m(-3). The values in the dental practice were between 200 and 600 CFU m(-3) and remain slightly but not significantly below the levels of the clinic (p > 0.05). In the common area, the CFUs were between 200 and 800 CFU m(-3). The proportion of micrococci was 56.8% in the clinic, 56.07% in the practice and 69.67% in the public area Coagulase-negative staphylococci constituted 35% at the dental clinic, 25% at the bank and 38% at the dental practice. No significant differences amongst the units were detected in the microbial composition of their dental aerosols (p > 0.05). CONCLUSION: Although, the bacterial counts in dental room were not significantly higher than the bacterial counts in a public area, the risk from dental clinic might be higher than a public area due to the type of micro-organisms, host susceptibility and the exposure time.