Inguinal microbiome in patients undergoing an endovascular aneurysm repair: Application of next-generation sequencing of the 16S-23S rRNA regions.

BACKGROUND: Surgical site infection (SSI) remains a hazardous complication after vascular surgery. In this pilot study we investigated the inguinal microbiome in skin biopsies using histology and 16S-23S rDNA Next Generation Sequencing (NGS). Our hypothesis was that causative microorganisms of SSI are present in the inguinal microbiome. METHODS: Data on surgical site infections and skin samples from the Percutaneous in Endovascular Repair versus Open (PiERO) trail were evaluated. Two patients with SSI were matched for age and comorbidity to eight matching patients of the PiERO trial. All patients were treated for an abdominal aortic aneurysm with endovascular repair. Nasal and perineal cultures were taken preoperatively to detect Staphylococcus aureus carriage. After disinfection with chlorhexidine, groin biopsies were taken to identify bacteria in deeper skin layers. All samples were subjected to histological analysis and culture-free 16S-23S rDNA NGS. RESULTS: Staphylococcus aureus species were cultured in 5 out of 20 preoperative nasal and perineal swaps. Histology detected only a few bacteria. NGS of the 16S-23S rRNA regions identified DNA of bacterial species in all biopsies (20/20). Most identified genera and species proved to be known skin flora bacteria. No relation was found between SSIs and the preoperative microbiome. CONCLUSION: In this pilot study, an innovative analysis of the preoperative microbiome using 16S-23S rDNA NGS did not show a relation with the occurrence of a surgical site infection. No pathogenic bacterial species were present in the inguinal skin after disinfection with chlorhexidine.

Vibrio vulnificus outbreaks in Dutch eel farms since 1996: strain diversity and impact.

Vibrio vulnificus is a potentially zoonotic bacterial pathogen of fish, which can infect humans (causing necrotic fasciitis). We analysed 24 V. vulnificus isolates (from 23 severe eel disease outbreaks in 8 Dutch eel farms during 1996 to 2009, and 1 clinical strain from an eel farmer) for genetic correlation and zoonotic potential. Strains were typed using biotyping and molecular typing by high-throughput multilocus sequence typing (hiMLST) and REP-PCR (Diversilab®). We identified 19 strains of biotype 1 and 5 of biotype 2 (4 from eels, 1 from the eel farmer), that were subdivided into 8 MLST types (ST) according to the international standard method. This is the first report of V. vulnificus biotype 1 outbreaks in Dutch eel farms. Seven of the 8 STs, of unknown zoonotic potential, were newly identified and were deposited in the MLST database. The REP-PCR and the MLST were highly concordant, indicating that the REP-PCR is a useful alternative for MLST. The strains isolated from the farmer and his eels were ST 112, a known potential zoonotic strain. Antimicrobial resistance to cefoxitin was found in most of the V. vulnificus strains, and an increasing resistance to quinolones, trimethoprim + sulphonamide and tetracycline was found over time in strain ST 140. Virulence testing of isolates from diseased eels is recommended, and medical practitioners should be informed about the potential risk of zoonotic infections by V. vulnificus from eels for the prevention of infection especially among high-risk individuals. Additional use of molecular typing methods such as hiMLST and Diversilab® is recommended for epidemiological purposes during V. vulnificus outbreaks.

Trapped in keratin; a comparison of dermatophyte detection in nail, skin and hair samples directly from clinical samples using culture and real-time PCR.

Traditionally, laboratory detection and identification of dermatophytes consists of culture and microscopy which yields results within approximately 2-6 weeks. In 2007 our medical microbiological diagnostic laboratory implemented a molecular method for the detection of dermatophytes. A real-time PCR assay was developed which simultaneously detects and identifies the most prevalent dermatophytes directly in nail, skin and hair samples and has a turnaround time of less than two days. For 1437 clinical samples, received by our diagnostic laboratory, we compared the results obtained from both culture and real-time PCR. This study showed that real-time PCR significantly increased the detection rate of dermatophytes compared to culture. Furthermore, excellent concordance between culture and real-time PCR identification was achieved.

Evaluation of a shortened QIAsymphony DNA extraction protocol for stool samples using a multiplex real-time PCR for the detection of enteric pathogens.

A shortened DNA extraction protocol for the QIAsymphony SP was evaluated by quantitative and qualitative comparison of real-time PCR results of 150 co-extracted stool samples. The average ∆Cycle threshold value for positive pathogenic targets was 0.28 Ct. A consensus of 96.91%, with a correlation coefficient of 0.9880 was recorded.

Comparison of the QIAsymphony automated nucleic acid extraction and PCR setup platforms with NucliSens easyMAG and Corbett CAS-1200 liquid handling station for the detection of enteric pathogens in fecal samples.

In this study we compared the QIAsymphony Sample Preparation and Assay Setup modules (Qiagen), which provide automated nucleic acid extraction and PCR setup, to the NucliSens easyMAG (bioMérieux) and CAS-1200 liquid handling station (Corbett) for a molecular screening approach of enteric pathogens in fecal samples using multiplex real-time PCR. The relative DNA recovery of both platforms, within- and between-run reproducibility and a prospective study, including 510 clinical fecal samples, were performed. The results demonstrated that the QIAsymphony Sample Preparation and Assay Setup modules were highly reproducible and achieve equal performance, quantitative and qualitative, when compared with the NucliSens easyMAG and CAS-1200 systems for the molecular screening analysis of enteric pathogens in fecal samples.

Prevention of Staphylococcus aureus burn wound colonization by nasal mupirocin.

BACKGROUND: There are two important routes for the transmission of Staphylococcus aureus to the burn wound. In the endogenous route, patients naturally carrying S. aureus colonize their own wounds, whereas in the exogenous route burn wounds are cross-infected from other sources. In this study we evaluated the effect of blocking the endogenous route on S. aureus burn wound colonization by mupirocin application in the nose of patients at the time of admission. METHODS: From September 2000 to January 2002 all patients with burns admitted to a single dedicated Burn Centre received nasal mupirocin upon admission. This period was compared to two control periods (C1: July 1999 to July 2000 and C2: January 2002 to January 2003) for S. aureus burn wound colonization. The colonization risk was analysed, adjusting for confounding, with Cox proportional hazard regression. RESULTS: A total of 98 patients did not have S. aureus burn wound colonization at the time of admission and were, thus, considered at risk for S. aureus acquisition during their stay. As compared to C1, the relative risk of acquiring S. aureus in their wound was 0.48 (95% CI: 0.24-0.97) in the mupirocin period and 0.55 (95% CI: 0.28-1.1) during the C2 period. S. aureus nasal/pharyngeal colonization was a significant independent risk factor for wound colonization (RR: 2.3; 95% CI: 1.2-4.2). CONCLUSION: Nasal mupirocin may contribute to risk reduction of S. aureus wound colonization in patients with burns.

Feasibility of a molecular screening method for detection of Salmonella enterica and Campylobacter jejuni in a routine community-based clinical microbiology laboratory.

Conventional diagnostic methods for the detection of Salmonella enterica and Campylobacter jejuni are laborious and time-consuming procedures, resulting in final results, for the majority of specimens, only after 3 to 4 days. Molecular detection can improve the time to reporting of the final results from several days to the next day. However, molecular assays for the detection of gastrointestinal pathogens directly from stool specimens have not made it into the routine clinical microbiology laboratory. In this study we have assessed the feasibility of a real-time PCR-based molecular screening method (MSM), aimed at S. enterica and C. jejuni, in the daily practice of a routine clinical microbiology laboratory. We have prospectively analyzed 2,067 stool specimens submitted for routine detection of gastrointestinal bacterial pathogens over a 7-month period. The MSM showed 98 to 100% sensitivity but routine culture showed only 77.8 to 86.8% sensitivity when an extended "gold standard" that included all culture-positive and all MSM-positive specimens, as confirmed by an independent secondary PCR of a different target gene, was used. An overall improvement in the rate of detection of both pathogens of 15 to 18% was observed. Both approaches performed nearly identically with regard to the specificity, positive predictive value, and negative predictive value, with the values for MSM being 99.7%, 93.1 to 96.6%, and 99.8 to 100%, respectively, and those for routine culture being 100%, 100%, and 97.6 to 99.5%, respectively. Finally, the final results were reported between 3 and 4 days earlier for negative specimens compared to the time of reporting of the results of routine culture. Positive specimens, on the other hand, required an additional 2 days to obtain a final result compared to the time required for routine culture, although preliminary MSM PCR-positive results were reported, on average, 2.9 to 3.8 days before the final routine culture results were reported. In conclusion, MSM can be incorporated into the daily practice of a routine clinical microbiology laboratory with ease. Furthermore, it provides an improvement in the screening for S. enterica and C. jejuni and substantially improves the time to the reporting of negative results.

Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats. I. Behavioral and biochemical studies.

Bilateral 6-hydroxydopamine (6-OHDA) lesions in the nucleus accumbens of rats induced motor hypoactivity 7 days after the lesion. Spontaneous functional recovery of this impaired behavior occurred in 3-4 weeks. Behavioral and biochemical studies suggest that the hypoactivity is due to damage of the dopamine systems in the nucleus accumbens. The 6-OHDA lesions induced a decrease in the nucleus accumbens levels of dopamine and its metabolites of about 30% both 7 and 20 days after the lesion. The in vitro uptake of [3H]dopamine in nucleus accumbens tissue of the 6-OHDA-lesioned rats was decreased to the same extent at 7, 14 and 28 days after the lesion. Scatchard analysis of [3H]haloperidol binding studies in nucleus accumbens tissue revealed a shift from one type of binding site in tissue of sham-lesioned rats to two types of binding sites in tissue of 6-OHDA-lesioned rats 29 days after the lesion. This shift was not present in nucleus accumbens tissue 8 days after a 6-OHDA lesion. The spontaneously recovered rats showed an enhanced behavioral response upon administration of the dopamine agonist apomorphine. The present data suggest that the spontaneous functional recovery of impaired motor activity is caused by the development of supersensitivity of the dopamine receptor systems in the nucleus accumbens. This supersensitivity may be the result of increased affinity of one type of binding site or an increased number of functional binding sites.

Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats. II. Facilitation by the ACTH-(4-9) analog ORG 2766.

Functional recovery from motor hypoactivity of rats with 6-OHDA lesions in the nucleus accumbens is accelerated by intra-accumbal or subcutaneous treatment with the ACTH-(4-9) analog ORG 2766. The spontaneous recovery period of 3 weeks is shortened to 7 days by daily treatment with this peptide during the first 6 days after the lesion. The 6-OHDA lesion induced a decrease of about 30-40% in the levels of dopamine, HVA and DOPAC as well as in the uptake of [3H]dopamine in nucleus accumbens tissue in vitro. Treatment with ORG 2766 during the first 6 days following the lesion did not affect the lesion-induced changes in these biochemical parameters. Binding studies with [3H]haloperidol in nucleus accumbens tissue of placebo or ORG 2766-treated sham-lesioned rats revealed a linear Scatchard plot 7 days after the sham lesion. In tissue of placebo-treated 6-OHDA lesioned animals a similar linear Scatchard plot was found but in tissue of ORG 2766-treated 6-OHDA-lesioned rats the Scatchard plot was curvilinear in shape indicating two types of binding sites. In the 6-OHDA-lesioned rats treated with ORG 2766 the behavioral response upon apomorphine challenge was enhanced suggesting the existence of functional supersensitivity of the DA system. Similar changes in Scatchard plots and apomorphine-induced behavioral changes have been previously reported after spontaneous recovery. The present study indicates that ORG 2766 accelerates the process of functional recovery from impaired motor behavior of rats with 6-OHDA lesions in the nucleus accumbens, which may be due to development of denervation supersensitivity.

Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats. IV. Delay by intra-accumbal treatment with ORG 2766- or alpha-MSH antiserum.

Rats with 6-OHDA lesions in the nucleus accumbens which were treated intra-accumbally with control serum during the first week following the lesion showed a similar level of motor activity 3 weeks after the lesion as sham-lesioned rats treated with control serum. In 6-OHDA-lesioned rats that were identically treated with antiserum against alpha-MSH or the ACTH-(4-9) analog ORG 2766 motor activity was decreased 3 weeks after the lesion. Intra-accumbal treatment with the antisera did not affect motor activity of sham-lesioned rats. The increased motor activity after apomorphine injection into the nucleus accumbens of control serum-treated 6-OHDA-lesioned rats was not observed in 6-OHDA-lesioned rats treated with the antisera. Furthermore, [3H]haloperidol binding studies showed that the changes in the DA receptor systems in the nucleus accumbens of 6-OHDA-lesioned rats treated with control serum, which may reflect denervation supersensitivity, were not observed in 6-OHDA-lesioned rats treated with the antisera. The present data indicate that the functional recovery and the concurrent development of supersensitive DA receptor systems in the nucleus accumbens of 6-OHDA-lesioned rats are delayed by intra-accumbal treatment with ORG 2766 or alpha-MSH antiserum. This suggests that endogenous ACTH/MSH-like factors may be mediating the recovery processes.

Perception of Indonesian vowels spoken in context.

The purpose of the present study is to test the perceptual reality of allophonic variation and harmony in Indonesian. Indonesian listeners with different regional backgrounds were asked to predict word identity (CVCV or CVCVC) based on the presence or absence of vowel allophony and harmony. The data show that listeners were sensitive to the spectrally different allophones, but were not in all cases able to use this information in predicting word identity, probably due to experimental artifact.

A "microtubule" in a bacterium.

A study of the anchorage of the flagella in swarmers of Proteus mirabilis led to the incidental observation of microtubules. These microtubules were found in thin sections and in whole mount preparations of cells from which most of the content had been released by osmotic shock before staining negatively with potassium phosphotungstate (PTA). The microtubules are in negatively stained preparations about 200 A wide, i.e. somewhat thicker than the flagella (approximately 130 A). They are thus somewhat thinner than most microtubules recorded for other cells. They are referred to as microtubules because of their smooth cylindrical wall, or cortex, surrounding a hollow core which is readily filled with PTA when stained negatively. Since this is probably the first time that such a structure is described inside a bacterium, we do not know for certain whether it represents a normal cell constituent or an abnormality, for instance of the type of "polysheaths" (16).

Basal bodies of bacterial flagella in Proteus mirabilis. II. Electron microscopy of negatively stained material.

This paper investigates further the question of whether the flagella of Proteus mirabilis emerge from basal bodies. The bacteria were grown to the stage of swarmer differentiation, treated lightly with penicillin, and then shocked osmotically. As a result of this treatment, much of the cytoplasmic content and also part of the plasma membrane were removed from the cells. When such fragmented organisms were stained negatively with potassium phosphotungstate, the flagella were found to be anchored-often by means of a hook-in rounded structures approximately 50 mmicro wide, thus confirming Part I of our study. In these rounded structures a more brilliant dot was occasionally observed, which we interpret as being part of the basal granule. A prerequisite for the demonstration of the basal granules within the cells was, however, the removal of both the cytoplasm and the plasma membrane from their vicinity. In some experiments, the chondrioids were "stained" positively by the incorporation into them of the reduced product of potassium tellurite. The chondrioids were here observed to be more or less circular areas from which rodlike structures extended. The chondrioids adhered so firmly to the plasma membrane that they were carried away with it during its displacement by osmotic shocking, while the basal bodies were left behind. This observation disproves our previous suggestion that the flagella might terminate in the chondrioids. The basal bodies often occur in pairs, which suggest that they could be self-reproducing particles.

Basal bodies of bacterial flagella in Proteus mirabilis. I. Electron microscopy of sectioned material.

Years ago (16, 18, 19), in a study of shadowed preparations of Proteus vulgaris that had been autolyzed in the cold, the observation was made that the flagella arose from basal bodies. However, recently (3, 7, 24, 33) doubt has been cast on the conclusion that the flagella of bacteria emerge from sizable basal bodies. This problem has, therefore, been reinvestigated with actively developing cultures of Proteus mirabilis, the cell walls of which had been expanded slightly by exposure to penicillin. Two techniques were applied: ultramicrotomy, and negative staining of whole mount preparations. This paper deals with the thin sections of bacteria after the usual fixation technique had been altered slightly: the cells were embedded in agar prior to their fixation and further processing. The flagella then remained attached to the cells and were seen to extend between the cell wall and the plasma membrane. Occasionally, the flagella appeared to be anchored in the cell by means of a hook-shaped ending. In sections of cells rich in cytoplasm, the basal bodies are particularly difficult to visualize due to their small size (25 to 45 mmicro) and the lack of properties that would enable one to distinguish them from the ribonucleoprotein structures; in addition, their boundary appears to be delicate. However, when the cytoplasm is sparse in the cells, either naturally or as a result of osmotic shocking in distilled water, the flagella can be observed to emerge from rounded structures approximately 25 to 45 mmicro wide. Contrary to a previous suggestion (21), the flagella do not terminate in the peripheral sites of reduced tellurite, i.e. the chondrioids. The observations in this part of the study agree with those described in the following paper (15) dealing with negatively stained preparations.