The tva(A) Gene from Brachyspira hyodysenteriae Confers Decreased Susceptibility to Pleuromutilins and Streptogramin A in Escherichia coli.

The tva(A) gene suspected to confer resistance to pleuromutilins in Brachyspira hyodysenteriae was tested for functionality in Escherichia coli AG100A and Staphylococcus aureus RN4220. Expression of the cloned tva(A) gene conferred decreased susceptibility to pleuromutilin (P) and streptogramin A (SA) antibiotics in E. coli and had a minor effect in S. aureus The finding provides evidence of the direct association of tva(A) with the PSA resistance phenotype.

Extended-spectrum ß-lactamase-producing Enterobacterales in diverse foodstuffs: a prospective, longitudinal study in the city of Basel, Switzerland.

Background: The involvement of non-human-to-human transmission of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) remains elusive. Foodstuffs may serve as reservoirs for ESBL-PE and contribute to their spread. Aim: We aimed to systematically investigate the presence and spatiotemporal distribution of ESBL-PE in diverse unprocessed foodstuffs of different origin purchased in a central European city. Methods: Chicken and green (herbs, salad, sprouts, vegetables) samples were collected monthly for two consecutive years, from June 2017 to June 2019, from large supermarket chains and small local food retailers, representing all ten postcode areas of the City of Basel (Switzerland), and the kitchen of the University Hospital Basel (Basel, Switzerland). After enrichment, presumptive ESBL-PE were isolated by selective culture methods and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. ESBL production was confirmed by phenotypic testing. Results: Among 947 food samples, 14.8% were positive for ESBL-PE isolate/s belonging to eight different ESBL-producing bacterial species. Escherichia coli and Serratia fonticola were predominant across samples (9 and 2%, respectively). Higher ESBL-PE prevalence was observed in chicken (25.9%) than in green (3.8%) samples (p < 0.001). Among greens, ESBL-PE were most frequently isolated from sprouts (15.2%). High ESBL-PE species diversity was observed among chicken samples, with E. coli as predominant (17.6%). ESBL-producing Enterobacter cloacae was detected among different greens. Yet, ESBL-producing Klebsiella pneumoniae was predominant in sprouts (12.1%). In total, 20.5% of samples from organic farming and 14.2% of samples from conventionally raised animals harbored an ESBL-producing isolate. Detection of ESBL-PE across samples differed between organic and non-organic when stratified by food source (p < 0.001), particularly among greens (12.5% organic, 2.4% conventional). High proportion of organic chicken samples was positive for ESBL-E. coli (33.3%), while the detection of several species characterized the conventional chicken samples. No significant differences in ESBL-PE frequences were detected between national (13.4%) and international samples (8.0%) (p = 0.122). Instead, differences were observed between regions of food production and countries (p < 0.001). No significant differences were found when comparing the proportion of ESBL-PE positive samples across districts, shop sizes and the hospital kitchen. The percentage of ESBL-PE positive samples did not differ monthly across the two-year sampling period (p = 0.107). Conclusion: Our findings indicate moderate dissemination of ESBL-PE in foodstuffs, especially between chicken products and sprouts. Chicken meat represents a source for several ESBL-producing Enterobacterales, especially E. coli, while greens are more prone to carry ESBL-K. pneumoniae and E. cloacae. We disclose the importance of food type, food production system and production origin when assessing the risk of contamination with different ESBL-PE species.

Within-host genetic diversity of extended-spectrum beta-lactamase-producing Enterobacterales in long-term colonized patients.

Despite recognition of the immediate impact of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) on human health, essential aspects of their molecular epidemiology remain under-investigated. This includes knowledge on the potential of a particular strain to persist in a host, mutational events during colonization, and the genetic diversity in individual patients over time. To investigate long-term genetic diversity of colonizing and infecting ESBL-Klebsiella pneumoniae species complex and ESBL-Escherichia coli in individual patients over time, we performed a ten-year longitudinal retrospective study and extracted clinical and microbiological data from electronic health records. In this investigation, 76 ESBL-K. pneumoniae species complex and 284 ESBL-E. coli isolates were recovered from 19 and 61 patients. Strain persistence was detected in all patients colonized with ESBL-K. pneumoniae species complex, and 83.6% of patients colonized with ESBL-E. coli. We frequently observed isolates of the same strain recovered from different body sites associated with either colonization or infection. Antimicrobial resistance genes, plasmid replicons, and whole ESBL-plasmids were shared between isolates regardless of chromosomal relatedness. Our study suggests that patients colonized with ESBL-producers may act as durable reservoirs for ongoing transmission of ESBLs, and that they are at prolonged risk of recurrent infection with colonizing strains.

Spatiotemporal dissemination of ESBL-producing Enterobacterales in municipal sewer systems: a prospective, longitudinal study in the city of Basel, Switzerland.

Background: The contribution of community and hospital sources to the transmission of extended-spectrum ß-lactamase producing Enterobacterales (ESBL-PE) remains elusive. Aim: To investigate the extent of community dissemination and the contribution of hospitals to the spread of ESBL-PE by exploring their spatiotemporal distribution in municipal wastewater of the central European city of Basel. Methods: Wastewater samples were collected monthly for two consecutive years throughout Basel, Switzerland, including 21 sites across 10 postcode areas of the city collecting either community wastewater (urban sites, n = 17) or community and hospital wastewater (mixed sites, n = 4). Presumptive ESBL-PE were recovered by selective culture methods. Standard methodologies were applied for species identification, ESBL-confirmation, and quantification. Results: Ninety-five percent (477/504) of samples were positive for ESBL-PE. Among these isolates, Escherichia coli (85%, 1,140/1,334) and Klebsiella pneumoniae (11%, 153/1,334) were most common. They were recovered throughout the sampling period from all postcodes, with E. coli consistently predominating. The proportion of K. pneumoniae isolates was higher in wastewater samples from mixed sites as compared to samples from urban sites, while the proportion of E. coli was higher in samples from urban sites (p = 0.003). Higher numbers of colony forming units (CFUs) were recovered from mixed as compared to urban sites (median 3.2 × 102 vs. 1.6 × 102 CFU/mL). E. coli-counts showed moderate correlation with population size (rho = 0.44), while this correlation was weak for other ESBL-PE (rho = 0.21). Conclusion: ESBL-PE are widely spread in municipal wastewater supporting that community sources are important reservoirs entertaining the spread of ESBL-PE. Hospital-influenced abundance of ESBL-PE appears to be species dependent.

Complete Circular Genome Sequences of Brachyspira hyodysenteriae Isolates of the Four Different Sequence Types Causing Swine Dysentery in Switzerland.

The complete genomes of four Brachyspira hyodysenteriae isolates of the four different sequence types (STs) (ST6, ST66, ST196, and ST197) causing swine dysentery in Switzerland were generated by whole-genome sequencing and de novo hybrid assembly of reads obtained from second (Illumina) and third (Oxford Nanopore Technologies and Pacific Biosciences) generation high-throughput sequencing.

Enhancing the production of hydroxyl radicals by Pleurotus eryngii via quinone redox cycling for pollutant removal.

The induction of hydroxyl radical (OH) production via quinone redox cycling in white-rot fungi was investigated to improve pollutant degradation. In particular, we examined the influence of 4-methoxybenzaldehyde (anisaldehyde), Mn(2+), and oxalate on Pleurotus eryngii OH generation. Our standard quinone redox cycling conditions combined mycelium from laccase-producing cultures with 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe(3+)-EDTA. The main reactions involved in OH production under these conditions have been shown to be (i) DBQ reduction to hydroquinone (DBQH(2)) by cell-bound dehydrogenase activities; (ii) DBQH(2) oxidation to semiquinone (DBQ(-)) by laccase; (iii) DBQ(-) autoxidation, catalyzed by Fe(3+)-EDTA, producing superoxide (O(2)(-)) and Fe(2+)-EDTA; (iv) O(2)(-) dismutation, generating H(2)O(2); and (v) the Fenton reaction. Compared to standard quinone redox cycling conditions, OH production was increased 1.2- and 3.0-fold by the presence of anisaldehyde and Mn(2+), respectively, and 3.1-fold by substituting Fe(3+)-EDTA with Fe(3+)-oxalate. A 6.3-fold increase was obtained by combining Mn(2+) and Fe(3+)-oxalate. These increases were due to enhanced production of H(2)O(2) via anisaldehyde redox cycling and O(2)(-) reduction by Mn(2+). They were also caused by the acceleration of the DBQ redox cycle as a consequence of DBQH(2) oxidation by both Fe(3+)-oxalate and the Mn(3+) generated during O(2)(-) reduction. Finally, induction of OH production through quinone redox cycling enabled P. eryngii to oxidize phenol and the dye reactive black 5, obtaining a high correlation between the rates of OH production and pollutant oxidation.

Induction of extracellular hydroxyl radical production by white-rot fungi through quinone redox cycling.

A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe(3+)-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H(2)O(2) + Fe(2+) --> OH + OH(-) + Fe(3+)). H(2)O(2) production, Fe(3+) reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H(2)O(2) levels and Fe(3+) reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.

VLA-4 mediated adhesion of melanoma cells on the blood-brain barrier is the critical cue for melanoma cell intercalation and barrier disruption.

Melanoma is the most aggressive skin cancer in humans. One severe complication is the formation of brain metastasis, which requires extravasation of melanoma cells across the tight blood-brain barrier (BBB). Previously, VLA-4 has been assigned a role for the adhesive interaction of melanoma cells with non-BBB endothelial cells. However, the role of melanoma VLA-4 for breaching the BBB remained unknown. In this study, we used a mouse in vitro BBB model and imaged the shear resistant arrest of melanoma cells on the BBB. Similar to effector T cells, inflammatory conditions of the BBB increased the arrest of melanoma cells followed by a unique post-arrest behavior lacking immediate crawling. However, over time, melanoma cells intercalated into the BBB and compromised its barrier properties. Most importantly, antibody ablation of VLA-4 abrogated melanoma shear resistant arrest on and intercalation into the BBB and protected the BBB from barrier breakdown. A tissue microarray established from human brain metastasis revealed that indeed a majority of 92% of all human melanoma brain metastases stained VLA-4 positive. We propose VLA-4 as a target for the inhibition of brain metastasis formation in the context of personalized medicine identifying metastasizing VLA-4 positive melanoma.

Predominance of a macrolide-lincosamide-resistant Brachyspira hyodysenteriae of sequence type 196 in Swiss pig herds.

Worldwide emergence of antimicrobial-resistant Brachyspira (B.) hyodysenteriae led us question whether specific clones are present in Switzerland. Fifty-one B. hyodysenteriae isolates originating from 27 different Swiss pig herds sampled between 2010 and 2017 were characterised. Multilocus sequence typing revealed the presence of four different sequence types (STs) ST6, ST66, ST196 and ST197 with ST196 being predominant. Antimicrobial susceptibility to six different antimicrobial agents was determined by measurement of the minimal inhibitory concentration by broth dilution. Isolates were examined for the presence of point mutations and genes known to be associated with antimicrobial resistance in B. hyodysenteriae by PCR and sequence analysis. Forty-one isolates belonging to ST6 (n = 1), ST66 (n = 4) and ST196 (n = 36) exhibited decreased susceptibility to macrolides and lincomycin associated with an A2058 T/G mutation in the 23S rRNA gene. One isolate of ST66 and five isolates of ST196 exhibited decreased susceptibility to doxycycline associated with a G1058C mutation in the 16S rRNA gene. The Swiss B. hyodysenteriae population is characterised by a low genetic diversity, with macrolide-lincosamide-resistant isolates of ST196 being predominant.

Seasonal and Sexual Differences in the Microbiota of the Hoopoe Uropygial Secretion.

The uropygial gland of hoopoe nestlings and nesting females hosts bacterial symbionts that cause changes in the characteristics of its secretion, including an increase of its antimicrobial activity. These changes occur only in nesting individuals during the breeding season, possibly associated with the high infection risk experienced during the stay in the hole-nests. However, the knowledge on hoopoes uropygial gland microbial community dynamics is quite limited and based so far on culture-dependent and molecular fingerprinting studies. In this work, we sampled wild and captive hoopoes of different sex, age, and reproductive status, and studied their microbiota using quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH) and pyrosequencing. Surprisingly, we found a complex bacterial community in all individuals (including non-nesting ones) during the breeding season. Nevertheless, dark secretions from nesting hoopoes harbored significantly higher bacterial density than white secretions from breeding males and both sexes in winter. We hypothesize that bacterial proliferation may be host-regulated in phases of high infection risk (i.e., nesting). We also highlight the importance of specific antimicrobial-producing bacteria present only in dark secretions that may be key in this defensive symbiosis. Finally, we discuss the possible role of environmental conditions in shaping the uropygial microbiota, based on differences found between wild and captive hoopoes.

Transposon-associated lincosamide resistance lnu(C) gene identified in Brachyspira hyodysenteriae ST83.

Treatment of Swine Dysentery (SD) caused by Brachyspira hyodysenteriae (B. hyodysenteriae) is carried out using antimicrobials such as macrolides, lincosamides and pleuromutilins leading to the selection of resistant strains. Whole genome sequencing of a multidrug-resistant B. hyodysenteriae strain called BH718 belonging to sequence type (ST) 83 revealed the presence of the lincosamide resistance gene lnu(C) on the small 1724-bp transposon MTnSag1. The strain also contains an A to T substitution at position 2058 (A2058T) in the 23S rRNA gene which is known to be associated with macrolide and lincosamide resistance in B. hyodysenteriae. Testing of additional strains showed that those containing lnu(C) exhibited a higher minimal inhibitory concentration (MIC) of lincomycin (MIC ≥ 64 mg/L) compared to strains lacking lnu(C), even if they also harbor the A2058T mutation. Resistance to pleuromutilins could not be explained by the presence of already reported mutations in the 23S rRNA gene and in the ribosomal protein L3. This study shows that B. hyodysenteriae has the ability to acquire mobile genetic elements conferring resistance to antibiotics.

ALCAM (CD166) is involved in extravasation of monocytes rather than T cells across the blood-brain barrier.

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to mediate leukocyte migration across the blood-brain barrier (BBB) in multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). Here, we confirmed vascular ALCAM expression in human brain tissue samples in situ and on two different human in vitro BBB models. Antibody-mediated inhibition of ALCAM reduced diapedesis of human CD4+ Th1 but not of Th17 cells across the human BBB in vitro. In accordance to human Th1 cells, mouse Th1 cells showed reduced diapedesis across an ALCAM-/- in vitro BBB model under static but no longer under flow conditions. In contrast to the limited role of ALCAM in T cell extravasation across the BBB, we found a contribution of ALCAM to rolling, adhesion, and diapedesis of human CD14+ monocytes across the human BBB under flow and static conditions. Taken together, our study highlights the potential differences in the CNS expression of ALCAM in mouse and human and supports a prominent role for ALCAM in the multi-step extravasation of monocytes across the BBB.

Contributions to a better comprehension of redox-mediated decolouration and detoxification of azo dyes by a laccase produced by Streptomyces cyaneus CECT 3335.

The ability of a laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 to decolourise and detoxify azo dyes was assessed. Results showed that a colour loss of 90% was achieved only in the presence of acetosyringone (0.1mM) acting as a redox mediator for the laccase. Toxicological analysis of the decolourised dyes revealed that there was no direct correlation between decolouration and detoxification; in fact, in the case of the dyes Methyl Orange and Orange II, a significant increase in toxicity was produced after the treatment. In contrast, a significant decrease in toxicity was observed after the decolouration of New Coccine and Chromotrope 2R. Finally, HPLC analysis of the dyes after treatment revealed the complete disappearance of both dyes and mediator and a concomitant appearance of new chromatographic peaks which could be responsible of the residual toxicity detected in some cases.