Editorial: Vaginal dysbiosis and biofilms

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Rapid identification and detection of β-lactamase-producing Enterobacteriaceae from positive blood cultures by MALDI-TOF/MS.

Abstract Objectives Current evidence suggests that early diagnosis of sepsis and timely detection of antimicrobial resistance are crucial to improve mortality rates among patients. The aim of this study was to evaluate a rapid method for the identification of Gram-negative bacteria from positive blood cultures (BCs), combined with the detection of extended spectrum β-lactamases (ESβL) and carbapenemases production, by means of MALDI-TOF/MS analysis. Methods During the study, all BCs positive for Gram-negative rods were selected. Starting from bacterial pellets obtained directly from BC broths, species identification and hydrolysis assays were achieved through MALDI-TOF/MS (Bruker). In particular, we performed a hydrolysis assays of cefotaxime (CTX) and ertapenem (ERT) for the rapid detection of resistance via ESβL and carbapenemases, respectively. These results were compared with the routine workflow, including BC subcultures and confirmation phenotypic methods. Finally, a comparison of the turnaround-time (TAT) between the two protocols was conducted. Results Overall, 185 BCs positive for Enterobacteriaceae were collected. In terms of species identification, we observed a concordance of 95.9% comparing MALDI-TOF/MS results to the subculture-based method. The sensitivity and specificity for CTX hydrolysis assay were 91.1% and 92%, respectively; ERT hydrolysis assay showed a sensitivity of 96.2% and a specificity of 99.2%. The TAT of the proposed MALDI TOF/MS-based protocol was significantly lower compared with the routine workflow (P  Conclusions The proposed protocol can provide reliable bacterial identification and data concerning β-lactam resistance in only 3 hours, positively improving management of patients in terms of antimicrobial stewardship

Real-Life Assessment of the Ability of an Ultraviolet C Lamp (SanificaAria 200, Beghelli) to Inactivate Airborne Microorganisms in a Healthcare Environment

Airborne-mediated microbial diseases represent one of the major challenges to public health. Ultraviolet C radiation (UVC) is among the different sanitation techniques useful to reduce the risk of infection in healthcare facilities. Previous studies about the germicidal activity of UVC were mainly performed in artificial settings or in vitro models. This study aimed to assess the sanitizing effectiveness of a UVC device (SanificaAria 200, Beghelli, Valsamoggia, Bologna, Italy) in 'real-life' conditions by evaluating its ability to reduce microbial loads in several hospital settings during routine daily activities. The efficacy of the UVC lamp in reducing the bacterial component was evaluated by microbial culture through the collection of air samples in different healthcare settings at different times (30 min-24 h) after turning on the device. To assess the anti-viral activity, air samplings were carried out in a room where a SARS-CoV-2-positive subject was present. The UVC device showed good antibacterial properties against a wide range of microbial species after 6 h of activity. It was effective against possible multi-drug resistant microorganisms (e.g., Pseudomonas spp., Acinetobacter spp.) and spore-forming bacteria (e.g., Bacillus spp.). In addition, the UVC lamp was able to inactivate SARS-CoV-2 in just one hour. Thanks to its effectiveness and safety, SanificaAria 200 could be useful to inactivate airborne pathogens and reduce health risks

Rectal screening of carbapenemase-producing Enterobacteriaceae: a proposed workflow

Abstract Objectives Active screening is a crucial element for the prevention of carbapenemase-producing Enterobacteriaceae (CPE) transmission in healthcare settings. Here, we proposed a culture-based protocol for rectal swab CPE screening that combines the detection of CPE and the identification of carbapenamase type. Methods The workflow integrates an automatic digital analysis of selective chromogenic media (WASPLab, Copan), with subsequent rapid tests for the confirmation of carbapenemase production (i.e. detection of Klebsiella pneumoniae KPC-specific peak by MALDI-TOF mass spectrometry; a multiplex immunochromatographic assay identifying the five commonest carbapenemase types). To in-depth evaluate the performance of this protocol, data about 21 162 rectal swabs submitted for CPE screening at the Microbiology of S. Orsola-Malpighi Hospital in Bologna were analyzed. Results Considering its ability to correctly segregate plates with/without Enterobacteriaceae, WASPLab Image Analysis Software showed globally a sensitivity and a specificity of 100% and 79.4%, respectively. Out of the plates with a bacterial growth (n = 901), 76.9% were found positive for CPE by MALDI-TOF (specific KPC-peak for K. pneumoniae) or by the immunochromatographic assay. Only 2.8% of KPC-positive K. pneumoniae strains were missed by the specific MALDI-TOF MS algorithm, being detected by the immunochromatographic assay. The mean turn-around-time needed from the sample arrival to the final report ranged between 18 to 24 hours, with a significant time saving compared to a manual reading. Conclusions This workflow proved to be fast and reliable, being particularly suitable for KPC-K. pneumoniae endemic areas and for high-throughput laboratories

Efecto de la colchicina y del amiprofos-metil en la producción in vitro de plantas dihaploides de cebolla y determinación de la correlación entre el nivel de ploidía y tamaño de los estomas

Doubled haploid onion (Allium cepa L.) plants allow the production of completely homozygous lines for a later production of hybrids. The haploid plants are normally produced using in vitro gynogenesis. The obtained haploid plantlets must be treated with different agents for doubling chromosomes. It is necessary to adjust the concentration and the length of treatment of the doubling agent. In this case, the effect of 250 and 500 mg.L-1 colchicine and 15.2; 30 and 60 mg.L- 1 amiprophos-methyl during 24 and 48 h was assessed over the rate of onion haploid plantlets chromosome doubling. The best duplication treatment was 250 mg.L-1 colchicine for 48 h, which yielded 100% of doubled haploid plants. On the other hand, a positive correlation resulted from the ploidy level and stomatal size, and a negative correlation between the level of ploidy and stomatal density. Significant differences between the stomatal length, width and density in haploid and doubled haploid plantlets were observed. An economical and quick method to test ploidy level in onion plantlets is proposed through the measurement of stomatal size and density.La producción de plantas dihaploides de cebolla (Allium cepa L.), permite obtener líneas completamente homocigotas para luego producir híbridos. A través de la ginogénesis in vitro se ha logrado producir plantas haploides, las cuales deben ser sometidas a tratamientos de duplicación de sus cromosomas, siendo necesario ajustar la concentración del agente duplicador y la duración del tratamiento. En este trabajo se evaluó el efecto de la aplicación de 250 y 500 mg.L-1 de colchicina y 15,2; 30 y 60 mg.L-1 de amiprofos-metil durante 24 y 48 h, sobre la tasa de duplicación de cromosomas en plántulas haploides de cebolla. El mejor tratamiento de duplicación fue 250 mg.L- 1 de colchicina, durante 48 h, logrando un 100% de plantas dihaploides. Por otra parte, se obtuvo una correlación positiva entre el nivel de ploidía y el tamaño de estomas y una correlación negativa entre el nivel de ploidía y la densidad estomática, registrándose diferencias significativas entre los valores promedio de largo, ancho y densidad de estomas para plantas haploides y dihaploides. Se propone un método económico y rápido para verificar el nivel de ploidía de las plántulas de cebolla mediante la medición del tamaño y densidad estomática.Fil: Foschi, María Laura. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Martínez, Liliana. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Ponce, María T.. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Galmarini, Claudio. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Bohanec, Borut. University of Ljubljana, Jamnikarjeva. Biotechnical Faculty

Lactobacilli extracellular vesicles: potential postbiotics to support the vaginal microbiota homeostasis

Background: Lactobacillus species dominate the vaginal microflora performing a first-line defense against vaginal infections. Extracellular vesicles (EVs) released by lactobacilli are considered mediators of their beneficial effects affecting cellular communication, homeostasis, microbial balance, and host immune system pathways. Up to now, very little is known about the role played by Lactobacillus EVs in the vaginal microenvironment, and mechanisms of action remain poorly understood. Results: Here, we hypothesized that EVs can mediate lactobacilli beneficial effects to the host by modulating the vaginal microbiota colonization. We recovered and characterized EVs produced by two vaginal strains, namely Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12. EVs were isolated by ultracentrifugation and physically characterized by Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS). EVs protein and nucleic acids (DNA and RNA) content was also evaluated. We explored the role of EVs on bacterial adhesion and colonization, using a cervical cell line (HeLa) as an in vitro model. Specifically, we evaluated the effect of EVs on the adhesion of both vaginal beneficial lactobacilli and opportunistic pathogens (i.e., Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Enterococcus faecalis). We demonstrated that EVs from L. crispatus BC5 and L. gasseri BC12 significantly enhanced the cellular adhesion of all tested lactobacilli, reaching the maximum stimulation effect on strains belonging to L. crispatus species (335% and 269% of average adhesion, respectively). At the same time, EVs reduced the adhesion of all tested pathogens, being EVs from L. gasseri BC12 the most efficient. Conclusions: Our observations suggest for the first time that EVs released by symbiotic Lactobacillus strains favor healthy vaginal homeostasis by supporting the colonization of beneficial species and preventing pathogens attachment. This study reinforces the concept of EVs as valid postbiotics and opens the perspective of developing postbiotics from vaginal strains to maintain microbiota homeostasis and promote women’s health

Effect of colchicine and amiprophos-methyl on the production of in vitro doubled haploid onion plants and correlation assessment between ploidy level and stomatal size = Efecto de la colchicina y del amiprofos-metil en la producción in vitro de plantas dihaploides de cebolla y determinación de la correlación entre el nivel de ploidía y tamaño de los estomas

Doubled haploid onion (Allium cepa L.) plants allow the production of completely homozygous lines for a later production of hybrids. The haploid plants are normally produced using in vitro gynogenesis. The obtained haploid plantlets must be treated with different agents for doubling chromosomes. It is necessary to adjust the concentration and the length of treatment of the doubling agent. In this case, the effect of 250 and 500 mg.L-1 colchicine and 15.2; 30 and 60 mg.L- 1 amiprophos-methyl during 24 and 48 h was assessed over the rate of onion haploid plantlets chromosome doubling. The best duplication treatment was 250 mg.L-1 colchicine for 48 h, which yielded 100% of doubled haploid plants. On the other hand, a positive correlation resulted from the ploidy level and stomatal size, and a negative correlation between the level of ploidy and stomatal density. Significant differences between the stomatal length, width and density in haploid and doubled haploid plantlets were observed. An economical and quick method to test ploidy level in onion plantlets is proposed through the measurement of stomatal size and density.La producción de plantas dihaploides de cebolla (Allium cepa L.), permite obtener líneas completamente homocigotas para luego producir híbridos. A través de la ginogénesis in vitro se ha logrado producir plantas haploides, las cuales deben ser sometidas a tratamientos de duplicación de sus cromosomas, siendo necesario ajustar la concentración del agente duplicador y la duración del tratamiento. En este trabajo se evaluó el efecto de la aplicación de 250 y 500 mg.L-1 de colchicina y 15,2; 30 y 60 mg.L-1 de amiprofos-metil durante 24 y 48 h, sobre la tasa de duplicación de cromosomas en plántulas haploides de cebolla. El mejor tratamiento de duplicación fue 250 mg.L- 1 de colchicina, durante 48 h, logrando un 100% de plantas dihaploides. Por otra parte, se obtuvo una correlación positiva entre el nivel de ploidía y el tamaño de estomas y una correlación negativa entre el nivel de ploidía y la densidad estomática, registrándose diferencias significativas entre los valores promedio de largo, ancho y densidad de estomas para plantas haploides y dihaploides. Se propone un método económico y rápido para verificar el nivel de ploidía de las plántulas de cebolla mediante la medición del tamaño y densidad estomática.EEA La ConsultaFil: Foschi, María Laura. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; ArgentinaFil: Martínez, Liliana Estela. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Ponce, María Teresa. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Galmarini, Claudio Romulo. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bohanec, Borut. University of Ljubljana. Biotechnical Faculty; Esloveni

Whole Genome Sequencing of a Chlamydia trachomatis Strain Responsible for a Case of Rectal Lymphogranuloma Venereum in Italy

Lymphogranuloma venereum (LGV) is a systemic sexually transmitted infection caused by Chlamydia trachomatis serovars L1 to L3. The current LGV cases in Europe are mainly characterized by an anorectal syndrome, spreading within men who have sex with men (MSM). Whole-genome sequencing of LGV strains is crucial to the study of bacterial genomic variants and to improve strategies for contact tracing and prevention. In this study, we described the whole genome of a C. trachomatis strain (LGV/17) responsible for a case of rectal LGV. LGV/17 strain was isolated in 2017 in Bologna (North of Italy) from a HIV-positive MSM, presenting a symptomatic proctitis. After the propagation in LLC-MK2 cells, the strain underwent whole-genome sequencing by means of two platforms. Sequence type was determined using the tool MLST 2.0, whereas the genovariant was characterized by an ompA sequence evaluation. A phylogenetic tree was generated by comparing the LGV/17 sequence with a series of L2 genomes, downloaded from the NCBI website. LGV/17 belonged to sequence type ST44 and to the genovariant L2f. Nine ORFs encoding for polymorphic membrane proteins A-I and eight encoding for glycoproteins Pgp1-8 were detected in the chromosome and in the plasmid, respectively. LGV/17 was closely related to other L2f strains, even in the light of a not-negligible variability. The LGV/17 strain showed a genomic structure similar to reference sequences and was phylogenetically related to isolates from disparate parts of the world, indicative of the long-distance dynamics of transmission

Prebiotic Activity of Vaginal Lactobacilli on Bifidobacteria: from Concept to Formulation

The gut of babies born vaginally is rapidly colonized by Bifidobacterium spp. after birth, while in infants born by cesarean section (C-section), the presence of bifidobacteria drops dramatically, increasing the risk of developing gastrointestinal disorders. Considering that newborns naturally come into contact with maternal lactobacilli as they pass through the birth canal, the aim of this work is to exploit for the first time the bifidogenic activity exerted by the cell-free supernatants (CFSs) from lactobacilli of vaginal origin, belonging to the species Lactobacillus crispatus, Lactobacillus gasseri, Limosilactobacillus vaginalis, and Lactiplantibacillus plantarum. CFSs were recovered after 7 h, 13 h, and 24 h of fermentation and assessed for the ability to stimulate the planktonic growth and biofilms of Bifidobacterium strains belonging to species widely represented in the gut tract. A bifidogenic effect was observed for all CFSs; such activity was maximal for CFSs recovered in exponential phase and was strongly dependent on the species of lactobacilli. Importantly, no stimulating effects on an intestinal Escherichia coli strain were observed. CFSs from L. vaginalis BC17 showed the best bifidogenic profile since they increased bifidobacterial planktonic growth by up to 432% and biofilm formation by up to 289%. The CFS at 7 h from BC17 was successfully formulated with a hyaluronic acid-based hydrogel aimed at preventing and treating breast sores in lactating women and exerting bifidogenic activity in infants born mainly by C-section. IMPORTANCE Bifidobacteria in the gut tract of infants play crucial roles in the prevention of gastrointestinal diseases and the maturation of the immune system. Consequently, strategies to trigger a bifidogenic shift in the infant gut are highly desirable. Evidences suggest that the presence of a maternal vaginal microbiota dominated by health-promoting lactobacilli and the development of a bifidobacterium-enriched gut microbiota in newborns are interconnected. In this context, we found out that the cell-free supernatants from lactobacilli of vaginal origin were able to effectively stimulate the proliferation of Bifidobacterium spp. grown in free-floating and biofilm forms. The cell-free supernatant from Limosilactobacillus vaginalis BC17 showed excellent bifidogenic behavior, which was preserved even after its incorporation into a nipple formulation for lactating women. Lactobacilli derivatives, such as cell-free supernatants, have gained increasing interest by virtue of their safer profile than that of living cells and can be proposed as an ecosustainable approach to favor gut colonization of infants by bifidobacteria

Prevalence and predictors of Lymphogranuloma venereum in a high risk population attending a STD outpatients clinic in Italy

We evaluated LGV prevalence and predictors in a high risk population attending a STI Outpatients Clinic in the North of Italy. METHODS: A total of 108 patients (99 MSM and 9 women), with a history of unsafe anal sexual intercourses, were enrolled. Anorectal swabs and urine samples were tested for Chlamydia trachomatis (CT) DNA detection by Versant CT/GC DNA 1.0 Assay (Siemens Healthcare Diagnostics Terrytown, USA). RFLP analysis was used for CT molecular typing. RESULTS: L2 CT genotype was identified in 13/108 (12%) rectal swabs. All LGV cases were from MSM, declaring high-risk sexual behaviour and complaining anorectal symptoms. Patients first attending the STI Outpatient Clinic received a significant earlier LGV diagnosis than those first seeking care from general practitioners or gastroenterologists (P\u2009=\u20090.0046). LGV prevalence and characteristics found in our population are in agreement with international reports. Statistical analysis showed that LGV positive patients were older (P\u2009=\u20090.0008) and presented more STIs (P\u2009=\u20090.0023) than LGV negative ones, in particular due to syphilis (P\u2009<\u20090.001), HIV (P\u2009<\u20090.001) and HBV (P\u2009=\u20090.001).Multivariate logistic regression analysis revealed that HIV and syphilis infections are strong risk factors for LGV presence (respectively, P\u2009=\u20090.001 and P\u2009=\u20090.010). CONCLUSIONS: Even if our results do not provide sufficient evidence to recommend routine screening of anorectal swabs in high-risk population, they strongly suggest to perform CT NAAT tests and genotyping on rectal specimens in presence of ulcerative proctitis in HIV and/or syphilis-positive MSM. In this context, CT DNA detection by Versant CT/GC DNA 1.0 Assay, followed by RFLP analysis for molecular typing demonstrated to be an excellent diagnostic algorithm for LGV identification