Anti-chlamydial activity of vaginal fluids: new evidence from an in vitro model.

Introduction: We assessed the in vitro anti-chlamydial activity of fresh vaginal secretions, deciphering the microbial and metabolic components able to counteract Chlamydia trachomatis viability. Methods: Forty vaginal samples were collected from a group of reproductive-aged women and their anti-chlamydial activity was evaluated by inhibition experiments. Each sample underwent 16S rRNA metabarcoding sequencing to determine the bacterial composition, as well as 1H-NMR spectroscopy to detect and quantify the presence of vaginal metabolites. Results: Samples characterized by a high anti-chlamydial activity were enriched in Lactobacillus, especially Lactobacillus crispatus and Lactobacillus iners, while not-active samples exhibited a significant reduction of lactobacilli, along with higher relative abundances of Streptococcus and Olegusella. Lactobacillus gasseri showed an opposite behavior compared to L. crispatus, being more prevalent in not-active vaginal samples. Higher concentrations of several amino acids (i.e., isoleucine, leucine, and aspartate; positively correlated to the abundance of L. crispatus and L. jensenii) lactate, and 4-aminobutyrate were the most significant metabolic fingerprints of highly active samples. Acetate and formate concentrations, on the other hand, were related to the abundances of a group of anaerobic opportunistic bacteria (including Prevotella, Dialister, Olegusella, Peptostreptococcus, Peptoniphilus, Finegoldia and Anaerococcus). Finally, glucose, correlated to Streptococcus, Lachnospira and Alloscardovia genera, emerged as a key molecule of the vaginal environment: indeed, the anti-chlamydial effect of vaginal fluids decreased as glucose concentrations increased. Discussion: These findings could pave the way for novel strategies in the prevention and treatment of chlamydial urogenital infections, such as lactobacilli probiotic formulations or lactobacilli-derived postbiotics.

Evaluation of an automated rapid phenotypic antimicrobial susceptibility testing (ASTar, Q-linea AB) applied directly on blood cultures bottles positive for Gram-negative pathogens.

We evaluated the performance of a new rapid phenotypic antimicrobial susceptibility test (ASTar; Q-linea AB) on Gram-negative bacilli, directly from positive blood cultures bottles. MIC values obtained by the routine reference method (Microscan, Beckman Coulter) were compared to the ones provided by the tested method (ASTar). ASTar demonstrated an overall essential agreement of 98% and a category agreement of 96.1%. The overall rate of major errors and very major errors was 2.5% and 3.3%, respectively. ASTar can represent a rapid, simple, and reliable method to speed up information about antimicrobial susceptibility of Gram-negative pathogens from positive blood culture bottles.

Antimicrobial resistance determinants in the oropharyngeal microbiome of 'men having sex with men' attending an sexually transmitted infection clinic.

BACKGROUND: 'Men having sex with men' (MSM) represent a key population with a significant prevalence of pharyngeal Neisseria gonorrhoeae (NG) infections and a high rate of antimicrobial resistance genes in the pharyngeal microbiome. As NG can acquire antibiotic resistance genes from other commensal oropharyngeal bacteria, monitoring the prevalence of these resistance determinants is critical to curtail the spread of NG-resistant strains. PURPOSE AND RESEARCH DESIGN: Here, we assessed the distribution of five resistance genes (pen (A), mtr (R), gyr (A), par (C), msr (D)) in the oropharynx of 164 MSM, attending an Outpatient clinic for STI screening. RESULTS: The most frequently detected resistance gene was msr (D) (88.4%), followed by gyr (A) (67.1%). The distribution of resistance genes was not influenced by pharyngeal gonorrhea nor by the HIV status, whereas a younger age was associated with mtr (R) presence (p = .008). Subjects using mouthwash exhibited significantly lower levels of mtr (R) (p = .0005). Smoking habit was associated with a higher prevalence of par (C) (p = .02). A noteworthy association was observed between the presence of msr (D) gene and the use of antibiotics (p = .014). CONCLUSIONS: Our findings reveal an enrichment of antimicrobial resistance genes in the oropharynx of MSM. These insights could aid in the development of screening programs and antimicrobial stewardship initiatives targeting populations at heightened risk of pharyngeal gonorrhea.

Chlamydia trachomatis and Neisseria gonorrhoeae rectal infections: Interplay between rectal microbiome, HPV infection and Torquetenovirus.

Men having sex with men (MSM) represent a key population, in which sexually transmitted rectal infections (STIs) caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and high-risk HPV (HR-HPV) are very common and linked to significant morbidity. Investigating the anorectal microbiome associated with rectal STIs holds potential for deeper insights into the pathogenesis of these infections and the development of innovative control strategies. In this study, we explored the interplay at the rectal site between C. trachomatis, N. gonorrhoeae, HR-HPV infection, and the anorectal microbiome in a cohort of 92 MSM (47 infected by CT and/or NG vs 45 controls). Moreover, we assessed the presence of Torquetenovirus (TTV), a non-pathogenic endogenous virus, considered as a possible predictor of immune system activation. We found a high prevalence of HR-HPV rectal infections (61%), especially in subjects with a concurrent CT/NG rectal infection (70.2%) and in people living with HIV (84%). In addition, we observed that TTV was more prevalent in subjects with CT/NG rectal infections than in non-infected ones (70.2% vs 46.7%, respectively). The anorectal microbiome of patients infected by CT and/or NG exhibited a reduction in Escherichia, while the presence of TTV was significantly associated with higher levels of Bacteroides. We observed a positive correlation of HR-HPV types with Escherichia and Corynebacterium, and a negative correlation with the Firmicutes phylum, and with Prevotella, Oscillospira, Sutterella. Our findings shed light on some of the dynamics occurring within the rectal environment involving chlamydial/gonococcal infections, HPV, TTV, and the anorectal microbiome. These data could open new perspectives for the control and prevention of STIs in MSM.

Human Campylobacter spp. infections in Italy.

PURPOSE: Campylobacter is a frequent cause of enteric infections with common antimicrobial resistance issues. The most recent reports of campylobacteriosis in Italy include data from 2013 to 2016. We aimed to provide national epidemiological and microbiological data on human Campylobacter infections in Italy during the period 2017-2021. METHODS: Data was collected from 19 Hospitals in 13 Italian Regions. Bacterial identification was performed by mass spectrometry. Antibiograms were determined with Etest or Kirby-Bauer (EUCAST criteria). RESULTS: In total, 5419 isolations of Campylobacter spp. were performed. The most common species were C. jejuni (n = 4535, 83.7%), followed by C. coli (n = 732, 13.5%) and C. fetus (n = 34, 0.6%). The mean age of patients was 34.61 years and 57.1% were males. Outpatients accounted for 54% of the cases detected. Campylobacter were isolated from faeces in 97.3% of cases and in 2.7% from blood. C. fetus was mostly isolated from blood (88.2% of cases). We tested for antimicrobial susceptibility 4627 isolates (85.4%). Resistance to ciprofloxacin and tetracyclines was 75.5% and 54.8%, respectively; resistance to erythromycin was 4.8%; clarithromycin 2% and azithromycin 2%. 50% of C. jejuni and C. coli were resistant to ≥ 2 antibiotics. Over the study period, resistance to ciprofloxacin and tetracyclines significantly decreased (p < 0.005), while resistance to macrolides remained stable. CONCLUSION: Campylobacter resistance to fluoroquinolones and tetracyclines in Italy is decreasing but is still high, while macrolides retain good activity.

Antimicrobial Resistance of Genital Mycoplasma and Ureaplasma: A Multicentre Study Over a 5-Year Period in Italy (2017-2021).

Updated data on genital Mollicutes prevalence and antimicrobial susceptibility can help provide guidance for antibiotic stewardship and set up effective strategies for infection control policies. In this multicentre study, we assessed the prevalence and the resistance profile of Mycoplasma hominis (MH) and Ureaplasma species (U. parvum/U. urealyticum), analyzing data from 21,210 subjects who provided urogenital samples for Mollicutes detection by culture over a 5-year period (2017-2021) in two high-density urban areas in the North of Italy (i.e., Bologna and Lecco). Overall prevalence of Mollicutes infection was 22.3%, with women showing a significantly higher detection rate than men (p < 0.00001). The prevalence decreased with age (highest prevalence <30 years) and over the years considered. Ureaplasma strains were much more frequently detected (62.3%) compared to MH (8.3%) and to mixed infections (29.4%). Ureaplasma species showed high levels of ciprofloxacin resistance (39.5%), whereas MH strains were nonsusceptible to azithromycin and roxithromycin in about 60% of cases. Over time, a significant decrease of resistance to azithromycin and doxycycline was detected (p < 0.0001 and 0.0004, respectively), in parallel with an important increase of ciprofloxacin-resistance levels (p < 0.0001). Overall, our results revealed that minocycline and josamycin can be first-line drugs for Mollicutes empirical treatment.

Non-pathogenic Neisseria species of the oropharynx as a reservoir of antimicrobial resistance: a cross-sectional study.

Commensal Neisseria species of the oropharynx represent a significant reservoir of antimicrobial resistance determinants that can be transferred to Neisseria gonorrhoeae. This aspect is particularly crucial in 'men having sex with men' (MSM), a key population in which pharyngeal co-colonization by N. gonorrhoeae and non-pathogenic Neisseria species is frequent and associated with the emergence of antimicrobial resistance. Here, we explored the antimicrobial susceptibility of a large panel of non-pathogenic Neisseria species isolated from the oropharynx of two populations: a group of MSM attending a 'sexually transmitted infection' clinic in Bologna (Italy) (n=108) and a group of males representing a 'general population' (n=119). We collected 246 strains, mainly belonging to N. subflava (60%) and N. flavescens (28%) species. Their antimicrobial susceptibility was evaluated assessing the minimum inhibitory concentrations (MICs) for azithromycin, ciprofloxacin, cefotaxime, and ceftriaxone using E-test strips. Overall, commensal Neisseria spp. showed high rates of resistance to azithromycin (90%; median MICs: 4.0 mg/L), and ciprofloxacin (58%; median MICs: 0.12 mg/L), whereas resistance to cephalosporins was far less common (<15%). Neisseria strains from MSM were found to have significantly higher MICs for azithromycin (p=0.0001) and ciprofloxacin (p<0.0001) compared to those from the general population. However, there was no significant difference in cephalosporin MICs between the two groups. The surveillance of the antimicrobial resistance of non-pathogenic Neisseria spp. could be instrumental in predicting the risk of the spread of multi-drug resistant gonorrhea. This information could be an early predictor of an excessive use of antimicrobials, paving the way to innovative screening and prevention policies.

Limosilactobacillus vaginalis Exerts Bifidogenic Effects: A Novel Postbiotic Strategy for Infant Prebiotic Supplementation.

Infant microbiota shaping strictly influences newborns' well-being and long-term health, and babies born by cesarean-section and formula-fed generally show low microbial gut diversity and are more prone to develop various disorders. The supplementation with beneficial microbes of vaginal origin or derivatives (postbiotics, including heat-inactivated cells) represents a valid strategy to drive the correct gut microbiota shaping. Here, we explored for the first time the bifidogenic activity of a heat-killed vaginal strain (Limosilactobacillus vaginalis BC17), in addition to the assessment of its safety. L. vaginalis BC17 whole genome was sequenced by Nanopore technology and highlighted the absence of antibiotic resistance genes and virulence factors, indicating the strain safety profile for human health. MIC values confirmed that L. vaginalis BC17 is susceptible to widely employed antibiotics. Heat-killed BC17 cells significantly enhanced the planktonic growth of Bifidobacterium spp. For the first time, stimulating effects were observed also toward biofilm formation of bifidobacteria and their pre-formed biofilms. Conversely, heat-killed BC17 cells exerted antibacterial and anti-biofilms activities against Gram-positive and Gram-negative pathogens. Lyophilized heat-killed BC17 cells were formulated in a sunflower oil suspension (1010 heat-killed cell/g) intended for infant oral intake. This possessed optimal technological (i.e., re-dispersibility and stability) and functional properties (i.e., bifidogenic activity) that were maintained even after pre-digestion in acidic conditions.

Role of D(-)-Lactic Acid in Prevention of Chlamydia trachomatis Infection in an In Vitro Model of HeLa Cells.

A vaginal microbiota dominated by certain Lactobacillus species may have a protective effect against Chlamydia trachomatis infection. One of the key antimicrobial compounds produced is lactic acid, which is believed to play a central role in host defense. Lactobacillus strains producing the D(-)-lactic acid isomer are known to exert stronger protection. However, the molecular mechanisms underlying this antimicrobial action are not well understood. The aim of this study was to investigate the role of D(-)-lactic acid isomer in the prevention of C. trachomatis infection in an in vitro HeLa cell model. We selected two strains of lactobacilli belonging to different species: a vaginal isolate of Lactobacillus crispatus that releases both D(-) and L(+) isomers and a strain of Lactobacillus reuteri that produces only the L(+) isomer. Initially, we demonstrated that L. crispatus was significantly more effective than L. reuteri in reducing C. trachomatis infectivity. A different pattern of histone acetylation and lactylation was observed when HeLa cells were pretreated for 24 h with supernatants of Lactobacillus crispatus or L. reuteri, resulting in different transcription of genes such as CCND1, CDKN1A, ITAG5 and HER-1. Similarly, distinct transcription patterns were found in HeLa cells treated with 10 mM D(-)- or L(+)-lactic acid isomers. Our findings suggest that D(-) lactic acid significantly affects two non-exclusive mechanisms involved in C. trachomatis infection: regulation of the cell cycle and expression of EGFR and α5ß1-integrin.

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.