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Infections caused by Staphylococcus aureus are particularly difficult to treat due to the high rate of antibiotic resistance. S. aureus also forms biofilms that reduce the effects of antibiotics and disinfectants. Therefore, new therapeutic approaches are increasingly required. In this scenario, plant waste products represent a source of bioactive molecules. In this study, we evaluated the antimicrobial and antibiofilm activity of the rice husk extract (RHE) on S. aureus clinical isolates. In a biofilm inhibition assay, high concentrations of RHE counteracted the formation of biofilm by S. aureus isolates, both methicillin-resistant (MRSA) and -sensitive (MSSA). The observation of the MRSA biofilm by confocal laser scanning microscopy using live/dead cell viability staining confirmed that the bacterial viability in the RHE-treated biofilm was reduced. However, the extract showed no or little biofilm disaggregation ability. An additive effect was observed when treating S. aureus with a combination of RHE and oxacillin/cefoxitin. In Galleria mellonella larvae treated with RHE, the extract showed no toxicity even at high concentrations. Our results support that the rice husk has antimicrobial and antibiofilm properties and could potentially be used in the future in topical solutions or on medical devices to prevent biofilm formation.

ABSTRACT The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the K. michiganensis 23999A2 isolate revealed that the strain belonged to the new sequence type 382 (ST382) and carried seven plasmid replicon sequences, including four IncF type plasmids (FII, FIIY, FIIk, and FIB), one IncHI1 plasmid, and two Col plasmids. The FIB and FIIk plasmids showed high homology to each other and to multireplicon pKpQIL-like plasmids that are found in epidemic KPC-K. pneumoniae clones worldwide. The strain carried multiple β-lactamase genes on the IncF plasmids: blaOXA-9 and blaTEM-1A on FIB, blaKPC-3 inserted in a Tn4401a on FIIK, and blaSHV-12 on FIIY. The IncHI1-ST11 harbored no resistance gene. The curing of the strain caused the loss of all of the bla genes and a rearrangement of the IncF plasmids. Conjugal transfer of the blaOXA-9, blaTEM-1A and blaKPC-3 genes occurred at a frequency of 5 × 10−7, using K. quasipneumoniae as a recipient, and all of the bla genes were transferred through a pKpQIL that originated from the recombination of the FIB and FIIk plasmids of the donor. A comparison with 31 K. michiganensis genomes that are available in the NCBI database showed that the closest phylogenetic relatives of K. michiganensis 23999A2 are an environmental isolate from soil in South Korea and a clinical isolate from human sputum in Japan. Finally, a pan-genome analysis showed a large accessory genome of the strain as well as the great genomic plasticity of the K. michiganensis species. IMPORTANCE Klebsiella michiganensis is an emerging nosocomial pathogen, and, so far, few studies describe isolates of clinical origin in the environment. This study contributes to the understanding of how the dissemination of carbapenem-resistance outside the hospital setting may be related to the circulation of pKpQIL-like plasmids that are derived from epidemic Klebsiella pneumoniae strains. The recovery of a carbapenem-resistant isolate in clams is of great concern, as bivalves could represent vehicles of transmission of pathogens and resistance genes to humans via the food chain. The study demonstrates the plasticity of K. michiganensis genome, which is probably useful to multiple environment adaptation and to the evolution of the species.

Achromobacter spp. lung infection in cystic fibrosis has been associated with inflammation, increased frequency of exacerbations, and decline of respiratory function. We aimed to evaluate in vivo the inflammatory effects of clinical isolates exhibiting different pathogenic characteristics. Eight clinical isolates were selected based on different pathogenic characteristics previously assessed: virulence in Galleria mellonella larvae, cytotoxicity in human bronchial epithelial cells, and biofilm formation. Acute lung infection was established by intratracheal instillation with 10.5 × 108 bacterial cells in wild-type and CFTR-knockout (KO) mice expressing a luciferase gene under control of interleukin-8 promoter. Lung inflammation was monitored by in vivo bioluminescence imaging up to 48 h after infection, and mortality was recorded up to 96 h. Lung bacterial load was evaluated by CFU count. Virulent isolates caused higher lung inflammation and mice mortality, especially in KO animals. Isolates both virulent and cytotoxic showed higher persistence in mice lungs, while biofilm formation was not associated with lung inflammation, mice mortality, or bacterial persistence. A positive correlation between virulence and lung inflammation was observed. These results indicate that Achromobacter spp. pathogenic characteristics such as virulence and cytotoxicity may be associated with clinically relevant effects and highlight the importance of elucidating their mechanisms.

We report a novel IncHI2 plasmid coharboring blaVIM-1blaKPC-3mcr-9.1Escherichia coliblaVIM-1mcr-9.1903blaKPC-34401

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

Recent findings indicate that the oral cavity acts as a bacterial reservoir and might contribute to the transmission of bacteria to the lower airways. Control of a potentially pathogenic microbiota might contribute to prevent the establishment of chronic infection in cystic fibrosis. We evaluated the presence of CF microorganisms in saliva and toothbrushes of CF patients and verify their possible transmission to lower airways. Methods: We assessed the presence of P. aeruginosa, S. aureus, S. maltophilia, A. xylosoxidans, S. marcescens, and yeasts in saliva, toothbrushes and sputum of 38 CF patients and assessed the clonal identity of the strains occurring contemporary in multiple sites by PFGE. Results: At least one of the investigated species was isolated from 60 saliva samples and 23 toothbrushes. S. aureus was the most abundant species, followed by Candida spp. 31 patients contemporary had the same species in sputum and saliva/toothbrush: in most cases, clonal identity of the strains among the different sites was confirmed. Conclusion: Toothbrushes may be sources of oral contamination and might act as reservoirs favoring transmission of potentially pathogenic microorganisms from the environment to the oral cavity and eventually to the LAW. Oral hygiene and toothbrush care are important strategies to prevent CF lung infections.

Achromobacter spp. is an opportunistic pathogen that can cause lung infections in patients with cystic fibrosis (CF). Although a variety of mobile genetic elements (MGEs) carrying antimicrobial resistance genes have been identified in clinical isolates, little is known about the contribution of Achromobacter spp. mobilome to its pathogenicity. To provide new insights, we performed bioinformatic analyses of 54 whole genome sequences and investigated the presence of phages, insertion sequences (ISs), and integrative and conjugative elements (ICEs). Most of the detected phages were previously described in other pathogens and carried type II toxin-antitoxin systems as well as other pathogenic genes. Interestingly, the partial sequence of phage Bcep176 was found in all the analyzed Achromobacter xylosoxidans genome sequences, suggesting the integration of this phage in an ancestor strain. A wide variety of IS was also identified either inside of or in proximity to pathogenicity islands. Finally, ICEs carrying pathogenic genes were found to be widespread among our isolates and seemed to be involved in transfer events within the CF lung. These results highlight the contribution of MGEs to the pathogenicity of Achromobacter species, their potential to become antimicrobial targets, and the need for further studies to better elucidate their clinical impact.

Achromobacter xylosoxidans can cause chronic infections in the lungs of patients with cystic fibrosis (CF) by adapting to the specific environment. The study of longitudinal isolates allows to investigate its within-host evolution to unravel the adaptive mechanisms contributing to successful colonization. In this study, four clinical isolates longitudinally collected from two chronically infected patients underwent whole genome sequencing, de novo assembly and sequence analysis. Phenotypic assays were also performed. The isolates coming from one of the patients (patient A) presented a greater number of genetic variants, diverse integrative and conjugative elements, and different protease secretion. In the first of these isolates (strain A1), we also found a large deletion in the mutS gene, involved in DNA mismatch repair (MMR). In contrast, isolates from patient B showed a lower number of variants, only one integrative and mobilizable element, no phenotypic changes, and no mutations in the MMR system. These results suggest that in the two patients the establishment of a chronic infection was mediated by different adaptive mechanisms. While the strains isolated from patient B showed a longitudinal microevolution, strain A1 can be clearly classified as a hypermutator, confirming the occurrence and importance of this adaptive mechanism in A. xylosoxidans infection.

BackgroundMycoplasma genitalium (MG) is one of the most warning emerging sexually transmitted pathogens also due to its ability in developing resistance to antibiotics. MG causes different conditions ranging from asymptomatic infections to acute mucous inflammation. Resistance-guided therapy has demonstrated the best cure rates and macrolide resistance testing is recommended in many international guidelines. However, diagnostic and resistance testing can only be based on molecular methods, and the gap between genotypic resistance and microbiological clearance has not been fully evaluated yet. This study aims at finding mutations associated with MG antibiotic resistance and investigating the relationship with microbiological clearance amongst MSM.MethodsFrom 2017 to 2021, genital (urine) and extragenital (pharyngeal and anorectal swabs) biological specimens were provided by men-who-have-sex-with-men (MSM) attending the STI clinic of the Infectious Disease Unit at the Verona University Hospital, Verona, Italy. A total of 1040 MSM were evaluated and 107 samples from 96 subjects resulted positive for MG. Among the MG-positive samples, all those available for further analysis (n=47) were considered for detection of mutations known to be associated with macrolide and quinolone resistance. 23S rRNA, gyrA and parC genes were analyzed by Sanger sequencing and Allplex™ MG and AziR Assay (Seegene).ResultsA total of 96/1040 (9.2%) subjects tested positive for MG in at least one anatomical site. MG was detected in 107 specimens: 33 urine samples, 72 rectal swabs and 2 pharyngeal swabs. Among them, 47 samples from 42 MSM were available for investigating the presence of mutations associated with macrolide and quinolone resistance: 30/47 (63.8%) showed mutations in 23S rRNA while 10/47 (21.3%) in parC or gyrA genes. All patients with positive Test of Cure (ToC) after first-line treatment with azithromycin (n=15) were infected with 23S rRNA-mutated MG strains. All patients undergoing second-line moxifloxacin treatment (n=13) resulted negative at ToC, even those carrying MG strains with mutations in parC gene (n=6).ConclusionOur observations confirm that mutations in 23S rRNA gene are associated with azithromycin treatment failure and that mutations in parC gene alone are not always associated with phenotypic resistance to moxifloxacin. This reinforces the importance of macrolide resistance testing to guide the treatment and reduce antibiotic pressure on MG strains.

UV radiation is used for sterilization but has adverse health effects in humans. UV-A radiation has lower antimicrobial effect than UV-B and UV-C but constitutes a lower health risk, opening up the possibility to sanitize environments with human presence in controlled exposure conditions. We investigated this possibility by identifying safe exposure conditions to a UV-A lamp along with efficient sanitization of the environment. The human exposure limits were calculated following the guidelines provided by the International Commission on Non-Ionizing Radiation Protection and the International Commission on Illumination. Antibacterial activity was evaluated on Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The maximum human exposure duration has been identified at different irradiation distance and angle, increasing with the increase of both parameters. Bactericidal activity was observed in all microorganisms and was higher with higher exposure time and at lower distance from the source. Noteworthily, in equal conditions of radiant exposure, the exposure time impacts on the bactericidal activity more than the distance from the source. The modulation of factors such as distance from the source, exposure time and irradiation angle can enable effective antibacterial activity and human safety. Prolonged direct irradiation of the surfaces associated with indirect human exposure represents the condition of greater efficacy and safety.

Background: The presence of carbapenemase-producing bacteria (CPB) in animal hosts and along the food chain may result in the development of reservoirs for human infections. Several CPB strains isolated from animals have been reported, suggesting that transmission and dissemination of the corresponding genes between humans and animals may occur. Animal and food samples have complex backgrounds that hinder the detection of CPB present in low concentrations by standard detection procedures. Methods: We evaluated the possibility of detecting blaKPC, blaVIM, and blaOXA-48-like carbapenemases in 286 animal and food samples (faeces from farm and companion animals, raw meat, bivalve molluscs) by culture-based and standard molecular methods and by ddPCR. Results: The proposed ddPCR managed to detect the target genes, also in samples resulting negative to standard methods. While the presence of blaKPC and blaVIM was detected in few samples (~3%), one third of the samples (n = 94/283) carried different variants of blaOXA-48-like genes. Conclusion: A specific and sensitive method such as ddPCR could be suitable to evaluate the current veterinarian and environmental situation and to assess the dynamic transmission and persistence of CPB between animals and humans and vice versa.

Background Paranasal sinuses act as bacterial reservoirs and contribute to transmitting bacteria to the lower airway of patients with cystic fibrosis (CF). Also, passage of bacteria from the oral cavity to the lungs may occur. Methods We evaluated the presence of Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and Serratia marcescens in sputum and nasal lavage of 59 patients with CF, and also collected saliva and used toothbrushes from 38 of them. We assessed the clonal identity of the strains isolated from the different samples by pulsed-field gel electrophoresis. Results About 80% of the patients were positive for at least one of the bacterial species examined in nasal lavage and sputum. Among the subjects with positive sputum, 74% presented the same species in the nasal lavage and saliva, and 26% on their toothbrush. S. aureus was the most abundant species in all samples. Clonal identity (≥80% similarity) of the strains isolated among the different samples from each patient was confirmed in almost all cases. Longitudinal observation helped to identify five patients who were colonised in the lower airways after an initial period of nasal or oral colonisation. Conclusion Nasal and oral sites act as bacterial reservoirs, favouring the transmission of potentially pathogenic microorganisms to the lower airway. The lack of eradication from these sites might undermine the antibiotic therapy applied to treat the lung infection, allowing the persistence of the bacteria within the patient if colonisation of these sites is not assessed, and no specific therapy is performed.

In the lungs of patients with cystic fibrosis (CF), the main pathogen Pseudomonas aeruginosa is often co-isolated with other microbes, likely engaging in inter-species interactions. In the case of chronic co-infections, this cohabitation can last for a long time and evolve over time, potentially contributing to the clinical outcome. Interactions involving the emerging pathogens Achromobacter spp. have only rarely been studied, reporting inhibition of P. aeruginosa biofilm formation. To evaluate the possible evolution of such interplay, we assessed the ability of Achromobacter spp. isolates to affect the biofilm formation of co-isolated P. aeruginosa strains during long-term chronic co-infections. We observed both competition and cohabitation. An Achromobacter sp. isolate secreted exoproducts interfering with the adhesion ability of a co-isolated P. aeruginosa strain and affected its biofilm formation. Conversely, a clonal Achromobacter sp. strain later isolated from the same patient, as well as two longitudinal strains from another patient, did not show similar competitive behavior against its P. aeruginosa co-isolates. Genetic variants supporting the higher virulence of the competitive Achromobacter sp. isolate were found in its genome. Our results confirm that both inter-species competition and cohabitation are represented during chronic co-infections in CF airways, and evolution of these interplays can happen even at the late stages of chronic infection.

Antimicrobial resistance is a major public health concern restricted not only to healthcare settings but also to veterinary and environmental ones. In this study, we analyzed, by whole genome sequencing (WGS) the resistome, mobilome and virulome of 12 multidrug-resistant (MDR) marine strains belonging to Shewanellaceae and Vibrionaceae families collected at aquaculture centers in Italy. The results evidenced the presence of several resistance mechanisms including enzyme and efflux pump systems conferring resistance to beta-lactams, quinolones, tetracyclines, macrolides, polymyxins, chloramphenicol, fosfomycin, erythromycin, detergents and heavy metals. Mobilome analysis did not find circular elements but class I integrons, integrative and conjugative element (ICE) associated modules, prophages and different insertion sequence (IS) family transposases. These mobile genetic elements (MGEs) are usually present in other aquatic bacteria but also in Enterobacteriaceae suggesting their transferability among autochthonous and allochthonous bacteria of the resilient microbiota. Regarding the presence of virulence factors, hemolytic activity was detected both in the Shewanella algae and in Vibrio spp. strains. To conclude, these data indicate the role as a reservoir of resistance and virulence genes in the environment of the aquatic microbiota present in the examined Italian fish farms that potentially might be transferred to bacteria of medical interest.