Achromobacter spp. adaptation in cystic fibrosis infection and candidate biomarkers of antimicrobial resistance

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

Convergent genetic and expression data implicate immunity in Alzheimer's disease

Background Late–onset Alzheimer's disease (AD) is heritable with 20 genes showing genome wide association in the International Genomics of Alzheimer's Project (IGAP). To identify the biology underlying the disease we extended these genetic data in a pathway analysis. Methods The ALIGATOR and GSEA algorithms were used in the IGAP data to identify associated functional pathways and correlated gene expression networks in human brain. Results ALIGATOR identified an excess of curated biological pathways showing enrichment of association. Enriched areas of biology included the immune response (p = 3.27×10-12 after multiple testing correction for pathways), regulation of endocytosis (p = 1.31×10-11), cholesterol transport (p = 2.96 × 10-9) and proteasome-ubiquitin activity (p = 1.34×10-6). Correlated gene expression analysis identified four significant network modules, all related to the immune response (corrected p 0.002 – 0.05). Conclusions The immune response, regulation of endocytosis, cholesterol transport and protein ubiquitination represent prime targets for AD therapeutics

A New Method to Easily Assess Bacteriostatic and Bactericidal Activity of Ultraviolet Radiation Using Quantitative Image Analysis

: Ultraviolet (UV) radiation can elicit both bactericidal and bacteriostatic activity depending on light parameters and targeted bacteria. Current methods based on bacterial growth on solid medium allow measurement of only bactericidal but not bacteriostatic activity, while liquid cultures exhibit low light penetration. Here, we propose a method to quantify both bactericidal and bacteriostatic activity of radiation based on (a) bacterial cultures on solid medium, (b) acquisition and quantitative analysis of photographic images of plates containing bacterial colonies, (c) application of two mathematical equations to evaluate bactericidal and bacteriostatic activity. The proposed method considers the differences in growth on test and control (unexposed) plates. The measurements performed on the plates image are the independent variables of the mathematical equations returning the values of bactericidal and bacteriostatic activity. Experimentally, a test was performed using Escherichia coli grown on a solid medium and exposed to UVA (365 nm) radiation. The standard method allowed quantifying bactericidal activity and evaluating only qualitatively bacteriostatic activity of the radiation. Differently, the new method here proposed allowed quantification of both activities. The proposed method proved to be simple, enabling deep assessment of the antibacterial effects of UV radiation directly on the solid medium through image acquisition and analysis

Achromobacter spp. prevalence and adaptation in cystic fibrosis lung infection

Bacteria belonging to the genus Achromobacter are widely distributed in natural environments and have been recognized as emerging pathogens for their contribution to a wide range of human infections. In particular, patients with cystic fibrosis (CF) are the subjects most frequently colonized by Achromobacter spp., which can cause persistent infections in their respiratory tract. Although many clinical aspects and pathogenic mechanisms still remain to be elucidated, Achromobacter spp. have been a source of expanding interest in recent years. This review examines the current literature regarding Achromobacter spp. role in CF, focusing on taxonomy, prevalence in CF lung infections, genomic characteristics, and adaptation strategies including modifications of metabolism and virulence, acquisition of antibiotic resistance, exchange of mobile genetic elements and development of hypermutation

Effect on Human Cells of Environmental Vibrio parahaemolyticus Strains Carrying Type III Secretion System 2▿

Vibrio parahaemolyticus is an inhabitant of estuarine and marine environments that causes seafood-borne gastroenteritis worldwide. Recently, a type 3 secretion system (T3SS2) able to secrete and translocate virulence factors into the eukaryotic cell has been identified in a pathogenicity island (VP-PAI) located on the smaller chromosome. These virulence-related genes have previously been detected only in clinical strains. Classical virulence genes for this species (tdh, trh) are rarely detected in environmental strains, which are usually considered to lack virulence potential. However, during screening of a collection of environmental V. parahaemolyticus isolates obtained in the North Adriatic Sea in Italy, a number of marine strains carrying virulence-related genes, including genes involved in the T3SS2, were detected. In this study, we investigated the pathogenic potential of these marine V. parahaemolyticus strains by studying their adherence ability, their cytotoxicity, their effect on zonula occludin protein 1 (ZO-1) of the tight junctions, and their effect on transepithelial resistance (TER) in infected Caco-2 cells. By performing a reverse transcription-PCR, we also tested the expression of the T3SS2 genes vopT and vopB2, encoding an effector and a translocon protein, respectively. Our results indicate that, similarly to clinical strains, marine V. parahaemolyticus strains carrying vopT and vopB2 and that other genes included in the VP-PAI are capable of adhering to human cells and of causing cytoskeletal disruption and loss of membrane integrity in infected cells. On the basis of data presented here, environmental V. parahaemolyticus strains should be included in coastal water surveillance plans, as they may represent a risk for human health

In Vivo Inflammation Caused by <i>Achromobacter</i> spp. Cystic Fibrosis Clinical Isolates Exhibiting Different Pathogenic Characteristics

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

The Emerging Nosocomial Pathogen Klebsiella michiganensis: Genetic Analysis of a KPC-3 Producing Strain Isolated from Venus Clam

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

Virulence and antibiotic resistance of Achromobacter spp. isolates from chronic and occasional lung infection in cystic fibrosis patients

Background: Achromobacter spp. are opportunistic pathogens that can establish both chronic or occasional infections in the lungs of cystic fibrosis (CF) patients. Chronic infections caused by these bacterial species have been associated with decline in respiratory function and lung inflammation, highlighting the need to identify markers of persistence. To this purpose, in this study virulence and antibiotic resistance of isolates from CF chronic and occasional infection were analyzed. Methods: Ninety-five Achromobacter spp. clinical isolates were collected from 38 patients followed at the CF centers of Verona and Rome (Italy): 24 patients presented a chronic colonization and 14 an occasional infection. A number of features were evaluated in vitro: virulence potential through inoculation of bacteria in Galleria mellonella larvae; susceptibility to selected antibiotics by Kirby-Bauer disk diffusion test; cytotoxicity by quantitative measurement of lactate dehydrogenase; biofilm formation by crystal violet staining of surface‐attached bacteria cultured in static conditions. Statistical analysis was performed to compare chronic and occasional isolates and ascertain the significance of results. Results: Virulence testing showed that isolates from occasionally infected patients induced significantly higher mortality of G. mellonella larvae than chronic infection isolates (Kaplan-Meier survival estimate p-value=0.02; Cox hazard ratio=1.32; 95% confidence interval (CI)=1.04-1.66). As concerns antibiotic susceptibility, isolates from chronically infected patients were significantly more resistant to sulfonamide and meropenem (respectively: Fisher’s exact test p-value=0.04 and 0.01 after 10000 permutations; CI=0.04-0.62 and 0-0.34; odds ratio=0.17 and 0) than occasional isolates. Cytotoxicity was tested in human bronchial epithelial cells: although no statistically significant difference was found, we observed that chronic infection isolates induced greater cytotoxicity than occasional isolates (Wilcoxon Mann Whitney p-value=0.05). Finally, no significant difference was observed in biofilm production among the two groups. Conclusion: Our results show that Achromobacter spp. isolates from chronic and occasional lung infection exhibit different virulence and antibiotic resistance characteristics, some of which might be linked to persistence in CF lungs. This highlights the potential to identify predictive markers of persistence that could be translated into the clinical setting. Acknowledgements: This study was supported by the Italian Cystic Fibrosis Research Foundation (project FFC#18/2019)

An Extensive investigation into the prevalence and the genetic and serological diversity of toxigenic <i>Vibrio parahaemolyticus</i> in Italian marine coastal waters

The relationship between Vibrio parahaemolyticus strains isolated from the aquatic environment and those isolated from cases of infection in humans is poorly understood due to the low prevalence of tdh- and/or trh-positive strains in the environment. To address this concern, it would be useful to analyse the genetic relationships among environmental and food strains and with reference to clinical isolates, also applying molecular typing methods. The aim of this study was to evaluate the prevalence of toxigenic V. parahaemolyticus in Italian coastal waters and seafood, to examine intra-species variability and to identify, using serotyping and pulsed-field gel electrophoresis (PFGE), relationships among strains from different sources, geographical origin and period of isolation. Of the 192 V. parahaemolyticus strains isolated in different Italian areas and examined in this study, 25 (13.0%) proved to carry the trh gene while none of the strains proved positive to the search by PCR for tdh and Group-Specific-toxRS genes. The prevalence of toxigenic strains in the Tyrrhenian Sea was significantly lower than that calculated for the Ligurian coasts. Regarding the sources of isolation, the higher prevalence of trh-positive V. parahaemolyticus was revealed in fish, followed by clams, plankton, oysters, mussels and lastly seawater. Within the toxigenic strains, 16 serotypes and 20 distinct PFGE patterns were identified. Two clusters, which included a total of 8 V. parahaemolyticus strains, were specifically associated with the North Adriatic Sea area and were stable over time. Our results demonstrate that trh-positive V. parahaemolyticus strains circulated in Italy in the period 2002–2009 with a prevalence higher than that reported from other European and extra-European countries, confirming that toxigenic V. parahaemolyticus is an emerging public health concern in Italy, regardless of its pandemic potential