Pseudomonas aeruginosa from river water: antimicrobial resistance, virulence and molecular typing.

Pseudomonas aeruginosa isolates were recovered from surface river water samples in La Rioja region (Spain) to characterise their antibiotic resistance, molecular typing and virulence mechanisms. Fifty-two P. aeruginosa isolates were isolated from 15 different water samples (45.4%) and belonged to 23 different pulsed-field electrophoresis (PFGE) patterns. All isolates were susceptible to all antibiotics tested, except one carbapenem-resistant P. aeruginosa that showed a premature stop codon in OprD porin. Twenty-two sequence types (STs) (six new ones) were detected among 29 selected P. aeruginosa (one strain with a different PFGE pattern per sample), with ST274 (14%) being the most frequent one. O:6 and O:3 were the predominant serotypes (31%). Seven virulotypes were detected, being 59% exoS-exoY-exoT-exoA-lasA-lasB-lasI-lasR-rhlAB-rhlI-rhlR-aprA-positive P. aeruginosa. It is noteworthy that the exlA gene was identified in three strains (10.3%), and the exoU gene in seven (24.1%), exoS in 18 (62.1%), and both exoS and exoU genes in one strain. High motility ranges were found in these strains. Twenty-seven per cent of strains produced more biofilm biomass, 90% more pyorubin, 83% more pyocyanin and 65.5% more than twice the elastase activity compared with the PAO1 strain. These results highlight the importance of rivers as temporary reservoirs and sources of P. aeruginosa transmission, and show the importance of their epidemiological surveillance in the environment.

Spread of Pseudomonas aeruginosa ST274 Clone in Different Niches: Resistome, Virulome, and Phylogenetic Relationship.

Pseudomonas aeruginosa ST274 is an international epidemic high-risk clone, mostly associated with hospital settings and appears to colonize cystic fibrosis (CF) patients worldwide. To understand the relevant mechanisms for its success, the biological and genomic characteristics of 11 ST274-P. aeruginosa strains from clinical and non-clinical origins were analyzed. The extensively drug-resistant (XDR/DTR), the non-susceptible to at least one agent (modR), and the lasR-truncated (by ISPsp7) strains showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity and low motility. Furthermore, the XDR/DTR and modR strains presented low pigment production and biofilm formation, which were very high in the lasR-truncated strain. Their whole genome sequences were compared with other 14 ST274-P. aeruginosa genomes available in the NCBI database, and certain associations have been primarily detected: blaOXA-486 and blaPDC-24 genes, serotype O:3, exoS+/exoU- genotype, group V of type IV pili, and pyoverdine locus class II. Other general molecular markers highlight the absence of vqsM and pldA/tleS genes and the presence of the same mutational pattern in genes involving two-component sensor-regulator systems PmrAB and CreBD, exotoxin A, quorum-sensing RhlI, beta-lactamase expression regulator AmpD, PBP1A, or FusA2 elongation factor G. The proportionated ST274-P. aeruginosa results could serve as the basis for more specific studies focused on better antibiotic stewardship and new therapeutic developments.

High Prevalence of GES-5 Variant and Co-Expression of VIM-2 and GES-45 among Clinical Pseudomonas aeruginosa Strains in Tunisia.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are a global health concern. The antimicrobial resistance, virulence, and molecular typing of 57 CRPA isolated from 43 patients who attended a specific Tunisian hospital from September 2018 to July 2019 were analyzed. All but one were multidrug-resistant CRPA, and 77% were difficult-to-treat-resistant (DTR) isolates. The blaVIM-2 gene was detected in four strains (6.9%), and among the 36 blaGES-positive CRPA (62%), the blaGES-5 gene was the predominant variant (86%). Three strains co-harbored the blaVIM-2 and blaGES-45 genes, and seven CRPA carried the blaSHV-2a gene (14%). OprD alterations, including truncations by insertion sequences, were observed in 18 strains. Regarding the 46 class 1 integron-positive CRPA (81%), the blaGES-5 gene was located in integron In717, while the blaGES-29 and blaGES-45 genes were found in two new integrons (In2122 and In4879), and the blaVIM-2 gene was found in In1183 and the new integron In2142. Twenty-four PFGE patterns and thirteen sequence types (three new ones) were identified. The predominant serotype O:11 and exoU (81%) were mostly associated with ST235 and the new ST3385 clones. The seven blaSHV-2a-CRPA from different patients belonged to ST3385 and the same PFGE pattern. The blaGES-5- and blaVIM-2 + blaGES-45-positive CRPA recovered mostly from ICU patients belonged to the high-risk clone ST235. Our results highlight the alarming prevalence of blaGES-5- and ST235-CRPA, the co-existence of blaGES-45 and blaVIM-2, and their location within integrons favoring their dissemination.

Resistance to Fluoroquinolones in Pseudomonas aeruginosa from Human, Animal, Food and Environmental Origin: The Role of CrpP and Mobilizable ICEs.

Fluoroquinolone resistance and the associated genetic mechanisms were assessed by antimicrobial susceptibility and whole genome sequencing in 56 Pseudomonas aeruginosa strains from human, animal, food and environmental origins. P. aeruginosa PAO1, PA7 and PA14 reference strains were also included in the study. Twenty-two strains (37%) were resistant to, at least, one fluoroquinolone agent. Correlation between the number of changes in GyrA and ParC proteins and the level of fluoroquinolone resistance was observed. Mutations or absence of genes, such as mexZ, mvaT and nalD encoding efflux pumps regulators, were also found in resistant strains. The crpP gene was detected in 43 strains (72.9%; 17 of them non-clinical strains), and coded seven different CrpP variants, including a novel one (CrpP-7). The crpP gene was located in 23 different chromosomal mobile integrative and conjugative elements (ICEs), inserted in two tRNAs integration sites. A great variety of structures was detected in the crpP-ICEs elements, e.g., the fimbriae related cup clusters, the mercury resistance mer operon, the pyocin S5 or S8 bacteriocin encoding genes, and mobilization genes. The location of crpP-like genes in mobilizable ICEs and linked to heavy metal resistance and virulence factors is of significant concern in P. aeruginosa. This work provides a genetic explanation of the fluoroquinolone resistance and crpP-associated pathogenesis of P. aeruginosa from a One-Health approach.

Occurrence of Pseudomonas spp. in Raw Vegetables: Molecular and Phenotypical Analysis of Their Antimicrobial Resistance and Virulence-Related Traits.

Pseudomonas is characterized by its great capacity to colonize different ecological niches, but also by its antimicrobial resistance and pathogenicity, causing human, animal, or plant diseases. Raw and undercooked food is a potential carrier of foodborne disease. The aim of this study was to determine the occurrence of Pseudomonas spp. among raw vegetables, analysing their antimicrobial resistance, virulence, and molecular typing. A total of 163 Pseudomonas spp. isolates (12 different species) were recovered from 77 of the 145 analysed samples (53.1%) and were classified into 139 different pulsed-field gel electrophoresis patterns. Low antimicrobial resistance levels, but one multidrug-resistant isolate, were found. Among the 37 recovered P. aeruginosa strains, 28 sequence-types and nine serotypes were detected. Eleven OprD patterns and an insertion sequence (ISPa1635) truncating the oprD gene of one imipenem-resistant strain were found. Ten virulotypes were observed, including four exoU-positive and thirty-one exoS-positive strains. The lasR gene was absent in three ST155 strains and was truncated by different insertion sequences (ISPre2, IS1411, and ISPst7) in other three strains. High biofilm, motility, pigment, elastase, and rhamnolipid production were detected. Our study demonstrated a low occurrence of P. aeruginosa (18%) and low antimicrobial resistance, but a high number of virulence-related traits in these P. aeruginosa strains, highlighting their pathological importance.

MotilityJ: An open-source tool for the classification and segmentation of bacteria on motility images.

BACKGROUND AND OBJECTIVES: Infectious diseases produced by antimicrobial resistant microorganisms are a major threat to human, and animal health worldwide. This problem is increased by the virulence and spread of these bacteria. Surface motility has been regarded as a pathogenicity element because it is essential for many biological functions, but also for disease spreading; hence, investigations on the motility behaviour of bacteria are crucial to understand chemotaxis, biofilm formation and virulence in general. To identify a motile strain in the laboratory, the bacterial spread area is observed on media solidified with agar. Up to now, the task of measuring bacteria spread was a manual, and, therefore, tedious and time-consuming task. The aim of this work is the development of a set of tools for bacteria segmentation in motility images. METHODS: In this work, we address the problem of measuring bacteria spread on motility images by creating an automatic pipeline based on deep learning models. Such a pipeline consists of a classification model to determine whether the bacteria has spread to cover completely the Petri dish, and a segmentation model to determine the spread of those bacteria that do not fully cover the Petri dishes. In order to annotate enough images to train our deep learning models, a semi-automatic annotation procedure is presented. RESULTS: The classification model of our pipeline achieved a F1-score of 99.85%, and the segmentation model achieved a Dice coefficient of 95.66%. In addition, the segmentation model produces results that are indistinguishable, and in many cases preferred, from those produced manually by experts. Finally, we facilitate the dissemination of our pipeline with the development of MotilityJ, an open-source and user-friendly application for measuring bacteria spread on motility images. CONCLUSIONS: In this work, we have developed an algorithm and trained several models for measuring bacteria spread on motility images. Thanks to this work, the analysis of motility images will be faster and more reliable. The developed tools will help to advance our understanding of the behaviour and virulence of bacteria.

Antimicrobial resistance and virulence of Pseudomonas spp. among healthy animals: concern about exolysin ExlA detection.

Pseudomonas is a ubiquitous genus that also causes human, animal and plant diseases. Most studies have focused on clinical P. aeruginosa strains from humans, but they are scarce on animal strains. This study was aimed to determine the occurrence of Pseudomonas spp. among faecal samples of healthy animals, and to analyse their antimicrobial resistance, and pathogenicity. Among 704 animal faecal samples analysed, 133 Pseudomonas spp. isolates (23 species) were recovered from 46 samples (6.5%), and classified in 75 different PFGE patterns. Low antimicrobial resistance levels were found, being the highest to aztreonam (50.3%). Five sequence-types (ST1648, ST1711, ST2096, ST2194, ST2252), two serotypes (O:3, O:6), and three virulotypes (analysing 15 virulence and quorum-sensing genes) were observed among the 9 P. aeruginosa strains. Type-3-Secretion System genes were absent in the six O:3-serotype strains that additionally showed high cytotoxicity and produced higher biofilm biomass, phenazine pigments and motility than PAO1 control strain. In these six strains, the exlAB locus, and other virulence genotypes (e.g. RGP69 pathogenicity island) exclusive of PA7 outliers were detected by whole genome sequencing. This is the first description of the presence of the ExlA exolysin in P. aeruginosa from healthy animals, highlighting their pathological importance.