Early transcriptional changes of heavy metal resistance and multiple efflux genes in Xanthomonas campestris pv. campestris under copper and heavy metal ion stress.

BACKGROUND: Copper-induced gene expression in Xanthomonas campestris pv. campestris (Xcc) is typically evaluated using targeted approaches involving qPCR. The global response to copper stress in Xcc and resistance to metal induced damage is not well understood. However, homologs of heavy metal efflux genes from the related Stenotrophomonas genus are found in Xanthomonas which suggests that metal related efflux may also be present. METHODS AND RESULTS: Gene expression in Xcc strain BrA1 exposed to 0.8 mM CuSO4.5H2O for 15 minutes was captured using RNA-seq analysis. Changes in expression was noted for genes related to general stress responses and oxidoreductases, biofilm formation, protein folding chaperones, heat-shock proteins, membrane lipid profile, multiple drug and efflux (MDR) transporters, and DNA repair were documented. At this timepoint only the cohL (copper homeostasis/tolerance) gene was upregulated as well as a chromosomal czcCBA efflux operon. An additional screen up to 4 hrs using qPCR was conducted using a wider range of heavy metals. Target genes included a cop-containing heavy metal resistance island and putative metal efflux genes. Several efflux pumps, including a copper resistance associated homolog from S. maltophilia, were upregulated under toxic copper stress. However, these pumps were also upregulated in response to other toxic heavy metals. Additionally, the temporal expression of the coh and cop operons was also observed, demonstrating co-expression of tolerance responses and later activation of part of the cop operon. CONCLUSIONS: Overall, initial transcriptional responses focused on combating oxidative stress, mitigating protein damage and potentially increasing resistance to heavy metals and other biocides. A putative copper responsive efflux gene and others which might play a role in broader heavy metal resistance were also identified. Furthermore, the expression patterns of the cop operon in conjunction with other copper responsive genes allowed for a better understanding of the fate of copper ions in Xanthomonas. This work provides useful evidence for further evaluating MDR and other efflux pumps in metal-specific homeostasis and tolerance phenotypes in the Xanthomonas genus. Furthermore, non-canonical copper tolerance and resistance efflux pumps were potentially identified. These findings have implications for interpreting MIC differences among strains with homologous copLAB resistance genes, understanding survival under copper stress, and resistance in disease management.

Crucifer Lesion-Associated Xanthomonas Strains Show Multi-Resistance to Heavy Metals and Antibiotics.

Copper resistance in phytopathogens is a major challenge to crop production globally and is known to be driven by excessive use of copper-based pesticides. However, recent studies have shown co-selection of multiple heavy metal and antibiotic resistance genes in bacteria exposed to heavy metal and xenobiotics, which may impact the epidemiology of plant, animal, and human diseases. In this study, multi-resistance to heavy metals and antibiotics were evaluated in local Xanthomonas campestris pv. campestris (Xcc) and co-isolated Xanthomonas melonis (Xmel) strains from infected crucifer plants in Trinidad. Resistance to cobalt, cadmium, zinc, copper, and arsenic (V) was observed in both Xanthomonas species up to 25 mM. Heavy metal resistance (HMR) genes were found on a small plasmid-derived locus with ~ 90% similarity to a Stenotrophomonas spp. chromosomal locus and a X. perforans pLH3.1 plasmid. The co-occurrence of mobile elements in these regions implies their organization on a composite transposon-like structure. HMR genes in Xcc strains showed the lowest similarity to references, and the cus and ars operons appear to be unique among Xanthomonads. Overall, the similarity of HMR genes to Stenotrophomonas sp. chromosomal genomes suggest their origin in this genus or a related organism and subsequent spread through lateral gene transfer events. Further resistome characterization revealed the presence of small multidrug resistance (SMR), multidrug resistance (MDR) efflux pumps, and bla (Xcc) genes for broad biocide resistance in both species. Concurrently, resistance to antibiotics (streptomycin, kanamycin, tetracycline, chloramphenicol, and ampicillin) up to 1000 µg/mL was confirmed.

Whole genome sequencing reveals complex resistome features of Klebsiella pneumoniae isolated from patients at major hospitals in Trinidad, West Indies.

OBJECTIVES: Antibiotic-resistant Klebsiella pneumoniae is a human pathogen of major global concern due to its ability to cause multiple severe diseases that are often difficult to treat therapeutically. This study aimed to investigate the resistome of local clinical K. pneumoniae isolates. METHODS: Herein, we used a whole genome sequencing approach and bioinformatics tools to reconstruct the resistome of 10 clinical K. pneumoniae isolates and one clinical isolate of the closely related Klebsiella quasipneumoniae obtained from patients from three major hospitals in Trinidad, West Indies. RESULTS: The results of the study revealed the presence of a complex antibiotic-resistant armoury among the local isolates with multiple resistance mechanisms involving (i) inactivation of antibiotics, (ii) efflux pumps, (iii) antibiotic target alteration, protection, and replacement against antibiotics, and (iv) altered porin protein that reduced the permeability to antibiotics. Several resistance genes such as blaCTX-M-15, blaTEM-1B, blaSHV-28, blaKPC-2, oqxA, sul1, tetD, aac(6')-Ib-cr5, aph(6)-Id, and fosA6, which are known to confer resistance to antibiotics used to treat K. pneumoniae infections. In most cases, the resistance genes were flanked by mobile elements, including insertion sequences and transposons, which facilitate the spread of these genetic features among related organisms. CONCLUSION: This is the first comprehensive study to thoroughly investigate the resistome of clinical K. pneumoniae isolates and K. quasipneumoniae from Trinidad, West Indies. These findings suggest that monitoring K. pneumoniae and its genome-wide antibiotic resistance features in clinical strains would be of critical importance for guiding antibiotic stewardship programs and improving regional disease management systems for this pathogen.

Multi-trait efficiency and interactivity of bacterial consortia used to enhance plant performance under water stress conditions.

Water stress is a major limiting factor for agricultural production under current and projected climate change scenarios. As a sustainable strategy, plant growth-promoting bacterial consortia have been used to reduce plant water stress. However, few studies have examined the effects of stress on multi-trait efficiency and interactivity of bacterial species. In this study, we used several in-vitro experiments, plant assays and greenhouse trials to investigate the effects of stress and bacterial consortia on 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) activities, indole-3-acetic acid (IAA) production and plant growth-promoting traits (Phosphate-solubilization, starch hydrolysis, siderophores and ammonium production). We further assessed biofilm formation and the chemotactic behaviour in response to ACC. A total of fifteen ACCD rhizobacteria with multiple growth-promoting traits from the dominant plant species from the hyperseasonal Aripo Savannas were screened in this study. Five of the isolates were further analyzed based on their ACCD activities and were tested in single and dual consortium to assess their abilities in promoting growth under simulated drought stress (-0.35 MPa) and chemically induced ACC conditions (0.03 mM). Our findings showed that bacteria which produce high concentrations of IAA affected the isolates' ability to promote growth under stress, irrespective of microbial combination with ACCD activity above the minimal threshold of 20 nmol α-ketobutyrate mg-1 h-1. Biofilm production with co-culture interaction varied greatly across treatments, however, the general trend showed an increase in biofilm under stress induce conditions. The best performing co-culture, UWIGT-83 and UWIGT-120 (Burkholderia sp.) showed enhanced growth in germination assays and in greenhouse trials with Capsicum chinense (Moruga red hot peppers) under drought stress, when compared to non-inoculated treatments. The findings highlight the importance of testing interactivity of bacterial species with multiple growth promoting traits under stress conditions; and proposed the use of ACC growth media as a novel biofilm screening method for selecting potential stress plant growth-promoting bacteria. Better screening strategies for appropriate plant growth-promoting bacteria may narrow the inconsistency observed between laboratory and field trials.

DNA Barcoding Analysis of Trinidad Haemagogus Mosquitoes Reveals Evidence for Putative New Species.

Background: Haemagogus janthinomys is a primary sylvan vector of yellow fever virus and the emerging Mayaro virus. However, despite its medical importance, there is a dearth of data on the molecular taxonomy of this mosquito species. Methods: In this study, DNA barcoding analysis was performed on 64 adult female mosquitoes from Trinidad morphologically identified as Hg. janthinomys. The mitochondrial cytochrome c oxidase I (COI) gene and ribosomal DNA internal transcribed spacer 2 (ITS2) region of the mosquitoes were PCR amplified and sequenced, and molecular phylogenies inferred. Results: The BLASTN analysis showed that only 20% (n = 13/66) of COI sequences had high similarity (>99% identity) to Hg. janthinomys and the remaining sequences had low similarity (<90% identity) to reference GenBank sequences. Phylogenetic analysis of COI sequences revealed the presence of four strongly supported groups, with one distinct clade that did not align with any reference sequences. Corresponding ITS2 sequences for samples in this distinct COI group clustered into three clades. Conclusions: These molecular findings suggest the existence of a putative new Haemagogus mosquito species and underscore the need for further, more in-depth investigations into the taxonomy and classification of the Haemagogus genus.