Effect of DMPEI coating against biofilm formation on PVC catheter surface.

Urinary tract infections (UTIs) are a significant cause of morbidity in healthcare systems and are prominently associated with applying urethral catheters, particularly in surgeries. Polyvinyl chloride (PVC) is extensively utilized in the fabrication of catheters. Biofilms, complex polymeric constructions, provide a protective milieu for cell multiplication and the enhancement of antibiotic resistance. Strategies to counteract biofilm development on medical apparatuses' surfaces incorporate antimicrobial agents such as N,N-dodecyl, and methyl polyethylenimine (DMPEI). This research endeavored to characterize the morphology of PVC and PVC-DMPEI surfaces utilizing Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) and to gauge hydrophobicity through contact angle measurements. Employing Escherichia coli, Staphylococcus aureus, and Candida albicans in adhesion assays enabled the assessment of DMPEI's efficacy in preventing microbial adherence to PVC. Butanol successfully solubilized 2 mg.mL-1 DMPEI without altering the PVC structure. SEM results substantiated the formation of a DMPEI layer on the PVC surface, which led to decreased surface roughness, as validated by AFM, and increased hydrophilicity, as demonstrated by contact angle evaluations. E. coli, S. aureus, and C. albicans exhibited significant adhesion reduction, 89.3%, 94.3%, and 86.6% on PVC-DMPEI surfaces. SEM visualizations confirmed reduced cellular colonization on PVC-DMPEI and highlighted considerable morphological modifications in E. coli. Consequently, DMPEI films effectively minimize the adhesion of E. coli, S. aureus, and C. albicans on PVC surfaces. DMPEI, with its potential as a protective coating for innovative medical devices, promises to inhibit biofilm adherence effectively.

Evaluation of Spore Acquisition, Spore Production, and Host Survival Time for Tea Shot-Hole Borer, Euwallacea perbrevis, Adults after Exposure to Four Commercial Products Containing Beauveria bassiana.

Euwallacea perbrevis, the tea shot-hole borer (TSHB), is an invasive ambrosia beetle that vectors several fungal pathogens that cause Fusarium branch dieback in avocado trees in southern Florida. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing adult TSHB beetles. Formulated products containing Bb strains to which adult beetles were exposed were BioCeres WP, BotaniGard WP, BotaniGard ES, and Velifer ES. Controls consisted of water only and BotaniGard ES and Velifer ES supernatant with spores removed. Acquisition of spores by adult beetles dipped in product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the Bb-based products in an in vivo avocado bark plug bioassay was determined. Production of Bb spores on beetles after being dipped in product suspensions and placed in a moistened bark-plug assay with water only was assessed. Significantly more spores were acquired by beetles exposed to Velifer ES and BotaniGard ES than beetles exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6-8 days) compared to beetles dipped in the other fungal products (10-11 days) and controls (12 days). Percentage of mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES (20 ± 6.4 × 105 spores/cadaver) was the highest among all treatments, whereas it was the lowest on cadavers of beetles dipped in BotaniGard ES (1 ± 0.2 × 105 spores/cadaver). All Bb-based products, especially Velifer ES, demonstrated potential to manage TSHB populations under laboratory conditions. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results.

Flexibility in the ambrosia symbiosis of Xyleborus bispinatus.

Introduction: Ambrosia beetles maintain strict associations with specific lineages of fungi. However, anthropogenic introductions of ambrosia beetles into new ecosystems can result in the lateral transfer of their symbionts to other ambrosia beetles. The ability of a Florida endemic ambrosia beetle, Xyleborus bispinatus, to feed and establish persistent associations with two of its known symbionts (Raffaelea subfusca and Raffaelea arxii) and two other fungi (Harringtonia lauricola and Fusarium sp. nov.), which are primary symbionts of invasive ambrosia beetles, was investigated. Methods: The stability of these mutualisms and their effect on the beetle's fitness were monitored over five consecutive generations. Surface-disinfested pupae with non-developed mycangia were reared separately on one of the four fungal symbionts. Non-treated beetles (i.e., lab colony) with previously colonized mycangia were used as a control group. Results: Xyleborus bispinatus could exchange its fungal symbionts, survive, and reproduce on different fungal diets, including known fungal associates and phylogenetically distant fungi, which are plant pathogens and primary symbionts of other invasive ambrosia beetles. These changes in fungal diets resulted in persistent mutualisms, and some symbionts even increased the beetle's reproduction. Females that developed on Fusarium sp. nov. had a significantly greater number of female offspring than non-treated beetles. Females that fed solely on Harringtonia or Raffaelea symbionts produced fewer female offspring. Discussion: Even though some ambrosia beetles like X. bispinatus can partner with different ambrosia fungi, their symbiosis under natural conditions is modulated by their mycangium and possibly other environmental factors. However, exposure to symbionts of invasive beetles can result in stable partnerships with these fungi and affect the population dynamics of ambrosia beetles and their symbionts.

Volatile Emissions and Relative Attraction of the Fungal Symbionts of Tea Shot Hole Borer (Coleoptera: Curculionidae).

Euwallacea perbrevis is an ambrosia beetle that vectors fungal pathogens causing Fusarium dieback in Florida avocado trees. Current monitoring lures contain quercivorol, a fungus-produced volatile, but the exact attractant is unknown since lures contain a mixture of p-menth-2-en-1-ol isomers and both α- and ß-phellandrene. This study used pure cultures of six symbiotic fungi isolated from E. perbrevis to document volatile emissions and determine the relative attraction of symbionts in binary choice assays. In a comparative test, headspace solid-phase microextraction followed by gas chromatography-mass spectroscopy was used to identify and quantify emissions from 3-week-old cultures. In a temporal study, Super-Q collection followed by gas chromatography-flame ionization detection was used to measure cis- and trans-p-menth-2-en-1-ol emissions for three months. A total of 15 compounds were detected, with monoterpene hydrocarbons and oxygenated monoterpenoids predominating. Only trans-p-menth-2-en-1-ol was common to all six symbionts. Peak levels of both isomers were observed at day 7, then gradually declined over a 90 day period. In choice tests, avocado sawdust disks inoculated with Fusarium sp. nov. were the most attractive. This symbiont produced only two volatiles, trans-p-menth-2-en-1-ol and limonene. The combined results indicate that trans-p-menth-2-en-1-ol is the primary female attractant emitted from symbiotic fungi, but limonene may be a secondary attractant of E. perbrevis.

Detection of the Lychee Erinose Mite, Aceria litchii (Keifer) (Acari: Eriophyidae) in Florida, USA: A Comparison with Other Alien Populations.

The lychee erinose mite (LEM), Aceria litchii (Keifer) is a serious pest of lychee (Litchi chinensis Sonn.). LEM causes a type of gall called 'erineum' (abnormal felty growth of trichomes from the epidermis), where it feeds, reproduces and protects itself from biotic and abiotic adversities. In February of 2018, LEM was found in a commercial lychee orchard on Pine Island, Florida. Infestations were recorded on young leaves, stems, and inflorescences of approximately 30 young trees (1.5-3.0 yrs.) of three lychee varieties presenting abundant new growth. Although LEM is present in Hawaii, this mite is a prioritized quarantine pest in the continental USA and its territories. Florida LEM specimens showed small morphological differences from the original taxonomic descriptions of Keifer (1943) and Huang (2008). The observed differences are probably an artifact of the drawings in the original descriptions. Molecular comparisons were conducted on the DNA of LEM specimens from India, Hawaii, Brazil, Taiwan, Australia and Florida. The amplified COI fragment showed very low nucleotide variation among the locations and thus, could be used for accurate LEM identification. The ITS1 sequences and partial 5.8S fragments displayed no nucleotide differences for specimens from any of the locations except Australia. Consistent differences were observed in the ITS2 and 28S fragments. The ITS1-ITS2 concatenated phylogeny yielded two lineages, with Australia in one group and Hawaii, India, Brazil, Florida and Taiwan in another. Specimens from Taiwan and Florida present identical ITS and rDNA segments, suggesting a common origin; however, analysis of additional sequences is needed to confirm the origin of the Florida population.

Xyleborus volvulus (Coleoptera: Curculionidae): Biology and Fungal Associates.

The ambrosia beetle Xyleborus volvulus Fabricius has been reported as a potential vector of the plant pathogen Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva that is affecting avocado orchards in South Florida. In this study, we examined its life cycle, process of gallery formation, gallery structure, and fungal associates by rearing one generation on avocado sawdust medium under control conditions. The adult foundress excavated a vertical tunnel that constituted the main gallery with a length of 2.5 cm, followed by the construction of up to six secondary galleries with a total length of 4.4 cm. The time period for one generation (egg to adult) was 28 days. Teneral males emerged 3 days after the emergence of the first females. The F1 generation did not significantly contribute to gallery expansion. Four species of Raffaelea and nine yeast species were recovered from galleries and beetles. Raffaelea arxii and Candida berthetii were the most frequent symbionts recovered from new adults and galleries. Candida berthetii dominated during the early stages of the gallery development, whereas R. arxii was most frequent in later stages. Other Raffaelea species were inconsistently isolated from galleries, which suggests a strong association between Xyleborus volvulus and both R. arxii and C. berthetii These results suggest that R. arxii is the primary nutritional symbiont of X. volvulus and that yeast species may be pioneer colonizers that assist with the growth of fungal symbionts.IMPORTANCE Ambrosia beetles cultivate fungi in tunnels bored into weakened host trees. This obligate interaction is required for their survival as beetles feed on these symbiotic fungi, and the fungi benefit from transportation by the beetles. Xyleborus volvulus carries many nonpathogenic symbionts; however, recently the acquisition of Raffaelea lauricola (the causal agent of a lethal vascular disease of lauraceous trees) by this beetle has altered its status from wood degrader to potential pest in avocado. We conducted a study to understand the relationship of this beetle and its fungal associates. Our results show that X. volvulus has a multipartite flexible association with different Raffaelea species. The lack of fidelity in the mutualistic association may explain the acquisition of R. lauricola Knowing the beetle biology and its mutualistic interactions furthers an understanding of the beetle's role as a potential vector and in disease transmission.

Xyleborus bispinatus Reared on Artificial Media in the Presence or Absence of the Laurel Wilt Pathogen (Raffaelea lauricola).

Like other members of the tribe Xyleborini, Xyleborus bispinatus Eichhoff can cause economic damage in the Neotropics. X. bispinatus has been found to acquire the laurel wilt pathogen Raffaelea lauricola (T. C. Harr., Fraedrich & Aghayeva) when breeding in a host affected by the pathogen. Its role as a potential vector of R. lauricola is under investigation. The main objective of this study was to evaluate three artificial media, containing sawdust of avocado (Persea americana Mill.) and silkbay (Persea humilis Nash.), for rearing X. bispinatus under laboratory conditions. In addition, the media were inoculated with R. lauricola to evaluate its effect on the biology of X. bispinatus. There was a significant interaction between sawdust species and R. lauricola for all media. Two of the media supported the prolific reproduction of X. bispinatus, but the avocado-based medium was generally more effective than the silkbay-based medium, regardless whether or not it was inoculated with R. lauricola. R. lauricola had a neutral or positive effect on beetle reproduction. The pathogen was frequently recovered from beetle galleries, but only from a few individuals which were reared on inoculated media, and showed limited colonization of the beetle's mycangia. Two media with lower water content were most effective for rearing X. bispinatus.

Rearing Xyleborus volvulus (Coleoptera: Curculionidae) on Media Containing Sawdust from Avocado or Silkbay, With or Without Raffaelea lauricola (Ophiostomatales: Ophiostomataceae).

Like other ambrosia beetles, Xyleborus volvulus Fabricius (Coleoptera: Curculionidae) lives in a mutualistic symbiotic relationship with fungi that serve as food source. Until recently, X. volvulus was not considered a pest, and none of its symbionts were considered plant pathogens. However, recent reports of an association between X. volvulus and Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva (Ophiostomatales: Ophiostomataceae), the cause of the laurel wilt disease of avocado (Persea americana Mill. [Laurales: Lauraceae]), and its potential role as vector of the pathogen merit further investigation. The objective of this study was to evaluate three artificial media containing sawdust obtained from avocado or silkbay (Persea humilis Nash) for laboratory rearing of X. volvulus. The effect of R. lauricola in the media on the beetle's reproduction was also evaluated. Of the three media, the one with the lowest content of sawdust and intermediate water content provided the best conditions for rearing X. volvulus. Reproduction on this medium was not affected by the sawdust species or the presence of R. lauricola. On the other two media, there was a significant interaction between sawdust species and R. lauricola. The presence of R. lauricola generally had a negative effect on brood production. There was limited colonization of the mycangia of X. volvulus by R. lauricola on media inoculated with the pathogen. From galleries formed within the best medium, there was 50% recovery of R. lauricola, but recovery was much less from the other two media. Here, we report the best artificial substrate currently known for X. volvulus.

The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a ß-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

Distribution, Pest Status and Fungal Associates of Euwallacea nr. fornicatus in Florida Avocado Groves.

Members of a complex of cryptic species, that correspond morphologically to the ambrosia beetle Euwallacea fornicatus (Eichhoff) (Coleoptera: Curculionidae: Scolytinae), were recently found attacking avocado (Persea americana Mill.) in Israel and California. In early 2016, an outbreak of another member of this species complex was detected infesting approximately 1500 avocado trees in an avocado orchard at Homestead, Florida. An area-wide survey was conducted in commercial avocado groves of Miami-Dade County, Florida to determine the distribution and abundance of E. nr. fornicatus, to identify different populations of E. nr. fornicatus and their fungal associates, and to assess the extent of damage to avocado trees. Ewallacea nr. fornicatus were captured in 31 of the 33 sampled sites. A sample of 35 beetles from six different locations was identified as E. nr. fornicatus sp. #2, which is genetically distinct from the species causing damage in California and Israel. Eleven fungal associates were identified: an unknown Fusarium sp., AF-8, AF-6, Graphium euwallaceae, Acremonium sp. Acremonium morum, Acremonium masseei, Elaphocordyceps sp. and three yeast species. The unknown Fusarium isolates were the most abundant and frequently found fungus species associated with adult beetles and lesions surrounding the beetle galleries. In addition to fungal associates, three bacteria species were found associated with adult E. nr. fornicatus. Visual inspections detected significant damage in only two orchards. A large number of beetles were captured in locations with no apparent damage on the avocado trees suggesting that E. nr. fornicatus are associated with other host(s) outside the groves or with dead trees or branches inside the groves. More research is needed to determine the potential threat E. nr. fornicatus and its fungal associates pose to the avocado industry and agricultural and natural ecosystems in Florida.

Presence of calcium-binding motifs in PilY1 homologs correlates with Ca-mediated twitching motility and evolutionary history across diverse bacteria.

Twitching motility, involving type IV pili, is essential for host colonization and virulence of many pathogenic bacteria. Studies of PilY1, a tip-associated type IV pili protein, indicate that PilY1 functions as a switch between pilus extension and retraction, resulting in twitching motility. Recent work detected a calcium-binding motif in PilY1 of some animal bacterial pathogens and demonstrated that binding of calcium to PilY1 with this motif regulates twitching. Though studies of PilY1 in non-animal pathogens are limited, our group demonstrated that twitching motility in the plant pathogen Xylella fastidiosa, which contains three PilY1 homologs, is increased by calcium supplementation. A study was conducted to investigate the phylogenetic relationship between multiple PilY1 homologs, the presence of calcium-binding motifs therein, and calcium-mediated twitching motility across diverse bacteria. Strains analyzed contained one to three PilY1 homologs, but phylogenetic analyses indicated that PilY1 homologs containing the calcium-binding motif Dx[DN]xDGxxD are phylogenetically divergent from other PilY1 homologs. Plant-associated bacteria included in these analyses were then examined for a calcium-mediated twitching response. Results indicate that bacteria must have at least one PilY1 homolog containing the Dx[DN]xDGxxD motif to display a calcium-mediated increase in twitching motility, which likely reflects an adaption to environmental calcium concentrations.

Calcium-Enhanced Twitching Motility in Xylella fastidiosa Is Linked to a Single PilY1 Homolog.

The plant-pathogenic bacterium Xylella fastidiosa is restricted to the xylem vessel environment, where mineral nutrients are transported through the plant host; therefore, changes in the concentrations of these elements likely impact the growth and virulence of this bacterium. Twitching motility, dependent on type IV pili (TFP), is required for movement against the transpiration stream that results in basipetal colonization. We previously demonstrated that calcium (Ca) increases the motility of X. fastidiosa, although the mechanism was unknown. PilY1 is a TFP structural protein recently shown to bind Ca and to regulate twitching and adhesion in bacterial pathogens of humans. Sequence analysis identified three pilY1 homologs in X. fastidiosa (PD0023, PD0502, and PD1611), one of which (PD1611) contains a Ca-binding motif. Separate deletions of PD0023 and PD1611 resulted in mutants that still showed twitching motility and were not impaired in attachment or biofilm formation. However, the response of increased twitching at higher Ca concentrations was lost in the pilY1-1611 mutant. Ca does not modulate the expression of any of the X. fastidiosa PilY1 homologs, although it increases the expression of the retraction ATPase pilT during active movement. The evidence presented here suggests functional differences between the PilY1 homologs, which may provide X. fastidiosa with an adaptive advantage in environments with high Ca concentrations, such as xylem sap.

Xylella fastidiosa differentially accumulates mineral elements in biofilm and planktonic cells.

Xylella fastidiosa is a bacterial plant pathogen that infects numerous plant hosts. Disease develops when the bacterium colonizes the xylem vessels and forms a biofilm. Inductively coupled plasma optical emission spectroscopy was used to examine the mineral element content of this pathogen in biofilm and planktonic states. Significant accumulations of copper (30-fold), manganese (6-fold), zinc (5-fold), calcium (2-fold) and potassium (2-fold) in the biofilm compared to planktonic cells were observed. Other mineral elements such as sodium, magnesium and iron did not significantly differ between biofilm and planktonic cells. The distribution of mineral elements in the planktonic cells loosely mirrors the media composition; however the unique mineral element distribution in biofilm suggests specific mechanisms of accumulation from the media. A cell-to-surface attachment assay shows that addition of 50 to 100 µM Cu to standard X. fastidiosa media increases biofilm, while higher concentrations (>200 µM) slow cell growth and prevent biofilm formation. Moreover cell-to-surface attachment was blocked by specific chelation of copper. Growth of X. fastidiosa in microfluidic chambers under flow conditions showed that addition of 50 µM Cu to the media accelerated attachment and aggregation, while 400 µM prevented this process. Supplementation of standard media with Mn showed increased biofilm formation and cell-to-cell attachment. In contrast, while the biofilm accumulated Zn, supplementation to the media with this element caused inhibited growth of planktonic cells and impaired biofilm formation. Collectively these data suggest roles for these minerals in attachment and biofilm formation and therefore the virulence of this pathogen.

Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility.

Xylella fastidiosa is a plant-pathogenic bacterium that forms biofilms inside xylem vessels, a process thought to be influenced by the chemical composition of xylem sap. In this work, the effect of calcium on the production of X. fastidiosa biofilm and movement was analyzed under in vitro conditions. After a dose-response study with 96-well plates using eight metals, the strongest increase of biofilm formation was observed when medium was supplemented with at least 1.0 mM CaCl(2). The removal of Ca by extracellular (EGTA, 1.5 mM) and intracellular [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), 75 µM] chelators reduced biofilm formation without compromising planktonic growth. The concentration of Ca influenced the force of adhesion to the substrate, biofilm thickness, cell-to-cell aggregation, and twitching motility, as shown by assays with microfluidic chambers and other assays. The effect of Ca on attachment was lost when cells were treated with tetracycline, suggesting that Ca has a metabolic or regulatory role in cell adhesion. A double mutant (fimA pilO) lacking type I and type IV pili did not improve biofilm formation or attachment when Ca was added to the medium, while single mutants of type I (fimA) or type IV (pilB) pili formed more biofilm under conditions of higher Ca concentrations. The concentration of Ca in the medium did not significantly influence the levels of exopolysaccharide produced. Our findings indicate that the role of Ca in biofilm formation may be related to the initial surface and cell-to-cell attachment and colonization stages of biofilm establishment, which rely on critical functions by fimbrial structures.