Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669—Potential Zoonotic Pathogens Isolated from Spotted Turtles

Antimicrobial resistance (AMR) is one of the biggest challenges of the 21st century, and biofilm formation enables bacteria to resist antibiotic at much higher concentrations than planktonic cells. Earlier, we showed that the Gram-negative Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669 (closely related to C. freundii NBRC 12681) from infected spotted turtles (Clemmys guttata), formed biofilms and upregulated toxin expression on plastic surfaces, and were predicted to possess multiple antibiotic resistance genes. Here, we show that they each resist several antibiotics in the planktonic phase, but were susceptible to neomycin, and high concentrations of tetracycline and cotrimoxazole. The susceptibility of their biofilms to neomycin and cotrimoxazole was tested using the Calgary device. For A. hydrophila, the minimum inhibitory concentration (MIC) = 500–1000, and the minimum biofilm eradication concentration (MBEC) > 1000 μg/mL, using cotrimoxazole, and MIC = 32.3–62.5, and MBEC > 1000 μg/mL, using neomycin. For C. freundii MIC = 7.8–15.6, and, MBEC > 1000 μg/mL, using cotrimoxazole, and MIC = 7.8, and MBEC > 1000 μg/mL, using neomycin. Both A. hydrophila and C. portucalensis activated an acyl homoserine lactone (AHL) dependent biosensor, suggesting that quorum sensing could mediate biofilm formation. Their multidrug resistance in the planktonic form, and weak biofilm eradication even with neomycin and cotrimoxazole, indicate that A. hydrophila and C. portucalensis are potential zoonotic pathogens, with risks for patients living with implants.

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Expression of a Shiga-Like Toxin during Plastic Colonization by Two Multidrug-Resistant Bacteria, Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669, Isolated from Endangered Turtles (Clemmys guttata)

Microorganisms ◽

10.3390/microorganisms8081172 ◽

2020 ◽

Vol 8 (8) ◽

pp. 1172 ◽

Cited By ~ 1

Author(s):

Seema G. Thomas ◽

Maryah A. Glover ◽

Anutthaman Parthasarathy ◽

Narayan H. Wong ◽

Paul A. Shipman ◽

...

Keyword(s):

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Phylogenetic Analyses ◽

Regulatory Protein ◽

Antibiotic Resistance Genes ◽

Fold Increase ◽

Resistant Bacteria ◽

Citrobacter Freundii ◽

Protein Subunit ◽

Clemmys Guttata

Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669 were isolated from endangered spotted turtles (Clemmys guttata). Whole-genome sequencing, annotation and phylogenetic analyses of the genomes revealed that the closest relative of RIT668 is A. hydrophila ATCC 7966 and Citrobacter portucalensis A60 for RIT669. Resistome analysis showed that A. hydrophila and C. freundii harbor six and 19 different antibiotic resistance genes, respectively. Both bacteria colonize polyethylene and polypropylene, which are common plastics, found in the environment and are used to fabricate medical devices. The expression of six biofilm-related genes—biofilm peroxide resistance protein (bsmA), biofilm formation regulatory protein subunit R (bssR), biofilm formation regulatory protein subunit S (bssS), biofilm formation regulator (hmsP), toxin-antitoxin biofilm protein (tabA) and transcriptional activator of curli operon (csgD)—and two virulence factors—Vi antigen-related gene (viaB) and Shiga-like toxin (slt-II)—was investigated by RT-PCR. A. hydrophila displayed a >2-fold increase in slt-II expression in cells adhering to both polymers, C. freundii adhering on polyethylene displayed a >2-fold, and on polypropylene a >6-fold upregulation of slt-II. Thus, the two new isolates are potential pathogens owing to their drug resistance, surface colonization and upregulation of a slt-II-type diarrheal toxin on polymer surfaces.

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Influence of Glucose Concentrations on Biofilm Formation, Motility, Exoprotease Production, and Quorum Sensing in Aeromonas hydrophila

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-321 ◽

2013 ◽

Vol 76 (2) ◽

pp. 239-247 ◽

Cited By ~ 51

Author(s):

IQBAL KABIR JAHID ◽

NA-YOUNG LEE ◽

ANNA KIM ◽

SANG-DO HA

Keyword(s):

Quorum Sensing ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Protease Production ◽

Acyl Homoserine Lactone ◽

Human Pathogen ◽

Initial Biofilm ◽

Motility Inhibition

Aeromonas hydrophila recently has received increased attention because it is opportunistic and a primary human pathogen. A. hydrophila biofilm formation and its control are a major concern for food safety because biofilms are related to virulence. Therefore, we investigated biofilm formation, motility inhibition, quorum sensing, and exoprotease production of this opportunistic pathogen in response to various glucose concentrations from 0.05 to 2.5% (wt/vol). More than 0.05% glucose significantly impaired (P < 0.05) quorum sensing, biofilm formation, protease production, and swarming and swimming motility, whereas bacteria treated with 0.05% glucose had activity similar to that of the control (0% glucose). A stage shift biofilm assay revealed that the addition of glucose (2.5%) inhibited initial biofilm formation but not later stages. However, addition of quorum sensing molecules N-3-butanoyl-DL-homoserine lactone and N-3-hexanoyl homoserine lactone partially restored protease production, indicating that quorum sensing is controlled by glucose concentrations. Thus, glucose present in food or added as a preservative could regulate acyl-homoserine lactone quorum sensing molecules, which mediate biofilm formation and virulence in A. hydrophila.

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Anti-quorum Sensing and Protective Efficacies of Naringin Against Aeromonas hydrophila Infection in Danio rerio

Frontiers in Microbiology ◽

10.3389/fmicb.2020.600622 ◽

2020 ◽

Vol 11 ◽

Author(s):

Ramanathan Srinivasan ◽

Kannan Rama Devi ◽

Sivasubramanian Santhakumari ◽

Arunachalam Kannappan ◽

Xiaomeng Chen ◽

...

Keyword(s):

Antibiotic Resistance ◽

Quorum Sensing ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Bacterial Load ◽

Antibiotic Resistance Genes ◽

Alternative Methods ◽

Zebrafish Model ◽

Virulence Gene Expression

It is now well known that the quorum sensing (QS) mechanism coordinates the production of several virulence factors and biofilm formation in most pathogenic microorganisms. Aeromonas hydrophila is a prime pathogen responsible for frequent outbreaks in aquaculture settings. Recent studies have also continuously reported that A. hydrophila regulates virulence factor production and biofilm formation through the QS system. In addition to the presence of antibiotic resistance genes, biofilm-mediated antibiotic resistance increases the severity of A. hydrophila infections. To control the bacterial pathogenesis and subsequent infections, targeting the QS mechanism has become one of the best alternative methods. Though very few compounds were identified as QS inhibitors against A. hydrophila, to date, the screening and identification of new and effective natural QS inhibitors is a dire necessity to control the infectious A. hydrophila. The present study endorses naringin (NA) as an anti-QS and anti-infective agent against A. hydrophila. Initially, the NA showed a concentration-dependent biofilm reduction against A. hydrophila. Furthermore, the results of microscopic analyses and quantitative virulence assays displayed the promise of NA as a potential anti-QS agent. Subsequently, the downregulation of ahh1, aerA, lip and ahyB validate the interference of NA in virulence gene expression. Furthermore, the in vivo assays were carried out in zebrafish model system to evaluate the anti-infective potential of NA. The outcome of the immersion challenge assay showed that the recovery rate of the zebrafish has substantially increased upon treatment with NA. Furthermore, the quantification of the bacterial load upon NA treatment showed a decreased level of bacterial counts in zebrafish when compared to the untreated control. Moreover, the NA treatment averts the pathogen-induced histoarchitecture damages in vital organs of zebrafish, compared to their respective controls. The current study has thus analyzed the anti-QS and anti-infective capabilities of NA and could be employed to formulate effective treatment measures against A. hydrophila infections.

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Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp

Marine Drugs ◽

10.3390/md17090499 ◽

2019 ◽

Vol 17 (9) ◽

pp. 499 ◽

Cited By ~ 2

Author(s):

Bao Zhang ◽

Xiyi Zhuang ◽

Liyun Guo ◽

Robert J. C. McLean ◽

Weihua Chu

Keyword(s):

Virulence Factors ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Crucian Carp ◽

Homoserine Lactone ◽

In Vivo Studies ◽

Acyl Homoserine Lactone ◽

Signal Molecule ◽

Promising Alternative ◽

Feed Supplementation

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.

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The Acyl Homoserine Lactone Receptor, SdiA, of Escherichia coli and Salmonella enterica Serovar Typhimurium Does Not Respond to Indole

Applied and Environmental Microbiology ◽

10.1128/aem.00046-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5424-5431 ◽

Cited By ~ 29

Author(s):

Anice Sabag-Daigle ◽

Jitesh A. Soares ◽

Jenée N. Smith ◽

Mohamed E. Elmasry ◽

Brian M. M. Ahmer

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Biofilm Formation ◽

Salmonella Enterica Serovar Typhimurium ◽

Homoserine Lactone ◽

Acyl Homoserine Lactone ◽

Content Type ◽

E Coli ◽

Serovar Typhimurium ◽

High Concentrations

ABSTRACTIn this study, we tested the hypothesis that the SdiA proteins ofEscherichia coliandSalmonella entericaserovar Typhimurium respond to indole. While indole was found to have effects on gene expression and biofilm formation, these effects were notsdiAdependent. However, high concentrations of indole did inhibitN-acyl-l-homoserine lactone (AHL) sensing by SdiA. We conclude that SdiA does not respond to indole but indole can inhibit SdiA activity inE. coliandSalmonella.

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Extract from phyllosphere bacteria with antibiofilm and quorum quenching activity to control several fish pathogenic bacteria

BMC Research Notes ◽

10.1186/s13104-021-05612-w ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Olivia Nathalia ◽

Diana Elizabeth Waturangi

Keyword(s):

Streptococcus Agalactiae ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Vibrio Harveyi ◽

Quorum Quenching ◽

Indicator Bacteria ◽

Antibiofilm Activity ◽

Fish Pathogen ◽

Phyllosphere Bacteria

Abstract Objective The objective of this research were to screen quorum quenching activity compound from phyllosphere bacteria as well as antibiofilm activity against several fish pathogen bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. Results We found eight phyllosphere bacteria isolates with potential quorum quenching activity to inhibit Chromobacterium violaceum as indicator bacteria. Crude extracts (20 mg/mL) showed various antibiofilm activity against fish pathogenic bacteria used in this study. Isolate JB 17B showed the highest activity to inhibit biofilm formation of A. hydrophila and V. harveyi, meanwhile isolate JB 3B showed the highest activity to inhibit biofilm of S. agalactiae. From destruction assay, isolate JB 8F showed the highest activity to disrupt biofilm of A. hydrophila isolate JB 20B showed the highest activity to disrupt biofilm of V. harveyi, isolate JB 17B also showed the highest activity to disrupt biofilm of S. agalactiae.

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Virulence Factors in Coagulase-Negative Staphylococci

Pathogens ◽

10.3390/pathogens10020170 ◽

2021 ◽

Vol 10 (2) ◽

pp. 170

Author(s):

Angela França ◽

Vânia Gaio ◽

Nathalie Lopes ◽

Luís D. R. Melo

Keyword(s):

Antibiotic Resistance ◽

Virulence Factors ◽

Resistance Genes ◽

Biofilm Formation ◽

Antibiotic Resistance Genes ◽

Coagulase Negative Staphylococci ◽

Healthcare Associated ◽

Major Pathogens

Coagulase-negative staphylococci (CoNS) have emerged as major pathogens in healthcare-associated facilities, being S. epidermidis, S. haemolyticus and, more recently, S. lugdunensis, the most clinically relevant species. Despite being less virulent than the well-studied pathogen S. aureus, the number of CoNS strains sequenced is constantly increasing and, with that, the number of virulence factors identified in those strains. In this regard, biofilm formation is considered the most important. Besides virulence factors, the presence of several antibiotic-resistance genes identified in CoNS is worrisome and makes treatment very challenging. In this review, we analyzed the different aspects involved in CoNS virulence and their impact on health and food.

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Prebiotic Combinations Effects on the Colonization of Staphylococcal Skin Strains

Microorganisms ◽

10.3390/microorganisms9010037 ◽

2020 ◽

Vol 9 (1) ◽

pp. 37

Author(s):

Silvia Di Lodovico ◽

Franco Gasparri ◽

Emanuela Di Campli ◽

Paola Di Fermo ◽

Simonetta D’Ercole ◽

...

Keyword(s):

Biofilm Formation ◽

Commensal Bacteria ◽

The Other ◽

Bacteriostatic Effect ◽

Skin Microbiota ◽

Relevant Effect ◽

Planktonic Phase ◽

Species Specific ◽

Biomass Quantification ◽

General Reduction

Background: An unbalanced skin microbiota due to an increase in pathogenic vs. commensal bacteria can be efficiently tackled by using prebiotics. The aim of this work was to identify novel prebiotic combinations by exerting species-specific action between S. aureus and S. epidermidis strains. Methods: First, the antimicrobial/antibiofilm effect of Xylitol-XYL and Galacto-OligoSaccharides–GOS combined with each other at different concentrations (1, 2.5, 5%) against S. aureus and S. epidermidis clinical strains was evaluated in time. Second, the most species-specific concentration was used to combine XYL with Fructo-OligoSaccharides–FOS, IsoMalto-Oligosaccharides–IMO, ArabinoGaLactan–LAG, inulin, dextran. Experiments were performed by OD600 detection, biomass quantification and LIVE/DEAD staining. Results: 1% XYL + 1% GOS showed the best species-specific action with an immediate antibacterial/antibiofilm action against S. aureus strains (up to 34.54% ± 5.35/64.68% ± 4.77) without a relevant effect on S. epidermidis. Among the other prebiotic formulations, 1% XYL plus 1% FOS (up to 49.17% ± 21.46/37.59% ± 6.34) or 1% IMO (up to 41.28% ± 4.88/36.70% ± 10.03) or 1% LAG (up to 38.21% ± 5.31/83.06% ± 5.11) showed antimicrobial/antibiofilm effects similar to 1% XYL+1% GOS. For all tested formulations, a prevalent bacteriostatic effect in the planktonic phase and a general reduction of S. aureus biofilm formation without loss of viability were recorded. Conclusion: The combinations of 1% XYL with 1% GOS or 1% FOS or 1% IMO or 1% LAG may help to control the balance of skin microbiota, representing good candidates for topic formulations.

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Attenuation of Aeromonas hydrophila Infection in Carassius auratus by YtnP, a N-acyl Homoserine Lactonase from Bacillus licheniformis T-1

Antibiotics ◽

10.3390/antibiotics10060631 ◽

2021 ◽

Vol 10 (6) ◽

pp. 631

Author(s):

Mengfan Peng ◽

Wentao Tong ◽

Zhen Zhao ◽

Ling Xiao ◽

Zhaoyue Wang ◽

...

Keyword(s):

Virulence Factors ◽

Bacillus Licheniformis ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Carassius Auratus ◽

Quorum Quenching ◽

Inhibition Rate ◽

Sequence Identity

In this experiment, the quorum quenching gene ytnP of Bacillus licheniformis T-1 was cloned and expressed, and the effect against infection of Aeromonas hydrophila ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the ytnP gene of T-1 and its homolog in B.subtilis sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with B.subtilis 3610, which was categorized as the Aidc cluster of the MBL family. The AHL lactonase activity of the purified YtnP was detected as 1.097 ± 0.7 U/mL with C6-HSL as the substrate. Otherwise, purified YtnP protein could significantly inhibit the biofilm formation of A.hydrophila ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against A. hydrophila reduced from 4 μg/mL and 0.5 μg/mL to 1 μg/mL and 0.125 μg/mL, respectively, in the presence of YtnP. In addition, YtnP significantly inhibited the expression of five virulence factors hem, ahyB, ast, ep, aerA of A. hydrophila ATCC 7966 as well (p < 0.05). The results of inhibition on virulence showed a time-dependence tendency, while the strongest anti-virulence effects were within 4–24 h. In vivo, when the YtnP protein was co-injected intraperitoneally with A. hydrophila ATCC 7966, it attenuated the pathogenicity of A. hydrophila and the accumulated mortality was 27 ± 4.14% at 96 h, which was significantly lower than the average mortality of 78 ± 2.57% of the Carassius auratus injected with 108 CFU/mL of A. hydrophila ATCC 7966 only (p < 0.001). In conclusion, the AHL lactonase in B. licheniformis T-1 was proven to be YtnP protein and could be developed into an agent against infection of A. hydrophila in aquaculture.

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Biofilm Formation, Communication and Interactions of Mesophilic Leaching Bacteria during Pyrite Oxidation

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.825.107 ◽

2013 ◽

Vol 825 ◽

pp. 107-110

Author(s):

Sören Bellenberg ◽

Robert Barthen ◽

Mario Vera ◽

Nicolas Guiliani ◽

Wolfgang Sand

Keyword(s):

Quorum Sensing ◽

Acidithiobacillus Ferrooxidans ◽

Biofilm Formation ◽

Pyrite Oxidation ◽

Cell Communication ◽

Homoserine Lactone ◽

Mixed Cultures ◽

Acyl Homoserine Lactone ◽

Leaching Bacteria ◽

Leaching Efficiency

A functional luxIR-type Quorum Sensing (QS) system is present in Acidithiobacillus ferrooxidans. However, cell-cell communication among various acidophilic chemolithoautotrophs growing on pyrite has not been studied in detail. These aspects are the scope of this study with emphasis on the effects exerted by the N-acyl-homoserine lactone (AHL) type signaling molecules which are produced by Acidithiobacillus ferrooxidans. Their effects on attachment and leaching efficiency by other leaching bacteria, such as Acidithiobacillus ferrivorans, Acidiferrobacter spp. SPIII/3 and Leptospirillum ferrooxidans in pure and mixed cultures growing on pyrite is shown.

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