Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps

ABSTRACT An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods. IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

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Droplet rather than Aerosol Mediated Dispersion is the Primary Mechanism of Bacterial transmission from Contaminated Hand Washing Sink Traps

10.1101/392431 ◽

2018 ◽

Author(s):

Shireen Kotay ◽

Rodney M. Donlan ◽

Christine Ganim ◽

Katie Barry ◽

Bryan E. Christensen ◽

...

Keyword(s):

Patient Care ◽

Sampling Methods ◽

Model Organism ◽

Air Sampling ◽

Multidrug Resistant ◽

Hand Washing ◽

Antibiotic Resistant ◽

E Coli ◽

Potential Reservoir ◽

Healthcare Associated

ABSTRACTAn alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgement that sinks are a major reservoir of antibiotic resistant pathogens in patient-care areas. An earlier study using a GFP-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilm in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events amending earlier theory that bacteria aerosolize from P-trap and disperse. Numbers of dispersed GFP-E. coli diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods.IMPORTANCEAmong the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as potential reservoir of multidrug resistant healthcare-associated pathogens to hospitalized patients. With increasing antimicrobial resistance limiting therapeutic options for patients, better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria colonizing sink drains.

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Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs

Applied and Environmental Microbiology ◽

10.1128/aem.03327-16 ◽

2017 ◽

Vol 83 (8) ◽

Cited By ~ 63

Author(s):

Shireen Kotay ◽

Weidong Chai ◽

William Guilford ◽

Katie Barry ◽

Amy J. Mathers

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Hand Washing ◽

Resistant Bacteria ◽

Droplet Dispersion ◽

Content Type ◽

E Coli ◽

Mode Of Transmission ◽

Green Fluorescent

ABSTRACT There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.

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Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01549-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2438-2441 ◽

Cited By ~ 107

Author(s):

Zeynep Baharoglu ◽

Didier Mazel

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Vibrio Cholerae ◽

Fluorescent Protein ◽

Resistance Development ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Green Fluorescent ◽

Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

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Mycelium-Like Networks Increase Bacterial Dispersal, Growth, and Biodegradation in a Model Ecosystem at Various Water Potentials

Applied and Environmental Microbiology ◽

10.1128/aem.03901-15 ◽

2016 ◽

Vol 82 (10) ◽

pp. 2902-2908 ◽

Cited By ~ 23

Author(s):

Anja Worrich ◽

Sara König ◽

Anja Miltner ◽

Thomas Banitz ◽

Florian Centler ◽

...

Keyword(s):

Fluorescent Protein ◽

Model Organism ◽

High Positive Correlation ◽

Poor Knowledge ◽

Content Type ◽

Model Ecosystem ◽

Microbial Ecosystems ◽

Benzoate Degradation ◽

Green Fluorescent ◽

Bacterial Movement

ABSTRACTFungal mycelia serve as effective dispersal networks for bacteria in water-unsaturated environments, thereby allowing bacteria to maintain important functions, such as biodegradation. However, poor knowledge exists on the effects of dispersal networks at various osmotic (Ψo) and matric (Ψm) potentials, which contribute to the water potential mainly in terrestrial soil environments. Here we studied the effects of artificial mycelium-like dispersal networks on bacterial dispersal dynamics and subsequent effects on growth and benzoate biodegradation at ΔΨoand ΔΨmvalues between 0 and −1.5 MPa. In a multiple-microcosm approach, we used a green fluorescent protein (GFP)-tagged derivative of the soil bacteriumPseudomonas putidaKT2440 as a model organism and sodium benzoate as a representative of polar aromatic contaminants. We found that decreasing ΔΨoand ΔΨmvalues slowed bacterial dispersal in the system, leading to decelerated growth and benzoate degradation. In contrast, dispersal networks facilitated bacterial movement at ΔΨoand ΔΨmvalues between 0 and −0.5 MPa and thus improved the absolute biodegradation performance by up to 52 and 119% for ΔΨoand ΔΨm, respectively. This strong functional interrelationship was further emphasized by a high positive correlation between population dispersal, population growth, and degradation. We propose that dispersal networks may sustain the functionality of microbial ecosystems at low osmotic and matric potentials.

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PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

Applied and Environmental Microbiology ◽

10.1128/aem.03720-13 ◽

2013 ◽

Vol 80 (4) ◽

pp. 1477-1481 ◽

Cited By ~ 8

Author(s):

Karina Klevanskaa ◽

Nadja Bier ◽

Kerstin Stingl ◽

Eckhard Strauch ◽

Stefan Hertwig

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Green Fluorescent Protein ◽

Vibrio Parahaemolyticus ◽

Fluorescent Protein ◽

Vibrio Vulnificus ◽

Shuttle Vector ◽

Content Type ◽

E Coli ◽

Green Fluorescent

ABSTRACTAn efficient electroporation procedure forVibrio vulnificuswas designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 106transformants per μg DNA were achieved. The vector stably replicated in bothV. vulnificusandEscherichia coliand was also successfully introduced intoVibrio parahaemolyticusandVibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and thevvhBAhemolysin operon were inserted into the vector and functionally expressed inVibrioandE. coli.

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TnFLX: a Third-Generation mariner-Based Transposon System for Bacillus subtilis

Applied and Environmental Microbiology ◽

10.1128/aem.02893-19 ◽

2020 ◽

Vol 86 (10) ◽

Author(s):

Felix Dempwolff ◽

Sandra Sanchez ◽

Daniel B. Kearns

Keyword(s):

Antibiotic Resistance ◽

Fluorescent Protein ◽

Protein Translation ◽

Delivery Vehicle ◽

Content Type ◽

E Coli ◽

Vector Construction ◽

Resistance Markers ◽

The Stability ◽

Green Fluorescent

ABSTRACT Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli. We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms. IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

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mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

Applied and Environmental Microbiology ◽

10.1128/aem.02110-13 ◽

2013 ◽

Vol 79 (20) ◽

pp. 6362-6368 ◽

Cited By ~ 12

Author(s):

Ying Xu ◽

Bing Chen ◽

Hongjun Chao ◽

Ning-Yi Zhou

Keyword(s):

Escherichia Coli ◽

Wild Type Strain ◽

Fluorescent Protein ◽

Gene Knockout ◽

Transport Activity ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Green Fluorescent ◽

K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

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Bestatin Inhibits Cell Growth, Cell Division, and Spore Cell Differentiation in Dictyostelium discoideum

Eukaryotic Cell ◽

10.1128/ec.05311-11 ◽

2012 ◽

Vol 11 (4) ◽

pp. 545-557 ◽

Cited By ~ 11

Author(s):

Yekaterina Poloz ◽

Andrew Catalano ◽

Danton H. O'Day

Keyword(s):

Cell Division ◽

Cell Differentiation ◽

Cell Growth ◽

Fluorescent Protein ◽

Model Organism ◽

Aminopeptidase Activity ◽

Content Type ◽

Aminopeptidase A ◽

Green Fluorescent ◽

Spore Cell

ABSTRACTBestatin methyl ester (BME) is an inhibitor of Zn2+-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have usedDictyosteliumas a model organism to study the effects of BME. Only two Zn2+-binding aminopeptidases have been identified inDictyosteliumto date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development ofDictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation inDictyosteliumlikely through inhibition of PsaA.

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Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.01147-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5831-5838 ◽

Cited By ~ 34

Author(s):

Yun Zhang ◽

Xiuling Shang ◽

Shujuan Lai ◽

Guoqiang Zhang ◽

Yong Liang ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Fluorescent Protein ◽

Expression System ◽

Carbon Sources ◽

Inducible Expression ◽

Gene Encoding ◽

Content Type ◽

E Coli ◽

Inducible Expression System ◽

Green Fluorescent

ABSTRACTCorynebacterium glutamicumis currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species uselac-derived promoters fromEscherichia colithat exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains thel-arabinose regulator AraC, thePBADpromoter from thearaBADoperon, and thel-arabinose transporter AraE, all of which are derived fromE. coli. The level of induciblePBAD-based expression could be modulated over a wide concentration range from 0.001 to 0.4%l-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control ofPBADpromoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case inE. coli,PBADinduction was not significantly affected in the presence of different carbon sources inC. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of theodhIgene fromC. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering ofC. glutamicum.

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Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽

10.3390/cells8111325 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1325 ◽

Cited By ~ 1

Author(s):

Ke Yue ◽

Tran Nam Trung ◽

Yiyong Zhu ◽

Ralf Kaldenhoff ◽

Lei Kai

Keyword(s):

Functional Analysis ◽

Membrane Proteins ◽

Fluorescent Protein ◽

Water Channel ◽

New Approach ◽

E Coli ◽

Straightforward Method ◽

Lipid Environment ◽

Green Fluorescent ◽

Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

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