Decay of Bacterial Pathogens, Fecal Indicators, and Real-Time Quantitative PCR Genetic Markers in Manure-Amended Soils

ABSTRACTThis study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green fluorescent protein-expressingEscherichia coliO157:H7/pZs and red fluorescent protein-expressingSalmonella entericaserovar Typhimurium/pDs were added to laboratory-scale manure-amended soil microcosms with moisture contents of 60% or 80% field capacity and incubated at temperatures of −20°C, 10°C, or 25°C for 120 days. A two-stage first-order decay model was used to determine stage 1 and stage 2 first-order decay rate coefficients and transition times for each organism and qPCR genetic marker in each treatment. Genetic markers for FIB (Enterococcusspp.,E. coli, andBacteroidales) exhibited decay rate coefficients similar to that ofE. coliO157:H7/pZs but not ofS. entericaserovar Typhimurium/pDs and persisted at detectable levels longer than both pathogens. Concentrations of these two bacterial pathogens, their counterpart qPCR genetic markers (stx1andttrRSBCA, respectively), and FIB genetic markers were also correlated (r= 0.528 to 0.745). This suggests that these qPCR genetic markers may be reliable conservative surrogates for monitoring fecal pollution from manure-amended land. Host-associated qPCR genetic markers for microbial source tracking decayed rapidly to nondetectable concentrations, long before FIB,Salmonella entericaserovar Typhimurium/pDs, andE. coliO157:H7/pZs. Although good indicators of point source or recent nonpoint source fecal contamination events, these host-associated qPCR genetic markers may not be reliable indicators of nonpoint source fecal contamination events that occur weeks following manure application on land.

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Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

Applied and Environmental Microbiology ◽

10.1128/aem.06644-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8310-8317 ◽

Cited By ~ 27

Author(s):

Joshua D. Morris ◽

Jessica L. Hewitt ◽

Lawrence G. Wolfe ◽

Nachiket G. Kamatkar ◽

Sarah M. Chapman ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescent Protein ◽

Nile Red ◽

Temporal Distribution ◽

Time Lapse ◽

Content Type ◽

Rhamnolipid Production ◽

Serovar Typhimurium ◽

Surface Motility ◽

Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

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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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An Improved Medium for Colistin Susceptibility Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.01950-17 ◽

2018 ◽

Vol 56 (5) ◽

Cited By ~ 10

Author(s):

Konrad Gwozdzinski ◽

Saina Azarderakhsh ◽

Can Imirzalioglu ◽

Linda Falgenhauer ◽

Trinad Chakraborty

Keyword(s):

Susceptibility Testing ◽

Acquired Resistance ◽

Multidrug Resistant ◽

Colistin Resistance ◽

Reliable Determination ◽

Content Type ◽

E Coli ◽

Resistant Species ◽

Mueller Hinton ◽

Serovar Typhimurium

ABSTRACTThe plasmid-located colistin resistance genemcr-1confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination ofmcr-1-containingEnterobacteriaceae. Colistin susceptibility testing was performed for 50mcr-1-containingEscherichia coliandKlebsiella pneumoniaeisolates, 7 intrinsically polymyxin-resistant species,K. pneumoniaeandE. coliisolates with acquired resistance to polymyxins due tomgrBandpmrBmutations, respectively, and 32mcr-1-negative, colistin-susceptible isolates ofAcinetobacter baumannii,Citrobacter freundii,Enterobacter cloacae,E. coli,K. pneumoniae, andSalmonella entericaserovar Typhimurium. A comparison of the colistin MICs determined in CA-MH medium and those obtained in CE-MH medium was performed using both the BMD and strip-based susceptibility test formats. We validated the data using an isogenic IncX4 plasmid lackingmcr-1. Use of the CE-MH broth provides clear separation between resistant and susceptible isolates in both BMD and gradient diffusion assays; this is true for bothmcr-1-containingEnterobacteriaceaeisolates and those exhibiting either intrinsic or acquired colistin resistance. CE-MH medium is simple to prepare and overcomes current problems associated with BMD and strip-based colistin susceptibility testing, and use of the medium is easy to implement in routine diagnostic laboratories, even in resource-poor settings.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01997-18 ◽

2018 ◽

Vol 85 (2) ◽

Cited By ~ 9

Author(s):

Shireen M. Kotay ◽

Rodney M. Donlan ◽

Christine Ganim ◽

Katie Barry ◽

Bryan E. Christensen ◽

...

Keyword(s):

Patient Care ◽

Sampling Methods ◽

Fluorescent Protein ◽

Model Organism ◽

Air Sampling ◽

Multidrug Resistant ◽

Hand Washing ◽

Content Type ◽

E Coli ◽

Green Fluorescent

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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Regulation of RamA by RamR in Salmonella enterica Serovar Typhimurium: Isolation of a RamR Superrepressor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01320-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 6037-6040 ◽

Cited By ~ 15

Author(s):

Vito Ricci ◽

Stephen J. W. Busby ◽

Laura J. V. Piddock

Keyword(s):

Transcription Factor ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Salmonella Enterica Serovar Typhimurium ◽

Promoter Sequence ◽

Palindromic Sequence ◽

Content Type ◽

Gfp Reporter ◽

Serovar Typhimurium ◽

Green Fluorescent

ABSTRACTRamA is a transcription factor involved in regulating multidrug resistance inSalmonella entericaserovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses theramApromoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to theramApromoter sequence more efficiently, thus exhibiting aramAinactivated phenotype.

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Facultative Anaerobes Shape Multispecies Biofilms Composed of Meat Processing Surface Bacteria and Escherichia coli O157:H7 or Salmonella enterica Serovar Typhimurium

Applied and Environmental Microbiology ◽

10.1128/aem.01123-19 ◽

2019 ◽

Vol 85 (17) ◽

Author(s):

Jeyachchandran Visvalingam ◽

Hui Wang ◽

Tim C. Ells ◽

Xianqin Yang

Keyword(s):

Food Processing ◽

Salmonella Enterica ◽

Biofilm Formation ◽

Single Species ◽

Meat Processing ◽

Content Type ◽

E Coli ◽

Facultative Anaerobes ◽

Serovar Typhimurium ◽

Multispecies Biofilms

ABSTRACT This study investigated the microbial dynamics in multispecies biofilms of Escherichia coli O157:H7 strain 1934 (O157) or Salmonella enterica serovar Typhimurium ATCC 14028 (ST) and 40 strains of meat processing surface bacteria (MPB). Biofilms of O157 or ST with/without MPB were developed on stainless steel coupons at 15°C for up to 6 days. Bacteria in suspensions (inoculum, days 2 and 6) and biofilms (days 2 and 6) were enumerated by plating. The composition of multispecies cultures was determined by 16S rRNA gene sequencing. In suspensions, levels of O157 and ST were ∼2 log higher in single-species than in multispecies cultures on both sampling days. ST was 3 log higher in single-species than in multispecies biofilms. A similar trend, though to a lesser extent, was observed for O157 in biofilms on day 2 but not on day 6. No difference (P > 0.05) in bacterial counts was noted for the two MPB-pathogen cocultures at any time during incubation. Bacterial diversity in multispecies cultures decreased with incubation time, irrespective of the pathogen or culture type. The changes in the relative abundance of MPB were similar for the two MPB-pathogen cocultures, though different interbacterial interactions were noted. Respective fractions of ST and O157 were 2.1% and 0.97% initially and then 0.10% and 0.07% on day 2, and 0.60% and 0.04% on day 6. The relative proportions of facultative anaerobes in both multispecies cultures were greater in both suspensions and biofilms than in the inoculum. Citrobacter, Hafnia, Aeromonas, and Carnobacterium predominated in biofilms but not always in the planktonic cultures. IMPORTANCE Results of this study demonstrate that Salmonella enterica serovar Typhimurium and E. coli O157:H7 can integrate into biofilms when cocultured with bacteria from meat plant processing surfaces. However, the degree of biofilm formation for both pathogens was substantially reduced in the presence of the competing microbiota, with S. Typhimurium more greatly affected than E. coli O157:H7. The expression of extracellular determinants such as curli and cellulose appears to be less important for biofilm formation of the pathogens in multispecies cultures than in monoculture. In contrast to previous reports regarding food processing surface bacteria, data collected here also demonstrate that facultative anaerobes may have a competitive edge over strict aerobes in establishing multispecies biofilms. It would be important to take into account the presence of background bacteria when evaluating the potential persistence of a pathogen in food processing facilities.

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F Plasmids Are the Major Carriers of Antibiotic Resistance Genes in Human-Associated Commensal Escherichia coli

mSphere ◽

10.1128/msphere.00709-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Craig Stephens ◽

Tyler Arismendi ◽

Megan Wright ◽

Austin Hartman ◽

Andres Gonzalez ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Transposable Elements ◽

Dna Sequencing ◽

Resistance Genes ◽

Bacterial Pathogens ◽

Acquired Resistance ◽

Antibiotic Resistance Genes ◽

Content Type ◽

E Coli

ABSTRACT The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an ∼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic. IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.

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Leaching of Cryptosporidium parvum Oocysts, Escherichia coli, and a Salmonella enterica Serovar Typhimurium Bacteriophage through Intact Soil Cores following Surface Application and Injection of Slurry

Applied and Environmental Microbiology ◽

10.1128/aem.05675-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 8129-8138 ◽

Cited By ~ 20

Author(s):

Anita Forslund ◽

Bo Markussen ◽

Lise Toenner-Klank ◽

Tina B. Bech ◽

Ole Stig Jacobsen ◽

...

Keyword(s):

Cryptosporidium Parvum ◽

Pore Volume ◽

Relative Concentration ◽

Weather Conditions ◽

Soil Cores ◽

Content Type ◽

E Coli ◽

Serovar Typhimurium ◽

Intact Soil Cores ◽

Intact Soil

ABSTRACTIncreasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching ofSalmonella entericaserovar Typhimurium bacteriophage 28B,Escherichia coli, andCryptosporidium parvumoocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148.C. parvumoocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching ofE. coliwas delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, butE. colicould be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenousE. coliwas not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.

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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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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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