scholarly journals Purification and Host Specificity of Predatory Halobacteriovorax Isolates from Seawater

2015 ◽  
Vol 82 (3) ◽  
pp. 922-927 ◽  
Author(s):  
Gary P. Richards ◽  
Johnna P. Fay ◽  
Joseph Uknalis ◽  
O. Modesto Olanya ◽  
Michael A. Watson
Keyword(s):  
Host Specificity ◽  
Vibrio Vulnificus ◽  
Plaque Assay ◽  
Tidal River ◽  
Host Cells ◽  
Content Type ◽  
E Coli ◽  
Cultural Methods ◽  
Serovar Typhimurium ◽  
Salinity Levels

ABSTRACTHalobacteriovorax(formerlyBacteriovorax) is a small predatory bacterium found in the marine environment and modulates bacterial pathogens in shellfish. Four strains ofHalobacteriovoraxoriginally isolated inVibrio parahaemolyticusO3:K6 host cells were separated from their prey by an enrichment-filtration-dilution technique for specificity testing in other bacteria. This technique was essential, since 0.45-μm filtration alone was unable to remove infectiousVibriominicells, as determined by scanning electron microscopy and cultural methods. PurifiedHalobacteriovoraxstrains were screened for predation against otherV. parahaemolyticusstrains and againstVibrio vulnificus,Vibrio alginolyticus,Escherichia coliO157:H7, andSalmonella entericaserovar Typhimurium DT104, all potential threats to seafood safety. They showed high host specificity and were predatory only against strains ofV. parahaemolyticus. In addition, strains ofHalobacteriovoraxthat were predatory forE. coliO157:H7 andS. Typhimurium DT104 were isolated from a tidal river at 5 ppt salinity. In a modified plaque assay agar, they killed their respective prey over a broad range of salinities (5 to 30 ppt). Plaques became smaller as the salinity levels rose, suggesting that the lower salinities were optimal for the predators' replication. These species also showed broader host specificity, infectious against each other's original hosts as well as againstV. parahaemolyticusstrains. In summary, this study characterized strains ofHalobacteriovoraxwhich may be considered for use in the development of broad-based biocontrol technologies to enhance the safety of commercially marketed shellfish and other foods.

scholarly journals Predatory Bacteria as Natural Modulators of Vibrio parahaemolyticus and Vibrio vulnificus in Seawater and Oysters

2012 ◽  
Vol 78 (20) ◽  
pp. 7455-7466 ◽  
Author(s):  
Gary P. Richards ◽  
Johnna P. Fay ◽  
Keyana A. Dickens ◽  
Michelle A. Parent ◽  
Douglas S. Soroka ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Regulatory Gene ◽  
Plaque Assay ◽  
Eastern Oyster ◽  
Host Cells ◽  
Natural Seawater ◽  
Content Type ◽  
Predatory Bacteria ◽  
Pathogenic Vibrios

ABSTRACTThis study shows that naturally occurringVibriopredatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence ofVibrio parahaemolyticusandVibrio vulnificuswere assessed in natural seawater and in the Eastern oyster,Crassostrea virginica. The pathogens examined wereV. vulnificusstrain VV1003,V. parahaemolyticusO1:KUT (KUT stands for K untypeable), andV. parahaemolyticusO3:K6 and corresponding O3:K6 mutants deficient in thetoxRSvirulence regulatory gene or therpoSalternative stress response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their enumeration. In natural seawater, oysters bioconcentrated eachVibriostrain for 24 h at 22°C; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, vibrios decreased more than 3 log units to near negligible levels within 72 h. NeithertoxRSnorrpoShad a significant effect onVibriolevels. In autoclaved seawater,V. parahaemolyticusO3:K6 counts increased 1,000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water samples for VPB on lawns ofV. parahaemolyticusO3:K6 host cells. Many VPB, includingBdellovibrioand like organisms (BALOs;Bdellovibrio bacteriovorusandBacteriovorax stolpii) andMicavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast, and Hawaiian seawater. WhenV. parahaemolyticusO3:K6 was added to natural seawater containing trace amounts of VPB,Vibriocounts diminished 3 log units to nondetectable levels, while VPB increased 3 log units within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.

scholarly journals An Improved Medium for Colistin Susceptibility Testing

2018 ◽  
Vol 56 (5) ◽  
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.

scholarly journals The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

2011 ◽  
Vol 80 (2) ◽  
pp. 493-505 ◽  
Author(s):  
Patrick D. Vigil ◽  
Travis J. Wiles ◽  
Michael D. Engstrom ◽  
Lev Prasov ◽  
Matthew A. Mulvey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Virulence Factors ◽  
Upper Urinary Tract ◽  
Host Cells ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Novel Target ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC.tosA, found in strains within the B2 phylogenetic subgroup ofE. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence oftosAin anE. coliisolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function oftosArevealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.

scholarly journals Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
Maarten F. de Jong ◽  
Neal M. Alto
Keyword(s):  
Escherichia Coli ◽  
Defense Mechanisms ◽  
Immune Defense ◽  
Effector Proteins ◽  
Innate Immune ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
E Coli ◽  
Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

scholarly journals The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT

2019 ◽  
Vol 87 (10) ◽  
Author(s):  
Tracy H. Hazen ◽  
David A. Rasko
Keyword(s):  
Escherichia Coli ◽  
Complete Genome ◽  
Genomic Analysis ◽  
Host Cells ◽  
Comparative Genomic ◽  
Content Type ◽  
E Coli ◽  
Type Iii ◽  
Severe Diarrhea ◽  
Typical Epec

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.

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

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.

scholarly journals Cold Shock Induces Chromosomal qnr in Vibrio Species and Plasmid-Mediated qnrS1 in Escherichia coli

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Hee-Chang Jang ◽  
Yin Wang ◽  
Chunhui Chen ◽  
Laura Vinué ◽  
George A. Jacoby ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Community ◽  
Vibrio Parahaemolyticus ◽  
Cold Shock ◽  
Vibrio Vulnificus ◽  
Wild Type ◽  
Induced Expression ◽  
Content Type ◽  
E Coli ◽  
Dna Damaging Agents

ABSTRACT qnr genes are found in aquatic bacteria and were present in the bacterial community before the introduction of synthetic quinolones. Their natural functions are unknown. We evaluated expression of chromosomal qnr in Vibrio species in response to environmental stresses and DNA-damaging agents. Subinhibitory concentrations of quinolones, but not other DNA-damaging agents, increased expression of chromosomal qnr by more than five times in Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio mytili. Cold shock also induced expression of qnr in V. parahaemolyticus, V. vulnificus, and V. mytili, as well as expression of qnrS1 in Escherichia coli. qnrS1 induction by cold shock was not altered in ΔihfA or ΔihfB mutants or in a strain overexpressing dnaA, all of which otherwise directly modulate qnrS1 induction by ciprofloxacin. In contrast, the level of qnrS1 induction by cold shock was reduced in a ΔcspA mutant in the cold shock regulon compared to the wild type. In conclusion, cold shock and quinolones induce expression of chromosomal qnr in Vibrio species and of the related qnrS1 gene in E. coli.

scholarly journals Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

2014 ◽  
Vol 197 (5) ◽  
pp. 905-912 ◽  
Author(s):  
Yuriy A. Knirel ◽  
Nikolai S. Prokhorov ◽  
Alexander S. Shashkov ◽  
Olga G. Ovchinnikova ◽  
Evelina L. Zdorovenko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Side Chain ◽  
Gram Negative Bacteria ◽  
Gene Encoding ◽  
Content Type ◽  
Surface Receptors ◽  
E Coli ◽  
O Antigen ◽  
Phage Sensitivity

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmentalEscherichia coliisolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen ofE. coliO22, differing only in side-chain α-d-glucosylation in the former, mediated by agtrlocus on the chromosome. Spontaneous mutations ofE. coli4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions ofE. coli4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

scholarly journals Role of Intracellular Carbon Metabolism Pathways in Shigella flexneri Virulence

2014 ◽  
Vol 82 (7) ◽  
pp. 2746-2755 ◽  
Author(s):  
E. A. Waligora ◽  
C. R. Fisher ◽  
N. J. Hanovice ◽  
A. Rodou ◽  
E. E. Wyckoff ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pentose Phosphate Pathway ◽  
Carbon Metabolism ◽  
Cultured Cells ◽  
Shigella Flexneri ◽  
Plaque Assay ◽  
Pentose Phosphate ◽  
Host Cells ◽  
Content Type ◽  
Pathway Gene

ABSTRACTShigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellularS. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkABandpykAFmutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway geneedaresulted in small plaques, but the doubleeda eddmutant formed normal-size plaques. This suggested that the plaque defect of theedamutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellularS. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-typeS. flexnerialso formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate ofS. flexneriin vitro, suggesting that it may be a preferred carbon source inside host cells.

scholarly journals 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

2011 ◽  
Vol 77 (22) ◽  
pp. 8129-8138 ◽  
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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