scholarly journals Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

2020 ◽  
Author(s):  
Fabienne Benz ◽  
Jana S. Huisman ◽  
Erik Bakkeren ◽  
Joana A. Herter ◽  
Tanja Stadler ◽  
...  
Keyword(s):  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Transfer Genes ◽  
Serovar Typhimurium ◽  
Mouse Intestine ◽  
Specific Factors ◽  
Natural Strains

Abstract Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.

scholarly journals Clinical extended-spectrum beta-lactamase antibiotic resistance plasmids have diverse transfer rates and can spread in the absence of antibiotic selection

2019 ◽  
Author(s):  
Fabienne Benz ◽  
Jana S. Huisman ◽  
Erik Bakkeren ◽  
Joana A. Herter ◽  
Tanja Stadler ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Conjugative Transfer ◽  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
Extended Spectrum ◽  
Resistance Plasmids ◽  
Serovar Typhimurium ◽  
Spreading Potential

AbstractHorizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global spread of antibiotic resistance. However, the relative contributions of factors that underlie the spread of clinically relevant plasmids are unclear. Here, we quantified conjugative transfer dynamics of Extended Spectrum Beta-Lactamase (ESBL) producing plasmids in the absence of antibiotics. We showed that clinical Escherichia coli strains natively associated with ESBL-plasmids conjugate efficiently with three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain, reaching final transconjugant frequencies of up to 1% within 24 hours in vitro. The variation of final transconjugant frequencies varied among plasmids, donors and recipients and was better explained by variation in conjugative transfer efficiency than by variable clonal expansion. We identified plasmid-specific genetic factors, specifically the presence/absence of transfer genes, that influenced final transconjugant frequencies. Finally, we investigated plasmid spread within the mouse intestine, demonstrating qualitative agreement between plasmid spread in vitro and in vivo. This suggests a potential for the prediction of plasmid spread in the gut of animals and humans, based on in vitro testing. Altogether, this may allow the identification of resistance plasmids with high spreading potential and help to devise appropriate measures to restrict their spread.

scholarly journals Effect of Inactivation of degS on Salmonella enterica Serovar Typhimurium In Vitro and In Vivo

2005 ◽  
Vol 73 (1) ◽  
pp. 459-463 ◽  
Author(s):  
Gary Rowley ◽  
Andrew Stevenson ◽  
Jan Kormanec ◽  
Mark Roberts
Keyword(s):  
Sigma Factor ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Wild Type ◽  
Alternative Pathways ◽  
E Coli ◽  
Mammalian Tissues ◽  
Serovar Typhimurium

ABSTRACT The alternative sigma factor (RpoE σE) enables Salmonella enterica serovar Typhimurium to adapt to stressful conditions, such as oxidative stress, nutrient deprivation, and growth in mammalian tissues. Infection of mice by Salmonella serovar Typhimurium also requires σE. In Escherichia coli, activation of the σE pathway is dependent on proteolysis of the anti-sigma factor RseA and is initiated by DegS. DegS is also important in order for E. coli to cause extraintestinal infection in mice. We constructed a degS mutant of the serovar Typhimurium strain SL1344 and compared its behavior in vitro and in vivo with those of its wild-type (WT) parent and an isogenic rpoE mutant. Unlike E. coli degS strains, the Salmonella serovar Typhimurium degS strain grew as well as the WT strain at 42°C. The degS mutant survived very poorly in murine macrophages in vitro and was highly attenuated compared with the WT strain for both the oral and parenteral routes of infection in mice. However, the degS mutant was not as attenuated as the serovar Typhimurium rpoE mutant: 100- to 1,000-fold more degS bacteria than rpoE bacteria were present in the livers and spleens of mice 24 h after intraperitoneal challenge. In most assays, the rpoE mutant was more severely affected than the degS mutant and a σE-dependent reporter gene was more active in the degS mutant than the rpoE strain. These findings indicate that degS is important for activation of the σE pathway in serovar Typhimurium but that alternative pathways for σE activation probably exist.

scholarly journals Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for Escherichia coli RfaH both In Vivo and In Vitro

2004 ◽  
Vol 186 (9) ◽  
pp. 2829-2840 ◽  
Author(s):  
Heather D. Carter ◽  
Vladimir Svetlov ◽  
Irina Artsimovitch
Keyword(s):  
Escherichia Coli ◽  
Expression Vectors ◽  
Divergent Evolution ◽  
Transcriptional Enhancer ◽  
Elongation Complex ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Transcript Elongation

ABSTRACT The transcriptional enhancer protein RfaH positively regulates production of virulence factors in Escherichia coli and Salmonella enterica serovar Typhimurium via a cis element, ops. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including Vibrio and Yersinia spp. We cloned the rfaH genes from Vibrio cholerae, Yersinia enterocolitica, S. enterica serovar Typhimurium, and Klebsiella pneumoniae into E. coli expression vectors. Purified RfaH orthologs, including the most divergent one from V. cholerae, were readily recruited to the E. coli transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to E. coli RfaH. V. cholerae RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the rfaH genes complemented the disruption of the chromosomal copy of the E. coli gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the E. coli system.

scholarly journals Salmonella enterica Serovar Typhimurium RamA, Intracellular Oxidative Stress Response, and Bacterial Virulence

2004 ◽  
Vol 72 (2) ◽  
pp. 996-1003 ◽  
Author(s):  
Tahar van der Straaten ◽  
Laurence Zulianello ◽  
Angela van Diepen ◽  
Donald L. Granger ◽  
Riny Janssen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Salmonella Enterica ◽  
Raw 264.7 Cells ◽  
Virulence Determinants ◽  
Multidrug Resistance Gene ◽  
Bacterial Gene ◽  
E Coli ◽  
Serovar Typhimurium

ABSTRACT Escherichia coli and Salmonella enterica serovar Typhimurium have evolved genetic systems, such as the soxR/S and marA regulons, to detoxify reactive oxygen species, like superoxide, which are formed as by-products of metabolism. Superoxide also serves as a microbicidal effector mechanism of the host's phagocytes. Here, we investigate whether regulatory genes other than soxR/S and marA are active in response to oxidative stress in Salmonella and may function as virulence determinants. We identified a bacterial gene, which was designated ramA (342 bp) and mapped at 13.1 min on the Salmonella chromosome, that, when overexpressed on a plasmid in E. coli or Salmonella, confers a pleiotropic phenotype characterized by increased resistance to the redox-cycling agent menadione and to multiple unrelated antibiotics. The ramA gene is present in Salmonella serovars but is absent in E. coli. The gene product displays 37 to 52% homology to the transcriptional activators soxR/S and marA and 80 to 100% identity to a multidrug resistance gene in Klebsiella pneumoniae and Salmonella enterica serovar Paratyphi A. Although a ramA soxR/S double null mutant is highly susceptible to intracellular superoxide generated by menadione and displays decreased Mn-superoxide dismutase activity, intracellular survival of this mutant within macrophage-like RAW 264.7 cells and in vivo replication in the spleens in Ityr mice are not affected. We concluded that despite its role in the protective response of the bacteria to oxidative stress in vitro, the newly identified ramA gene, together with soxR/S, does not play a role in initial replication of Salmonella in the organs of mice.

scholarly journals Evaluation of Thymol-β-d-Glucopyranoside as a Potential Prebiotic Intervention to Reduce Carriage of Zoonotic Pathogens in Weaned and Feeder Pigs

2021 ◽  
Vol 9 (4) ◽  
pp. 860
Author(s):  
Gizem Levent ◽  
Robin C. Anderson ◽  
Branko Petrujkić ◽  
Toni L. Poole ◽  
Haiqi He ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
In Vivo Studies ◽  
Intestinal Microbes ◽  
E Coli ◽  
Antimicrobial Sensitivity ◽  
Current Thinking ◽  
Bactericidal Effects ◽  
Serovar Typhimurium

The gut of food-producing animals is a reservoir for foodborne pathogens. Thymol is bactericidal against foodborne pathogens but rapid absorption of thymol from the proximal gut precludes the delivery of effective concentrations to the lower gut where pathogens mainly colonize. Thymol-β-d-glucopyranoside is reported to be more resistant to absorption than thymol in everted jejunal segments and could potentially function as a prebiotic by resisting degradation and absorption in the proximal gut but being hydrolysable by microbial β-glycosidase in the distal gut. Previous in vitro studies showed bactericidal effects of thymol-β-d-glucopyranoside against Campylobacter, Escherichia coli, and Salmonella enterica serovar Typhimurium in the presence but not absence of intestinal microbes expressing β-glycosidase activity, indicating that hydrolysis was required to obtain antimicrobial activity. Presently, the oral administration of thymol-β-d-glucopyranoside was studied to examine the effects on intestinal carriage of Campylobacter, E. coli, and S. Typhimurium in swine. The effects of thymol-β-d-glucopyranoside or thymol on antimicrobial sensitivity of representative E. coli isolates and characterized Salmonella strains were also explored. Results from two in vivo studies revealed little antimicrobial effects of thymol-β-d-glucopyranoside on Campylobacter, E. coli, or S. Typhimurium in swine gut. These findings add credence to current thinking that hydrolysis and absorption of thymol-β-d-glucopyranoside and thymol may be sufficiently rapid within the proximal gut to preclude delivery to the distal gut. Antibiotic susceptibilities of selected bacterial isolates and strains were mainly unaffected by thymol. Further research is warranted to overcome obstacles, preventing the delivery of efficacious amounts of thymol-β-d-glucopyranoside to the lower gut.

scholarly journals Loss of RpoS results in attenuated Escherichia coli colonization of human intestinal organoids and a competitive disadvantage within the germ-free mouse intestine

2020 ◽  
Author(s):  
Madeline R. Barron ◽  
Roberto J. Cieza ◽  
David R. Hill ◽  
Sha Huang ◽  
Veda K. Yadagiri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
General Stress Response ◽  
E Coli ◽  
Germ Free ◽  
General Stress ◽  
Intestinal Organoids ◽  
Mouse Intestine

AbstractPluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a previously uncolonized, naïve intestinal epithelium in an in vitro system. We recently demonstrated that microinjection of the non-pathogenic Escherichia coli strain, ECOR2, into HIOs induced morphological and functional maturation of the HIO epithelium, including increased secretion of mucins and cationic antimicrobial peptides. In the current work, we use ECOR2 as a biological probe to investigate the bacterial response to colonization of the HIO lumen. In E. coli and other Gram-negative bacteria, adaptation to environmental stress is regulated by the general stress response sigma factor, RpoS. We generated an isogenic ∆rpoS ECOR2 mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine, which is currently the best available system for studying the initial establishment of bacterial populations within the gut. We demonstrate that loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, though it does not prevent colonization of germ-free mice. Rather, the ∆rpoS ECOR2 exhibits a fitness defect in the germ-free mouse intestine only in the context of microbial competition. These results indicate that HIOs pose a differentially restrictive luminal environment to E. coli during colonization, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses.ImportanceTechnological advancements have and will continue to drive the adoption of organoid-based systems for investigating host-microbe interactions within the human intestinal ecosystem. Using E. coli deficient in the RpoS-mediated general stress response, we demonstrate that the type or severity of microbial stressors within the HIO lumen differ from those of the in vivo environment of the germ-free mouse gut. This study provides important insight into the nature of the HIO microenvironment from a microbiological standpoint.

scholarly journals l-Fucose Stimulates Utilization of d-Ribose by Escherichia coli MG1655 ΔfucAO and E. coli Nissle 1917 ΔfucAO Mutants in the Mouse Intestine and in M9 Minimal Medium

2007 ◽  
Vol 75 (11) ◽  
pp. 5465-5475 ◽  
Author(s):  
Steven M. Autieri ◽  
Jeremy J. Lins ◽  
Mary P. Leatham ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Minimal Medium ◽  
Wild Type ◽  
E Coli ◽  
Commensal Strain ◽  
Mouse Intestine ◽  
Level 2 ◽  
Growth Experiments

ABSTRACT Escherichia coli MG1655 uses several sugars for growth in the mouse intestine. To determine the roles of l-fucose and d-ribose, an E. coli MG1655 ΔfucAO mutant and an E. coli MG1655 ΔrbsK mutant were fed separately to mice along with wild-type E. coli MG1655. The E. coli MG1655 ΔfucAO mutant colonized the intestine at a level 2 orders of magnitude lower than that of the wild type, but the E. coli MG1655 ΔrbsK mutant and the wild type colonized at nearly identical levels. Surprisingly, an E. coli MG1655 ΔfucAO ΔrbsK mutant was eliminated from the intestine by either wild-type E. coli MG1655 or E. coli MG1655 ΔfucAO, suggesting that the ΔfucAO mutant switches to ribose in vivo. Indeed, in vitro growth experiments showed that l-fucose stimulated utilization of d-ribose by the E. coli MG1655 ΔfucAO mutant but not by an E. coli MG1655 ΔfucK mutant. Since the ΔfucK mutant cannot convert l-fuculose to l-fuculose-1-phosphate, whereas the ΔfucAO mutant accumulates l-fuculose-1-phosphate, the data suggest that l-fuculose-1-phosphate stimulates growth on ribose both in the intestine and in vitro. An E. coli Nissle 1917 ΔfucAO mutant, derived from a human probiotic commensal strain, acted in a manner identical to that of E. coli MG1655 ΔfucAO in vivo and in vitro. Furthermore, l-fucose at a concentration too low to support growth stimulated the utilization of ribose by the wild-type E. coli strains in vitro. Collectively, the data suggest that l-fuculose-1-phosphate plays a role in the regulation of ribose usage as a carbon source by E. coli MG1655 and E. coli Nissle 1917 in the mouse intestine.

scholarly journals Competition and Resilience between Founder and Introduced Bacteria in the Caenorhabditis elegans Gut

2011 ◽  
Vol 80 (3) ◽  
pp. 1288-1299 ◽  
Author(s):  
Cynthia Portal-Celhay ◽  
Martin J. Blaser
Keyword(s):  
Caenorhabditis Elegans ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
C Elegans ◽  
Serovar Typhimurium ◽  
Colonization Factors

The microbial communities that reside within the intestinal tract in vertebrates are complex and dynamic. In this report, we establish the utility ofCaenorhabditis elegansas a model system for identifying the factors that contribute to bacterial persistence and for host control of gut luminal populations. We found that for N2 worms grown on mixed lawns of bacteria,Salmonella entericaserovar Typhimurium substantially outcompetedEscherichia coli, even whenE. coliwas initially present at 100-fold-higher concentrations. To address whether innate immunity affects the competition, thedaf-2anddaf-16mutants were studied; their total gut bacterial levels reflect overall capacity for colonization, butSalmonellaoutcompetedE. colito an extent similar to wild-type worms. To address the role of virulence properties,SalmonellaΔspi-1Δspi-2was used to compete withE. coli. The net differential was significantly less than that for wild-typeSalmonella; thus,spi-1 spi-2encodesC. eleganscolonization factors. AnE. colistrain with repeatedin vivopassage had an enhanced ability to compete against anin vitro-passedE. colistrain and againstSalmonella. Our data provide evidence of active competition for colonization niches in theC. elegansgut, as determined by bacterial factors and subject toin vivoselection.

scholarly journals Determination of the In Vivo Pharmacodynamic Profile of Cefepime against Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli at Various Inocula

2004 ◽  
Vol 48 (6) ◽  
pp. 1941-1947 ◽  
Author(s):  
Dana Maglio ◽  
Christine Ong ◽  
Mary Anne Banevicius ◽  
Qiuming Geng ◽  
Charles H. Nightingale ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Infection Model ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Extended Spectrum ◽  
Pharmacodynamic Profile ◽  
Significant Difference ◽  
Bactericidal Effects

ABSTRACT Cefepime was evaluated in vivo against two inoculum sizes of four strains of Escherichia coli that produced extended-spectrum beta-lactamases (ESBLs) in a murine neutropenic thigh infection model to characterize the pharmacodynamic activity of cefepime in the presence of ESBL-producing bacteria and to evaluate if differences in lengths of cefepime exposure are required with various inocula. Three strains possessed a single enzyme each: TEM-10, TEM-12, and TEM-26. The fourth strain possessed two TEM-derived ESBLs and a third uncharacterized enzyme. Two non-ESBL-producing E. coli strains were included for comparison. Mice received various doses of cefepime to achieve a spectrum of percentages of time the drug was above the MIC (%T>MICs) for each isolate at both inocula. No significant difference in cefepime exposure was required to achieve similar bactericidal effects for ESBL- and non-ESBL-producing isolates when the starting inoculum was 105 CFU of E. coli per thigh. The increased MICs observed in vitro for the ESBL-producing strains at 107 CFU/ml did not predict the amount of exposure required to achieve a comparable level of bactericidal activity in vivo at the corresponding starting inoculum of 107 CFU/thigh. Compared to the cefepime exposure in tests with the lower inoculum (105 CFU/thigh), less exposure was required when the starting inoculum was 107 CFU/thigh (%T>MIC, 6% versus 26%), such that similar doses (in milligrams per kilogram of body weight) produced similar bactericidal effects with both inocula of ESBL-producing isolates. Equivalent exposures of cefepime produced similar effects against the microorganisms regardless of the presence of ESBL production. Pharmacodynamic profiling undertaken with conventional cefepime MIC determinations predicted in vivo microbial outcomes at both inoculum sizes for the ESBL-producing isolates evaluated in this study. These data support the use of conventional MIC determinations in the pharmacodynamic assessment of cefepime.

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