ESBL-plasmid carriage in E. coli enhances in vitro bacterial competition fitness and serum resistance in some strains of pandemic sequence types without overall fitness cost

ABSTRACT Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum β-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

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Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a UropathogenicEscherichia coliIsolate

mBio ◽

10.1128/mbio.00603-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 11

Author(s):

Jingyu Diao ◽

Catrien Bouwman ◽

Donghong Yan ◽

Jing Kang ◽

Anand K. Katakam ◽

...

Keyword(s):

Outer Membrane ◽

Capsular Polysaccharide ◽

Cell Envelope ◽

Bloodstream Infections ◽

Serum Resistance ◽

E Coli ◽

Serum Killing ◽

Group 2

ABSTRACTMurein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins inEscherichia coli. Their roles in cell-envelope integrity have been documented inE. colilaboratory strains, and while Lpp has been linked to serum resistancein vitro, the underlying mechanism has not been established. Here,lppandpalmutants of uropathogenicE. colistrain CFT073 showed reduced survival in a mouse bacteremia model, but only thelppmutant was sensitive to serum killingin vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysisin vitroand complement-mediated clearancein vivo. Compared to the wild-type strain, thelppmutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenicE. coliisolates.IMPORTANCEUropathogenicE. coli(UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistancein vitroand for complement-mediated bacterial clearancein vivo. This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of “group 2” capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis.

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Prospective Multicenter Study of Carbapenemase-Producing Enterobacteriaceae from 83 Hospitals in Spain Reveals HighIn VitroSusceptibility to Colistin and Meropenem

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00086-15 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3406-3412 ◽

Cited By ~ 85

Author(s):

Jesús Oteo ◽

Adriana Ortega ◽

Rosa Bartolomé ◽

Germán Bou ◽

Carmen Conejo ◽

...

Keyword(s):

Health Concern ◽

Inclusion Criteria ◽

Prospective Multicenter Study ◽

Content Type ◽

E Coli ◽

New Finding ◽

Phenotypic Methods ◽

The Impact ◽

Sequence Types

ABSTRACTThe aim of this study was to determine the impact of carbapenemase-producingEnterobacteriaceae(CPE) in Spain in 2013 by describing the prevalence, dissemination, and geographic distribution of CPE clones, and their population structure and antibiotic susceptibility. From February 2013 to May 2013, 83 hospitals (about 40,000 hospital beds) prospectively collected nonduplicateEnterobacteriaceaeusing the screening cutoff recommended by EUCAST. Carbapenemase characterization was performed by phenotypic methods and confirmed by PCR and sequencing. Multilocus sequencing types (MLST) were determined forKlebsiella pneumoniaeandEscherichia coli. A total of 702Enterobacteriaceaeisolates met the inclusion criteria; 379 (54%) were CPE. OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequent carbapenemases, andK. pneumoniae(74.4%),Enterobacter cloacae(10.3%), andE. coli(8.4%) were the species most affected. Susceptibility to colistin, amikacin, and meropenem was 95.5%, 81.3%, and 74.7%, respectively. The most prevalent sequence types (STs) were ST11 and ST405 forK. pneumoniaeand ST131 forE. coli. Forty-five (54.1%) of the hospitals had at least one CPE case. ForK. pneumoniae, ST11/OXA-48, ST15/OXA-48, ST405/OXA-48, and ST11/VIM-1 were detected in two or more Spanish provinces. ST11 isolates carried four carbapenemases (VIM-1, OXA-48, KPC-2, and OXA-245), but ST405 isolates carried OXA-48 only. A wide interregional spread of CPE in Spain was observed, mainly due to a few successful clones of OXA-48-producingK. pneumoniae(e.g., ST11 and ST405). The dissemination of OXA-48-producingE. coliis a new finding of public health concern. According to the susceptibilities determinedin vitro, most of the CPE (94.5%) had three or more options for antibiotic treatment.

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The Microbiological Characteristics of Carbapenem-Resistant Enterobacteriaceae Carrying the mcr-1 Gene

Journal of Clinical Medicine ◽

10.3390/jcm8020261 ◽

2019 ◽

Vol 8 (2) ◽

pp. 261 ◽

Cited By ~ 3

Author(s):

Chih-Wei Chen ◽

Hung-Jen Tang ◽

Chi-Chung Chen ◽

Ying-Chen Lu ◽

Hung-Jui Chen ◽

...

Keyword(s):

Clinical Significance ◽

Resistance Mechanisms ◽

E Coli ◽

Carbapenem Resistant ◽

Microbiological Characteristics ◽

Carbapenemase Gene ◽

Susceptible Isolate ◽

Sequence Types ◽

Carbapenem Resistant Enterobacteriaceae

Objectives: This study aims to assess the prevalence of the mcr-1 gene among carbapenem-resistant Enterobacteriaceae (CRE) isolated from clinical specimens and to further investigate the clinical significance and microbiological characteristics of CRE carrying the mcr-1 gene. Methods: Four hundred and twenty-three CRE isolates were screened for the presence of the mcr-1 gene. After identification, their clinical significance, antibiotic susceptibility, and antibiotic resistance mechanisms including the ESBL gene, carbapenemase gene, outer membrane protein (OMP), and plasmid sequencing were assessed. Results: Only four (0.9%) isolates of carbapenem-resistant Escherichia coli (E. coli) were found to carry the mcr-1 gene and demonstrated different pulsed-field gel electrophoresis (PFGE) patterns and sequence types (ST). While one patient was considered as having mcr-1-positive carbapenem-resistant E. coli (CREC) colonization, the other three mcr-1-positive CREC-related infections were classified as nosocomial infections. Only amikacin and tigecycline showed good in vitro activity against these four isolates, and three of them had a minimum inhibitory concentration with colistin of ≥4 mg/L. In the colistin-susceptible isolate, mcr-1 was nonfunctional due to the insertion of another gene. In addition, all of the mcr-1-positive CREC contained various resistant genes, such as AmpCCMY, blaNDM, blaTEM, blaSHV, and blaCTX. In addition, one strain (EC1037) had loss of the OMP. Conclusions: The emergence of the mcr-1 gene among CRE, especially E. coli, remains worth our attention due to its resistance to most antibiotics, and a further national survey is warranted.

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Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut

Applied and Environmental Microbiology ◽

10.1128/aem.00892-19 ◽

2019 ◽

Vol 85 (17) ◽

Cited By ~ 1

Author(s):

Egil A. J. Fischer ◽

Cindy M. Dierikx ◽

Alieda van Essen-Zandbergen ◽

Dik Mevius ◽

Arjan Stegeman ◽

...

Keyword(s):

Escherichia Coli ◽

Competitive Exclusion ◽

Antibiotic Use ◽

Beta Lactamase ◽

Content Type ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Extended Spectrum ◽

Plasmid Carriage

ABSTRACT Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1. At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro. Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro. Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation. IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro. We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.

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Carriage trends and fitness cost of MDROs in Lebanese nursing homes

The Journal of Infection in Developing Countries ◽

10.3855/jidc.9996 ◽

2018 ◽

Vol 12 (02.1) ◽

pp. 22S

Author(s):

Caren Challita ◽

Nourhane Hafza ◽

Elias Dahdouh ◽

Michel Attieh ◽

Iman Dandachi ◽

...

Keyword(s):

Nursing Homes ◽

Klebsiella Pneumoniae ◽

Fitness Cost ◽

Pcr Analysis ◽

Competitive Growth ◽

Medical Treatments ◽

E Coli ◽

Klebsiella Pneumoniae Carbapenemase ◽

Competition Assays

Introduction: Nowadays, medical treatments efficiency is challenged by multi drug resistant organisms (MDROs). Lebanese nursing homes’ residents revealed high fecal carriage rates of MDR Enterobacteriacea. Previous studies claim that bacteria with resistant genes experience fitness cost. This study assesses the competitive growth of MDR Escherichia coli and Klebsiella pneumoniae. Methodology: Fecal swabs were collected, during six consecutive months, from ten elderly residing in a Lebanese nursing home. All isolates were subject to API 20E (bioMerieux, Marcy L’ Etoile, France) and antimicrobial susceptibility (Kirby–Bauer method) testing. Phenotypically, ESBL (extended spectrum β-lactamase), MBL (metallo β-lactamase), AmpC and KPC (Klebsiella pneumoniae carbapenemase) were detected using EDTA, PBA, cloxacillin, and DDSTs (Biorad, Hercules, USA). Selected ESBL producing E. coli and K. pneumoniae underwent multiplex PCR analysis. Intra and inter-species in-vitro competition assays were conducted in multiple combinations. Results: Among 117 collected isolates, E. coli was predominant (71.8%); 7.7% were ESBL and 5.1% AmpC producers. With E. coli intra-species assays, sensitive isolates out-competed all others, followed by ESBL, AmpC, and OXA-48 (oxacillin) producers. Inter-species assays, demonstrated a decreased fitness of ESBL producing K. pneumoniae in presence of sensitive E. coli. While out-competing ESBL producing E. coli required 2 sensitive K. pneumoniae isolates. Conclusion: This study highlights resistant E. coli and K. pneumoniae frequency decrease in presence of sensitive isolates, endorsing the fitness cost hypothesis. Hence, competing supplementary species reproducing gut flora, would ensure further steps in the fight against MDROs.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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Антимикробная активность лиофилизированного водного извлечения Caragana jubata (Pall.) Poir.

Химико-фармацевтический журнал ◽

10.30906/0023-1134-2020-54-3-42-44 ◽

2020 ◽

Vol 54 (3) ◽

pp. 42-44

Author(s):

Павел Алексеевич Какорин ◽

Татьяна Владимировна Фатеева ◽

Ольга Ивановна Терешкина ◽

Ирина Борисовна Перова ◽

Галина Владиславовна Раменская ◽

...

Keyword(s):

E Coli

На основании ранее проведенных исследований установлен профиль флавоноидов лиофилизированного водного извлечения, полученного из побегов C. jubata. В связи с тем, что, согласно данным литературы, флавоноиды являются потенциальными ингибиторами микроорганизмов, проведено изучение антимикробной активности лиофилизата в опытах in vitro с использованием скринигового метода определения антимикробной активности для препаратов растительного происхождения. При изучении бактериостатической и фунгистатической активности в опытах in vitro использовали метод двукратного серийного разведения препаратов в жидких питательных средах. В результате исследования лиофилизированного водного извлечения караганы гривастой установлено наличие умеренной антимикробной активности в отношении всех изученных штаммов патогенных микроорганизмов: грамположительных и грамотрицательных бактерий (S. aureus, E. coli, P. vulgaris, P. aeruginosa), дрожжеподобных и мицелиальных грибов (C. albicans, M. canis). Полученные данные позволяют рекомендовать лиофилизированное водное извлечение караганы гривастой для создания на его основе лекарственных форм наружного применения для лечения заболеваний кожи и слизистых оболочек, связанных с бактериальным воспалительным процессом.

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Влияние in vitro изолированного и сочетанного с антибактериальными средствами применения бовгиалуронидазы азоксимер на целостность бактериальной биопленки и жизнеспосоность микроорганизмов

Экспериментальная и клиническая фармакология ◽

10.30906/0869-2092-2020-83-2-38-44 ◽

2020 ◽

Vol 83 (2) ◽

pp. 38-44

Author(s):

Е. Ю. Тризна ◽

Д. Р. Байдамшина ◽

Александр А. Виницкий ◽

А. Р. Каюмов

Keyword(s):

E Coli

Исследована способность лиофилизата бовгиалуронидазы азоксимера («Лонгидаза») разрушать бактериальные биопленки S. aureus, E. faecalis, E. coli, а также сочетанное действие препарата с антибактериальными средствами. Показано, что 2 ч инкубации бовгиалуронидазы азоксимер в концентрации 750 – 1500 МЕ/мл вызывает двукратное снижение биомассы матрикса зрелых биопленок E. faecalis и E. coli, и на 60 % — S. aureus. Данный ферментный препарат не влияет на образование бактериальных биопленок. При сочетанном применении с антибактериальными средствами препарат повышает их эффективность в отношении бактерий в составе биопленок. Так, концентрация ципро-флоксацина и амоксициллина, необходимая для снижения количества КОЕ на 3 порядка в биопленке E. faecalis, в присутствии бовгиалуронидазы азоксимера снижается в 16 раз (p < 0,05). В присутствии фермента в 16 раз меньшие концентрации цефуроксима, фосфомицина, ципрофлоксацина и амикацина достаточны для снижения количества КОЕ на 3 порядка в биопленке E. coli (p < 0,05), и в значительно меньшей концентрации цефуроксим оказывает бактерицидное действие на клетки в биопленке S. aureus (p < 0,05). Вероятно, бовгиалуронидаза азоксимер увеличивает проникновение антибактериальных средств к клеткам бактерий в биопленке, что обеспечивает потенцирование их антибактериального эффекта. Такое действие ферментного препарата позволяет снизить дозу и повысить безопасность антибактериальных средств при сохранении их эффективности.

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Properties and Biotechnological Application of mutant Derivatives of Mini-intein PRP8 from Penicillium chrysogenum with Improved Control of C-terminal Processing

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-91-101 ◽

2019 ◽

Vol 35 (6) ◽

pp. 91-101

Author(s):

F.A. Klebanov ◽

S.E. Cheperegin ◽

D.G. Kozlov

Keyword(s):

Penicillium Chrysogenum ◽

Protein Denaturation ◽

Biologically Active ◽

Cultivation Temperature ◽

Target Proteins ◽

E Coli ◽

Complete Inactivation ◽

Target Molecules ◽

Proteins And Peptides

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing were characterized. The presented variants can serve as a basis for self-removed polypeptide tags capable of carrying an affine label and allowing to optimize the process of obtaining target proteins and peptides in E. coli cells. They allow to synthesize target molecules in the composition of soluble and insoluble hybrid proteins (fusions), provide their afnne purification, autocatalytic processing and obtaining mature target products. The presented variants have a number of features in comparison with the known prototypes. In particular the mutant mini-intein Int4bPRO, containing the L93P mutation, has temperature-dependent properties. At cultivation temperature below 30 °C it allows the production of target molecules as part of soluble fusions, but after increasing of cultivation temperature to 37 °C it directs the most of synthesized fusions into insoluble intracellular aggregates. The transition of Int4bPRO into insoluble form is accompanied by complete inactivation of C-terminal processing. Further application of standard protein denaturation-renaturation procedures enable efficiently reactivate Int4bPRO and to carry out processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, containing a point mutation T62N or combination of mutations D144N and L146T respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows to optimize the biosynthesis of biologically active target proteins and peptides in the composition of soluble fusions, suitable for afnne purification and subsequent intein-dependent processing without the use of protein denaturation-renaturation procedures. intein, fusion, processing, processing rate, gelonin The work was supported within the framework of the State Assignment no. 595-00003-19 PR.

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