Selective Inhibition ofNeisseria gonorrhoeaeby a Dithiazoline in Mixed Infections withLactobacillus gasseri

ABSTRACTThe Gram-negative human pathogenNeisseria gonorrhoeaehas progressively developed resistance to antibiotic monotherapies, and recent failures of dual-drug therapy have heightened concerns that strains resistant to all available antibiotics will begin circulating globally. Targeting bacterial cell wall assembly has historically been effective at treating infections withN. gonorrhoeae, but as the effectiveness of β-lactams (including cephalosporins) is challenged by increasing resistance, research has expanded into compounds that target the numerous other enzymes with roles in peptidoglycan metabolism. One example is the dithiazoline compound JNJ-853346 (DTZ), which inhibits the activity of anEscherichia coliserine proteasel,d-carboxypeptidase (LdcA). Recently, the characterization of an LdcA homolog inN. gonorrhoeaerevealed localization and activity differences from the characterizedE. coliLdcA, prompting us to explore the effectiveness of DTZ againstN. gonorrhoeae. We found that DTZ is effective at inhibitingN. gonorrhoeaein all growth phases, unlike the specific stationary-phase inhibition seen inE. coli. Surprisingly, DTZ does not inhibit gonococcal LdcA enzyme activity, and DTZ sensitivity is not significantly decreased inldcAmutants. While effective against numerousN. gonorrhoeaestrains, including recent multidrug-resistant isolates, DTZ is much less effective at inhibiting growth of the commensal speciesLactobacillus gasseri. DTZ treatment during coinfections of epithelial cells resulted in significant lowering of gonococcal burden and interleukin-8 secretion without significantly impacting recovery of viableL. gasseri. This selective toxicity presents a possible pathway for the use of DTZ as an effective antigonococcal agent at concentrations that do not impact vaginal commensals.

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Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

Applied and Environmental Microbiology ◽

10.1128/aem.00599-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7104-7112 ◽

Cited By ~ 59

Author(s):

Maria Karczmarczyk ◽

Yvonne Abbott ◽

Ciara Walsh ◽

Nola Leonard ◽

Séamus Fanning

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Gene Array ◽

Multidrug Resistant ◽

Genetic Determinants ◽

Gene Encoding ◽

Content Type ◽

E Coli ◽

Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

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Molecular Characterization of Escherichia coli Isolates Carrying mcr-1, fosA3, and Extended-Spectrum-β-Lactamase Genes from Food Samples in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00064-17 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 10

Author(s):

Xiaobo Liu ◽

Ruichao Li ◽

Zhiwei Zheng ◽

Kaichao Chen ◽

Miaomiao Xie ◽

...

Keyword(s):

Public Health ◽

Escherichia Coli ◽

Molecular Characterization ◽

Food Products ◽

Multidrug Resistant ◽

Food Samples ◽

Content Type ◽

E Coli ◽

Extended Spectrum

ABSTRACT This study surveyed the prevalence of mcr-1 in extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains of food origin in China and identified strains that carried mcr-1, fosA3, and ESBL genes, which were carried in various plasmids. The mcr-1 and ESBL genes could be cotransferred by one or more types of plasmids. The presence of these multidrug-resistant E. coli strains in food products might pose a huge threat to public health.

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Houseflies (Musca domestica) as Vectors for Extended-Spectrum β-Lactamase-Producing Escherichia coli on Spanish Broiler Farms

Applied and Environmental Microbiology ◽

10.1128/aem.04252-14 ◽

2015 ◽

Vol 81 (11) ◽

pp. 3604-3611 ◽

Cited By ~ 27

Author(s):

Marc Solà-Ginés ◽

Juan José González-López ◽

Karla Cameron-Veas ◽

Nuria Piedra-Carrasco ◽

Marta Cerdà-Cuéllar ◽

...

Keyword(s):

Escherichia Coli ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Ecological Niches ◽

Potential Contribution ◽

Content Type ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Extended Spectrum

ABSTRACTFlies may act as potential vectors for the spread of resistant bacteria to different environments. This study was intended to evaluate the presence ofEscherichia colistrains resistant to cephalosporins in flies captured in the areas surrounding five broiler farms. Phenotypic and molecular characterization of the resistant population was performed by different methods: MIC determination, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylotyping. The presence of extended-spectrum beta-lactamase (ESBL) genes, their plasmid location, and the mobile genetic elements involved in their mobilization were studied. Additionally, the presence of 35 genes associated with virulence was evaluated. Out of 682 flies captured, 42 yielded ESBL-producingE. coli. Of these isolates, 23 containedblaCTX-M-1, 18 containedblaCTX-M-14, and 1 containedblaCTX-M-9. ESBL genes were associated mainly with the presence of the IncI1 and IncFIB replicons. Additionally, all the strains were multiresistant, and five of them also harboredqnrS. Identical PFGE profiles were found forE. coliisolates obtained from flies at different sampling times, indicating a persistence of the same clones in the farm environment over months. According to their virulence genes, 81% of the isolates were considered avian-pathogenicE. coli(APEC) and 29% were considered extraintestinal pathogenicE. coli(ExPEC). The entrance of flies into broiler houses constitutes a considerable risk for colonization of broilers with multidrug-resistantE. coli. ESBLs in flies reflect the contamination status of the farm environment. Additionally, this study demonstrates the potential contribution of flies to the dissemination of virulence and resistance genes into different ecological niches.

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Screening and Characterization of Multidrug-Resistant Gram-Negative Bacteria from a Remote African Area, São Tomé and Príncipe

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01021-18 ◽

2018 ◽

Vol 62 (9) ◽

Cited By ~ 10

Author(s):

Laurent Poirel ◽

Marta Aires-de-Sousa ◽

Patrick Kudyba ◽

Nicolas Kieffer ◽

Patrice Nordmann

Keyword(s):

Epidemiological Data ◽

Multidrug Resistant ◽

Content Type ◽

E Coli ◽

Sao Tome And Principe ◽

Carbapenemase Gene ◽

High Prevalence ◽

Methylase Gene ◽

16S Rrna Methylase

ABSTRACT The occurrence of resistance to last-resort antibiotics was evaluated among Enterobacteriaceae isolates recovered from hospitalized children in a remote African archipelago, São Tomé and Príncipe, where there is limited access to those antibiotics. Fifty patients were screened for colonization by carbapenem-, pan-aminoglycoside-, or polymyxin-resistant Enterobacteriaceae. A total of 36 isolates (including 30 Escherichia coli and 4 Klebsiella pneumoniae) were recovered from 23 patients, including 26 isolates harboring the blaOXA-181 carbapenemase gene, a single isolate harboring the 16S rRNA methylase gene rmtB encoding pan-resistance to aminoglycosides, and 8 isolates coharboring both genes. A single isolate possessed the plasmid-borne colistin resistance gene mcr-1. A high clonal relationship was found for OXA-181-producing E. coli (4 clones), and conversely, three of the four OXA-181-producing K. pneumoniae isolates were clonally unrelated. This study overall showed a high prevalence of resistance to last-resort antibiotics in this country, where no epidemiological data were previously available.

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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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Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

Journal of Bacteriology ◽

10.1128/jb.00433-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 7

Author(s):

Charles T. Lauhon

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Genomic Context ◽

Similar Reduction ◽

Content Type ◽

E Coli ◽

Wobble Position ◽

Fertile Ground ◽

And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

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Molecular Characterization of Plasmid-Mediated Non-O157 Verotoxigenic Escherichia coli Isolated from Infants and Children with Diarrhea

Baghdad Science Journal ◽

10.21123/bsj.2020.17.3.0710 ◽

2020 ◽

Vol 17 (3) ◽

pp. 0710

Author(s):

Md Fazlul Karim Khan ◽

Shah Samiur Rashid

Keyword(s):

Escherichia Coli ◽

Virulence Gene ◽

Multidrug Resistant ◽

Plasmid Curing ◽

Health Issues ◽

E Coli ◽

Disk Diffusion Assay ◽

Microbiological Techniques ◽

Polymerase Chain

A significant increase in the incidence of non-O157 verotoxigenic Escherichia coli (VTEC) infections have become a serious health issues, and this situation is worsening due to the dissemination of plasmid mediated multidrug-resistant microorganisms worldwide. This study aims to investigate the presence of plasmid-mediated verotoxin gene in non-O157 E. coli. Standard microbiological techniques identified a total of 137 E. coli isolates. The plasmid was detected by Perfectprep Plasmid Mini preparation kit. These isolates were subjected to disk diffusion assay, and plasmid curing with ethidium bromide treatment. The plasmid containing isolates were subjected to a polymerase chain reaction (PCR) for investigating the presence of plasmid mediated verotoxin gene (VT1 and VT2) in non-O157 E. coli. Among the 137 E. coli isolates, 49 isolates were non-O157 E. coli while 29 (59.1%) isolates were verotoxin producing non-O157 serotypes and 26 non-O157 VTEC isolates possessed plasmids. Certain isolates harboured single sized plasmid while others had multiple plasmids with different size varied from 1.8kb to 7.6kb. A plasmid containing all (100%) the isolates was multidrug-resistant. Eight isolates changed their susceptibility patterns while three isolates were found to lose plasmid after post plasmid curing treatment and the rest of the isolates (15) remained constant. Different PCR sets characterized 3 plasmid-mediated verotoxins producing non-O157 E. coli. This current study demonstrated the occurrence of plasmid mediated verotoxin gene in non-O157 E. coli. To the best of our knowledge, this is the first report in the global literature on plasmid-mediated verotoxin gene in non-O157 E. coli. Timely diagnosis and surveillance of VTEC infections should prioritize to stop or slow down the virulence gene for dissemination by plasmid-mediated gene transfer amongst the same bacteria or other species.

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Molecular Characterization of Commensal Escherichia coli Adapted to Different Compartments of the Porcine Gastrointestinal Tract

Applied and Environmental Microbiology ◽

10.1128/aem.01688-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 6799-6803 ◽

Cited By ~ 12

Author(s):

Sam Abraham ◽

David M. Gordon ◽

James Chin ◽

Huub J. M. Brouwers ◽

Peter Njuguna ◽

...

Keyword(s):

Escherichia Coli ◽

Gastrointestinal Tract ◽

Random Amplified Polymorphic Dna ◽

Content Type ◽

E Coli ◽

Plasmid Replicon ◽

Different Strains ◽

Different Characteristics

ABSTRACTThe role ofEscherichia colias a pathogen has been the focus of considerable study, while much less is known about it as a commensal and how it adapts to and colonizes different environmental niches within the mammalian gut. In this study, we characterizeEscherichia coliorganisms (n= 146) isolated from different regions of the intestinal tracts of eight pigs (dueodenum, ileum, colon, and feces). The isolates were typed using the method of random amplified polymorphic DNA (RAPD) and screened for the presence of bacteriocin genes and plasmid replicon types. Molecular analysis of variance using the RAPD data showed thatE. coliisolates are nonrandomly distributed among different gut regions, and that gut region accounted for 25% (P< 0.001) of the observed variation among strains. Bacteriocin screening revealed that a bacteriocin gene was detected in 45% of the isolates, with 43% carrying colicin genes and 3% carrying microcin genes. Of the bacteriocins observed (H47, E3, E1, E2, E7, Ia/Ib, and B/M), the frequency with which they were detected varied with respect to gut region for the colicins E2, E7, Ia/Ib, and B/M. The plasmid replicon typing gave rise to 25 profiles from the 13 Inc types detected. Inc F types were detected most frequently, followed by Inc HI1 and N types. Of the Inc types detected, 7 were nonrandomly distributed among isolates from the different regions of the gut. The results of this study indicate that not only may the different regions of the gastrointestinal tract harbor different strains ofE. colibut also that strains from different regions have different characteristics.

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Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽

10.1099/mic.0.001087 ◽

2021 ◽

Vol 167 (9) ◽

Author(s):

Nirbhay Singh ◽

Anu Chauhan ◽

Ram Kumar ◽

Sudheer Kumar Singh

Keyword(s):

Amino Acids ◽

Mycobacterium Tuberculosis ◽

Type Species ◽

Low Ph ◽

Stress Conditions ◽

Starvation Stress ◽

Content Type ◽

E Coli ◽

Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

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