Culture of Urine Specimens by Use of chromID CPS Elite Medium Can Expedite Escherichia coli Identification and Reduce Hands-On Time in the Clinical Laboratory

Urine is one of the most common specimen types submitted to the clinical microbiology laboratory; the use of chromogenic agar is one method by which the laboratory might expedite culture results and reduce hands-on time and materials required for urine culture analysis. The objective of our study was to compare chromID CPS Elite (bioMérieux), a chromogenic medium, to conventional primary culture medium for evaluation of urine specimens. Remnant urine specimens (n= 200) were inoculated into conventional media and into chromID CPS Elite agar (chromID). The time to identification and consumables used were documented for both methods. Clinically significant pathogen(s) were recovered from 51 cultures using conventional media, withEscherichia colibeing the most frequently recovered organism (n= 22). The rate of exact uropathogen agreement between conventional and chromogenic media was 82%, while overall categorical agreement was 83.5% The time interval between plating and final organism identification was decreased with chromID agar versus conventional media forE. coli(mean of 24.4 h versus 27.1 h,P< 0.001). Using chromID, clinically significant cultures required less hands-on time per culture (mean of 1 min and 2 s [1:02 min]) compared to conventional media (mean of 1:31 min). In addition, fewer consumables (2.4 versus 3.3 sticks and swabs) and rapid biochemical tests (1.0 versus 1.9) were necessary using chromID versus conventional media. Notably, antimicrobial susceptibility testing demonstrated good overall agreement (97.4%) between the chromID and conventional media for all antibiotics tested. chromID CPS Elite is accurate for uropathogen identification, reduces consumable usage, and may expedite the identification ofE. coliin clinical specimens.

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Simplified scheme for identification of prompt lactose-fermenting members of the Enterobacteriaceae

Journal of Clinical Microbiology ◽

10.1128/jcm.4.6.511-514.1976 ◽

1976 ◽

Vol 4 (6) ◽

pp. 511-514

Author(s):

M J Hicks ◽

K J Ryan

Keyword(s):

Escherichia Coli ◽

Ornithine Decarboxylase ◽

Clinical Microbiology ◽

Clinical Microbiology Laboratory ◽

Microbiology Laboratory ◽

Biochemical Tests ◽

Gram Negative ◽

E Coli ◽

Colonial Morphology ◽

Level Identification

A brief, simplified scheme involving the spot indole test and colonial morphology was evaluated for genus level identification of prompt lactose-fermenting (PLF) members of the Enterobacteriaceae. One hundred and ninety-four consecutive, clinically important PLF gram-negative rods isolated in a clinical microbiology laboratory were identified by this simplified scheme, as well as by standard biochemical tests, and the API 20E (Analytab Products, Inc., Plainview, N.Y.) system. In the simplified scheme a flat, spot indole-positive colony was identified as Escherichia coli. Spot indole-negative organisms forming nucoid colonies were identified as Klebsiella sp. or Enterobacter sp. on the basis of semisolid motility and ornithine decarboxylase tests. Approximately 94% of the study isolates followed reactions typical for E. coli, Klebsiella sp., and Enterobacter sp. as defined by this simplified scheme. When compared with the standard and Analytab Products Inc. identifications, the overall accuracy was 97.4%. The accuracy of identification of E. coli, Klebsiella sp., and Enterobacter sp. was 98.1%, 95.6%, and 87.5%, respectively. This simplified scheme is recommended for identification of selected PLF isolates in the clinical microbiology laboratory.

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AnIn VitroDeletion inribEEncoding Lumazine Synthase Contributes to Nitrofurantoin Resistance in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03952-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7225-7233 ◽

Cited By ~ 13

Author(s):

Jascha Vervoort ◽

Basil Britto Xavier ◽

Andrew Stewardson ◽

Samuel Coenen ◽

Maciek Godycki-Cwirko ◽

...

Keyword(s):

Escherichia Coli ◽

Flavin Mononucleotide ◽

Content Type ◽

E Coli ◽

Lumazine Synthase ◽

Oxygen Sensitive ◽

Clinically Significant ◽

Essential Enzyme ◽

Tract Infections

ABSTRACTNitrofurantoin has been used for decades for the treatment of urinary tract infections (UTIs), but clinically significant resistance inEscherichia coliis uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinalE. colistrains (ST540-p and ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 μg/ml) harbored mutations in the known nitrofurantoin resistance determinantsnfsAand/ornfsB, which encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC of 32 μg/ml) and exhibited remarkable growth deficits but did not harbornfsA/nfsBmutations. We identified a 12-nucleotide deletion inribE, encoding lumazine synthase, an essential enzyme involved in the biosynthesis of flavin mononucleotide (FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-RE. coliisolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations innfsAand/ornfsBbut notribE. Sequencing of theribEgene in six intestinal and three urinaryE. colistrains showing reduced nitrofurantoin susceptibility (MICs of 16 to 48 μg/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generatedin vitro, presumably because of additional mutations in oxygen-sensitive nitroreductases.

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Occurrence of Intestinal and Extraintestinal Virulence Genes in Escherichia coli Isolates from Rainwater Tanks in Southeast Queensland, Australia

Applied and Environmental Microbiology ◽

10.1128/aem.06047-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7394-7400 ◽

Cited By ~ 29

Author(s):

W. Ahmed ◽

L. Hodgers ◽

N. Masters ◽

J. P. S. Sidhu ◽

M. Katouli ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Genes ◽

Phylogenetic Groups ◽

Content Type ◽

E Coli ◽

Rainwater Tank ◽

Clinically Significant ◽

Treatment Procedures ◽

Southeast Queensland ◽

Rainwater Tanks

ABSTRACTIn this study, 200Escherichia coliisolates from 22 rainwater tank samples in Southeast Queensland, Australia, were tested for the presence of 20 virulence genes (VGs) associated with intestinal and extraintestinal pathotypes. In addition,E. coliisolates were also classified into phylogenetic groups based on the detection of thechuA,yjaA, and TSPE4.C2 genes. Of the 22 rainwater tanks, 8 (36%) and 5 (23%) were positive for theeaeA(belonging to enteropathogenicE. coli[EPEC] and Shiga-toxigenicE. coli[STEC]) and ST1 (belonging to enterotoxigenicE. coli[ETEC]) genes, respectively. VGs (cdtB,cvaC,ibeA,kpsMTallele III, PAI,papAH, andtraT) belonging to extraintestinal pathogenicE. coli(ExPEC) were detected in 15 (68%) of the 22 rainwater tanks. Of the 22 samples, 17 (77%) and 11 (50%) containedE. colibelonging to phylogenetic groups A and B1, respectively. Similarly, 10 (45%) and 16 (72%) containedE. colibelonging to phylogenetic groups B2 and D, respectively. Of the 96 of the 200 strains from 22 tanks that were VG positive, 40 (42%) were carrying a single VG, 36 (37.5%) were carrying two VGs, 17 (18%) were carrying three VGs, and 3 (3%) had four or more VGs. This study reports the presence of multiple VGs inE. colistrains belonging to the STEC, EPEC, ETEC, and ExPEC pathotypes in rainwater tanks. The public health risks associated with potentially clinically significantE. coliin rainwater tanks should be assessed, as the water is used for drinking and other, nonpotable purposes. It is recommended that rainwater be disinfected using effective treatment procedures such as filtration, UV disinfection, or simply boiling prior to drinking.

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Isolation and Characterization of Escherichia coli Sequence Type 131 and Other Antimicrobial-Resistant Gram-Negative Bacilli from Clinical Stool Samples from Veterans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00383-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4638-4645 ◽

Cited By ~ 3

Author(s):

Muhanad Mohamed ◽

Connie Clabots ◽

Stephen B. Porter ◽

Paul Thuras ◽

James R. Johnson

Keyword(s):

Escherichia Coli ◽

Clinical Laboratory ◽

Medical Center ◽

Multidrug Resistant ◽

Sequence Type ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Isolation And Characterization ◽

Stool Samples

ABSTRACTEmerging multidrug-resistant (MDR) Gram-negative bacilli (GNB), includingEscherichia colisequence type 131 (ST131) and its resistance-associatedH30 subclone, constitute an ever-growing public health threat. Their reservoirs and transmission pathways are incompletely defined. To assess diarrheal stools as a potential reservoir for ST131-H30 and other MDR GNB, we cultured 100 clinical stool samples from a Veterans Affairs Medical Center clinical laboratory (October to December 2011) for fluoroquinolone- and extended-spectrum cephalosporin (ESC)-resistantE. coliand other GNB, plus totalE. coli. We then characterized selected resistant and susceptibleE. coliisolates by clonal group, phylogenetic group, virulence genotype, and pulsotype and screened all isolates for antimicrobial resistance. Overall, 79 of 100 stool samples yielded GNB (52E. coli; 48 other GNB). Fifteen samples yielded fluoroquinolone-resistantE. coli(10 were ST131, of which 9 wereH30), 6 yielded ESC-resistantE. coli(2 were ST131, both non-H30), and 31 yielded susceptibleE. coli(1 was ST131, non-H30), for 13 total ST131-positive samples. Fourteen non-E. coliGNB were ESC resistant, and three were fluoroquinolone resistant. Regardless of species, almost half (46%) of the fluoroquinolone-resistant and/or ESC-resistant non-E. coliGNB were resistant to at least three drug classes. Fecal ST131 isolates closely resembled reference clinical ST131 isolates according to virulence genotypes and pulsed-field gel electrophoresis (PFGE) profiles. Thus, a substantial minority (30%) of veterans with diarrhea who undergo stool testing excrete antibiotic-resistant GNB, includingE. coliST131. Consequently, diarrhea may pose transmission risks for more than just diarrheal pathogens and may help disseminate clinically relevant ST131 strains and other MDR GNB within hospitals and the community.

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Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v1i2.21506 ◽

2016 ◽

Vol 1 (2) ◽

pp. 38-42 ◽

Cited By ~ 4

Author(s):

Khairun Nessa ◽

Dilruba Ahmed ◽

Johirul Islam ◽

FM Lutful Kabir ◽

M Anowar Hossain

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Clinical Laboratory ◽

Proteinase K ◽

Microbiology Laboratory ◽

Pcr Assay ◽

E Coli ◽

Diarrheagenic Escherichia Coli ◽

Multiplex Pcr Assay ◽

Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

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Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00781-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 9

Author(s):

Dongfei Han ◽

Ji-Young Ryu ◽

Robert A. Kanaly ◽

Hor-Gil Hur

Keyword(s):

Escherichia Coli ◽

Pseudomonas Putida ◽

Hypothetical Protein ◽

Aldehyde Group ◽

Agrobacterium Vitis ◽

T7 Promoter ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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Autotransporter Protein-Encoding Genes of Diarrheagenic Escherichia coli Are Found in both Typical and Atypical Enteropathogenic E. coli Strains

Applied and Environmental Microbiology ◽

10.1128/aem.02635-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 411-414 ◽

Cited By ~ 21

Author(s):

Afonso G. Abreu ◽

Vanessa Bueris ◽

Tatiane M. Porangaba ◽

Marcelo P. Sircili ◽

Fernando Navarro-Garcia ◽

...

Keyword(s):

Escherichia Coli ◽

Content Type ◽

E Coli ◽

Diarrheagenic Escherichia Coli ◽

Virulence Markers ◽

Protein Encoding ◽

Encoding Genes ◽

Specific Virulence

ABSTRACTAutotransporter (AT) protein-encoding genes of diarrheagenicEscherichia coli(DEC) pathotypes (cah,eatA,ehaABCDJ,espC,espI,espP,pet,pic,sat, andtibA) were detected in typical and atypical enteropathogenicE. coli(EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

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A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

Author(s):

Kristin R. Baker ◽

Bimal Jana ◽

Henrik Franzyk ◽

Luca Guardabassi

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Gram Negative Bacteria ◽

Chromogenic Substrate ◽

Intrinsic Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽

10.1128/mbio.01105-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 7

Author(s):

Christopher W. Lennon ◽

Kimberly C. Lemmer ◽

Jessica L. Irons ◽

Max I. Sellman ◽

Timothy J. Donohue ◽

...

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Rhodobacter Sphaeroides ◽

Fatty Acid Content ◽

Regulatory Protein ◽

Growth Conditions ◽

Globular Domain ◽

Content Type ◽

E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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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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