scholarly journals Arr-cb Is a Rifampin Resistance Determinant Found Active or Cryptic in Clostridiumbolteae Strains

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Jean-Christophe Marvaud ◽  
Thierry Lambert
Keyword(s):  
Escherichia Coli ◽  
Gut Microbiota ◽  
Reverse Transcription ◽  
Recent Analysis ◽  
Pcr Analysis ◽  
Directed Mutagenesis ◽  
Human Gut ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Rifampin Resistance

ABSTRACT Clostridium bolteae, which belongs to the Clostridium clostridioforme complex, is a member of the human gut microbiota. Recent analysis of seven genomes of C. bolteae revealed the presence of an arr-like gene. Among these strains, only 90A7 was found to be resistant to rifampin in the absence of alteration of RpoB. Cloning of arr-cb from 90A7 in Escherichia coli combined with directed mutagenesis demonstrated that Arr-cb was functional but that a Q127→R variant present in 90A9 and 90B3 was inactive. Quantitative reverse transcription-PCR analysis indicated that arr-cb was silent in the four remaining strains because of defective transcription. Thus, two independent mechanisms can make the probably intrinsic arr-cb gene of C. bolteae cryptic.

scholarly journals Impact of Ceftiofur Injection on Gut Microbiota and Escherichia coli Resistance in Pigs

2015 ◽  
Vol 59 (9) ◽  
pp. 5171-5180 ◽  
Author(s):  
M. A. Fleury ◽  
G. Mourand ◽  
E. Jouy ◽  
F. Touzain ◽  
L. Le Devendec ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gut Microbiota ◽  
Treated Group ◽  
Conjugative Plasmid ◽  
Health Concern ◽  
Pcr Analysis ◽  
Contamination Level ◽  
Content Type ◽  
E Coli ◽  
The Impact

ABSTRACTResistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance inEscherichia coli. Pigs were orally inoculated with an ESC-resistantE. coliM63 strain harboring a conjugative plasmid carrying a gene conferring resistance,blaCTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids.E. coliand ESC-resistantE. coliwere determined by culture methods, and the ESC-resistantE. coliisolates were characterized. The copies of theblaCTX-M-1gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in theE. colipopulation. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups,E. coliM63 persisted in most pigs, and theblaCTX-M-1gene was transferred to otherE. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistantE. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

scholarly journals The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Felizza F. Gunderson ◽  
Nicholas P. Cianciotto
Keyword(s):  
Reverse Transcription ◽  
Legionella Pneumophila ◽  
Host Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Content Type ◽  
Intracellular Infection ◽  
Reverse Transcription Pcr ◽  
Crispr Array ◽  
Agar Media

ABSTRACTRecent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of theLegionella pneumophilastrain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that containscas9,cas1,cas2,cas4, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels ofcastranscripts, especially those ofcas1andcas2, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media.cas9,cas1,cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However,cas2mutants, although they grew typically in macrophages, were significantly impaired for infection of bothHartmannellaandAcanthamoebaspecies. A complementedcas2mutant infected the amoebae at wild-type levels, confirming thatcas2is required for intracellular infection of these host cells.IMPORTANCEGiven that infection of amoebae is critical forL. pneumophilapersistence in water systems, our data indicate thatcas2has a role in the transmission of Legionnaires’ disease. Because our experiments were done in the absence of added phage, plasmid, or nucleic acid, the event that is facilitated by Cas2 is uniquely distinct from current dogma concerning CRISPR-Cas function.

scholarly journals Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg
Keyword(s):  
Reverse Transcription ◽  
Membrane Filtration ◽  
Limit Of Detection ◽  
Mineral Waters ◽  
Pcr Analysis ◽  
Rna Sequences ◽  
Natural Mineral ◽  
Reverse Transcription Pcr ◽  
Viral Particles ◽  
Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

scholarly journals Evolution of Rifampin Resistance in Escherichia coli and Mycobacterium smegmatis Due to Substandard Drugs

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Zohar B. Weinstein ◽  
Muhammad H. Zaman
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Smegmatis ◽  
Degradation Products ◽  
Treatment Options ◽  
Poor Quality ◽  
Cross Resistance ◽  
Standard Drug ◽  
Content Type ◽  
Drug Degradation ◽  
Rifampin Resistance

ABSTRACT Poor-quality medicines undermine the treatment of infectious diseases, such as tuberculosis, which require months of treatment with rifampin and other drugs. Rifampin resistance is a critical concern for tuberculosis treatment. While subtherapeutic doses of medicine are known to select for antibiotic resistance, the effect of drug degradation products on the evolution of resistance is unknown. Here, we demonstrate that substandard drugs that contain degraded active pharmaceutical ingredients select for gene alterations that confer resistance to standard drugs. We generated drug-resistant Escherichia coli and Mycobacterium smegmatis strains by serially culturing bacteria in the presence of the rifampin degradation product rifampin quinone. We conducted Sanger sequencing to identify mutations in rifampin-resistant populations. Strains resistant to rifampin quinone developed cross-resistance to the standard drug rifampin, with some populations showing no growth inhibition at maximum concentrations of rifampin. Sequencing of the rifampin quinone-treated strains indicated that they acquired mutations in the DNA-dependent RNA polymerase B subunit. These mutations were localized in the rifampin resistance-determining region (RRDR), consistent with other reports of rifampin-resistant E. coli and mycobacteria. Rifampin quinone-treated mycobacteria also had cross-resistance to other rifamycin class drugs, including rifabutin and rifapentine. Our results strongly suggest that substandard drugs not only hinder individual patient outcomes but also restrict future treatment options by actively contributing to the development of resistance to standard medicines.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

scholarly journals Dietary Fatty Acids Sustain the Growth of the Human Gut Microbiota

2018 ◽  
Vol 84 (21) ◽  
Author(s):  
Richard Agans ◽  
Alex Gordon ◽  
Denise Lynette Kramer ◽  
Sergio Perez-Burillo ◽  
José A. Rufián-Henares ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Small Intestine ◽  
Fatty Acid ◽  
Gut Microbiota ◽  
Short Chain Fatty Acids ◽  
Dietary Fatty Acids ◽  
Dietary Fats ◽  
High Fat ◽  
Human Gut ◽  
Content Type

ABSTRACTWhile a substantial amount of dietary fats escape absorption in the human small intestine and reach the colon, the ability of resident microbiota to utilize these dietary fats for growth has not been investigated in detail. In this study, we used anin vitromultivessel simulator system of the human colon to reveal that the human gut microbiota is able to utilize typically consumed dietary fatty acids to sustain growth. Gut microbiota adapted quickly to a macronutrient switch from a balanced Western diet-type medium to its variant lacking carbohydrates and proteins. We defined specific genera that increased in their abundances on the fats-only medium, includingAlistipes,Bilophila, and several genera of the classGammaproteobacteria. In contrast, the abundances of well-known glycan and protein degraders, includingBacteroides,Clostridium, andRoseburiaspp., were reduced under such conditions. The predicted prevalences of microbial genes coding for fatty acid degradation enzymes and anaerobic respiratory reductases were significantly increased in the fats-only environment, whereas the abundance of glycan degradation genes was diminished. These changes also resulted in lower microbial production of short-chain fatty acids and antioxidants. Our findings provide justification for the previously observed alterations in gut microbiota observed in human and animal studies of high-fat diets.IMPORTANCEIncreased intake of fats in many developed countries has raised awareness of potentially harmful and beneficial effects of high fat consumption on human health. Some dietary fats escape digestion in the small intestine and reach the colon where they can be metabolized by gut microbiota. We show that human gut microbes are able to maintain a complex community when supplied with dietary fatty acids as the only nutrient and carbon sources. Such fatty acid-based growth leads to lower production of short-chain fatty acids and antioxidants by community members, which potentially have negative health consequences on the host.

Norovirus Detection at Oualidia Lagoon, a Moroccan Shellfish Harvesting Area, by Reverse Transcription PCR Analysis

2019 ◽  
Vol 11 (3) ◽  
pp. 268-273
Author(s):  
N. El Moqri ◽  
F. El Mellouli ◽  
N. Hassou ◽  
M. Benhafid ◽  
N. Abouchoaib ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Oualidia Lagoon

scholarly journals N-Hydroxyarylamine O-Acetyltransferases Catalyze Acetylation of 3-Amino-4-Hydroxyphenylarsonic Acid in the 4-Hydroxy-3-Nitrobenzenearsonic Acid Transformation Pathway of Enterobacter sp. Strain CZ-1

2019 ◽  
Vol 86 (2) ◽  
Author(s):  
Ke Huang ◽  
Fan Gao ◽  
X. Chris Le ◽  
Fang-Jie Zhao
Keyword(s):  
Growth Promotion ◽  
Functional Characterization ◽  
Major Product ◽  
Site Directed Mutagenesis ◽  
Feed Additive ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Arsanilic Acid ◽  
Relative Expression Level

ABSTRACT The organoarsenical feed additive 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone [ROX]) is widely used and released into the environment. We previously showed a two-step pathway of ROX transformation by Enterobacter sp. strain CZ-1 involving the reduction of ROX to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) and the acetylation of 3-AHPAA to N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA) (K. Huang, H. Peng, F. Gao, Q. Liu, et al., Environ Pollut 247:482–487, 2019, https://doi.org/10.1016/j.envpol.2019.01.076). In this study, we identified two nhoA genes (nhoA1 and nhoA2), encoding N-hydroxyarylamine O-acetyltransferases, as responsible for 3-AHPAA acetylation in Enterobacter sp. strain CZ-1. The results of genetic disruption and complementation showed that both nhoA genes are involved in ROX biotransformation and that nhoA1 is the major 3-AHPAA acetyltransferase gene. Quantitative reverse transcription-PCR analysis showed that the relative expression level of nhoA1 was 3-fold higher than that of nhoA2. Each of the recombinant NhoAs was overexpressed in Escherichia coli BL21 and homogenously purified as a dimer by affinity chromatography. Both purified NhoAs catalyzed acetyl coenzyme A-dependent 3-AHPAA acetylation. The Km values of 3-AHPAA for NhoA1 and NhoA2 were 151.5 and 428.3 μM, respectively. Site-directed mutagenesis experiments indicated that two conserved arginine and cysteine residues of each NhoA were necessary for their enzyme activities. IMPORTANCE Roxarsone (ROX) is an organoarsenic feed additive that has been widely used in poultry industries for growth promotion, coccidiosis control, and meat pigmentation improvement for more than 70 years. Most ROX is excreted in the litter and dispersed into the environment, where it is transformed by microbes into different arsenic-containing compounds. A major product of ROX transformation is N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), which is also used as a clinical drug for treating refractory bacterial vaginosis. Here, we report the cloning and functional characterization of two genes encoding N-hydroxyarylamine O-acetyltransferases, NhoA1 and NhoA2, in Enterobacter sp. strain CZ-1, which catalyze the acetylation of 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) formed by the reduction of ROX to N-AHPAA. This study provides new insights into the function of N-hydroxyarylamine O-acetyltransferase in the transformation of an important organoarsenic compound.

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