THE TENTH C. L. OAKLEY LECTURE * : The success of plasmid-encoded resistance genes in clinical bacteria: An examination of plasmid-mediated ampicillin and trimethoprim resistance genes and their resistance mechanisms

Race-specific resistance against powdery mildews is well documented in small grains but, in other crops such as grapevine, controlled analysis of host–pathogen interactions on resistant plants is uncommon. In the current study, we attempted to confirm powdery mildew resistance phenotypes through vineyard, greenhouse, and in vitro inoculations for test cross-mapping populations for two resistance sources: (i) a complex hybrid breeding line, ‘Bloodworth 81-107-11', of at least Vitis rotundifolia, V. vinifera, V. berlandieri, V. rupestris, V. labrusca, and V. aestivalis background; and (ii) Vitis hybrid ‘Tamiami’ of V. aestivalis and V. vinifera origin. Statistical analysis of vineyard resistance data suggested the segregation of two and three race-specific resistance genes from the two sources, respectively. However, in each population, some resistant progeny were susceptible in greenhouse or in vitro screens, which suggested the presence of Erysiphe necator isolates virulent on progeny segregating for one or more resistance genes. Controlled inoculation of resistant and susceptible progeny with a diverse set of E. necator isolates clearly demonstrated the presence of fungal races differentially interacting with race-specific resistance genes, providing proof of race specificity in the grape powdery mildew pathosystem. Consistent with known race-specific resistance mechanisms, both resistance sources were characterized by programmed cell death of host epidermal cells under appressoria, which arrested or slowed hyphal growth; this response was also accompanied by collapse of conidia, germ tubes, appressoria, and secondary hyphae. The observation of prevalent isolates virulent on progeny with multiple race-specific resistance genes before resistance gene deployment has implications for grape breeding strategies. We suggest that grape breeders should characterize the mechanisms of resistance and pyramid multiple resistance genes with different mechanisms for improved durability.

Download Full-text

Detection of resistance genes from Pseudomonas aeruginosa using immunomagnetic isolation and chemiluminescence

Materials Express ◽

10.1166/mex.2020.1650 ◽

2020 ◽

Vol 10 (3) ◽

pp. 412-418

Author(s):

Fei Xu ◽

Cheng Chen ◽

Xing Li ◽

Bo Zhang

Keyword(s):

Pseudomonas Aeruginosa ◽

Resistance Genes ◽

High Efficiency ◽

Bacterial Pathogen ◽

Resistance Mechanisms ◽

Clinical Samples ◽

Specific Gene ◽

Beta Lactamases ◽

Immunomagnetic Isolation ◽

Encoding Gene

Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic and nosocomial bacterial pathogen. Various multi-resistance mechanisms present across numerous P. aeruginosa strains counteract conventional antimicrobial therapy, thereby becoming a great challenge. This study aimed to establish the application of immunomagnetic isolation and chemiluminescence to detect the presence of extended spectra of β-lactamases encoding genes: blaTEM and blaVEB; metallo-beta-lactamases encoding gene: blaVIM; aminoglycoside modifying enzymes encoding gene: aac(6)II, ant(3)I; and the specific gene for P. aeruginosa, gyrB. P. aeruginosa was specifically selected using the immunomagnetic nanoparticles (IMNPs) in the six parallel bacterial plates counting, proving that they are reliable. Then, the high efficiency of IMNPs@Probes in targeting the resistance genes of P. aeruginosa was demonstrated using the results of chemiluminescent intensities of blaTEM, blaVEB, blaVIM aac(6)II, ant(3)I, and gyrB (more than 10 times higher than that of the control). Sixty-eight in situ clinical samples were tested for the presence of these resistance genes, and one more blaTEM and three more blaVIM individuals were detected using this method compared to the traditional PCR. Thus, the application of our method in clinical screening is specific, accurate, and reliable, and it could be useful in the administration of appropriate treatment.

Download Full-text

480. Molecular Characterization of Multidrug-Resistant Gram-Negative Pathogens in Three Tertiary Care Hospitals in Egypt

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.553 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S235-S235

Author(s):

Amani Kholy ◽

Samia A Girgis ◽

Arwa R Elmanakhly ◽

Mervat A F Shetta ◽

Dalia El- Kholy ◽

...

Keyword(s):

Molecular Characterization ◽

Resistance Genes ◽

Genetic Basis ◽

Tertiary Care ◽

Carbapenem Resistance ◽

Resistance Mechanisms ◽

Gram Negative ◽

E Coli ◽

Tertiary Care Hospitals

Abstract Background High rates of AMR among Gram-negative bacilli (GNB) have been reported from Egypt for almost 2 decades. Surveillance and identifying the genetic basis of AMR provide important information to optimize patient care. As there is no adequate data on the genetic basis of AMR in Egypt, we aimed to identify the molecular characterization of multi-drug-resistant (MDR) Gram-negative pathogens (GNP). Methods Three major tertiary-care hospitals in Egypt participated in the “Study for Monitoring Antimicrobial Resistance Trends” (SMART) from 2014 to 2016. Consecutive GNPs were identified and their susceptibility to antimicrobials were tested. Molecular identification of ESBL, AmpC, and carbapenemase resistance genes was conducted on MDR isolates. Results We enrolled 1,070 consecutive Gram-negative isolates; only one isolate per patient according to the standard protocol of (SMART). During 2014–2015, 578 GNP were studied. Enterobacteriaceae comprised 66% of the total isolates. K. pneumoniae and E. coli were the most common (29.8% and 29.4%). K. pneumoniae and E. coli were the predominant organisms in IAI (30.5% and 30.1%, respectively) and UTI (and 38.9% and 48.6%, respectively), while Acinetobacter baumannii was the most prevalent in RTI (40.2%). ESBL producers were phenotypically detected in 53% of K. pneumoniae, and 68% of E. coli. During 2016, 495 GNP were studied. ESBL continued to be high. For E. coli and K. pneunomiea, the most active antimicrobials were amikacin (≥93%), imipenem/meropenem (≥87%) and colistin (97%). Genetic study of ertapenem-resistant isolates and 50% of isolates with ESBL phenotype revealed ESβL production in more than 90% of isolates; blaCTXM-15 was detected in 71.4% and 68.5% in K. pneumoniae and E. coli, respectively, blaTEM-OSBL in 48.5% and47.5% of K. pneumoniae and E. coli, respectively. Carbapenem resistance genes were detected in 45.4% of isolates. In K. pneumoniae, OXA-48 dominated (40.6%), followed by NDM1 (23.7%) and OXA-232 (4.5%). Conclusion Our study detected alarming rates of resistance and identified many resistance mechanisms in clinical isolates from Egyptian hospitals. These high rates highlight the importance of continuous monitoring of the resistance trend and discovering the novel resistant mechanisms of resistance, and the underscores a national antimicrobial stewardship plan in Egypt. Disclosures All authors: No reported disclosures.

Download Full-text

Antibiotic Resistances of Starter and Probiotic Strains of Lactic Acid Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02105-06 ◽

2006 ◽

Vol 73 (3) ◽

pp. 730-739 ◽

Cited By ~ 188

Author(s):

Anja S. Hummel ◽

Christian Hertel ◽

Wilhelm H. Holzapfel ◽

Charles M. A. P. Franz

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Resistance Genes ◽

Evaluation System ◽

Genetic Basis ◽

Safety Evaluation ◽

Resistance Mechanisms ◽

Resistance Testing ◽

Gene Probe ◽

Erythromycin Resistance

ABSTRACT The antibiotic resistances of 45 lactic acid bacteria strains belonging to the genera Lactobacillus, Streptococcus, Lactococcus, Pediococcus, and Leuconostoc were investigated. The objective was to determine antibiotic resistances and to verify these at the genetic level, as is currently suggested by the European “qualified presumption of safety” safety evaluation system for industrial starter strains. In addition, we sought to pinpoint possible problems in resistance determinations. Primers were used to PCR amplify genes involved in β-lactam antibiotic, chloramphenicol, tetracycline, and erythromycin resistance. The presence of ribosomal protection protein genes and the ermB gene was also determined by using a gene probe. Generally, the incidences of erythromycin, chloramphenicol, tetracycline, or β-lactam resistances in this study were low (<7%). In contrast, aminoglycoside (gentamicin and streptomycin) and ciprofloxacin resistances were higher than 70%, indicating that these may constitute intrinsic resistances. The genetic basis for ciprofloxacin resistance could not be verified, since no mutations typical of quinolone resistances were detected in the quinolone determining regions of the parC and gyrA genes. Some starter strains showed low-level ampicillin, penicillin, chloramphenicol, and tetracycline resistances, but no known resistance genes could be detected. Although some strains possessed the cat gene, none of these were phenotypically resistant to chloramphenicol. Using reverse transcription-PCR, these cat genes were shown to be silent under both inducing and noninducing conditions. Only Lactobacillus salivarius BFE 7441 possessed an ermB gene, which was encoded on the chromosome and which could not be transferred in filter-mating experiments. This study clearly demonstrates problems encountered with resistance testing, in that the breakpoint values are often inadequately identified, resistance genes may be present but silent, and the genetic basis and associated resistance mechanisms toward some antibiotics are still unknown.

Download Full-text

Co-selection of antibiotic and heavy metal resistance in freshwater bacteria

Journal of Limnology ◽

10.4081/jlimnol.2016.1198 ◽

2016 ◽

Vol 75 (s2) ◽

Cited By ~ 34

Author(s):

Andrea Di Cesare ◽

Ester Eckert ◽

Gianluca Corno

Keyword(s):

Heavy Metal ◽

Resistance Genes ◽

Heavy Metal Resistance ◽

Resistance Mechanisms ◽

Metal Resistance ◽

Resistant Bacteria ◽

Human Pathogens ◽

Antibiotic Resistant Bacteria ◽

Antibiotic Resistant ◽

Selection Of

Antibiotic resistant bacteria are found in most environments, especially in highly anthropized waters. A direct correlation between human activities (e.g., pollution) and spread and persistence of antibiotic resistant bacteria (ARB) and resistance genes (ARGs) within the resident bacterial communities appears more and more obvious. Furthermore, the threat posed for human health by the presence of ARB and ARGs in these environments is enhanced by the risk of horizontal gene transfer of resistance genes to human pathogens. Although the knowledge on the spread of antibiotic resistances in waters is increasing, the understanding of the driving factors determining the selection for antibiotic resistance in the environment is still scarce. Antibiotic pollution is generally coupled with contamination by heavy metals (HMs) and other chemicals, which can also promote the development of resistance mechanisms, often through co-selecting for multiple resistances. The co-selection of heavy metal resistance genes and ARGs in waters, sediments, and soils, increases the complexity of the ecological role of ARGs, and reduces the effectiveness of control actions. In this mini-review we present the state-of-the-art of the research on antibiotic- and HM-resistance and their connection in the environment, with a focus on HM pollution and aquatic environments. We review the spread and the persistence of HMs and/or ARB, and how it influences their respective gene co-selection. In the last chapter, we propose Lake Orta, a system characterized by an intensive HM pollution followed by a successful restoration of the chemistry of the water column, as a study-site to evaluate the spread and selection of HMs and antibiotic resistances in heavily disturbed environments.

Download Full-text

Feeding responses of the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), to resistant and susceptible host-plants

Bulletin of Entomological Research ◽

10.1017/s0007485300018290 ◽

1989 ◽

Vol 79 (2) ◽

pp. 309-318 ◽

Cited By ~ 8

Author(s):

D. E. Padgham ◽

S. Woodhead ◽

H. R. Rapusas

Keyword(s):

Resistance Genes ◽

Host Plants ◽

Brown Planthopper ◽

Nilaparvata Lugens ◽

Resistance Mechanisms ◽

Population Development ◽

Rice Varieties ◽

Susceptible Host ◽

Insect Activity ◽

Nilaparvata Lugens Stål

AbstractThe feeding responses, growth and population development of Nilaparvata lugens (Stål) are quantified on a range of twelve susceptible or resistant host-plants. Paris of rice varieties carrying the Bph1, bph2 and Bph3 resistance genes are compared as hosts for N. lugens, and it is concluded that such notations do not adequately describe the diversity of plant resistance mechanisms. Evidence is presented for resistance mechanisms involving enhanced insect activity and gustatory responses to unacceptable phloem.

Download Full-text

Mapping the malaria parasite drug-able genome using in vitro evolution and chemogenomics

10.1101/139386 ◽

2017 ◽

Cited By ~ 1

Author(s):

Annie N. Cowell ◽

Eva S. Istvan ◽

Amanda K. Lukens ◽

Maria G. Gomez-Lorenzo ◽

Manu Vanaerschot ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Resistance Genes ◽

Genome Analysis ◽

Drug Targets ◽

Malaria Parasite ◽

Resistance Mechanisms ◽

Whole Genome

AbstractChemogenetic characterization through in vitro evolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262 Plasmodium falciparum parasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of the P. falciparum resistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.One Sentence SummaryWhole genome sequencing reveals how Plasmodium falciparum evolves resistance to diverse compounds and identifies new antimalarial drug targets.

Download Full-text

Elucidating Anthracnose Resistance Mechanisms in Sorghum—A Review

Phytopathology ◽

10.1094/phyto-04-20-0132-rvw ◽

2020 ◽

Vol 110 (12) ◽

pp. 1863-1876

Author(s):

Lauren R. Stutts ◽

Wilfred Vermerris

Keyword(s):

Resistance Genes ◽

Draft Genome ◽

Resistance Mechanisms ◽

Anthracnose Resistance ◽

Sequencing Technologies ◽

Recent Developments ◽

Fuels And Chemicals ◽

Sorghum Production ◽

Sorghum Germplasm ◽

Identify Gene Expression

Sorghum (Sorghum bicolor) is the fifth most cultivated cereal crop in the world, traditionally providing food, feed, and fodder, but more recently also fermentable sugars for the production of renewable fuels and chemicals. The hemibiotrophic fungal pathogen Colletotrichum sublineola, the causal agent of anthracnose disease in sorghum, is prevalent in the warm and humid climates where much of the sorghum is cultivated and poses a serious threat to sorghum production. The use of anthracnose-resistant sorghum germplasm is the most environmentally and economically sustainable way to protect sorghum against this pathogen. Even though multiple anthracnose resistance loci have been mapped in diverse sorghum germplasm in recent years, the diversity in C. sublineola pathotypes at the local and regional levels means that these resistance genes are not equally effective in different areas of cultivation. This review summarizes the genetic and cytological data underlying sorghum’s defense response and describes recent developments that will enable a better understanding of the interactions between sorghum and C. sublineola at the molecular level. This includes releases of the sorghum genome and the draft genome of C. sublineola, the use of next-generation sequencing technologies to identify gene expression networks activated in response to infection, and improvements in methodologies to validate resistance genes, notably virus-induced and transgenic gene silencing approaches.

Download Full-text

New Determinants of Aminoglycoside Resistance and Their Association with the Class 1 Integron Gene Cassettes in Trueperella pyogenes

International Journal of Molecular Sciences ◽

10.3390/ijms21124230 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4230

Author(s):

Ewelina Kwiecień ◽

Ilona Stefańska ◽

Dorota Chrobak-Chmiel ◽

Agnieszka Sałamaszyńska-Guz ◽

Magdalena Rzewuska

Keyword(s):

Resistance Genes ◽

Resistance Mechanisms ◽

Aminoglycoside Resistance ◽

Class 1 Integron ◽

Gene Cassettes ◽

Genetic Elements ◽

Trueperella Pyogenes ◽

Broth Microdilution Method ◽

Class 1 ◽

Aminoglycoside Resistance Genes

Trueperella pyogenes is an important opportunistic animal pathogen. Different antimicrobials, including aminoglycosides, are used to treat T. pyogenes infections. The aim of the present study was to evaluate aminoglycoside susceptibility and to detect aminoglycoside resistance determinants in 86 T. pyogenes isolates of different origin. Minimum inhibitory concentration of gentamicin, streptomycin, and kanamycin was determined using a standard broth microdilution method. Genetic elements associated with aminoglycoside resistance were investigated by PCR and DNA sequencing. All studied isolates were susceptible to gentamicin, but 32.6% and 11.6% of them were classified as resistant to streptomycin and kanamycin, respectively. A total of 30 (34.9%) isolates contained class 1 integrons. Class 1 integron gene cassettes carrying aminoglycoside resistance genes, aadA11 and aadA9, were found in seven and two isolates, respectively. Additionally, the aadA9 gene found in six isolates was not associated with mobile genetic elements. Moreover, other, not carried by gene cassettes, aminoglycoside resistance genes, strA-strB and aph(3’)-IIIa, were also detected. Most importantly, this is the first description of all reported genes in T. pyogenes. Nevertheless, the relevance of the resistance phenotype to genotype was not perfectly matched in 14 isolates. Therefore, further investigations are needed to fully explain aminoglycoside resistance mechanisms in T. pyogenes.

Download Full-text

Characterization of Fosfomycin and Nitrofurantoin Resistance Mechanisms in Escherichia coli Isolated in Clinical Urine Samples

Antibiotics ◽

10.3390/antibiotics9090534 ◽

2020 ◽

Vol 9 (9) ◽

pp. 534

Author(s):

Antonio Sorlozano-Puerto ◽

Isaac Lopez-Machado ◽

Maria Albertuz-Crespo ◽

Luis Javier Martinez-Gonzalez ◽

Jose Gutierrez-Fernandez

Keyword(s):

Escherichia Coli ◽

Resistance Genes ◽

Resistance Mechanisms ◽

Surveillance Studies ◽

Epidemiologic Surveillance ◽

Genetic Mechanisms ◽

Polymerase Chain ◽

Plasmid Genes ◽

Tract Infections

Fosfomycin and nitrofurantoin are antibiotics of choice to orally treat non-complicated urinary tract infections (UTIs) of community origin because they remain active against bacteria resistant to other antibiotics. However, epidemiologic surveillance studies have detected a reduced susceptibility to these drugs. The objective of this study was to determine possible mechanisms of resistance to these antibiotics in clinical isolates of fosfomycin- and/or nitrofurantoin-resistant UTI-producing Escherichia coli. We amplified and sequenced murA, glpT, uhpT, uhpA, ptsI, cyaA, nfsA, nfsB, and ribE genes, and screened plasmid-borne fosfomycin-resistance genes fosA3, fosA4, fosA5, fosA6, and fosC2 and nitrofurantoin-resistance genes oqxA and oqxB by polymerase chain reaction. Among 29 isolates studied, 22 were resistant to fosfomycin due to deletion of uhpT and/or uhpA genes, and 2 also possessed the fosA3 gene. Some modifications detected in sequences of NfsA (His11Tyr, Ser33Arg, Gln67Leu, Cys80Arg, Gly126Arg, Gly154Glu, Arg203Cys), NfsB (Gln44His, Phe84Ser, Arg107Cys, Gly192Ser, Arg207His), and RibE (Pro55His), and the production of truncated NfsA (Gln67 and Gln147) and NfsB (Glu54), were associated with nitrofurantoin resistance in 15/29 isolates; however, the presence of oqxAB plasmid genes was not detected in any isolate. Resistance to fosfomycin was associated with the absence of transporter UhpT expression and/or the presence of antibiotic-modifying enzymes encoded by fosA3 plasmid-mediated gene. Resistance to nitrofurantoin was associated with modifications of NfsA, NfsB, and RibE proteins. The emergence and spread of these resistance mechanisms, including transferable resistance, could compromise the future usefulness of fosfomycin and nitrofurantoin against UTIs. Furthermore, knowledge of the genetic mechanisms underlying resistance may lead to rapid DNA-based testing for resistance.

Download Full-text