Low Frequency of Ceftazidime-Avibactam Resistance among Enterobacteriaceae Isolates Carrying blaKPC Collected in U.S. Hospitals from 2012 to 2015

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae isolates have been increasingly reported worldwide, and therapeutic options to treat infections caused by these organisms are limited. We evaluated the activity of ceftazidime-avibactam and comparators against 456 Enterobacteriaceae isolates carrying bla KPC collected from 79 U.S. hospitals during 2012 to 2015. Overall, ceftazidime-avibactam (MIC50/90, 0.5/2 μg/ml; 99.3% susceptible) and tigecycline (MIC50/90, 0.5/1 μg/ml; 98.9% susceptible at ≤2 μg/ml) were the most active agents. Only 80.5% and 59.0% of isolates were susceptible to colistin and amikacin, respectively. All three isolates (0.7%) displaying resistance to ceftazidime-avibactam (K. pneumoniae; MICs, ≥16 μg/ml) were evaluated using whole-genome sequencing analysis and relative quantification of expression levels of porins and efflux pump. Two isolates carried metallo-β-lactamase genes, bla NDM-1 or bla VIM-4, among other β-lactam resistance mechanisms, and one displayed a premature stop codon in ompK35 and decreased expression of ompK36. Ceftazidime-avibactam was active against 100.0 and 99.3% of isolates carrying bla KPC-3 (n = 221) and bla KPC-2 (n = 145), respectively. Isolates carrying bla KPC were more commonly recovered from pneumonia (n = 155), urinary tract (n = 93), and skin/soft tissue (n = 74) infections. Ceftazidime-avibactam (97.8 to 100.0% susceptible) was consistently active against isolates from all infection sites. K. pneumoniae (83.3% of the collection) susceptibility rates were 99.2% for ceftazidime-avibactam, 98.9% for tigecycline, and 80.1% for colistin. Ceftazidime-avibactam susceptibility did not vary substantially when comparing isolates from intensive care unit (ICU) patients to those from non-ICU patients. Ceftazidime-avibactam was active against this large collection of isolates carrying bla KPC and represents a valuable addition to the armamentarium currently available for the treatment of infections caused by KPC-producing Enterobacteriaceae.

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In VitroActivities of the Novel Investigational Tetrazoles VT-1161 and VT-1598 Compared to the Triazole Antifungals against Azole-Resistant Strains and Clinical Isolates ofCandida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00341-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 4

Author(s):

Andrew T. Nishimoto ◽

Nathan P. Wiederhold ◽

Stephanie A. Flowers ◽

Qing Zhang ◽

Steven L. Kelly ◽

...

Keyword(s):

Fungal Infections ◽

Stop Codon ◽

Geometric Mean ◽

Premature Stop Codon ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Azole Resistance ◽

Content Type ◽

Mutant Strains

ABSTRACTThe fungal Cyp51-specific inhibitors VT-1161 and VT-1598 have emerged as promising new therapies to combat fungal infections, includingCandidaspp. To evaluate theirin vitroactivities compared to other azoles, MICs were determined by Clinical and Laboratory Standards Institute (CLSI) method for VT-1161, VT-1598, fluconazole, voriconazole, itraconazole, and posaconazole against 68 C. albicansclinical isolates well characterized for azole resistance mechanisms and mutant strains representing individual azole resistance mechanisms. VT-1161 and VT-1598 demonstrated potent activity (geometric mean MICs ≤0.15 μg/ml) against predominantly fluconazole-resistant (≥8 μg/ml) isolates. However, five of 68 isolates exhibited MICs greater than six dilutions (>2 μg/ml) to both tetrazoles compared to fluconazole-susceptible isolates. Four of these isolates likewise exhibited high MICs beyond the upper limit of the assay for all triazoles tested. A premature stop codon inERG3likely explained the high-level resistance in one isolate. VT-1598 was effective against strains with hyperactive Tac1, Mrr1, and Upc2 transcription factors and against mostERG11mutant strains. VT-1161 MICs were elevated compared to the control strain SC5314 for hyperactive Tac1 strains and two strains with Erg11 substitutions (Y132F and Y132F&K143R) but showed activity against hyperactive Mrr1 and Upc2 strains. While mutations affecting Erg3 activity appear to greatly reduce susceptibility to VT-1161 and VT-1598, the elevated MICs of both tetrazoles for four isolates could not be explained by known azole resistance mechanisms, suggesting the presence of undescribed resistance mechanisms to triazole- and tetrazole-based sterol demethylase inhibitors.

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Resistance to nitrofurantoin is an indicator of extensive drug-resistant (XDR) Enterobacteriaceae

Journal of Medical Microbiology ◽

10.1099/jmm.0.001347 ◽

2021 ◽

Vol 70 (4) ◽

Author(s):

Balaram Khamari ◽

Prakash Kumar ◽

Bulagonda Eswarappa Pradeep

Keyword(s):

Antimicrobial Agents ◽

Efflux Pump ◽

Treatment Options ◽

Strong Association ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Resistant Bacteria ◽

Drug Resistant ◽

Content Type ◽

Link Type

Introduction. Nitrofurantoin is one of the preferred antibiotics in the treatment of uropathogenic multidrug-resistant (MDR) infections. However, resistance to nitrofurantoin in extensively drug-resistant (XDR) bacteria has severely limited the treatment options. Gap statement. Information related to co-resistance or collateral sensitivity (CS) with reference to nitrofurantoin resistant bacteria is limited. Aim. To study the potential of nitrofurantoin resistance as an indicator of the XDR phenotype in Enterobacteriaceae . Methods. One hundred (45 nitrofurantoin-resistant, 21 intermediately resistant and 34 nitrofurantoin-susceptible) Enterobacteriaceae were analysed in this study. Antibiotic susceptibility testing (AST) against nitrofurantoin and 17 other antimicrobial agents across eight different classes was performed by using the Vitek 2.0 system. The isolates were screened for the prevalence of acquired antimicrobial resistance (AMR) and efflux pump genes by PCR. Results. In total, 51 % of nitrofurantoin-resistant and 28 % of intermediately nitrofurantoin resistant isolates exhibited XDR characteristics, while only 3 % of nitrofurantoin-sensitive isolates were XDR (P=0.0001). Significant co-resistance was observed between nitrofurantoin and other tested antibiotics (β-lactam, cephalosporin, carbapenem, aminoglycoside and tetracycline). Further, the prevalence of AMR and efflux pump genes was higher in the nitrofurantoin-resistant strains compared to the susceptible isolates. A strong association was observed between nitrofurantoin resistance and the presence of bla PER-1, bla NDM-1, bla OXA-48, ant(2) and oqxA-oqxB genes. Tigecycline (84 %) and colistin (95 %) were the only antibiotics to which the majority of the isolates were susceptible. Conclusion. Nitrofurantoin resistance could be an indicator of the XDR phenotype among Enterobacteriaceae , harbouring multiple AMR and efflux pump genes. Tigecycline and colistin are the only antibiotics that could be used in the treatment of such XDR infections. A deeper understanding of the co-resistance mechanisms in XDR pathogens and prescription of AST-based appropriate combination therapy may help mitigate this problem.

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Prevalence and Distribution of Listeria monocytogenesinlAAlleles Prone to Phase Variation andinlAAlleles with Premature Stop Codon Mutations among Human, Food, Animal, and Environmental Isolates

Applied and Environmental Microbiology ◽

10.1128/aem.02752-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8339-8345 ◽

Cited By ~ 18

Author(s):

Clyde S. Manuel ◽

Anna Van Stelten ◽

Martin Wiedmann ◽

Kendra K. Nightingale ◽

Renato H. Orsi

Keyword(s):

Stop Codon ◽

Premature Stop Codon ◽

Phase Variation ◽

Virulence Gene ◽

Farm Animals ◽

Nucleotide Substitutions ◽

Content Type ◽

Niche Adaptation ◽

Frameshift Deletion ◽

Animal Isolates

ABSTRACTInListeria monocytogenes, 18 mutations leading to premature stop codons (PMSCs) in the virulence geneinlAhave been identified to date. While most of these mutations represent nucleotide substitutions, a frameshift deletion in a 5′ seven-adenine homopolymeric tract (HT) ininlAhas also been reported. This HT may play a role in phase variation and was first identified amongL. monocytogeneslineage II ribotype DUP-1039C isolates. In order to better understand the distribution of differentinlAmutations in this ribotype, a newly developed multiplex real-time PCR assay was used to screen 368 DUP-1039C isolates from human, animal, and food-associated sources for three known 5′inlAHT alleles: (i) wild-type (WT) (A7), (ii) frameshift (FS) (A6), and (iii) guanine interruption (A2GA4) alleles. Additionally, 228 DUP-1039C isolates were screened for allinlAPMSCs; data on the presence of allinlAPMSCs for the other 140 isolates were obtained from previous studies. The statistical analysis based on 191 epidemiologically unrelated strains showed that strains withinlAPMSC mutations (n= 41) were overrepresented among food-associated isolates, while strains encoding full-length InlA (n= 150) were overrepresented among isolates from farm animals and their environments. Furthermore, the A6allele was overrepresented and the A7allele was underrepresented among food isolates, while the A6allele was underrepresented among farm and animal isolates. Our results indicate that genetic variation ininlAcontributes to niche adaptation within the lineage II subtype DUP-1039C.

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IS5Element Integration, a Novel Mechanism for RapidIn VivoEmergence of Tigecycline Nonsusceptibility in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03053-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 6151-6156 ◽

Cited By ~ 24

Author(s):

Lindsey E. Nielsen ◽

Erik C. Snesrud ◽

Fatma Onmus-Leone ◽

Yoon I. Kwak ◽

Ricardo Avilés ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Insertion Element ◽

Content Type ◽

Reverse Transcription Quantitative Pcr ◽

Antimicrobial Susceptibility Tests ◽

Susceptibility Tests

ABSTRACTTigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem- or colistin-resistant organisms. InEnterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated inEnterobacteriaceaeandAcinetobacterisolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR)Klebsiella pneumoniaeisolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 μg/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression oframA,ramR,oqxA,acrB,marA, orrarAgenes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here namedkpgABC, previously unknown to be involved in resistance. Introduction of thekpgABCgenes in a non-kpgABCbackground increased the MIC of tigecycline 4-fold and is independent of a functional AcrAB-TolC pump. This is the first report to propose a function forkpgABCand identify an insertion element whose presence correlated with thein vivodevelopment of tigecycline nonsusceptibility inK. pneumoniae.

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Characterization of AreABC, an RND-type efflux system involved in antimicrobial resistance of Aliarcobacter butzleri

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00729-21 ◽

2021 ◽

Author(s):

Susana Ferreira ◽

Ana L. Silva ◽

Joana Tomás ◽

Cristiana Mateus ◽

Fernanda Domingues ◽

...

Keyword(s):

Antimicrobial Agents ◽

Efflux Pump ◽

Stop Codon ◽

Premature Stop Codon ◽

Efflux System ◽

Erythromycin Resistance ◽

Efflux Pump Inhibitor ◽

Underlying Mechanisms ◽

Multiple Drugs ◽

Increased Susceptibility

Aliarcobacter butzleri is an emergent enteropathogen for which resistance to several classes of antimicrobial agents has been described, although the underlying mechanisms have been poorly addressed. We aimed to evaluate the contribution of the resistance-nodulation-division-type (RND) efflux system, AreABC, to drug resistance in A. butzleri . A. butzleri strains were first tested against several antimicrobials, with and without an efflux pump inhibitor. Then, erythromycin resistant strains were screened for the presence of a premature stop codon in a putative transcriptional regulator of the AreABC system, areR . Lastly, antimicrobial susceptibility and ethidium bromide (EtBr) accumulation were evaluated using an areB -knockout strain and a strain overexpressing the AreABC system through areR truncation. The presence of the efflux pump inhibitor resulted in increased susceptibility to most of the antimicrobials tested. A correlation between erythromycin resistance and the presence of premature stop codons in areR was observed. The truncation of areR resulted in increased expression of the AreABC system and decreased susceptibility to various antimicrobials. In contrast, areB inactivation resulted in increased susceptibility and a higher intracellular accumulation of EtBr. In conclusion, the AreABC efflux pump plays a role in the resistance of A. butzleri to multiple drugs and is regulated by a putative transcriptional repressor areR . Our results support the importance of efflux pumps in this bacterium's resistance to major classes of antibiotics and other antimicrobials.

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Sequencing Analysis and Enzyme Activity Assay of SrUGT76G1 Revealed the Mechanism Toward on/off Production of Rebaudioside-A in Stevia Plants

10.21203/rs.3.rs-987472/v1 ◽

2021 ◽

Author(s):

Simone Ribeiro Lucho ◽

Marcelo do Amaral ◽

Valmor Bianchi ◽

Lorena Almagro ◽

Maria Ferrer ◽

...

Keyword(s):

Stop Codon ◽

Premature Stop Codon ◽

Binding Pocket ◽

Structural Features ◽

Steviol Glycosides ◽

Sequencing Analysis ◽

Structural Level ◽

Rebaudioside A ◽

Enzyme Activity Assay ◽

Structure Activity

Abstract Stevia plants is known for its ability to synthesize steviol glycosides (SGs), a natural sweetener blend. Stevioside (STEV) and Rebaudioside-A (Reb-A) are the main SGs. However, Reb-A is more palatable than STEV and shows reduced bitter aftertaste. SrUGT76G1 catalyzes the conversion of STEV to Reb-A, improving their organoleptic properties. The better understanding of the structure/activity of SrUGT76G1 would allow shedding light up on on/off production of Reb-A in stevia plants. Thus, we analyzed the STEV and Reb-A content in stevia leaves of plants from Brazil and Spain and did not find detectable levels of Reb-A in Brazilian samples (off production). For this reason, we used a sequencing tool to study at the genetic and structural level the SrUGT76G1 gene. Changes in key amino acid residues in Brazilian samples were found, such as Leu204Phe, Thr284Leu and Leu126Ile. Leu204Phe mutants can narrow substrate-binding pocket to favor flavonoids recognition and decrease SGs synthesis, while Thr284 is considered key for 1,3-glucosylation of SGs, including Reb-A. These punctual mutations may partly explain the lack of functionality of UGT76G1 enzyme and off production of Reb-A in stevia plants from Brazil. Following this trend, Brazilian samples exhibited a T-to-A substitution, resulting in premature stop codon. As expected, the relative expression of SrUGT76G1 gene showed a higher level in Spanish samples than in Brazilian ones. Collectively, the results presented here show the structure-activity interplay of SrUGT76G1 enzyme and provide new insights on structural features and its role toward Reb-A synthesis.

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Deletion of Rv2571c Confers Resistance to Arylamide Compounds in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02334-20 ◽

2021 ◽

Vol 65 (5) ◽

Author(s):

Catherine D. Shelton ◽

Matthew B. McNeil ◽

Julie V. Early ◽

Thomas R. Ioerger ◽

Tanya Parish

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Stop Codon ◽

Fusaric Acid ◽

Premature Stop Codon ◽

Acid Resistance ◽

New Drugs ◽

Wild Type ◽

Content Type ◽

Global Health Problem

ABSTRACT Tuberculosis, caused by Mycobacterium tuberculosis, is an urgent global health problem requiring new drugs, new drug targets, and an increased understanding of antibiotic resistance. We have determined the mode of resistance to be a series of arylamide compounds in M. tuberculosis. We isolated M. tuberculosis resistant mutants to two arylamide compounds which are inhibitory to growth under host-relevant conditions (butyrate as a sole carbon source). Thirteen mutants were characterized, and all had mutations in Rv2571c; mutations included a premature stop codon and frameshifts as well as nonsynonymous polymorphisms. We isolated a further 10 strains with mutations in Rv2571c with resistance. Complementation with a wild-type copy of Rv2571c restored arylamide sensitivity. Overexpression of Rv2571c was toxic in both wild-type and mutant backgrounds. We constructed M. tuberculosis strains with an unmarked deletion of the entire Rv2571c gene by homologous recombination and confirmed that these were resistant to the arylamide series. Rv2571c is a member of the aromatic amino acid transport family and has a fusaric acid resistance domain which is associated with compound transport. Since loss or inactivation of Rv2571c leads to resistance, we propose that Rv2571c is involved in the import of arylamide compounds.

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The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity

Infection and Immunity ◽

10.1128/iai.00319-19 ◽

2019 ◽

Vol 87 (12) ◽

Author(s):

Stephen R. Coats ◽

Nutthapong Kantrong ◽

Thao T. To ◽

Sumita Jain ◽

Caroline A. Genco ◽

...

Keyword(s):

Cell Surface ◽

Immune Responses ◽

Host Cell ◽

Porphyromonas Gingivalis ◽

Stop Codon ◽

Premature Stop Codon ◽

Innate Immune ◽

Surface Proteins ◽

Innate Immune Responses ◽

Content Type

ABSTRACT The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1β (IL-1β) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1β production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

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PA3225 Is a Transcriptional Repressor of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02114-16 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 5

Author(s):

Clayton W. Hall ◽

Li Zhang ◽

Thien-Fah Mah

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Regulatory Region ◽

Resistance Mechanisms ◽

Wild Type ◽

Type Vi Secretion System ◽

Planktonic Cells ◽

Content Type ◽

Dependent Protein

ABSTRACT The tssABC1 locus is part of the Hcp secretion island I (HSI-I) type VI secretion system (T6SS) in Pseudomonas aeruginosa. Previous work implicated the tssC1 gene in P. aeruginosa biofilm-specific antibiotic resistance, and tssC1 is preferentially expressed in biofilms compared to planktonic cells. Using a DNA-dependent protein pulldown approach, we discovered that PA3225, an uncharacterized LysR-type transcriptional regulator, specifically bound to the tssABC1 upstream regulatory region. The deletion of PA3225 led to a 2-fold decrease in tssA1 expression levels in planktonic cells compared to the wild type, and tssA1 expression was slightly reduced in ΔPA3225 biofilms compared to wild-type biofilms. Intriguingly, further investigations revealed that the ΔPA3225 mutant was less susceptible to multiple, structurally unrelated antibiotics with various mechanisms of action when grown planktonically. The ΔPA3225 mutant was additionally more resistant to ciprofloxacin when grown in a biofilm. The decreased antibiotic susceptibility of the ΔPA3225 strain was linked to the transcriptional upregulation of the MexAB-OprM efflux pump. By using transcriptome sequencing (RNA-seq), other PA3225-regulated genes were identified, and the products of these genes, such as the putative ABC transporter PA3228, may also contribute to antibiotic resistance.

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Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.00541-17 ◽

2017 ◽

Vol 85 (11) ◽

Cited By ~ 41

Author(s):

Mylène M. Maury ◽

Viviane Chenal-Francisque ◽

Hélène Bracq-Dieye ◽

Lei Han ◽

Alexandre Leclercq ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Stop Codon ◽

Premature Stop Codon ◽

Host Cells ◽

Single Amino Acid ◽

Listeriolysin O ◽

Loss Of Function ◽

Phenotypic Marker ◽

Content Type ◽

Virulence Attenuation

ABSTRACT The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA−/LLO−) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA−/LLO− mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.

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