Fluoroquinolone resistance in Achromobacter spp.: substitutions in QRDRs of GyrA, GyrB, ParC and ParE and implication of the RND efflux system AxyEF-OprN

Abstract Background Achromobacter are emerging pathogens in cystic fibrosis patients. Mechanisms of resistance to fluoroquinolones are unknown in clinical isolates. Among non-fermenting Gram-negative bacilli, fluoroquinolone resistance is mostly due to amino acid substitutions in localized regions of the targets (GyrA, GyrB, ParC and ParE) named QRDRs, but also to efflux. Objectives To explore quinolone resistance mechanisms in Achromobacter. Methods The putative QRDRs of GyrA, GyrB, ParC and ParE were sequenced in 62 clinical isolates, and in vitro one-step mutants obtained after exposure to fluoroquinolones. An in vitro mutant and its parental isolate were investigated by RNASeq and WGS. RT–qPCR and gene inactivation were used to explore the role of efflux systems overexpression. Results We detected seven substitutions in QRDRs (Q83L/S84P/D87N/D87G for GyrA, Q480P for GyrB, T395A/K525Q for ParE), all in nine of the 27 clinical isolates with ciprofloxacin MIC ≥16 mg/L, whereas none among the in vitro mutants. The RND efflux system AxyEF-OprN was overproduced (about 150-fold) in the in vitro mutant NCF-39-Bl6 versus its parental strain NCF-39 (ciprofloxacin MICs 64 and 1.5 mg/L, respectively). A substitution in AxyT (putative regulator of AxyEF-OprN) was detected in NCF-39-Bl6. Ciprofloxacin MIC in NCF-39-Bl6 dropped from 64 to 1.5 mg/L following gene inactivation of either axyT or axyF. Substitutions in AxyT associated with overexpression of AxyEF-OprN were also detected in seven clinical strains with ciprofloxacin MIC ≥16 mg/L. Conclusions Target alteration is not the primary mechanism involved in fluoroquinolone resistance in Achromobacter. The role of AxyEF-OprN overproduction was demonstrated in one in vitro mutant.

Download Full-text

Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01252-08 ◽

2009 ◽

Vol 53 (6) ◽

pp. 2463-2468 ◽

Cited By ~ 29

Author(s):

Patrizia Spigaglia ◽

Fabrizio Barbanti ◽

Thomas Louie ◽

Frédéric Barbut ◽

Paola Mastrantonio

Keyword(s):

Clostridium Difficile ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Toxin B ◽

Relevant Role ◽

Vitro Development ◽

Selection Of

ABSTRACT Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of Clostridium difficile infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the C. difficile toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five C. difficile parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10−6 to 6.6 × 10−5 for MX and from 1.0 × 10−6 to 2.4 × 10−5 for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant C. difficile clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant C. difficile strains also in vivo.

Download Full-text

Chlorhexidine reduced susceptibility associated to tetracycline resistance in clinical isolates of Escherichia coli

10.1101/2021.10.05.463149 ◽

2021 ◽

Author(s):

Guilhem ROYER ◽

Jose-Manuel Ortiz de la Rosa ◽

Xavier Vuillemin ◽

Beatrice Lacombe ◽

Francoise Chau ◽

...

Keyword(s):

Escherichia Coli ◽

Transport System ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Cross Resistance ◽

E Coli ◽

K 12 ◽

Phospholipid Transport

Chlorhexidine is a widely used antiseptic in hospital and community healthcare. Decreased susceptibility to this compound has been recently described in Klebsiella pneumoniae and Pseudomonas aeruginosa, together with cross-resistance to colistin. Surprisingly, few data are available for Escherichia coli, the main species responsible for community and healthcare-associated infections. In order to decipher chlorhexidine resistance mechanisms in E. coli, we studied both in vitro derived and clinical isolates through whole-genome sequence analysis. Comparison of strains grown in vitro under chlorhexidine pressure identified mutations in the gene mlaA coding for a phospholipid transport system. Phenotypic analyses of single-gene mutant from the Keio collection confirmed the role of this mutation in the decreased susceptibility to chlorhexidine. However, mutations in mlaA were not found in isolates from large clinical collections. In contrast, genome wide association studies (GWAS) showed that, in clinical strains, chlorhexidine reduced susceptibility was associated with the presence of tetA genes of class B coding for efflux pumps and located in a Tn10 transposon. Construction of recombinant strains in E. coli K-12 confirmed the role of tetA determinant in acquired resistance to both chlorhexidine and tetracycline. Our results reveal two different evolutionary paths leading to chlorhexidine decreased susceptibility: one restricted to in vitro evolution conditions and involving a retrograde phospholipid transport system; the other observed in clinical isolates associated with efflux pump TetA. None of these mechanisms provides cross-resistance to colistin or to the cationic surfactant octenidine. This work demonstrates the GWAS power to identify new resistance mechanisms in bacterial species.

Download Full-text

1280. In-Vitro Activity of Cefiderocol, Imipenem/Relebactam, & Ceftazidime/Avibactam in Ceftolozane/Tazobactam Resistant Strains of Multidrug Resistant Pseudomonas aeruginosa

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1463 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S655-S655

Author(s):

Daniel Navas ◽

Angela Charles ◽

Amy Carr ◽

Jose Alexander

Keyword(s):

Multidrug Resistant ◽

Resistance Mechanisms ◽

Vitro Activity ◽

Clinical Isolates ◽

Resistance Testing ◽

Clinical Samples ◽

In Vitro Activity ◽

Carbapenemase Gene ◽

Resistant Strains

Abstract Background The activity of imipenem/relebactam (I/R), ceftazidime/avibactam (CZA) and cefiderocol (FDC) were evaluated against clinical isolates of multidrug resistant (MDR) strains of P. aeruginosa which was resistant to ceftolozane/tazobactam (C/T). The recent increase of MDR P. aeruginosa strains isolated from clinical samples has prompted research and development of new antimicrobials that can withstand its multiple resistance mechanisms. C/T is an effective option for treatment of MDR P. aeruginosa in our facility with only 10% of resistance in MDR strains, but the emergence of resistance may occur due to the presence of a carbapenemase gene or an ampC mutation. Methods Antimicrobial susceptibility testing for C/T Etest® (bioMérieux, Inc.) were performed on all MDR strains initially screened by the VITEK2® (bioMérieux, Inc.). 10% (n=20) of all MDR isolates were resistant to C/T by the CLSI 2019 breakpoints. These resistant isolates were tested for presence of a carbapenemase gene using the GeneXpert CARBA-R (Cepheid®) PCR and against CZA Etest® (bioMérieux, Inc.) I/R gradient strips (Liofilchem®) and FDC broth microdilution (Thermo Scientific™ Sensititre™). Results A total of 20 clinical isolates of MDR P. aeruginosa resistant to C/T were tested following standardized CLSI protocols and techniques. All 20 isolates were screened for the presence of a carbapenemase gene (blaVIM, blaNDM, blaKPC, blaOXA-48, blaIMP). A blaVIM gene was detected in 6 (30%) out of 20 isolates. FDC demonstrated the greatest activity with 85% (n=17) of susceptible isolates (CLSI MIC <4µg/dL). CZA (CLSI MIC <8µg/dL) and I/R (FDA MIC <2µg/dL) showed 15% (n=3) and 10% (n=2) of susceptible isolates respectively. FDC was active against all 6 blaVIM isolates, where all 6 strains were resistant to CZA and I/R as expected. 3 isolates tested non-susceptible against FDC; additional characterization was not performed at this time. Conclusion Based on these results, FDC demonstrated the greatest in-vitro activity against C/T resistant strains of MDR P. aeruginosa. FDC also demonstrated activity against all 6 MDR P. aeruginosa carrying blaVIM gene. FDC is a strong option to consider on MDR P. aeruginosa strains based on a resistance testing algorithm and a cost/effective protocol. Disclosures All Authors: No reported disclosures

Download Full-text

1593. Antimicrobial Activity of Ceftazidime-Avibactam and Comparator Agents Against OXA-48 β-lactamase–Producing Enterobacterales Collected in International Medical Centers, Including the United States, in 2017–2018

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1773 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S793-S793

Author(s):

Lynn-Yao Lin ◽

Dmitri Debabov ◽

William Chang

Keyword(s):

Antimicrobial Agents ◽

The United States ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Surveillance Program ◽

Multiple Resistance ◽

In Vitro Susceptibility ◽

Medical Centers ◽

Custom Made

Abstract Background OXA-48 is a carbapenemase with low-level hydrolytic activity toward cephalosporins. This study evaluated in vitro activities of ceftazidime-avibactam (CAZ-AVI), meropenem (MEM), meropenem-vaborbactam (MVB), ceftolozane-tazobactam (C/T), and other antimicrobial agents against 113 OXA-48-producing Enterobacterales with multiple resistance mechanisms collected in a 2017–2018 global surveillance program. Methods Nonduplicate clinical isolates of 113 Enterobacterales were collected from medical centers in 25 countries in 2017–2018. In vitro susceptibility tests were performed by broth microdilution with a custom-made panel consisting of CAZ-AVI, ceftazidime (CAZ), MEM, MVB, C/T, colistin (COL), gentamicin (GEN), levofloxacin (LEV), and amikacin (AMK). Whole genome sequencing or quantitative PCR data were used to analyze resistance mechanisms, such as OXA-48, extended-spectrum β-lactamase (ESBL), original-spectrum β-lactamase (OSBL), and AmpC β-lactamase. Clinical and Laboratory Standards Institute breakpoints were applied for susceptibility interpretations. Results Of 113 OXA-48–producing clinical isolates, 20 carried OXA-48 alone. The remaining 93 isolates carried additional β-lactamases, including 63 with ESBL (CTX-M-15) + OSBL (SHV, TEM), 15 with AmpC (DHA, AAC, CMY) + ESBL (CTX-M-15), and 15 with OSBL (SHV, TEM). 99.1% (all but 1) of all isolates tested were susceptible to CAZ-AVI, whereas 71.7%, 17.7%, and 14.2% were susceptible to MVB, MEM, and C/T, respectively. Among isolates harboring multiple resistance mechanisms (OXA-48 + ESBL + OSBL; n=63), 98.4%, 69.8%, 11.1%, and 7.9% were susceptible to CAZ-AVI, MVB, MEM, and C/T, respectively. Among isolates carrying OXA-48 + AmpC + ESBL + OSBL (n=15), 100%, 66.7%, 13.3%, and 13.3% were susceptible to CAZ-AVI, MVB, MEM, and C/T, respectively (Table). Aminoglycosides (AMK and GEN) and other β-lactams (eg, CAZ) were 20%–90% active against these isolates. COL was the second most effective comparator, inhibiting 83.2% of these isolates. Table Conclusion CAZ-AVI was the most effective agent in this study compared with other antibiotics, including β-lactams, β-lactam–β-lactamase inhibitor combinations, aminoglycosides, and COL, against OXA-48-producing Enterobacterales carrying multiple β-lactamases. Disclosures Lynn-Yao Lin, MS, AbbVie (Employee) Dmitri Debabov, PhD, AbbVie (Employee) William Chang, BS, AbbVie (Employee)

Download Full-text

C3 Promotes Clearance of Klebsiella pneumoniae by A549 Epithelial Cells

Infection and Immunity ◽

10.1128/iai.72.3.1767-1774.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1767-1774 ◽

Cited By ~ 26

Author(s):

Beatriz de Astorza ◽

Guadalupe Cortés ◽

Catalina Crespí ◽

Carles Saus ◽

José María Rojo ◽

...

Keyword(s):

Epithelial Cells ◽

Klebsiella Pneumoniae ◽

Alveolar Epithelial Cells ◽

Clinical Isolates ◽

Complement Components ◽

Innate Defense ◽

Alveolar Epithelial

ABSTRACT The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen, Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulated K. pneumoniae clinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonize K. pneumoniae. CD46 could competitively inhibit the internalization of K. pneumoniae by the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonized K. pneumoniae to these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.

Download Full-text

Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis

Cells ◽

10.3390/cells9112450 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2450

Author(s):

Lucy Urwin ◽

Katarzyna Okurowska ◽

Grace Crowther ◽

Sanhita Roy ◽

Prashant Garg ◽

...

Keyword(s):

Ex Vivo ◽

Bacterial Species ◽

Resistance Mechanisms ◽

Bacterial Keratitis ◽

Corneal Surface ◽

Corneal Infection ◽

Infection Models

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.

Download Full-text

Association of Alterations in ParC and GyrA Proteins with Resistance of Clinical Isolates of Enterococcus faecium to Nine Different Fluoroquinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.10.2513 ◽

1999 ◽

Vol 43 (10) ◽

pp. 2513-2516 ◽

Cited By ~ 22

Author(s):

Sylvain Brisse ◽

Ad C. Fluit ◽

Ulrich Wagner ◽

Peter Heisig ◽

Dana Milatovic ◽

...

Keyword(s):

Enterococcus Faecium ◽

Resistance Mechanisms ◽

Clinical Isolates

ABSTRACT The parC and gyrA genes of 73 ciprofloxacin-resistant and 6 ciprofloxacin-susceptibleEnterococcus faecium clinical isolates were partly sequenced. Alterations in ParC and GyrA, possibly in combination with other resistance mechanisms, severely restricted the in vitro activities of the nine quinolones tested. For all isolates, clinafloxacin and sitafloxacin showed the best activities.

Download Full-text

681. In vitro Activity of the β-Lactamase Inhibitor QPX7728 in Combination with Several β-Lactams Against Acinetobacter baumannii (AB) and Pseudomonas aeruginosa (PSA)

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.749 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S310-S311 ◽

Cited By ~ 1

Author(s):

Olga Lomovskaya ◽

Jill Lindley ◽

Debora Rubio-Aparicio ◽

Kirk J Nelson ◽

Mariana Castanheira

Keyword(s):

Resistance Mechanisms ◽

Vitro Activity ◽

Clinical Isolates ◽

In Vitro Activity ◽

Microdilution Method ◽

Carbapenem Resistant ◽

Broth Microdilution Method ◽

Data Representative ◽

Lactamase Inhibitor

Abstract Background QPX7728 (QPX) is a novel broad-spectrum boron-containing inhibitor of serine- and metallo-β-lactamases (MBLs). We evaluated the in vitro activity of QPX combined with several β-lactams against carbapenem-resistant AB (CRAB) and PSA clinical isolates with varying β-lactam resistance mechanisms. Methods A total of 503 CRAB (meropenem [MEM] MIC ≥8 µg/mL) and 762 PSA clinical isolates were tested by the reference broth microdilution method against β-lactams alone and combined with QPX (4 µg/mL and 8 µg/mL). PSA isolates were selected to represent the normal distribution of MEM, ceftazidime–avibactam (CAZ-AVI), and ceftolozane-tazobactam (TOL-TAZ) resistance according to 2017 surveillance data (representative panel). Additionally, 262 PSA isolates that were either nonsusceptible (NS) to MEM (MIC, ≥4 µg/mL) or to TOL-TAZ (MIC, ≥8 µg/mL), or resistant (R) to CAZ-AVI (MIC, ≥16 µg/mL) (challenge panel) were also tested. Within this 262 strain challenge set, 56 strains carried MBLs and the majority also had nonfunctional OprD. Results Against CRAB, QPX at 4 and 8 µg/mL increased the potency of all β-lactams tested. MEM-QPX was the most potent combination (table) displaying MIC50/MIC90 at 1/8 and 0.5/4 µg/mL with QPX at fixed 4 and 8 µg/mL, respectively. Susceptibility (S) to MEM was restored in >95% of strains. Against the 500 PSA from the representative panel, S for all QPX combinations was >90%. For the challenge panel, TOL-QPX and piperacillin (PIP)-QPX were the most potent combinations, restoring S in 76–77% of strains. TOL-QPX and MEM-QPX or cefepime (FEP)-QPX restored the MIC values to S rates when applying the CLSI breakpoint for the compound alone (comparison purposes only) in ~90% and ~75% of non-MBL-producing strains, respectively, vs. 60–70% for TOL-TAZ and CAZ-AVI. PIP-QPX reduce the MIC values to S values for PIP-TAZ in ~60% of MBL-producing strains vs. 20–30% and 3–7% for other QPX combinations and non-QPX tested combinations, respectively. Conclusion Combinations of QPX with various β-lactam antibiotics displayed potent activity against CRAB and resistant PSA isolates and warrant further investigation. Disclosures All authors: No reported disclosures.

Download Full-text

Role of Erythropoietin in Megakaryocyte Colony Formation in Man

10.1055/s-0039-1684860 ◽

1979 ◽

Author(s):

W. Vainehenker ◽

J. Breton-Gorius

Keyword(s):

Bone Marrow ◽

Linear Relationship ◽

Colony Formation ◽

Colony Stimulating Factor ◽

Plasma Clot ◽

In Vitro Technique ◽

One Step ◽

Stimulating Factor

We have recently realized megakaryocyte (MK) colony formation in culture from blood and bone marrow progenitors using the plasma clot technique. In this study, the MK stimulating factor was an erythropoietin (Epo) either a poorly purified one(step III from anaemic sheep serum, a crude serum from anaemic mice, an urinary human Epo) or a highly purified one (GOLDWASSER). Similar results were obtained with all these Epo. A linear relationship was found between the number of colonies and seeded cells. However with less than 5.105 plated cells from the blood, no MK colonies were obtained, although erythroid colonies could be grown. In contrast, without Epo, spontaneous colonies could be observed which represented 1/5 th of the maximum plating efficiency , in these eases no erythroid colonies were present. These data suggest that Epo itself acts an a MK colony stimulating factor; but is not the only factor involved in the formation of MK colonies. This in vitro technique will be useful of in determining the factors regulating megakaryocytopoiesis.

Download Full-text