Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe

The European Study Group on Clostridium difficile (ESGCD) conducted a prospective study in 2005 to monitor and characterize C. difficile strains circulating in European hospitals, collecting 411 isolates. Eighty-three of these isolates, showing resistance or intermediate resistance to moxifloxacin (MX), were selected for this study to assess susceptibility to other fluoroquinolones (FQs) and to analyse the gyr genes, encoding the DNA gyrase subunits GyrA and GyrB. Twenty MX-susceptible isolates from the surveillance study were included for comparison. Overall, one amino acid substitution in GyrA (Thr82 to Ile) and four different substitutions in GyrB (Ser416 to Ala, Asp426 to Asn, Asp426 to Val and Arg447 to Lys) were identified. A high level of resistance (MIC ≥32 μg ml−1) to MX, ciprofloxacin (CI), gatifloxacin (GA) and levofloxacin (LE) was found in 68 isolates showing the amino acid substitution Thr82 to Ile in GyrA, in eight isolates with the substitutions Thr82 to Ile in GyrA and Ser416 to Ala in GyrB, in two isolates showing the substitution Asp426 to Asn in GyrB and in one isolate with Asp426 to Val in GyrB. The remaining four isolates showed high MICs for CI and LE, but different MIC levels for MX and GA. In particular, intermediate levels of resistance to MX were shown by two isolates, one with the substitution Thr82 to Ile in GyrA, and one showing Asp426 to Asn in GyrB. The substitution Arg447 to Lys in GyrB was found in two strains resistant to MX, CI and LE but susceptible to GA. No substitutions in GyrA were found in the FQ-susceptible strains, whereas two strains showed the amino acid change Ser416 to Ala in GyrB. Thr82 to Ile was the most frequent amino acid change identified in the C. difficile isolates examined. In contrast to previous observations, 10 % of the isolates showed this substitution in association with Ser416 to Ala in GyrB. The other amino acid changes found were characteristic of a few strains belonging to certain types and/or countries. Two new substitutions for C. difficile, Ser416 to Ala and Arg447 to Lys, were found in GyrB. Whereas the former does not seem to have a key role in resistance, since it was also detected in susceptible strains, the latter substitution occurred in the same position where other amino acid variations take place in resistant Escherichia coli and other C. difficile strains. A large number of C. difficile isolates now show an alarming pattern of resistance to the majority of FQs currently used in hospitals and outpatient settings, therefore judicious use of these antibiotics and continuous monitoring of in vitro resistance are necessary.

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Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates ofStreptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3517-3523.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3517-3523 ◽

Cited By ~ 56

Author(s):

L. M. Weigel ◽

G. J. Anderson ◽

R. R. Facklam ◽

F. C. Tenover

Keyword(s):

Amino Acid ◽

Antimicrobial Agents ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Cross Resistance ◽

Genetic Analyses ◽

High Level ◽

Susceptibility Profiles ◽

Level Resistance

ABSTRACT Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA,gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrAwas associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.

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Effects of Subinhibitory Concentrations of Antibiotics on Colonization Factor Expression by Moxifloxacin-Susceptible and Moxifloxacin-Resistant Clostridium difficile Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00532-09 ◽

2009 ◽

Vol 53 (12) ◽

pp. 5155-5162 ◽

Cited By ~ 24

Author(s):

Cécile Denève ◽

Sylvie Bouttier ◽

Bruno Dupuy ◽

Frédéric Barbut ◽

Anne Collignon ◽

...

Keyword(s):

Clostridium Difficile ◽

Fluoroquinolone Resistance ◽

Reverse Transcription Pcr ◽

Colonization Factor ◽

Expression Of Genes ◽

Genes Encoding ◽

Resistant Strains ◽

Subinhibitory Concentrations ◽

The Impact

ABSTRACT Recent outbreaks of Clostridium difficile infection have been related to the emergence of the NAP1/027 epidemic strain. This strain demonstrates increased virulence and resistance to the C-8-methoxyfluoroquinolones gatifloxacin and moxifloxacin. These antibiotics have been implicated as major C. difficile infection-inducing agents. We investigated by real-time reverse transcription-PCR the impact of subinhibitory concentrations of ampicillin, clindamycin, ofloxacin, and moxifloxacin on the expression of genes encoding three colonization factors, the protease Cwp84, the high-molecular-weight S-layer protein, and the fibronectin-binding protein Fbp68. We have previously shown in six non-NAP1/027 moxifloxacin-susceptible strains that the presence of ampicillin or clindamycin induced an upregulation of these genes, whereas the presence of fluoroquinolones did not. The objective of this study was to analyze the expression of these genes under the same conditions in four NAP1/027 strains, one moxifloxacin susceptible and three moxifloxacin resistant. Two in vitro-selected moxifloxacin-resistant mutants were also analyzed. Moxifloxacin resistance was associated with the Thr82→Ile substitution in GyrA in all but one of the moxifloxacin-resistant strains. The expression of cwp84 and slpA was strongly increased after culture with ampicillin or clindamycin in NAP1/027 strains. Interestingly, after culture with fluoroquinolones, the expression of cwp84 and slpA was only increased in four moxifloxacin-resistant strains, including the NAP1/027 strains and one of the in vitro-selected mutants. The overexpression of cwp84 was correlated with increased production of the protease Cwp84. The historical NAP1/027 moxifloxacin-susceptible strain and its mutant appear to be differently regulated by fluoroquinolones. Overall, fluoroquinolones appear to favor the expression of some colonization factor-encoding genes in resistant C. difficile strains. The fluoroquinolone resistance of the NAP1/027 epidemic strains could be considered an ecological advantage. This could also increase their colonization fitness and promote the infection.

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Fluoroquinolone Resistance in Clinical Isolates ofStreptococcus pneumoniae: Contributions of Type II Topoisomerase Mutations and Efflux to Levels of Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.3049-3054.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 3049-3054 ◽

Cited By ~ 91

Author(s):

Darrin J. Bast ◽

Donald E. Low ◽

Carla L. Duncan ◽

Laurie Kilburn ◽

Lionel A. Mandell ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Amino Acid Substitutions ◽

Type Ii ◽

Low Level ◽

High Level ◽

The Impact ◽

Level Resistance

ABSTRACT We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates ofS. pneumoniae for which the ciprofloxacin MIC is ≥4 μg/ml and 28 isolates for which the ciprofloxacin MIC is ≤2 μg/ml. The amino acid substitutions in ParC conferring low-level resistance (MICs, 4 to 8 μg/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with intermediate-level (MICs, 16 to 32 μg/ml) and high-level (MICs, 64 μg/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr). Glu-85-Lys was found exclusively in isolates with high-level resistance. Efflux contributed primarily to low-level resistance in isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.

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In vitro susceptibility of Clostridium difficile to rifaximin and rifampin in 359 consecutive isolates at a university hospital in Houston, Texas

Journal of Clinical Pathology ◽

10.1136/jcp.2009.071688 ◽

2010 ◽

Vol 63 (4) ◽

pp. 355-358 ◽

Cited By ~ 35

Author(s):

Z-D Jiang ◽

H L DuPont ◽

M La Rocco ◽

K W Garey

Keyword(s):

Clostridium Difficile ◽

In Vitro Study ◽

University Hospital ◽

Binary Toxin ◽

In Vitro Susceptibility ◽

Toxin Genes ◽

Genes Encoding ◽

Agar Dilution ◽

High Level

AimThis was an in vitro study to analyse the susceptibility of Clostridium difficile isolates to rifampin and rifaximin.MethodsStool samples from patients who had nosocomial diarrhoea and C difficile toxin B at a university hospital between August 2006 and December 2007 were cultured for C difficile. Susceptibility of C difficile isolates to rifaximin and rifampin was determined by agar dilution and E strips, respectively. C difficile isolates were analysed via PCR for genes encoding toxins A and B, for binary toxin (BT), and for partial deletions of the tcdC gene (tcdC-del).ResultsRifaximin exhibited high-level activity against 359 C difficile isolates, with MIC50 <0.01 μg/ml and MIC90 0.25 μg/ml; rifampin had MIC50 <0.002 μg/ml and MIC90 4 μg/ml. Among isolates analysed, 55 (15%) were positive for BT and tcdC-del. 28 (8% of 359) isolates were resistant to rifampin (≥32 μg/ml), of which 6 (2% of 359) were resistant to rifaximin and rifampin with MIC values ≥32 μg/ml. 2 of the 28 isolates resistant to rifampin were A+/B+/BT+/tcdC-del+, 5 were A+/B+/BT−/tcdC-del+, 4 were A+/B+/BT+/tcdC-del−, 13 were A+/B+/BT−/tcdC-del−, and 4 had no detectable toxin genes. Of the 11 isolates resistant to rifaximin alone, 1 was A+/B+/BT−/tcdC-del+, 2 were A+/B+/BT+/tcdC-del−, 6 were A+/B+/BT−/tcdC-del−, and 2 had no detectable toxin genes.ConclusionsThe study demonstrates that rifaximin has high-level activity against C difficile in vitro. Determination of resistance to rifampin by E strip did not predict rifaximin resistance.

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Multiclonal Expansion and High Prevalence of β-Lactamase-Negative Haemophilus influenzae with High-Level Ampicillin Resistance in Japan and Susceptibility to Quinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00851-18 ◽

2018 ◽

Vol 62 (9) ◽

Cited By ~ 9

Author(s):

Hiroyuki Honda ◽

Toyotaka Sato ◽

Masaaki Shinagawa ◽

Yukari Fukushima ◽

Chie Nakajima ◽

...

Keyword(s):

Amino Acid ◽

Haemophilus Influenzae ◽

Amino Acid Substitution ◽

University Hospital ◽

Fluoroquinolone Resistance ◽

Content Type ◽

Group Iii ◽

High Prevalence ◽

High Level ◽

Sequence Types

ABSTRACT β-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of β-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were β-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

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Characterization of Mycoplasma hominis mutations involved in resistance to fluoroquinolones.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.2.269 ◽

1997 ◽

Vol 41 (2) ◽

pp. 269-273 ◽

Cited By ~ 37

Author(s):

C M Bebear ◽

J M Bové ◽

C Bebear ◽

J Renaudin

Keyword(s):

Reference Strain ◽

Bacterial Species ◽

Mycoplasma Hominis ◽

Base Change ◽

Fluoroquinolone Resistance ◽

Genes Encoding ◽

One Step ◽

Resistant Mutants ◽

High Level

Fluoroquinolone-resistant mutants of Mycoplasma hominis were selected in vitro from the PG21 susceptible reference strain either by multistep selection on increasing concentrations of various fluoroquinolones or by one-step selection on agar medium with ofloxacin. The quinolone resistance-determining regions (QRDR) of the structural genes encoding the A and b subunits of DNA gyrase were amplified by PCR, and the nucleotide sequences of eight multistep-selected resistant strains were compared to those of susceptible strain PG21. Four high-level resistant mutants that were selected on norfloxacin or ofloxacin contained a C-to-T transition in the gyrA QRDR, leading to substitution of Ser-83 by Leu in the GyrA protein. Analysis of the sequence of the gyrB QRDR of the eight multistep-selected mutants did not reveal any difference compared to that of the gyrB QRDR of the reference strain M. hominis PG21. Similar analyses of eight one-step-selected mutants did not reveal any base change in the gyrA and gyrB QRDRs. These results suggest that in M. hominis, like in other bacterial species, a gyrA mutation at Ser-83 is associated with fluoroquinolone resistance.

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Analysis of amino acid substitution mutations of gyrA and parC genes in the clonal lineage of Klebsiella pneumoniae conferring high-level quinolone resistance

10.26226/morressier.56d5ba32d462b80296c94d38 ◽

2016 ◽

Author(s):

Mohammad Reza Shakibaie

Keyword(s):

Amino Acid ◽

Klebsiella Pneumoniae ◽

Amino Acid Substitution ◽

Quinolone Resistance ◽

Clonal Lineage ◽

Substitution Mutations ◽

High Level

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Improved human oocyte in vitro maturation in co-culture with cumulus cells from mature oocytes in the same patient: a prospective study

10.26226/morressier.573c1514d462b80296c98d07 ◽

2016 ◽

Author(s):

Irma Virant - Klun

Keyword(s):

Prospective Study ◽

In Vitro Maturation ◽

Cumulus Cells ◽

Human Oocyte ◽

A Prospective Study

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Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

Journal of General Virology ◽

10.1099/vir.0.009449-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1741-1747 ◽

Cited By ~ 11

Author(s):

Tahir H. Malik ◽

Candie Wolbert ◽

Laura Nerret ◽

Christian Sauder ◽

Steven Rubin

Keyword(s):

Amino Acid ◽

Clinical Isolate ◽

Amino Acid Change ◽

Mumps Virus ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Supporting Evidence ◽

L Protein ◽

Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

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