Thomas Guillard

Rapid detection of qnr and qepA plasmid-mediated quinolone resistance genes using real-time PCR.

Plasmid-mediated quinolone resistance genes in clinical strains cannot be detected by phenotypic traits but require gene detection. We developed a multiplex real-time polymerase chain reaction (PCR) assay using high-resolution melting master mix with ResoLight dye to detect qnr genes and a simplex real-time PCR assay using SYBR Green I to detect qepA genes. Using qnr-positive and qepA1-positive control strains, the ResoLight method was able to rapidly identify qnrA, qnrB, qnrS, qnrC, and qnrD genes; the SYBR Green I method identified qepA genes. Among 118 extended spectrum beta-lactamase-producing Enterobacteriaceae isolates, the 2 new assays efficiently detected and identified qnr in 9 strains, but no qepA gene. To our knowledge, this is the first study describing the detection of all 5 qnr and qepA genes using real-time PCR. The 2 tests constitute a significant step forward for screening for plasmid quinolone resistance genes in clinical strains.

Rapid Detection of aac(6′)-Ib-cr Quinolone Resistance Gene by Pyrosequencing

ABSTRACT Pyrosequencing was used to rapidly detect aac(6′)-Ib and aac(6′)-Ib-cr genes. This plasmid-mediated quinolone resistance determinant is increasing in extended-spectrum beta-lactamase-producing Enterobacteriaceae. This method is faster and more cost-effective than the methods previously described. Sequences obtained with this pyrosequencing method showed 100% concordance with conventional sequencing.

Rapid detection of quinolone resistance gene aac(6')-Ib-cr by pyrosequencing

ABSTRACT Pyrosequencing was used to rapidly detect aac(6′)-Ib and aac(6′)-Ib-cr genes. This plasmid-mediated quinolone resistance determinant is increasing in extended-spectrum beta-lactamase-producing Enterobacteriaceae. This method is faster and more cost-effective than the methods previously described. Sequences obtained with this pyrosequencing method showed 100% concordance with conventional sequencing.

Gastroscopy-associated transmission of extended-spectrum beta-lactamase-producing Pseudomonas aeruginosa

An unusual multi-drug-resistant Pseudomonas aeruginosa (MDR-PA) was isolated in four patients whilst hospitalized in a French teaching hospital between May and August 2011. All four patients had undergone an oesophago-gastro-duodenoscopy with the same gastroscope over a five-month period. This endoscope was associated with a culture positive for the MDR-PA. Observations of endoscope reprocessing identified deviations from the agreed processes: insufficient initial cleaning, shortening of the immersion time and brushing time, insufficient channel flushing, and inadequate drying prior to storage. Since withdrawing the gastroscope and institution of strict adherence to the agreed processes, no other MDR-PA cases have been isolated.

Diversity of Individual Dynamic Patterns of Emergence of Resistance to Quinolones in Escherichia coli From the Fecal Flora of Healthy Volunteers Exposed to Ciprofloxacin

BACKGROUND Emergence of quinolone-resistant Escherichia coli (QREC) is an increasing clinical challenge mostly originating in fecal microbiota. The dynamics of the emergence of QREC in feces from individuals exposed to ciprofloxacin is unknown. METHODS A total of 48 healthy volunteers received oral ciprofloxacin for 14 days. Fecal specimens were collected on days 0, 8, 14, and 42. Subpopulations of QREC were detected on selective agar, genetically characterized, and compared with quinolone-susceptible E. coli (QSEC) strains collected on different days. RESULTS On day 42, 34 subjects carried QSEC, and 14 carried QREC. Of the 14 who carried QREC, 9 carried quinolone-susceptible E. coli on day 0, 1 carried E. coli with a lower level of quinolone resistance on day 0, and 4 carried E. coli with similar levels of resistance and RAPD-genotypes on days 0 and 42. No plasmid acquisition and no selection of resistant mutants from the initial microbiota was evidenced in any case. CONCLUSIONS In QREC emerging under ciprofloxacin pressure in the fecal microbiota, no proof of selection of quinolone-resistant mutants from the initial microbiota was evidenced, suggesting that QREC strains on day 42 were either present at undetectable levels in the initial microbiota or that exogenous acquisition of QREC strains occurred. Clinical Trials Registration. NCT00190151.

Diversity of the Bacterial and Fungal Microflora from the Midgut and Cuticle of Phlebotomine Sand Flies Collected in North-Western Iran

Background Phlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies. Methodology/Principal Findings Five sand fly species: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates. Conclusion/Significance Our study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies.

Description of a 2,683-base-pair plasmid containing qnrD in two Providencia rettgeri isolates.

ABSTRACT qnr genes are plasmid-mediated quinolone resistance genes mainly harbored on large conjugative multiresistant plasmids. The qnrD gene was recently observed in Salmonella enterica on a small nonconjugative plasmid (p2007057). We describe two strains of Providencia rettgeri harboring qnrD on nonconjugative plasmids. The plasmids were 99% identical, with 2,683 bp and four open reading frames, including qnrD, but exhibited only 53% identity with the plasmid found in S. enterica.

High-resolution melting analysis for rapid characterization of qnr alleles in clinical isolates and detection of two novel alleles, qnrB25 and qnrB42

OBJECTIVES qnr genes are plasmid-mediated quinolone resistance genes. Five qnr families have been described with several alleles (7 alleles of qnrA, 53 alleles of qnrB, 1 allele of qnrC, 1 allele of qnrD and 6 alleles of qnrS). Their detection requires a PCR specific for each qnr family and further sequencing for allele characterization. METHODS High-resolution melt curve analysis (HRMA) was coupled to multiplex and simplex real-time PCR assays for detection and characterization of qnrA, qnrB and qnrS alleles. The protocol was set using 27 reference strains harbouring the most frequent alleles and was applied to 55 clinical isolates unknown for qnr positivity. RESULTS Out of the 27 reference strains tested, 21 alleles showed distinct profiles using HRMA: 6 qnrA, 12 qnrB and 3 qnrS. For the qnrB alleles showing similar profiles, we gathered them into four groups that were easily distinguished. For the alleles that we could not test, in silico analysis showed that they would be identified using the HRMA protocol set. Among the clinical isolates, 28 qnr-positive isolates were detected and the qnr allele was characterized as 8 qnrA1, 4 qnrB1, 5 qnrB2, 3 qnrB4, 1 qnrB8, 1 qnrB5, 3 qnrS1 and 1 qnrS2, with concordant results with PCR sequencing. Two new qnrB alleles were detected and distinguished using HMRA. They were further designated as qnrB25 and qnrB42. CONCLUSIONS We developed an HRMA assay for characterizing the qnr alleles in clinical isolates. This high-throughput method can be used to screen a large number of isolates. This method allowed the detection of new qnrB alleles.

Mobile Insertion Cassette Elements Found in Small Non-Transmissible Plasmids in Proteeae May Explain qnrD Mobilization

qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36–60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae.

Ciprofloxacin Treatment Failure in a Murine Model of Pyelonephritis Due to an AAC(6′)-Ib-cr-Producing Escherichia coli Strain Susceptible to Ciprofloxacin In Vitro

ABSTRACT AAC(6′)-Ib-cr is a plasmid-mediated quinolone resistance mechanism described worldwide for Escherichia coli. Since it confers in vitro only a low level of resistance to ciprofloxacin, we evaluated its impact on the in vivo activity of ciprofloxacin. Isogenic strains were obtained by transferring plasmid p449, harboring aac(6′)-Ib-cr, into the quinolone-susceptible strain E. coli CFT073-RR and its D87G gyrA mutant. MICs were 0.015, 0.06, 0.25, and 0.5 μg/ml against E. coli strains CFT073-RR, CFT073-RR/p449, CFT073-RR GyrAr, and CFT073-RR GyrAr/p449, respectively. Bactericidal activity was reduced at 1× the MIC for the three resistant derivatives, while at a fixed concentration of 0.5 μg/ml, 99.9% killing was observed for all strains except E. coli CFT073-RR GyrAr/p449. In the murine model of pyelonephritis, an optimal regimen of ciprofloxacin (10 mg/kg of body weight twice a day [b.i.d.]) significantly decreased the bacterial count in the kidneys of mice infected with E. coli CFT073 (1.6 versus 4.3 log10 CFU/g of kidney compared to untreated controls; P = 0.0001), while no significant decrease was observed for E. coli CFT073-RR/p449 (2.7 versus 3.1 log10 CFU/g; P = 0.84), E. coli CFT073-RR GyrAr (4.2 versus 4.1 log10 CFU/g; P = 0.35), or E. coli CFT073-RR GyrAr/p449 (2.9 versus 3.6 log10 CFU/g; P = 0.47). While pharmacokinetic and pharmacodynamic (PK/PD) parameters accounted for ciprofloxacin failure against gyrA-containing mutants, this was not the case for the aac(6′)-Ib-cr-containing strains, suggesting an in situ hydrolysis of ciprofloxacin in the latter case.