Fluoroquinolone Resistance Linked to GyrA, GyrB, and ParC Mutations in Salmonella enterica Typhimurium Isolates in Humans

We report two cases of infection with clonally unrelated, high-level ciprofloxacin-resistant, β-lactamase–producing strains of Salmonella enterica Typhimurium. Resistance was caused by four topoisomerase mutations, in GyrA, GyrB, and ParC and increased drug efflux. Ciprofloxacin treatment failed in one case. In the second case, reduced susceptibility to third-generation cephalosporins occurred after initial treatment with these drugs and may explain the treatment failure with ceftriaxone

Identification par PCR simplex et multiplex des campylobacter isolés des hémocultures de patients hospitalisés à l'hôpital Saint-Louis

PARIS6-Bibl. St Antoine CHU (751122104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Salmonella enterica Serovar Typhimurium bla(PER-1)-Carrying Plasmid pSTI1 Encodes an Extended-Spectrum Aminoglycoside 6′-N-Acetyltransferase of Type Ib

We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 β-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6′)-Ib(11), was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the β-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of aac(6′)-Ib(11) translation. AAC(6′)-Ib(11) had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of aac(6′)-Ib(11) and two other acetyltransferase gene variants, aac(6′)-Ib(7) and -Ib(8), which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119

Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant Staphylococcus aureus (MRSA): a Disk Diffusion Method with Cefoxitin and Moxalactam, the Vitek 2 System, and the MRSA-Screen Latex Agglutination Test

Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the “gold standard.” We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-μg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter(−1) for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-μg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-μg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA

Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii

Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6′-N acetyltransferase type II [AAC(6′)-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6′) which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Both compounds, however, as well as isepamicin, retained good bactericidal activity against the three strains. The aac genes were borne by conjugative plasmids (pLMM562 and pLMM564 of ca. 100 kb and pLMM563 of ca. 20 kb). By PCR mapping and nucleotide sequence analysis, an aac(6′)-Ib gene was found in each strain upstream of an ant(3")-I gene in a sulI-type integron. The size of the AAC(6′)-Ib variant encoded by pLMM562 and pLMM564, AAC(6′)-Ib(7), was deduced to be 184 (or 177) amino acids long, whereas in pLMM563 a 21-bp duplication allowing the recruitment of a start codon resulted in the translation of a variant, AAC(6′)-Ib(8), of 196 amino acids, in agreement with size estimates obtained by Western blot analysis. Both variants had at position 119 a serine instead of the leucine typical for the AAC(6′)-Ib variants conferring resistance to amikacin. By using methods that predict the secondary structure, these two amino acids appear to condition an α-helical structure within a putative aminoglycoside binding domain of AAC(6′)-Ib variants

Multiple-Antibiotic Resistance in Salmonella enterica Serotype Paratyphi B Isolates Collected in France between 2000 and 2003 Is Due Mainly to Strains Harboring Salmonella Genomic Islands 1, 1-B, and 1-C

This study was conducted to investigate the occurrence of multiple-antibiotic resistance among 261 clinical isolates of Salmonella enterica serotype Paratyphi B strains collected between 2000 and 2003 through the network of the French National Reference Center for Salmonella. The 47 multidrug-resistant (MDR) isolates identified (18%), were characterized on the basis of the presence of several resistance genes (bla(TEM), bla(PSE-1), bla(CTX-M), floR, aadA2, qacEΔ1, and sul1), the presence of Salmonella genomic island 1 (SGI1) by PCR mapping and hybridization, and the clonality of these isolates by several molecular (ribotyping, IS200 profiling, and pulsed-field gel electrophoresis [PFGE]) and phage typing methods. The results of PCR and Southern blot experiments indicated that 39 (83%) of the 47 S. enterica serotype Paratyphi B biotype Java MDR isolates possessed the SGI1 cluster (MDR/SGI1). Among these 39 MDR/SGI1 isolates, only 3 contained variations in SGI1, SGI1-B (n = 1) and SGI1-C (n = 2). The 39 MDR/SGI1 isolates showed the same specific PstI-IS200 profile 1, which contained seven copies from 2.6 to 18 kb. Two PstI ribotypes were found in MDR/SGI1 isolates, RP1 (n = 38) and RP6 (n = 1). Ribotype RP1 was also found in two susceptible strains. Analysis by PFGE using XbaI revealed that all the MDR/SGI1 isolates were grouped in two related clusters, with a similarity percentage of 82%. Isolation of MDR/SGI1 isolates in France was observed mainly between the second quarter of 2001 and the end of 2002. The source of the contamination has not been identified to date. A single isolate possessing the extended-spectrum β-lactamase bla(CTX-M-15) gene was also identified during the study

Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance

The emergence of multi-resistant pathogenic bacteria is a worldwide health issue. Recently, clinical variants of a single antibiotic-modifying acetyltransferase, AAC(6′)-Ib—a variant of aminoglycoside 6′-N-acetyltransferase—have been identified that confer extended resistance to most aminoglycosides and, more surprisingly, to structurally unrelated fluoroquinolones. The corresponding gene is carried by mobile genetic elements and is present in most multi-resistant pathogenic strains, hence making it a serious threat to current therapies. Here, we report the crystal structures of both narrow- and broad-spectrum resistance variants of this enzyme, which reveal the structural basis for the emergence of extended resistance. The active site shows an important plasticity and has adapted to new substrates by a large-scale gaping process. We have also obtained co-crystals with both substrates, and with a simple transition state analogue, which provides new clues for the design of inhibitors of this resistance mechanism

Molecular and Biochemical Characterization of the Natural Chromosome-Encoded Class A -Lactamase from Pseudomonas luteola

International audiencePseudomonas luteola (formerly classified as CDC group Ve-1 and named Chryseomonas luteola) is an unusual pathogen implicated in rare but serious infections in humans. A novel beta-lactamase gene, bla(LUT-1), was cloned from the whole-cell DNA of the P. luteola clinical isolate LAM, which had a weak narrow-spectrum beta-lactam-resistant phenotype, and expressed in Escherichia coli. This gene encoded LUT-1, a 296-amino-acid Ambler class A beta-lactamase with a pI of 6 and a theoretical molecular mass of 28.9 kDa. The catalytic efficiency of this enzyme was higher for cephalothin, cefuroxime, and cefotaxime than for penicillins. It was found to be 49% to 59% identical to other Ambler class A beta-lactamases from Burkholderia sp. (PenA to PenL), Ralstonia eutropha (REUT), Citrobacter sedlakii (SED-1), Serratia fonticola (FONA and SFC-1), Klebsiella sp. (KPC and OXY), and CTX-M extended-spectrum beta-lactamases. No gene homologous to the regulatory ampR genes of class A beta-lactamases was found in the vicinity of the bla(LUT-1) gene. The entire bla(LUT-1) coding region was amplified by PCR and sequenced in five other genetically unrelated P. luteola strains (including the P. luteola type strain). A new variant of bla(LUT-1) was found for each strain. These genes (named bla(LUT-2) to bla(LUT-6)) had nucleotide sequences 98.1 to 99.5% identical to that of bla(LUT-1) and differing from this gene by two to four nonsynonymous single nucleotide polymorphisms. The bla(LUT) gene was located on a 700- to 800-kb chromosomal I-CeuI fragment, the precise size of this fragment depending on the P. luteola strain