Thomas A. Wichelhaus

NDM-2 carbapenemase in Acinetobacter baumannii from Egypt

OBJECTIVES To analyse the mechanisms responsible for carbapenem resistance in one Acinetobacter baumannii isolate recovered from a patient transferred to Germany from an Egyptian hospital. METHODS PCR and sequencing were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of the isolate. RESULTS Sequencing of the PCR product obtained using primers for bla(NDM-1) revealed a variant of NDM-1 that had a C to G substitution at position 82 resulting in an amino acid substitution of proline to alanine at position 28. This variant was designated NDM-2. Genes encoding extended-spectrum β-lactamases or 16S RNA methylase were not detected. The strain lacked detectable plasmids and bla(NDM-2) was not transferred by conjugation. MLST showed that the isolate belonged to a new ST, ST103. CONCLUSIONS This work further underlines the spread of NDM carbapenemases in A. baumannii, and the spread of the corresponding gene in the Middle East. It also describes the first variant of NDM-1.

Molecular characterization of blaNDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007

OBJECTIVES To investigate the genetic environment of the metallo-β-lactamase gene bla(NDM-1) in an Acinetobacter baumannii isolated in 2007 in a German hospital. METHODS Antimicrobial susceptibility testing was performed and resistance genes were characterized by PCR amplification and sequencing. Transferability of β-lactam resistance was tested by broth mating assays and transformation of plasmids. The genetic background of bla(NDM-1) was analysed by primer walking. Typing of the A. baumannii strain was performed by repetitive extragenic palindromic sequence-based PCR (rep-PCR) using the DiversiLab system. RESULTS The multidrug-resistant A. baumannii isolate harboured β-lactamase genes bla(NDM-1) and intrinsic bla(OXA-64), but without the insertion sequence ISAba1 often located upstream. Transfer of carbapenem resistance by conjugation and transformation failed. Hybridization of isolated plasmid DNA with bla(NDM) probes was not successful. Shotgun cloning of whole genomic DNA and sequence analyses revealed that bla(NDM-1) was located between two insertion elements of ISAba125. Furthermore, this bla(NDM-1)-containing transposon structure was integrated into a chromosomal gene encoding a putative A. baumannii major facilitator superfamily (MFS) metabolite/H+ symporter. CONCLUSIONS The metallo-β-lactamase gene bla(NDM-1) in this A. baumannii strain was integrated in the chromosome on a new transposon structure composed of two copies of insertion sequence ISAba125. The variability of the genetic environment of bla(NDM-1) likely facilitates the rapid dissemination of this gene within many Gram-negative bacterial species.

Linezolid Resistance in Staphylococcus aureus: Gene Dosage Effect, Stability, Fitness Costs, and Cross-Resistances

ABSTRACT Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

Biological Cost of Rifampin Resistance from the Perspective of Staphylococcus aureus

ABSTRACT Resistance determinants that interfere with normal physiological processes in the bacterial cell usually cause a reduction in biological fitness. Fitness assays revealed that 17 of 18 in vitro-selected chromosomal mutations within the rpoB gene accounting for rifampin resistance in Staphylococcus aureus were associated with a reduction in the level of fitness. There was no obvious correlation between the level of resistance to rifampin and the level of fitness loss caused by rpoB mutations. Among 23 clinical rifampin-resistant S. aureus isolates from six countries, only seven different rpoB genotypes could be identified, whereby the mutation 481His→Asn was present in 21 (91%) of these 23 isolates. The mutation 481His→Asn, in turn, which confers low-level rifampin resistance on its own, was not shown to be associated with a cost of resistance in vitro. The restriction to distinct mutations that confer rifampin resistance in vivo, as demonstrated here, appears to be determined by the Darwinian fitness of the organisms.

Molecular analysis of fusidic acid resistance in Staphylococcus aureus

Fusidic acid is a potent antibiotic against severe Gram‐positive infections that interferes with the function of elongation factor G (EF‐G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF‐G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid‐resistant clinical S. aureus strains as well as in 10 fusidic acid‐resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF‐G. To confirm the importance of observed amino acid exchanges in EF‐G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF‐G derivatives with the exchanges P406L, H457Y and L461K were constructed by site‐directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid‐susceptible S. aureus strain RN4220 caused a fusidic acid‐resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid‐resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF‐G for the generation of fusidic acid resistance in S. aureus.

Detection of NDM-7 in Germany, a new variant of the New Delhi metallo-β-lactamase with increased carbapenemase activity

OBJECTIVES This study characterized a new variant of the New Delhi metallo-β-lactamase (NDM). METHODS A multidrug-resistant Escherichia coli isolate was recovered from the wounds, throat and rectum of a Yemeni patient who presented at the Frankfurt University Hospital in Germany. The presence of β-lactamase genes was analysed by PCR and sequencing. The isolate was further characterized by susceptibility testing, conjugation and transformation assays and plasmid analysis. RESULTS The E. coli isolate was resistant to all β-lactams including carbapenems. By PCR analysis, the β-lactamase genes blaCMY-2, blaCTX-M-15, blaTEM-1 and blaNDM were identified. Sequencing revealed a blaNDM gene that differed from blaNDM-1 by two point mutations at positions 388 (G→A) and 460 (A→C) corresponding to amino acid substitutions Asp130Asn and Met154Leu, respectively. This NDM variant was identified as NDM-7. The blaNDM-7 gene was located on a self-transferable IncX3 plasmid of 60 kb. E. coli TOP10 transformants harbouring NDM-7 showed higher MICs of β-lactams including carbapenems compared with transformants harbouring NDM-1. Multilocus sequence typing analysis revealed that the E. coli isolate belonged to a novel sequence type (ST599). CONCLUSIONS This study identified a novel NDM variant in E. coli, NDM-7, possessing a high ability to hydrolyse β-lactam antibiotics. Given the diversity of NDM variants located on self-transferable plasmids found in different Gram-negative species and isolated in different countries, the blaNDM gene will most likely efficiently disseminate worldwide.

The Thymidine-Dependent Small-Colony-Variant Phenotype Is Associated with Hypermutability and Antibiotic Resistance in Clinical Staphylococcus aureus Isolates

ABSTRACT Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus can be isolated from the airway secretions of patients suffering from cystic fibrosis (CF) and are implicated in persistent and treatment-resistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthase-encoding thyA gene which are responsible for thymidine dependency, suggest that these morphological variants are hypermutable. To prove this hypothesis, we analyzed the mutator phenotype of different S. aureus phenotypes, in particular CF-derived TD-SCVs, CF-derived isolates with a normal phenotype (NCVs), and non-CF NCVs. The comparative analysis revealed that the CF isolates had significantly higher mutation rates than the non-CF isolates. The TD-SCVs, in turn, harbored significantly more strong hypermutators (mutation rate ≥ 10−7) than the CF and non-CF NCVs. In addition, antimicrobial resistance to non-beta-lactam antibiotics, including gentamicin, ciprofloxacin, erythromycin, fosfomycin, and rifampin, was significantly more prevalent in TD-SCVs than in CF and non-CF NCVs. Interestingly, macrolide resistance, which is usually mediated by mobile genetic elements, was conferred in half of the macrolide-resistant TD-SCVs by the point mutation A2058G or A2058T in the genes encoding the 23S rRNA. Sequence analysis of mutS and mutL, which are involved in DNA mismatch repair in gram-positive bacteria, revealed that in hypermutable CF isolates and especially in TD-SCVs, mutL was often truncated due to frameshift mutations. In conclusion, these data provide direct evidence that TD-SCVs are hypermutators. This hypermutability apparently favors the acquisition of antibiotic resistance and facilitates bacterial adaptation during long-term persistence.

Multidrug-resistant bacteria in geriatric clinics, nursing homes, and ambulant care – Prevalence and risk factors

Colonization/infection with multidrug-resistant bacteria (MDRB) such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, is an increasing problem not only in hospitals but also in long-term care facilities. The aim of this study was to determine the prevalence as well as the risk factors of colonization/infection with MRSA, VRE, and ESBL producing Enterobacteriaceae in geriatric clinics, nursing homes, and ambulant care in Frankfurt am Main, Germany. 288 patients from 2 geriatric clinics (n=46), 8 nursing homes (n=178), and 2 ambulant care facilities (n=64) as well as 64 staff members were screened for MDRB in the time period from October 2006 to May 2007. 58 patients (20.1%) and 4 staff members (6.2%) were colonized with MDRB. Among patients, 27 (9.4%) were colonized with MRSA, 11 (3.8%) were screened positive for VRE, and 25 (8.7%) were found to be colonized with ESBL producing Enterobacteriaceae. Prevalence of MDRB in geriatric clinics, nursing homes, and ambulant care facilities were 32.6%, 18.5%, and 15.6%, respectively. Significant risk factors for MDRB were immobility (OR: 2.7, 95% CI: 1.5-4.9; p=0.002), urinary catheter (OR: 3.1, 95% CI: 1.7-5.9; p<0.001), former hospitalization (OR: 2.1, 95% CI: 1.1-4.0; p=0.033), and wounds/decubiti (OR: 2.3, 95% CI: 1.5-4.9; p=0.03). Finally, the high level of MDRB in geriatric clinics, nursing homes, and ambulant care points to the importance of these institutions as a reservoir for dissemination.

Removal of PCR Inhibitors by Silica Membranes: Evaluating the Amplicor Mycobacterium tuberculosis Kit

ABSTRACT The effectiveness of PCR inhibitor removal by silica membranes in combination with the Amplicor Mycobacterium tuberculosiskit was analyzed for 655 respiratory and nonrespiratory specimens. The overall inhibition rate was reduced from 12.5%, when applying the Amplicor kit alone, to 1.1% with the addition of silica membrane DNA purification.

Rapid Molecular Typing of Methicillin-Resistant Staphylococcus aureus by PCR-RFLP

OBJECTIVE To establish a new, rapid, and reliable genotypic fingerprinting technique for methicillin-resistant Staphylococcus aureus (MRSA) typing in routine epidemiological surveillance. DESIGN The method is based on polymerase chain reaction (PCR) restriction fragment-length polymorphism (RFLP) following HaeII digestion of simultaneously amplified parts of the protein A gene, the coagulase gene, and the hypervariable region adjacent to mecA. A total of 46 MRSA initial isolates were analyzed, including 14 isolates from five countries; the six German epidemic strains; 16 isolates from the Frankfurt metropolitan area, which were known to be heterogeneous by pulsed-field gel electrophoresis (PFGE); and 10 isolates obtained during three epidemics, all of which displayed an identical genotype. RESULTS Restriction analysis by PCR-RFLP permitted discrimination of 10 of 14 international isolates, all six German epidemic strains, and 15 of 16 national isolates. It also confirmed the homogeneous character of the 10 outbreak isolates. CONCLUSIONS This new and rapid PCR-RFLP typing method is an attractive tool in routine epidemiological surveillance. Its impressive characteristics are ease of performance and interpretation, while at the same time guaranteeing good discriminatory power, reproducibility, and typeability.