Experimental Legionella longbeachae infection in intratracheally inoculated mice.

This study established an experimental model of replicative Legionella longbeachae infection in A/J mice. The animals were infected by intratracheal inoculation of 10(3)-10(9) c.f.u. L. longbeachae serogroup 1 (USA clinical isolates D4968, D4969 and D4973). The inocula of 10(9), 10(8), 10(7) and 10(6) c.f.u. of all tested L. longbeachae serogroup 1 isolates were lethal for A/J mice. Inoculation of 10(5) c.f.u. L. longbeachae caused death in 90 % of the animals within 5 days, whilst inoculation of 10(4) c.f.u. caused sporadic death of mice. All animals that received 10(3) c.f.u. bacteria developed acute lower respiratory disease, but were able to clear Legionella from the lungs within 3 weeks. The kinetics of bacterial growth in the lungs was independent of inoculum size and reached a growth peak about 3 logarithms above the initial inoculum at 72 h after inoculation. The most prominent histological changes in the lungs were observed at 48-72 h after inoculation in the form of a focal, neutrophil-dominant, peribronchiolar infiltration. The inflammatory process did not progress towards the interstitial or alveolar spaces. Immunohistological analyses revealed L. longbeachae serogroup 1 during the early phase of infection near the bronchiolar epithelia and later co-localized with inflammatory cells. BALB/c and C57BL/6 mice strains were also susceptible to infection with all L. longbeachae serogroup 1 strains tested and very similar changes were observed in the lungs of infected animals. These results underline the infection potential of L. longbeachae serogroup 1, which is associated with high morbidity and lethality in mice.

DNA microarray for genotyping multidrug-resistant Pseudomonas aeruginosa clinical isolates.

The management of infections with multidrug-resistant Pseudomonas aeruginosa needs fast and reliable methods of antibiotic susceptibility testing for a therapy improvement. For this purpose, we developed a DNA microarray for genotyping antibiotic resistance and a few virulence factors. The array covers mutations in the efflux regulators mexR, nfxB, mexT, gyrase gyrA, and parC, as well as plasmid-encoded vim, imp, oxa, aph, aac, and aad genes, and virulence-associated mucA and exoU, exoT, and exoS genes, respectively. The whole procedure can be performed in less than 5 h and consists of DNA isolation, target gene amplification, fluorescence labeling, fragmentation, and array hybridization. Concerning the genotype-phenotype comparison in the test collection, the coverage of relevant resistance determinants for antibiotics used in a calculated therapy of critical ill patients was 87.8%.

Rapid and sensitive detection of fluoroquinolone-resistant Escherichia coli from urine samples using a genotyping DNA microarray.

Urinary tract infections (UTI) are among the most common bacterial infections in humans, with Escherichia coli being the major cause of infection. Fluoroquinolone resistance of uropathogenic E. coli has increased significantly over the last decade. In this study a microarray-based assay was developed and applied, which provides a rapid, sensitive and specific detection of fluoroquinolone-resistant E. coli in urine. The capture probes were designed against previously identified and described hotspots for quinolone resistance (codons 83 and 87 of gyrA). The key goals of this development were to reduce assay time while increasing the sensitivity and specificity as compared with a pilot version of a gyrA genotyping DNA microarray. The performance of the assay was demonstrated with pure cultures of 30 E. coli isolates as well as with urine samples spiked with 6 E. coli isolates. The microarray results were confirmed by standard DNA sequencing and were in full agreement with the phenotypic antimicrobial susceptibility testing using standard methods. The DNA microarray test displayed an assay time of 3.5h, a sensitivity of 100CFU/ml, and the ability to detect fluoroquinolone-resistant E. coli in the presence of a 10-fold excess of fluoroquinolone-susceptible E. coli cells. As a consequence, we believe that this microarray-based determination of antibiotics resistance has a true potential for the application in clinical routine laboratories in the future.

First detection of a VIM-1 metallo-beta-lactamase in a carbapenem-resistant Citrobacter freundii clinical isolate in an acute hospital in Germany.

We report the first detection of a carbapenem-resistant Citrobacter freundii clinical strain in Germany. It was isolated from an abscess of a patient with acute necrotic pancreatitis in an acute hospital. PCR and sequencing experiments revealed that the carbapenem resistance was mediated by a VIM-1 metallo-beta-lactamase, located on a plasmid encoded class 1 integron. Carbapenem resistance in Enterobacteriaceae is so far a rare event and 1 of the major therapeutic concerns in the future.

Development and validation of a diagnostic DNA microarray to detect quinolone-resistant Escherichia coli among clinical isolates.

The incidence of resistance against fluoroquinolones among pathogenic bacteria has been increasing in accordance with the worldwide use of this drug. Escherichia coli is one of the most relevant species for quinolone resistance. In this study, a diagnostic microarray for single-base-mutation detection was developed, which can readily identify the most prevalent E. coli genotypes leading to quinolone resistance. Based on genomic sequence analysis using public databases and our own DNA sequencing results, two amino acid positions (83 and 87) on the A subunit of the DNA gyrase, encoded by the gyrA gene, have been identified as mutation hot spots and were selected for DNA microarray detection. Oligonucleotide probes directed against these two positions were designed so that they could cover the most important resistance-causing and silent mutations. The performance of the array was validated with 30 clinical isolates of E. coli from four different hospitals in Germany. The microarray results were confirmed by standard DNA sequencing and were in full agreement with phenotypic antimicrobial susceptibility testing.

Use of DNA microarrays for rapid genotyping of TEM beta-lactamases that confer resistance.

Standard clinical procedures for pathogen resistance identification are laborious and usually require 2 days of cultivation before the resistance can be determined unequivocally. In contrast, clinicians and patients face increasing threats from antibiotic-resistant pathogenic bacteria in terms of their frequencies and levels of resistance. A major class of microbial resistance stems from the occurrence of beta-lactamases, which, if mutated, can cause the severe extended-spectrum beta-lactamase (ESBL) or inhibitor-resistant TEM (IRT) phenotype, which cause resistance to extended-spectrum cephalosporins, monobactams, and beta-lactamase inhibitors. We describe an oligonucleotide microarray for identification of the single nucleotide polymorphisms (SNPs) of 96% of the TEM beta-lactamase variants described to date which are related to the ESBL and/or IRT phenotype. The target DNA, originating from Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae cells isolated from clinical samples, was amplified and fluorescently labeled by PCR with consensus primers in the presence of cyanine 5-labeled nucleotides. The total assay, including PCR, hybridization, and image analysis, could be performed in 3.5 h. The microarray results were validated by standard clinical procedures. The microarray outperformed the standard procedures in terms of assay time and the depth of information provided. In conclusion, this array offers an attractive option for the identification and epidemiologic monitoring of TEM beta-lactamases in the routine clinical diagnostic laboratory.

The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga.

We have shown previously that the five rib (release of intracellular bacteria) mutants of Legionella pneumophila are competent for intracellular replication but defective in pore formation-mediated cytolysis and egress from protozoan and mammalian cells. The rib phenotype results from a point mutation (deletion) DeltaG544 in icmT that is predicted to result in the expression of a protein truncated by 32 amino acids from the C-terminus. In contrast to the rib mutants that are capable of intracellular replication, an icmT null mutant was completely defective in intracellular replication within mammalian and protozoan cells, in addition to its defect in pore formation-mediated cytolysis. The icmT wild-type allele complemented the icmT null mutant for both defects of intracellular replication and pore formation-mediated cytolysis and egress from mammalian cells. In contrast, the icmTDeltaG544 allele complemented the icmT null mutant for intracellular growth, but not for the pore-forming activity. Consistent with their defect in pore formation-mediated cytotoxicity in vitro, both mutants failed to cause pulmonary inflammation in A/J mice. Interestingly, the rib mutant was severely defective in intracellular growth within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy confirmed that the rib mutant and the icmT null mutant were severely and completely defective, respectively, in intracellular growth in A. polyphaga, and the respective defects correlated with fusion of the bacterial phagosomes to lysosomes. Taken together, the data showed that the C-terminus domain of IcmT is essential for the pore-forming activity and is required for intracellular trafficking and replication within A. polyphaga, but not within mammalian cells.