Isolation and in-vitro and in-vivo characterisation of a mutant of Pseudomonas aeruginosa PAO1 that exhibited a reduced postantibiotic effect in response to imipenem

The postantibiotic effect (PAE) is the persistent inhibition of bacterial growth after a brief exposure to an antibiotic. Most β-lactams do not induce a PAE for Gram-negative bacteria, but PAEs have been reported for carbapenems and penems. This study investigated the effect of sequential doses of imipenem on the PAE for Pseudomonas aeruginosa and Escherichia coli cultures in a chemostat. The PAE for the bacterial population did not change even after six successive exposures to imipenem. Nevertheless, screening of colonies isolated after repeated drug exposure identified a single P. aeruginosa mutant whose imipenem PAE was shortened, although the MIC was unchanged. The PAEs for the parent and mutant were studied in vitro in batch culture by monitoring: (i) viable counts; (ii) electrical impedance of the culture medium; (iii) incorporation of radiolabelled N-acetyl-D-glucosamine and (iv) cell volume changes. PAEs for the parent and mutant were found to be significantly different by all in-vitro methods used. Moreover, the median cell volume in antibiotic-exposed cultures remained much smaller and less heterogeneous than in the control cultures, even though both cultures were growing at the same rate. The mutant was found to have a reduced expression of a 52 kDa outer membrane protein. These observations suggest that factors in addition to suppression of bacterial growth should be considered when studying the PAE. The PAEs of imipenem for the parent and mutant were studied in a thigh infection model in leucopenic mice. Similar PAEs were observed in vivo for both parent and mutant in one experiment and no PAEs for either organism were found in a second experiment. This study showed that although the PAE is a stable in-vitro phenomenon, the lack of correlation between the in-vitro and in-vivo results warrants caution in attributing clinical significance to the PAE of imipene

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Clinically Relevant Bacteria

Background: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens. Methodology/Principal Findings: We compared the identification efficiency of MALDI-TOF MS with that of PhoenixH, APIH and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7 % of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63 % of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn’s kappa values: 0.9 to 0.93). Conclusions/Significance: MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria

Importance of the Collagen Adhesin Ace in Pathogenesis and Protection against Enterococcus faecalis Experimental Endocarditis

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFΔace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFΔace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis

Lysozyme M deficiency leads to an increased susceptibility to Streptococcus pneumoniae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity.</p> <p>Methods</p> <p>Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M^-/-mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of <it>S. pneumoniae </it>6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation.</p> <p>Results</p> <p>Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M^-/-mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M^-/-mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with <it>S. pneumoniae </it>6B and resulted in severe middle ear inflammation, compared to wild type mice.</p> <p>Conclusion</p> <p>The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.</p

Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system

Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops

Heterocyclic aromatic amines as chemical hazard in thermally processed meat products

Niektóre sposoby przetwarzania żywności mogą powodować powstawanie w niej szkodliwych związków chemicznych, w tym mutagennych i kancerogennych heterocyklicznych amin aromatycznych (HCA). Substancje te tworzą się podczas obróbki termicznej produktów o dużej zawartości białka, przy czym ich ilość i rodzaj są warunkowane głównie temperaturą, czasem i rodzajem tego procesu. W niniejszej pracy przedstawiono ogólną charakterystykę oraz podział HCA, z uwzględnieniem ich budowy chemicznej i warunków powstawania. Omówiono również metody obróbki termicznej mięsa pod względem tworzenia się heterocyklicznych amin aromatycznych oraz hamowania procesów ich powstawania. Istnieje bowiem związek pomiędzy częstym spożywaniem przetworzonego mięsa a częstotliwością występowania nowotworów u ludzi. Ostatnia cześć pracy stanowi przegląd technik analitycznych oczyszczania i ekstrakcji próbek zawierających wymienione związki, a także metod ich analizy jakościowej i ilościowej.Some food processing methods can cause many chemical compounds to form in food, including mutagenic and carcinogenic heterocyclic aromatic amines (HAA). Those substances are formed during the thermal treatment of products with a high content of protein, whereby the quantity and the type thereof are mainly dependent on the temperature and, sometimes, on the type of process. The paper presents the general profile of HCAs and classes they are divided into based on their chemical structure and formation conditions. Furthermore, various thermal processing methods of meat were discussed in terms of the formation of heterocyclic aromatic amines and methods to inhibit those formation processes, because there is a correlation between frequent consumption of processed meat and cancer incidences in humans. The last part of the paper is a review of analytical techniques used to purify and extract samples containing the compounds listed as well as the most popular techniques used to qualitatively and quantitatively analyze them

Antibacterial and mutagenic activity of inhaled bronchodilators on the respiratory pathogen Haemophilus influenzae

The U.K. prevalence of non-beta-lactamase-mediated resistance to ampicillin among Haemophilus influenzae reached 4% in 1986. The majority (70%) of isolates of this type come from sputa of patients with chronic obstructive airways disease. This study investigated whether bronchodilator drugs delivered directly to the respiratory tract have any antibacterial activity and/or play a role in promoting selection of organisms with this type of resistance. Antibacterial activity was detected in two out of six pharmaceutical preparations for nebulization examination but was entirely attributable to the preservative (benzalkonium chloride) in them. Exposure of ampicillin-susceptible H. influenzae (minimum inhibitory concentration 0.25 mg l-1) to concentrations of salbutamol, fenoterol and beclomethasone theoretically attainable in vivo resulted, after 48 h, in isolation of colonies with reduced susceptibility to ampicillin (minimum inhibitory concentration 1-4 mg l-1) but reversion to beta-lactam susceptibility occurred following serial subculture on chocolate agar. Organisms with stable reduced susceptibility to ampicillin were obtained when exposure to one of these three bronchodilators in broth was followed by serial subculture on agar containing the same preparations at equivalent concentrations and when the period of exposure to salbutamol at 100 mg l-1 in broth was extended to 5 days. The occurrence of these phenomena in vivo might be contributing to failures in treatment of exacerbations with ampicillin and to an increasing prevalence of beta-lactamase-negative, ampicillin-resistant H. influenzae

Muropeptide signature of inhibitors of protein synthesis correlates with β-lactam synergism against methicillin-resistant Staphylococcus aureus.

Quinupristin/dalfopristin (Q/D) and β-lactams interact positively against methicillin-resistant Staphylococcus aureus (MRSA). The effect extends to other inhibitors of protein synthesis, but not to inhibitors of polynucleotide synthesis or assembly, or to Q/D plus non-β-lactam cell wall inhibitors. Moreover, electron microscopy studies have correlated this effect with a thickened cell wall. In this study, we sought to determine whether inhibitors of protein synthesis might produce a specific peptidoglycan muropeptide signature that would correlate with their positive β-lactam interaction. The muropeptides of six S. aureus isolates (three methicillin-susceptible and three MRSA) were analysed using high-performance liquid chromatography and mass spectrometry. Exposure to 0.25× the minimum inhibitory concentration of inhibitors of protein synthesis consistently produced three main alterations irrespective of methicillin resistance: (i) an increase in peak 12 (a cyclic dimer of glycine-containing disaccharide-tetrapeptide); (ii) an increase in poorly resolved late-eluting materials; and (iii) a decrease in peak 1 (a disaccharide-pentapeptide). Eventually, the rate of autolysis was also decreased, supporting the structural alteration of the peptidoglycan. Other drug classes did not produce these anomalies. An increase in peak 12 was also observed in staphylococci treated with fosfomycin, which decreases expression of the native penicillin-binding protein (PBP) 2 and 4. Parallel blockage of normal PBPs with β-lactams abolished the anomalies, indicating that they resulted from altered function of native PBPs. This underlines the potential of inhibiting both protein synthesis and transpeptidation simultaneously and suggests that such a drug combination strategy might be efficaciously exploited