Effect of sub-lethal challenge with Photodynamic Antimicrobial Chemotherapy (PACT) on the antibiotic susceptibility of clinical bacterial isolates

This study aimed to determine the effect of sub-lethal challenge with Photodynamic Antimicrobial Chemotherapy (PACT) on the susceptibility of clinical Staphylococcus aureus and Pseudomonas aeruginosa isolates to both PACT and a range of antibiotics used in the treatment of infection caused by these bacteria. Clinical S. aureus and P. aeruginosa isolates were exposed to sub-lethal PACT with meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) and methylene blue (MB) over a 72 h period. After exposure, susceptibility of surviving organisms to a range of antibiotics was determined and compared with the susceptibility of an untreated control. Surviving bacteria were also exposed to previously lethal photosensitizer-light combinations, to determine if susceptibility to PACT was affected by sub-lethal exposure. Exposure to sub-lethal PACT did not decrease susceptibility to antibiotics with the minimum inhibitory concentrations for 95% and 100% of P. aeruginosa and S. aureus isolates, respectively, within two doubling dilutions of the MIC of the untreated control. Similarly, habituation with sub-lethal PACT did not reduce susceptibility of P. aeruginosa isolates to PACT levels previously determined as lethal. A reduction in susceptibility to PACT following habituation was apparent for two S. aureus isolates with MB and for 1 S. aureus isolate with IMP. However, for two of these three isolates, the log reduction for habituated cells was still greater than 4 log(10). PACT remains an attractive potential treatment for infection caused by these bacteria. (C) 2010 Elsevier B.V. All rights reserved.

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed. (C) 2004 Elsevier B.V. All rights reserved.

Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells

Aim: The aim of this study was to compare both the antimicrobial activity of terpinen-4-ol and tea tree oil (TTO) against clinical skin isolates of meticillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) and their toxicity against human fibroblast cells.

Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae

Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA–ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA–ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA–ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA–ramA genes, strongly suggesting that a secondary regulator may control ramA expression.

Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.

Differential cytopathogenesis of respiratory syncytial virus prototypic and clinical isolates in primary pediatric bronchial epithelial cells.

Background Human respiratory syncytial virus (RSV) causes severe respiratory disease in infants. Airway epithelial cells are the principle targets of RSV infection. However, the mechanisms by which it causes disease are poorly understood. Most RSV pathogenesis data are derived using laboratory-adapted prototypic strains. We hypothesized that such strains may be poorly representative of recent clinical isolates in terms of virus/host interactions in primary human bronchial epithelial cells (PBECs). Methods To address this hypothesis, we isolated three RSV strains from infants hospitalized with bronchiolitis and compared them with the prototypic RSV A2 in terms of cytopathology, virus growth kinetics and chemokine secretion in infected PBEC monolayers. Results RSV A2 rapidly obliterated the PBECs, whereas the clinical isolates caused much less cytopathology. Concomitantly, RSV A2 also grew faster and to higher titers in PBECs. Furthermore, dramatically increased secretion of IP-10 and RANTES was evident following A2 infection compared with the clinical isolates. Conclusions The prototypic RSV strain A2 is poorly representative of recent clinical isolates in terms of cytopathogenicity, viral growth kinetics and pro-inflammatory responses induced following infection of PBEC monolayers. Thus, the choice of RSV strain may have important implications for future RSV pathogenesis studies.

Fasciola hepatica: Comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study. F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response. (C) 2011 Elsevier B.V. All rights reserved.

Factors affecting in vitro adherence of ureteral stent biofilm isolates to polyurethane

Adherence of bacteria to biomaterials is the first stage in the development of a device-related infection. The adherence of bacterial cells to biomaterials may be influenced by surface characteristics of the cell, its growth conditions and the biomaterial surface chemistry. Following growth in human urine, the cell surface,hydrophobicity and zeta potential of two ureteral stent biofilm isolates, Enterococcus faecalis and Escherichia coli, were significantly altered. In addition, the adherence of human urine-grown Enterococcus faecalis and Escherichia coli to polyurethane was significantly increased by up to 52.1% and 58.6%, respectively. Treatment of the polyurethane with human urine rendered the polymer surface more hydrophilic (mean advancing water contact angle reduced from 97.59 degrees to 26.37 degrees). However, organisms grown in human urine showed less adherence (up to 90.4%) to the treated polymer than those grown in Mueller-Hinton broth. The results presented in this study indicate that in vivo conditions should be simulated as far as possible when carrying out in vitro bacterial adherence assays, especially if assessing novel methods for reduction of adherence. (C) 1997 Elsevier Science B.V.

Complete Genome Sequences of Three Propionibacterium acnes Isolates from the Type IA(2) Cluster

Propionibacterium acnes is an anaerobic Gram-positive bacterium that has been linked to a wide range of opportunistic human infections and conditions, most notably acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821-832, 2009). We now present the whole-genome sequences of three P. acnes strains from the type IA(2) cluster which were recovered from ophthalmic infections (A. McDowell et al., Microbiology 157:1990-2003, 2011).