Mechanism of Escherichia coli resistance to Pyrrhocoricin.

Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10(-7). Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials.

Implementation of evidence-based practice for a pediatric pain assessment instrument.

PURPOSE/OBJECTIVES: The purpose of this project was to facilitate the successful implementation of an evidence-based practice (EBP) change to the Faces Pain Scale-Revised (FPS-R) using the Promoting Action on Research Implementation in Health Services (PARIHS) framework. BACKGROUND/RATIONALE: Accurate pain assessment is a high priority in the clinical setting. Despite the availability of valid pain assessment instruments, use in practice remains deficient. Compared with the Wong-Baker FACES Pain Scale, the FPS-R has stronger evidence for validity and reliability and is identified as a preferred instrument to measure children's pain. The PARIHS framework, developed to guide the change process of EBP implementation through strong evidence, quality of context, and type of facilitation, was used in the development and implementation of the project. DESCRIPTION: The clinical nurse specialist implemented an education program about EBP and FPS-R for nurses, followed by a 2-week pilot using FPS-R to assess children's pain. We measured perceptions of strength of evidence, quality of context, and barriers to research utilization. OUTCOME: Nurses reported positive perceptions about the strength of evidence for FPS-R and identified unit context as moderately receptive to change before the project. Nurses' perceived barriers to research utilization did not significantly change after the project. CONCLUSION: Nurses' positive perceptions about the use of FPS-R were based on strong evidence, quality context, and type of facilitation. The PARIHS framework supported this successful EBP change. The PARIHS framework can be applied by the clinical nurse specialist in the clinical setting, with minimal effort, to facilitate EBP implementation.

The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.

Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.

We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS) in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.

Neisserial Correia repeat-enclosed elements do not influence the transcription of pil genes in Neisseria gonorrhoeae and Neisseria meningitidis.

Two human-specific neisserial pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, require the expression of type IV pili (tfp) for initial attachment to the host during infection. However, the mechanisms controlling the assembly and functionality of tfp are poorly understood. It is known that the gonococcal pilE gene, encoding the major subunit, is positively regulated by IHF, a multifunctional DNA binding protein. A neisserial specific repetitive DNA sequence, termed the Correia repeat-enclosed element (CREE) is situated upstream of three pil loci: pilHIJKX (pilH-X), pilGD, and pilF. CREEs have been shown to contain strong promoters, and some CREE variants contain a functional IHF binding site. CREEs might therefore be involved in the regulation of tfp biogenesis in pathogenic Neisseria. Site-directed and deletion mutagenesis on promoter::cat reporter constructs demonstrated that transcription of pilH-X and pilGD is from a σ(70) promoter and is independent of the CREE. The insertion of a CREE in the pilF promoter region in N. meningitidis generated a functional σ(70) promoter. However, there is also a functional promoter at this position in N. gonorrhoeae, where there is no CREE. These results suggest CREE insertion in these three pil loci does not influence transcription and that IHF does not coordinately regulate tfp biogenesis.

Ecf, an alternative sigma factor from Neisseria gonorrhoeae, controls expression of msrAB, which encodes methionine sulfoxide reductase.

A DNA microarray was used to identify genes transcribed in Neisseria gonorrhoeae using Ecf, an alternative sigma factor. No differences between the transcriptional profiles of strain FA1090 and a mutant where ecf had been inactivated could be detected when both were grown in vitro. We therefore constructed a gonococcal strain in which Ecf can be overexpressed. Some differentially expressed genes are clustered with ecf on the genome and appear to form a single transcriptional unit. Expression of the gene encoding MsrAB, which possesses methionine sulfoxide reductase activity, was also dependent on Ecf, suggesting that the regulon responds to oxidative damage. Western blotting confirmed that the increased level of MsrAB protein is dependent on the presence of Ecf.

Comparative overview of the genomic and genetic differences between the pathogenic Neisseria strains and species.

The availability of complete genome sequences from multiple pathogenic Neisseria strains and species has enabled a comprehensive survey of the genomic and genetic differences occurring within these species. In this review, we describe the chromosomal rearrangements that have occurred, and the genomic islands and prophages that have been identified in the various genomes. We also describe instances where specific genes are present or absent, other instances where specific genes have been inactivated, and situations where there is variation in the version of a gene that is present. We also provide an overview of mosaic genes present in these genomes, and describe the variation systems that allow the expression of particular genes to be switched ON or OFF. We have also described the presence and location of mobile non-coding elements in the various genomes. Finally, we have reviewed the incidence and properties of various extra-chromosomal elements found within these species. The overall impression is one of genomic variability and instability, resulting in increased functional flexibility within these species.

The RpoH-mediated stress response in Neisseria gonorrhoeae is regulated at the level of activity.

The general stress response in Neisseria gonorrhoeae was investigated. Transcriptional analyses of the genes encoding the molecular chaperones DnaK, DnaJ, and GrpE suggested that they are transcribed from sigma32 (RpoH)-dependent promoters upon exposure to stress. This was confirmed by mutational analysis of the sigma32 promoter of dnaK. The gene encoding the gonococcal RpoH sigma factor appears to be essential, as we could not isolate viable mutants. Deletion of an unusually long rpoH leader sequence resulted in elevated levels of transcription, suggesting that this region is involved in negative regulation of RpoH expression during normal growth. Transcriptional analyses and protein studies determined that regulation of the RpoH-mediated stress response is different from that observed in most other species, in which regulation occurs predominantly at the transcriptional and translational levels. We suggest that an increase in the activity of preformed RpoH is primarily responsible for induction of the stress response in N. gonorrhoeae.

Chloramphenicol-resistant Neisseria meningitidis containing catP isolated in Australia.

Earlier workers have described chloramphenicol resistance in meningococci isolated from cerebrospinal fluid sampled in patients in Vietnam (11 cases) and France (one case) during 1987-1996. Here we describe two distinct serogroup B strains isolated in Australia in 1994 and 1997, and found among approximately 1400 invasive meningococcal isolates examined in Australia over a 9 year period. Both were phenotypically chloramphenicol resistant on disc, Etest and agar inclusion MIC and acetylated chloramphenicol examination. DNA amplification and sequencing confirmed the presence of catP and the 3' end of tnpV from Tn4451, a mobilizable element from Clostridium perfringens, although other sequences were not present. Tn4451 has inserted into a gene designated TIGR locus NMB1350 in both isolates with no loss of DNA and no apparent interruption of virulence genes. This second report of chloramphenicol-resistant meningococci is in a setting with a very low volume of systemic chloramphenicol use, but where the high topical use may contribute to recombination events in vivo. Methods for screening for chloramphenicol resistance in meningococci and the in vitro parameters that define this resistance are ill defined.