Ultraviolet C inactivation of dermatophytes: implications for treatment of onychomycosis.

BACKGROUND: Onychomycosis responds to systemic antifungals and sometimes to topical lacquers, but alternative treatments are desirable. Topical application of germicidal ultraviolet (UV) C radiation may be an acceptable and effective therapy for infected nails. OBJECTIVES: To test the ability of UVC to inactivate dermatophyte suspensions in vitro and to sterilize a novel ex vivo model of nail infection. METHODS: Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum and Microsporum canis suspensions were irradiated with UVC (254 nm) at a radiant exposure of 120 mJ cm(-2) and surviving colony-forming units quantified. T. rubrum infecting porcine hoof slices and human toenail clippings was irradiated with UVC at radiant exposures of 36-864 J cm(-2). RESULTS: In vitro studies showed that 3-5 logs of cell inactivation in dermatophyte suspensions were produced with 120 mJ cm(-2) UVC irradiation. Depending on factors such as the thickness and infectious burden of the ex vivo cultures, the radiant exposure of UVC needed for complete sterilization was usually in the order of tens to hundreds of J cm(-2). Resistance of T. rubrum to UVC irradiation did not increase after five cycles of subtotal inactivation in vitro. CONCLUSIONS: UVC irradiation may be a less invasive treatment option for onychomycosis, when the appropriate consideration is given to safety.

Detecting the Hidden Properties of Immunological Data and Predicting the Mortality Risks of Infectious Syndromes.

BACKGROUND: To extract more information, the properties of infectious disease data, including hidden relationships, could be considered. Here, blood leukocyte data were explored to elucidate whether hidden information, if uncovered, could forecast mortality. METHODS: Three sets of individuals (n = 132) were investigated, from whom blood leukocyte profiles and microbial tests were conducted (i) cross-sectional analyses performed at admission (before bacteriological tests were completed) from two groups of hospital patients, randomly selected at different time periods, who met septic criteria [confirmed infection and at least three systemic inflammatory response syndrome (SIRS) criteria] but lacked chronic conditions (study I, n = 36; and study II, n = 69); (ii) a similar group, tested over 3 days (n = 7); and (iii) non-infected, SIRS-negative individuals, tested once (n = 20). The data were analyzed by (i) a method that creates complex data combinations, which, based on graphic patterns, partitions the data into subsets and (ii) an approach that does not partition the data. Admission data from SIRS+/infection+ patients were related to 30-day, in-hospital mortality. RESULTS: The non-partitioning approach was not informative: in both study I and study II, the leukocyte data intervals of non-survivors and survivors overlapped. In contrast, the combinatorial method distinguished two subsets that, later, showed twofold (or larger) differences in mortality. While the two subsets did not differ in gender, age, microbial species, or antimicrobial resistance, they revealed different immune profiles. Non-infected, SIRS-negative individuals did not express the high-mortality profile. Longitudinal data from septic patients displayed the pattern associated with the highest mortality within the first 24 h post-admission. Suggesting inflammation coexisted with immunosuppression, one high-mortality sub-subset displayed high neutrophil/lymphocyte ratio values and low lymphocyte percents. A second high-mortality subset showed monocyte-mediated deficiencies. Numerous within- and between-subset comparisons revealed statistically significantly different immune profiles. CONCLUSION: While the analysis of non-partitioned data can result in information loss, complex (combinatorial) data structures can uncover hidden patterns, which guide data partitioning into subsets that differ in mortality rates and immune profiles. Such information can facilitate diagnostics, monitoring of disease dynamics, and evaluation of subset-specific, patient-specific therapies.

Identification of a promoter region on the Halomonas elongata cryptic plasmid pHE1 employing the inaZ reporter gene of Pseudomonas syringae.

A native promoter located on the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata was identified employing a promoterless ice nucleation gene inaZ of Pseudomonas syringae by direct subcloning and assaying for ice nucleation activity. The presence of the promoter was verified by inserting the corresponding intact or deleted pHE1 fragment in the promoter analysis vector pKK232-8 upstream of the promoterless cat or inaZ gene. Only constructs carrying the intact pHE1 fragment gave CAT phenotype (chloramphenicol resistance) or ice nucleation activity, respectively. Comparative evaluation of the sequence analysis data of the intact and deleted fragment suggested the localization of an Escherichia coli-type promoter region.

Genetic organization of the mobilization region of the plasmid pHE1 from Halomonas elongata.

The mobilization (mob) region of the non-self transmissible 4.2-kb plasmid pHE1 from the moderately halophilic bacterium Halomonas elongata ATCC 33174 has been identified and characterized. Analysis of the sequence revealed the presence of four open reading frames (mobCABD) which show a complex organization with two of them (mobB and mobD) entirely overlapped by a third (mobA). The deduced proteins appeared to have a high degree of homology to Mob proteins of CoIE1 and closely related plasmids. To assess the functionality of the mob region, the hybrid vector pHS134 was constructed, consisting of the complete plasmid pHEI, the E. coli vector pKS(-) and a streptomycin-resistance gene for positive selection in Halomonas. Vector pHS134 was found to be mobilizable from E. coli to H. elongata assisted by pRK600. Upstream of the mob genes, an oriT region with a putative nick sequence highly homologous to that of CoIE1 plasmids was identified. To our knowledge, this is the first mobilizable plasmid found in moderate halophiles. This property, together with its small size, the availability of its complete sequence, and its broad host range in moderately halophilic strains, makes pHE1 a good candidate for the construction of cloning and expression vectors for these extremophiles.

Analysis of the replication region of the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata.

The basic replicon of the narrow-host-range plasmid pHE1 from the moderately halophilic bacterium Halomonas elongata ATCC 33174 has been identified and characterized. The replicon consists of a 1.7-kb DNA fragment, which contains the genetic information required for autonomous replication and stable maintenance. Analysis of its sequence revealed the presence of two ORFs which seem to form one transcription unit. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology to the theta-replicase (RepA) protein of ColE2 plasmid and to the RepA proteins of a family of replicons from gram-positive and gram-negative bacteria, also related to ColE2. The product encoded by ORF2 showed a certain similarity to the RepB proteins of the same family of replicons and perhaps represents the pHE1 RepB function. Deletion analysis suggests that the pHE1 origin of replication (ori) is located in an 800-bp region upstream of repA. A third putative gene, incA, was found on the complementary strand to the leader region of the repA mRNA. This, together with the presence in the 5' untranslated region of the repA mRNA of inverted repeats that could form stable stem-loop structures, suggests that the incA gene encodes a small antisense RNA. A possible control mechanism for pHE1 replication is proposed, involving an RNA molecule which sequesters the translational initiation region of the replication protein RepA. The basic replicon characterized here shows very interesting properties that should allow it to be used in the construction of cloning and expression vectors for moderate halophiles.

Release of cell-free ice nuclei from Halomonas elongata expressing the ice nucleation gene inaZ of Pseudomonas syringae.

Release of ice nuclei in the growth medium of recombinant Halomonas elongata cells expressing the inaZ gene of Pseudomonas syringae was studied in an attempt to produce cell-free active ice nuclei for biotechnological applications. Cell-free ice nuclei were not retained by cellulose acetate filters of 0.2 microm pore size. Highest activity of cell-free ice nuclei was obtained when cells were grown in low salinity (0.5-5% NaCl, w/v). Freezing temperature threshold, estimated to be below -7 degrees C indicating class C nuclei, was not affected by medium salinity. Their density, as estimated by Percoll density centrifugation, was 1.018 +/- 0.002 gml(-1) and they were found to be free of lipids. Ice nuclei are released in the growth medium of recombinant H. elongata cells probably because of inefficient anchoring of the ice-nucleation protein aggregates in the outer membrane. The ice+ recombinant H. elongata cells could be useful for future use as a source of active cell-free ice nucleation protein.

Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae.

The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata. One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H. elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters. A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies. The ice nucleation gene was also expressed in H. elongata and V. eurihalina under the control of Pbla (the promoter of the beta-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis). One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila. In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species.