Asymmetric MSM sub-bandgap all-silicon photodetector with low dark current.

Design, fabrication, and characterization of an asymmetric metal-semiconductor-metal photodetector, based on internal photoemission effect and integrated into a silicon-on-insulator waveguide, are reported. For this photodetector, a responsivity of 4.5 mA/W has been measured at 1550 nm, making it suitable for power monitoring applications. Because the absorbing metal is deposited strictly around the vertical output facet of the waveguide, a very small contact area of about 3 µm2 is obtained and a transit-time-limited bandwidth of about 1 GHz is demonstrated. Taking advantage of this small area and electrode asymmetry, a significant reduction in the dark current (2.2 nA at -21 V) is achieved. Interestingly, applying reverse voltage, the photodetector is able to tune its cut-off wavelength, extending its range of application into the MID infrared regime.

Surface plasmon resonance optical cavity enhanced refractive index sensing.

We report on a method for surface plasmon resonance (SPR) refractive index sensing based on direct time-domain measurements. An optical resonator is built around an SPR sensor, and its photon lifetime is measured as a function of loss induced by refractive index variations. The method does not rely on any spectroscopic analysis or direct intensity measurement. Time-domain measurements are practically immune to light intensity fluctuations and thus lead to high resolution. A proof of concept experiment is carried out in which a sensor response to liquid samples of different refractive indices is measured. A refractive index resolution of the current system, extrapolated from the reproducibility of cavity-decay time determinations over 133 s, is found to be about 10(-5) RIU. The possibility of long-term averaging suggests that measurements with a resolution better than 10(-7) RIU/√Hz are within reach.

Evaluation by real-time PCR of the expression of S. flexneri virulence-associated genes ospB and phoN2 under different genetical backgrounds.

Under conditions of activated type III secretion Shigella flexneri up-regulates the expression of numerous genes, including the virulence plasmid (pINV)-encoded ospB and phoN2 genes. ospB and phoN2 are virulence-associated genes which are part of a bicistronic transcriptional unit encoding OspB, a protein (effector) of unknown function secreted by the type III secretion (TTS) apparatus, and PhoN2 (apyrase or ATP-diphosphohydrolase), a periplasmic protein involved in polar IcsA localization on the surface of S. flexneri. In this work we used real-time PCR to measure transcription of ospB and phoN2 of wild-type S. flexneri strain M90T as well as of derivative mutants impaired in definite virulence traits. The results obtained confirmed and extended previous reports indicating that the expression of ospB and phoN2 genes is modulated in a virB-dependent, mxiE-independent manner under conditions of non-activated secretion, while their expression is considerably induced in a mxiE-dependent manner under conditions of activated secretion. That the expression of the ospB-phoN2 operon is up-regulated in condition of activated secretion, indicates that probably the expression of these two genes might be important, especially during the later stages of infection of S. flexneri.

Molecular characterization of virulence determinants of Stenotrophomonas maltophilia strains isolated from patients affected by cystic fibrosis.

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen which is currently isolated with increasing frequency from the airways of cystic fibrosis (CF) patients. In this study 13 S. maltophilia strains (11 isolated from the airways of independent CF patients, and two non-CF respiratory reference strains) have been characterized for the expression of several virulence-associated factors. In particular, the ability to form biofilm on abiotic surfaces has been determined and correlated with different features, such as motility, adherence and the ability to invade A549 respiratory epithelial cells. Moreover, the presence of a flagellum-associated gene as well as that of the StmPr1 gene, which encodes an extracellular protease, have been determined by Southern blot hybridization. Our data indicate that the different degree of biofilm formation exhibited by the 11 CF isolates does not correlate with motility, ability to adhere to and invade A549 cells, or with the presence of flagella. On the other hand, among the CF isolates the StmPr1 gene was found only in two strains, both able to establish chronic lung infections in CF patients. Moreover, only four of the strains analyzed show a temperature-independent antibiotic-resistance profile, suggesting either a different origin of these strains or an intervening adaptation to host tissues.

A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli.

We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the expression of the virF gene present on the large virulence plasmid of Shigella and enteroinvasive Escherichia coli in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the VirG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of H-NS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30 degrees C. The expression of a virF-lacZ translational fusion in E.coli wild type and in an hns-defective derivative grown at 30 degrees or 37 degrees C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virF expression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controls virF expression by binding to the virF promoter and by repressing its expression at low temperature and at low pH.

A two-year study of enteric infections associated with diarrhoeal diseases in children in urban Somalia.

A hospital-based systematic sample of 1667 children with severe diarrhoeal disease was studied in Mogadishu, Somalia, throughout 1983 and 1984. One or more enteric pathogens were found in 61% of the patients. Rotavirus (25%), enterotoxigenic Escherichia coli (11%), Shigella spp. (9%), Aeromonas hydrophila (9%), Giardia lamblia trophozoites (8%), Campylobacter jejuni (8%), and Vibrio cholerae non-O1 (6%) were the most frequently identified pathogens. Age-specific detection rates of enteric pathogens and helminths, seasonal patterns, and relationship of some specific infections with feeding status and main clinical features have been defined for all the sample examined.

Intrauterine growth retardation: a neonatologist's approach.

Early recognition of intrauterine growth retardation, and recognition of these compromised infants at birth, is essential, to correct, if possible, any adverse intrauterine influence; then, to provide proper nursery care, screening for abnormalities, and proper post-natal nutrition. Improved management of both the in-utero and the ex-utero environment may offer these neonates a more favorable prognosis for their physical, neurological, and intellectual development.

Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread.

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.

Composite IS1-tetracycline resistance elements in aerobactin-encoding FIme plasmids from epidemic Salmonella wien.

Class B tetracycline resistance determinants have been identified in two aerobactin-encoding FIme plasmids representative of those isolated from epidemic Salmonella wien. Genetic data, restriction enzyme analysis of recombinant and mutant plasmids, and Southern blot hybridizations indicate that in both plasmids the class B determinant so far found and described only on Tn10-like transposons is part of a different genetic element. This composite insertion sequence element is about 7 kilobases long and has copies of insertion sequence IS1 at the ends.

Stability of plasmid content in Salmonella wien in late phases of the epidemic history.

Prevalence, genetic characteristics, and EcoRI cleavage analysis of plasmids identified in clinical strains of Salmonella wien isolated in recent years showed that the plasmid content in this serotype has remained uniform and stable over more than a decade and also late in the epidemic history. No correlation between decrease in S. wien isolations and naturally occurring systematic changes in the DNA of its most common FIme plasmid was structurally detectable.

Virulence plasmids of enteroinvasive Escherichia coli and Shigella flexneri integrate into a specific site on the host chromosome: integration greatly reduces expression of plasmid-carried virulence genes.

The ability of enteroinvasive Escherichia coli and Shigella flexneri to cause disease depends on the presence of a large virulence plasmid (pINV). In this report we show that pHN280, the pINV of the O135:K-:H- enteroivasive strain E. coli HN280, and pWR100, the pINV of S. flexneri serotype 5 strain M90T, are able to integrate into a specific site on the host chromosome. pINV-integrated HN280 and M90T strains required methionine (Met-) to grow in minimal medium, were noninvasive, did not produce contact-mediated hemolysin, and had lost the ability to bind Congo red (Crb-) at 37 degrees C. Immunoblots of whole bacterial extracts from pHN280-integrated HN280 derivatives revealed that integration severely reduced the expression of ipa and virG (icsA) plasmid genes. Met- HN280 and M90T derivative strains spontaneously generated Met+ revertants that either contained excised forms of pINV or had lost pINV. Restriction analysis of excised pINVs showed that they either were virtually identical to parental pINVs (precise excision) or had suffered some deletion (imprecise excision). Precisely excised pINVs expressed the full pattern of virulence, whereas imprecisely excised pINVs were always Crb- and noninvasive. The revertion to Met+ was shown to be recA dependent, indicating that homologous plasmid and chromosomal DNA sequences are involved in the integration-excision process. The maintainance of pINV through integration and downregulation of its virulence genes may represent an advantageous mechanism for enteroinvasive bacteria, particularly when they are outside host cells and/or have to face adverse environmental conditions.

Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose-negative Escherichia coli strains of serotype O4 isolated from children with diarrhea.

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin.

Comparison among enterotoxigenic strains of Escherichia coli isolated in Italy and Somalia.

Nine strains of ETEC isolated in Italy have been compared with 13 isolates from Somalia with respect to toxin production, serotype and antimicrobial resistance pattern. None of the strains isolated from Italy belonged to any serotype or serogroups found among the strains from Somalia. Remarkable differences between the two groups of isolates were also observed with regard to the susceptibility to antimicrobials and the presence of R-plasmids. These findings suggest that ETEC strains isolated in Italy are not related to the strains widespread in Somalia and, generally, in developing countries.

The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936-kanamycin resistance element.

Tn1935, a 23.5-kb transposon mediating resistance to ampicillin, kanamycin, mercury, spectinomycin, and sulfonamide was isolated from pZM3, an IncFIme virulence plasmid from Salmonella wien. Tn1935 possesses the entire sequence of Tn21 and contains two additional DNA segments of 0.95 and 2.7 kb carrying the ampicillin and kanamycin resistance genes, respectively. The latter is part of a composite element since it is flanked by two IS15-like insertion sequences (IS1936) in direct orientation. IS1936 is about 800 bp long and is closely related to IS15 delta, IS26, IS46, IS140, and IS176. Functional analysis of IS1936-mediated cointegrates shows that both insertion sequences are active and able to form cointegrates at the same frequency. Resolution of the cointegrates requires the presence of the host Rec system. The presence of the composite IS1936-element within Tn1935 supports the hypothesis that multidrug resistance transposons evolved by insertion of antibiotic determinants which are themselves transposable.

Plasmids and transposable elements in Salmonella wien.

The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.

Expression of the virulence plasmid-carried apyrase gene (apy) of enteroinvasive Escherichia coli and Shigella flexneri is under the control of H-NS and the VirF and VirB regulatory cascade.

The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37 degreesC and repressed at 30 degreesC. In this work, we report that the O135: K-:H- EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Deltahns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb-) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb- mutants of HN280 and SFZM53, respectively. While all Crb- apy+ mutants of HN280 failed to produce apyrase, Crb- apy+ phoN-Sf+ mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb- mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.

Composite IS1 elements encoding hydroxamate-mediated iron uptake in FIme plasmids from epidemic Salmonella spp.

Eleven FIme plasmids representative of those identified in epidemic strains of Salmonella wien and Salmonella typhimurium isolated in North Africa, Europe, and the Middle East have been examined for the presence of determinants of toxigenicity, adherence, and iron-sequestering mechanisms. Chemical and genetic data indicated that all plasmids code for a hydroxamate-mediated iron assimilation system. Detailed analysis of derivative plasmids and cloned fragments of FIme plasmid pZM61 demonstrated that the general genetic and structural organization of the DNA region containing the genes for hydroxamate biosynthesis and cloacin DF13 receptor was virtually identical to that described for the aerobactin-mediated iron uptake system of pColV-K30. This DNA region is part of a composite element that is 16.7 kilobases long and carries its IS1 modules as inverted repeats. A very similar element is present in either orientation in all nine FIme plasmids analyzed.