The latency-associated UL138 gene product of human cytomegalovirus sensitizes cells to tumor necrosis factor alpha (TNF-alpha) signaling by upregulating TNF-alpha receptor 1 cell surface expression.

Many viruses antagonize tumor necrosis factor alpha (TNF-α) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-α receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-α has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb' region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb'-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb' gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-α responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-α on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-α-mediated reactivation of HCMV.

Rapid and accurate diagnosis of human intestinal spirochetosis by fluorescence in situ hybridization.

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.

Efficacy of calcium hydroxide, Er:YAG laser or gaseous ozone against Enterococcus faecalis in root canals.

PURPOSE: To evaluate the efficacy of Ca(OH)2, Er:YAG laser or gaseous ozone (either alone or combined with instrumentation and various irrigants) against Enterococcus faecalis in root canals. METHODS: 180 extracted, human, single-rooted teeth were divided into four groups of 45 teeth each. In Group 1 root canal enlargement up to ISO-size 60 (MAF) was performed, whereas only initial shaping (MAF ISO-size 40) was carried out in Groups 2 to 4. After sterilization all teeth were inoculated with E. faecalis and incubated for 3 days, followed by evaluation of CFU. Subsequently, root canal enlargement up to ISO-size 60 was performed in Groups 2 to 4 using NaCl solution (0.9%) in Group 2, NaOCl (1%) in Group 3 and CHX (0.2%) in Group 4. Finally, each group of 45 teeth was subdivided into three groups (n= 15 each) either applying Ca(OH)2 for 7 days, using Er:YAG laser radiation for 30 seconds or gaseous ozone for 120 seconds, followed by final evaluation of CFU. RESULTS: Both in Groups 1 and 2 the median reduction of bacteria after application of Ca(OH)2 (factor 10(4) each) and ozone (in Group 1: factor 5 x 10(3); in Group 2: factor 5 x 10(4)), respectively, was significantly higher than after Er:YAG laser treatment (factor 102 each, Mann-Whitney test). The antibacterial efficacy was significantly increased by the additional use of NaOCl or CHX as irrigants in all subgroups (Groups 3 and 4) compared to corresponding subgroups of Group 1 (Mann-Whitney test).

High rate of resistance to locally used antibiotics among enteric bacteria from children in Northern Ghana.

OBJECTIVES: Information on antimicrobial susceptibility of bacterial pathogens is scarce in resource-poor settings. We determined the susceptibility of bacterial enteric pathogens and faecal Escherichia coli isolates obtained from children in urban Tamale, Northern Ghana, to antibiotics widely used in the that area [ampicillin or amoxicillin, trimethoprim/sulfamethoxazole (SXT) and chloramphenicol] and to alternative drugs. METHODS: Five Shigella spp., 6 Salmonella spp. and 318 E. coli were isolated from stool specimens obtained from 367 children with or without acute diarrhoea. Isolates were differentiated using standard laboratory procedures and tested using a breakpoint microbroth dilution method for their susceptibility to 18 antimicrobials and by disc diffusion for their susceptibility to chloramphenicol. RESULTS: Although the salmonellae showed an acceptable resistance pattern, E. coli isolates and the closely related shigellae were highly resistant. About 91% and 81% of E. coli isolates from patients or controls, respectively, were resistant to ampicillin (MICs > or = 8 mg/L), 88% and 76% to trimethoprim/sulfamethoxazole (MICs > or = 80/4 mg/L) and 46% and 41% to chloramphenicol (inhibition zones < or = 12 mm). Resistance to beta-lactam antibiotics or chloramphenicol was observed more frequently among isolates obtained from infants when compared with older children (1-4 years of age). CONCLUSIONS: Enteric bacteria from children in urban Northern Ghana are highly resistant to antibiotics used in that area. Therefore, new antibiotics should be introduced for the treatment of infections caused by these bacteria. Additionally, the establishment of a surveillance of the prevalence of the main bacterial infectious agents and their antimicrobial resistance is desirable.

Community-acquired Legionnaires' disease caused by Legionella pneumophila serogroup 10 linked to the private home.

We describe the case of a 66-year-old man with a culture-proven Legionella pneumonia after kidney transplantation. The patient developed the infection 15 days after discharge from a university hospital. Legionella pneumonia caused by Legionella pneumophila serogroup 5/10 was established by positive direct fluorescence assay, positive urinary-antigen detection and isolation of the causative agent. The infection was successfully treated by giving appropriate antibiotics, but the further course was complicated by invasive aspergillosis, cytomegalovirus pneumonia, failure of the transplanted kidney and development of septic anaemia. Four months after the diagnosis of Legionella pneumonia the patient died of multi-organ failure. The microbiological and epidemiological investigation revealed that strains from the water supply of the patient's private home were indistinguishable from the patient's isolate by amplified fragment length polymorphism analysis and sequence-based typing (SBT). Unrelated strains of serogroups 4, 5, 8 and 10 from the Dresden strain collection were of different SBT types. Thus, SBT is a very useful tool for epidemiological investigation of infections by L. pneumophila serogroups other than serogroup 1.

Human cytomegalovirus blocks tumor necrosis factor alpha- and interleukin-1beta-mediated NF-kappaB signaling.

NF-kappaB plays an important role in the early cellular response to pathogens by activating genes involved in inflammation, immune response, and cell proliferation and survival. NF-kappaB is also utilized by many viral pathogens, like human cytomegalovirus (HCMV), to activate their own gene expression programs, reflecting intricate roles for NF-kappaB in both antiviral defense mechanisms and viral physiology. Here we show that the NF-kappaB signaling pathway stimulated by proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) becomes inhibited in HCMV-infected cells. The block to NF-kappaB signaling is first noticeable during the early phase of infection but is fully established only at later times. Biochemical and genetic evidence demonstrates that the viral inhibition of proinflammatory signaling by distinct cytokines occurs upstream of the convergence point of NF-kappaB-activating pathways, i.e., the IkappaB kinase complex, and that it is mediated via different mechanisms. Consistent with this, we further show that an HCMV variant that has lost the ability to downregulate TNF-alpha-induced NF-kappaB signaling also fails to downregulate surface expression of TNF receptor 1, thereby mechanistically linking the inhibition of TNF-alpha-induced NF-kappaB signaling by HCMV to TNF receptor targeting. Our data support a model whereby HCMV inhibits cytokine-induced NF-kappaB signaling at later times during infection, and we suggest that this contributes to the inhibition of the cell's antiviral defense program.

High frequency of multidrug-resistant Mycobacterium tuberculosis isolates in Georgetown, Guyana.

Emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates constitutes a threat to public health worldwide. This study aimed at acquiring first epidemiological data for Guyana. Thirty-six M. tuberculosis isolates from patients of the Georgetown Chest Clinic were subjected to susceptibility testing on solid agar and in broth media. Resistance to at least one first-line drug was observed in 8 (22.2%, 95% confidence interval 8.3-36.1%) and simultaneous resistance to rifampicin and isoniazid (MDR) in 4 (11.1%, 95% confidence interval 0.6-21.6%) of the 36 isolates. The risk of infection with resistant isolates was significantly related to earlier antituberculosis therapy (P=0.040). These data indicate a high proportion of resistant M. tuberculosis isolates in Guyana and call for the implementation of control strategies based on an improved laboratory diagnosis of TB.

Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2001-2002 from poultry and humans in Berlin, Germany.

The susceptibilities of 430 Campylobacter jejuni strains and 79 C. coli strains to six antimicrobial agents were tested and analyzed. The two sets of strains originated from retail market chicken and turkey samples and from humans, respectively, in Berlin, Germany. Two groups of isolates, one dating from 1991 and the other dating from 2001-2002, were tested. Of the Campylobacter sp. isolates recovered from humans in 2001-2002, 45.1% were resistant to ciprofloxacin, 37.8% were resistant to tetracycline, 12.8% were resistant to ampicillin, and 50.0% were resistant to trimethoprim-sulfamethoxazole. All isolates were susceptible to gentamicin, while the overall rate of resistance to erythromycin was 6.1%. During the 10 years between the two sampling times, the rates of resistance to ciprofloxacin (P<0.001), ampicillin (P=0.035), and tetracycline (P=0.01) increased significantly among strains isolated from humans. Furthermore, among human C. coli strains the rate of resistance to erythromycin rose from 7.1% in 1991 to 29.4% in 2001-2002. In comparison, Campylobacter sp. isolates from poultry already had high rates of resistance in 1991. Different rates of resistance to tetracycline among isolates from chickens and turkeys suggested the development of resistance during antimicrobial treatment in food animals. Thus, discrepancies in the antimicrobial resistance rates among Campylobacter isolates originating from poultry and humans support the hypothesis that at least some of the resistant Campylobacter strains causing infection in humans come from sources other than poultry products.

Comparison of broth microdilution, E Test, and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli.

A standardized broth microdilution method was compared to the E test and an agar dilution method for the antimicrobial susceptibility testing of Campylobacter jejuni and C. coli isolates. A group of 47 human clinical isolates, 37 isolates from retail poultry, and 29 isolates from living turkeys (total, 113 isolates) was included in the study. These encompassed 92 C. jejuni and 21 C. coli strains. The MICs of six antimicrobial agents were determined by the broth microdilution and E test methods, and the strains of human origin were additionally tested by the agar dilution method. In general, broth microdilution MICs agreed within 1 log(2) MIC increment with 90.0% of E test results and 78.7% of agar dilution test results. The agar dilution method gave much lower gentamicin MICs than the broth microdilution method, but the data were significantly (P < 0.01) correlated and there was 100% agreement in the sensitivities and specificities in the comparison of the tests. The broth microdilution method had the highest sensitivity for analysis of the susceptibilities of Campylobacter to nalidixic acid and trimethoprim-sulfamethoxazole. The MICs of ciprofloxacin and erythromycin complied numerically by all three methods. The classification of the results and the correlation of the data demonstrated a high degree of agreement. All methods were equally suitable for the testing of the sensitivity of Campylobacter to tetracycline. Thus, the broth microdilution method appears to be an easy and reliable method for determination of the MICs of antibiotics for C. jejuni and C. coli, and it may offer an interesting alternative to MIC determination by the agar dilution technique or the E test.

Susceptibilities of Campylobacter jejuni isolates from Germany to ciprofloxacin, moxifloxacin, erythromycin, clindamycin, and tetracycline.

To elucidate Campylobacter jejuni resistance to antibiotics in Germany, MICs of ciprofloxacin, moxifloxacin, erythromycin, clindamycin, and tetracycline were determined (using agar dilution) for 144 clinical isolates. The data indicate a considerable ciprofloxacin resistance (45.1%) without a clonal relationship of the strains and a greater in vitro activity of moxifloxacin, erythromycin, and clindamycin.

High frequency of multidrug-resistant Mycobacterium tuberculosis isolates in Georgetown, Guyana

Emergence of multi-drug resistance (MDR) Mycobacterium tuberculosis isolates constitutes a threat to public health worldwide. This study aimed at acquiring first epidemiological data for Guyana. Thirty-six M. tuberculosis isolates from patients of the Georgetown Chest Clinic were subjected to susceptibility testing on solid agar and in broth media. Resistance to at least one first-line drug was observed in 8 (22.2 percent, 95 percent confidence interval 8.3 -36.1 percent) and simultaneous resistance to rifampicin and isoniazid (MDR) in 4 (11.1 percent, 95 percent confidence interval 0.6-21.6 percent) of the 36 isolates. The risk of infection with resistant isolates was significantly related to earlier antituberculosis therapy (P=0.040). These data indicate a high proportion of resistant M.tuberculosis isolates in Guyana and call for the implementation of control strategies based on an improved laboratory diagnosis of TB(AU)