Differential susceptibility of different cell lines to swine-origin influenza A H1N1, seasonal human influenza A H1N1, and avian influenza A H5N1 viruses.

BACKGROUND: The novel swine-origin influenza A H1N1 virus (S-OIV) causes the current pandemic. Its tissue tropism and replication in different cell lines are not well understood. OBJECTIVE: Compare the growth characteristics of cell lines infected by S-OIV, seasonal influenza A H1N1 (sH1N1) and avian influenza A H5N1 (H5N1) viruses and the effect of temperature on viral replication. STUDY DESIGN: Cytopathic effect (CPE), antigen expression by immunofluorescence (IF) and viral load profile by quantitative RT-PCR in 17 cell lines infected by S-OIV, sH1N1 and H5N1 were examined. Comparison of their replication efficiency in chick embryo was performed. The effect of temperature on viral replication in Madin-Darby canine kidney (MDCK) cells was determined by TCID(50) at 33 degrees C, 37 degrees C and 39 degrees C for 5 consecutive days. RESULTS: S-OIV replicated in cell lines derived from different tissues or organs and host species with comparable viral load to sH1N1. Among 13 human cell lines tested, Caco-2 has the highest viral load for S-OIV. S-OIV showed a low viral load with no CPE or antigen expression in pig kidney cell PK-15, H5N1 demonstrated the most diverse cell tropism by CPE and antigen expression, and the highest viral replication efficiency in both cell lines and allantoic fluid. All three viruses demonstrated best growth at 37 degrees C in MDCK cells. CONCLUSION: Cell line growth characteristics of S-OIV, sH1N1 and H5N1 appear to correlate clinically and pathologically with involved anatomical sites and severity. Low replication of S-OIV in PK-15 suggests that this virus is more adapted to human than swine.

Outbreak of intestinal infection due to Rhizopus microsporus.

Sinopulmonary and rhinocerebral zygomycosis has been increasingly found in patients with hematological malignancies and bone marrow transplantation, but intestinal zygomycosis remains very rare in the literature. We investigated an outbreak of intestinal infection due to Rhizopus microsporus in 12 patients on treatment for hematological malignancies over a period of 6 months in a teaching hospital. The intake of allopurinol during hospitalization (P < 0.001) and that of commercially packaged ready-to-eat food items in the preceding 2 weeks (P < 0.001) were found to be independently significant risk factors for the development of intestinal zygomycosis. A total of 709 specimens, including 378 environmental and air samples, 181 food samples, and 150 drug samples, were taken for fungal culture. Among them, 16 samples of allopurinol tablets, 3 prepackaged ready-to-eat food items, and 1 pair of wooden chopsticks were positive for Rhizopus microsporus, which was confirmed by ITS1-5.8S-ITS2 rRNA gene cluster (internal transcribed spacer [ITS]) sequencing. The mean viable fungal counts of allopurinol obtained from wards and pharmacy were 4.22 x 10(3) CFU/g of tablet (range, 3.07 x 10(3) to 5.48 x 10(3)) and 3.24 x 10(3) CFU/g of tablet (range, 2.68 x 10(3) to 3.72 x 10(3)), respectively, which were much higher than the mean count of 2 x 10(2) CFU/g of food. Phylogenetic analysis by ITS sequencing showed multiple clones from isolates of contaminated allopurinol tablets and ready-to-eat food, of which some were identical to patients' isolates, and with one isolate in the cornstarch used as an excipient for manufacture of this drug. We attempted to type the isolates by random amplification of polymorphic DNA analysis, with limited evidence of clonal distribution. The primary source of the contaminating fungus was likely to be the cornstarch used in the manufacturing of allopurinol tablets or ready-to-eat food. Rhizopus microsporus is thermotolerant and can multiply even at 50 degrees C. The long holding time of the intermediates during the manufacturing process of allopurinol amplified the fungal load. Microbiological monitoring of drugs manufactured for highly immunosuppressed patients should be considered.

Phylogenetic and recombination analysis of coronavirus HKU1, a novel coronavirus from patients with pneumonia.

Phylogenetic trees constructed using predicted amino acid sequences of putative proteins of coronavirus HKU1 (CoV-HKU1) revealed that CoV-HKU1 formed a distinct branch among group 2 coronaviruses. Of the 14 trees from p65 to nsp10, nine showed that CoV-HKU1 was clustered with murine hepatitis virus. From nsp11, the topologies of the trees changed dramatically. For the eight trees from nsp11 to N, seven showed that the CoV-HKU1 branch was the first branch. The codon usage patterns of CoV-HKU1 differed significantly from those in other group 2 coronaviruses. Split decomposition analysis revealed that recombination events had occurred between CoV-HKU1 and other coronaviruses.

Isolation and characterization of a Salmonella enterica serotype Typhi variant and its clinical and public health implications.

We report the isolation and characterization of a member of the family Enterobacteriaceae isolated from the gallbladder pus of a food handler. Conventional biochemical tests suggested Salmonella enterica serotype Typhi, but the isolate agglutinated with poly(O), 2O, 9O, and Vi Salmonella antisera but not with poly(H) or any individual H Salmonella antisera. 16S rRNA gene sequencing showed that there were two base differences between the isolate and Salmonella enterica serotype Montevideo, four base differences between the isolate and serotype Typhi, five base differences between the isolate and Salmonella enterica serotype Typhimurium, and six base differences between the isolate and Salmonella enterica serotype Dublin, indicating that the isolate was a strain of S. enterica. Electron microscopy confirmed that the isolate was aflagellated. The flagellin gene sequence of the isolate was 100% identical to that of the H1-d flagellin gene of serotype Typhi. Sequencing of the rfbE gene, which encoded the CDP-tyvelose epimerase of the isolate, showed that there was a point mutation at position +694 (G-->T), leading to an amino acid substitution (Gly-->Cys). This may have resulted in a protein of reduced catalytic activity and hence the presence of both 2O and 9O antigens. We therefore concluded that the isolate was a variant of serotype Typhi. Besides antibiotic therapy and cholecystectomy, removal of all stones in the biliary tree was performed for eradication of the carrier state.

Identification of Mycobacterium neoaurum isolated from a neutropenic patient with catheter-related bacteremia by 16S rRNA sequencing.

A rapidly growing pigmented mycobacterial strain with an ambiguous biochemical profile was isolated from the blood culture taken through the Hickman catheter of a 9-year-old girl with acute lymphoblastic leukemia. Whole-cell fatty acid analysis showed that the best match profile was that of Mycobacterium aurum, but the similarity index was only 0.217, meaning that there were no good matches between the isolate and the organisms in the database of the Microbial Identification System. The 16S rRNA gene of the mycobacterial strain was amplified, agarose gel purified, and sequenced. There were 44 base differences between the gene sequence of the isolate and that of M. aurum but only one base difference between the sequence of the isolate and that of Mycobacterium neoaurum, showing that the isolate was indeed a strain of M. neoaurum by using this "gold standard." This represents the first case of M. neoaurum infection documented by 16S rRNA sequencing.

Enhancement of humoral and protective immune response induced by live-attenuated Salmonella typhi by ampicillin.

Efficacy of live-attenuated Salmonella vaccines delivered by the mucosal route is limited by the dose and interference from mucosal flora of the alimentary tract. In a mouse model, the total antibody response towards lipopolysaccharide of S. typhi was significantly enhanced at day 21 post-immunization with live-attenuated S. typhi (Ty21a) when ampicillin was concomitantly administered (p<0.005), and the lethal dose 50 of mice in the ampicillin and control groups immunized with Ty21a after wild-type S. typhi challenge on day 24 was 4x10(7) and 1x10(7), respectively. The faecal bacterial counts of the ampicillin group at days 1 and 3 were significantly lower than those of the control group (p<0.01 and <0.05). On day 1, the number of mice with > or =10 Ty21a colonies isolated from the spleen was significantly higher in the ampicillin group than the control group (p<0.05). Furthermore, on the same day, Ty21a was isolated from the faeces of three mice from the ampicillin group, but only one from the control group. We conclude that ampicillin may have enhanced the humoral and protective immune responses by giving the Ty21a a selective advantage over the normal bacterial flora. This concept of antibiotic enhancement of immunization could have important implications for other live-attenuated vaccines, as well as the delivery of microbial antigens and DNA vaccines by live-attenuated Salmonella carriers.

Enhancement by ampicillin of antibody responses induced by a protein antigen and a DNA vaccine carried by live-attenuated Salmonella enterica serovar Typhi.

Live-attenuated Salmonella species are effective carriers of microbial antigens and DNA vaccines. In a mouse model, the immunoglobulin M (IgM) and total antibody levels directed toward the lipopolysaccharide of Salmonella enterica serovar Typhi were significantly enhanced at day 21 after oral immunization with live-attenuated serovar Typhi (strain Ty21a) when ampicillin was concomitantly administered (P < 0.05 and P < 0.005, respectively). The heat-killed Ty21a-stimulated lymphocyte proliferation indices for the ampicillin group at day 21 were significantly higher than those for the normal saline (NS) group (P < 0.005, P < 0.001, and P < 0.01) for all three doses of antigen (10(4), 10(5), and 10(6) heat-killed Ty21a per well, respectively). The 50% lethal doses for mice from the ampicillin and NS groups immunized with Ty21a with pBR322 after wild-type serovar Typhi challenge on day 24 were 3.4 x 10(7) and 5.0 x 10(6) CFU, respectively. The fecal bacterial counts for the ampicillin group at days 1, 3, and 5 were significantly lower than those for the NS group (P < 0.01, P < 0.01, and P < 0.05, respectively), and there was a trend toward recovery of Ty21a in a larger number of mice from the ampicillin group than from the NS group. Furthermore, the IgG2a levels directed toward tetanus toxoid were significantly enhanced at days 7 and 21 after oral immunization with Ty21a that carried the fragment c of tetanus toxoid when ampicillin was concomitantly administered (P < 0.05 and P < 0.005, respectively), and the IgM and total hepatitis B surface antibody levels were significantly enhanced at days 7 (P < 0.005 and P < 0.05, respectively) and 21 (P < 0.01 and P < 0.05, respectively) after oral immunization with Ty21a that carried the DNA vaccine that encodes hepatitis B surface antigen when ampicillin was concomitantly administered. The present observation may improve the efficacy of the protein antigens and DNA vaccines carried in live-attenuated bacteria, and further experiments should be carried out to determine the best antibiotics and dosage regimen to be used, as well as the best carrier system for individual protein antigens and DNA vaccines.

Clarithromycin attenuates cyclophosphamide-induced mucositis in mice.

No universally recognized agent is available for prophylaxis or therapy of mucositis induced by chemotherapy or chemo-radiotherapy. The effect of clarithromycin on the severity of mucositis induced by cyclophosphamide was investigated using a mouse model. Four cross-sections of small intestine (levels A, B, C, and D) were taken at equivalent intervals at day 5 after cyclophosphamide (400 mg kg(-1)) administration. The sections were stained with haematoxylin and eosin, and were graded for the degree of mucositis histologically. At section level B, the number of mice with no mucositis (grade 0) in the clarithromycin group was significantly greater than that in the ceftriaxone group (P<0.05). At levels B and C, the number of mice with no mucositis (grade 0) in the clarithromycin group was significantly greater than that in the normal saline (NS) group (P<0.05). At level C, the number of mice with grade 2 mucositis in the ceftriaxone group was significantly greater than that in the NS group (P<0.05). When the number of sections at all levels were analyzed together, the number of mice with no mucositis (grade 0) in the clarithromycin group was significantly greater than that in the ceftriaxone and NS groups (P<0.05). The present observation suggests that clarithromycin and ceftriaxone attenuates and aggravates cyclophosphamide-induced mucositis. It prompts clinical trials in bone marrow transplant (BMT) recipients receiving cyclophosphamide for conditioning, and reconsideration in the use of ceftriaxone in the treatment of neutropenic fever in BMT recipients.

Antibiotics modulate vaccine-induced humoral immune response.

The effects of antibiotics on the antigen-specific humoral immune response are not known. Macrolides, tetracyclines, and beta-lactams are commonly prescribed antibiotics. The first two are known to have immunomodulatory activities. The effects of clarithromycin, doxycycline, and ampicillin on the primary and secondary antibody responses to tetanus toxoid, a pneumococcal polysaccharide vaccine, a hepatitis B virus surface antigen (HBsAg) vaccine, and live attenuated Salmonella typhi (Ty21a) were investigated using a mouse model. For the mice receiving the tetanus toxoid, the immunoglobulin M (IgM) level of the clarithromycin group at day 7 was significantly lower than the corresponding antibody level of the normal saline (NS) group. For the mice receiving the pneumococcal polysaccharide vaccine, the total antibody and IgM levels of the clarithromycin group and the IgM level of the doxycycline group at day 7 were significantly lower than the corresponding antibody levels of the ampicillin and NS groups. For the mice receiving the HBsAg vaccine, the IgM level of the doxycycline group at day 7 was significantly lower than the corresponding antibody levels of the clarithromycin and NS groups, while the IgM level of the clarithromycin group at day 28 was significantly lower than the corresponding antibody levels of the doxycycline, ampicillin, and NS groups. For the mice receiving all three vaccines, there were no statistically significant differences between any of the antibody levels of the ampicillin group and the corresponding antibody levels of the NS group. For the mice receiving Ty21a, the total antibody levels of the ampicillin group at days 7 and 21 were significantly higher than the corresponding antibody levels of the NS group. Moreover, the IgM levels of the clarithromycin, doxycycline, and ampicillin groups at days 7 and 21 were significantly higher than the corresponding antibody levels of the NS group. Furthermore, the total antibody level of the ampicillin group at day 21 was significantly higher than the corresponding antibody level of the doxycycline group. For all four vaccines, there were no statistically significant differences among the serum levels of interleukin-10 and gamma interferon for the mice treated with the various antibiotics. We conclude that clarithromycin and doxycycline, but not ampicillin, suppress the antibody responses of mice to T-cell-dependent and T-cell-independent antigens, whereas all three antibiotics enhance the antibody response to live attenuated mucosal bacterial vaccines.