First detection of the mcr-1 colistin resistance gene in Escherichia coli from a patient with urinary tract infection in Myanmar.

Colistin-resistance gene mcr-1 was detected in an Escherichia coli sample among 442 clinical isolates collected in a tertiary-care hospital in Yangon, Myanmar, in 2018. This isolate was classified into phylogroup A-ST23 complex and harboured bla CTX-M-15 and bla TEM-1, associated with multiple mutations in quinolone-resistance-determining regions in gyrA and parC.

Drug resistance and genetic characteristics of clinical isolates of staphylococci in Myanmar: high prevalence of PVL among methicillin-susceptible Staphylococcus aureus belonging to various sequence types.

Prevalence, drug resistance and genetic characteristics were analysed for a total of 128 clinical isolates of staphylococci obtained from a tertiary hospital in Myanmar. The dominant species were S. aureus (39%) and S. haemolyticus (35%), followed by S. epidermidis (6%) and S. saprophyticus (5%). The majority of S. haemolyticus isolates (71.1%) harboured mecA, showing high resistance rates to ampicillin, cephalosporins, erythromycin and levofloxacin, while methicillin-resistant S. aureus (MRSA) was only 8% (four isolates) among S. aureus with type IV SCCmec. Panton-Valentine leukocidin (PVL) genes were detected in 20 isolates of S. aureus (40%), among which only one isolate was MRSA belonging to sequence type (ST) 88/agr-III/coa-IIIa, and the other 19 methicillin-susceptible S. aureus (MSSA) isolates were classified into six STs (ST88, ST121, ST1153, ST1155, ST1930, ST3206). An ST1153 MSSA isolate with PVL was revealed to belong to a novel coa type, XIIIa. ST121 S. aureus was the most common in the PVL-positive MSSA (47%, 9/19), harbouring genes of bone sialoprotein and variant of elastin binding protein as a distinctive feature. Although PVL-positive MSSA was susceptible to most of the antimicrobial agents examined, ST1930 isolates were resistant to erythromycin and levofloxacin. ST59 PVL-negative MRSA and MSSA had more resistance genes than other MRSA and PVL-positive MSSA, showing resistance to more antimicrobial agents. This study indicated higher prevalence of mecA associated with multiple drug resistance in S. haemolyticus than in S. aureus, and dissemination of PVL genes to multiple clones of MSSA, with ST121 being dominant, among hospital isolates in Myanmar.

Fast track pathway reduces sight loss in giant cell arteritis: results of a longitudinal observational cohort study.

OBJECTIVES: To investigate the effectiveness of a fast track pathway (FTP) on sight loss in patients with suspected giant cell arteritis (GCA). METHODS: A longitudinal observational cohort study was conducted in the secondary care rheumatology department. One hundred and thirty-five newly referred suspected GCA patients seen via the FTP (Jan. 2012-Dec. 2013) were compared to 81 patients seen through the conventional referral and review system (Jan. 2009-Dec. 2011). RESULTS: The FTP resulted in significant reduction in irreversible sight loss from 37.0% (as seen in the historical cohort 2009-2011) to 9.0 % (2012-2013, OR 0.17, p=0.001). Adjustment for clinical and demographic parameters including known risk factors for GCA associated blindness did not significantly change the primary result (OR 0.08, p=0.001). FTP resulted in a reduction of time from symptom onset to diagnosis, particularly by reduction of time from general practitioner's (GP) referral to the rheumatology review (79% of FTP patients were seen within one working day compared to 64.6 % in the conventional pathway, p=0.023). The FTP has seen a reduction in number of GP appointments. CONCLUSIONS: There was a significant reduction of permanent sight loss with a fast track GCA pathway. The effect may be due to multiple factors including better GP education and reduction in delayed diagnosis. These results need verification at other sites.

Roles of tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), and IL-10 in the modulation of intercellular adhesion molecule-1 (ICAM-1) expression by macrophages during mycobacterial infection.

Profiles of ICAM-1 expression on cultured murine peritoneal macrophages infected with Mycobacterium avium complex (MAC) were examined, with special reference to modulating roles of TNF-alpha, TGF-beta, and IL-10. When macrophages were infected with MAC, ICAM-1 expression, measured by microscopic counting of ICAM-1+ macrophages stained with anti-ICAM-1 antibody, ELISA, and flow cytometric analysis, was rapidly increased, peaking at day 3 (early-phase up-regulation) due to endogenous TNF-alpha, and thereafter gradually declined to the normal level within 1 week or more (late-phase down-regulation). The late-phase ICAM-1 down-regulation was also seen in macrophages phagocytosing heat-killed MAC and those stimulated with lipopolysaccharide but not in macrophages phagocytosing latex beads. ICAM-1 mRNA expression was augmented markedly at day 1 after MAC infection and thereafter decreased. While TNF-alpha and IL-10 production by MAC-infected macrophages was observed during the first 3 days, TGF-beta production was initiated from day 3 and continued until day 14. Exogenously added TGF-beta strongly inhibited the early-phase increase in ICAM-1 expression by infected macrophages, and the blockade of endogenous TGF-beta with anti-TGF-beta antibody markedly inhibited late-phase ICAM-1 down-regulation. Moderate blocking effect was also observed for anti-IL-10 antibody. On the other hand, late-phase ICAM-1 down-regulation was not prevented by the addition of exogenous TNF-alpha. Therefore, TGF-beta and IL-10, especially the former, appear to play active roles in the late-phase down-regulation of ICAM-1 in MAC-infected macrophages during long-term cultivation.

Roles of tumor necrosis factor-alpha and transforming growth factor-beta in regulating intercellular adhesion molecule-1 expression on murine peritoneal macrophages infected with M. leprae.

Profiles of intercellular adhesion molecule-1 (ICAM-1) expression on murine peritoneal macrophages (M phi s) infected with Mycobacterium leprae during cultivation were examined with special reference to the regulatory effects of tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). When M phi s were infected with M. leprae or stimulated with heat-killed M. leprae at day 0, their ICAM-1 expression, measured in terms of the ratio of M phi s positively stained with anti-ICAM-1 antibody (Ab), rapidly increased, peaking during days 1 to 3 and thereafter fell, returning to the normal level by day 7. The addition of TNF-alpha or anti-TGF-beta Ab inhibited the middle phase (day 7) downregulation of M phi ICAM-1 expression, although the late-phase (day 14) downregulation of ICAM-1 was not prevented by them. M. leprae-infected M phi s released small amounts of TNF-alpha and significant amounts of TGF-beta into the culture medium. This may indicate that M. leprae-infected M phi s produced the majority of TNF-alpha in a membrane-bound form. Alternatively, endogenous TNF-alpha might upregulate M phi ICAM-1 expression even at very low concentrations. In any case, these findings indicate the central roles of TNF-alpha and TGF-beta in the early phase upregulation and the middle-to-late phase downregulation, respectively, of ICAM-1 expression by M. leprae-infected M phi s.

Studies on therapeutic activity of benzoxazinorifamycin KRM-1648 in combination with other antimicrobial agents and biological response modifiers interferon-gamma and granulocyte-macrophage colony-stimulating factor against M. leprae infection in athymic nude mice.

In the present study, we evaluated the in vivo anti-Mycobacterium leprae activities of KRM-1648 (KRM) given at long intervals in combination with ofloxacin (OFLX), clofazimine (CFZ), and dapsone (DDS). We also examined the combined effects of two biological response modifiers (BRMs), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF), on the therapeutic efficacy of KRM. KRM exhibited potent therapeutic efficacy against M. leprae infection in mice even when given at 4-week intervals. KRM displayed increased efficacy in combination with OFLX, CFZ, and DDS (given three or six times per week) when given to mice in the multidrug combination KRM + OFLX + CFZ + DDS. The therapeutic efficacy of KRM given at 4-week intervals was increased by combined use with IFN-gamma but not by GM-CSF. Adoptive transfer of M. leprae antigen-primed lymphocytes of euthymic mice to recipient athymic nude mice with progressive M. leprae infection markedly enhanced host resistance.

Further study on the roles of the effector molecules of immunosuppressive macrophages induced by mycobacterial infection in expression of their suppressor function against mitogen-stimulated T cell proliferation.

Previously, we found that phospholipids and reactive nitrogen intermediates (RNI) collaborated in expression of the T cell mitogenesis-inhibitory activity of immunosuppressive macrophages induced by Mycobacterium avium-intracellulare complex (MAIC) infection. In this study, we examined the roles of free fatty acids (FFA) and prostaglandins (PG) as effectors of MAIC-induced macrophages, and moreover, their collaborating effects with RNI. First, treatment of MAIC-induced macrophages with quinacrine (phospholipase A2 (PLA2) inhibitor), dexamethasone (inhibitor of PLA2 and PG synthesis) or indomethacin (PG synthesis inhibitor) attenuated their suppressor activity against concanavalin A (Con A)-induced mitogenesis of splenocytes (SPC), indicating important roles of FFA liberated from membrane phospholipids and PG, as effectors. Second, oleic acid, PGE2, RNI generated from NOR 4 (a new nitric oxide (NO) donor), and phosphatidylserine (PS) exhibited suppressor activity against SPC mitogenesis without showing significant cytotoxicity, in an irreversible manner. Third, the suppressor activities of RNI and PGE2 were potentiated by combined use with oleic acid in a synergistic manner. Fourth, a dual-chamber experiment in which target SPC were separated from MAIC-induced macrophages by a Millipore filter revealed a requirement for cell-to-cell contact for expression of the suppressor function of MAIC-induced macrophages. These findings indicate that RNI, FFA, PG, and phospholipids (presumably PS) and their collaboration play central roles in expression of the T cell mitogenesis-inhibitory function of MAIC-induced suppressor macrophages.

[The expression of ICAM-1 on macrophages stimulated with Mycobacterium avium complex and its control by some regulatory cytokines].

The interaction of LFA-1 on T lymphocytes with ICAM-1 on antigen presenting cells (APCs) is critical in determining conjugate formation between the APCs and T cells as well as activation of T cells. Recently, it was found that stimulation of THP-1 cells, a human monocyte M phi cell line, with Mycobacterium tuberculosis or its lipoarabinomannan, elicited the increase in the ICAM-1 expression. In addition, in cases of lepromatous leprosy patients with a serious defect in the M. leprae antigen-specific cellular immunity, keratinocytes in the leprosy lesions were lacking in the ICAM-1 expression. Therefore, ICAM-1 seems to participate in the host response to mycobacterial infections. Here, we studied the mode of the expression of ICAM-1 molecules on murine peritoneal M phis in response to stimulation with M. avium complex (MAC). In addition, the regulatory effect of some cytokines including TNF-alpha, IL-10, and transforming growth factor-beta (TGF-beta) on the ICAM-1 expression was studied. Monolayer cultures of peptone-starch induced murine peritoneal M phis were cultured in RPMI-1640 medium in the presence of MAC with or without test agents. At intervals, the M phis were stained with anti-ICAM-1 antibody (Ab) and then treated with alkaline phosphatase (Ap)-conjugated anti-1g Ab. After color development with NBT-BCIP substrate, percentage of the blue-stained (ICAM-1 positive) M phis was determined microscopically. The concentrations of TNF-alpha, IL-10, and TGF-beta in the M phi culture fluid was measured by ELISA using capture Ab, biotin-labelled capping Ab, and Ap-conjugated streptavidin. When M phis infected with MAC organisms were cultured in RPMI medium containing 10% fetal bovine serum or in serum-free GPI medium, a significant increase in their ICAM-1 expression was observed, reaching the peak at days 1 to 3, thereafter rapidly decreased and returned to the normal level by day 14. Further addition of TNF-alpha caused no significant change in the mode of the MAC-induced expression of ICAM-1. A transient increase in the IL-10 production of MAC-infected M phis was observed during the first 3-days cultivation, as in the case of TNF-alpha. On the other hand, TGF-beta production of the Mø population was initiated from day 3, and therafter increased gradually until day 14. ICAM-1 expression of the MAC-infected M phis was not influenced by the addition of IL-10, while anti-IL-10 Ab retarded the decline of ICAM-1 expression at day 7. On the other hand, the addition of TCF-beta attenuated the MAC infection-induced increase in the ICAM-1 expression significantly. In addition, anti-TGF-beta Ab significantly delayed the reduction of the ICAM-1 expression of MAC-infected M phis during days 3 to 7. These results indicate that, in the MAC-infected M phis. TGF-beta rather than IL-10 play important roles as a mediators for the late-phase down-regulation of ICAM-1 expression which had been transiently elevated by the action of TNF-alpha in the early phase of the Mø cultivation.

The role of tumour necrosis factor-alpha in combination with interferon-gamma or interleukin-1 in the induction of immunosuppressive macrophages because of Mycobacterium avium complex infection.

The role of some cytokines including tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta) and interleukin-6 (IL-6) in the generation of immunosuppressive macrophages (M phi s) in host spleen cells of Mycobacterium avium complex (MAC)-infected mice was studied. M phi populations with potent suppressor activity against concanavalin A (Con A)-induced mitogenesis of splenocytes (SPCs) were elicited not only in euthymic but also in athymic nude mice during MAC infection. The suppressor M phi s are, therefore, inducible not only through a T-cell-dependent mechanism but also through T-cell-independent mechanism. However, MAC-induced M phi s of athymic mice displayed about four times lower suppressor activity than those of euthymic mice, indicating that mature T cells are important for M phi activation to the highly immunosuppressive state. Anti-TNF, anti-IFN-gamma, and anti-TGF-beta antibodies (Abs) but not anti-IL-6 Ab inhibited in vivo generation of MAC-induced immunosuppressive M phi s, and the neutralizing efficacy was in the order of anti-IFN-gamma Ab > anti-TNF Ab > anti-TGF-beta Ab. The effects of TNF-alpha, IL-1 alpha, IL-6, and IFN-gamma alone or combinations of them upon the acquisition of the suppressor activity by cultured splenic M phi s were studied. When normal splenic M phi s were treated with each cytokine for 3 days, TNF-alpha, IFN-gamma, and IL-1 alpha alone caused a slight elevation of their suppressive activity. Treatment of the normal M phi s with the combination of either TNF-alpha+IL-1 alpha or TNF-alpha+IFN-gamma yielded a marked increase in the suppressor activity, followed by IL-1 alpha+IFN-gamma. These findings indicate the important roles of TNF-alpha, IFN-gamma, and IL-1 alpha in the generation of MAC-induced suppressor M phi s.

Phospholipids and reactive nitrogen intermediates collaborate in expression of the T cell mitogenesis-inhibitory activity of immunosuppressive macrophages induced in mycobacterial infection.

We studied the role of phospholipids and nitric oxide in expression of the suppressor activity of splenic macrophages induced by Mycobacterium avium-intracellulare complex infection (MAIC-induced macrophages) in mice against mitogenic response of concanavalin A (Con A)-stimulated splenocytes (SPC) as follows. First, phosphatidylserine (PS) and phosphatidylinositol were found to suppress Con A-induced mitogenesis of SPC via inhibition of IL-2 production and acquisition of IL-2 reactivity in Con A-stimulated T cells. The mitogenesis-inhibitory activity of PS was increased when SPC were cultured under mildly acidic condition (pH 6.3). When SPC were pretreated with PS for 24 h prior to Con A blastogenesis, their mitogenic response was irreversibly abrogated. Second, NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthase, was found to attenuate in part the expression of the suppressor activity of MAIC-induced macrophages. Third, reactive nitrogen intermediates (RNI) including NO generated from acidified NO2- exerted potent inhibitory activity against SPC mitogenic response, and the suppressive activity of RNI was significantly augmented by the combination with PS. These findings indicate that phospholipids and RNI plays an important role in the expression of suppressor activity of MAIC-induced macrophages as the effector molecules.

The role of tumor necrosis factor, interferon-gamma, transforming growth factor-beta, and nitric oxide in the expression of immunosuppressive functions of splenic macrophages induced by Mycobacterium avium complex infection.

In order to verify the participation of some cytokines in the expression of the suppressor activity of splenic macrophages (M phi s) induced by Mycobacterium avium complex (MAC) infection, we studied whether anticytokine antibodies were capable of blocking their suppressor activity against concanavalin A (ConA)-induced mitogenesis of splenocytes (SPCs). When either anti-tumor necrosis factor (TNF), anti-transforming growth factor-beta (TGF-beta), or anti-interferon-gamma (IFN-gamma) antibody was added to culture medium, suppressor activity was markedly reduced, in the order of anti-TNF, anti-IFN-gamma, and anti-TGF-beta antibodies. By contrast, neither anti-interleukin-6 (IL-6) nor anti-IL-10 antibody exerted such a blocking effect. Therefore, TNF, IFN-gamma, and TGF-beta seem to be related to the full display of the suppressor function of MAC-induced M phi s. However, TNF-alpha and IFN-gamma but not TGF-beta were substantially lacking in inhibitory action against SPC mitogenesis, when added exogenously. Hence, it is unlikely that TNF-alpha and INF-gamma directly modulated the proliferative response of T cells. On the other hand, both TNF-alpha and IFN-gamma potentiated the effector function of the suppressor M phi s. Because their suppressor activity was severely reduced by NG-monomethyl-L-arginine and aminoguanidine, nitric oxide (NO) synthase inhibitors, an NO-dependent mechanism is important for the expression of the immunosuppressive function of MAC-induced M phi s. Moreover, because these M phi s seem to produce a substantial amount of TNF-alpha in membrane-bound form, cell-to-cell contact might be needed for efficient expression of their suppressor action on target T cells.

Study on the roles of CD4+ and CD8+ T cells in the expression of host resistance to Mycobacterium leprae infection induced in athymic nude mice.

The contribution of CD4+ or CD8+ T cells in the host resistance to Mycobacterium leprae was investigated. Athymic BALB/c nude mice were infected subcutaneously with M. leprae into the foot pads 1 or 3 weeks after adoptive transfer with whole, CD4 T cell-depleted, or CD8 T cell-depleted lymphocyte fractions prepared from spleen cells (SPCs) of euthymic BALB/c mice. SPCs from all of the recipient mice showed nearly the same levels of proliferative responses to phytohemagglutinin (PHA) and lipopolysaccharide, except that those of mice transferred with CD4 T cell-depleted lymphocytes showed somewhat reduced mitogenic responses to PHA. Significantly potentiated host resistance to the infection was seen, as evidenced by the obviously reduced bacterial growth in all three groups of recipient mice compared to that seen in control mice, that is, 4.7- to 6.4-log unit decreases in the bacterial loads were observed. Levels of the reduction conferred by CD 4 T cell-depleted lymphocytes were comparable to that of CD8 T cell-depleted lymphocytes. This suggests that both CD4 and CD8 T cells are important for the expression of resistance to M. leprae infection.

[Mechanism of bacterial regrowth at the sites of infection in Mycobacterium avium complex-infected mice during treatment with chemotherapeutic agents].

Although various antimicrobial drugs show appreciable bactericidal activity in the early phase (2 to 4 weeks after infection) of Mycobacterium avium complex (MAC) infections in mice, no drug, as far as we know, can continue to exert the growth inhibiting activity against the bacteria at the site of infection in the progressed stage. Here, we studied the mechanisms of the bacterial regrowth which usually starts around 2-4 weeks after infection. First, the changes in the level of TNF-alpha, IFN-gamma, IL-6 and IL-10 in the lungs and spleen during the course of MAC infections was examined. Tissue levels of TNF-alpha and IL-10 increased around weeks 2 to 4, then rapidly decreased thereafter, and returned to the normal levels by week 8, while levels of IFN-gamma and IL-6 remained very low throughout the observation period. In this experiment, the bacterial CFUs rapidly decreased during the first 2 weeks of the treatment with a rifamycin derivative, KRM-1648, and thereafter the regrowth of the organisms was observed even in mice treated continuously with KRM-1648, although the rate of bacterial growth in the treated mice was much lower than in untreated control mice. Second, effect of either anti-TGF-beta or anti-IL-10 antibody on intracellular growth of MAC in human monocytes cultured in vitro in the medium with or without addition of TNF-alpha or IFN-gamma were examined. Anti-TGF-beta and anti-IL-10 antibodies potently reduced the bacterial growth in monocytes. Effects of TNF-alpha and IFN-gamma in reducing the bacterial growth was potentiated by the addition of either anti-TGF-beta or anti-IL-10 antibody. Third, anti-IL-10 antibody augmented to some extent the chemotherapeutic efficacy of KRM-1648 against MAC infection, when the drug was given to mice at weeks 2 and 4 after infection. From these results, it is suggested that IL-10 derived from MAC-infected macrophages in response to stimulation with some bacterial components, such as lipoarabinomannan, might downregulate the antimicrobial function of host macrophages against MAC.