Pacemaker-associated infection caused by ST81/SCCmec IV methicillin-resistant, vancomycin-intermediate Staphylococcus aureus in Japan.

A 76-year-old Japanese man was admitted to hospital for treatment of fever and skin lesion at the implantation site of his pacemaker. During his hospitalization, vancomycin-intermediate Staphylococcus aureus (MIC 4 µg/mL) with reduced susceptibility to daptomycin was isolated from venous blood. This isolate was identified as methicillin-resistant S. aureus with SCCmec IV and was genotyped as sequence type 81, coa VIIa and spa type t7044, harbouring blaZ, aac(6')-aph(2″) and enterotoxin(-like) genes sea, seb, sek, sel, selx and selw. The patient was successfully treated with daptomycin, minocycline and sulfamethoxazole/trimethoprim. We describe the identification of sequence type 81/SCCmec IV vancomycin-intermediate S. aureus from pacemaker-associated septicaemia.

Genetic characteristics of VanA-type vancomycin-resistant Enterococcus faecalis and Enterococcus faecium in Cuba.

VanA-type vancomycin-resistant enterococci isolates from bloodstream infections in Cuba were genetically characterized. Enterococcus faecalis isolates were assigned to sequence type (ST) 28, closely related to Eastern Europe, while Enterococcus faecium belonged to ST262, ST656 and ST1349, and showed different genetic profiles.

Nationwide prevalence of Rickettsia felis infections in patients with febrile illness in Bangladesh.

From July 2015 to December 2016, the presence of rickettsial pathogens was investigated for 414 patients with unknown fever in eight places in all the divisions of Bangladesh. Rickettsia felis was identified in blood samples from all the regions (overall detection rate, 19.6%), suggesting nationwide prevalence of R. felis infections.

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.

Detection of ST772 Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (Bengal Bay clone) and ST22 S. aureus isolates with a genetic variant of elastin binding protein in Nepal.

Genetic characteristics were analysed for recent clinical isolates of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA respectively) in Kathmandu, Nepal. MRSA isolates harbouring Panton-Valentine leukocidin (PVL) genes were classified into ST1, ST22 and ST88 with SCCmec-IV and ST772 with SCCmec-V (Bengal Bay clone), while PVL-positive MSSA into ST22, ST30 and ST772. ST22 isolates (PVL-positive MRSA and MSSA, PVL-negative MRSA) possessed a variant of elastin binding protein gene (ebpS) with an internal deletion of 180 bp, which was similar to that reported for ST121 S. aureus previously outside Nepal. Phylogenetic analysis indicated that the ebpS variant in ST22 might have occurred independently of ST121 strains. This is the first report of ST772 PVL-positive MRSA in Nepal and detection of the deletion variant of ebpS in ST22 S. aureus.

Emerging non-PCV13 serotypes of noninvasive Streptococcus pneumoniae with macrolide resistance genes in northern Japan.

In Japan, the 7-valent pneumococcal conjugate vaccine (PCV7) was introduced to the nation's routine immunization program in April 2013 and was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13) in November 2013. Distribution of serotypes and macrolide resistance genotypes was investigated for a total of 1097 (975 children, 122 adults) and 960 (873 children, 87 adults) clinical isolates of Streptococcus pneumoniae from noninvasive infections in Hokkaido (northern main island of Japan) in the routine immunization periods for PCV7 and PCV13 (April-October 2013 and November 2013-November 2014, respectively). Serotype was determined by sequential multiplex PCR and additional genetic analyses. Macrolide resistance genes erm(B) and mef(A/E) were detected by multiplex PCR. Although the most prevalent serotypes in children were 23A and 6C in the PCV7 period, after replacement with PCV13, 19A became the most common, followed by 6C, 15A and 23A. Among adults, serotype 3 was consistently the most frequent throughout the study periods. Compared with values from the pre-PCV7 routine immunization period, PCV7 serotypes decreased from 48.3 to 3.3% in the PCV13 period among children, while the rates of non-PCV13 serotypes (particularly 15A, 23A, 11A, 10A and 35B) increased from 39.7 to 75.1% (p < 0.001). In the PCV13 period, erm(B), mef(A/E) and both of these genes were detected in 75.8, 31.6 and 11.3% of all isolates, respectively. Serotype 19A accounted for 76.9% of the isolates with both the macrolide resistance genes, and emerging non-PCV13 serotypes 15A, 15C and 23A mostly harboured erm(B).

Emergence of Klebsiella pneumoniae clinical isolates producing KPC-2 carbapenemase in Cuba.

The emergence of Klebsiella pneumoniae producing carbapenemase (KPC) has now become a global concern. As a part of a nationwide multicentre surveillance study in Cuba, three K. pneumoniae clinical isolates resistant to carbapenems were detected for a 1-month period (September to October 2011). PCR and sequence analysis revealed that the three strains harboured bla KPC-2. They showed resistance or intermediate susceptibility to expanded-spectrum cephalosporins, other ß-lactams, a ß-lactam/ß-lactamase inhibitor combination, and gentamicin. Two strains were susceptible only to colistin, whereas the other strain showing colistin resistance was susceptible to fluoroquinolones. These bla KPC -2-positive K. pneumoniae strains were classified into ST1271 (CC29), a novel clone harbouring bla KPC -2, and were revealed to be genetically identical by PCR-based DNA fingerprinting. The three patients infected with the KPC-producing K. pneumoniae had common risk factors, and had no overseas travel experience outside Cuba, suggesting local acquisition of the resistant pathogen. This is the first report of a KPC-producing K. pneumoniae in Cuba. Although detection of KPC in Enterobacteriaceae is still rare in Cuba, our finding indicated that KPC-producing bacteria are a global concern and highlighted the need to identify these microorganisms in clinical laboratories.

Heterogeneity of platelet responsiveness to anti-CD36 in plasma associated with adverse transfusion reactions.

BACKGROUND AND OBJECTIVES: Antibodies to CD36 (anti-CD36) are clinically important. As some platelet immunoglobulins produced by transfusion or pregnancy have been shown to induce platelet activation and to play roles in non-haemolytic transfusion reactions (NHTRs), we investigated the in vitro response of platelets to plasma containing anti-CD36. MATERIALS AND METHODS: Plasma containing anti-CD36, implicated in the development of NHTRs and subsequent thrombocytopenia, was incubated with CD36-positive platelets. Plasma-induced platelet activation was examined by evaluating platelet aggregation and RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) release. RESULTS: Platelet activation was induced by plasma alone in four out of 20 CD36-positive subjects. In seven subjects, platelet activation was synergistically induced by the combination of epinephrine priming and the plasma. The platelets of the nine remaining subjects failed to respond to the plasma. Platelet activation induced by either the plasma alone or by synergy with epinephrine required the involvement of Fc gamma RIIa. The different responsiveness of the platelets was partially associated with the surface levels of CD36 and Fc gamma RIIa, but not with Fc gamma RIIa polymorphisms. CONCLUSIONS: Plasma containing anti-CD36, implicated in the development of NHTRs, exhibited a platelet-activating capability. Additionally, platelets from healthy human subjects exhibited a considerable degree of heterogeneity in their responsiveness to this plasma. The heterogeneity of these responses may determine the occurrence of anti-CD36-related NHTRs.

The selective reduction in PTPdelta expression in hepatomas.

The mRNA levels for receptor-like protein tyrosine phosphatases (PTPases), PTPalpha, PTPdelta, PTPgamma and LAR, were evaluated by Northern blot analysis in two types of chemically-induced rat primary hepatomas. In the four PTPases the PTPdelta mRNA was selectively reduced in these hepatoma tissues. It was also diminished in HepG2 hepatoblastoma cell line and in all of the poorly differentiated ascites hepatoma cells examined. PTPalpha, PTPgamma and LAR did not show such a characteristic decrease. This selective reduction in PTPdelta expression strongly suggests PTPdelta plays an important role in hepatocarcinogenesis, possibly as a tumor suppressor gene.

The characteristic gene expressions of MAPK phosphatases 1 and 2 in hepatocarcinogenesis, rat ascites hepatoma cells, and regenerating rat liver.

mRNA levels of mitogen-activated protein kinase phosphatases, MKP-1 and MKP-2, were determined during chemical hepatocarcinogenesis and during regeneration of rat liver. In chemical hepatocarcinogenesis, the mRNA levels of MKP-1 were increased in primary hepatomas but decreased in rat ascites hepatomas as compared with normal liver. MKP-2 was undetectable in normal liver but strongly expressed in hepatomas. The MKP-2 mRNA level was increased with expression of malignant phenotypes in hepatomas. In regenerating liver, the mRNA level of MKP-1 increased immediately but transiently after partial hepatectomy, and peaked again on day 10, the time when hepatocytes cease proliferation. The elevated expression of MKP-1 on day 10 suggests some roles of MKP-1 as a negative regulator in hepatocyte proliferation.

Dissociation between upper and lower neck N13 potentials following paired median nerve stimuli.

Cervical N13 potential in response to the median nerve stimulation can be recorded either from upper (Cv2) or lower (Cv6) neck with almost equal amplitudes and latencies. It has long been debated whether they represent the same or different generator sources. Using a conditioning-test paired stimuli paradigm, we examined the differences of recovery function of Cv2- and Cv6-N13, anterior neck (AN)-P13, and scalp recorded P13/P14 in 6 healthy subjects. All cervical electrodes were referenced to the non-cephalic site. Scalp response was recorded with linked ear reference. The inter-stimulus intervals ranged from 4 to 20 ms with 2 ms increments. Throughout 4 to 18 ms ISI, Cv6-N13, AN-P13 and scalp P13/P14 were suppressed, whereas Cv2-N13 was facilitated. All but scalp P13/P14 returned close to the control at 20 ms ISI. The findings indicate that Cv2-N13, Cv6-N13 and scalp P13/P14 are independent each other and arise from different generator sources. The suppression of Cv6-N13 is consistent with a postsynaptic nature of this potential and may indeed be mediated through dorsal horn interneurons creating a current field orientation in the posterior-anterior direction. The facilitation of Cv2-N13 suggests that this is a presynaptic potential and may travel through the dorsal column with vertical orientation. The longer period of suppression of scalp P13/P14 suggests it to be of polysynaptic origin and to arise at least rostral to the cuneate nucleus.

A novel gene (drad-1) expressed in hematopoiesis-supporting stromal cell lines, ST2, PA6 and A54 preadipocytes: use of mRNA differential display.

We established a differentiation-inducible preadipocyte cell line, designated A54 preadipocytes, from C3H10T1/2 (10T1/2) mouse embryo fibroblasts. A54 preadipocytes had marked hematopoiesis-supporting ability in vitro but this ability was lost after terminal differentiation to adipocytes. In this study, to identify molecules that contribute to the hematopoiesis-supporting ability of A54 preadipocytes, we screened genes that were differentially expressed in A54 preadipocytes and isolated seven novel genes by reverse transcriptase polymerase chain reaction mRNA differential display. An RNase protection assay confirmed that one of these genes was expressed at high levels in parent 10T1/2 cells and A54 preadipocytes but to a much lesser extent in fully differentiated A54 adipocytes. This gene was defined as a gene that was downregulated during adipocyte differentiation-1 (drad-1). The size of drad-1 mRNA was 8.2 kb, and the gene was expressed in other mouse preadipocytes, namely, ST2 and PA6 cells, that have hematopoiesis-supporting ability. Moreover, drad-1 was also found to be expressed in bone marrow in vivo. The function of the protein encoded by drad-1 is unknown, but the expression of the gene may be useful as a molecular marker of adipocyte differentiation.

Human cytomegalovirus DNA is not detectable with nested double polymerase chain reaction in healthy blood donors.

The PCR method was introduced to detect cytomegalovirus (CMV) DNA from 189 peripheral blood samples of volunteer donors. We adopted the nested double PCR method with primers specific for immediate early gene 1 followed by electrophoresis and ethidium bromide staining. This nested double PCR method was sensitive enough to detect approximately a single copy of CMV DNA. However, we failed to obtain positive amplification of CMV DNA from any of these donor samples. In contrast, CMV DNA could be detected in all 3 tested immunocompromised patients who had undergone bone marrow transplantation. These results support our previous report that the frequency of CMV DNA is of an order lower than 1 copy/10(5) leucocytes in the peripheral blood of healthy seropositive individuals.

[Effect of pyramidal tract stimulation on segmental spinal cord potentials].

The author reports his results of the effect of pyramidal tract stimulation on the segmental spinal cord potentials (seg-SCP) using a total of 50 Wistar rats. Forty two Wistar rats were used to observe the effects of a pyramidal tract stimulation on the seg-SCP evoked by hindpaw stimulation. Another 5 Wistar rats were used to observe dorsal column stimulation as the conditioning stimuli instead of pyramidal tract stimulation. And the other 3 Wistar rats were used for both pyramidal tract and dorsal column stimulation as a separate trial. The seg-SCP of the rats consisted of three major peaks, P1, N1 and P2. P2 was separated into a first (P2F) and second component (P2S). The P2F peak was constantly preceded by a small notch, considered to be the onset of P2F, and named the P2F initial peak. The P2F peak amplitude of the seg-SCP was significantly decreased by pyramidal tract stimulation, while the P2F initial peak amplitude remained unchanged. This effect persisted until the concentration of sevoflurane was reduced to below 1.5% or until the peak arterial blood pressure was decreased to below 80 mmHg. The amplitude of both the P2F initial peak and the P2F peak were decreased significantly by dorsal column stimulation. These findings indicated that inhibition of the P2F peak amplitude was a characteristic effect of the pyramidal tract stimulation on the seg-SCP, while inhibition of the overall P2F amplitude was characteristic of the dorsal column stimulation.

UV irradiation can induce in vitro clonal anergy in alloreactive cytotoxic T lymphocytes.

The alloreactive cytotoxic T lymphocytes (CTL) were generated by coculturing peripheral blood mononuclear cells (PBMC) with allogeneic Sa cells (an Epstein-Barr virus [EBV]-transformed B-cell line). The CTL did not proliferate in response to UV-B-irradiated Sa cells unless exogenous interleukin-2 (IL-2) was present, although they could kill the UV-B-irradiated Sa cells. The results indicate that UV-B-irradiated Sa cells do not provide sufficient signals for the proliferation of the CTL while they can be recognized by CTL and induce high-affinity IL-2 receptor (IL-2R) expression on them. The alloreactive CTL could be rendered anergic by previous exposure to UV-B-irradiated Sa cells. The alloreactive CTL previously stimulated with UV-B-irradiated Sa cells failed to proliferate in response to nontreated Sa cells. Proliferation of the anergic CTL could not be restored by Sa cells and exogenous IL-2 but by the combination of phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (A23187). The anergic CTL showed a considerably low cytotoxic activity against Sa target cells. The expression of TCR on the anergic CTL was downregulated while expression of high-affinity IL-2R was upregulated. Their CD28 and CD8 expression were unchanged. In addition, the proliferative response and cytotoxicity of the anergic CTL to Sa cells could be restored after the cells had been rested for 7 days to allow reexpression of TCR. These results suggest that downregulation of T-cell receptor (TCR) and impairment in the post-IL-2/IL-2R signaling pathway are relevant to the clonal anergy induced in the alloreactive CTL by stimulation of UV-B-irradiated Sa cells.

Ultraviolet irradiation inhibits killer-target cell interaction.

The effects of ultraviolet (UV) irradiation on cell-mediated cytolysis were examined in order to clarify the inhibitory mechanisms of allosensitization by UV irradiation. UV-B-irradiated target cells (Sa; an Epstein-Barr virus-transformed B cell line) exhibited more resistance against alloreactive cytotoxic T lymphocytes (CTL) than mitomycin C (MMC)-treated target cells. In the conjugate formation assay, UV-B-irradiated target cells showed a considerably lower binding to alloreactive CTL than MMC-treated target cells. UV-B irradiation induced a reduction of HLA-class I, -DR, CD54 (ICAM-1) and CD58 (LFA-3) expression on target cells. However, it does not seem to contribute to the inhibition of cell adhesion induced by UV-B irradiation because a similar reduction of cell surface antigens was observed in MMC-treated target cells. Number of cells capped with anti-HLA-class I, -DR, CD54 or CD58 monoclonal antibody were markedly reduced by UV-B irradiation compared to that by MMC treatment. These findings suggest the possibility that the inhibition of cell adhesion between UV-B-irradiated Sa target cells and alloreactive CTL is due to the impaired mobility of cell surface antigens which will affect the early process of cell-mediated cytolysis.

Hemoglobin-binding site on human haptoglobin. Identification of lysyl residues participating in the binding.

The locus of human haptoglobin (Hp) molecules that interacts with human hemoglobin (Hb) was examined by the differential labeling technique. First, amino groups of Hp were extensively labeled with ethyl acetimidate (EAI) either in the free state (experiment A) or in the equimolar complex with Hb (experiment B). Each of the labeled Hp samples was reduced and S-carboxymethylated, and the remaining amino groups were then allowed to react with trinitrobenzenesulfonic acid (TNBS) under denatured conditions. Only 0.6 mol/mol of the trinitrophenyl (TNP) group was introduced into the heavy chain of Hp in experiment A, whereas 2.5 mol/mol of TNP groups were into the same chain in experiment B. The extent of TNP modification for the light chain was very low in either of the experiments. The amino acid residues carrying TNP groups in the heavy chain were identified by the peptide mapping procedure. They were Ile1, Lys136, and Lys218 in experiment B, and only Ile1 in experiment A. These findings suggest that epsilon-amino groups of Lys136 and Lys218 in the heavy chain are both situated within the Hb-binding locus of Hp, and that the alpha-amino group of Ile1 is buried inside the molecule either in the presence or absence of Hb.

Amino acid sequence and disulfide-bridge location of canine haptoglobin.

The complete amino acid sequence and disulfide-bridge location of canine haptoglobin (Hp) were determined by analyzing various fragments produced chemically and/or enzymatically. Canine Hp consists of two light (L) and two heavy (H) chains with 83 and 245 amino acid residues, respectively. It has three potential oligosaccharide-binding sequences, Asn-X-Ser/Thr, one in the L chain and two in the H chain. All of them are glycosylated. Comparison of the amino acid sequences between canine Hp and human Hp shows 68 and 85% homology for L chains and H chains, respectively. About 20% of the canine L chain still retains a carboxyl-terminal arginine residue, which is completely removed during maturation in human L-chain. The half-cystine residue at position 15 in the L chain, which participates in the inter-L chain disulfide bridging in human Hp, has been replaced by a leucine residue in canine Hp. Therefore, an LH unit in canine Hp may be joined to another LH unit by a noncovalent (mainly hydrophobic) interaction to form the complete molecule. The disulfide bridges in canine Hp link Cys-34L to Cys-68L, Cys-72L to Cys-105H, Cys-148H to Cys-179H, and Cys-190H to Cys-220H, as in the case of human Hp.