SCCmec typing of PVL-positive community-acquired Staphylococcus aureus (CA-MRSA) at a Japanese hospital.

The epidemiology of Panton-Valentine leukocidin (PVL)-positive MRSA in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was examined. Three hundred and forty-two CA-MRSA strains that were susceptible to imipenem and cefazolin were isolated from 1107 samples (intravenous catheter, blood, sputum, urine, skin, wound, and pharynx) from outpatients at Showa University Hospital in Japan between September 2009 and March 2017. The PVL gene was detected in 46 of 342 CA-MRSA strains, accounting for 13.5%. The type of SCCmec was determined by detection of each SCCmec-specific region, class complex, and ccr. SCCmec type IV comprised 33 strains, type V comprised 5 strains, type VII comprised 4 strains, and the unclassified type comprised 4 strains. Among the type IV strains, subtype IVa was dominant, comprising 23 of 33 strains, and the remaining 10 strains were of varying subtypes. The SCCmec type III-specific region, CZ049, was amplified in 2 type V strains, 4 type VII strains, and 4 unclassified strains. In 4 unclassified strains, CZ049 and ccr5 were detected, but neither the SCCmec-specific region nor class complex was detected. The PVL-positive rate was lower than that in Western countries. The SCCmec types of PVL-positive CA-MRSA strains were found to vary, indicating a diverse spreading route.

Analysis of OXA23 and AmpCß-Lactamase Genes in Clinically Isolated Multidrug-Resistant Acinetobacter baumannii Clonal Complex 92.

Multidrug-resistant Acinetobacter baumannii, which is resistant to carbapenems, amino glycosides, and fluoroquinolones, was isolated from the wound of an outpatient. We performed Multi Locus Sequence Typing and analyzed the structures of the AmpC ß-lactamase and OXA23 carbapenemase genes. This strain was assigned as ST451, a member of clonal complex 92, by its MLST scheme. The structures of ISAba1-AmpC ß-lactamase and ISAba1-OXA23 carbapenemase were revealed and the major globally prevalent clone of carbapenem-resistant A. baumannii was identified.

Detection of metallo-ß-lactamase genes in clinically isolated Klebsiella pneumoniae and Klebsiella oxytoca.

We detected and characterized metallo-ß-lactamase genes (blaIMP-1 and blaIMP-11) in third generation cephalosporin-resistant Klebsiella pneumoniae and Klebsiella oxytoca isolated at Showa University Hospital between January 1, 2011 and December 31, 2012. The cephalosporin-resistant K pneumoniae strains were frequently isolated from the urine, while one strain of K. pneumoniae, which was resistant to carbapenem, was isolated from the stool. We analyzed the phenotypes and genotypes of the metallo-ß-lactamase genes from the 16 strains of cephalosporin-resistant-K pneumoniae and 6 strains of -K. oxytoca isolated from the same ward. The minimum inhibitory concentrations of imipenem were below 4 µg/ml in 21 out of the 22 isolated strains. The double disc synergy test using ceftazidime and sodium mercaptoacetic acid revealed enlargements in the inhibitory zones of 14 of the 16 strains of K. pneumoniae and all 6 strains of K. oxytoca. Metallo-ß-lactamase genes were detected in all of the tested strains, with blaIMP-1 in 3 K. pneumoniae and 1 K. oxytoca, blaIMP-11 in 13 K pneumoniae and 4 K. oxytoca, and both blaIMP-1 and blaIMP-11 in one K. oxytoca. Our results indicate that third generation cephalosporin-resistant and imipenem-susceptible K. pneumoniae and K. oxytoca possess the metallo-ß-lactamase gene. The active surveillance of metallo-ß-lactamase genes should be performed in clinical laboratories. (Original).

MLST analysis of multiple antimicrobial resistant Acinetobacter baumannii.

Multi-locus sequencing typing (MLST) of Acinetobacter baumannii, isolated at Showa University Hospital, was performed between November 2010 and March 2011. A. baumannii was isolated from 15 patients. Among the 15 isolates, the STs of three isolates were able to be determined, ST76, ST92, and ST146, and belonged to Clonal Complex (CC) 92, the global epidemic clone among carbapenem resistant A. baumannii. The other 12 strains were not applicable to the MLST classification. The ST76 strain was resistant to carbapenems, aminoglycosides, and fluoroquinolones. The ST92 strain was resistant to aminoglycosides and fluoroquinolones. The ST146 strain was resistant to fluoroquinolones. The other 12 strains were susceptible to either of the drugs. Neither the metallo beta lactamase gene (IMP type or VIM2) nor the OXA23 gene was detected in carbapenem resistant A. baumannii. These results indicate that A. baumannii of CC92 has spread as the drug resistant strain in Japan. Monitoring A. baumannii using molecular epidemiology is necessary.

[Analysis of the antibiotic resistant gene in multidrug-resistant Enterobacter cloacae isolated at Showa University Hospital].

We analyzed the antibiotic resistant genes, metallo-beta-lactamase and extended-spectrum beta-lactamase (ESBL), in highly resistant Enterobacter cloacae isolated at Showa University Hospital from April to June 2008. Thirty eight strains of E. cloacae were isolated. Of these strains, 35 demonstrating resistant to either cefotaxime (Minimum inhibitory concentration: MIC > or = 32 microg/ml), ceftadizime (MIC > or = 16 microg/ml), or aztreonam (MIC > or = 16 microg/ml) were selected for the analysis. The strains were subjected to a Double-disk synergy test (DDST) using sodium mercaptoacetic acid (SMA) and ceftadizime. As a result, 7 strains showed an enlargement of inhibition zone diameter by SMA. Among 7 SMA responded strains, 6 strains carried the bla(IMP-1) or bla(IMP-11) gene. There were 2 strains sensitive to imipenem (MIC < 1 microg/ml) in SMA responded strains, and one carried bla(IMP-11) gene. ESBL genes were detected in 5 strains, bla(CTX-M3) gene from 3 strains, and bla(CTX-M14) gene from 2 strains. All of the ESBL harboring strains showed an enlargement of inhibition zone diameter in DDST using clavulanic acid and ceftadizime, cefotaxime, aztreonam or cefepime. Among 35 strains, one strain carried both bla(IMP-1) and bla(CTX-M3) genes. In addition to the AmpC beta-lactamase, increase of the E. cloacae strains carrying these resistant genes cause the complex antibiotic resistant patterns. Especially, strains carrying bla(IMP) gene, which exhibit low imipenem MIC value may be induced to be resistant strains under the existence of imipenem. Analysis of resistant genes would be a beneficial for the measure the spread of drug-resistant E. cloacae.

[Analysis of the relevance of the mutations of the precore and basic core promoter in HBV genome with the DNA amount of HBV viremia].

In the course of hepatitis B, serum concentrations of the virus usually fall following sero-conversion, characterized by the loss of HBe antigen and appearance of detectable anti-HBe. However, hepatitis B viremia may persist even after sero-conversion. We assessed the association of continuous viremia with the precore (PC) (G1896A) mutation, basic core promoter (BCP) (A1762T, G1764A) mutations, the viral genotype and the quantity of viral DNA. Neither PC nor BCP mutations were detected in the viral DNA isolated from cases in which HBV became negative during the course of infection. The virus quantity increased after sero-conversion in all of the cases of persistent viremia with HBV genotype C harboring BCP mutations, indicating that the BCP mutation in genotype C is a determinant of continuous viremia. This feature was not evident in infections with HBV genotype B. Although the PC mutation was detected in the viral DNA from both genotypes B and C in continuous viremia, the mutation was not relevant to the quantity of virus. Our data suggest that HBV genotype C has a predisposition to acquire BCP mutations and these mutations activate virus replication after sero-conversion. This mechanism may be a cause of the poor prognosis of hepatitis with HBV genotype C. In conclusion, analyses of HBV genotype and BCP mutations are imperative to determine the prognosis of hepatitis B.

[Evaluation of the anti-TP antibody latex agglutination immunoassay in routine testing and a clinical viewpoint].

We evaluated TP-LAIA (anti-TP latex agglutination immunoassay) and compared the results with those obtained using Serological Tests for Syphilis (STS), namely Venereal Disease Research Laboratory (VDRL) method and Rapid Plasma Reagin (RPR) test. We also examined early-stage antibody reaction using rabbits infected with active pathogen, and analyzed early-stage syphilis patient serum with IgM class anti-TP antibody. Based on routine test results and case history reviews for possible syphilis infection, TP-LAIA showed high specificity, 0.64% false positive results in comparison with 13.5% by VDRL method. Sensitivity was also significantly higher than TP-Hemagglutination Assay(TPHA). In the examination of TP early-stage infection, the fastest positive antibody reaction was observed with TP-LAIA, indicating its significance in the diagnosis of early-stage syphilis. TP-LAIA was confirmed to give a reliable reaction with IgM class anti-TP antibody. TP-LAIA results coincided with VDRL method results in the decrease in anti-treponemal antibody titers following medical treatment, suggesting that TP-LAIA will be a valuable tool for monitoring the effect of medical treatments. We concluded that not only the high sensitivity and specificity of TP-LAIA assay and its suitability for automation make it an ideal screening test, but also the assay performs sufficiently and satisfactorily for its use in monitoring medical treatments.

[Isolation of beta-lactamase non-producing ampicillin-resistant Haemophilus influenzae (BLNAR) and analysis of its PBP3 gene].

We analyzed the incidence of beta-lactamase non-producing ampicillin-resistance (BLNAR) Haemophilus influenzae in Showa University Hospital from June 2004 to March 2005. The ratio of BLNAR in total H. influenzae isolates was 44.5%. BLNAR accounted for 89.6% of the ampicillin non-susceptible strains displaying a minimal inhibitory concentration(MIC) of ampicillin of more than 2 microg/ml, which is the break point advocated by the Clinical and Laboratory Standards Institute. Of the total BLNARs, 71.4% were isolated from children of six and under. We analyzed mutations of the SSN and KTG motif of penicillin binding protein 3 encoded by the ftsI gene, since mutations in these regions are considered to be responsible for the drug-resistance of BLNAR. Mutations in SSN and KTG motifs were identified in all BLNAR isolates. We also detected mutations in the ampicillin susceptible strains displaying an ampicillin MIC of 0.5 or microg/ml. When a noticeable MIC increase of ABPC or other beta-lactams in the routine clinical laboratory practice, gene analysis of ftsI gene of the isolates displaying increased MIC is required to measure the spreading of BLNAR.

[Analysis of the macrolide resistance gene in penicillin-resistant Streptococcus pneumoniae].

We examined the penicillin and macrolide resistance of 496 strains of Streptococcus pneumoniae (S. pneumoniae) isolated at Showa University Hospital from November 2004 to May 2005. According to the classification established by the Clinical and Laboratory Standards Institute, the ratio of penicillin susceptible S. pneumoniae (PSSP) was 25.8%, penicillin intermediate S. pneumoniae was 35.9% and penicillin resistant S. pneumoniae (PRSP) was 38.3%. The ratios of macrolide resistant S. pneumoniae were 85.3% for erythromycin and 76.2% for clarithromycin. S. pneumoniae strains were isolated mainly from pediatric patients, and the ratios of PRSP were similar between outpatients (39.8%) and inpatients (45.6%). We screened for mutations in pbp1a, pbp2b and pbp2x, and the retention rate of the macrolide resistance genes, ermB and mefA in 90 strains isolated in the same period. Seventy two strains had at least one mutation in the pbp genes. Interestingly, some of the penicillin susceptible strains had one or two pbp mutations, suggesting a progressive genetic acquirement of penicillin resistance. In screening for retention of the macrolide resistance genes, we found that 42 strains(46.7%) had ermB, 19 strains (21.1%) had mefA and 13 strains (14.4%) had both ermB and mefA. The possession of resistance genes and the minimal inhibitory concentration indicate that the resistance to erythromycin and clarithromycin were induced by ermB or mefA, and the resistance to clindamycin was induced only by ermB. Among the 72 strains with pbps mutations, 65 strains (90.3%) had ermB or mefA or both. Together, these results show that the strains with pbp mutations were being selected and, after acquiring the macrolide resistance gene, transform to multidrug resistant S. pneumoniae.

[The relation between virus genotypes and coexisting surface antigen and antibody in patients with hepatitis B, and the development of preS region deletions].

In recent years, there has been renewed interest in hepatitis B virus (HBV) genotypes. Our previous data have shown the importance of in-frame deletions in the preS region in cases of coexisting hepatitis B surface antigen (HBsAg) and anti-hepatitis B surface antibody (HBsAb). The aim of the present study was to investigate the relation between HBV genotypes and coexisting HBsAg and HBsAb, preS deletion mutants. We investigated the HBV genotypes in 9 patients with coexisting HBsAg and HBsAb. Viral DNA was extracted from the patients' sera and the HBV S gene region was amplified by polymerase chain reaction (PCR). HBV genotypes were then investigated by restriction fragment length polymorphism(RFLP) analysis. All 9 cases were found to have genotype C. This result clearly indicates that the unique finding of coexisting HBsAg and HBsAb depends on the HBV genotype. After genotypic screening was performed for HBV-positive samples from randomly selected 60 cases. The results of the 60 cases we investigated showed 26 cases of genotype B (43.3%), 31 cases of genotype C (51.7%), 1 case of coexisting genotype B and C (1.7%), and 2 cases of other genotypes (3.3%). Of the 60 cases, 45 cases consisting of 21 with genotype B and 24 with genotype C were subject to direct DNA sequencing of PCR products in the preS region to determine the presence or absence of preS deletion mutants. PreS deletion mutants were found in a total of 7 of the 45 HBV cases that underwent sequencing(7/45; 15.6%), and 6 of these had genotype C (6/24 cases, 25.0%), whereas only 1 had genotype B (1/21 cases, 4.8%). These results demonstrate a greater frequency of preS deletion mutants with genotype C. Interestingly, many preS deletion mutants showed deletions at the same point, namely the amino terminal side of the preS2 region. These results indicate that the HBV genotype is involved in the molecular pathogenesis of hepatitis B.

[Detection of novel nucleotide substitutions resulting amino acid substitutions in TEM type beta-lactamase gene isolated from a clinically isolated extended spectrum beta-lactamases (ESBLs) productive Escherichia coli].

We screened Escherichia coli (E. coli) from specimens submitted to the Showa University hospital clinical laboratory that showed an advanced resistance to third generation cephems (Cefotaxime or Ceftazidime), as candidate extended spectrum beta-lactamases(ESBLs) producing strains. Among the candidates, two strains showed the characteristics of class A ESBLs in their susceptibility to Cefmetazole, a second generation cephem, and their resistance was inhibited by the addition of clavulanic acid. Further, we detected the TEM-type gene in one of the two E. coli strains. By determining the nucleotide sequence of the whole coding region of the TEM gene, two nucleotide substitutions with amino acid substitutions 82Val-->Ile, and 182Ala-->Val were identified.

[Arbekacin resistant gene, aacA/aphD, in Staphylococcus aureus is lost during in vitro passage].

We analyzed for the presence of the aacA/aphD gene in arbekacin resistant-Staphylococcus aureus. Tested strains were 50 clinically isolated Staphylococcus aureus that had an MIC of more than 2 micrograms/ml for arbekacin. Among the primary cultures, aacA/aphD genes were detected in 42 of 50 strains(84%) overall, including 34 of 41 Methicillin resistant Staphylococcus aureus(MRSA) strains, and 8 of 9 strains of Methicillin sensitive Staphylococcus aureus(MSSA). After 10 or 20 passages, the aacA/aphD gene was lost in 5 strains of MRSA and 2 strains of MSSA. In the strains that lost the gene, the MIC of arbekacin was reduced remarkably in 3 strains of MRSA and 2 strains of MSSA. Further, MICs of arbekacin decreased along with passages even in the strains that did not loose the aacA/aphD gene, as well as in the strains that did not possess the aacA/aphD gene originally. The fact that MIC values are easily reduced with in vitro passage is important to consider when performing the arbekacin-resistance test for Staphylococcus aureus.