Hidehito Matsui

Development of an Immunochromatographic Strip for Simple Detection of Penicillin-Binding Protein 2′

ABSTRACT Infections with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MR-CNS) are a serious problem in hospitals because these bacteria produce penicillin-binding protein 2′ (PBP2′ or PBP2a), which shows low affinity to β-lactam antibiotics. Furthermore, the bacteria show resistance to a variety of antibiotics. Identification of these pathogens has been carried out mainly by the oxacillin susceptibility test, which takes several days to produce a reliable result. We developed a simple immunochromatographic test that enabled the detection of PBP2′ within about 20 min. Anti-PBP2′ monoclonal antibodies were produced by a hybridoma of recombinant PBP2′ (rPBP2′)-immunized mouse spleen cells and myeloma cells. The monoclonal antibodies reacted only with PBP2′ of whole-cell extracts and showed no detectable cross-reactivity with extracts from other bacterial species tested so far. One of the monoclonal antibodies was conjugated with gold colloid particles, which react with PBP2′, and another antibody was immobilized on a nitrocellulose membrane, which captures the PBP2′-gold colloid particle complex on a nitrocellulose strip. This strip was able to detect 1.0 ng of rPBP2′ or 2.8 × 105 to 1.7 × 107 CFU of MRSA cells. The cross-reactivity test using 15 bacterial species and a Candida albicans strain showed no detectable false-positive results. The accuracy of this method in the detection of MRSA and MR-CNS appeared to be 100%, compared with the results obtained by PCR amplification of the PBP2′ gene, mecA. This newly developed immunochromatographic test can be used for simple and accurate detection of PBP2′-producing cells in clinical laboratories.

Rapid Depletion of Free Vancomycin in Medium in the Presence of β-Lactam Antibiotics and Growth Restoration in Staphylococcus aureus Strains with β-Lactam-Induced Vancomycin Resistance

ABSTRACT A class of methicillin-resistant Staphylococcus aureus strains shows vancomycin resistance in the presence of β-lactam antibiotics (β-lactam-induced VAN-resistant methicillin-resistant S. aureus [BIVR]). Two possible explanations may be offered: (i) vancomycin in culture medium is depleted, and (ii) the d-Ala-d-Ala terminal of the peptidoglycan network is replaced with d-Ala-d-lactate. We tested these hypotheses by quantifying free vancomycin in the medium through the course of cell growth and by PCR amplification of the van genes. Growth of the BIVR cells to an absorption level of ∼0.3 at 578 nm required about 24 h in the presence of vancomycin alone at the MIC (4.0 μg/ml). However, growth was achieved in only about 10 h when 1/1,000 to 1/2,000 the MIC of β-lactam antibiotic was added 2 h prior to the addition of vancomycin, suggesting that the β-lactams shortened the time to recovery from vancomycin-mediated growth inhibition. Free vancomycin in the culture medium decreased to 2.3 μg/ml in the first 8 h in the culture containing vancomycin alone, yet cell growth was undetectable. When the vancomycin concentration dropped below ∼1.5 μg/ml at 24 h, the cells began to grow. In the culture supplemented with the β-lactam 2 h prior to the addition of vancomycin, the drug concentration continuously dropped from 4 to 0.5 μg/ml in the first 8 h, and the cells began to grow at a vancomycin concentration of ∼1.7 μg/ml or at 4 h of incubation. The gene encoding the enzyme involved in d-Ala-d-lactate synthesis was undetectable. Based on these results, we concluded that BIVR is attributable mainly to a rapid depletion of vancomycin in the medium triggered or promoted by β-lactam antibiotics.

Identification of the active component that induces vancomycin resistance in MRSA

A fraction of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin (VCM) in the presence of β-lactam antibiotics (BIVR) at low concentrations. We hypothesized that the BIVR phenomenon might be exerted by a peptidoglycan derivative(s) generated as a consequence of β-lactam antibiotic action. To verify this hypothesis, we isolated the fraction that mimicked the effect of β-lactam antibiotics by the enzymatic treatment of the crude cell wall. The active components were purified by a combination of reverse phase chromatographies, mass spectrum and amino-acid analyses, and were identified to be a muropeptide with the following formula: N-acetyglucosamyl-N-acetylmuramyl--Ala-D-isoGln-L-Lys-(ɛ-NH-4Gly)-D-Ala-2Gly. This is the very first identification of the active component, which induces VCM resistance in MRSA. We found that the BIVR cells are highly sensitive to this compound rendering the cells resistant to VCM compared with non-BIVR MRSA.

Aranorosin circumvents arbekacin-resistance in MRSA by inhibiting the bifunctional enzyme AAC(6')/APH(2″).

Aranorosin circumvents arbekacin-resistance in MRSA by inhibiting the bifunctional enzyme AAC(6′)/APH(2″)

Rapid Detection of Vaginal Candida Species by Newly Developed Immunochromatography

ABSTRACT For the diagnosis of vulvovaginal candidiasis, we developed a simple immunochromatographic method that enables the detection of vaginal Candida spp. within about 30 min. Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of this method appeared to be 80.3, 99.3, 98.0, and 92.0%, respectively.

Antimalarial C-9 oxime derivatives from desmycosin, produced by click chemistry

Plasmodium falciparum parasites remain the major cause of malaria, a serious and potentially fatal infectious disease worldwide, despite the intermittent introduction of several different successive classes of potent and effective drugs. According to the World Health Organization’s Malaria Report (2011), an estimated 3.3 billion people were still at risk of malaria in 2010.1 Worldwide, in 2010, 99 countries and territories reported ongoing malaria transmission and a further 7 countries were trying to prevent the reintroduction of the disease, comprising a total of 106 countries in which malaria was considered endemic.1 The prevention and successful treatment of malaria is heavily dependent on antimalarial drugs. Chloroquine proved to be a remarkable safer, cheap and extremely effective antimalarial drug, serving as the frontline antimalarial treatment from its introduction in the mid-1940s to the 1990s.2 However, the malaria parasite has continually proved to be particularly efficient at developing resistance to virtually all drugs used to control it. Chloroquine-resistant parasites quickly emerged and spread worldwide, rendering the drug useless in many locations. The same scenario is being witnessed with the latest in the long line of potent antimalarials, artemisinin. Therefore, there is a continuing need for new antimalarial drugs that are effective, safe, affordable and easy to use and which, ideally, have a novel mode of action. In our institute, we have focused on the screening and synthesis of antimalarial agents from microbial metabolites including antibacterial macrolides.3–5 In 2007, azithromycin, a 15-membered antibacterial macrolide, was found to possess potent antimalarial activity by Fidock and co-workers.6 Azithromycin is a slow-acting antimalarial that targets the parasite apicoplast and its prokaryotic ribosomes. The effects of its action increase with prolonged incubation time (from one to two generations), giving an IC50 in the nano-molar range. Continuously, new derivatives of azithromycin have been developed with the aim to improve activity and selectivity for the malaria parasite.7 Another macrolide antibiotic, tylosin, was first isolated by McGuire and co-workers8 in 1961, and is a 16-membered macrolide isolated from a culture broth of Streptomyces fradiae. In 1984, McColm and McHardy9 reported that tylosin demonstrated antimalarial activity against P. falciparum strain Liverpool (IC501⁄4 0.1mg ml 1). However, there have been no subsequent reports concerning the antimalarial activity of tylosin or its derivatives. In this short note, we report that novel C-9 oximes of desmycosin express antimalarial activity against chloroquine-resistant P. falciparum K110 malaria parasites. The removal of mycarose from tylosin can be accomplished under mild acidic hydrolysis to give desmycosin, a known minor component in the fermentation broth of the tylosin-producing strain.11 The antimicrobial activity of desmycosin is potent but slightly lower than that of tylosin, but it has almost no efficacy in vivo when given orally.11 Likewise, C-9 oxime derivatives of tylosin and desmycosin have been evaluated in bioassays, but did not show better antimicrobial activity than tylosin.12 Our work on tylosin analog synthesis produced C-9 oximes of desmycosin (3, 6 and 7), prepared from dimethoxydesmycosin (1)13 by oxime formation with corresponding hydroxylamines HCl14 and acid hydrolysis of dimethoxyacetal moiety in good yields, respectively. These analogs showed antimicrobial activities, similar to or slightly lower than that of desmycosin, (Scheme 1, Table 1). We specifically tested for antimalarial activity in all new analogs derived from tylosin. Moreover, as the discovery of the antimicrobial alkyne-bearing lead compounds (6 and 7) for antimicrobial activities, our efforts have focused on the preparation of new analogs in this series utilizing ‘click chemistry’, which provides an important approach for simple and rapid evaluation of functional activity. The concept of ‘click chemistry’ was originally introduced by Kolb, Finn and Sharpless in 2001.15 It incorporates powerful and selective reactions for efficient synthesis of interesting compounds and combinatorial libraries through heteroatom links. The advantages of ‘click chemistry’ in biological studies of macrolides have recently been clearly demonstrated in our laboratory.16,17 The alkyne-bearing oximes (6 and 7) are intermediates allowing generation of targeted triazole compounds. Anti-selective triazole formation was carried out with a catalytic amount of Cu(MeCN)4PF6

In silico analysis of AHJD-like viruses, Staphylococcus aureus phages S24-1 and S13′, and study of phage S24-1 adsorption

Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor‐binding proteins and host receptor molecules. However, little information is available on the receptor‐binding protein of S. aureus phages. S. aureus virulent phages S24‐1 and S13′ (family Podoviridae, genus AHJD‐like viruses) were isolated from sewage. In the present study, we investigated the receptor‐binding protein of AHJD‐like viruses using phage S24‐1. First, based on a comparative genomic analysis of phages S24‐1 and S13′, open reading frame 16 (ORF16) of phage S24‐1 was speculated to be the receptor‐binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor‐binding protein of phage S24‐1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24‐1. Finally, the C‐terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24‐1 and its homologs may be the receptor‐binding proteins of AHJD‐like viruses.

Low level β-lactamase production in methicillin-resistant staphylococcus aureus strains with β-lactam antibiotics-induced vancomycin resistance

BackgroundA class of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin only in the presence of β-lactam antibiotics (BIVR). This type of vancomycin resistance is mainly attributable to the rapid depletion of free vancomycin in the presence of β-lactam antibiotics. This means that β-lactam antibiotics remain active or intact in BIVR culture, although most MRSA cells are assumed to produce β-lactamase. We hypothesised that the BIVR cells either did not harbour the β-lactamase gene, blaZ, or the gene was quiescent. We tested this hypothesis by determining β-lactamase activity and conducting PCR amplification of blaZ.ResultsFive randomly selected laboratory stock BIVR strains showed an undetectable level of β-lactamase activity and were blaZ-negative. Five non-BIVR stock strains showed an average β-lactamase activity of 2.59 ± 0.35 U. To test freshly isolated MRSA, 353 clinical isolates were collected from 11 regionally distant hospitals. Among 25 BIVR strains, only 16% and 8% were blaZ positive and β-lactamase-positive, respectively. In contrast, 95% and 61% of 328 non-BIVR strains had the blaZ gene and produced active β-lactamase, respectively. To know the mechanism of low β-lactamase activity in the BIVR cells, they were transformed with the plasmid carrying the blaZ gene. The transformants still showed a low level of β-lactamase activity that was several orders of magnitude lower than that of blaZ-positive non-BIVR cells. Presence of the β-lactamase gene in the transformants was tested by PCR amplification of blaZ using 11 pairs of primers covering the entire blaZ sequence. Yield of the PCR products was consistently low compared with that using blaZ-positive non-BIVR cells. Nucleotide sequencing of blaZ in one of the BIVR transformants revealed 10 amino acid substitutions. Thus, it is likely that the β-lactamase gene was modified in the BIVR cells to downregulate active β-lactamase production.ConclusionsWe concluded that BIVR cells gain vancomycin resistance by the elimination or inactivation of β-lactamase production, thereby preserving β-lactam antibiotics in milieu, stimulating peptidoglycan metabolism, and depleting free vancomycin to a level below the minimum inhibitory concentration of vancomycin.

Rapid and easy detection of low-level resistance to vancomycin in methicillin-resistant Staphylococcus aureus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Vancomycin-intermediately resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are associated with treatment failure. hVISA contains only a subpopulation of cells with increased minimal inhibitory concentrations, and its detection is problematic because it is classified as vancomycin-susceptible by standard susceptibility testing and the gold-standard method for its detection is impractical in clinical microbiology laboratories. Recently, a research group developed a machine-learning classifier to distinguish VISA and hVISA from vancomycin-susceptible S. aureus (VSSA) according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data. Nonetheless, the sensitivity of hVISA classification was found to be 76%, and the program was not completely automated with a graphical user interface. Here, we developed a more accurate machine-learning classifier for discrimination of hVISA from VSSA and VISA among MRSA isolates in Japanese hospitals by means of MALDI-TOF MS data. The classifier showed 99% sensitivity of hVISA classification. Furthermore, we clarified the procedures for preparing samples and obtaining MALDI-TOF MS data and developed all-in-one software, hVISA Classifier, with a graphical user interface that automates the classification and is easy for medical workers to use; it is publicly available at https://github.com/bioprojects/hVISAclassifier. This system is useful and practical for screening MRSA isolates for the hVISA phenotype in clinical microbiology laboratories and thus should improve treatment of MRSA infections.

Paraphaeosphaeride D and berkleasmin F, new circumventors of arbekacin resistance in MRSA, produced by Paraphaeosphaeria sp. TR-022

Two new compounds, designated paraphaeosphaeride D (1) and berkleasmin F (2) together with a previously known compound, berkleasmin A (3), isolated from a culture broth of the fungus Paraphaeosphaeria sp. TR-022, proved to be new circumventors of arbekacin (ABK) resistance in methicillin-resistant Staphylococcus aureus (MRSA). The structures of 1 and 2 were elucidated by spectroscopic analyses, including various NMR experiments. All compounds showed 10–100 times ABK circumvention activities using the paper disc method and reduced the MIC values of ABK against MRSA from 16 μg ml−1 to 4 μg ml−1 (fourfold) using the agar dilution method. These new compounds might be promising lead compounds for developing circumventors of ABK resistance in MRSA.