Hideaki Hanaki

Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin

BACKGROUND Since the discovery of the vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (minimum inhibitory concentration [MIC] 8 mg/L), there has been concern about the potential spread of such strains throughout Japanese hospitals. Two important questions need to be answered: (1) what is the prevalence of VRSA, and (2) by what mechanism does vancomycin resistance occur. METHODS The vancomycin susceptibilities of three methicillin-resistant S aureus (MRSA) strains (Mu50, Mu3, and H1) and the methicillin-susceptible S aureus type strain FDA209P were compared by MIC determinations and population analysis. Mu3 (MIC 3 mg/L) was isolated from the sputum of a patient with pneumonia after surgery who had failed vancomycin therapy. H1 (MIC 2 mg/L), which is a representative vancomycin-susceptible MRSA strain, was isolated from a patient with pneumonia who responded favourably to vancomycin therapy. Subclones of Mu3 with increased resistance against vancomycin were selected with serial concentrations of vancomycin and their MICs were determined. The prevalence of VRSA and Mu3-like strains in Japanese hospitals was estimated by population analysis from 1149 clinical MRSA isolates obtained from 203 hospitals throughout Japan. The genetic traits of the Mu3 and Mu50 strains were compared with clonotypes of MRSA from around the world. FINDINGS Mu3 and Mu50 had an identical pulsed-field gel electrophoresis banding pattern. When grown in a drug-free medium, Mu3 produced subpopulation of cells with varying degrees of vancomycin resistance, thus demonstrating natural heterogeneity, or variability, in susceptibility to vancomycin. In the presence of vancomycin, Mu3 produced subclones with resistance roughly proportional to the concentrations of vancomycin used. Selection of Mu3 with 8 mg/L or more of vancomycin gave rise to subclones with vancomycin resistance equal to that of Mu50 (MIC 8 mg/L) at a frequency of 1/1,000,000. During screening of Japanese MRSA strains, no strain of VRSA additional to Mu50 was found. The prevalence of MRSA isolates heterogeneously resistant to vancomycin was 20% in Juntendo University Hospital, 9.3% in the other seven university hospitals, and 1.3% in non-university hospitals or clinics. INTERPRETATION Heterogeneously resistant VRSA is a preliminary stage that allows development into VRSA upon exposure to vancomycin. Heterogeneously resistant VRSA was found in hospitals throughout Japan. This finding could explain, at least partly, the frequent therapeutic failure of MRSA infection with vancomycin in Japan.

Contribution of a Thickened Cell Wall and Its Glutamine Nonamidated Component to the Vancomycin Resistance Expressed by Staphylococcus aureus Mu50

ABSTRACT Staphylococcus aureus Mu50, which has reduced susceptibility to vancomycin, has a remarkably thickened cell wall with an increased proportion of glutamine nonamidated muropeptides. In addition, Mu50 had enhanced glutamine synthetase andl-glutamine d-fructose-6-phosphate aminotransferase activities, which are involved in the cell-wall peptidoglycan synthesis pathway. Furthermore, significantly increased levels of incorporation of 14C-labeledd-glucose into the cell wall was observed in Mu50. Unlike afemC mutant S. aureus strain, increased levels of production of nonamidated muropeptides in Mu50 was not caused by lower levels of glutamine synthetase activity but was considered to be due to the glutamine depletion caused by increased glucose utilization by the cell to biosynthesize increased amounts of peptidoglycan. After the cells were allowed to synthesize cell wall in the absence or presence of glucose and glutamine, cells with different cell-wall thicknesses and with cell walls with different levels of cross-linking were prepared, and susceptibility testing of these cells demonstrated a strong correlation between the cell-wall thickness and the degree of vancomycin resistance. Affinity trapping of vancomycin molecules by the cell wall and clogging of the outer layers of peptidoglycan by bound vancomycin molecules were considered to be the mechanism of vancomycin resistance of Mu50. The reduced cross-linking and the increased affinity of binding to vancomycin of the Mu50 cell wall presumably caused by the increased proportion of nonamidated muropeptides may also contribute to the resistance to some extent.

Nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy in 2007: general view of the pathogens' antibacterial susceptibility.

For the purpose of nationwide surveillance of the antimicrobial susceptibility of bacterial respiratory pathogens collected from patients in Japan, the Japanese Society of Chemotherapy conducted a third year of nationwide surveillance during the period from January to April 2008. A total of 1,097 strains were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections. Susceptibility testing was evaluable with 987 strains (189 Staphylococcus aureus, 211 Streptococcus pneumoniae, 6 Streptococcus pyogenes, 187 Haemophilus influenzae, 106 Moraxella catarrhalis, 126 Klebsiella pneumoniae, and 162 Pseudomonas aeruginosa). A total of 44 antibacterial agents, including 26 β-lactams (four penicillins, three penicillins in combination with β-lactamase inhibitors, four oral cephems, eight parenteral cephems, one monobactam, five carbapenems, and one penem), three aminoglycosides, four macrolides (including a ketolide), one lincosamide, one tetracycline, two glycopeptides, six fluoroquinolones, and one oxazolidinone were used for the study. Analysis was conducted at the central reference laboratory according to the method recommended by the Clinical and Laboratory Standard Institute (CLSI). The incidence of methicillin-resistant S. aureus (MRSA) was as high as 59.8%, and those of penicillin-intermediate and penicillin-resistant S. pneumoniae (PISP and PRSP) were 35.5 and 11.8%, respectively. Among H. influenzae, 13.9% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately resistant (BLNAI), 26.7% to be β-lactamase-non-producing ABPC-resistant (BLNAR), and 5.3% to be β-lactamase-producing ABPC-resistant (BLPAR) strains. A high frequency (76.5%) of β-lactamase-producing strains was suspected in Moraxella catarrhalis isolates. Four (3.2%) extended-spectrum β-lactamase-producing K. pneumoniae were found among 126 strains. Four isolates (2.5%) of P.aeruginosa were found to be metallo β-lactamase-producing strains, including three (1.9%) suspected multidrug-resistant strains showing resistance to imipenem, amikacin, and ciprofloxacin. Continual national surveillance of the antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis.

Occurrence and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus pseudintermedius in an Academic Veterinary Hospital

ABSTRACT Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.

TOC-39, a novel parenteral broad-spectrum cephalosporin with excellent activity against methicillin-resistant Staphylococcus aureus.

TOC-39, a new parenteral cephalosporin, is a hydroxyimino-type cephem antibiotic with vinylthio-pyridyl moiety at the 3 position. TOC-39 was evaluated for antibacterial activity against various clinically isolated strains. TOC-39 had excellent activity, stronger than that of methicillin, oxacillin, the cephalosporins tested, imipenem, gentamicin, minocycline, tobramycin, ofloxacin, and ciprofloxacin against methicillin-resistant Staphylococcus aureus (MRSA) and had an MIC comparable to that of vancomycin (the MICs of TOC-39 and vancomycin for 90% of the strains tested were 3.13 and 1.56 micrograms/ml, respectively). Against Enterococcus faecalis strains, which are resistant to cephalosporins, TOC-39 was twice as active as ampicillin. Against methicillin-susceptible S. aureus, coagulase-negative Staphylococcus spp., and Streptococcus pneumoniae, TOC-39 was twice as active as or more active than cefotiam, ceftazidime, flomoxef, and cefpirome. Against Streptococcus pyogenes, TOC-39 was superior to cefotiam, ceftazidime, and flomoxef and was similar to cefpirome. In addition, the activity of TOC-39 was equal to or greater than that of cefotiam, ceftazidime, flomoxef, and cefpirome against Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Morganella morganii. In terms of bactericidal effect against MRSA, TOC-39 was superior to vancomycin. No mutant resistant to TOC-39 or vancomycin was obtained from susceptible MRSA strains. In murine systemic infection models, TOC-39 showed potent activity against S. aureus and E. coli. Against highly MRSA, the activity of TOC-39 was comparable to that of vancomycin.

Cyslabdan, a New Potentiator of Imipenem Activity against Methicillin-resistant Staphylococcus aureus, Produced by Streptomyces sp. K04-0144 : II. Biological Activities

AbstractCyslabdan produced by Streptomyces sp. K04-0144 was found to potentiate imipenem activity against methicillin-resistant Staphylococcus aureus (MRSA). The MIC value of imipenem against MRSA was reduced from 16 to 0.015 μg/ml in combination with cyslabdan. Study on anti-MRSA activity of other typical antibiotics in combination with cyslabdan showed that the potentiating activity was limited to β-lactam antibiotics. Furthermore, among β-lactam antibiotics, the activity of carbapenems was most remarkably poteintiated by cyslabdan.

Emergence of the community-acquired methicillin-resistant Staphylococcus aureus USA300 clone in a Japanese child, demonstrating multiple divergent strains in Japan

In 2008 we isolated methicillin-resistant Staphylococcus aureus (MRSA) from an 11-month-old Japanese girl who lived in Saitama, Japan, and suffered from cellulitis of the lower thigh and sepsis. The MRSA (strain NN47) belonged to multilocus sequence type (ST) 8 and exhibited spa363 (t024), agr1, staphylococcal cassette chromosome mec (SCCmec) type IVa, and coagulase type III. It was positive for Panton–Valentine leukocidin (PVL) and the arginine catabolic mobile element (ACME). Pulsed-field gel electrophoresis (PFGE) demonstrated that the MRSA was the USA300 clone, which is the predominant community-acquired MRSA (CA-MRSA) in the US. Strain NN47 was divergent, in terms of the spa type and patterns of PFGE and plasmids, from the USA300-0114 type strain or USA300 strain NN36, previously isolated from a visitor (Indian girl) from the US. Strain NN47 was resistant to erythromycin, in addition to β-lactam agents (e.g., oxacillin). These data demonstrate the first emergence of the USA300 clone in Japanese children who have never been abroad and have had no contact with foreigners (and therefore, the first USA300 spread in Japan), and also emergence of multiple divergent strains of the USA300 clone in Japan. Because the USA300 clone is highly transmissible and virulent, surveillance of the USA300 clone is needed.

Combination Effect of Vancomycin and β-Lactams against a Staphylococcus aureus Strain, Mu3, with Heterogeneous Resistance to Vancomycin

ABSTRACT We tested the combined activity of vancomycin and seven β-lactam antibiotics against Staphylococcus aureus clinical strain Mu3, which displays heterogeneous resistance to vancomycin. When combined with vancomycin, four of the seven tested β-lactams exhibited an additive effect at or near their MICs, while all showed an antagonistic effect at lower, sub-MIC levels. This study implicated the unpredictable nature of combination therapy of β-lactams and vancomycin against S. aureus with reduced susceptibility to vancomycin.

The first nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy. Part 1: a general view of antibacterial susceptibility

The Japanese Society of Chemotherapy (JSC) conducted the first nationwide surveillance of bacterial respiratory pathogens during the period from January to August 2006. With the cooperation of 32 medical institutions throughout Japan, a total of 924 strains belonging to seven clinically relevant bacterial species were collected from adult patients with well-diagnosed respiratory tract infections (RTIs). Antimicrobial susceptibility testing of the 887 evaluable strains (205 Staphylococcus aureus, 200 Streptococcus pneumoniae, 9 Streptococcus pyogenes, 165 Haemophilus influenzae, 91 Moraxella catarrhalis, 74 Klebsiella pneumoniae, and 143 Pseudomonas aeruginosa) to 42 antibacterial agents was conducted at the Central Laboratory of the Research Center for Anti-infective Drugs of the Kitasato Institute, according to recommendations issued by the Clinical and Laboratory Standards Institute (CLSI). The antibacterial agents employed were 25 β-lactams, three aminoglycosides, four macrolides (including one azalide and one ketolide), one lincosamide, one tetracycline, two glycopeptides, five fluoroquinolones, and one oxazolidinone. The incidence of methicillin-resistant S. aureus (MRSA) was 63.4%, and the incidences of penicillin-intermediately resistant S. pneumoniae (PISP) and penicillin-resistant S. pneumoniae (PRSP) were 35.0% and 4.0%, respectively. Among H. influenzae, 21.2% of the strains were found to be β-lactamase-nonproducing ampicillin (ABPC)-intermediately resistant (BLNAI), 29.1% to be β-lactamase-nonproducing ABPC-resistant (BLNAR), and 4.8% to be β-lactamaseproducing ABPC-resistant (BLPAR) strains. The incidence of extended-spectrum β-lactamase-producing K. pneumoniae was 2.7% (2 of 74 strains). Three (2.1%) of the 143 P. aeruginosa strains were found to be metallo-β-lactamaseproducing, including 1 (0.7%) multidrug-resistant strain. Through the nationwide surveillance, we obtained fundamental antimicrobial susceptibility data of clinically relevant bacterial pathogens in adult RTI to various antibacterial agents. These data will be a useful reference for future periodic surveillance studies, as well as for investigations to control antimicrobial-resistant pathogens.

Emergence of vancomycin resistance during therapy against methicillin-resistant Staphylococcus aureus in a burn patient—importance of low-level resistance to vancomycin

OBJECTIVES Staphylococcus aureus with low-level resistance to vancomycin (VLSA) which could develop into vancomycin-resistant S. aureus (VRSA) is most important. However, VLSA is difficult to detect by standard laboratory methods. We describe here improved methods to detect VLSA. METHODS Three methicillin-resistant S. aureus (MRSA) strains, designated Fu6, Fu10, and Fu18, were sequentially isolated from the burn wound site of a patient, during vancomycin therapy. The properties of these strains were compared with those of reference strains Mu3 and Mu50 (previous resistant isolates from other patients). RESULTS The isolated strains, Fu10 and Fu18, had identical phenotypes and genotypes. The vancomycin resistance of Fu10 was equivalent to that of strain Mu3, whereas Fu18 had much higher vancomycin resistance than Fu10 and Mu3, although reaching the level of Mu50. Fu18 showed similar growth to Mu50 on gradient gels and on Mu3 medium. CONCLUSIONS Our data indicate that the VLSA developed vancomycin resistance during exposure to vancomycin in vivo. The population analysis of tested VLSA and vancomycin intermediately resistant S. aureus (VISA) indicates that a penem at relatively low concentrations induced a significant increase in the number of vancomycin-resistant subpopulations. Furthermore, we confirmed that gradient gel analysis and Mu3 medium are simple and useful methods for the detection of VLSA judged as VSSA by its conventional MIC alone.