Development of a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF-MS)-based typing method to identify meticillin-resistant Staphylococcus aureus clones.

BACKGROUND: Mass spectrum analysis enables species- and subspecies-level identification, and can be used as an epidemiological tool in outbreak management. However, its reliability at clonal level has yet to be established. AIM: To establish a matrix-assisted laser desorption/ionization time-of-flight mass-spectrum-based method that enables bacterial clone identification with accuracy equivalent to pulsed-field gel electrophoresis/phage open-reading frame typing (PFGE/POT). METHODS: Meticillin-resistant Staphylococcus aureus (MRSA) was used in this study. Mass spectra were obtained from a standard strain of S. aureus (ATCC29213) and 57 clinically isolated strains, categorized according to POT. Peaks associated with MRSA clone identification (N = 67) were extracted. Based on this peak information, the feasibility of MRSA clone identification was examined by cluster analysis. FINDINGS: In addition to the 58 strains used for peak extraction, mass spectrum analysis of 24 clinically isolated outbreak strains revealed that peak data could be used for successful identification of clones. These typing results were fully consistent with the PFGE and POT results. CONCLUSION: This novel method enables simple and rapid typing with accuracy equivalent to PFGE/POT. This method would be suited to rapid outbreak analysis, offering accurate information to combat infectious diseases.

Current practice of management of bacteremic sepsis: a study in a tertiary care teaching hospital in Japan.

OBJECTIVE: To investigate how patients with bacteremic sepsis are managed in a tertiary care teaching hospital. PATIENTS AND METHODS: Prospective observational study on patients with bacteremic sepsis. Clinical and microbiological characteristics of bacteremic sepsis were analyzed in relation to prognosis. Severity of the illness was quantitatively analyzed by the APACHE (Acute Physiology, Age, Chronic Health Evaluation) III scoring system. Also investigated was how closely physicians paid attention to acute physiological alterations in patients. RESULTS: The 28-day mortalities in fifty hemodynamically stable patients and in twenty-three septic shock patients were 26% and 52%, respectively (p=0.028). Gram-positive organisms accounted for 54% of all organisms, with the mortality and incidence of septic shock being the same as with Gram-negative infections. The mean APACHE III score was 42.9 in survivors, and 76.5 in non-survivors (p < 0.001). Although serum levels of C-reactive protein and acute physiology score (APS) was significantly higher in non-survivors than in survivors, the correlation with APACHE III score was more prominent in APS. The number of vital signs recorded was 1.67 in physicians and 3.6 in nurses (p < 0.001). CONCLUSIONS: The present study proved that the APACHE III score accurately discriminates between survivors and non-survivors of patients with sepsis. By addressing the need for an objective evaluation of severity of illness, it strongly recommends that physicians should be made aware of physiologically defined sepsis and that they should pay closer attention to patients' physiological alterations to identify the development of sepsis in critically ill patients.

[A trial of infection control measure by clinical microbiology laboratory].

In 1998, we developed a Total Infection Control System in Saga Medical School Hospital, and would like to introduce it for the practical use. This system was named "Dr. FLEMING" (Flexible Microbiological Test & Information System for the New Generation) and is expected to help physicians by providing highly valuable test results and useful information. For example, bacterial identification and drug susceptibility test can be completed within 4-6 hrs after bacterial colony is isolated, and the test report the contains full-colored pictures to enhance understanding. In addition, we have made an information center for infectious disease, where physicians can have access to various data bases outside our hospital. Furthermore, we offer many kinds of useful information to physicians working at other medical facilities to assist their clinical practice of infectious diseases.

Prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from Japanese patients.

BACKGROUND: Although cagD and cagE (cagDE) identified upstream of cagA have been shown to be involved in the induction of interleukin (IL)-8 expression, the relationship between cagDE status and gastroduodenal diseases still remains to be examined. Thus we investigated prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from patients with peptic ulcer or gastritis. METHODS: We analyzed 73 H. pylori strains isolated from Japanese patients (gastritis (GA), 15; gastric ulcer (GU), 28; duodenal ulcer (DU), 23; GU and DU, 7). The presence of cagDE was evaluated by polymerase chain reaction (PCR) and Southern hybridization. The vacA genotype was examined by PCR, using type-specific primers. RESULTS: cagDE was present in 13 (86.7%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 21 (91.3%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.89). vacA signal sequence type s1 was found in 14 (93.3%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 22 (95.7%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.84). Sequences of cagDE and vacA in our Japanese strains were highly homologous with one another, and there were no disease-specific mutations. CONCLUSIONS: Most of the H. pylori strains in Japan were cagDE-positive, vacA s1 type, regardless of clinical outcome. The present study also indicated that these genes were conserved well among our H. pylori isolates.

[In vitro activities of 23 antimicrobial agents against 4,993 gram-positive and gram-negative bacterial strains isolated from multicenter of Japan during 1994--in vitro susceptibility surveillance.Levofloxacin-Surveillance Group].

In a surveillance study conducted during 1994 at 24 medical institutes from different geographical areas of Japan, the susceptibility of clinical isolates to twenty three comparative agents, such as ofloxacin, levofloxacin, ciprofloxacin, tosufloxacin, ampicillin, clavulanic acid/amoxicillin, oxacillin, piperacillin, cefaclor, cefotiam, cefdinir, cefclidin, ceftazidime, cefpirome, imipenem, aztreonam, vancomycin, minocycline, chloramphenicol, clarithromycin, sulfamethoxazole/trimethoprim, amikacin, and gentamicin, were tested by the standard broth micro-dilution method. A total of 4,993 isolates tested in this study included Streptococcus pneumoniae, methicillin susceptible Staphylococcus aureus (MSSA), methicillin resistant Staphylococcus aureus (MRSA), coagllase negative streptococci (CNS), Enterococcus faecalis, Enterococcus faecium, Enterobactericeae, Pseudomonas aeruginosa from patients with urinary tract infections or respiratory tract infections, and Haemophilus influenzae. For MSSA, S. pneumoniae, Enterobacteriaceae, and H. influenzae, more than 70% of the isolates was susceptible to fluoloquinolones. However, resistance occurred in more than 50% of MRSA and P. aeruginosa isolated from UTI. Fluoroquinolones were found to be effective against high level penicillin-resistant S. pneumoniae, the third generation cephem-resistant Enterobacteriaceae and ampicillin-resistant H. influenzae.

Education as a means of reducing medical expenses.

In treating patients with infectious diseases, key knowledge and experience are essential in making appropriate clinical decisions. Medical students in Japan receive limited tuition in clinical pharmacology and microbiology in their undergraduate curriculum. Education and guidance in these fields are not always provided, even during postgraduate training. To help overcome this problem, we have devised a quick medical reference system to support antimicrobial chemotherapy, and this has been operating in our hospital since May 1994. This system is integrated in order to convey the maximal significance of test results by providing detailed information on various kinds of pathogens and antibiotics immediately on every computer display. This is a unique system in Japan, and aims to help doctors provide effective therapy. Using this system, we have succeeded in reducing medical expenditure for antimicrobials by around 10%.

Anionic properties of beta-lactam-enhancing factor on methicillin-resistant Staphylococcus aureus.

We found a Factor (factor T) in aged mixtures of tungstate and phosphate which greatly enhanced the antibacterial effects of beta-lactams upon methicillin-resistant Staphylococcus aureus (MRSA). Factor T suppressed penicillinase production and the amount of penicillin-binding protein-2' in the membrane fraction, thus sensitizing MRSA strains to beta-lactams. In addition, Factor T caused a metachromatic reaction and prolonged the blood coagulation time, indicating that it is a heparin-like polyanion. Since Factor T becomes ineffective in the presence of a polycation, a charge interaction may play an important role in the enhancing effect. One possibility is that Factor T non-specifically inhibits several anion-sensitive enzymes. Factor T inhibited several nucleotide-interacting enzymes, but not most serum enzymes.

[Clinical bacteriological analysis of Vibrio vulnificus infection--a report of five case].

Clinical features in Vibrio infection are generally represented by gastrointestinal involvements such as food poisoning, and its prognosis is usually good. However, Vibrio vulnificus infection not uncommonly causes serious problems including sepsis, necrotizing fasciitis of the extremities, and other conditions, sometimes resulting in fatal outcome. In the present study, we analyzed clinical microbiological aspects of five cases with V. vulnificus infection. All the strains of V. vulnificus isolated in five patients are oxidase-positive Gram negative rods presenting comma-like configuration, which were yielded on TCBS agar forming green colonies; they were grayish-white in color and viscous in texture on 5% sheep blood agar, identification of bacteria were done using VITEK AMS (BioMérieux). Piperacillin and third-generation cephalosporins were found to have bactericidal activities against these strains. All five cases we experienced have primary ailments, and three cases out of the five had taken perishable sea-food before showing disease symptoms. V. vulnificus has two infection channels; one is external wound and the other is oral intake. The latter is said that it may become serious. This has a rather short period from the starting the symptoms to death, and there is high death rate. For life-saving, it is inevitably necessary to dose an effective antibacterial medicine in the early stage. If we suspect this bacteria in the test laboratory, it is important to report this to the clinical doctor. In other words, this is one of the bacteria that needs prompt treatment and further microbiology testing.

Possible mechanism of action of beta-lactam-enhancing factor on methicillin-resistant Staphylococcus aureus.

We have recently found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of beta-lactams on methicillin-resistant strains of staphylococcal species such as methicillin-resistant Staphylococcus aureus (MRSA), but shows only weak effects on methicillin-susceptible S. aureus and bacterial strains other than staphylococci. Factor T alone did not strongly inhibit cell metabolism and bacterial growth unless an excess amount was added. When Factor T was added to the culture medium beforehand, the growth of MRSA cells was rapidly suppressed just after addition of oxacillin (MPIPC). However, the growth of the cells was inhibited gradually when these two reagents were added in reverse order. For full expression of the enhancing effect, it seemed necessary for cells of MRSA strains to be incubated with Factor T for at least 2-3 hr. When the cells were washed after being sensitized by incubating them for 5 hr with Factor T, it took approximately 1 hr for the cells to recover their resistance to MPIPC. Factor T reduced the amount of penicillin-binding protein-2' (PBP 2'), and thus sensitized the MRSA strains to beta-lactams.

[Isolation of penicillin-resistant Streptococcus pneumoniae in Saga Medical School Hospital].

A recent nationwide increase in beta-lactams-resistant Streptococcus pneumoniae has attracted a great deal of attention. We studied the drug sensitivity of S. pneumoniae isolated from various clinical specimens in Saga Medical School Hospital between April 1988 and December 1991. To determine the drug sensitivity of the strains, we used a micro-dilution method and determined the MIC. Drug resistance was evaluated using MIC of ampicillin (ABPC) as a reference MIC, and the results were roughly classified into the following three groups: sensitive (< or = 0.1 microgram/ml), moderately resistant (0.2-3.13 micrograms/ml) and highly resistant (> or = 6.25 micrograms/ml). The isolation frequency was calculated on the basis of one strain from one patient. No strain of S. pneumoniae with high resistance against ABPC was found in 1988 (94 strains of S. pneumoniae were isolated) and 1990 (115 strains isolated), but one such strain (0.8%) was found among 129 strains isolated in 1989, and 2 such strains (2.4%) among 84 strains isolated in 1991. Moderately resistant strains were isolated at the frequencies of 12.8%, 15.5%, 22.6%, and 21.4% respectively, in 1988, 1989, 1990, and 1991. A sum of the frequencies of "moderately resistant" and "highly resistant" (2.4%) strains was 23.8% in 1991. The frequency of resistant strains is increasing and the intensity of resistance is also being elevated.

[Hospital infection with methicillin-resistant Staphylococcus aureus (MRSA) in Saga Medical School Hospital, a rapid increase in coagulase type-VIII strains].

Hospital infection with MRSA has increased in Saga Medical School Hospital. The causative MRSA consisted predominantly of coagulase type-II strain before 1989, but after 1990, coagulase type-VII MRSA increased rapidly. This type-VII strain has marked multiple drug-resistance, and the pattern of drug sensitivity of MRSA in this hospital was different from that of MRSA detected in other facilities, which are clinically serious problems, therefore, we conducted an etiological study of the background of the increase in MRSA infection in our hospital. The results of the study are summarized as follows: 1) The proportions of MRSA (on strain from one patient) to all types of S. aureus detected in the hospital were 26% for 1986, 23% for 1988, 37% for 1989, 30% for 1990 and 60% for 1991. The proportion increased greatly in 1991. 2) Coagulase type VII-MRSA was first detected only in 5 patients in 1989, then it tended to spread, and this type (probably derived from the same strain) accounted for 47% of MRSA infection in patients examined in 1991. 3) The study of the drug sensitivity pattern and etiological survey of the infection showed that coagulase type VII-MRSA prevalent in the hospital consisted of two types: CLDM, and EM-sensitive, IPM/CS, and MINO-resistant and TSST-1 non-producing and enterotoxin non-producing type, and CLDM, and EM-sensitive, IPM/CS, and MINO-resistant and TSST-1 non-producing type with enterotoxin serotype A. 4) Coagulase type VII-MRSA (Probably derived from the same strain) was detected in physicians and nurses working in affected wards and also in the patients's room.(ABSTRACT TRUNCATED AT 250 WORDS)

A factor found in aged tungstate solution enhanced the antibacterial effect of beta-lactams on methicillin-resistant Staphylococcus aureus.

We have found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of beta-lactams on both inducible and constitutive methicillin-resistant Staphylococcus aureus, but not on methicillin-susceptible S. aureus. Factor T alone did not strongly inhibit bacterial growth. There was no synergism of Factor T with other classes of antibiotics, nor with other groups of bacteria, and it reduced the efficacy of amino-glycosides and tetracycline. Upon preparation of Factor T, acidifying and heating the mixture of tungstate and phosphate resulted in a high yield and reproducibility, and no enhancing effect was observed when other anions such as sulfate or molybdate were used instead. Factor T is heat- and acid-stable but labile to alkalization, and is probably a complex of phosphate and tungstate.

An improved method for the serotyping of free coagulase from Staphylococcus aureus.

The serotyping of free coagulase, one of the most reliable ways to identify strains of Staphylococcus aureus, and widely employed in Japan, has been improved by adding magnetite sand to the reaction mixture. Culture medium supernatant and a type-specific antibody are mixed in a well of a microtiter plate, and plasma-enriched bovine fibrinogen is treated with magnetite sand. The use of tranexamic acid and gum arabic in the reaction mixture also increases the sensitivity of the reaction. Finally, the plate is placed on a magnetic stirrer. If the type of the coagulase corresponds to that of the antibody, no clot formation will occur, and this is easily confirmed by the movement of the sand. Although the amount of reaction mixture required is much less than that for the conventional tube method, our new method is able to detect slight increases in viscosity of the reaction mixture due to fibrin formation even before complete clotting occurs, thus providing very high sensitivity. Clot formation can also be judged by observing a turbid mass of fibrin in the well (Hwang's method), but this approach is a little slower than our method involving immobilization of magnetite sand.

[A case of Pasteurella multocida infection in bronchiectasis].

Pasteurella multocide (P. multocida), a small gram-negative bacillus, has been known to be the causative agent of hemorrhagic septicemia in animals. P. multocida infection in human was reported as skin abscess and/or septicemia after an animal bit or scratch. Pulmonary infections of P. multocida have been developed in the patients with chronic pulmonary diseases such as bronchiectasis. In Japan, however, P. multocida respiratory tract infections are rare. In this report, a 80-year-old female with bronchiectasis was admitted on August, 1985. She had a productive cough, hemosputum, and a low grade fever. The chest X-P on admission showed an atelectasis of the left middle lobe and severe bronchiectatic changes of the left lower lobe. P. multocida was isolated from her sputa. The chemotherapy of CTM resulted in clinical improvement. On May 1988, she complained of a productive cough and a low grade fever again. P. multocida was isolated from the sputum on several occasions in significant numbers (1 x 10(8)/ml). Recently, the cases of the chronic respiratory diseases have been increasing. We think, P. multocida is important and should be considered as a pathogen in the care of chronic pulmonary diseases.

[A case of pulmonary nocardiosis associated with middle lobe atelectasis].

Pulmonary nocardiosis is a rare disease. We reported a case of pulmonary nocardiosis associated with middle lobe atelectasis. A 66-year-old female was admitted to Saga Medical School Hospital with complaints of fever, productive cough and hemosputum. Nocardia asteroides was isolated from her sputum and bronchial lavage fluid. Administration of sulfamethoxazole-trimethoprim (ST) improved her symptoms and Nocardia asteroides were rapidly eliminated from her sputum. However, she had severe side effects of ST: toxic dermatitis, granulocytopenia and liver dysfunction, whereupon administration of ST was stopped. About two weeks later, Nocardia asteroides appeared again in her sputum. Either netilmicin (NTL) or minocycline (MINO) given consecutively could not eliminate Nocardia asteroides from her sputa. Subsequently a combination therapy of OFLX administration and gentamicin inhalation was tried. This treatment improved her symptoms and eradicated Nocardia asteroides from her sputa completely. In vitro study, ST, MINO and aminoglycosides showed good susceptibility against Nocardia asteroides. In this case, it was revealed that GM inhalation therapy is also very useful for pulmonary Nocardiosis.

[Susceptibility of clinical isolates to aztreonam].

In vitro antibacterial activities of 9 antibiotics including aztreonam (AZT) against clinically isolated Gram-negative bacteria were determined using MIC-2000 plus system. Bacteria were isolated from clinical materials in Saga Medical School during a period from May 1987 to March 1988. Summarized results were as follows: 1. AZT showed excellent antibacterial activities against Escherichia coli, Klebsiella pneumoniae, Proteus sp. and Haemophilus influenzae, and MIC80 values of AZT against these organisms were lower than 0.20 microgram/ml. 2. Antibacterial activities of AZT were superior to cephem antibiotics compared against Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii and Serratia marcescens. 3. The MIC50 and MIC80 of AZT against Pseudomonas aeruginosa were 12.5 micrograms/ml and 25 micrograms/ml, respectively. 4. AZT did not show any antibacterial activity against Acinetobacter sp. and Xanthomonas maltophilia.

[Evaluation of in vitro antibacterial potencies of oral antibiotics against sputum isolates].

Susceptibility of 162 sputum isolates to oral antibiotics was measured by an agar dilution method. The sputum isolates included S. pneumoniae 25 strains, S. aureus 30 strains, H. influenzae 37 strains, K. pneumoniae 51 strains and E. coli 19 strains. Minimal inhibitory concentration (MIC) values of cefaclor (CCL), cephalexin (CEX), ampicillin (ABPC) and minocycline (MINO) were measured for each strains. Eighty percent of S. pneumoniae strains were inhibited at 0.024 to 0.05 micrograms/ml of ABPC, 0.39 to 0.78 micrograms/ml of CCL, and 1.56 to 3.13 micrograms/ml of CEX and MINO. ABPC, CCL and CEX were considered to be effective clinically when they were used with the usual dosage. However, about 30% of strains were resistant to the usual dosage of orally administrated MINO. Eighty percent of S. aureus strains were inhibited at 0.20 to 0.39 microgram/ml of MINO and 3.13 to 6.25 micrograms/ml of the other 3 drugs. MINO is the most effective with the usual dosage. Twenty to 40% of strains showed resistance to CCL, CEX and ABPC. Eighty percent of H. influenzae strains were inhibited at 0.39 micrograms/ml of ABPC, 0.78 to 1.56 micrograms/ml of MINO, 3.13 micrograms/ml of CCL and 12.5 to 25 micrograms/ml of CEX. ABPC should be selected as the first choice antibiotic. However, there were 2 ABPC-resistant strains that were highly susceptible to CCL. Eighty percent of K. pneumoniae strains were inhibited at 0.39 to 0.78 micrograms/ml of CCL, 3.13 to 6.25 micrograms/ml of MINO and CEX, and 12.5 to 25 micrograms/ml of ABPC. CCL seemed to be only effective oral antibiotic for K. pneumoniae infection.(ABSTRACT TRUNCATED AT 250 WORDS)