The multilocus variable-number tandem repeat analysis (MLVA) method to target eight variable-number tandem repeat loci, based on agarose gel electrophoresis separation of multiplexed PCR products, and the PFGE method were applied to clinical isolates of Escherichia coli O157 : H7 with the aim of comparing their performance as methods of typing this bacterium. Using MLVA, a total of 57 isolates from patients in Shizuoka prefecture, Japan, were divided into 20 types and classified into 23 PFGE types. Twenty-four isolates from four sporadic infections, four household contact infections and one outbreak that occurred in central parts of Shizuoka prefecture during August to November in 2005 were shown to be the same MLVA type, and most of the isolates had identical PFGE banding patterns, suggesting the diffuse outbreak in these parts of Japan. Thus, there was a good correlation between MLVA types and PFGE types, with both methods displaying broadly similar discriminatory powers. However, the MLVA typing proved to be a much easier and more rapid method for the analysis of E. coli O157 : H7 strain relatedness to identify transmission routes. Hence, our MLVA method would be a suitable technique for routine typing in many laboratories, including public health agencies, and even in hospitals.
In recent years, many public bathhouses introduced bathing water circulating systems for extended use, in which a sand filtration unit was installed. However, this resulted in several large scale outbreaks of legionellosis due to the microbiologically insufficient maintenance of the bathing facilities. Occurrence of Legionella in bathing water circulating systems, appears to be common and is a serious public health concern in Japan. The authors constructed a life-size model plant of a bathing water circulating system for the simulation experiment. These experiments are aimed at monitoring changes in the microbial constituents, especially a possible occurrence of Legionella in a bathing water circulating system, and developing preventive measures and intervention strategies. As a result of experiment 1, Legionella was detected in both the bathing water and the filter water at concentrations of 6.6 X 102 CFU/100 ml on the 3rd day after residual chlorine disappeared. The number of amoebae in the filter water fluctuated and amounted to 12 cells/ml at the end of the experiment. In the experiments, it was clearly demonstrated that Legionella occurred in the bathing water circulating system within a short period in a sequential manner of microbial growth. Namely, concentration of organic matter (dirt) in the bathing water can be monitored as the KMnO4 consumption value increased in correlation to the number of bathers. The deposited dirt allows bacteria to rapidly undergo multiplication in the bathing water, which consequently supports the occurrence of a large number of host amoebae.
Thirty-one Holstein cows were placed in pasture in Shimane Prefecture over a period from April to August, 1970. Each of them was inoculated with 1ml of defibrinated blood of indigenous cattle infected with piroplasrnas to a severity of -H-F, in April, 1970. As a result, piroplasmas were detected from all the cows by 45 days after inoculation. Finally, 13 cows (41.9%) were proved to have acquired immunity to the protozoa, without receiving any treatment during the proper period of grazing. In general, 70 days seemed to be insufficient and at least 4 months required for effective immunity to be established.
In a newly-built layer processing plant in Shizuoka prefecture, a survey of bacterial contamination was conducted and the adequacy of improvements aimed at minimizing the level of contamination was assessed. The carcasses after defeathering and after chilling as well as finished chicken products showed rather heavy bacterial contamination, a mean standard plate count (SPC) of log 3.7 CFU/cm2 (3.7), 3.1 and 4.3, respectively. After improving in the defeathering machines and maintaining chlorine concentration at a level of 0.4 ppm in the chilling water, SPC on the carcasses after defeathering was adequately decreased to 3.0, but that in chiken products was inadequately decreased to 4.2. When the products were processed by workers wearing disposable rubber gloves, SPC of the chicken products decreased to 3.4. These findings suggested that the bacterial contamination of the products was due to the workers wearing cotton gloves, and also indicated that bacterial control management of the gloves worn is indispensable to the production of sanitary chicken products at this plant.
A direct polymerase chain reaction method to EC cultured broth (named EC-PCR) for screening detection of enteropathogenic E. coli (ETEC, VTEC and EIEC) in foods and feces is described.Eleven E. coli strains belonging to three groups (4ETEC: 06, 025, 0128, 0148; 4VTEC: 026, 0111, 0145, 0157; and 3EIEC: 028ac, 0124, 0164) were used for this study. Four pairs of oligonucleotide primers homologous to LT, ST, VT and EIEC genes were used in combination. The culture condition at 37-43°C for 16-20 h in EC broth was most suitable for the EC-PCR method, and no cross readings were observed with other bacteria, substances in meats or feces.After a 20-h enrichment step, it was possible to detect fewer than 10 to 102 bacteria per g of the aritificially inoculated meat. E. coli was easily detected because of low contamination with other bacteria which disturb the detection of E. coli. However, in feces, it ranged from 103 to 104 cfu per g. The large number of bacteria with feces are the main limiting factor of the EC-PCR detection assay. All E. coli strains examined were detected from all the enrichment cultures on both the EC-PCR and the culture methods. In two sporadic cases and two food poisoning cases, the enteropathogenicity of E. coli isolates from patients was rapidly judged by the EC-PCR method.These findings were consistent with those of the culture method. Thus, the findings suggest that the EC-PCR method is a suitable, sensitive and rapid method for detection of the potentially enteropathogenic E. coli.
In most of hot spring baths in Japan, bathing water is circulated for extended use to conserve hot spring water. In recent years, massive outbreaks of Legionnaires' disease among hot spring bath users have been reported in many districts in Japan. In the present chapter, the authors used a bath model to investigate the effectiveness of backwashing the filtering medium using a high concentration of chlorine for disinfection and growth inhibition of Legionella. They then assessed the usefulness of this method from the perspective of hygiene control of circulating bathing water. Chlorine backwashing by the filter refreshment method (5 to 10 mg/liter) was performed once a day for 9 days, and the bathing water and the water from the filter unit were collected every day prior to backwashing to determine the presence of Legionella and amoebae. The number of Legionella in both bathing water and filter water was maintained at a level lower than 10 to 70 CFU/100 ml by repeated backwashing with chlorinated water alone once a day; the Legionella growth was greatly inhibited compared to that under non-disinfection conditions. On the basis of these results, daily backwashing by the filter refreshment method is considered to be effective for growth inhibition of both Legionella and host amoebae in circulating bathing water. Together with daily use of the filter refreshment method, addition of chlorine into the bathing water to a minimum concentration of 0.2 to 0.4 mg/liter may ensure the supply of circulating bathing water with increased microbial safety.
