Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples

Isolation of Legionella Species from Hot Spring Bath Water Samples in Japan, and the Antibiotic Susceptibility of the L. pneumophila Isolates

In order to obtain an understanding of the inhabitation of Legionella spp. in hot spring bath water, the isolation of Legionella spp. was attempted in 30 prefectures nationwide in 2009-2010, and the following results have been obtained. 1) Isolation of the bacteria was achieved for 89 out of 366 samples (24.3%). When this was broken down into individual regions, the isolation rate in Kyushu and Okinawa regions was the highest at 58.6%, followed by Hokkaido at 42.4%. With regard to isolation by facility, isolation was achieved at 49 out of 124 facilities (39.5%). 2) In the 89 samples form which genus Legionella were isolated, counts of Legionella spp. in 100 ml of hot spring water were less than 102 CFU in 45 samples (50.6%), followed by 28 samples within the 102 CFU range (31.5%). 3) Of the bacterial species of the 111 strains isolated from the 89 samples of hot spring water collected from all over the country, the bacterial strain that was most frequently identified was Legionella pneumophila, and it accounted for 78 strains (69.6%). Among these strains by serogroup, group 1 was the most common with 25 strains (32.1%), followed by group 4 with 17 strains (21.8%). Moreover, out of the bacterial strains that were identified other than Legionella pneumophila, Legionella londiniensis was the second most common, with 20 strains (17.9%), followed by Legionella micdadei with 5 strains (4.5%). 4) The 90% MIC rate (MIC90) of Legionella pneumophila isolates (82 strains) was tested with 10 drugs. Out of the 10 drugs, rifampicin showed the best antimicrobial activity of 0.016 μg/ml, followed by imipenem at 0.5 μg/ml. However, isolates showed high resistance to minocycline and piperacillin with the MIC90 of minocycline at 16 μg/ml, and that of piperacillin at >256 μg/ml

A novel nairovirus associated with acute febrile illness in Hokkaido, Japan

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014–2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan

A novel nairovirus associated with acute febrile illness in Hokkaido, Japan

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014–2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan