The usefulness of semi‐solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka, Japan

Abstract

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi-solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira-positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi-solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semisolid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.

Leptospira and Leptospirosis

“…Furthermore, there are only a few reports describing the isolation of virulent Leptospira from environmental samples; as discussed above, the main difficulty in the isolation of these species is the overgrowth of environmental bacteria or the fast-growing saprophytic Leptospira (Barragan et al, 2011;Benacer et al, 2013;Saito et al, 2013). Culture-based protocols include the use EMJH (Ellinghausen and McCullough modified by Johnson and Harris) culture medium containing cocktails of antibacterial and anti-fungal compounds such as sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5-fluorouracil (Benacer et al, 2013;Saito et al, 2015;Saito et al, 2013). Alternatively bacterial contamination in water samples can be reduced by utilizing 0.22 µm filtrate before enriching in EMJH medium, then isolating colonies on solid medium (Saito et al, 2015;Wuthiekanun et al, 2013 Kumar et al, 2016b;Aviat et al, 2010) or pathogenic specific sequences of genes, such as flaB (Villanueva et al, 2014), secY (Perez and Goarant, 2010;Guernier et al, 2018) and the 16S rRNA gene (Ganoza et al, 2006).…”

“…Culture-based protocols include the use EMJH (Ellinghausen and McCullough modified by Johnson and Harris) culture medium containing cocktails of antibacterial and anti-fungal compounds such as sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5-fluorouracil (Benacer et al, 2013;Saito et al, 2015;Saito et al, 2013). Alternatively bacterial contamination in water samples can be reduced by utilizing 0.22 µm filtrate before enriching in EMJH medium, then isolating colonies on solid medium (Saito et al, 2015;Wuthiekanun et al, 2013 Kumar et al, 2016b;Aviat et al, 2010) or pathogenic specific sequences of genes, such as flaB (Villanueva et al, 2014), secY (Perez and Goarant, 2010;Guernier et al, 2018) and the 16S rRNA gene (Ganoza et al, 2006). Because of sequence polymorphisms, PCR targeting lipL32 or secY fail to detect virulent Leptospira of the intermediate cluster (e.g.…”

Advances and challenges in barcoding pathogenic and environmentalLeptospira

“…Another bacterial gene that is widely used in phylogenetic studies is the gyrB gene, which is reported to have a higher nucleotide divergence in Leptospira species than the 16S RNA rrs gene (Huang, 1996; Slack et al , 2006). This gene target has also been used for Leptospira diagnostic assays (Slack et al , 2006) and typing of isolates carried by rodents in Japan and the Philippines (Kawabata et al , 2006; Saito et al , 2015; Villanueva et al , 2014).…”