Takahashi Eizo, Ochi Sadayuki, Mizuno Tamaki, Morita Daichi, Morita Masatomo, Ohnishi Makoto, Koley Hemanta, Dutta Moumita, Chowdhury Goutam, Mukhopadhyay Asish K, Dutta Shanta, Miyoshi Shin-Ichi, Okamoto Keinosuke
Collaborative Research Center of Okayama University for Infectious Diseases in India, NICED-JICA Building, Kolkata, India.
Department of Health Pharmacy, Yokohama University of Pharmacy, Yokohama, Japan.
Front Microbiol. 2021 Aug 20;12:726273. doi: 10.3389/fmicb.2021.726273. eCollection 2021.
Cholera toxin (CT)-producing O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium at two temperatures, 25°C and 37°C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25°C, but that was low when cultured at 37°C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of . However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of O1, may emerge in various parts of the world and cause epidemics in the future.
产霍乱毒素（CT）的O1群和O139群霍乱弧菌可引发急性腹泻疾病，是霍乱流行和大流行已被证实的病原体。另一方面，非O1/非O139群霍乱弧菌被归类为不可凝集（NAG）弧菌，与霍乱流行无关。大多数NAG弧菌不具备CT基因（）。在本研究中，我们从印度加尔各答的池塘水中分离出三株NAG菌株（菌株1、2和3），并检测了它们的致病特性。使用兔回肠肠袢试验检测了这三株NAG菌株的肠毒性。菌株1可导致肠袢中液体蓄积，同时向肠袢中注射抗CT抗血清可使液体量减少。菌株2和3导致的肠袢中液体量分别很少且无法检测到。然后，我们在25°C和37°C两个温度下在液体培养基中培养这三株菌株，并检测培养上清液中积累的CT量。当菌株1在25°C培养时，CT在培养上清液中积累，但在37°C培养时积累量较低。在检测的培养条件下，菌株2和3的培养上清液中积累的CT量在两个温度下都极低。为了阐明这些菌株的毒力特性，对这三株菌株的基因组序列进行了分析。分析表明，在毒力因子基因和的调控基因方面，三株分离株之间没有明显差异。然而，菌株1保留了霍乱弧菌第七次大流行岛-II（VSP-II），而菌株2和3没有。此外，还发现菌株1中CT B亚基的基因型为1型，菌株2和3的基因型为8型。组织病理学检查显示，暴露于菌株1的肠道组织中绒毛消失。此外，由于菌株1的作用，肠袢中蓄积的液体具有溶血活性。这表明菌株1在感染人类时可能具有诱导严重综合征的毒力因子，并且居住在加尔各答池塘水中的一些NAG弧菌菌株已经获得了毒力，可导致人类发病。这些获得了编码O1群霍乱弧菌毒力相关因子基因的有毒NAG弧菌有可能在世界各地区出现并在未来引发疫情。
