Wastewater Knows Pathogen Spread: Analysis of Residential Wastewater for Infectious Microorganisms including SARS-CoV-2.

BACKGROUND: We aimed to identify the genes of 35 pathogens, including viruses, bacteria, and protozoa that cause waterborne infectious diseases, and to assess the feasibility of a wastewater-based surveillance system. MATERIALS AND METHODS: Wastewater was aseptically sampled once a month from 2 sites. A total of 1 L of wastewater from each site underwent 0.2 µm filtration to generate the sample A. Subsequently, 200 ul of the filtered water was ultra-filtered and concentrated to generate the sample B, which was mixed with sample A in a 1:1 ratio. We performed a Filmarray® Gastrointestinal (GI) panel (BioFire Diagnnostics', Salt Lake City, UT, USA) test to simultaneously detect 13 enterobacteria, 5 enterovirus, and 4 protozoa. RNA was extracted to assess 18 types of viruses. RESULTS: Severe acute respiratory syndrome coronavirus 2 adenovirus, bocavirus, and rhinovirus was detected at both site. Norovirus GI/GII was continuously detected at both sites. Moreover, adenovirus, group A rotaviruses, and hepatitis A virus were frequently detected; however, hepatitis E virus was absent at either site. Campylobacter, enteroaggregative Escherichia coli, enterotoxigenic E. coli, Shiga toxin-producing E. coli, and Clostridioides difficile toxin A/B were detected at both sites. Giardia lamblia was also detected in both sites. CONCLUSION: We analyze multiple infectious disease pathogens under sample surveillance with incidence. Further in-depth studies on wastewater-based surveillance will be feasible and important.

Effects of Non-Pharmacological Interventions on Respiratory Viruses Other Than SARS-CoV-2: Analysis of Laboratory Surveillance and Literature Review From 2018 to 2021.

BACKGROUND: Since the global coronavirus disease 2019 (COVID-19) pandemic, non-pharmacological interventions (NPIs) such as extensive and comprehensive hand hygiene, mask-wearing, and social distancing have been implemented globally. This study aimed to investigate changes in respiratory viruses other than severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that occurred following the implementation of these NPIs. METHODS: From January 2018 to December 2021, influenza-like illness patient specimens and specimens from the Korea Influenza and Respiratory Viruses Surveillance System were analyzed at the Incheon Metropolitan City Institute of Public Health and Environment. Oropharyngeal or nasopharyngeal swab samples from respiratory infection patients were transferred in a virus transport medium at 4°C. After RNA or DNA extraction, respiratory virus-specific genes for human influenza virus (IFV), adenovirus (ADV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), human rhinovirus (hRV), human coronavirus, human bocavirus, and human metapneumovirus were detected by individual real-time reverse transcription polymerase chain reaction. RESULTS: A total 3,334 samples were collected. After NPI was implemented, the detection of respiratory viruses other than SARS-CoV-2 decreased overall. The yearly detection rate of respiratory viruses was decreased from 69.5% (399/574) in 2018 and 73.3% (505/689) in 2019 to 19.8% (206/1,043) in 2020 and 34.9% (365/1,028) in 2021. The epidemic was more prominent in respiratory viruses such as IFV and RSV, which were considered dominant viruses, especially those with viral envelopes. Among viruses that were not considered dominant, hRV showed no clear change before and after NPI, while PIV showed a rapid increase compared to the existing dominant viruses between October-December 2021, after the increase in the number of gatherings started at the end of September and the "Relaxing COVID19 and mitigation policy," which was implemented on November 1. CONCLUSION: NPI seems to have influenced the isolation and transmission of respiratory viruses in South Korea. In the future, additional studies focusing on the isolation and transmission patterns of respiratory viruses following NPI are needed.

Investigation of Pathogenic Escherichia coli Contamination of Vegetables Distributed in a Korean Agricultural Wholesale Market.

The purpose of this study was to investigate the monthly contamination rate of pathogenic Escherichia coli, a major cause of food poisoning, in vegetables sold in agricultural wholesale markets, which distribute vegetables from all over the country, in the Incheon Metropolitan City area, South Korea, and to identify a source of the pathogen. In total, 1739 vegetables of 80 types, along with 109 soil, 67 manure, and 33 livestock feces samples, were tested for pathogenic E. coli using polymerase chain reaction, from September 2016 through August 2017. The average annual prevalence rate of vegetables was 5.8%, and the prevalence rate was above 5% from June through October. The highest prevalence rate (15.7%) was recorded in July. Water dropwort showed the highest prevalence rate (28.6%) among the vegetables examined. Pathogenic E. coli was detected in >20 types of the vegetables that were to be consumed without cooking. Among these, the prevalence rates of ponytail radish (n = 21), crown daisy (n = 86), young radish (n = 68), romaine lettuce (n = 133), perilla leaf (n = 103), Korean leek (n = 43), young Chinese cabbage (n = 68), and Chinese cabbage (n = 30) were 9.5%, 8.1%, 7.4%, 6.8%, 4.9%, 4.7%, 4.4%, and 3.3%, respectively. Among the vegetables cooked before consumption, prevalence rates were 28.6%, 27.3%, and 25.0% in wormwood, sweet potato stalk, and edible mountain vegetables (Saussurea sp., etc.), respectively. In soil, manure, and livestock feces, 36.7%, 26.9%, and 90.6% prevalence rates were confirmed, respectively. This study confirmed the pathogenic E. coli contamination of vegetables to be consumed without cooking. Therefore, to produce agricultural products that do not induce food poisoning and are safe for consumption, it is important to develop a process for killing the pathogenic microorganisms and set up a sanitary environment for effectively managing compost. In addition, it is necessary to establish surveillance systems to monitor the production chain.

Multidrug-Resistant Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated from Vegetable Farm Soil in South Korea.

The populations of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) have increasingly disseminated in humans, animals, and the environment. This study aimed to determine the prevalence, antimicrobial susceptibilities, and molecular characteristics of ESBL-EC isolates obtained from vegetable farm soil. In total, 200 soil samples were collected from vegetable farms in Incheon, South Korea, between 2018 and 2019 and cultured on MacConkey screening plates supplemented with 2 µg/mL cefotaxime. Cefotaxime-resistant ESBL-EC isolates were recovered from 4.0% (8/200) of the soil samples. All eight isolates were nonsusceptible to ampicillin, piperacillin, cefazolin, cefotaxime, and cefepime and harbored blaCTX-M-type ESBL genes, including blaCTX-M-15 (50.0%), blaCTX-M-55 (25.0%), and blaCTX-M-14 (25.0%). Phylogenetic analysis showed that the B1 lineage was predominant (75.0%), followed by A (12.5%) and B2 (12.5%) lineages. Multilocus sequence typing revealed eight different E. coli sequence types (STs), including ST10, ST73, ST155, ST847, ST2521, ST3285, ST5173, and ST9479. Notably, ST10 and ST73 belong to the global extraintestinal pathogenic E. coli lineages. Our findings demonstrated that the farm soil environment may serve as a reservoir of human-associated multidrug-resistant ESBL-producing pathogens.

Extended-spectrum ß-lactamase-producing Escherichia coli isolated from raw vegetables in South Korea.

The increasing prevalence of oxyimino-cephalosporin-resistant Enterobacteriaceae has become a global concern because of their clinical impact on both human and veterinary medicine. The present study determined the prevalence, antimicrobial susceptibility, and molecular genetic features of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) isolates from raw vegetables. A total of 1324 samples were collected from two agricultural wholesale markets in Incheon, South Korea in 2018. The ESBL-EC strains were isolated from 0.83% (11/1324) samples, and all of them were resistant to ampicillin, piperacillin, cefazoline, cefotaxime, and nalidixic acid and yielded CTX-M-type ESBL, including CTX-M-14, CTX-M-15, CTX-M-55, CTX-M-27, and CTX-M-65. The isolates belonged to phylogenetic subgroups D (n = 5), A (n = 4), and B1 (n = 2). Multilocus sequence typing revealed nine known E. coli sequence types (STs), including ST10, ST38, ST69, ST101, ST224, ST349, ST354, ST2509, ST2847, and two new STs. Notably, ST69, ST10, ST38, and ST354 belong to the major human-associated extraintestinal pathogenic E. coli lineages. Our results demonstrate that ESBL-producing multidrug-resistant pathogens may be transmitted to humans through the vegetable intake, highlighting the importance of resistance monitoring and intervention in the One Health perspective.

Monitoring Culicine Mosquitoes (Diptera: Culicidae) as a Vector of Flavivirus in Incheon Metropolitan City and Hwaseong-Si, Gyeonggi-Do, Korea, during 2019.

The flaviviruses are small single-stranded RNA viruses that are typically transmitted by mosquitoes or tick vectors and are etiological agents of acute zoonotic infections. The viruses are found around the world and account for significant cases of human diseases. We investigated population of culicine mosquitoes in central region of Korean Peninsula, Incheon Metropolitan City and Hwaseong-si. Aedes vexans nipponii was the most frequently collected mosquitoes (56.5%), followed by Ochlerotatus dorsalis (23.6%), Anopheles spp. (10.9%), and Culex pipiens complex (5.9%). In rural regions of Hwaseong, Aedes vexans nipponii was the highest population (62.9%), followed by Ochlerotatus dorsalis (23.9%) and Anopheles spp. (12.0%). In another rural region of Incheon (habitat of migratory birds), Culex pipiens complex was the highest population (31.4%), followed by Ochlerotatus dorsalis (30.5%), and Aedes vexans vexans (27.5%). Culex pipiens complex was the predominant species in the urban region (84.7%). Culicine mosquitoes were identified at the species level, pooled up to 30 mosquitoes each, and tested for flaviviral RNA using the SYBR Green-based RT-PCR and confirmed by cDNA sequencing. Three of the assayed 2,683 pools (989 pools without Anopheles spp.) were positive for Culex flaviviruses, an insect-specific virus, from Culex pipiens pallens collected at the habitats for migratory birds in Incheon. The maximum likelihood estimation (the estimated number) for Culex pipiens pallens positive for Culex flavivirus was 25. Although viruses responsible for mosquito-borne diseases were not identified, we encourage intensified monitoring and long-term surveillance of both vector and viruses in the interest of global public health.

Clinically Relevant Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates From Food Animals in South Korea.

Extended-spectrum ß-lactam antimicrobials have been broadly used in food animals and humans to control infectious diseases. However, the emergence and rapid spread of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, mainly Escherichia coli, have seriously threatened global health in recent decades. In this study, we determined the prevalence, antimicrobial susceptibility, and genetic properties of ESBL-producing E. coli (ESBL-EC) strains isolated from food animals in South Korea. A total of 150 fecal samples from healthy chickens (n = 34), pigs (n = 59), and cattle (n = 57) were screened from January to July 2018. Among these, 77 non-duplicate cefotaxime-resistant ESBL-EC strains were isolated from 32 chicken, 41 pig, and 4 cattle samples, with the corresponding occurrence rates of 94.1, 69.5, and 7.0%, respectively. All the isolates showed multidrug resistance (MDR) and produced at least one type of ß-lactamase, including CTX-M (98.7%) and TEM (40.3%). CTX-M-14 (53.1%), CTX-M-55 (53.7%), and CTX-M-65 (50.0%) were the predominant genotypes in the chicken, pig, and cattle samples, respectively. Multilocus sequence typing revealed 46 different sequence types (STs), including the human-associated extraintestinal pathogenic E. coli ST131 (n = 2), ST10 (n = 5), ST38 (n = 1), ST410 (n = 4), ST354 (n = 2), ST58 (n = 3), ST117 (n = 1), and ST457 (n = 1). To the best of our knowledge, this is the first report of pandemic E. coli ST131 in non-human isolates in South Korea. Our results demonstrate the high prevalence and diversity of MDR-ESBL-EC in food animals and highlight them as potential pathogenic ESBL-EC reservoirs that may pose a high risk to human health.

Increasing prevalence of multidrug-resistant mcr-1-positive Escherichia coli isolates from fresh vegetables and healthy food animals in South Korea.

Colistin is a last-resort antimicrobial against multidrug-resistant gram-negative bacteria. The occurrence and spread of colistin resistance in humans and animals have been reported globally. This study was performed to investigate the prevalence and antimicrobial susceptibility of mcr-harboring colistin-resistant Enterobacteriaceae from retail vegetables and food animals in South Korea in 2018. The mcr-1 gene was detected in Escherichia coli isolates from 0.076% (1/1324) of vegetables, 5.9% (2/34) of chickens, 6.8% (4/59) of pigs, and 0% (0/57) of cattle. Other mcr genes were not detected. All seven of the mcr-1-positive isolates showed multidrug resistance and co-produced ß-lactamases. Multilocus sequence typing analysis revealed five known E. coli sequence types (STs), including ST10 in the vegetable sample. The study findings demonstrated that the mcr-1 gene has emerged in vegetables and is increasingly detected in food animals in South Korea, highlighting the importance of continuous monitoring and control of colistin-resistant Enterobacteriaceae to prevent them from being transmitted to humans.

Molecular Detection of Severe Fever with Thrombocytopenia Syndrome Virus in Korean Domesticated Pigs.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus in China, Japan, and Korea. Although the incidence of SFTS has been increasing annually since the first SFTSV case was reported in Korea, the prevalence of SFTSV in Korean livestock has not conducted. The purpose of this study was to investigate the prevalence of SFTSV in domesticated pigs (Sus scrofa domesticus) throughout Korea. METHODS: A total of 240 sera were collected from 30 domesticated pigs in eight provinces. To detect SFTSV, one-step reverse transcription RT nested PCR was conducted using SFTSV genome-specific primer sets. The amplified PCR products were cloned and then sequenced. RESULTS: Four of 240 (1.7%) pig sera tested positive for SFTSV through one-step RT nested PCR. Two of the four obtained sequences were included in the Japanese clade, whereas the other two sequences were related to the Chinese clade based on phylogenetic analysis. Moreover, the obtained SFTSV sequences from this study were distinct from human SFTSV strains. CONCLUSIONS: This study is the first to attempt the identification of SFTSV sequences from domesticated pigs and the first molecular detection of SFTSV in Korean livestock. Our findings indicate that a new subclade of SFTSV-different from that in humans-may be present in domesticated pigs; the surveillance of SFTSV in livestock is required to better understand the life cycle of SFTSV.

Prevalence of severe fever with thrombocytopenia syndrome virus in black goats (Capra hircus coreanae) in the Republic of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen in China, Japan, and the Republic of Korea (ROK). The aim of this study was to investigate the prevalence of SFTSV antigens and anti-SFTSV antibodies in black goats (Capra hircus coreanae) throughout the ROK. Sera were collected from 737 black goats in nine provinces in the ROK. Eighteen of 737 (2.4%) goat sera were positive for SFTSV on one-step reverse transcription nested polymerase chain reaction. The amplified 346-bp S segments of SFTSV sequences were classified into three genotypes (BG1, BG2, and BG3), and were included in the Japanese clade rather than the Chinese clade, based on phylogenetic analysis. Forty-three of 624 (6.9%) serum samples were seropositive for anti-SFTSV antibodies on enzyme-linked immunosorbent assay analysis. This study is the first to examine the molecular prevalence of SFTSV in goats and the first to perform serological detection of anti-SFTSV antibodies in livestock in the ROK. Moreover, the results indicate that SFTSV is widely distributed in goats and that additional monitoring for SFTSV is needed in livestock in the ROK.

A waterborne outbreak involving hepatitis A virus genotype IA at a residential facility in the Republic of Korea in 2015.

BACKGROUND: Hepatitis A virus (HAV), a major cause of acute hepatitis, has had the highest occurrence among group 1 nationally notifiable infectious diseases in Korea since 2010.Recently,the annual increase in the HAV infection rate among young adults has become a public health concern. OBJECTIVES: The aim of this study was to describe an outbreak of acute hepatitis in a residential facility in April 2015 and to identify potential sources of this outbreak. STUDY DESIGN: Sera from all exposed residents were tested for anti-HAV IgM or IgG antibodies by ELISA. Clinical (sera and stool) and environmental samples were screened for the presence of HAV RNA using one-step RT-PCR and nested PCR. The VP3-VP1 regions of HAV were analyzed using the BLAST database and MEGA7 software. RESULTS: Of the 82 persons in the facility, 12 (14.6%, including 10 residents and 2 health care workers) were diagnosed with hepatitis A. Clinical symptoms were evident in 9 individuals, one of whom died, and the remaining four patients were asymptomatic. Traceback investigation revealed that HAV-RNA (genotype IA) was detected in the patients' stools and the groundwater used in the facility. CONCLUSIONS: We described an HAV outbreak in a facility for the disabled due to using a water supply that was mixed with contaminated groundwater. Therefore, HAV vaccination and periodic water inspections in group facilities should be emphasized to prevent HAV infection.

Outbreak of CTX-M-15-Producing Enterotoxigenic Escherichia coli O159:H20 in the Republic of Korea in 2016.

We investigated an outbreak of enterotoxigenic Escherichia coli (ETEC) O159:H20 associated with the consumption of a tossed-noodle dish in a high school in 2016. Thirty-three ETEC strains isolated from clinical and food samples were genetically indistinguishable. The outbreak strains were resistant to third-generation cephalosporins and harbored a blaCTX-M-15 gene on a 97-kb self-transferable IncK plasmid. This is the first outbreak caused by CTX-M-15-producing ETEC strains.

Molecular detection of severe fever with thrombocytopenia syndrome virus (SFTSV) in feral cats from Seoul, Korea.

This study tested serum samples of feral cats from a highly urbanized habitat, Seoul, Korea to determine the infection to severe fever with thrombocytopenia syndrome virus (SFTSV). From 126 samples tested, SFTSV was detected by RT-PCR in 22 (17.5%) cats from various sites of Seoul. Sequences identified from this study were grouped with clusters from China and Japan. Our result provides data that SFTSV may have been circulating in settings that were suspected to have relatively low risk, such as highly urbanized habitats. Thus it warrants further study to investigate the ecology of SFTSV in urban-dwelling animals including ticks, human and other potential host species.

Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Wild Animals and Ixodidae Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic to central-northeastern China, southern Japan, and the Republic of Korea (ROK). To investigate SFTSV infections, we collected serum samples and ticks from wild animals. Using serum samples and ticks, SFTSV-specific genes were amplified by one-step RT-PCR and nested PCR and sequenced. Indirect immunofluorescence assay (IFA) was performed to analyze virus-specific antibody levels in wild animals. Serum samples were collected from a total of 91 animals: 21 Korean water deer (KWD), 3 Siberian roe deer, 5 gorals, 7 raccoon dogs, 54 wild boars (WBs), and 1 carrion crow. The SFTSV infection rate in wild animals was 3.30% (3 of 91 animals: 1 KWD and 2 WBs). The seropositive rate was 6.59% (6 of 91 animals: 5 KWD and 1 WB). A total of 891 ticks (3 species) were collected from 65 wild animals (9 species). Of the attached tick species, Haemaphysalis longicornis (74.86%) was the most abundant, followed by Haemaphysalis flava (20.20%) and Ixodes nipponensis (4.94%). The average minimum infection rate (MIR) of SFTSV in ticks was 4.98%. The MIRs of H. longicornis, H. flava, and I. nipponensis were 4.51%, 2.22%, and 22.73%, respectively. The MIRs of larvae, nymphs, and adult ticks were 0.68%, 6.88%, and 5.53%, respectively. In addition, the MIRs of fed and unfed ticks were 4.67% and 4.96%, respectively. We detected a low SFTSV infection rate in wild animals, no differences in SFTSV infection rate with respect to bloodsucking in ticks, and SFTSV infection for all developmental stages of ticks. This is the first report describing the detection of SFTSV in wild animals in the ROK.

Detection of Tick-Borne Pathogens in the Korean Water Deer (Hydropotes inermis argyropus) from Jeonbuk Province, Korea.

The objective of this study was to investigate the prevalence of tick-borne pathogens in the Korean water deer (Hydropotes inermis argyropus). Pathogens were identified using PCR which included Anaplasma, Ehrlichia, Rickettsia, and Theileria. Rickettsia was not detected, whereas Anaplasma, Ehrlichia, and Theileria infections were detected in 4, 2, and 8 animals, respectively. The most prevalent pathogen was Theileria. Of the 8 Theileria-positive animals, 2 were mixed-infected with 3 pathogens (Anaplasma, Ehrlichia, and Theileria) and another 2 animals showed mixed-infection with 2 pathogens (Anaplasma and Theileria). Sequencing analysis was used to verify the PCR results. The pathogens found in this study were identified as Anaplasma phagocytophilum, Ehrlichia canis, and Theileria sp. To the best of our knowledge, this is the first report identifying these 3 pathogens in the Korean water deer. Our results suggest that the Korean water deer may serve as a major reservoir for these tick-borne pathogens, leading to spread of tick-borne diseases to domestic animals, livestock, and humans. Further studies are needed to investigate their roles in this respect.

Malaria vector surveillance in Ganghwa-do, a malaria-endemic area in the Republic of Korea.

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.