Therya_notes_NE7

THERYA NOTES 2024, Vol. 5 : 133-138 DOI: 10.12933/therya_notes-24-161 ISSN 2954-3614

Enteropathogenic bacteria isolated in Sturnira hondurensis from central México

Aislamiento de bacterias enteropatógenas en Sturnira hondurensis del centro de México

Daniela Segura-Trejo1,2, Norberto A. Angel-Ruiz2,3, and Pablo Colunga-Salas2*

1Facultad de Ciencias, Universidad Nacional Autónoma de México. Investigación Científica s/n, C. P. 04510, Ciudad Universitaria, Coyoacán. Ciudad de México, México. E-mail: daniela.st@ciencias.unam.mx (DS-T).

2Instituto de Biotecnología y Ecología Aplicada (INBIOTECA), Universidad Veracruzana. Av. de las Culturas Veracruzanas 101, C. P. 91090, Xalapa - Enríquez. Veracruz, México. E-mail: norbertoalejandroangel@gmail.com (NAA-R); pcolunga@uv.mx (PC-S).

3Posgrado en Ciencias en Ecología y Biotecnología, Instituto de Biotecnología y Ecología Aplicada (INBIOTECA), Universidad Veracruzana. Av. de las Culturas Veracruzanas 101, C. P. 91090, Xalapa - Enríquez. Veracruz, México.

*Corresponding author

About 45 % of human deaths from infectious diseases are caused by enteropathogens, among them Salmonella, Shigella and Escherichia stand out. This group of bacteria has been poorly studied in bats and their ecosystem. The aim of this work was to evaluate the presence of enterobacteria in bats associated with a contaminated body of water in Xalapa, México. Two mist nets were placed along the stream bank of the Honduras River. The bat species were identified, and fecal samples were obtained from them, which were grown in triplicate in Salmonella-Shigella medium, incubated at 35 °C for 48 hr. Additionally, a sample of water from the stream was isolated, also in triplicate. The bacterial species were identified by colorimetry. Four specimens of Sturnira hondurensis were collected. Strains from Salmonella enterica (2 individuals), Shigella flexneri (2 individuals), and colony-forming unit (CFU) suspected of Escherichia coli (3 individuals) were isolated. Two coinfections of S. enterica - CFU suspected of E. coli was reported. All these enterobacteria were isolated from the stream water. This study represents the first report of the isolation of S. enterica and S. flexneri in Mexican bats, specifically in S. hondurensis. Finally, the importance of good wastewater management is highlighted to prevent bodies of water from being potential sources of infection for wildlife, as well as the need for multidisciplinary studies with the “One Health” approach.

Key words: Anthropozoonosis; contaminated water bodies; Escherichia coli; Salmonella; Shigella.

Cerca del 45 % de las muertes humanas causadas por enfermedades infecciosas son causadas por enteropatógenos, entre ellos destacan los géneros Salmonella, Shigella y Escherichia. Este grupo de bacterias han sido poco estudiadas en murciélagos y sus ecosistemas. El objetivo de este trabajo fue evaluar la presencia de enterobacterias en murciélagos asociados a un cuerpo de agua contaminado en Xalapa, México. Se colocaron 2 redes de niebla en el riachuelo Honduras, Xalapa, México. Los murciélagos se identificaron a nivel especie y de ellos se obtuvieron muestras de heces, las cuales se inocularon por triplicado en medio Salmonella-Shigella, se incubaron a 35 °C por 48 hr, adicionalmente se aisló, también por triplicado, una muestra de agua del riachuelo. Las especies bacterianas se identificaron mediante colorimetría. Se colectaron 4 individuos de Sturnira hondurensis. Dos individuos fueron positivos para Salmonella enterica, 2 positivos para Shigella flexneri y unidades formadoras de colonia (UFC) sospechosas a Escherichia coli se detectaron en 3 individuos. En 2 individuos se observó la coinfección de S. enterica - UFC sospechosas a E. coli. Todas estas especies se obtuvieron del cultivo del agua del riachuelo. Este estudio representa el primer reporte de aislamiento de S. enterica y S. flexneri en murciélagos mexicanos, específicamente en S. hondurensis. Finalmente, se resalta la importancia del buen manejo de aguas residuales, para evitar que cuerpos de agua sean potenciales fuente de infección para la fauna silvestre, así como la necesidad de estudios multidisciplinarios con el enfoque de “Una Salud”.

Palabras clave: Antropozoonosis; cuerpos de agua contaminados; Escherichia coli; Salmonella; Shigella.

In recent decades there has been an increase in the study of zoonotic diseases from different perspectives, such as prevention (Quaresma et al. 2023), their effect on biological conservation (Galindo-González 2023), the role of wildlife as reservoirs (Wood et al. 2012), epidemiological studies (van der Westhuizen et al. 2023), evaluation of transmission routes between hosts (Colunga-Salas et al. 2022), among other aspects. However, since the transmission cycles of zoonotic pathogens include animals, humans, and ecosystems, the “One Health” approach has been widely proposed and used in multidisciplinary studies (Cunningham et al. 2017). This approach seeks to study the emergence of zoonotic pathogens from a holistic point of view, bringing together various areas aiming to assess the veterinary, human and ecosystem health together to address and prevent among other emergencies, those caused by zoonotic pathogens (Lerner and Berg 2015; Cunningham et al. 2017; Colunga-Salas et al. 2021).

One of the groups of zoonotic pathogens that cause ~ 45 % of human deaths due to infectious diseases are enteropathogens, mainly bacteria and viruses, that causes diarrhea (Ecker et al. 2005). Particularly, enterobacteria stand out, a family of Gram-negative bacilli of medical importance, according to the World Health Organization (WHO 2023). The species of genus Salmonella, Shigella, and Escherichia are noteworthy due to the number of pathogenic strains and the presence of drugs-resistant strains; their transmission is mainly carried out through infected drinks and foods (Pérez-Guerrero et al. 2014). Paradoxically, this group of enterobacteria has been widely studied in the human population given the high mortality, but poorly studied in terms of its presence and effect on wild mammals’ health, as well as ecosystem health (Colunga-Salas et al. 2021). Currently, the genus Salmonella comprises only 2 species, S. enterica and S. bongori, both with several serovars. Of this group, S. enterica serovar Typhi stands out, whom natural reservoirs are only humans, with an annual estimate of ~ 9 million typhoid cases, of which, about 110,000 are fatal (WHO 2023). On the other hand, S. enterica serovar Typhimurium also highlight among the rest of the serotypes for its high morbidity and mortality in humans, which are its only natural reservoirs. The symptoms appear between 6 and 72 hr after ingestion of contaminated food or water and the infection persists between 2 to 7 days (Mellado-Ferreiro et al. 2016). On the other hand, the genus Shigella comprises 4 species, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. Around, ~164,000 annual deaths are attributable to Shigella species (Kotloff et al. 2018; Bennish et al. 2020). The most important species are S. flexneri and S. sonnei, which are characterized by fulminant dysenteries with a wide range of intestinal complications. Like Salmonella, this genus only has humans as its natural reservoirs (Kotloff et al. 2018). Escherichia coli stands out as the agent that causes the most infections and diarrhea in humans. It is part of the normal intestinal microbiota of several mammalian species, but due to its great capacity for mutation, it can generate resistance to immune mechanisms, causing various episodes of reinfection, reaching the circulatory system and urinary tract (Mueller and Tainter 2023). Within this species, several serotypes have been described with a wide human pathogenesis such as enterohaemorragic diarrhea (Campos et al. 2004) or hemolytic-uremic syndrome (Freedman et al. 2023).

A total 12 of the 18 families of bats throughout all continents have been associated with these enterobacteria (Colunga-Salas et al. 2021). The alimentary guild most affected are frugivores, due to the spread of bacteria through contaminated fruits (Colunga-Salas et al. 2021). Also, there is a report of antibiotic-resistant enterobacteria in bats, which could provide evidence that humans and these organisms coexist closely, which can favor the spread of these pathogens (Cláudio et al. 2018). Recently, the presence of antibiotic-resistant strains of Salmonella, Staphylococcus, and E. coli in the digestive tract of fruit bats which were captured near a contaminated river in Bangladesh, were reported (Uddin et al. 2020). For this reason, the objective of this work was to record and isolate enterobacteria from the digestive tract of bats associated with a water body that receives wastewater discharges in eastern México.

During a 2-day sampling (June 29 and 30, 2023), 2 12-meter mist nets were placed from 18:30 to 23:00 hr along the stream bank of the Honduras River (19° 30' 36" N, 96° 55' 12" W; and 19° 30' 0" N, 96° 55' 12" W) in the central area of Xalapa - Enríquez, Veracruz, México (Figure 1a, b). The mist nets were checked every 30 min, all individuals were manually releasing and morphologically identified using the field guide of Medellín et al. (2008) and then were released at the same sampling site. Handling of vertebrate hosts was done following all requirements specified in the General Wildlife Federal Law of México (Ley General de Vida Silvestre) under collection permit SGPA/DGVS/03821/22, issued to P. F. Colunga Salas. From each released bat, a feces sample was taken directly from the anus and immediately collected in a 1.5 µl low-adherence tubes (LoBind®, Eppendorf AG, Hamburg, Germany) previously sterilized and with 500 µl of isotonic sodium chloride (NaCl) saline solution at 0.9 % NaCl (PiSA, Lot P23E701). Previously to isolation, fecal samples were homogenized using a vortex and incubated at 25 °C for 12 hr as previously reported (Dusch and Altwegg 1995; Mikoleit 2010). Additionally, in a previously sterilized 50 ml tube, a water sample from the Honduras River in the adjacent site where the nets were placed was taken.

As the gold standard test for detection of Salmonella and Shigella in mammalian feces still being the isolation and culture in selective media (Andrews and Ryan 2015; Tai et al. 2016; CDC 2019; CDC 2024), the isolation and identification of enteropathogens from the fecal samples was carried out by triplicate in Petri dishes with Salmonella-Shigella agar (SS) (MCD LAB, Lot 716122E002, a certificate culture medium for clinical testing; de la Garza-Garza et al. 2020; Flores-Alfonso et al. 2021; Parra-Flores et al. 2022) following the manufacturer's instructions, incubating the culture media at 35 °C for 48 hr. This culture medium reports a recovery percentage greater than 80 % for S. flexneri, S. enterica serovar Typhi, and S. enterica serovar Typhimurium, as well as 25 % for E. coli and the inhibition of Enterococcus faecalis and Staphylococcus aureus (data sheet available: https://mcd.com.mx/medios-de-cultivo/45-158-agar-salmonella-shigella.html). This identification of the colony-forming units was carried out based on the color, as mentioned by the manufacturer, with colorless or transparent colonies for S. flexneri, colorless or transparent colonies with black center for S. enterica serovar Typhimurium and serovar Typhi and pink colonies with pink precipitate zone for CFU suspected of E. coli (see data sheet of the culture medium previously mentioned). As a positive control, the stream water sample was also cultured in triplicate. Finally, 2 negative controls were included. The first was a triplicate culture of the stock of saline solution used as a collection buffer and the second was a Petri dish with SS medium open throughout the entire sample cultivation process. All cultures were performed in a laminar flow hood previously disinfected with 0.01 % NaClO followed by 70 % ethanol.

Four female individuals were captured (1 juvenile and 3 adults), all identified as Sturnira hondurensis and without signs of reproductive activity (Figure 1c). The presence of transparent-slightly beige colonies with a black dot characteristic of S. enterica was identified in 2 samples (Figure 2a, 2b). Similarly, the presence of S. flexneri colonies was identified in 2 samples (Figure 2c, 2d), given the transparent-slightly beige color and no black dot, as well as the presence of CFU suspected of E. coli, given the pinky color in 3 individuals (Figure 2a, 2b). Coinfection with S. enterica and CFU suspected of E. coli was recorded in 2 individuals (Figure 2b). No coinfections of the 3 bacteria were found in the same bat. No colonies were observed in any of the negative controls (Figure 3a, 3b). On the other hand, the presence of S. enterica, CFU suspected of E. coli, and S. flexneri was confirmed in the sample from the stream (Figure 3c). The total infection frequency of S. enterica and S. flexneri was 0.5 for both cases, while for CFU suspected of E. coli it was 0.75.

This study represents the first attempt to identify and isolate enteropathogens, specifically S. enterica and S. flexneri from Mexican bats. However, a limitation of this work is the impossibility of identifying the S. enterica serovar, because the 2 serovars that can be isolated in the culture medium have similar morphology (de la Garza-Garza et al. 2020). Therefore, it is important to consider additional methods in the future, whether serological or molecular, for the complete identification. On the other hand, it represents the third report of E. coli in Mexican bats (Souza et al. 1999; Sandner et al. 2001; Galicia et al. 2014), and the first report of E. coli, Salmonella sp., and S. flexneri in S. hondurensis.

It is possible that the source of infection of these bacteria occurs at the bats' feeding sites. The above considering that some species of bats defecate near their roosts (Twente 1955; Klite 1965; Staliński 1994). For instance, there is a report of Glossophaga mutica individuals infected with S. enterica serovar Copenhagen, possibly through the consumption of food contaminated with feces from other individuals of the same species, as they tend to defecate around foraging sites (Klite 1965). Therefore, it is possible that a similar transmission mechanism may occur in S. hondurensis, considering their behavior of roosting and foraging in groups (Cortés-Delgado and Sosa 2014; García-García et al. 2014).

Regarding the initial focus of infection of the isolated enteropathogens in S. hondurensis, although it is difficult to infer with our results, it cannot be ruled out that because the Honduras River receives daily discharges of residential wastewater, this could act as a source of a continuous infection. A possible indication of this is that the same bacterial species recovered form fecal samples were also recovered from the stream water, as a previous study suggests may have occurred with fruit bats from Bangladesh (Uddin et al. 2020). However, to corroborate this hypothesis, it is necessary to carry out a study whose methodology allows us to verify if this happens in the sampling area. It is important to highlight that human activities can affect the health of bats. Therefore, promoting good ecosystem health – throughout proper wastewater management, taking actions to prevent the emergence of drug-resistant strains and the responsibly disposing of medical drugs – can positively support the conservation of not only bats but also wildlife associated with human settlements.

Finally, it is important to highlight the necessity of conducting multidisciplinary studies focused on recognizing infection routes and potential factors that may favor the transmission of zoonotic pathogens between humans and wild mammals, as well as among wild mammals themselves. These studies are crucial for developing sanitary and veterinary management plans aimed at promoting optimal ecosystem health and preventing future zoonotic outbreaks (Cunningham et al. 2017).

Acknowledgements

We thank to L. Álvarez-Castillo for their comments that helped to improve the first version of the manuscript. We also thank the two anonymous reviewers who helped improve earlier versions of this note.

Literature cited

Andrews, J.R., and E.T. Ryan. 2015. Diagnostics for invasive Salmonella infections: Current challenges and future directions. Vaccine 33: C8–C15.

Bennish, K. L., et al. 2020. 48 – Shigellosis. Pp. 492–499 in Hunter’s Tropical Medicine and Emerging Infectious Diseases (Ryan, E. T., et al., eds.). Elsevier. Amsterdam, The Netherlands.

Campos, L. C., M. R. Franzolin, and L. R. Trabuli. 2004. Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E. coli O serogroups - A review. Memórias do Instituto Oswaldo Cruz 99:545–552.

Centers for Disease Control and Prevention (CDC). 2019. Salmonella: Diagnostic and Public Health testing. Available at: https://www.cdc.gov/salmonella/general/diag-testing-salmonella.html. Accessed on April 10, 2023.

Centers for Disease Control and Prevention (CDC). 2024. Shigellosis: CDC Yellow Book 2024. Available at: https://wwwnc.cdc.gov/travel/yellowbook/2024/infections-diseases/shigellosis#diagnosis. Accessed on May 20, 2023.

Cláudio, V. C., et al. 2018. Bacteria richness and antibiotic-resistance in bats from a protected area in the Atlantic Forest of Southeastern Brazil. PloS ONE 13:e0203411.

Colunga-Salas, P., et al. 2021. Bats as hosts of important unicellular endoparasites. Pp. 331–348 in 50 Years of Bat Research. Fascinating Life Sciences (Lim, B. K., et al., eds.). Springer. Cham, Switzerland.

Colunga-Salas, P., et al. 2022. Is vertical transmission the only pathway for Rickettsia felis? Transboundary and Emerging Diseases 69:e3352–e3356.

Cortés-Delgado, N., and V. J. Sosa. 2014. Do bats roost and forage in shade coffee plantations? A perspective from the frugivorous bat Sturnira hondurensis. Biotropica 46:624–632.

Cunningham, A. A., P. Daszak, and J. L. N. Wood. 2017. One Health, emerging infectious diseases and wildlife: two decades of progress? Philosophical Transactions of the Royal Society B: Biological Sciences 372:20160167.

de la Garza-Garza, J. A., et al. 2020. Frequency of contamination and serovars of Salmonella enterica and Escherichia coli in an integrated cattle slaughtering and deboning operation. Revista Mexicana de Ciencias Pecuarias 11:971–990.

Dusch, H., and M. Altwegg. 1995. Evaluation of five new plating media for isolation of Salmonella species. Journal of Clinical Microbiology 33:802–804.

Ecker, D. J., et al. 2005. The Microbial Rosetta Stone Database: A compilation of global and emerging infectious microorganisms and bioterrorist threat agents. BMC Microbiology 5:19.

Flores-Alfonso, P. A., et al. 2021. Prevalencia y caracterización molecular de Escherichia coli y Salmonella spp. en gallinas de guinea (Numida meleagris) en dos ranchos de Chiapas, México. Espacio I+D, Innovación más Desarrollo 10:160–170.

Freedman, S. B., et al. 2023. Shiga Toxin–Producing Escherichia coli and the Hemolytic–Uremic Syndrome. The New England Journal of Medicine 389:1402–11414.

Galicia, J., et al. 2014. Diversidad específica bacteriana en murciélagos de distintos gremios alimenticios en la sierra sur de Oaxaca, México. Revista de Biología Tropical 62:1673–1681.

Galindo-González, J. 2023. Avoiding novel, unwanted interactions among species to decrease risk of zoonoses. Conservation Biology 19:e14232.

García-García, J. L., A. Santos-Moreno, and C. Kraker-Castañeda. 2014. Ecological traits of phyllostomid bats associated with sensitivity to tropical forest fragmentation in Los Chimalapas, Mexico. Tropical Conservation Science 7:457–474.

Klite, P. D. 1965. Intestinal bacterial flora and transit time of three teotropical bat species. Journal of Bacteriology 90:375–379.

Kotloff, K. L., et al. 2018. Shigellosis. Lancet 391:801–812.

Lerner, H., and C. Berg. 2015. The concept of health in One Health and some practical implications for research and education: what is One Health? Infection, Ecology and Epidemiology 5:25300.

Medellín, R., H. Arita, and O. Sánchez. 2008. Identificación de los murciélagos de México, clave de campo. Instituto de Ecología, UNAM. México City, México.

Mellado-Ferreiro, M., et al. 2016. Diarrea crónica por Salmonella typhimurium en paciente inmunocompentente. Anales del Sistema Sanitario de Navarra 39:139–141.

Mikoleit, M. L. 2010. WHO Global Foodborne Infections Network. A WHO network building capacity to detect, control and prevent foodborne and other enteric infections from farm to table laboratory protocol: “Isolation of Salmonella and Shigella from Faecal specimens”. Enteric diseases laboratory branch, Centers for Disease Control and Prevention. Atlanta, U.S.A.

Mueller, M., and C. R. Tainter. 2023. Escherichia coli infection. https://www.ncbi.nlm.nih.gov/books/NBK564298/. Accessed on February 8, 2024.

Parra-Flores, J., et al. 2022. Genomic characterization of Cronobacter spp. and Salmonella spp. strains isolated from powdered infant formula in Chile. Frontiers in Microbiology 12:884721.

Pérez-Guerrero, P., et al. 2014. Infecciones por enterobacteria. Medicine - Programa de Formación Médica Continuada Acreditado 11:3276–3282.

Quaresma, P. F., E. S. Martins-Duarte, and L. C. Soares-Medeiros. 2023. Editorial: One Health Approach in Zoonosis: strategies to control, diagnose and treat neglected diseases. Frontiers in Cellular and Infection Microbiology 13:1227865.

Staliński, J. 1994. Digestion, defecation and food passage rate in the insectivorous bat Myotis myotis. Acta Theriologica 39:1–11.

Sandner, L., et al. 2001. The elements of the locus of enterocyte effacement in human and wild mammal isolates of Escherichia coli: evolution by assemblage or disruption? Microbiology 147:3149–3158.

Souza, V., et al. 1999. Genetic structure of natural populations of Escherichia coli in wild hosts on different continents. Australian and New Zealand Journal of Public Health 40:588–591.

Tai, M., et al. 2016. A review of the public health management of Shigellosis in Australia in the era of culture‐independent diagnostic testing. Australian and New Zealand Journal of Public Health 40:588–591.

Twente, J. W. 1955. Some aspects of habitat selection and other behavior of cavern-dwelling bats. Ecology 36:706–732.

Uddin, M., et al. 2020. Ecology of bat drinking behavior and AMR patterns of Sal spp., Staphylococcus spp. and E. coli recovered from fecal droppings of bats and water in BD. International Journal of Infectious Diseases 101:1.

van der Westhuizen, C. G., et al. 2023. Prevalence and occupational exposure to zoonotic diseases in high-risk populations in the Free State Province, South Africa. Frontiers in Microbiology 14:1196044.

World Health Organization (WHO). 2023. Typhoid. https://www.who.int/news-room/fact-sheets/detail/typhoid Accessed on February 8, 2024.

Wood, J. L. N., et al. 2012. A framework for the study of zoonotic disease emergence and its drivers: spillover of bat pathogens as a case study. Philosophical Transactions of the Royal Society B: Biological Sciences 367:2881–2892.

Associated editor: Itandehui Hernández Aguilar.

Submitted: March 6, 2024; Reviewed: May 16, 2024.

Accepted: May 28, 2024; Published on line: June 7, 2024.