Molecular Detection and Multidrug Resistance of Shigella spp. Isolated from Wild Waterfowl and Migratory Birds in Bangladesh

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Introduction
Every year, thousands of migratory birds travel worldwide for suitable weather and food. Bangladesh hosts millions of migratory birds because of its suitable weather and vast water lands during the winter [1]. Tey are apt reservoirs for spreading pathogenic and antimicrobial-resistant bacteria to the aquatic environment, including wild waterfowl. Moreover, poor sanitation and water management systems help bacterial agents to spread through the fecal and environmental samples into the water bodies. Rural people often come close to these wild waterfowl and migratory bird habitats as they are intimately associated with the water lands for fshing, farming, or bathing. Te same water used by the farmer or fsherman and wild waterfowl and migratory birds enhances the chances of spreading antimicrobial-resistant bacteria to people and poses health risks to humans and animals [2].
Antimicrobial resistance (AMR) may be the next catastrophe of the twenty-frst century that global health will face in the coming days [3]. It has already become a global threat to humans, animals, and the environment. Bacterial pathogens have developed AMR or multidrug resistance due to the selective pressure caused by the abuse and overuse of antibiotics [4]. AMR poses a signifcant risk to both the wellbeing of humans and the growth of the economy. Low-and middle-income countries (LMICs) in Africa and Asia, such as Bangladesh, would be afected the most strongly. Tey spread in numerous ways, mainly through environmental sources, food chains, and human and animal waste [5].
Shigella is a Gram-negative facultative rod-shaped anaerobe with four species responsible for shigellosis which causes watery diarrhea, vomiting, and dysentery with mucoid stool, cramps, and abdominal pain [6]. Moreover, bacillary dysentery caused by Shigella spp. is the most abundant disease for children in middle-and low-income countries worldwide, resulting in thousands of deaths every year [7]. Shigella was frst found in chickens in 2004, and 3day-old chicks showed signs of human dysentery [8]. Recently, Shigella exhibited AMR in the newer or atypical stains by harboring integrons that are more signifcant than any other enteric bacteria [9]. It carries several AMR genes, showing resistance to multiple antimicrobial classes, including beta-lactams, tetracyclines, fuoroquinolones, folate pathway antagonists, and others [10]. Te ability to horizontally transfer genes through mobile genetic materials is the essential factor that makes these bacteria develop antimicrobial-resistant strains [11]. Te fact that Shigella has developed resistance to multiple antimicrobial agents in the modern period highlights how critical it is to maintain constant surveillance of the pathogen. Shigella causes epidemics by contaminating food and water supplies [12]. Wild bird species, such as wild waterfowl and migratory birds have been recognized as major transport modes of pathogens, highlighting the importance of unrestrained animals in the natural setting as a key driver in the spread of infections [13].
Humans, animals, and poultry have all been subjected to considerable research into AMR. Despite the prevalence of AMR, there is still a lot we don't know about it when it occurs in nontypical hosts like wild waterfowl and migratory birds. Previously, several studies identifed bacterial isolates from wild waterfowl and migratory birds in Bangladesh [14][15][16][17][18][19], but, to our knowledge, none focused on the detection of Shigella spp. from wild waterfowl and migratory birds in Bangladesh. Terefore, we conducted this study to detect multidrug-resistant (MDR) Shigella spp. from fecal materials of wild waterfowl and migratory birds in Bangladesh.

Ethical Approval.
All the methods and procedures followed in this study were approved by the institutional ethical committee (AWEEC/BAU/2019 (14)).

Sample Collection and
Processing. Eighty freshly dropped wet fecal materials of wild waterfowl (n � 50) and migratory birds (n � 30) were collected from Jahangir Nagar University (23.8796°N, 90.2726°E), Savar, Bangladesh ( Figure 1), from December 2020 to March 2021. In this study, two types of wild waterfowl (Asian Openbill Stork: Anastomus oscitans, n � 30; Oriental Darter: Anhinga melanogaster, n � 20) and one type of migratory birds (White Stork: Ciconia ciconia, n � 30). Fecal materials were collected by swirling a sterilized cotton bud into the wet fecal materials [1] and shifted to sterilized zip-locked bags with unique identifcation name badges. All the samples were then transferred to the laboratory by maintaining a proper cooling chain. Immediately after transferring them to the laboratory, a sample containing a sterile cotton bud was taken into 5 ml of sterile nutrient broth (HiMedia, India) and left overnight in a shaker incubator at 37°C.

Isolation of Bacteria.
After overnight enrichment, a loopful of broth was transferred and streaked on a Salmonella-Shigella (SS) Agar (HiMedia, India) plate. Subsequently, the streaked plates were transferred to the incubator and left for 18-24 hours at 37°C. Serial subcultures were performed to acquire pure colonies of the target bacteria. Large, circular, convex, and transparent colonies on SS agar plates were suspected to be Shigella spp. Culture-positive samples were then subjected to Gram staining and diferent biochemical tests (oxidase, urease, carbohydrate fermentation test or mannitol, H 2 S, methyl red, motility, sucrose, citrate utilization, lysine decarboxylase, and indole tests) [20].

Molecular Detection of Shigella spp.
Finally, the suspected isolates were confrmed as Shigella by polymerase chain reaction (PCR) targeting the genus-specifc gene, as mentioned in Table 1.
Before performing PCR, the genomic DNA of isolated bacteria was extracted by the boiling and chilling method [24]. In brief, a pure colony of Shigella was inoculated in one ml of sterile nutrient broth contained in an Eppendorf tube and placed in a shaker incubator for overnight growth at 37°C. Overnight incubated culture was centrifuged at 5,000 rpm for 5 min and the supernatant was discarded. Ten, one ml of phosphate bufer solution (PBS) was added in the same Eppendorf tube, and the same centrifugation procedure was followed. After the subsequent centrifugation, the supernatant was discarded, and 250 μl of PBS was added and mixed with the vortex. Te suspension was then boiled for 10 minutes and chilled for another 10 minutes. After cooling, the tube was centrifuged at 10,000 rpm for 10 minutes, followed by the collection of the supernatant as genomic DNA. Te fnal product was stored at −20°C until subsequent use.
To amplify the specifc gene of Shigella, a total of 20 μl of PCR volume was prepared with 10 μl of PCR master mix 2X (Promega, USA), 1 μl of each forward and reverse primer (100 pmol), 4 μl of nuclease-free water, and 4 μl of genomic DNA. Te amplifed product was analyzed through gel electrophoresis (100 volts) in 1.5% agarose gel (HiMedia, India) and visualized under an ultraviolet transilluminator (Biometra, Germany). A 100 bp DNA ladder (Promega, Madison, WI, USA) was utilized to distinguish the target amplicon size of the amplifed gene.

MAR �
The number of antimicrobial agents to which a particular Shigella isolate shows resistance The total number of antibiotics that were utilized in treating an isolate . (1)

Discussion
Migratory birds can act as carriage and spreader of antibiotic-resistant Shigella spp. during migration, but their resistant bacteria disseminating activities are still frequently neglected. Tey have the potential to disseminate bacterial pathogens to other waterfowl in Bangladesh. Terefore, we screened fecal materials of wild waterfowl and migratory birds in Bangladesh in this study to fnd MDR Shigella spp.
In this study, to our knowledge, we detected Shigella spp. in wild waterfowl and migratory birds for the frst time in Bangladesh, showing an occurrence rate of 18.75%. Te high detection rate of Shigella spp. indicates that wild waterfowl and migratory birds are vastly associated with the dissemination of Shigella spp. in the environment and from environmental sources. As wild waterfowl and migratory birds are directly linked to environmental sources, they have the potential to contaminate the environment with Shigella species. During the various types of migration, migrating birds typically occupy a diversity of ecological niches and develop a range of distinct feeding behaviors. In the course of these migrations, these birds can become hosts for Shigella spp. and aid in the pathogen's spread from one location to another. Moreover, Shigella spp. are increasingly isolated in migratory birds, and the possibility of their movement and transmission by wild birds is a growing public health concern. Because of this, it is even more crucial to keep an eye on migrating bird populations so that we may anticipate an epizootic state of the Shigella to one-health components. Previously, Alam et al. [13] detected Shigella spp. (36% of the samples) from watering sites of migratory birds in Pakistan, which is higher than the present study. However, a lower detection rate (4.1%) was also recorded in Ghana by Modupe et al. [30]. Moreover, Zhao et al. [31] reported Shigella spp. (11.02%) in duck-type waterfowl. Te disparities in results might be related to variances in the climate and environment of locations, particularly temperature variations that afect bacterial development, as well as the types of wild waterfowl and migratory species, sample types, and sample size. Furthermore, migrating birds may have a change in their Veterinary Medicine International rate of bacterial shedding owing to the stressful situations they endure throughout migration [32].
Resistance to antimicrobial agents is an urgent public health concern. In the present study, Shigella isolates exhibited high to moderate resistance to imipenem, tetracycline, azithromycin, ampicillin, cotrimoxazole, ciprofoxacin, meropenem, and streptomycin. Surprisingly, a high percentage of resistance was observed in Shigella isolates against imipenem (93.33%) and meropenem (26.67%), showing an alarming situation for public health. Carbapenem antibiotics, of which imipenem and meropenem are members, are reserved for the direst of human medical emergencies [33,34]. However, further confrmation of this resistance using MIC and molecular approaches is required before any defnitive conclusion can be drawn. Moreover, it is noted that Shigella isolates showed a higher resistance to azithromycin (80%) and ciprofoxacin (40%), indicating a treatment limitation in humans. Te Centers for Disease Control and Prevention (CDC) has recommended that ciprofoxacin (for adults) and azithromycin (for children) be considered frst-line antibiotics for the treatment of shigellosis [35]. Moreover, as a member of the quinolone antimicrobial class, the use of ciprofoxacin in animals has been severely limited since the 1990s, when fuoroquinolone resistance began to spread rapidly [36]. Terefore, the presence of ciprofoxacin resistant Shigella spp. in wild waterfowl and migratory birds suggests a serious issue for human health. Furthermore, statistical analysis using a bivariate analysis demonstrated a signifcant positive correlation existed between the resistance patterns of ciprofoxacin and cotrimoxazole, tetracycline and imipenem, tetracycline and ampicillin, and tetracycline and imipenem. Possible causes for the strong antimicrobial connections discovered include the indiscriminate use of antibiotics in livestock and poultry in regions frequented by wild waterfowl and migrating birds. Possibly related factors include environmental contamination, particularly in water sources.
In our current study, the tetA (69.23%) and SHV (50%) genes responsible for resistance to their corresponding antibiotics were found in Shigella spp. isolates. Te tetB gene was not detected in any of the isolates. Tere is a possibility that mobile genetic elements are to blame for the existence of a variety of resistance genes in Shigella spp. isolates [37]. Moreover, the existence of these resistance genes in Shigella spp. can be attributed to many mechanisms, including a decrease in cellular permeability, the extrusion of drugs by active efux pumps, the overexpression of drug-modifying and inactivating enzymes, or target modifcation by mutation [10]. Te detection of resistant Shigella spp. in wild waterfowl and migratory birds might be associated with the transmission of antibiotic resistance genes from environmental sources. Water contaminated with feces of wild waterfowl and migratory birds might be deemed a signifcant risk factor for the dissemination of resistant Shigella spp. pathogens and their resistance genes.
Te efects of infections caused by MDR bacteria are extremely serious for human health and may even be fatal. In this study, 86.67% of the Shigella isolates were phenotypically MDR in nature, showing resistance to up to seven diferent classes of antibiotics. Previously, Alam et al. [13] reported that all the Shigella isolates from migratory birds in Pakistan showed multidrug resistance, which is higher than this study. Tese diferences might be due to variations in geographical locations, types of birds, sample sizes, detection methods, and others. Moreover, 12 out of 15 isolates had more than 0.2 MAR indices. Sources with a MAR value greater than 0.2 indicate heavy antibiotic usage, suggesting the presence of MDR-prone Shigella spp. [28]. Te detection of high levels of MDR Shigella spp. along with their high MAR indices in wild waterfowl and migratory birds revealed an emerging situation. Tis might be

Conclusion
To the best of our knowledge, this is the frst study in Bangladesh to isolate and identify Shigella spp. from fecal materials of wild waterfowl and migratory birds. Te results of this study revealed the frequent occurrence of MDR Shigella spp. and their resistance genes in wild waterfowl and migratory birds in Bangladesh. Te identifcation of MDR Shigella spp. in wild waterfowl and migrating birds raises signifcant concerns for the general public's health due to the potential of these pathogens to contaminate ecosystems and spread to One Health components. Tese birds therefore need to be kept under active strict surveillance with a One Health approach as a crucial step in combating the transmission of zoonotic potential Shigella spp. and their associated AMR hazards.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.