Molecular Epidemiology of Staphylococcus aureus in Oral and Rectal Swabs from Bats in Pakistan

1. Introduction

Pakistan has a high diversity of bat species with 50 recognized species belonging to 26 genera and 8 families [1,2]. There are numerous ecological and epidemiological traits of bats that make them suitable as reservoirs of pathogens [3,4]. Some bat species can be highly mobile which could facilitate the movement of pathogens between populations, such as Australian flying-fox individuals have been observed flying up to 50 km in a night [5,6]. Different bat species have varied social structures, some are solitary roosters and other species roost in colonies of hundreds to thousands of individuals and form mixed species roosts, a potential factor of transmitting pathogens to co-habituating bats. Furthermore, bats occupy a wide range of habitats from caves, woodlands, and forests to house openings, roof trusses, and crevices in urban areas. During the early migration season fruit bats such as Eidolon helvum often roost by the thousands in trees located in dense urban areas which increases human exposure to the bats’ excreta [7,8,9].

Bats are reservoir hosts to zoonotic and non-zoonotic pathogens such as bacteria, fungi, and viruses [9,10]. However, most research has focused on viruses associated with bats but relatively little research has been conducted on bacterial associations [13]. Previous studies have shown that the nasopharynx and digestive system of fruit bats are probably colonized by the S. aureus complex which showed resistance against tetracycline and erythromycin [14]. Members of the S. aureus complex include S. aureus, S. argenteus, and S. schweitzeri [15]. In humans, S. argenteus can cause skin and soft tissue infections, as well as bacteremia [16,17]. Staphylococcus schweitzeri, on the other hand, mostly colonizes non-human primates and bats [14,18]. However, three cases of S. schweitzeri infection in humans have been documented, with a suspected zoonotic source [19]. Apart from Staphylococcus spp., one study identified 34 bacterial species isolated from fecal swabs of bats kept in captivity. Most of these bacteria are not known to cause sickness in animals and humans except Leptospira sp., Bartonella, and Barrelia, of which some may be pathogenic to humans [20]. Some enteric bacteria such as Shigella sp., Yersinia sp., and Salmonella sp., which are causative agents of human and animal diseases that were also isolated from fecal and tissue samples (i.e., kidney, heart, spleen, liver, lung) of different species of bats [19,20,21].

Some bat species can be a source of multidrug resistance bacteria which pose a risk human and animal health [24,25,26,27]. Antimicrobial resistance genes of bacterial species have been isolated from bat guano samples of Myotis blythii and M. myotis [23], and antibiotic resistance genes were detected in fecal samples of wild and captive grey-headed flying foxes [28]. Antibiotic resistant genes have also been detected in other volant wildlife species [29]. For example, antibiotic resistant genes such as tet(M), erm(A), blaZ, erm(T), fexA, str, eta, and tst were detected in tracheal samples from white storks captured in residential areas [30]. Tetracycline is the drug of choice for the control of certain infections in dairy cattle and poultry, however, excessive use of such widely used antibiotics can result in the development of resistance. The prevalence of antimicrobial resistance (AMR) genes in wildlife species, including bats, could contribute to the epidemiology of what is a major threat to human populations all over the globe. In this circumstance, it is important to study AMR from a ‘One Health’ perspective that includes the diversity of free-ranging wildlife [29,30].

There are few studies on the occurrence of antibiotic resistant bacteria associated with oral and rectal microbiota in bats, however, such studies are lacking in Pakistan. The present study was conducted using molecular and bacteriological diagnostics to identify the presence and antibiotic resistance profile of bacteria in oral and rectal samples collected from bat species captured in KPK and Punjab regions of Pakistan. We also assess the environmental and demographic factors associated with S. aureus prevalence in sampled bat populations.

2. Materials and Methods

2.1. Study area

The present study was conducted at three sites in two different provinces of Pakistan specifically Khyber Pakhtunkhwa and Punjab (Figure 1). Bats were captured and sampled from Malakand caves and a nearby livestock shed in August 2018 and Sheikhupura fort in October 2019. Additional details on the three sampling sites is as follows:

Site I: A cave in district Malakand of Khyber Pakhtunkhwa province (34.4897° N, 71.7978° E), previously used for the mining of chromite but now has become a roosting site for bats after the government banned mining. The cave consisted of five passages which were interconnected mainly by a narrow passage internally. Forty-eight greater mouse-tailed bats (Rhinopoma microphyllum) and 42 fulvous fruit bats (Rousettus leschenaultii) were captured from this site.

Site II: Bats were captured from the cracks of a dilapidated brick shed traditionally used to house livestock. The shed was in Malakand district of Khyber Pakhtunkhwa province (34.07757 N° E71.83435° E), located near to Site I. We captured 10 common pipistrelles (Pipistrellus pipistrellus).

Site III: Sheikhupura fort dates back to 1607 AD. Sheikhupura fort is a very old multi-story building, and due to its dilapidated condition bats occupied crevices in the brick buildings as their roosting site (31.70813° N, 73.99078° E). We captured 17 Java pipistrellus (Pipistrellus javanicus), 82 fulvous fruit bats (Rousettus leschenaultii), and 1 Asiatic lesser yellow house bat (Scotophilus kuhlii).

2.2. Collection of samples and data

Bats were captured (n = 200) by using different types of nets (hand and mist net) and were kept in hygienic cotton bags to prevent cross-contamination. Oral (n = 200) and rectal (n = 200) swabs were collected from each bat using a sterile cotton swab and transferred into an Eppendorf tube (1.5 mL) containing phosphate buffer solution and transported in a dry shipper to the laboratory for bacteriological studies. Bat species, age class, reproductive status, and sex were recorded in addition to the habitat type (natural vs. manmade). All bats were released after non-lethal sample collection.

2.3. Isolation and phenotypic identification of bacteria

Samples were enriched in nutrient broth and incubated at 37^oC for 24 hours. After enrichment, samples were streaked on mannitol salt agar and kept at 37°C for 24 hours. Suspected colonies were subculture on tryptic soya agar plates for purification. After 24 hours of incubation, individual S. aureus colonies were identified colony morphology, shape, and color. Each isolate was identified using Gram staining, oxidase, catalase, indole, voges Proskauer, and urease tests according to standard procedure [33]. Finally, bacterial isolates were preserved in 50% glycerol till further analysis.

2.4. Molecular confirmation of bacteria

Nucleic acid was extracted from isolated bacteria using the FavorPerp TM DNA extraction mini kit (Favorgen Biotech Crop, Taiwan). Molecular confirmation of S. aureus using 16S rRNA gene of 484 base pairs was done [34]. For PCR initial denaturation was done at 94 ºC for 3 min, followed by 35 cycles of amplification with 94 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 30 sec. Five microliters of the amplified product were subjected to 1.5% agarose gel having ethidium bromide stain. Gel documentation system was used for the confirmation of PCR products (Gel Doc EZ imager). PCR products were sequenced in one direction only using primer 16S rRNA (1ST BASE, Malaysia) and sequences were checked using BioEdit (Version 7.2.5) and EMBOSS Seqret. New sequences were submitted to GenBank (accession numbers: OP001633- OP001637).

2.5. Phenotypic and genotypic antibiotic resistance profiling

After confirmation, Staphylococcus aureus isolates were further tested for antibiotic sensitivity against commonly used antimicrobials agents i.e. Tetracycline (30 µg), erythromycin (15 µg), and gentamicin (10 µg) by disc diffusion method [35]. Moreover, antibiotic-resistant genes of tetracycline such as Tetk and TetM were detected with modification as described previously [36]. PCR conditions for the detection of Tetk resistance gene were initial denaturation at 94°C for 3 min, followed by denaturation at 94°C for 30 sec, annealing 55°C for 30 sec, elongation at 72°C for 30 sec, and final elongation at 72°C for 4 min for 30 cycles [36]. In the case of TetM gene PCR conditions were initial denaturation at 96°C for 3 min, denaturation at 96°C for 30 sec, annealing at 54°C for 30 sec, elongation at 72°C for 30 sec, and final elongation at 72°C for 4 min for 30 cycles using a thermocycler (T100^TM Thermal cycler, BioRad, Singapore). The detection of erythromycin resistance gene (ermA) was done as described by [37] with certain modifications; PCR conditions were initial denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, elongation 72°C for 30 sec, and final elongation at 72°C for 4 min for 30 cycles. Gentamicin resistance gene (aacA-D) was detected with modifications as described by[38]. PCR conditions were initial denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, elongation at 72°C for 30 sec, and final elongation 72°C for 4 min for 30 cycles.

2.6. Statistical analysis

The role of biological (bat species, age class, sex, and reproductive condition) and environmental (i.e., habitat type - natural, manmade) factors on the prevalence of S. aureus in oral and rectal swabs were analyzed using Chi-square tests. Post hoc pairwise Wilcox tests were performed if a factor had a significant influence on prevalence. A p-value less than 0.05 was considered significant. All statistical analyses were conducted using R ver. 4.2.2. with base functions [39]

3. Results

In our study, four hundred (200 oral and 200 rectal) swabs from 5 bat species were collected for isolation of Staphylococcus aureus. Overall, fifty-eight (14.5%) swabs were positive for Staphylococcus aureus and were found positive for voges proskauer, oxidase, and urease while negative for Gram stain, catalase, and indole test. We sequenced PCR products of the 16S rRNA gene for 5 samples, confirming these isolates were S. aureus (Genbank Accession numbers: OP001633- OP001637). All five sequences were identical across the 484 base pairs. The highest prevalence of Staphylococcus aureus was isolated from Scotophilus kuhlii (n = 1, 100%), followed by Rousettus leschenaultii (n = 47, 37.90%), and Pipistrellus javanicus (n = 4, 23.52%). Isolates of Staphylococcus sp. were recovered more frequently from rectal swabs (n=31/200, 15.50%) vs. oral swabs (n=19/200, 9.50%) of bats. Isolates were recovered from both rectal and oral swabs collected from 8 individual bats (4% of animals). For oral samples, the highest prevalence of Staphylococcus aureus was recorded in Rousettus leschenaultii (n = 16, 12.90%), followed by Pipistrellus javanicus (n = 2, 11.76%) and Rhinopoma microphyllum (n = 1, 2.08%), respectively. However, rectal samples positive for Staphylococcus aureus were highest in Scotophilus kuhlii (n = 1, 100%), followed by Rousettus leschenaultii (n = 25, 20.16%) and Pipistrellus javanicus (n = 2, 11.76%), respectively. Only two bat species had both swab types positive for Staphylococcus aureus, specifically Rousettus leschenaultii (n = 6, 4.84%) and Rhinopoma microphyllum (n = 2, 4.17%).

Table 1. Prevalence of Staphylococcus aureus isolated from oral and rectal swabs from bat species sampled at sites across northern Pakistan.

Bat species	Swabs collected	S. aureusPrevalence (%)
	Oral/Rectal (no. each)	Oral (95% CI)	Rectal (95% CI)	Total (95% CI)
Pipistrellus javanicus	17/17	11.76 (1.46 – 36.44)	11.76 (1.46 – 36.44)	23.52 (6.81 – 49.89)
Pipistrellus pipistrellus	10/10	0	0	0
Rhinopoma microphyllum	48/48	2.08 (1.31 – 17.20)	10.42 (3.47 – 22.58)	12.5 (4.73 – 25.25)
Rousettus leschenaultii	124/124	12.90 (11.47 – 25.62)	20.16 (17.66 – 33.57)	37.90 (29.35 – 47.05)
Scotophilus kuhlii	1/1	0	100 (2.50 – 100)	100 (2.50 – 100)
Total	200/200	13.50 (9.10 – 19.03)	19.50 (14.25 – 25.68)	29.00 (22.82 – 35.82)

Table 1. Prevalence of Staphylococcus aureus isolated from oral and rectal swabs from bat species sampled at sites across northern Pakistan.

Bat species	Swabs collected	S. aureusPrevalence (%)
	Oral/Rectal (no. each)	Oral (95% CI)	Rectal (95% CI)	Total (95% CI)
Pipistrellus javanicus	17/17	11.76 (1.46 – 36.44)	11.76 (1.46 – 36.44)	23.52 (6.81 – 49.89)
Pipistrellus pipistrellus	10/10	0	0	0
Rhinopoma microphyllum	48/48	2.08 (1.31 – 17.20)	10.42 (3.47 – 22.58)	12.5 (4.73 – 25.25)
Rousettus leschenaultii	124/124	12.90 (11.47 – 25.62)	20.16 (17.66 – 33.57)	37.90 (29.35 – 47.05)
Scotophilus kuhlii	1/1	0	100 (2.50 – 100)	100 (2.50 – 100)
Total	200/200	13.50 (9.10 – 19.03)	19.50 (14.25 – 25.68)	29.00 (22.82 – 35.82)

The association between prevalence of S. aureus and factors such as sampling site, bat species, age, reproductive status, and sex is given in Table 2. The prevalence of S. aureus was significantly different (p < 0.05) amongst sampling sites and bat species. Sampling sites were also a contributing factor for the occurrence of S. aureus in oral and rectal samples which were collected from bats at roosting sites in caves (23.3%) and castle (37 %) while all bats collected from animal sheds were negative. We observed significant variation in prevalence by bat species: Rousettus leschenaultii (35.90 %), followed by Pipistrellus javanicus (23.52 %), Pipistrellus pipistrellus (0 %), and Rhinopoma microphyllum (12.5 %). While only one sample of Scotophilus kuhlii was captured and tested positive (Table 2). Male individuals (36.8 %) tended to be positive for S. aureus more often than female bats (24.1%), but the trend was not significant. There were no significant differences in infection status based on age class or reproductive status.

Figure 1. PCR-based confirmation using primers for 16S rRNA of Staphylococcus isolated from oral and rectal swabs from five bat species. Sequences of these PCR products confirmed all samples were S. aureus. Lane M: (left to right) 100 bp ladder, Lane A to E: Field isolates of S. aureus from bat oral and rectal swab samples.

Figure 1. PCR-based confirmation using primers for 16S rRNA of Staphylococcus isolated from oral and rectal swabs from five bat species. Sequences of these PCR products confirmed all samples were S. aureus. Lane M: (left to right) 100 bp ladder, Lane A to E: Field isolates of S. aureus from bat oral and rectal swab samples.

Based on the disc diffusion method, 58 isolates of S. aureus were subjected to antimicrobial susceptibility tests against three antibiotics: tetracycline, erythromycin, and gentamicin. S. aureus isolates were found to be highly resistant to tetracycline (43.10 %) and gentamicin (43.10 %). While 27.58 % isolates were also resistant to erythromycin (Table 3).

Further, all 58 isolates of S. aureus were analyzed for the presence of resistance genes using PCR assays. Among 58 isolates, 24 oral and 34 rectal isolates were subjected to tetracycline, erythromycin and gentamicin resistance genes detection. A comparatively higher number of oral S. aureus isolates were resistant against TetM (n = 18, 75 %), followed by TetK (n = 17, 70.08 %), aacA-D (n = 15, 62.5 %) and ermA (n = 8, 33.33 %) genes, respectively. Similarly in rectal isolates TetM and TetK genes were detected in 14 (41.17 %) and 12 (35.29 %) isolates while 13 (38.23 %) isolates of S. aureus carried aacA-D genes. In the case of erythromycin resistance gene; ermA was detected only in 6 (17.64 %) isolates (Table 4).

5. Conclusions

In conclusion, the present study confirmed that multidrug resistance S. aureus is prevalent in the oral and rectal microbiota of bats in Pakistan. Sampling site (an environmental factor) and bat species (a biological factor) contributed significantly to differences in prevalence of S. aureus. We provide important baseline data and a first study for Pakistan and the locations and species we investigated. Together with other studies, our finding provide evidence that bats across the globe can harbor multidrug resistance bacteria which could pose human and animal health risks, and if associated with bat morbitity or mortality could be a threat to bat populations themselves. A deeper understanding of these bacterial associations and the animal and human interactions with bats are needed to understand and minimize the chance of cross-species transmission of such pathogens.