Antibiotic-resistant salmonellae in pet reptiles in Saudi Arabia

Abstract

We investigated the occurrence rate of antibiotic-resistant salmonellae in exotic pet reptiles in Saudi Arabia. Salmonellae samples were collected from eight different genera of pet reptiles (snakes and lizards). Selective enrichment and selective plating procedures were carried out in order to detect salmonellae. Isolated bacteria were identified using biochemical tests, API 20E strips, and the VITEK compact system. Antimicrobial susceptibility testing was performed using the disc diffusion method. Salmonella spp. belonging to subspecies I (Salmonella enterica ssp. enterica) were detected in 29.2% of the samples. All of the detected salmonellae showed multidrug resistance (p<0.001, χ²). The results demonstrated that pet reptiles in private households could present health hazards to humans. Therefore, these animals should be carefully handled to avoid infection. To the best of our knowledge, this is the first report regarding the occurrence rate of antibiotic-resistant salmonellae in pet reptiles in Saudi Arabia. The detected Salmonella serovars should be subjected to further in-depth molecular analyses in order to understand the overall epidemiology of salmonellosis in Saudi Arabia.

Main article text

INTRODUCTION

Human gastrointestinal infections that are caused by Salmonella species are of global concern. Salmonellae are ubiquitous in the environment, especially in the intestinal tract. Salmonella enetrica subsp. enterica (ssp. I) and subsp. salame (ssp. II) are commonly found in the intestinal tract of warm-blooded animals, whereas Salmonella enterica subsp. arizonae (ssp. IIIa), diarizonae (ssp. IIIb), houtenae (ssp. IV), and indica (ssp. VI) are known to inhabit the intestinal tract of cold-blooded animals, including amphibians and reptiles [1, 2]. The presence of Salmonellae in captive and free-living reptiles has been reported worldwide. Salmonella could infect reptiles with obvious clinical manifestations [3, 4]. Similarly, captive lizards, snakes, and turtles could also serve as asymptomatic reservoirs of these bacteria [5]. Salmonellae infections in pet reptiles could pose health risks to family members and pet handlers in zoos and pet shops. Adults of all ages, immunocompromised individuals, and children could contract salmonellosis either through direct contact with animals or with their fecal droppings [4, 5, 6, 7]. Reptile pets are becoming increasingly popular in Saudi Arabia but there is a lack of awareness regarding their link to salmonellosis. Therefore, the current study was conducted to assess the presence of salmonellae in pet reptiles in Saudi Arabia. Special attention was paid to the multidrug-resistant Salmonella strains and their potential health hazards to the animal handlers.

MATERIALS AND METHODS

Eight pet reptile species were obtained from a private household in Makkah, Saudi Arabia. The imported reptilian species included common boa (Boa constrictor) (n=1), Burmese python (Python bivittatus) (n=2), Schneider’s skink (Eumeces schneiderii) (n=2), and mountain skink (Plestiodon callicephalus) (n=1). The hissing sand snake (Psammophis sibilans) (n=1) and the diadem snake (Spalerosophis diadema) (n=1) represented the local species. All of the reptiles belong to the same owner and were kept in separate cages with the exception of the two Schneider’s skinks. Samples for the study were collected from the fecal droppings, cage swabs, and cloacal swabs of reptiles. All of the samples were collected in aseptic conditions. Three samples were collected for each reptile. In total, 24 samples were examined: eight fecal dropping samples, eight cloacal swab samples, and eight cage swab samples.

Salmonella was isolated by the pre-enrichment of samples at 37°C in buffered peptone water (BPW) (Oxoid, Basingstoke, UK) for 24 h. Positive BPW cultures were further subjected to selective enrichment at 41.5°C in Rappaport-Vassiliadis broth (Molecule-On, Auckland, New Zealand) for 24 h. All of the selective enrichment cultures were streaked on xylose lysine deoxycholate (XLD) (Oxoid) and CHROM agar Salmonella Plus plates (CHROMagar, Paris, France). Plates were aerobically incubated at 37°C for 24 h [8, 9].

The colonies that showed up as red with black centers on XLD agar and as mauve on CHROMagar Salmonella Plus were considered as presumptive salmonellae isolates. All of the presumptive salmonellae samples were subcultured on triple sugar iron agar (TSI) (Molecule-On) and Simmons’ citrate slants (HiMedia, Mumbai, India) and incubated at 37°C for 24 h. The API 20E strips and VITEK 2 Compact system (bioMerieux, Marcy-I’Etoil, France) were used to confirm the salmonellae according to the manufacturer’s instructions [9].

Then, the disc diffusion method was used for testing the antimicrobial susceptibility of all the confirmed Salmonella isolates according to the guidelines of the Clinical & Laboratory Standards Institute [10]. A 0.5 McFarland suspension of each isolate was spread on Mueller-Hinton agar (HiMedia) plates, incubated for 18 h at 37°C, and tested against antimicrobial agents that belong to nine different classes of antibiotics. Antimicrobial agents included ampicillin (10 μg), piperacillin (100 μg), ticarcillin (75 μg), amoxicillin/clavulanic acid (20/10 μg), piperacillin/tazobactam (30/6 μg) [penicillins], cefepime (30 μg), ceftazidime (30 μg) [cephalosporins], aztreonam (30 μg) [monobactams], imipenem (10 μg) [carbapenems], amikacin (30 μg), gentamicin (10 μg) [aminoglycosides], ciprofloxacin (1 μg) [fluoroquinolones], tetracycline (30 μg) [tetracyclines], chloramphenicol (30 μg) [phenicols], and nitrofurantoin (300 μg) [misc. agent]. Escherichia coli ATCC 25922, Pseudomonas aeruginosa NCTC 10662, and Salmonella enterica subspecies enterica serovar Typhimurium ATCC 14028 served as controls.

RESULTS

Salmonellae were detected in seven (29.2%) samples out of the total 24. For most of the pet reptiles, salmonellae were found in the fecal droppings. The diadem snake was an exception – in that case, bacteria were also found in the cloacal and cage swabs. The cage swabs of all other reptiles were negative for salmonellae (Table 1). A higher salmonellae presence in lizards was noted since salmonellae were detected in both lizard species (100%), whereas only 2 snake species (40%) tested positive for salmonellae (Table 1). All of the detected salmonellae belonged to Salmonella enterica ssp. enterica (subspecies I) and exhibited 100% antimicrobial resistance to ampicillin and ticarcillin (penicillins), cefepime and ceftazidime (cephalosporins), and aztreonam (monobactams) (Table 2). Two (8.3%) Salmonella isolates exhibited resistance to amoxicillin/clavulanic acid (penicillins), whereas five (20.8%) were resistant to gentamicin (aminoglycosides), ciprofloxacin (fluoroquinolones), tetracycline (tetracyclines), chloramphenicol (phenicols), and nitrofurantoin (misc. agent) (Table 2). However, none of the isolates demonstrated resistance against imipenem (carbapenems), piperacillin and piperacillin-tazobactam (penicillins), and amikacin (aminoglycosides).

Table 1.

Prevalence of Salmonella in pet reptiles

Reptile species	Origin	Sample type (number)	Number of positive samples	API ID	VITEK ID
Common boa	Imported	Feces (n=1)	1	Salmonella sp.	Salmonella enterica ssp. enterica
		Cloacal swab (n=1)	0	-	-
		Cage swab (n=1)	0	-	-
Burmese python	Imported	Feces (n=1)	0		-
		Cloacal swab (n=1)	0	-	-
		Cage swab (n=1)	0	-	-
Burmese python	Imported	Feces (n=1)	0	-	-
		Cloacal swab (n=1)	0	-	-
		Cage swab (n=1)	0	-	-
Hissing sand snake	Local	Feces (n=1)	0	-	-
		Cloacal swab (n=1)	0	-	-
		Cage swab (n=1)	0	-	-
Diadem snake	Local	Feces (n=1)	1	Salmonella sp.	Salmonella enterica ssp. enterica
		Cloacal swab (n=1)	1	Salmonella sp.	Salmonella enterica ssp. enterica
		Cage swab (n=1)	1	Salmonella sp.	Salmonella enterica ssp. enterica
Mountain skink	Imported	Feces (n=1)	1	Salmonella sp.	Salmonella enterica ssp. enterica
		Cloacal swab (n=1)	0	-	-
		Cage swab (n=1)	0	-	-
Schneider’s skink	Imported	Feces (n=1)	1	Salmonella sp.	Salmonella enterica ssp. enterica
		Cloacal swab (n=1)	0	-	-
		Cage swab (n=1)	0	-	-
Schneider’s skink	Imported	Feces (n=1)	1	Salmonella sp.	Salmonella enterica ssp. enterica
		Cloacal swabs (n=1)	0	-	-
		Cage swabs (n=1)	0	-	-
Total		24	7 (29.2%)

Table 2.

Antimicrobial susceptibility profiles of salmonellae isolated from pet reptiles

Salmonella isolates	Origin	Antimicrobial susceptibility profiles (zone diameter/mm)
		AM	PRL	TC	AMC	TZP	CPM	CAZ	ATM	IMI	AK	GM	CIP	T	C	FT
A1	Common boa	R (0)	S (24)	R (0)	S (21)	S (25)	R (0)	R (0)	R (20)	S (30)	S (19)	S (19)	S (29)	S (18)	S (21)	R (10)
B1	Diadem snake	R (0)	S (21)	R (0)	R (12)	S (28)	R (0)	R (0)	R (19)	S (35)	S (20)	S (20)	S (25)	S (20)	S (20)	S (13)
B2	Diadem snake	R (0)	S (21)	R (0)	R (13)	S (28)	R (0)	R (0)	R (19)	S (35)	S (20)	S (19)	R (21)	S (20)	S (20)	S (13)
B3	Diadem snake	R (0)	S (21)	R (0)	S (16)	S (28)	R (0)	R (0)	R (19)	S (35)	S (20)	R (13)	S (25)	S (20)	S (20)	S (13)
C1	Mountain skink	R (0)	S (26)	R (0)	S (29)	S (30)	R (0)	R (0)	R (20)	S (38)	S (23)	S (21)	S (26)	R (13)	R (14)	S (19)
G1	Schneider’s skink	R (0)	S (20)	R (0)	S (21)	S (34)	R (0)	R (0)	R (19)	S (32)	S (17)	S (19)	S (24)	S (16)	S (18)	S (11)
G2	Schneider’s skink	R (0)	S (20)	R (0)	S (21)	S (34)	R (0)	R (0)	R (19)	S (32)	S (17)	S (19)	S (24)	S (16)	S (18)	S (11)
Number of resistant (total)		7	0	7	2	0	7	7	7	0	0	1	1	1	1	1

The resistance was determined by disc diffusion. S – sensitive, R – resistant, AM – ampicillin, PRL – piperacillin, TC – ticarcillin, AMC – amoxicillin-clavulanic acid, TZP – piperacillin-tazobactam, CPM – cefepime, CAZ – ceftazidime, ATM – aztreonam, IMI – imipenem, AK – amikacin, GM – gentamicin, CIP – ciprofloxacin, T – tetracycline, C – chloramphenicol, FT – nitrofurantoin

Multidrug resistance (resistance to three or more antimicrobial classes) (p<0.001, χ²) was observed in all of the isolated reptilian salmonellae in this study (100%, n=7). The resistance of salmonellae isolates to ampicillin, ticarcillin, cefepime, ceftazidime, and aztreonam was the most frequent resistance pattern (Table 3). One of the isolates exhibited a multidrug-resistance pattern to drugs that belong to five different antimicrobial classes, whereas two groups of three isolates demonstrated multidrug-resistance to drugs that belong to three and four antimicrobial classes (Table 3).

Table 3.

Multidrug-resistance patterns of pet reptile-associated salmonellae

Salmonella isolates	Origin	Resistance pattern	Number of antimicrobial classes
A1	Common boa	AM, TC, CPM, CAZ, ATM, FT	4
B1	Diadem snake	AM, TC, AMC, CPM, CAZ, ATM,	3
B2	Diadem snake	AM, TC, AMC, CPM, CAZ, ATM, CIP	4
B3	Diadem snake	AM, TC, CPM, CAZ, ATM, GN	4
C1	Mountain skink	AM, TC, CPM, CAZ, ATM, T, C	5
G1	Schneider’s skink	AM, TC, CPM, CAZ, ATM	3
G2	Schneider’s skink	AM, TC, CPM, CAZ, ATM	3
P			p<0.001

AM – ampicillin, TC – ticarcillin, AMC – amoxicillin-clavulanic acid, CPM – cefepime, CAZ – ceftazidime, ATM – aztreonam, GM – gentamicin, CIP – ciprofloxacin, T – tetracycline, C – chloramphenicol, FT – nitrofurantoin.

The testing of the hypothesis that pet reptiles carry less abundant multidrug-resistant salmonellae isolates than non-multidrug-resistant ones was done by Pirson χ².

DISCUSSION

Wild and captive reptiles (turtles, snakes, and lizards) serve as symptomatic or asymptomatic carriers of exotic (subspecies diarizonae and arizonae) and human pathogenic (subspecies enterica) Salmonella serovars [2, 11, 12]. This study revealed the presence of Salmonella in two pet snakes (diadem snake and common boa) and two pet lizards (Schneider’s skink and mountain skink) in a private household in Makkah, Saudi Arabia. Lukac et al. [11] have reported a higher salmonellae presence in lizards – 31 (48.4%) compared to 90 (8.9%) snakes. However, the relatively low number of samples examined, and the low number of isolates detected in the current study does not make it feasible to compare the prevalence of salmonellae in the reptiles reported in this study with other published results. The majority of the reptile-associated Salmonella serovars especially belonging to the subspecies diarizonae, arizonae, houtenae, and salamae are not pathogenic to humans [13]. However, human salmonellosis-associated serovars belonging to the subspecies (I) Salmonella enterica ssp. enterica, including Typhimurium, Entertidis, Paratyphi B, Kentucky, and Guinea, have been frequently reported in zoo and pet reptiles [2, 4, 11, 12, 14, 15]. All of the reptile salmonellae isolated in this study were identified as Salmonella enterica subsp. enterica. This subspecies poses potential hazards of containing human salmonellosis-associated serovars.

Multidrug resistance (MDR) – resistance to three or more antimicrobial classes – was noted in all of the bacteria isolated from the reptiles in this study. All of the Salmonella isolates showed resistance to ampicillin, ticarcillin, cefepime, ceftazidime, and aztreonam. Marin et al. [14] have reported multidrug resistance in 72% Salmonella species isolated from reptiles that are kept in private households and pet shops in Spain. Several studies have reported the reptile-associated Salmonella resistance to at least one antimicrobial agent [2, 14, 16, 17, 18]. Similarly, the results of the current study showed that resistance to ampicillin is common for the reptile-associated salmonellae [14, 16, 18]. The presence of the MDR reptile-salmonellae is associated with the animal diet [14]. A high abundance of salmonellae in the pet shop environment, which has been commonly reported, could easily lead to the infection of animals [14].

Contaminated animal feed can be a source of the Salmonella serovars in reptiles that can eventually lead to the salmonellosis of humans who take care of these animals [19]. The transfer of reptiles-associated salmonellosis via direct animal contact or fecal droppings have been reported in several studies [6, 13, 20]. Indirect reptile-associated salmonellosis transmission is also possible during the free-roaming of reptiles in the home, animal kissing, and cage cleaning [15]. In the UK, pet reptiles were reported to cause 27% of the Salmonella cases in children under the age of five [21]. Salmonella Guinea infections in patients younger than 5 years old in the US were found to be linked to the bearded dragon pet [15]. Approximately 7% of the salmonellosis cases in children in 16 US states (93,000 cases annually) in the period of 1996–1998 occurred through direct or indirect contact with pet reptiles (snakes, turtles, and lizards) [22]; that remains relevant today.

The pet reptiles containing multidrug-resistant Salmonella serovars that can lead to human salmonellosis are potentially hazardous in private households. The potential health risks related to improper reptile pet handling should be conveyed to the owners. The owners and pet handlers should also follow hygienic practices when handling pet reptiles. The areas in the house as well as the chairs and tables where the pet reptiles freely roam must be properly cleaned and disinfected. The feed of pet reptiles should also be kept away from the kitchen. To the best of our knowledge, this is the first study on the presence of antibiotic-resistant salmonellae in the pet reptiles of private households in Saudi Arabia. The identification of salmonellae serovars isolated during this study requires further molecular confirmation that will contribute to the further development of salmonellosis epidemiology in Saudi Arabia.

Footnotes

Conflict of interest: All of the authors declare no conflict of interest.

Funding: None

Acknowledgements:

We are grateful to Mohammed Khalid Albar for allowing us to collect samples from the pet reptiles of a private household in Makkah.

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