Reptiles have been shown to be natural reservoirs of Salmonella and other enteric bacteria and the reptile species close to homes and eateries are house lizards. This study aimed to determine the occurrence of Salmonella in house lizards at residential and eateries 

premises. Fresh faecal samples were collected from live 25 house lizards and 20 pooled dried droppings were collected around premises. None of the samples were positive for Salmonella. It was probable that a number of lizards may be carrying Salmonella as shown by other previous studies and in this case they were not shedding the bacteria in the faeces at the time and in the dried faecal droppings, Salmonellae was probably absent or did not survive the dry and hot condition. Enteric bacteria that were frequently isolated from fresh droppings were Klebsiella pneumoniae and Citrobacter freundii which were found resistant to amoxicillin + clavulanic and tetracycline

Abdul Aziz, Saleha

Lo, Gwen

Universiti Putra Malaysia Institutional Repository

J. Vet. Malaysia (2013) 25 (1&2):11-14   11  Journal Article  OCCURRENCE OF SALMONELLA AND OTHER ENTERIC MICROBES IN FAECES OF  HOUSE LIZARDS (Hemidactylus frenatus)  Gwen Lo and Saleha A.A*  Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Malaysia  SUMMARY  Reptiles have been shown to be natural reservoirs of Salmonella and other enteric bacteria and the reptile species close to homes and eateries are house lizards. This study aimed to determine the occurrence of Salmonella in house lizards at residential and eateries premises. Fresh faecal samples were collected from live 25 house lizards and 20 pooled dried droppings were collected around premises. None of the samples were positive for Salmonella. It was probable that a number of lizards may be carrying Salmonella as shown by other previous studies and in this case they were not shedding the bacteria in the faeces at the time and in the dried faecal droppings, Salmonellae was probably absent or did not survive the dry and hot condition. Enteric bacteria that were frequently isolated from fresh droppings were Klebsiella pneumoniae and Citrobacter freundii which were found resistant to amoxicillin + clavulanic and tetracycline.  Keywords: Salmonella, enteric bacteria, house lizards / geckos, antibiotic resistance   INTRODUCTION  Salmonella lives in the intestines of vertebrates and has been frequently reported in herpatofauna, particularly reptiles. The presence of Salmonella in reptiles was first reported in Gila monster and the regal horned lizard (Chambers et al, 2006). The increase in popularity of exotic reptiles, such as turtles, snakes and iguanas, as pet animals has led to increase in reptile-associated salmonellosis, such as in United States, which is estimated at 93,000 cases annually (Schroter et al, 2004; Pasmans et al, 2005). One reptile species that is of interest is the house lizards or geckos because they are a common sight in residential, commercial and eateries premises. These house lizards are native to Southeast Asia but are now invasively distributed worldwide in other tropical regions and also subtropical regions such as northern parts of Australia, Africa and America. They can be seen at night on walls and ceilings of houses, buildings, eateries, porches and balconies to hunt for insects that are attracted to lights.  Salmonella presences in reptiles are often asymptomatic and they shed the organisms, continuously or intermittently, in the faeces. Humans and pet animals may be infected if they are in contact with Salmonella-laden faecal materials. Conditions associated with salmonellosis in humans include gastroenteritis, bacteraemia, meningitis, osteomyelitis, peritonitis and pleuritis (Chambers et al, 2006).  Salmonella are ubiquitous in nature and able to survive for weeks in water and for years in soil and dust in environmental conditions (such as temperature, humidity and pH) that are favourable (Todar, 2006). Thus, the bacteria can survive in dried faeces in the environment for a period of time. However, the occurrence of Salmonella in    *Corresponding author: Prof. Dr. Saleha Abdul Aziz (Saleha A.A.) Phone: +603 8609 3459; Email: saleha@upm.edu.my. the faeces was low compared to geckos that were actually carrying the bacteria (Chan et al, 1982). Nonetheless, these faeces or droppings are still considered as route of transmission of the infection. Little is known on the faecal carrier status of Salmonella in the house lizards. Apart from Salmonella, not much is known regarding the occurrence of other pathogenic microorganisms in their faeces. This is because the droppings may contaminate food or water supply or the premises environments and that humans may be in contact with these faeces. Thus, the aim of this study was to determine the occurrence of Salmonella and other enteric microbes in the faeces of house lizards and the antibiotic resistance of the bacteria isolated.  MATERIALS AND METHODS  Samples collection  The premises identified to trap the live house lizards were two blocks of a residential college and two cafeterias in UPM, an apartment block and a water tank on the highest floor of an apartment building.  A total of 25 live lizards were caught. These lizards which crept on ceilings and walls were swept down and then captured using gloved hands. The lizards were then individually placed in a bottle (5L) containing a small box in which the lizard can hide inside. The lizards were kept until they defecated. Fresh faeces were collected within 12 hr after defecation using a sterile glove, placed in a plastic bag and brought to the Veterinary Public Health Laboratory, UPM. Most of the lizard defecated within a day after they were captured but a small number took two days. Those that did not defecate within two days were fed with a meal worm until they defecated. Once the faeces were collected, the lizard was released.  J. Vet. Malaysia (2013) 25 (1&2):11-14   12  Twenty (20) pooled lizard droppings were collected from different residential and eateries premises. The lizard droppings could not be differentiated physically in terms of age but were estimated to be more than 5 - 7 days old. Ten (10) of the droppings were collected from several eateries in Seri Kembangan and the other 10 were collected from various houses in Serdang. These droppings could be found on walls, furniture tops, window panes and corners and edges of rooms. The droppings on the floor were not collected.   Bacteria culture, isolation and identification  Unlike fresh faeces, isolation of enteric microbes was not done on the dried droppings, that is, isolation of Salmonella was only conducted on the dried faeces. Each faecal dropping was added into 10ml of buffered peptone water in a bottle for pre-enrichment. The mixture was incubated at 37°C for 24 hr. In the enrichment stage, 1ml of the preenriched sample was transferred into Rappaport Vassiliadis Enrichment Broth (Oxoid). The mixture was then incubated at 37°C for 24 hr. After incubation, a loopful of the enriched culture was inoculated onto Brilliant Green Agar (BGA) and Xylose Lysine Tergital 4 (XLT4) agar (Oxoid) and the plates were incubated at 37°C for 24 hr. Then, the plates were examined for bacterial growths and colonial morphology and the cellular morphology was examined by Gram staining. Different colonies were subcultured onto the same agar for purification. On BGA, typical Salmonella colonies appeared red whereas on XLT4, the bacteria colonies appeared black or black-centred with a yellow periphery. The suspected Salmonella colonies from pure cultures were subjected to biochemical tests for identification which consisted of inoculation into Triple Sugar Iron agar, Lysine Iron agar, Sulphide Indole Motility agar and subjected to citrase and urease tests. Then, suspected Salmonella colonies were subjected to a serological test, that is, a slide agglutination test using polyvalent O Salmonella antisera. To isolate other enteric microbes, the faecal samples were cultured on Blood agar (Oxoid) and MacConkey agar (Oxoid) to isolate Gram positive and Gram negative bacteria respectively. All the different types of colonies obtained were subjected to appropriate biochemical tests to identify the Gram positive and Gram negative bacteria as described in Jang et al (2008).   Antibiotic susceptibility test  Bacteria that were found predominant in the lizards caught in each premises were subjected to antibiotic susceptibility test using the disc diffusion method of Kirby Bauer as described by NCCLS (2002), now known as Clinical and Laboratory Standards Institute or CLSI. Six (6) antibiotics were used, namely streptomycin (S, 10µg), chloramphenicol (C, 30µg), amoxycillin + clavulanic acid (AMC, 30µg), tetracycline (TE, 30µg), trimethoprim-sulfamethoxazole (SXT, 25µg), and enrofloxacin (ENR, 5µg). Briefly, each isolate was suspended into 2mL nutrient broth and standardized to 0.5 Mac Farland standard. A sterile swab was dipped into the suspension and swabbed over the entire surface of Mueller Hinton (MH) agar (Oxoid) plates in three overlapping directions - horizontally, vertically and obliquely. The plates were allowed to dry for approximately 5 min. The six antibiotic discs were dispensed onto the inoculated MH agar plate by using an antibiotic disc dispenser. After incubation, the plates were examined against a dark background to clearly visualize the zone of inhibition around each antibiotic disc. The zone of inhibition was measured using a calliper. The measured zones were compared to the zone diameter interpretive standards breakpoints (NCCLS, 2002) or following the manufacturer instructions to determine the susceptibility of the isolates to the antibiotics.   RESULTS AND DISCUSSION  From the study, Salmonella was not isolated from all the fresh and dried samples. Nine (9) out of 20 dried droppings that were collected from various residential and eateries premises showed no bacteria growth. It is probable that the lizards may be carrying Salmonella but not shedding the bacteria in the faeces at the time, hence it would not be present in the sampled faecal droppings. This is because the study by Fazhana et al (2007) on 32 house geckos found 31.0% positive, with 43.0% and 28.0% from houses and eateries, respectively. In that study, Salmonella was isolated from the gastrointestinal tracts of the house lizards. A study by Calaway et al (2011) in Townsville, North Australia found 7% of the house geckos carried salmonellae in the large intestines. Otokune for et al (2003) reported Salmonella carriage rate of 32% in the gastrointestinal tracts of wall gecko (Geckonidae) and 35% in pooled lizard droppings from various sources. A number of works had reported that Salmonella is able to survive in adverse environment for a considerable period of time; however in the dried lizard droppings sampled, Salmonellae was probably absent or did not survive the dry and hot condition. However, according to Otokune for et al (2003), Salmonella can survive longer in dry as compared to wet environments – 4 weeks in tap water and up to 6 and 8 weeks in droppings left exposed directly to air and mixed with dry sand respectively. The frequency of isolation of different species of enteric bacteria from the faeces of the house lizards sampled is shown in Table 1. The predominant enteric bacteria from house lizards at the residential and eateries premises were Klebsiella pneumoniae, followed by Citrobacter freundii. Other  bacteria that were isolated from the residential premises were Klebsiella oxytoca and Proteus penneri whereas from the eateries were Enterobacter cloacae, Staphylococcus sp and Bacillus sp. Apart from Salmonella, the enteric organisms which Gugnani et al (1986) isolated from wall geckos (Hemidactylus brookei) in Nigeria, included Proteus J. Vet. Malaysia (2013) 25 (1&2):11-14   13  mirabilis, Pseudomonas aeruginosa,  Escherichia coli, K. pneumoniae and E. cloacae. A number of these enteric bacteria are opportunistic pathogens that can cause infections to those who are immunocompromised such as the young, old, people with debilitating disease, cancer or Acquired Immunodeficiency Syndrome. Figure 1 depicts the antimicrobial resistance among all the K. pneumoniae and C. freundii isolates. The two bacteria were found resistant to amoxicillin + clavulanic acid while 80% of K. pneumoniae isolates were resistant to tetracycline. The bacteria were susceptible to enrofloxacin, chloramphenicol, trimethoprim + sulfamethoxazole and streptomycin. The isolated bacteria showed low antibiotic resistance with no multiple drug resistance (resistant to four or more antibiotics) detected; it is most likely due to lack of exposures of the house lizards to environment that were contaminated with resistant bacteria (Pasmans et al, 2005). The house lizards are a common sight in residential, commercial and eateries premises and they indiscriminately litter the premises with their droppings, also reported by Otokune et al (2003).  Although in this study Salmonella was not isolated, other studies had shown otherwise. The presence of house lizards in close proximity to man and the enteropathogens they may carry could cause infections in man should man come in contact with enteropathogen-laden faeces or by contaminating food, water and the floors. A more detailed epidemiological study with larger sample size, more premises and wider locations and to include the zoonotic viruses and enteric parasites would paint a better picture into the possible role of the house lizards in sporadic zoonotic infections such as salmonellosis in households and eateries.    Table 1: Enteric microbes isolated from faeces of house lizards  Bacteria No. of sample with isolates Percentage (%) Klebsiella pneumoniae 15 44.1 Klebseilla oxytoca 3 8.9 Citrobacter freundii 10 29.4 Proteus penneri 1 2.9 Enterobacter clocae 2 5.9 Staphylococcus sp 2 5.9 Bacillus sp 1 2.9 TOTAL 34 100.0                      Figure 1: Antimicrobial resistance among Klebsiella pneumoniae and Citrobacter freundii isolated from house geckos        J. Vet. Malaysia (2013) 25 (1&2):11-14  14  REFERENCES  Calaway, Z., A. Thomas, W. Melrose, P. Buttner and R. Speare (2011).  Salmonella Virchow and Salmonella Weltevreden in a random survey of the Asian house gecko, Hemidactylus frenatus, in houses in Northern Australia. Vector-Borne and Zoonotic Diseases, 11: 621-625. Chambers, D.L. and A. C. Hulse (2006). Salmonella serovars in the herpatofauna of Indiana County, Pennsylvania. Applied and Environmental Microbiology, 72 : 3771 – 3773. Chan, J.G., C., Shero, L., Young and B.  Bereng, B. (1982). Salmonella in two gecko species on the island of Hawaii, Retrieved 29 December 2012, from http://scholarspace.manoa. hawaii.edu /bitstream/handle/10123/18 428/fourth-41-47.pdf.  Fazhana, I, N. Salim and A.A. Saleha  (2007). A study on the presence of Salmonella spp. in house geckos (Hemidactylus frenatus). 2nd Proceedings of the Seminar on Veterinary Sciences, Faculty of Veterinary Medicine, UPM, 15 – 19 January 2007, pp. 24-26.  Gugnani, H.C., J.U. Oguike and R. Sakazaki (1986). Salmonellae and other enteropathogenic bacteria in the intestines of wall geckos in Nigeria. Antonie van Leeuwenhoek, 52: 117 – 120. Jang, S.S., E.L. Biberstein, and D.C. Hirsch. (2008)  A Diagnostic Manual of Veterinary Clinical Bacteriology and Mycology. Davis : University of California.  NCCLS, National Committee for Clinical Laboratory Standards  (2002). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests For bacteria Isolated from Animals; Approved Standard – 2nd Edition. Pennsylvania, USA : NCCLS Otokunefor, T.V., B.I. Kindzeka, I.O Ibiteye, G.U. Osuji, F.O. Obi and A.W.K. Jack (2003). Salmonella in gut and droppings of three pest lizards in Nigeria. World Journal of Microbiology & Biotechnology, 19: 545 – 548. Pasmans, F., A, Martel, F. Boyen,  D. Vandekerchove, I. Wybo., F. Van Immerseel, M. Heyndrickx, J.M. Collard, R. Ducatelle and F. Haesebrouck (2005). Characterization of Salmonella isolates from captive lizards. Veterinary Microbiology, 110:285-291. Schroter, M., P. Roggentin, J. Hofmann, A. Speicher, R. Laufs and D. Mack (2004). Pet snakes as a reservoir for Salmonella enteric subsp. Diarizonae (Serogroup IIIb): a prospectice study. Applied and Environmental Microbiology, 70: 613 – 615. Todar, K. 2006. Todar's Online Textbook of Bacteriology. University of Wisconsin, Madison Department of Bacteriology. Available at: http://www.textbookofbacteriology.net/.                                               

Occurrence of Salmonella and other enteric microbes in faeces of house lizards (Hemidactylus frenatus)

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Occurrence of Salmonella and other enteric microbes in faeces of house lizards (Hemidactylus frenatus)

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