Document Type : Infectious agents- Diseases
1 Department of Avian Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
2 Department of Avian Diseases Faculty of Veterinary Medicine University of Tehran Tehran, Iran
Article Title [Persian]
زمینه مطالعه: استافیلوکوکوس اورئوس به عنوان عامل عفونتهای استافیلوکوکی در بسیاری از حیوانات از جمله پرندگان شناخته می شود. علیرغم اهمیت این پاتوژن در پرندگان زینتی و حیات وحش، تاکنون مطالعه ای در مورد عفونت های استافیلوکوکی در پرندگان زینتی ایران انجام نشده است.
هدف: هدف از این مطالعه بررسی الگوی مقاومت دارویی جدایه استافیلوکوکوس اورئوس از پرندگان زینتی ارجاعی به کلینیک تخصصی پرندگان دانشکده دامپزشکی دانشگاه تهران و همچنین تعیین الگوی RAPD-PCRاین جدایه ها می باشد.
مواد و روش کار: طی این مطالعه از پرندگان ارجاعی به کلینیک مذکور نمونه گیری به عمل آمد. پس از تایید با روش های باکتریولوژی، نهایتاً 53 نمونه استافیلوکوکوس اورئوس جداسازی گردید. روش استاندارد دیسک دیفوژیون برای تعیین حساسیت جدایه ها به 30 عامل ضد میکروبی مورد استفاده قرار گرفت. همچنین جدایه های استافیلوکوکوس اورئوس توسط روش RAPD-PCR با دو جفت پرایمر 10 نوکلئوتیدی تعیین تیپ شدند.
نتایج: مطالعه ی حاضر نشان داد که جدایههای استافیلوکوکوس اورئوس پرندگان زینتی، بیشترین درصد مقاومت را به اگزاسیلین (58 درصد)، کلیندامایسین (53 درصد) و متیسیلین (53 درصد) نشان دادند. در بین جدایه های استافیلوکوکوس اورئوس، وقوع مقاومت چندگانه بسیار شایع بود به طوری که آنها حداقل به صفر و حداکثر به 17 دارو مقاوم بودند. همچنین 43 الگوی مقاومت دارویی شناسایی شد. پس از انجام تستRAPD-PCR بر روی 53 نمونه استافیلوکوکوس اورئوس پرندگان، 5 الگو بدست آمد. از مجموع 53 نمونه ای که مورد آزمایش قرار گرفته بودند 20 درصد نمونه ها الگوی ،A ۶۲ درصد الگوی ،B ۳ درصد الگوی ،C ۹ درصد الگویD و ۳ درصد الگویE را نشان دادند.
نتیجه گیری نهایی: نتایج مطالعه حاضر مقاومت آنتی بیوتیکی گسترده را در بین جدایه های استافیلوکوکوس اورئوس در پرندگان زینتی به ویژه حضور جدایه هایMRSA را نشان داد. همچنین ارزش روش RAPD-PCR برای پایش اپیدمیولوژیک استافیلوکوکوس اورئوس در پرندگان زینتی مورد تایید قرار گرفت.
The genus Staphylococcus contains ap- proximately 45 species and 24 subspecies of which Staphylococcus aureus is the most important cause of infections (Andreasen, 2013). However, other species are occa- sionally involved. The infections, sporadic or enzootic, may occur in different avian species with various clinical manifestations including acute septicemia or subacute to chronic arthritis, osteomyelitis and osteitis. Vesicular dermatitis or omphalitis also have been less commonly reported (Andreasen, 2013). Staphylococci, particularly S. aureus, are gram positive bacteria that, primarily or secondarily, can infect humans and are of major concern in human medicine. It has been associated with various life-threatening conditions such as pneumonia, osteomyeli- tis, endocarditis, and septicemia in humans (Watkins et al., 2012). Drug resistance re- mains a major threat to public health, there- fore, understanding the epidemiology of
S. aureus and its prevalent drug resistance pattern is very important in human and vet- erinary medicine. Understanding the meth- icillin resistant S. aureus existence in com- panion animals including pet birds, due to its zoonotic concerns, must be noticed (Si- moons-Smit et al., 2000).
In order to type S. aureus isolates, various pheno- and genotypic methods have been investigated by different researchers (Lui- jendijk et al., 1996; Tambic et al., 1999; But- terworth et al., 2001; Lee, 2003; Reinoso et al., 2004). The random-amplified polymor- phic DNA-PCR (RAPD-PCR) is a method for S. aureus typing and has been designed based on single primer of arbitrary nucle- otide sequence, attaching to their possible sites throughout genome, and resulting in a different pattern of amplified DNA segments
on the gel. The RAPD-PCR is an appropri- ate, simple, inexpensive and efficient tool for
S. aureus typing, and is applicable for further
S. aureus features recognition.
There is still limited information on the drug resistance and RAPD-PCR pattern of
S. aureus in birds. Hence, the aim of this study was to genotype 53 S. aureus isolates by RAPD-PCR and to determine the drug re- sistance pattern of S. aureus isolates of birds referred to the pet birds’ clinic of University of Tehran.
During a 4-month period, various species of companion and wild birds referred to the pet birds’ clinic of University of Tehran that were suspected of Staphylococcus infection were sampled by swabbing of the related site. Each sample was cultured on 5% defi- brinated sheep blood agar and MacConkey agar and observed after 18 and 36 h of in- cubation at 37.8 ºC. Bacterial growth of all samples were characterized based on mor- phology, Gram’s stain, catalase test, tube co- agulase reaction and their ability to ferment mannitol anaerobically (Andreasen, 2013). In total, 53 S. aureus isolates were identified, frozen at -70 ºC and kept for future use.
The susceptibility of the S. aureus isolates to a panel of antimicrobial agents was de- termined by the agar disk diffusion method and the interpretation of results was carried out according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2008). The antimicrobial agents that were tested and their concentrations (μg) were as follows: oxacillin (1), methicillin (10), clindamycin
(2), cefixime (5), penicillin (10), rifampicin
(5), ceftazidime (30), vancomycin (30), ka-
namycin, (30), erythromycin (15), norfloxa-
cin (10), amoxicillin (25), streptomycin (10),
ceftizoxime (30), danofloxacin (10), ofloxa-
cin (5), enrofloxacin (5), ciprofloxacin (5),
ampicillin (10), ceftriaxone (30), cefotaxime
(30), meropenem (10), cefepime (30), ami-
kacin (30), neomycin (30), trimethoprim/ sulfamethoxazole (1.25/23.75), azithro- mycin (15), gentamicin (10), linco-spectin (15/200), chloramphenicol (30). All antibac- terial disks were provided from Padtan Teb Co. (Tehran, Iran) except methicillin (Hime- dia, Mumbai, India). The ATCC reference strain S. aureus ATCC 25923 was used for quality control purposes. The isolates were classified as susceptible, intermediate sus- ceptible, or resistant based on the standard interpretation chart updated according to the CLSI guidelines (CLSI, 2008).
To extract bacterial DNA, each S. au- reus isolate was individually cultured on Luria-Bertani (LB) agar and incubated over- night at 37 ºC. Template DNA was prepared from each S. aureus isolate grown overnight at 37 ºC on LB agar using the MBST Genom- ic DNA extraction kit (MBST Co., Tehran, Iran). DNA concentration was estimated us- ing spectrophotometry at 260 nm.
Two primers, A (5’-TGCGCCCTTC) and B (5′-GGTGACGCAG) were used for RAPD typing in this study (Butterworth et al., 2001). The primers and other materials used in PCR reaction were provided by SinaClon (Tehran, Iran). Amplifications were carried out in a 25 μl reaction volume containing 2.5 μl 10 x PCR buffer, 1 μl 10 mM dNTP mix, 0.8 μl 50 mM MgCl2, 1 μl (100 ng) of each prim- er, 0.25 μl (1 unit) of Taq polymerase DNA,
16.45 μl dH2O and approximately 2 μl (200
ng) of template DNA. Negative controls (dH2O instead of template DNA) were in- cluded in all PCR reaction sets. Amplification was programmed in a thermocycler (Senso- Quest, Germany) as follows: 94 °C for 105 s followed by 40 cycles of 94 °C for 60 s, 37 °C for 60 s, 72 °C for 180 s, and a final extension at 72 °C for 120 s. The amplified products were detected by gel electrophoresis in 1.5% agarose gel at 100 V for 90 min in 1 x TAE buffer. A commercial DNA ladder, GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific, Germany), was used as the molecular- weight marker in each gel running. Reproducibility of the RAPD patterns was confirmed using triplet runs on separate days but on the same thermocycler.
The percentages of S. aureus isolates that were resistant to the antimicrobial agents were as follows: 58 to oxacillin, 53 to clin- damycin, 53 to methicillin, 47 to cefixime, 45 to penicillin, 36 to rifampicin, 34 to cef- tazidime, 32 to vancomycin, 32 to kanamy- cin, 30 to erythromycin, 23 to norfloxacin, 23 to amoxicillin, 23 to streptomycin, 21 to ceftizoxime, 19 to danofloxacin, 19 to ofloxacin, 19 to ampicillin, 17 to ceftriax- one, 17 to cefotaxime, 17 to meropenem, 15 to enrofloxacin, 13 to ciprofloxacin, 13 to amikacin, 13 to neomycin, 11 to trimetho- prim/sulfamethoxazole, 11 to cefepime, 11 to azithromycin, 9 to gentamicin, 8 to lin- co-spectin and 4 to chloramphenicol. For- ty-three multi-drug resistance (MDR) pat- terns were observed among 53 S. aureus isolates (Table 1). However, it is noteworthy to mention that the observed MDR patterns were variable, ranging from being resistant to 0 to 17 drugs (Table 2).
Table 1. Drug resistance patterns among 53 companion birds S. aureus isolates
#Pattern Resistant to No. of isolates (%)
1 V, NOR 3 (5.66)
2 K, CFM, S, P 3 (5.66)
V, MET, OX, E, CC, RA, P
4 DFX, MET, OX, CFM, NFX, OFX, LS, CP, CAZ, NOR
8 K, AN, CRO, MET, OX, CT, CC, N, CFM, CTX, MEN, CAZ
9 V, DFX, MET, OX, E, RA, NFX, AMX, OFX, S, AM, SXT, P, CP, NOR
12 CC, CXT
13 CFM, CAZ
14 OFX, CAZ
15 CC, SXT, GN
16 CFM, RA, AM
17 ,MET, OX, CC, AMX
18 MET, OX, CT, CC
19 K, CFM, S, AM, P
20 V, MET, OX, CC, RA, P
21 V, DFX, E, CTX, NOR
22 C, OX, CT, CC, AMX, P
23 OX, CC, CFM, RA, P, CAZ
24 V, OX, E, CC, RA, AMX, P
25 V, MET, OX, CC, RA, AMX, P
26 MET, OX, CC, CFM, AM, MEN, CAZ
27 E, CC, NFX, OFX, LS, CP, NOR
28 V, MET, OX, E, CC, RA, AMX, P
29 V, MET, OX, E, CC, RA, AMX, AM, P
30 V, AZM, MET, OX, E, CC, RA, AMX, AM, P
31 MET, OX, CC, CFM, AM, P, MEN, CAZ
32 K, DFX, MET, OX, CT, CFM, SXT, MEN, CAZ
33 DFX, MET, OX, CFM, NFX, OFX, CP, CAZ, NOR
Each pattern included two isolates (3.77)
Each pattern included only one isolate (1.88)
#Pattern Resistant to No. of isolates (%)
34 K, AN, AZM, MET, OX, CT, E, CC, N, CFM, GM
35 K, AN, AZM, MET, OX, E, CC, N, CFM, CTX, S, MEN, CAZ
36 CRO, AZM, OX, CT, E, N, CFM, RA, AMX, S, FEP, P, CAZ
37 K, CRO, MET, CC, CFM, RA, CTX, OFX, FEP, P, CAZ
38 V, CRO, MET, OX, CT, E, CC, RA, AMX, S, AM, GM, P, NOR
39 K, CRO, DFX, AZM, MET, OX, E, CC, N, CTX, S, FEP, CAZ
40 V, K, DFX, MET, C, OX, E, CC, RA, AMX, OFX, SXT, P, LS, CP, NOR
41 K, AN, CRO, MET, OX, CT, CC, N, CFM, RA, CTX, S, AM, FEP, P, MEN, CAZ
42 K, AN, CRO, MET, OX, CT, CC, CFM, RA, CTX, AM, S, GM, FEP, P, MEN, CAZ
43 K, AN, CRO, DFX, MET, OX, CT, CC, CFM, RA, NFX, CTX, OFX, GM, FEP, P, MEN, CP, CAZ, NOR
Each pattern included only one isolate (1.88)
OX = Oxacillin, MET = Methicillin, CC = Clindamycin, CFM = Cefixime, P= Penicillin, RA = Rifampicin, CAZ = Ceftazidime, V
= Vancomycin, K= Kanamycin, E = Erythromycin, NOR = Norfloxacin, AMX = Amoxicillin, S = Streptomycin, CT = Ceftizoxime, DFA = Danofloxacin, OFX = Ofloxacin, AM = Ampicillin, CRO = Ceftriaxone, CTX = Cefotaxime, MEN = Meropenem, NFX = Enrofloxacin, CP = Ciprofloxacin, AN = Amikacin, N = Neomycin, SXT = Trimethoprim/sulfamethoxazole, FEP = Cefepime, AZM
= Azithromycin, GM = Gentamicin, LS = Lincospectin, C = Chloramphenicol
Table 2. Multi-drug resistance level among 53 S. aureus isolates from companion birds
No. (%) of resistant isolates
No. of antimicrobial drugs
Using primers A and B, five differ- ent RAPD types (A to E) were observed among 53 isolates (Fig. 1). The RAPD pro- files differed in the number of fragments and ranged from 0.2 to 2.5 kb in molecular weight. Among 53 isolates, 11 (20.76%),
33 (62.27%), 2 (3.77%), 5 (9.43%) and 2
(3.77%) showed RAPD patterns of A, B, C, D and E, respectively. The pattern B was the most frequent one which were mostly taken from the cloaca and choanal cleft samples. The pattern D was observed in two isolates, both from canary conjunctivitis. The pattern E was also observed in two isolates obtained from European nightjar (Caprimulgus eu-
ropaeus) and domestic chicken (Gallus do- mesticus) foot pad abscess. All five of the samples taken from African gray parrots (Psittacus erithacus) showed pattern A. In this study, four isolates were detected from sulphur-crested cockatoo (Cacatua galer- ita) and sparrow hawk with the pattern C. In the current study, nine isolates were from common myna (Acridotheres tristis) and except one isolate from pododermatitis that belonged to pattern A, the rest belonged to pattern B. Additionally, out of six isolates from pododermatitis four isolates were clas- sified as pattern A, whereas all isolates from domesticated ducks (Anas platyrhyncha) showed pattern B.
Figure1. RAPD-PCR sample pattern derived from avian S. aureus isolates separated by electrophoresis on 1.5% agarose gel.
This study determined the drug resistance patterns and RAPD profiles of 53 S. aureus isolates recovered from pet birds. Staphylo- coccus aureus may be the cause of fetal loss, omphalitis, yolk sac inflammation, arthritis, synovitis, septicemia, osteomyelitis, vesic- ular dermatitis, gangrenous dermatitis and pododermatitis in birds (Andreasen, 2013).
Antimicrobial resistance determination of
bacterial isolates recovered from pet birds is of great importance due to the close re- lationships that exist between pet birds and human beings. The patterns of antimicrobial resistance among S. aureus isolates is critical because of the possible presence of methi- cillin- resistant isolates. In this study, more than 50% of isolates were resistant to methi- cillin. The methicillin antibiotic is extensive- ly used against human staphylococcal infec-
tions (Harkins et al., 2017). Furthermore, the methicillin-resistant S. aureus is frequently reported in human nosocomial infections (Wang and Ruan, 2017; Lounsbury et al., 2019). Therefore, S. aureus infection among pet birds is a matter of concern because keeping companion birds is very popular in Iran. The highest resistance rate among iso- lates of this study was observed in penicillin family. Resistance to clindamycin was also very high (53%) among isolates. Clindamy- cin is one of the most effective drugs against staphylococcal infections and is being wide- ly administered in companion bird in Iran.
Antimicrobial susceptibility pattern of S. aureus isolates originating from avian spe- cies have been reported by various research- ers. Lee (2003) found 15 PCR mecA-posi- tive MRSA isolates in which 12 were from dairy cows and 3 were from chickens. All isolates were resistant to members of the penicillin family, such as ampicillin, oxa- cillin, and penicillin (Lee, 2003). In another study, Susa et al. (2014) analyzed the anti- microbial resistance determinants of staph- ylococcal nasal microbiota in 16 birds of prey and their contents and found that six of the 16 tested animals carried staphylococci (37.5%). The S. aureus isolates were penicil- lin-resistant but methicillin-susceptible. Due to possible contact that may occur among wildlife, domestic animals, humans, insects and even non-living facilities, an increased possibility of interchange of these micro- organisms in the different ecosystems may lead to transmission of antimicrobial resis- tance, including MRSA isolates, to pet birds (Simoons-Smit et al., 2000; El-Mokhtar and Hetta, 2018; Kwok et al., 2018; Abdolmale- ki et al., 2019). In a German study, the re- sistance pheno- and genotypes of 37 MRSA isolates from various sources in four broil-
er farms were investigated. Except for one farm, isolates from chickens, broiler hous- es, the farm residences and humans living/ working on the same farm were often closely related or indistinguishable. MRSA isolates from the same farm showed apparent iden- tity indicating transmission among broilers, humans and their environment (Wendlandt et al., 2013).
Random amplified polymorphic DNA (RAPD)-PCR has been frequently used to determine molecular epidemiological relat- edness of S. aureus isolates originated from various sources (Tambic et al., 1999; Butter- worth et al., 2001; Lee, 2003; Reinoso et al. 2004). Tambic et al. (1999) in Zagreb detect- ed four RAPD profiles among 36 S. aureus isolates from inpatients and staff in a hospi- tal and found that the most common profile involved 15 of 36 tested strains and indicat- ed the RAPD-PCR typing as a useful aid to epidemiological investigations of MRSA (Tambic et al., 1999). In a study in England (Butterworth et al., 2001), 111 S. aureus iso- lates from chickens were typed by RAPD- PCR and four main groups were found based on the observed banding patterns. The pre- dominance of a restricted number of RAPD types in association with pathologies causing lameness was noticed and it was concluded that the putative RAPD groupings may pro- vide a basis for epidemiological studies of S. aureus in broiler production systems (Butter- worth et al., 2001). Lee (2003) determined molecular epidemiological relatedness of 15 animal MRSA isolates from humans and an- imals by RAPD patterns, and found a close relationship between the genomes of the six animal MRSA isolates to those of some hu- man MRSA isolates and suggested that those animals may be the possible source of hu- man infections caused by consuming con-
taminated food products. Using RAPD-PCR with three primers, Reinoso et al. (2004) as- sessed successfully the genetic relationship of S. aureus isolates from different hosts, bovine and human in this case (Reinoso et al., 2004). The findings of the present study were comparable to those of other studies. In this study, RAPD-PCR was used for molec- ular typing of S. aureus isolates from com- panion birds leading to identification of five different RAPD types from A to E, in which the type B was the most frequent one. How- ever, we were not able to exactly correlate a specific RAPD type to a specific pathology. In conclusion, this study showed that the resistance to antimicrobial agents among S. aureus in pet birds is widespread and it is very important to choose the right antimi- crobial agent for treatment purposes. The presence of MRSA among S. aureus isolates indicates that serious measures need to be taken against such pathogens due to its pub- lic health concerns. The present investiga- tion also found the value of RAPD-PCR for epidemiologic monitoring of S. aureus in pet
This research was supported by grant no. 7508007-6-25 from the Research Council of the University of Tehran.
The authors declare that there is no con- flict of interest.