Introduction

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.

Materials and Methods

Sampling and bacterial  isolation

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.

Drug susceptibility test

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).

Random-amplified polymorphic DNA (RAPD) analysis

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.

Results

Drug susceptibility test

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)

3         
V, MET, OX, E, CC, RA, P

4    DFX, MET, OX, CFM, NFX, OFX, LS, CP, CAZ, NOR

5    CFM

6    C

7    K

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

10   CAZ

11 V

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

(100) 53

(98) 52

(86) 46

(75) 40

(71) 38

(60) 32

(60) 32

(52) 28

(41) 22

(37) 20

(32) 17

(26) 14

(22) 12

(18) 10

(13) 7

(11) 6

(7) 4

(7) 4

(0) 0

No. of antimicrobial drugs

0

> 1

> 2

> 3

> 4

> 5

> 6

> 7

> 8

> 9

> 10

> 11

> 12

> 13

> 14

> 15

> 16

> 17

> 18

RAPD analysis

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.

Discussion

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-