MOLECULAR CHARACTERIZATION OF STREPTOCOCCUS AND ENTEROCOCCUS SPECIES ISOLATED FROM BROILER CHICKENS

Assiut University web-site: www.aun.edu.eg

MOLECULAR CHARACTERIZATION OF STREPTOCOCCUS AND ENTEROCOCCUS SPECIES ISOLATED FROM BROILER CHICKENS

AHMED H. ABED ¹; ASMAA S. A. SEDIK ¹ AND SALAMA A. S. SHANY ²

¹ Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.

² Poultry Diseases Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.

Received: 19 September 2021;     Accepted: 3 October 2021

INTRODUCTION

The poultry industry is considered one of main sources of animal protein (meat and

Corresponding author: Asmaa S.A. Sedik

E-mail address: asmaa.saad91@yahoo.com

Present address: Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.

egg) to man also it is a good source of  manure for crops. (Mohammed and Sunday, 2015). Streptococci and Enterococci are intestinal inhabitants of birds and mammals and they may accidentally enter circulation and causing disease in poultry (Smyth and McNamee, 2001). Streptococci are Gram-positive cocci, arranged in short chains catalase-negative organisms. Recently more than 40 species are documented, most of these species are contributed with causing disease in human and animals (Collins et al., 2001). Enterococci are Gram-positive cocci facultative anaerobes  and non sporulated also, they are able to hydrolyze esculin in the presence of bile salts, and are catalase negative (Dubin and Pamer, 2017). Since 2000, several new species have been identified and currently more than 50 species of streptococci and at least 21 species of enterococci are recognized and the most common species isolated from poultry are Streptococcus gallinaceus, Streptococcus zooepidemicus, Enterococcus durans, Enterococcus faecalis and Enterococcus hirae (Smyth and McNamee, 2001).

In chicken husbandry, antimicrobial agents used for treatment and growth promotion in broilers more than layers, so resistant enterococci usually  recovered from broilers (Klare et al., 1995; Butaye et al., 1999). Enterococcus isolates from poultry subsequently acquired resistance against macrolides, chloramphenicol, β-lactams, and tetracycline has been described (Maasjost et al., 2015). High resistance to aminoglycosides recorded in Enterococci found related to different genes such as (aac(6′) aph(2′′), ant(6))  (Hegstad et al., 2010).Moreover, tetM and tetO were the most common tetracycline resistance genes detected in different Streptococcus species(Oppegaard et al., 2020). Some recent studies established that, the genes encoding certain Enterococcus virulence factors such as asa1, gelE and cylA in addition to different antibiotic resistance genes are associated with causing nosocomial infection (Ngbede et al., 2017). The present study was designed for detection of genotypic characterization of Streptococcus and Enterococcus species isolated from broiler chickens achieved by determination of some virulence and antimicrobial resistance associated genes in the MDR isolates using PCR technique.

MATERIALS AND METHODS

1. Ethical approval

The present study was approved by the Institutional Animal Care and Use committee Beni-Suef University (BSU-IACUC, 021-191), Egypt.

2. Chicken Samples

A total of 272 pooling samples were aseptically collected from 272 diseased broiler chickens aged from 2-5 weeks from different farms in Beni-Suef Governorate during duration from December 2018 untill December 2019. The pooling samples were collected aseptically from lesions (septicemic organs, enlarged organs, necrotic focci) in the internal organs; liver, lung, heart, and kidney of diseased slaughtered chickens and freshly dead ones.

3. Bacteriological isolation

Isolation of both Streptococci and Enterococci was done according to Collee et al. (1996) and Quinn et al. (2002).

4. Identification of Streptococci and Enterococci isolates

4.1. Morphological identification

Pure culture from each isolate was identified morphologically according to its staining reaction, shape, size, and arrangement. these colonies that revealed to be Gram positive cocci  medium  size  and non- sporulated were further examiened biochemically.

4.2. Biochemical identification

1. catalase test: used to differentiate between catalase positive and catalase ngative cocci. Colonies which revealed to be catalase negative were further examiened.  

2. Other non-biochemical tests: were performed on catalase negative colonies including,

• · growth on MacConkey agar
• cultivation on bile aesculin agar  
• · detection of hemolytic activity of isolates using sheep blood agar (7%) this was done according to Collee et al. (1996).

4.3. Biochemical identification of isolates using Vitek2 compact system: (Using ID-GP kits) according to (BioMérieux, 2013)

The Vitek2 compact system using ID-GP (Gram positive cocci) identification kits was applied on pure cultures for complete identification according to BioMérieux (2013).

5. Antibiograms sensetivity testing

The isolated Enterococci and Streptococci were investigated for their susceptibility aganist 12 different antimicrobial agents of veterinary and human significance. Antimicrobial discs included amoxicillin (10μg), apramycin (15μg), cefotaxime sodium (30μg), ceftriaxone (30μg), cephalexine (30μg), cefepime (30μg), enrofloxacin (5μg), sulphamethoxazol-trimethoprim (25μg), amoxicillin-clavulanic (30μg), tetracycline (30μg), gentamicin (10μg) and kanamycin (30μg). All antimicrobial discs used in this study were obtained from (Oxoid, Basing Stoke, UK). Antimicrobial susceptibility profiling and reults interpretation were performed according to (CLSI, 2019). Resistance to more than three antimicrobials of different classes was recorded as multidrug resistance (MDR) according to Chandran et al. (2008).

6. Polymerase chain reaction (PCR) for Streptococcus and Enterococcus isolates

PCR used for detection 3 resistance-associated genes (tetO,aac(6')aph(2'') and pbp1A) and 3 virulence-associated genes (cylE, hyl and brpA) in 3 streptococci isolates. Moreover, it was applied on 3 enterococcus isolates for detection of 3 resistance genes (tetO,aac(6')aph(2'') and blaZ) and 4 virulence genes (hyl, cylA, Asa1 and gelE). Extraction of Genomic DNA was done by using QIAamp DNA extraction Mini prep Kit. Extracted DNA was stored at -80˚C for later using in PCR amplification. Table (1) reveals the used Primers sequences and amplified products for the targeted genes for Streptococcus and Enterococcus isolates. Cycling conditions (temperature & time) of the primers during PCR were displayed in table (2).

Table 1: Primers of virulence and resistance genes used in PCR.

Table 2: Cycling conditions of the different primers during PCR.

RESULTS

1. Prevalence of bacterial isolation from different samples

Out of 272 samples from broiler chickens, a total of 49 bacterial isolates suspected (morphologically and by biochemical tests) to be streptococci or enterococci were recovered; with a total prevalence of 18%.

According to Vitek2 compact system, the bacterial isolates were arranged as 26 Streptococcus spp. (53.1%) and 21 Enterococcus spp. (42.8%) while there were 2 unidentified isolates (4.1%). Streptococcus isolates (n= 26) were identified as 12 S. gallinaceous (24.5%), 8 S. dysgalactiae (16.3%) and 6 S. durans (12.2%). On the other hand, all Enterococcus isolates (n=21) were identified as E. faecalis (Table 3).

Table 3: Prevalence of Streptococcus and Enterococcus isolated from the diseased broiler chickens.

%: was calculated according to the corresponding number (No.) of isolates

2. Antibiogram sensetivity testing

The in-vitro antimicrobial susceptibility testing revealed that the tested isolates (n=49) showed high sensitivity to amoxicillin (77.6%), sulfamethoxazole-trimethoprim (73.5%) and amoxicillin-clavulanic acid (65.3 %). On the other hand, they were completely resistant to cephalexin, cefotaxime, cefipime, cefotriaxone, tetracycline, kanamycin and apramycin (100% for each) and were highly resistant to gentamicin (87.8%) and enrofloxacin (63.2%) (Table 4). More over, all investigated isolates showed presence of multidrug resistance.

Table 4: Results of in-vitro antimicrobial susceptibility testing of recovered isolates.

% was calculated according to the number of the tested isolates (n=49).

3. Polymerase chain reaction (PCR) analyses of streptococcus and enterococcus isolates

Concerning Streptococcus isolates (n=3), all the tested resistance associated genes (tetO,aac(6')aph(2'') and pbp1A) were detected in all the tested isolates (n=3; 100%). On the other hand, among the tested virulence genes; cylE and brpA genes were detected in all the tested isolates (n=3; 100%) while hyl gene was not found in any isolates (Table 5 and Figs. 1, 2& 3).

Table 5: Distribution and prevalence of resistance and virulence -associated genes in the examined Streptococcus isolates.

%: was calculated according to the number (No.) of the tested isolates (n=3).

Moreover, it was applied on 3 enterococcus isolates for detection of 3 resistance genes (tetO,aac(6')aph(2'') and blaZ) and 4 virulence genes (hyl, cylA, Asa1 and gelE).

Regarding Enterococcus isolates (n=3), all the tested resistance genes (tetO,aac(6')aph(2'') andblaZ) were detected in all the tested isolates (n=3; 100%). On the other hand, among the tested virulence genes; hyl, cylA and Asa1 genes were detected in all the tested isolates (n=3; 100%) while gelE gene was not found in any isolates (Tables 6 and Figs. 1, 2, 4, 5& 6).

Table 6: Distribution and prevalence of resistance and virulence -associated genes in the examined Enterococcus isolates.

%: was calculated according to the number (No.) of the tested isolates (n=3).

Fig. (6): PCR amplification of the 276 and 375 bp fragments of hyl and asa1 virulence genes, respectively, from 3 Enterococci (E1-E3) showing positive amplicons migrates with the molecular DNA size ladder (L)., P (control positive), and N (control negative).

DISCUSSION

Agreat attention has been paid to poultry-based industries due to its importance as a source of animal protein in Egypt. Poultry are regarded the most appropriate source of animal protein supply of high nutritive value for humans all over the world. This is due to the efficiency cost of production.

Infectious diseases such as (Streptococci and Enterococci) are important in the broiler industry due to high mortality, retardation of growth, as well as the preventive and therapeutic use of antimicrobials. Moreover, economic losses may result from the loss of uniformity of the flock and condemnations in the slaughterhouse (McKissick, 2006). Streptococcus and Enterococcus  are considered to cause  disease in human and animals (Collins et al., 2001). Also, the enterococci are important agents in human nosocomial infections (Cardona et al., 1993). Streptococcus and Enterococcus have been considered as normally inhabitant, Gram-positive fastidus microorganisms of chickens. Additionally, they may cause disease conditions as endocarditis and urinary tract, intra- abdominal infections in broilers. (Tankson et al., 2001). In the present study, the incidence of Streptococci and Enterococci were identified in broilers in Beni Suef Governorate. The data illustrated in the table (3) revealed that the total prevalence of Streptococcus and Enterococcus speciesin the diseased broiler chickens was 18% where 49isolates were recovered from 272 diseased broiler chickens. According to Vitek2 compact system, the bacterial isolates were arranged as 26 Streptococcus spp. (53.1%) and 21 Enterococcus spp. (42.8%) and isolates while there were 2 unidentified isolates (4.1%). Streptococcus isolates (n= 26) were identified as 12 S. gallinaceous (24.5%), 8 S. dysgalactiae (16.3%) and 6 S. durans (12.3%). On the other hand, all Enterococcus isolates (n=21) were identified as E. faecalis. These results were higher than those recovered by Cauwerts et al. (2007) who found E. faecalis with  a prevalence  rate of 13.6% in broilers, Diarra et al. (2010); who remarked that prevalence rate of E.faecalis  was 10.1%.While the lowest result recorded by (Chadfield et al., 2004) who collected 227 samples from broiler chickens  and recovered 15 E. faecalis isolates (6.6%). Also, Aslantaş (2019) isolated E.durans with prevalence of 2.4% and Cauwerts et al. (2007) who recorded E. durans with prevelace of 9.5%. Results in present study was noted to be less than those recorded by (Chadfield et al., 2004) who documented S. gallinaceous with prevelace of 37.4%  and Abd El-Hafeez et al. (2018) who recorded S. dysgalactae with prevalace rate 34.7%. Higher rates of isolation were achived by Petersen et al. (2008); 77.5%, and 46.5%. Meanwhile, much higher prevalence was recorded by Aslantaş (2019); 87.8%.

In poultry rearing Antimicrobials are used for treatment infecous microbial diseases also they play an important role in growth promotion. Its exessive use in animal production leads to spread of antibiotic resistance (Gosh and LaPara 2007). In-vitro antimicrobial suscebtibility testing of different veterinary pathogens helps the veterinarian in the choice of the most suitable drug for treatment (Radwan et al., 2016). In the present study, the isolated Enterococci and Streptococci were investigated for their susceptibility aganist 12 different antimicrobial agents of veterinary and human significance. The in-vitro antibiogram sensetivity testing results for both Streptococcus and Enterococcus isolates were showed in table (4). Isolates were highly sensitive to amoxicillin (77.6%) followed by sulfamethoxazole- trimethoprim (73.5%) and amoxicillin- clavulanic acid (65.3 %). Meanwhile, they were completely resistant to cephalexin, cefotaxime sodium, cefipime, cefotriaxone, tetracyclin, kanamycin and apramycin (100%) and were highly resistant to gentamicin (87.8%) and enrofloxacin (63.2%). Also, growing of resistance was observed by the intermediate behavior of the tested isolates against the tested antimicrobial agents. The percentages of the intermediate zones were 2, 4.1 and 18.4 % against gentamicin, trimethoprim- sulfamethoxazole and enrofloxacin, respectively. Additionally, multidrug resistance was detected in all tested isolates. Higher prevelance rates of resistance were reported against tetracycline and kanamycin by Diarra et al. (2010) 91.3, 59.4% ; (Tremblay et al., 2011) 95.6, 25.2% and Nowakiewicz et al. (2017) 60.5, 42.1%, respectively. Also, Hershberger et al. (2005) recorded resistance against gentamicin in 32% of isolates. On the other hand, Rehman et al. (2018); Aslantaş (2019); Obeng et al. (2013) and Liu et al. (2013) reported closely matching resistance rates with those detected in present study. B eacuse of misuse antmicrobials which might leads to high resistance rates, it was difficult to found an effective drug aganist the Streptococci and Enterococci infections. (Sharada et al., 2001). More over, all investigated isolates showed presence of multidrug resistance. Our results were nearly similar to previous reports all over the world. Aslam et al. (2012) founded that multidrug resistance were detected in 91% of isolates. Meanwhile, lower percentages of MDR were recorded by Nowakiewicz et al. (2017); 56.8% and (Ngbede et al., 2017); 53.1%.

In the present study, PCR was used for detection of 3 resistance-associated genesincluding resistance to tetracycline (tetO), resistance to aminoglycosides (aac(6')aph(2'') and resistance to β-lactams (pbp1A) in 3 Streptococcus isolates. Moreover, it was applied on 3 Enterococcus isolates for detection of 3 resistance genes including (tetO, aac(6')aph(2'') and blaZ). The data illsturated in (tables 5-6 and Figs. (1:4) revealed that 100% of the tested isolates harbored teto, (aac(6')aph(2'') genes on the other hand 100% of streptococci isolates harbored (Pbp1A) gene also, (blaZ) gene were detected in all investigated  Enterococci isolates. 

Many genes were detected for tetracycline resistance including tetK, tetL, tetM and tetO genes Ngbede et al. (2017). Tet(O) gene which resposible for tetracycline resistance was detected in enterococci isolated from broilers  byAarestrup et al. (2000) and, when studing  tetracycline resistance determinants in raw food, Wilcks et al. (2005) founded that  this gene only occur in enterococci isolated from poultry meat. Also, this gene has been described in human E. faecalis, but is rare Aarestrup et al. (2000). The efflux proteins have been the best studied of the Tet determinants including tetA, tetB, tetC, tetD, tetE, tetG, tetH, tetK, tetL and tetA(P) genes which have been identified. All of the following Tet determinants (tetA, tetB, tetC, tetD, tetE) protected the bacterial ribosomes because they ecoded for energy-dependent membrane associated proteins which release tetracycline out of the cell reducing the intercellular tetracycline concentration (El-Seedy et al. 2019). Regarding the obtained results of tetO which was detected in all tested isolates (100%), they were higher than those recorded by Cauwerts et al. (2007) who found tetO and tetM in 30% of tested isolates. Meanwhile, much lower prevalence was recorded by Diarra et al. (2010) who founded that 7.2% of tested isolates harbored tetO also, tetL and tetM were detected in 57.15% of isolates. Moreover, (Tremblay et al., 2011; Ngbede et al., 2017; Nowakiewicz et al., 2017) detected this gene in Enterococci isolates from broilers. On the other hand, the obtained results of aminoglycosides resistance encoding gene (aac(6')aph(2'')) which is detected in all tested isolate (100%), were higher than those obtained by Diarra et al. (2010) who found (aac(6')aph(2'')) gene in 30.4% of tested isolates. Also, Rehman et al. (2018) recorded (aac(6')aph(2'')) gene in 8.3% of Enterococci isolates from broilers.

 In the present  study, PCR was applied on 3 MDR  Streptococci and 4 Enterococcci   isolates  to detect the following virulence associated genes including β-haemolysin cytolysin gene (cylE), hyalurinidase encoded by (hly)andbiofilm production (brpA) for Streptococci isolates. Also, the following genes asa1 (aggregation substance), which associated with adherence and conjugation; cylA encodes (cytolysin-haemolysin) which lyses red blood cells, hly (hyalurindase) while gelatinase, encoded by (gelE) which can hydrolyze gelatin,  were investigated in Enterococci isolates  using PCR. The results illustrated in tables (5-6) and Figs. (3, 5& 6) revealed that all the tested isolates (100%) harbored both cylE and brpA genes meanwhile no isolates (0%) harbored hly gene in case of Streptococci isolates. On the other hand, all Enterococci tested isolates (100%) harbored asa1, cylA and hly genes meanwhile no isolates (0%) harbored gelE gene. Regarding the obtained results of cylA and cylE which were detected in all tested isolates (100%), this result was higher than those recorded by Diarra et al. (2010) who found cylA and cylB genes in 28.5 % of tested isolates. Also, Ngbede et al. (2017) recorded cylA gene in 28.3% of tested isolates. Meanwhile, Song et al. (2019) found cylA in 16% isolates. The lower prevelances were recorded by (Champagne et al., 2011) who detected cylA and cylB in 6% of isolates and Aslantaş (2019) who found cylA in 0.7 %. On the contrary, Nowakiewicz et al. (2017) reported that none of tested isolates (0%) exhibeted genes responsible for haemolysin – cytolysin production. Regarding the obtained results of gelE which were not detected in any tested isolate,this result is lower than those detected by Ngbede et al. (2017) who found gelE gene in 11.3 % and Aslantaş (2019) recorded this gene in 40.3% of tested isolates. While (Diarra et al., 2010; Champagne et al. 2011; Nowakiewicz et al., 2017) recorded gelE in 100% of investegated isolates. Regarding the obtained results of asa1 gene which was found in all investigated isolates (100%). This result is higher than those recorded by Aslantaş (2019) who found asa1 gene in 6.1% of isolates. While   Song et al. (2019) found asa1 gene in 44% of tested  isolates.

CONCLUSION

Streptococcus and Enterococcus spp. are important infectious agents which can cause disease in broilers, and affect on morbidity and mortality rates. The exessive use of antibiotics resulting in multidrug resistance pathogens and this is considered a great problem. The in-vitro antimicrobial sensetivity testing revealed that all tested isolates were highly sensitive to amoxicillin, sulfamethoxazole- trimethoprim and amoxicillin- clavulanic acid meanwhile they  were completely resistant to cephalexin, cefotaxime, cefipime, cefotriaxone, tetracycline, kanamycin and apramycin. All the testedisolates were MDR. PCR results revealed  that tetO, aac(6')aph(2''), blaZ, pbp1A, cylE, brpA, cylA asa1 genes were detected in all the investigated  isolates meanwhile, hyl gene was not detected in any Streptococcusisolates and gelE gene was not detected in Enterococcus isolates.

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