First Characterization of Class 1 Integron in Corynebacterium bovis Isolated from Subclinical Bovine Mastitis

| Integrons play major roles in spreading and expression of antibiotic resistance genes among bacteria especially Gram-negative bacteria. However, the incidences of class 1 integrons in Gram-positive bacteria remained elusive. The present study was conducted to monitor the prevalence of class 1 integrons in Corynebacterium bovis (C. bovis) isolated from cases of bovine mastitis in Egypt. A total of 35 Corynebacteria spp., isolated from milk samples of subclinical mastitis, were examined using PCR analysis with 16S rRNA specific primers. The results revealed a prevalence of 8 (22.8%) C. bovis strains in the examined samples. All isolates of C. bovis showed an elevated level of resistance, mostly against spectinomycin, streptomycin followed by tetracycline, ampicillin, kanamycin, chloramphenicol and sulfamethoxazole-trimethoprim. Using PCR and DNA sequencing, two isolates of C. bovis were identified containing class 1 integrons that encoded aminoglycoside adenyltransferase type A (aadA1) as a gene cassette. The aadA1 confers resistance to spectinomycin/streptomycin. Additionally, class 1 integron resistance genes in C. bovis isolates were described utilizing PCR and DNA sequencing. These resistance genes conferred resistance to several antimicrobial agents frequently used in dairy farming. This study reports, for the first time, the detection and identification of class 1 integron containing aadA1 gene in C. bovis of animal origin.


INTRODUCTION
C orynebacteria spp. are one of the most isolated pathogens responsible for subclinical mastitis in dairy cows. However, quick, reliable and simple techniques for the identification of species of the genus Corynebacterium are not currently available ( Juliano et al., 2014).
The wide utilization or misuse of antibiotics in veterinary medicine initiates the development of resistance and increase incidence of multidrug resistance (MDR) in bacteria. This leads to implication of substantial public health impacts (Pyorala, 2009;Richet, 2012;Saini et al., 2012;Chen et al., 2017).
Integrons are portable hereditary components having a system of site-specific recombination, which permits capture the genes responsible for antibiotic resistance (Partridge et al., 2009;Chen et al., 2011). Integrons play important roles in spreading and expression of antibiotic resistance genes among bacterial populations through transposons or plasmids (Mazel, 2006). The integron gene sequence arrays are formed when multiple gene cassettes are integrated at one site on a plasmid or on the The class 1 integrons genes are mostly detected in Gramnegative bacteria (Martinez-Freijo et al., 1998;Partridge et al., 2001). However, the incidence of class 1 integrons in Gram-positive bacteria remained least explored (Nesvera et al., 1998;Clark et al., 1999;Tauch et al., 2002).
The present study aim to detect C. bovis strains isolated from subclinical cases of bovine mastitis using PCR techniques and to genetically characterize class 1 integron resistance genes using advanced genomic approaches.

sAMpliNg ANd isolATioN
A total of 150 mastitis milk samples, obtained from cases of subclinical mastitis (positive for California mastitis test), were gathered from different farms of dairy cattle at Giza governorate in the Egypt. At these farms, cows suffered from decreased milk production with no clinical signs. A total of 35 isolates were identified as Corynebacteria species through microbiological cultures according to the method described by Oliver et al. (2004).
Analysis of PCR products was carried out through gel electrophoresis (SCIE-PLAS, UK) on a 1% agarose gel prepared with Tris/Acetate/EDTA (TAE) (Intron Biotechnology, Inc) buffer plus agarose powder (Biotechnology grade AGA001.100 -Bioshop® Canada Inc) stained with ethidium bromide (0.5 µg/ml). 10-µL aliquot of a molecular marker containing a DNA Ladder (Intron Biotechnology, Inc) was added to the first well of each gel, control negative in the second, control positive (K. pneumoniae KPB 127 contain two integrons (dfrA12 -aadA2; and aadA2) isolated by (Hazim et al., 2016) in the third well and 10-µL of samples in the other wells ( Figure 2) at 80 V for 1 h, after that single DNA bands were visualized by ultraviolet trans-illumination (UV) according to target size of gene as showed in Table 2. A QIA quick Gel Extraction Kit (Qiagen, Germany) was used for purification of PCR fragments from the agarose gel according to the manufacturer's instructions. Finally, the sequencing for PCR product with both DNA strands was performed by using an ABI automatic DNA sequencer (Model 373; Perkin-Elmer).

coMpuTEr ANAlysis of ThE sEquENcE dATA
The data of DNA sequenced data of amplified gene was identified by the database of GenBank using the BLAST program available at the NCBI BLAST homepage (http:// www.ncbi.nlm.nih.gov/BLAST/).

Advances in Animal and Veterinary Sciences
May 2020 | Volume 8 | Issue 5 | Page 454
In the present study, 35 isolates of Corynebacteria spp. were inspected using PCR with 16S rRNA gene, which is a primer specific for C. bovis. The result showed 8 positive strains of C. bovis out of 35 (22.8%) tested samples (Table 3) with a target PCR amplicon size of 1250 bp (Figure 1, Table 1). Further, PCR and DNA sequencing of 8 positive C. bovis fragments using primers specific to class 1 integrons revealed two isolates (5.7%) of C. bovis which have one

Advances in Animal and Veterinary Sciences
May 2020 | Volume 8 | Issue 5 | Page 455 type of class 1 integron with middle size (974 bp), as shown in (Figure 2) and contains a single gene cassette of aadA1 (aminoglycoside adenyltransferase type A), which confers resistance to streptomycin/spectinomycin. Up to date the aadA1 gene has not been previously identified in C. bovis.

DISCUSSION
In this study, 8 out of 35 isolates were identified as C. bovis using PCR with a faire percentage (22.8%). This rate is lower than 54.0% strains as C. bovis reported by Watts et al. (2000) and also Theel et al. (2012) which detects C. bovis with a percent 88.5%. Higher incidence of C. bovis was reported from sub-clinical mastitis (96.1% and 97.2%) by Juliano et al., 2014 andHuxely et al., 2004, respectively. The class 1 integrons were first reported in 1989 (Stokes and Hall, 1989) and were described as reservoirs and an essential source of antimicrobial resistance genes within microbial populations (Fujimura et al., 2004;Xu et al., 2007Xu et al., , 2009. However, these studies have been conducted in Gram-negative bacteria with few exceptions (Tauch et al., 2002;Nandi et al., 2004). While in recent studies, class 1 integron had been observed as antibiotic resistance gene in Gram-positive bacteria, particularly in methicillinresistant coagulase negative staphylococci (MRCNS) and methicillin-resistant Staphylococcus aureus (MRSA) (Xu et al., 2007(Xu et al., , 2008(Xu et al., , 2010. Class 1 integron have been found mainly in Gram-negative bacteria (Mokracka et al., 2012;Malek et al., 2015). Upto-date, only few reports on class 1 integron in Gram-positive bacteria are available. The previously published papers of class 1 integron in Gram-positive bacteria with gene cassettes are as following; (1) Class 1 integron containing aadA gene was first report in E. faecalis strain and confers resistance to streptomycin/spectinomycin (Clark et al., 1999). (2) The genome of some Gram-positive bacteria such as Staphylococcus aureus, coagulase-negative staphylococci, Staphylococcus warneri, Staphylococcus haemolyticus and Staphylococcus epidermidis carries class 1 integrons containing gene cassettes of dhfrXII-orfF-aadA2 which confers resistance to trimethoprim, unknown function protein and aminoglycoside adenyltransferase type A, respectively (Lei et al., 2006). (3) The aadA2 gene cassette on a 29-kb plasmid pCG4 in C. glutamicum, which confers resistance to streptomycin/spectinomycin (Nesívera et al., 1998). (4) Class 1 integrons containing aadA9 detected previously in C. glutamicum on a 27.8kb R-plasmid pTET3, which confers resistance to spectinomycin, streptomycin and tetracycline (Tauch et al., 2002). (5) The aadA2 gene cassette in S. aureus, which conferred resistance to aminoglycoside (Xu et al., 2007). (6) Class 1 integron of different gene cassettes with 2 att sites and 3 arrays of cassettes were detected in methicillinresistant S. aureus (MRSA) (Shengjie et al., 2011).

CONCLUSION
In the present study, class 1 integron was identified with gene cassette (aadA1) in two isolates of C. bovis (5.7 %) obtained from milk samples of subclinical mastitis cows using PCR and DNA sequencing. These resistance genes confer resistance to several antimicrobial agents frequently used in dairy farming. This study is the first report for detection and identification of class 1 integron containing aadA1 gene in C. bovis of animal origin.

sTudy liMiTATioN
Findings of this study were limited to a small sample size, also class 1 integron incidence is little in Gram positive bacteria. In addition, few Egyptian studies were accessed in this area of research.
Moreover, there were some difficulties related to the expense of chemicals and kits which restricted the application of PCR and DNA sequencing primers of multi drug resistance.