Gentamicin enhances toxA expression in Pseudomonas aeruginosa isolated form cow mastitis

| The present study was undertaken in order to investigate the role of gentamicin in the gene expression of toxA in Pseudomonas aeruginosa isolated from cow mastitis. A total of ten P. aeruginosa strains originally isolated from cows infected with mastitis. Agar dilution methodology was performed to determine the minimal inhibitory concentration of gentamicin, all of which developed resistance toward gentamicin. The findings presented here demonstrated that all these strains harboured toxA depending on PCR-based assay. Nonetheless, RT-PCR technique revealed a wide variation in expression of toxA. Moreover, the cultivation of P. aeruginosa in the presence of gentamicin, significantly (P< 0.05), induced the expression of toxA, in addition to the possibility of enhancing the virulence of this bacterium. In conclusion, using gentamicin to treat infections caused by P. aeruginosa may participate in more severe outcomes.


Advances in Animal and Veterinary Sciences
December 2018 | Volume 6 | Issue 12 | Page 527 nosa responsible for regulating exotoxin A synthesis, has been extensively used in molecular detection of P. aeruginosa (Dong et al., 2015). Subinhibitory concentrations of antibiotics are known to provoke extensive transcriptional changes in bacteria (Ohlsen et al., 1998), to our knowledge, this is the first report that investigating the influence of gentamicin at sub minimal inhibitory (sub MIC) on gene expression of toxA. Therefore, the main goal of this study is investigating the effect of gentamicin at sub MIC on the gene expression of toxA of P. aeruginosa isolated form cow mastitis.

MAtErIAlS AnD MEthoDS
A total of ten P. aeruginosa isolates were obtained from microbiology laboratory, College of veterinary medicine, University of Baghdad. Originally, these isolates were isolated from milk samples obtained from mastitis-affected cows. Nevertheless, re-identification was accomplished using the conventional biochemical tests (Gram stain, lactose fermentation, catalase, oxidase, IMViC, motility, and hemolysis pattern) in addition to VITEK 2 compact and API 20NE (bioMe´rieux, France) identification systems. determinAtion of minimum inhibitory ConCentrAtion of GentAmiCin Different concentrations of gentamicin (2 -q1024 µg/ml) were used to estimate the MIC following agar dilution method and the results were interpreted in accordance to the guidelines of Clinical Laboratory Standards Institute (2016), P. aeruginosa ATCC 27853was used as a quality control strain.

Culture method
Tryptone soy broth (TSB; HiMedia, India) with or without subinhibitory concentrations of gentamicin were inoculated with each isolate at concentration compatible to MacFarland (1-1.5 × 10 8 CFU/ml) at 37°C for 24 hr. thereafter, they were sent for RT-PCR study.

reAl-time quAntitAtive PolymerAse ChAin reACtion
Extraction of RNA was performed using protocol of TRI-zol™ Reagent (Rio et al., 2010). cDNA was created following the manufacturer's instructions of the RT-PCR kit supplied byInvitrogen.
The gene (toxA) expression in P. aeruginosa culture was accomplished via RT-q PCR technique by Magnetic induction cycler (Bimolecular system, Australia) using primers listed in Table 1. Nonetheless, the reaction mixture is demonstrated in Table 2. Control samples were created by using 10.5 µl nuclease-free water and no cDNA template. However, 16SrRNA was employed to normalized the gene expression of toxA.  Reaction conditions for 16SrRNA and toxA genes amplification were achieved as described by Goldsworthy (11). For every qPCR a plate read-out was taken after each cycle. Moreover, a melting curve using 1°C increments was also performed following the 40 cycles in order to determine that the correct gene was amplified.
The relative expression ratios were calculated by using the cycle threshold (C t ) of the 16SrRNA as the calibrator (n-fold expression = 2 −ΔCt , where ΔC t represents the difference between the C t of the toxA and the C t of the 16SrR-NA).

stAtistiCAl AnAlysis
Data were presented as the mean of three replicates ± standard error of the mean. The statistical significance of toxA gene expression differences between P. aeruginosa isolates was determined based on repeated analysis of variance (ANOVA) using the statistical program IBMSPSS version 25. P values of <0.05 were regarded as significant (Paulson, 2008).

rESultS
The identification was confirmed for all isolates included

Advances in Animal and Veterinary Sciences
December 2018 | Volume 6 | Issue 12 | Page 528 in the present work depending on the results summarized in Table 3. In regard to MIC, all ten isolates were resistant to gentamicin (MIC ≥ 16 µg/ml) in accordance to CLSI breakpoints (Table 4).  The current findings revealed that all isolates of P. aeruginosa harboured toxA ( Figure 1). Moreover, these ten isolates developed very low expression levels of mean C t value of 28.0 ± 1.02.
Pseudomonas aeruginosa 16SrRNA displayed, similarly, moderate levels of expression, giving mean C t values of 22.93 ± 0.67.
When P. aeruginosa isolates grown in the presence of gentamicin; toxA expression mean has increased significantly (P< 0.05) over their spouses before the addition of gentamicin. Fold change ranged from 5.11 -323.36 and it varied significantly (P < 0.05) among the isolates as it is depicted in Figure 2.

DIScuSSIon
Observational data investigated the correlation between virulence and antibiotic resistance has established a simultaneous rising of both of them (Schroeder et al., 2017). However, the current findings are in agreement with a study done by Neamah, (2017) as he stated the toxA was detected in all P. aeruginosa strains that isolated from cow mastitis. While, Raziq, (2017) has detected toxA gene in 84% of the isolates.In compatible with our results, Banerjee et al. (2017) in a study done in South Bengal reported that,nearly, 5.4% of bovine subclinical mastitis were due to toxA containing P. aeruginosa.
Markedly, the link between toxin production and antibi-

Advances in Animal and Veterinary Sciences
December 2018 | Volume 6 | Issue 12 | Page 529 otic resistance has been analyzed by many authors. Ohlsen et al. (1998) demonstrated that some methicillin-resistant S. aureus isolates produced up to 30-fold more alpha-toxin in the presence of 10 mg of methicillin per ml than in its absence. The authors explained such an interaction may induce signal transduction mechanisms, resulting in activation of the hla promoter. However, it cannot be explained by increased levels of the regulatory molecule RNA III. Moreover, Tofik (2011) confirmed that toxin production by S. aureus in the presence of oxacillin was significantly up-regulated. Another hypothesis was adopted by Kimmitt et al. (2000), SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression.
The regulatory mechanisms govern the interconnection between virulence determinants and genes responsible for the antibiotic resistance is highly complicated. Occasionally these mechanisms are believed of as distinct events. Yet, form the genetic point of view, such regulatory mechanisms are interweaved and connected. Frequently, the expression of antibiotic resistance genes can be influenced by the regulatory mechanisms of virulence determinants and vice versa. Unluckily, the host can affect this gene regulation directly or indirectly via many forms. Of these, stress response, environmental sensing, post-transcriptional modifications, ribo-regulation, multi-networks of regulation, and formation of biofilm govern by quorum sensing (Schroeder et al., 2017).

concluSIon
The findings demonstrated in the present work confirmed that growing P. aeruginosa in the presence of gentamicin induced toxA expression and might enhance the virulence of this pathogen; upon that, including gentamicin in the treatment regimen of infections caused by P. aeruginosa might severely worsen the outcomes.