Multidrug-resistant Escherichia coli isolated from a dog with a history of urolithiasis: case report

Bacterial resistance is a reality in both human and veterinary health, it limits the therapeutic arsenal and raises the costs of the patient’s treatment. A dog with signs of cystitis received treatment with 5mg/kg enrofloxacin at three consecutive times, with low effectiveness. The presence of urethral uroliths was identified and urohydropulsion was done. The animal presented a new obstruction, for which a cystotomy was performed, but continued with signs of infection. Uroculture and antimicrobial susceptibility test were then performed. Escherichia coli was identified, which was resistant to 13 antibiotics, being sensitive only to piperacillin-tazobactam and amikacin. In the screening test for β-lactamase, the production of ESβL was detected. The qPCR indicated the presence of the bla CTXm, bla DHA , bla OXA , bla IMP , bla TEM , bla GIM , bla SIM , bla SPM and bla SME genes, which may lead to a phenotypic resistance profile for ampicillin, amoxicillin-clavulanate, aztreonam, cefepime cefoxitin, cefuroxime, ceftazidime, ceftriaxone, imipenem, and piperacillin-tazobactam. This case reaffirms the value that laboratory analysis adds to the diagnosis and treatment of cystitis and urolithiasis, which can define the direction of evolution of the prognosis and the speed at which the patient's health will be restored.


INTRODUCTION
Bacterial resistance to antibiotics is a reality in both human and veterinary health and has been reported frequently in dogs (Shaheen et al., 2010). Escherichia coli is a bacterium capable of developing resistance phenotypes from gene expression (Gandolfi-Decristophoris et al., 2013), and the production of enzymes called betalactamases is common.
One of the enzymes produced is extended spectrum beta-lactamase (ESβL), which can inactivate penicillin and cephalosporins, as well as antibiotics from other classes such fluoroquinolones, aminoglycosides, trimethoprim-sulfamethoxazole and tetracyclines. This is due to the co-selection process in which plasmids carrying ESβL genes can often carry a wide range of resistance genes to other non-βlactam antimicrobials (Winokur et al. 2001). Thus, bacteria such as E. coli, carriers of ESβL genes, limit the therapeutic arsenal, which makes patient treatment more difficult and expensive (Hordijk et al., 2013), especially in cases of urinary tract infections (UTI).
As an opportunistic microorganism, E. coli can be found in the urinary tract of animals such as dogs and cats (Ewers et al., 2012) and is constantly related to UTI, which may arise as a result of urolithiasis. Urolithiasis results from the formation of uroliths, which are mineral crystals formed mainly due to supersaturation of the urine, which can result in urinary flow obstruction (Oyafuso et al., 2010) and, consequently, lead to UTI.
Due to the clinical status of patients coming to veterinary care with signs of UTI and because of the socioeconomic profile of their tutors, the treatment protocol is not always standardized. Thus, the use of fluoroquinolones is common due to its broad spectrum against gram positive and negative bacteria. However, even being common, treatment of UTI can become challenging (Weese et al., 2011) due to bacterial resistance to routine antibiotics.
Laboratory techniques have been used to integrate diagnosis to improve treatment. An example is the phenotypic detection of antibiotic resistance by screening tests, as well as molecular detection of resistance genes by the real time polymerase chain reaction technique (qPCR).

CASUISTRY
An undefined breed male dog was seen at a private veterinary hospital, with difficulty urinating, and when he did, the urine was dark and fetid. The clinical suspicion was cystitis. Initially, the proposed treatment was enrofloxacin antibiotic therapy at a dose of 5mg / kg for seven days. After one month, the dog had the same symptoms and was prescribed the same therapy. As there was no improvement, the animal was sent to the Veterinary Hospital of the School of Veterinary and Animal Science of the Federal University of Goiás (HV / EVZ / UFG).
After clinical evaluation, the same treatment was prescribed. However, the condition remained unchanged and the dog was taken back to HV / EVZ / UFG, and the presence of urethral uroliths was diagnosed by means of x-ray. An urohydropropulsion was performed to remove the crystals, and new antibiotic therapy with enrofloxacin was performed.
After a few days, the dog presented new obstruction and, when taken again to the HV / EVZ / UFG, a cystotomy was performed. After surgery, a bladder catheter was introduced into the dog, which remained for 15 days. However, the animal continued with signs of infection.

DISCUSSION
In the past decade, urolithiasis was the third most common canine urinary tract health problem (Sosnar et al., 2005), which may be associated with UTI cases, which was detected in the present study. Among the most commonly isolated bacteria in cases of UTI is E. coli, considered the largest reservoir of resistance genes today and may be responsible for treatment failures in both human and veterinary medicine. A growing number of resistance genes have been identified in E. coli isolates in recent decades and many of these genes have been acquired by horizontal transfer, being able to both donate and receive them from other bacteria. Thus, resistance in E. coli has been one of the biggest challenges worldwide in the treatment of human and animal infections and should be considered a real public health concern (Poirel et al., 2018).
The results of the antibiogram and qPCR reinforced, in this case, the low effectiveness of the strategy adopted for the treatment, which calls attention to the prescription of the same antibiotic, although without obtaining a desirable response, with gradual worsening of the animal. This highlights the importance of microbiological examination and antibiogram testing that, once applied at the beginning of treatment, could have contributed to a good prognosis and positive patient outcome.
These analyzes showed that although the gene encoding piperacillin-tazobactam resistance was identified in qPCR, the antibiogram indicated bacterial sensitivity to this combination and there was no phenotypic expression of the gene. In addition, E. coli was resistant to cefazolin, trimethoprim-sulfamethoxazole, tetracycline, ciprofloxacin and gentamicin. The genetic profile of resistance to such antibiotics (Perreten et al., 2010) was probably also present. However, only testing for β-lactamase coding genes was performed.
In 2017, researchers in China (Li et al., 2017) isolated three strains of E. coli ESβL from the urinary tract of three dogs in a group of 80 dogs with acute or chronic cystitis. PCR was performed to analyze 3 genes (blaCTXm, blaTEM and blaSHV), which were also analyzed in this case. The phenotype found by the authors -resistance to ampicillin, tetracycline, trimethoprimsulfamethoxazole and sensitivity to amikacinwas also detected in the present work.
It is understood that the aggravating factors of veterinary treatments vary, ranging from the difficulty of a quick and accurate diagnosis to the high costs that come with the therapy. However, it is essential to reinforce the importance of performing laboratory tests in the early stages of clinical follow-up, as well as the reformulation or implementation of care protocols that encourage prescription based on clinical evidence and laboratory results.

CONCLUSION
Bacterial resistance is a reality in the veterinary field, and treating infectious diseases today is a challenge. The present case reaffirms the value that laboratory analysis adds to the diagnosis and treatment of cystitis and urolithiasis and may define the direction in which the prognosis will evolve and the speed at which the patient's health will be restored.