{'fraction_non_alphanumeric': 0.03889689236468409, 'fraction_numerical': 0.019139740687384235, 'mean_word_length': 5.256652360515021, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Prevalence, Genetic Diversity and Antimicrobial Resistance of Proteus mirabilis Isolated from Dogs Hospitalized in Beijing

Received: Revised: Accepted: Published online: June 23, 2019 August 20, 2019 August 22, 2019 September 17, 2019 Proteus mirabilis is an opportunistic pathogen that causes diarrhea and urinary tract infections in humans and companion animals. Emergence of antimicrobial resistance in P. mirabilis increases risk of failure of antimicrobial therapy in companion animals. The current study determined prevalence, genetic diversity and phenotypic antimicrobial resistance of P. mirabilis isolated from feces of 35 dogs with diarrhea hospitalized in Beijing, China. Random amplified polymorphic DNA (RAPD), pulse-field gel electrophoresis (PFGE), PCR and antimicrobial disk diffusion were used to characterize these isolates. Prevalence of P. mirabilis in feces from hospitalized dogs was 28%. RAPD and PFGE demonstrated a great diversity of isolates. Thirteen (37%) of 35 isolates produced extended-spectrum betalactamases (ESBLs), with blaTEM being detected in all ESBLs-producing isolates. All isolates were susceptible to imipenem, cefoxitin and cefotaxime/clavulanic. rmtB (with 51% prevalence rate) was the only aminoglycoside 16S rRNA methylase resistance gene identified. Among plasmid-mediated quinolones resistance genes, qnrB (14%) and aac(6’)-Ib (26%) were detected. In conclusion, P. mirabilis isolates from feces of dogs with diarrhea in China had great genetic diversity and a high prevalence produced ESBLs. Consequently, antimicrobial stewardship programs should also target companion animals to reduce emergence of antimicrobial resistance. ©2019 PVJ. All rights reserved


INTRODUCTION
The genus Proteus belongs to the Enterobacteriaceae family. Bacteria in this genus are motile, Gram-negative and can thrive in soil, water and intestinal tracts of mammals (Drzewiecka, 2016). Several species of this genus are known to colonize and infect human and animal hosts, with Proteus mirabilis being the most prevalent one (Wang et al., 2014;Yazdi et al., 2018).
Proteus mirabilis has gained increasing importance as an emerging opportunistic pathogen causing nosocomial infections in humans and animals (Jacobsen and Shirtliff, 2011;Adams-Sapper et al., 2012). In companion animals, they have been implicated as causative agents of urinary tract infections (UTIs) and chronic otitis (Zamankhan Malayeri et al., 2010;Marques et al., 2019). There is supporting evidence that human patients with UTIs caused by P. mirabilis will have the same strain in their stool (Schaffer and Pearson, 2015). Likewise, presence of Proteus spp. in fecal content of dogs is associated with an increased risk of UTIs caused by the same bacteria (Harada et al., 2014). However, whether there is any genetic similarity between isolates that colonize dogs is apparently unknown. Therefore, identification of major clonal complexes colonizing dogs would provide valuable insights into the epidemiology of UTIs and other diseases caused by this important opportunistic pathogen.
In general, infections caused by P. mirabilis are treated with extended-spectrum cephalosporins (ESC), although aminoglycosides or fluoroquinolones are also used. Recently, the emergence of extended-spectrum betalactamases (ESBLs) in P. mirabilis has become an increasing public health concern (Schultz et al., 2017). Whereas it is still uncertain whether and how these bacteria promote dissemination of antimicrobial resistance (AMR), ESBL-producing isolates may be clinically resistant to beta-lactams such as penicillin, aztreonam and cephalosporins (Lin et al., 2019). Currently, prevalence of AMR and associated genes in P. mirabilis isolated from hospitalized dogs in China with diarrhea remains unknown. In this study, we aimed to investigate prevalence, genetic diversity and AMR profile of P. mirabilis isolated from fecal samples of hospitalized dogs with diarrhea. In addition, presence of ESBL-encoding genes in P. mirabilis was also determined.

MATERIALS AND METHODS
Ethics statement: This study was conducted following ethical guidelines of China Agricultural University (CAU), Beijing. Prior to start of the study, ethical approval was granted by the Departmental committee of College of Veterinary Medicine, CAU. Sampling was carried out according to standardized protocols (Jay-Russell et al., 2014) and with prior consent of each owner.

Collection of samples, isolation and identification of Proteus mirabilis isolates:
A total of 125 fecal samples was collected aseptically from 125 randomly selected hospitalized dogs with diarrhea at the Small Animal Teaching Hospital, College of Veterinary Medicine, China Agricultural University, from October 2014 to May 2015. A cotton swab was used to inoculate each sample on MacConkey agar (DifcoTM, Becton Dickinson, Sparks, MD USA). Bacterial identification was done based on colony characteristics, triple sugar iron test and gram staining, and isolates were confirmed to be P. mirabilis by API20E biochemical test (BioMerieux, Lyon, France).

RAPD genotyping:
For RAPD genotyping, OPX13 5'-ACGGGAGCAA-3 primers (Beijing Sunbiotech) were used as reported (Michelim et al., 2008). Amplifications were repeated three times (independent DNA extractions and PCR) to evaluate reproducibility for each isolate. PFGE fingerprinting analysis: Genotyping of P. mirabilis isolates was conducted by macro restriction, using XbaI digestion of bacterial DNA, followed by separation of resulting fragments by PFGE. Resulting maps were prepared using Quantity OneTM software and cluster analysis performed using the UPGMA method based on Dice coefficients, with optimization and tolerance set at 0.5%. Analyses were done in BioNumerics V 5.1 software. Clusters were defined at the 80% similarity level. Degree of relatedness was considered as defined (Tenover et al., 1995).
ESBLs phenotyping: All isolates were screened for presence of ESBLs using the disk diffusion method (CLSI, 2015). Briefly, an ESBLs producer was detected with discs containing ceftazidime (CAZ, 30 µg) or cefotaxime (CTX, 30 µg) alone or in combination with clavulanic acid (CLA, 10 µg). Production of ESBLs was confirmed by an increase of ≥5 mm in zones of inhibition of combined disks when compared to agents tested alone.

Identification of antimicrobial resistance genes:
All isolates were screened for presence of AMR genes (Table  2). PCR conditions were used as described (Kim et al., 2005). PCR-positive products were sequenced and compared to existing databases for further confirmation.
Statistical analyses: Confidence intervals (CI) were estimated using q binomial distribution. Fisher's Exact test was used to compare antimicrobial resistance rates between antimicrobials, and also between ESBLproducers and non-producers. Statistical significance was considered at the 5% level.

Samples collected and prevalence of Proteus mirabilis:
A total of 35 Proteus mirabilis were isolated from 125 fecal samples based on colony characteristics, microscopic appearance, biochemical tests and 16S rRNA, with an estimated prevalence of 28% (95% exact confidence interval: 20-37%). Round transparent or translucent colonies with smooth surface and migrating growth at colony edges were visualized on SS agar plate ( Fig. 1-A). Colonies were round, smooth and black on HE agar plates ( Fig. 1-B). Hydrogen sulfide and gasproducing characteristics of Proteus mirabilis were readily detected in triple sugar iron testing ( Fig. 1-C). In addition, when examined with an optical microscope, they were gram negative, polymorphic or rod-shaped bacilli, without spore or capsule formation ( Fig. 1-D).

Random amplification of polymorphic DNA (RAPD):
Seventeen distinct RAPD profiles were detected (Fig. 2). One profile (RAPD-A) was predominant (composed of 9 isolates). Six isolates were typed as RAPD-B; type RAPD-C and D contained 3 isolates each and 2 isolates were classified as type RAPD-E. The remaining 12 isolates were identified as 12 distinct types (1 isolate per type).
Proteus mirabilis PFGE analysis: Thirty-five P. mirabilis isolates were divided into 9 clusters by PFGE, revealing greater diversity than initially detected using RAPD. From the 9 clusters detected, there were 3 with 3 isolates and 6 with 2 isolates. The remaining 14 isolates were considered unrelated. Isolates from clusters were considered either to be the same strain (all clusters containing 2 isolates and 2 clusters of 3 isolates) or were closely related (2 clusters of 3 isolates) (Fig. 3).

Fig. 3:
Analysis of Pulsed-Field Gel Electrophoresis (PFGE) patterns obtained from the relevant Proteus mirabilis isolates. PFGE profile were compared by BioNumerics V 5.1 software. In this experiment, a global reference strain Salmonella enterica serotype Braenderup strain H9812, XbaI digestion, was used as the molecular marker (Marker) (Hunter et al., 2005).

DISCUSSION
To the best of our knowledge, this was the first report of the prevalence, antimicrobial susceptibility and genomic diversity of P. mirabilis isolated from canine fecal samples in China. Prevalence of P. mirabilis in feces from hospitalized diarrheic dogs in Beijing was estimated to be 28%. In the current study, despite the great diversity in P. mirabilis isolated, clustering of isolates was present using either RAPD or PFGE results. Five RAPD types (A, B, C, D and E) containing >1 isolate and indistinguishable isolates were present; consequently, we speculate that there is a common source of P. mirabilis in dogs with diarrhea (e. g. some RAPD types commonly isolated from UTIs and, therefore, being more prevalent in fecal content). Our data also demonstrated that 1 predominant type (RAPD-A) existed among tested isolates. Furthermore, potential ESBLs genes were identified in all type A isolates.
Antimicrobial resistance (AMR) in P. mirabilis isolated from dogs has been reported (Zhang et al., 2018). Broad-spectrum antimicrobials are frequently prescribed for treating bacterial infections in small animals. Overall prevalence of antimicrobial resistance in P. mirabilis isolated from dogs seemed higher than reported for human isolates (Siebor and Neuwirth, 2013). Prevalence of resistance to ampicillin (71%) was higher than the reported (Harada et al., 2014), probably due to presence of TEM penicillinases (Hordijk et al., 2013). The high prevalence of resistance to quinolone, trimethoprim/ sulfamethoxazole and aminoglycosides limits therapeutic options for infections caused by P. mirabilis, as these classes of antimicrobials are frequently used to treat dogs with infections caused by these bacteria (Wong et al., 2015).
ESBLs lead to extensive beta-lactam resistance, undermining efficacious therapeutic options for treating bacterial infections (Schultz et al., 2017). AMR rates differed between ESBL-positive and negative isolates. Among the non-beta-lactam antimicrobials, ESBL isolates also had higher resistance rates to chloramphenicol and some fluoroquinolones. The exact mechanism of increased resistance to non-beta-lactam antimicrobials in ESBL-producing strains remains unknown.
Proteus mirabilis is amongst the most common cephalosporin-resistant bacterial isolates from dogs (Fritsche et al., 2008). In our study, the blaTEM gene was detected in all ESBL-positive isolates. TEM-type ESBLs are among the most prevalent in P. mirabilis isolated from hospitalized patients (Ahn et al., 2017;Rajivgandhi et al., 2018). Despite being commonly present in members of the Enterobacteriaceae family (Alonso et al., 2017), blaSHV genes were not detected in any strain, corroborating recent results (Zhang et al., 2018). Prevalence of OXA-1 positive isolates in this study was much higher than in a French report (Bonnet et al., 2002).
Aminoglycosides bind irreversibly to the 30S small subunit of bacterial ribosomes, inhibiting mRNA transcription and protein synthesis. Resistance against amikacin (20%) was lower than that of gentamicin (46%) and tobramycin (51%), consistent with previous reports (Wieczorek et al., 2008). The rmtB gene was detected in 18 P. mirabilis isolates; half of these isolates were resistant to all aminoglycosides evaluated (gentamicin, kanamycin, tobramycin, streptomycin, amikacin). Perhaps high resistance to aminoglycosides was due to presence of the rmtB gene (Fritsche et al., 2008).
qnrB and aac(6')-Ib are important genes associated with decreased susceptibility to fluoroquinolones. In our study, presence of PMQR genes was always followed by resistance to fluoroquinolones. It is well-established that resistance against fluoroquinolones may also occur due to mutations in the quinolone-resistance determining region of specific genes (de Jong et al., 2018); therefore, mechanisms other than those screened could have been present for fluoroquinolone-resistant P. mirabilis isolates where no resistance genes were detected.
A previous study reported that companion animals and humans could be infected with closely related P. mirabilis strains, suggesting a potential role for companion animals as reservoirs of P. mirabilis to humans (Marques et al., 2019). Therefore, higher frequency of elimination of P. mirabilis to the environment through diarrhea in dogs, combined with high antimicrobial resistance of P. mirabilis in this study, represented a potential threat for human health.
This study had some limitations. Although Proteus spp. can be recovered from healthy individuals, they are more frequently isolated from feces of patients suffering from diarrhea. Whereas it is tempting to assume that P. mirabilis were the causative agents of diarrhea in studied dogs, especially due to their apparent high prevalence, there were not definitively implicated as the cause. Presence of enteropathogenic bacteria causing any intestinal disorder in dogs is clouded by presence of normal indigenous flora. Therefore, in the present study, no causal association can be established between presence of P. mirabilis and diarrhea in dogs. Additionally, as ESBL genes such as blaTEM were not sequenced, we cannot be sure whether variants detected were ESBLassociated genes or not.
Conclusions: P. mirabilis was commonly isolated from diarrheic dogs hospitalized in Beijing. A predominant RAPD type (A) was observed. Clustering of isolates was relatively common; several isolates were either indistinguishable or closely related. Isolates had high resistance to a majority of common antimicrobials. blaTEM gene was detected in all ESBL-producing isolates.