Assessment of the gradient diffusion method for fosfomycin susceptibility testing in Staphylococcus spp. and Enterococcus spp. isolated from the urine of companion dogs in Thailand

Background and Aim: The agar dilution method is the approved method for determining the minimum inhibitory concentration (MIC) in fosfomycin susceptibility testing, whereas the broth dilution method is not recommended. This study aimed to investigate the potential of the gradient diffusion method as a more convenient alternative to agar dilution method for MIC evaluation, particularly for the susceptibility testing of Staphylococcus spp. and Enterococcus spp. to fosfomycin. Materials and Methods: A total of 194 isolates of Staphylococcus spp. and Enterococcus spp. were collected from urine samples of dogs diagnosed with bacterial cystitis. Bacterial identification and susceptibility to multiple antibiotics were tested using the Vitek 2 automated system. The susceptibility to fosfomycin was compared between agar dilution (reference method) and the gradient diffusion method. We assessed the agreement rates and errors between the two approaches by analyzing the MIC data. Results: Staphylococcus pseudintermedius (98.7%) and Enterococcus faecalis (80.0%) exhibited high fosfomycin susceptibility rates, whereas Enterococcus faecium exhibited a lower susceptibility rate (38.5%). The gradient diffusion method demonstrated unacceptably low essential agreement (EA) rates (>90%) but acceptable categorical agreement (CA) rates (≥ 90%) for S. pseudintermedius (83.54% EA and 97.47% CA) and coagulase-negative staphylococci (CoNS) such as Staphylococcus chromogenes, Staphylococcus hominis, and Staphylococcus simulans (85.00% EA and 95.00% CA). Enterococcus spp. had an acceptable EA of 93.75%, but an unacceptably low CA rate of 82.81%, with a minor error rate of 17.19%. No significant errors were observed for Staphylococcus and Enterococcus spp. Conclusion: The gradient diffusion method reliably determines MICs and interpretative breakpoints (S, I, R) for S. pseudintermedius. However, its applicability to CoNS and enterococci may be limited due to unacceptable errors.


Introduction
Fosfomycin is a broad-spectrum bactericidal drug.This drug does not undergo any metabolic process and is excreted in its active form through the kidneys [1,2].The high concentrations of fosfomycin in urine make it a favorable choice for treating uncomplicated urinary tract infections (UTIs) in humans [3,4].However, fosfomycin has not yet been approved for veterinary use in many countries, including the European Union, and its administration to individual companion animals is restricted to exceptional circumstances due to concerns related to public health and the growing problem of antimicrobial resistance [5,6].Therefore, there are limited data on the use of fosfomycin in animals.Fosfomycin has been suggested as an alternative antibacterial drug for treating bacterial cystitis in dogs when other antibacterial drugs are ineffective or unavailable [7,8].
Bacterial cystitis is widely recognized as one of the most common diseases among dogs.In Thailand, Staphylococcus spp.and Escherichia coli are the main uropathogens of these infections [9,10].The antimicrobial resistance of methicillin-resistant Staphylococcus pseudintermedius (MRSP) is of particular concern.Several studies have indicated that MRSP strains display multidrug resistance (MDR) to three or more antibacterial classes [11,12].Enterococci commonly cause UTIs and are naturally resistant to many antibacterial drugs [13,14].Therefore, the susceptibility data of Staphylococcus spp.and Enterococcus spp., which are the main Gram-positive uropathogens in Copyright: Jariyapamornkoon, et al.Open Access.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
dogs, may provide supportive information for the clinical treatment of fosfomycin.
Based on the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the agar dilution method is the approved method for minimum inhibitory concentration (MIC) measurement in the susceptibility testing of fosfomycin, whereas the broth dilution method is not recommended [15,16].Automated susceptibility testing should therefore be avoided [17].However, the agar dilution method has certain disadvantages, such as being time-consuming, laborious, and inconvenient for routine laboratory work.Alternatively, gradient diffusion method may offer a more convenient practice for determining MIC values, but its reliability needs to be evaluated.The agreement between agar dilution and gradient diffusion methods has been extensively studied in bacteria such as Klebsiella pneumoniae and E. coli [17][18][19].However, it does not agree with Gram-positive bacteria, especially in animals, and remains poorly explored.To address this research gap, the present study focuses on investigating Staphylococcus spp.and Enterococcus spp.found in dog urine.
The main objectives of this study were to determine the susceptibility of these bacteria to fosfomycin and other antibacterial drugs and to evaluate the reliability of the gradient diffusion method compared to the reference method for susceptibility testing.

Ethical approval
This study does not require ethical approval.All urine samples were obtained from the Veterinary Diagnostic Laboratory of the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.

Study period and location
This study was conducted from July to December 2022.The samples were processed at the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.

Bacterial identification
A total of 194 Staphylococcus spp.and Enterococcus spp.isolates were obtained from the Veterinary Diagnostic Laboratory of the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.Bacterial samples were isolated from urine samples of dogs diagnosed with bacterial cystitis at the Small Animal Teaching Hospital of Veterinary Medicine of Chulalongkorn University from 2018 to 2021.Bacterial isolates were identified using a Vitek 2 automated system equipped with Gram-positive identification cards (ID-GP card) (BioMerieux, Marcy L'Étoile, France).For further analysis, all bacterial samples were stored in 30% glycerol storage media at −80°C.

Susceptibility testing to fosfomycin using agar dilution method
Agar dilution was performed in accordance with the CLSI standard for determining the MIC of fosfomycin [16,20].The inoculum was cultured on Mueller-Hinton agar (Difco, Franklin Lakes, NJ, USA) supplemented with 25 mg/L glucose-6-phosphate (Sigma-Aldrich, Saint Louis, MO, USA) plates containing 0.125-256 mg/L fosfomycin (Sigma-Aldrich) using a 48-pin replicator (Sigma-Aldrich).The final inoculum concentration was approximately 1 × 10 4 colony forming unit/spot.All isolate analyses were performed in triplicate.Quality control testing was conducted by examining the reference strains of Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 [16].The lowest concentration of fosfomycin-inhibited visible colonies was recorded and interpreted based on MIC breakpoints [15,16].

Susceptibility testing to fosfomycin using gradient diffusion method
The gradient diffusion technique (Liofilchem, Roseto degli Abruzzi, Italy) was performed according to the manufacturer's instructions [21].Isolated bacterial colonies were suspended in a 0.85% sodium chloride solution to achieve a McFarland standard turbidity of 0.5.The prepared inoculum was spread onto Mueller-Hinton II agar (MHA) using a sterile cotton swab.A gradient diffusion strip ranging from 0.016 to 256 mg/L supplemented with glucose-6-phosphate (G6P) was placed onto the MHA surface before incubation at 37°C for 16-20 h or extended for up to 48 h for slow-growing bacteria.We recorded and interpreted the MIC strip's scale that intersected the inhibition zone according to MIC breakpoints [15,16].All of the isolates were tested in triplicate.Staphylococcus aureus ATCC 29213 and E. faecalis ATCC 29212 [21] were used as quality control isolates.

Interpretive criteria for MIC of fosfomycin
The EUCAST criteria were applied with MIC breakpoints for Staphylococcus spp. as follows: susceptible (S) 32 mg/L and resistance (R) >32 mg/L [15].The CLSI criteria for E. faecalis were implemented with MIC breakpoints: S <64 mg/L, intermediate (I) 128 mg/L, and R >256 mg/L [16].In the absence of approved fosfomycin breakpoints for E. faecium, the MIC breakpoints for E. faecalis were utilized to interpret the susceptibility of E. faecalis, following a similar approach as in a previous study [19].

Comparative analysis of susceptibility testing methods
We analyzed the degree of agreement and error between the MICs obtained using agar dilution (reference) and gradient diffusion methods.The analysis focused on the following parameters: (1) the essential agreement (EA) refers to the MIC of gradient diffusion method equal to or within ±1 dilution of the reference method MIC, (2) the categorical agreement (CA) refers to the agreement of susceptibility categories (S, I, R) between the gradient dilution method and the reference method, (3) major errors (ME) refer to the result of gradient diffusion was resistant but the result of reference method was susceptible, (4) very major errors (VMEs) refer to the result of gradient diffusion was susceptible but the result of reference method was resistant, (5) minor error (mE) refers to when the result of gradient diffusion was susceptible or resistant but the result of reference method was intermediate, or when the result of gradient diffusion was intermediate but the result of reference method was susceptible or resistant [19,23].
On the basis of CLSI standards, an acceptable rate of >90% for the EA and the CA should be adopted.The acceptable ME and VME should be <3.0%[23].Regarding the gradient diffusion method, the susceptibility rates observed for MSSP, S. aureus, and E. faecalis (Table-2) were similar to those obtained from the agar dilution technique.However, the susceptibility rates for MRSP, CoNS, and E. faecium were lower (95.4%,87.5%, and 33.3%, respectively) than the agar dilution method (97.9%, 90.0%, and 38.5%, respectively).

Comparative analysis of susceptibility testing methods
The MIC 50 and MIC 90 values of fosfomycin obtained from agar dilution and gradient diffusion methods were similar or within ±1 doubling dilution for S. pseudintermedius, CoNS, and Enterococcus spp.(Table -4).The EA between the gradient dilution and agar dilution methods for Enterococcus spp.achieved an acceptable rate (90%), whereas it was below 90% for S. pseudintermedius and CoNS.Both S. pseudintermedius and CoNS achieved an acceptable rate of CA (90%), whereas Enterococcus spp.had an unacceptably low CA.Staphylococcus pseudintermedius and Enterococcus spp.exhibited a ME rate within an acceptable range (3%), whereas CoNS showed unacceptably high ME rates.No significant errors were identified in Staphylococcus spp.and Enterococcus spp.Minor errors were observed exclusively in Enterococcus spp.testing.

Discussion
Staphylococcus pseudintermedius has been frequently identified as the predominant uropathogen causing UTIs in dogs across many countries, including Thailand [9,24,25].MRSP isolates display MDR to three or more different antibacterial classes [11,12].Therefore, antimicrobial resistance in S. pseudintermedius poses a significant challenge in terms of treatment options for dogs.Coagulase-negative staphylococci are typically considered opportunistic or contaminant pathogens, but they have also been found to be causative agents of infections [26,27].
In the present study, Staphylococcus spp.isolates, including MRSP, displayed high susceptibility rates to fosfomycin.These findings are consistent with a previous study that reported an approximately 84% susceptibility rate in MRSP from dogs, with an MIC 50 of 0.125 mg/L [28].Although CoNS isolates showed a 90% susceptibility rate to fosfomycin, approximately 20% of CoNS isolates (n=8/40) exhibited a MIC of 32 mg/L, which closely approached the interpretative breakpoints of S 32 and R > 32 mg/L [16].Therefore, the use of fosfomycin for CoNS may require intensive monitoring for developing resistance.With regard to Enterococcus spp., we observed a high susceptibility rate of 80% for E. faecalis, which is consistent with the results reported for humans (94.4%) [29].However, regarding E. faecium, only 38.5% of samples were susceptible to fosfomycin.A previous study indicated that more than 90% of Enterococcus spp.isolated from dog wounds and dermatitis are resistant to fosfomycin [30].These results suggest that fosfomycin may be a suitable treatment for E. faecalis but not for E. faecium, emphasizing the importance of bacterial identification and susceptibility testing before fosfomycin administration.
Our results indicate that S. pseudintermedius isolates exhibit low susceptibility to trimethoprim, sulfamethoxazole, and fluoroquinolones.In addition, MRSP isolates displayed low susceptibility to amoxicillin/clavulanic acid (64.6%), which is a recommended empirical drug for bacterial cystitis in dogs, as well as trimethoprim/sulfamethoxazole [31].These findings are consistent with the previous studies reporting low susceptibility rates of MRSP strains to amoxicillin/clavulanic acid (35%) [32] and high resistance rates to enrofloxacin (38%) [33].According to CLSI guidelines, MRSP isolates are generally resistant to all beta-lactam antibacterial drugs except ceftaroline [16].Considering the high susceptibility rates to fosfomycin (>97%) observed in this study, fosfomycin may be a reasonable alternative antibiotic for S. pseudintermedius, including MRSP strains.
The susceptibility rate of E. faecalis to amoxicillin/clavulanic acid (96%) was consistent with that reported in the previous studies (95%) [34].Enterococcus faecium isolates from canine UTIs have been reported to exhibit high resistance rates to ampicillin (67.9%), enrofloxacin (91.8%), and nitrofurantoin (90.9%) [13].Enterococcus spp. is known to be intrinsically resistant to various antibacterial drugs [14], with E. faecium isolates being more resistant than E. faecalis [35].Therefore, selecting appropriate antibacterial drugs for E. faecium infection may be difficult.
While the use of gradient diffusion strips offers a convenient alternative for measuring MIC, several studies have reported low agreement between MIC results obtained through agar dilution (reference method) and other methods, particularly in K. pneumoniae and E. coli [17,18].The presence of colonies on the agar makes reading difficult and may affect the accuracy of the results [17].On the other hand, the use of E test to determine MICs in S. aureus and E. faecalis isolates from humans has shown good concordance with the agar dilution method, as reported in early publications [19,36].At present, however, there is no available data on S. pseudintermedius.
In the present study, the fosfomycin susceptibility rate of S. pseudintermedius obtained using the gradient diffusion method decreased by approximately 1% compared to that obtained using the agar dilution method.The EA rate between these two methods is unacceptably low (83.54%).However, the CA and ME rates fell within acceptable ranges (≥90% and ≤3%, respectively).These findings are consistent with those of an earlier study on S. aureus, where the EA, CA, and ME rates were 84.1%, 98.7%, and 1.3%, respectively [19].The low EA rate suggests that reporting MIC values alone without interpreting them as S, I, or R using the gradient diffusion method may not be appropriate [19].Regarding CoNS, the agreement results were similar to those of S. pseudintermedius (unacceptable EA and acceptable CA), but the ME rate was unacceptably high (5.56%,n=2).The two isolates that exhibited ME results were identified as S. chromogenes and S. warneri.These findings emphasize the need to exercise caution when interpreting the MIC values obtained using the gradient diffusion method for CoNS.With regard to Enterococcus spp., the study showed an acceptable EA rate; however, the CA rates exceeded the acceptable range (>90%) with a relatively high rate of MEs (17.19%).These results are consistent with those of a previous study on E. faecalis, which reported rates of 98.5% for EA, 81.2% for CA, and 18.8% for mE [19].Unsatisfactory CA and mE rates indicate the importance of interpreting MIC values obtained by gradient diffusion for Enterococcus spp.cautiously.
This study has certain limitations.The small number of bacterial samples used in this study may have affected the accuracy of the percentage data.Therefore, agreements and errors were evaluated between different methods using groups of bacteria rather than individual species.In the assessment study, S. aureus isolates were not included alongside S. pseudintermedius as coagulase-positive staphylococci because S. pseudintermedius, the main uropathogen in dogs, was the primary focus of this study.In addition, the interpretation of susceptibility to fosfomycin was based on human guidelines since there are currently no MIC breakpoints for veterinary use.

Conclusion
The high susceptibility rates of Staphylococcus spp.and E. faecalis to fosfomycin indicate the efficacy of fosfomycin as an alternative antibiotic for the treatment of canine bacterial cystitis caused by susceptible strains.However, the low susceptibility rate of E. faecium suggests that fosfomycin may not be effective against this pathogen.Minimum inhibitory concentration determination in S. pseudintermedius with interpretative breakpoints (S, I, and R) proved reliable using the gradient diffusion method.In addition, caution should be exercised when using this method for CoNS and Enterococcus spp.due to the presence of unacceptable errors.

Table - 1
: Distribution of MICs and the susceptibility rates to fosfomycin (FOS) using agar dilution method.

Table - 4
: Comparative analysis of susceptibility testing methods.