Urinary tract infection due to Staphylococcus schleiferi biofilm formation in the subcutaneous ureteral bypass system in a cat

Abstract

Background

Staphylococcus schleiferi is mainly isolated from dogs and occasionally infects cats. We recently encountered a case of a biofilm-related urinary tract infection (UTI) caused by S. schleiferi in a cat with a subcutaneous ureteral bypass (SUB) system. This report presents a case of biofilm formation by S. schleiferi in the SUB system and discusses the causes of UTIs.

Case presentation

A 9-year-old female cat had been using the SUB system since 4 years-of-age. The cat had no significant clinical history or UTIs for 4 years after the first implantation of the SUB system. The SUB system was flushed once per month. When the cat was 8 years-of-age, the subcutaneous port of the SUB system was contaminated with Staphylococcus pseudintermedius and replaced with a new one. Subsequently, the SUB system had no particular problem for 8 months. The SUB system was flushed once every 2 months. However, the cat occasionally developed gross haematuria. Additionally, S. schleiferi was detected in urine. Although doxycycline was administered to the cat, 6 weeks later, the cat had cutaneous wounds with abscesses caused by excessive grooming of the skin in contact with the subcutaneous port of the SUB system. S. schleiferi was detected in a severe abscess in the cutaneous wound, and skin necrosis was observed. As bacterial contamination of the SUB system was suspected, the SUB system was removed from the cat. Scanning electron microscopy analysis revealed biofilm formation inside the locking loop catheters and outside the subcutaneous port of the SUB system. In an in vitro assay, S. schleiferi isolated from a catheter of the SUB system had low biofilm-forming ability. After the SUB system was removed, S. schleiferi was not detected in the urine and the infection was completely cured.

Conclusion

Considering these results, bacterial infections in cats with SUB systems should be carefully monitored, as contamination by biofilm-forming bacteria can occur regardless of flushing frequency.

Background

Although Staphylococcus schleiferi colonises the healthy skin of dogs and cats [1, 2], it is pathogenic, causing ear and skin infections in dogs [3, 4]. Although S. schleiferi infections appear to be much less common in cats than in dogs [3], clinical outcomes of cats with suppurative otitis media have occasionally been reported [3, 5]. Among these infections, the increased prevalence of Staphylococcus spp., including methicillin-resistant S. schleiferi, is a concern in the veterinary field [6]. However, clinical outcomes associated with the pathogenicity of S. schleiferi, except for ear and skin infections, have not been well reported. Over the past decade, the subcutaneous ureteral bypass (SUB) system has revolutionised the treatment of upper urinary tract urolithiasis in cats [7]. This allows urine to flow from the kidney to the bladder, bypassing the obstructed ureter and allowing renal decompression and function to return. However, chronic bacteriuria is a common complication of SUB placement [8, 9]. This report describes a urinary tract infection (UTI) caused by S. schleiferi in a cat using the SUB system, and details the biofilms formed in the SUB system. In addition, we evaluated the biofilm-forming ability of S. schleiferi isolated from the SUB system.

Case presentation

A 9-year-old female cat had been using the SUB system since 4 years-of-age. The cat was kept indoors with another cat and did not cohabitate with dogs. At the pet clinic, the SUB system was cleaned by veterinarians once a month with alternating saline and saline with 2% tetra-ethylenediaminetetraacetic acid (EDTA), and hygiene protocols were followed during the procedure. Suitable function of the SUB system was maintained for 4 years. However, when the cat was 8 years-of-age, it experienced cutaneous wounds with S. schleiferi infection caused by excessive grooming of the skin in contact with the subcutaneous port of the SUB system [10]. Although orbifloxacin (4.0 mg/kg body weight [BW] p.o. semel in die [SID]), i.e., a fluoroquinolone, was administered to the cat to treat the infection, a fluoroquinolone-resistant Staphylococcus pseudintermedius with biofilm-forming ability (strain 2306K1) was isolated from the wound abscess in contact with the subcutaneous port of the SUB system [10]. Surgery for replacement of the new subcutaneous port was performed, and the cutaneous wound healed. On the other hand, many scraps by the needle were observed on the septum after 4 years in-use by the cat (Fig. 1). Increased flushing frequency can lead to scratches on the septum of the SUB system, thereby bacterial infection may be caused. Therefore, the SUB system was cleaned with saline containing 2% tetra-EDTA, and the cleaning interval was extended from once a month to once every two months. The cat showed no significant clinical signs for 8 months. However, the cat occasionally developed gross haematuria, and S. schleiferi was detected in urine. Although doxycycline (8 mg/kg BW p.o. SID) was administered to the cat, 6 weeks later the cat developed cutaneous wounds with abscesses caused by excessive grooming of the skin in contact with the subcutaneous port of the SUB system. S. schleiferi was also detected in severe abscesses in the cutaneous wound, and skin necrosis was observed. As bacterial contamination of the SUB system was suspected, the SUB system was removed from the cat. The drug susceptibility of S. schleiferi was as follows: ampicillin, amoxycillin, piperacillin, and gentamycin were resistant, and cefpodoxime proxetil, cefcapene pivoxil, and cefepime were intermediate. Other major antibiotics were susceptible. To prevent infection, cefalexin (18‒20 mg/kg BW p.o. SID) was administered; however, methicillin-resistant S. pseudintermedius (strain 2406K1) and Pseudomonas aeruginosa were detected in surgical wounds after 46 days of surgery. The wounds were continuously cleaned with electrolysed water every other day for 70 days, and the cat entered complete remission with no UTIs. Coincidentally, the ureteral obstruction healed completely and kidney function was preserved. Since then, the condition of kidney of the cat has been monitored every two to three months.

Fig. 1.

The septum of the SUB system collected from the cat after 4 years in-use. The septum of the SUB system was collected from the cat during replacement surgery for the new SUB system

To clarify the cause of the UTI, we isolated S. schleiferi from the SUB system collected from the cat. The inside of the locking loop catheters of the SUB system appeared dirty (Figs. 2a, b). Additionally, to isolate bacteria from the catheters, both the kidney and bladder sides of the catheter (1. 5-cm long) were washed with 0.5 mL of saline, and gram-positive cocci were observed (Figs. 2c and d). Higher bacterial counts were observed on the bladder side than on the kidney side of the locking loop catheter. The bacteria isolated from these catheters included only one strain, which was reidentified as S. schleiferi (strain 2404K2) using MALDI-TOF MS (ICLAS Monitoring Centre, Central Institute for Experimental Medicine and Life Science, Kanagawa, Japan). These results suggested that S. schleiferi contaminated the bladder. Next, we observed the locking loop catheter and the subcutaneous port of the SUB system using scanning electron microscopy (SEM) (Hitachi TM-3030, Hitachi High-Tech Corporation, Tokyo, Japan). Biofilm formation was observed on the kidney side (Figs. 3a‒d), near the port (Figs. 3e‒h), and on the bladder side of the catheter (Figs. 3i‒l), and was more visible and mature in the bladder catheter than in the kidney catheter (Figs. 3a, i). Additionally, biofilm formation was observed on the septum (Figs. 4a‒c) and metal parts of the subcutaneous port (Figs. 4d‒f). These results indicated that the UTI and contamination of the subcutaneous port were caused by biofilms formed by S. schleiferi.

Fig. 2.

Gram-stained S. schleiferi isolated from the locking loop catheter of the SUB system after 8 months in-use. The SUB system was collected from a cat with a UTI. Photographs of the: (a) kidney side, and; (b) bladder side of the locking loop catheter in the SUB system. Gram-positive cocci were observed from the: (c) kidney side, and; (d) bladder sides of the catheter (1. 5-cm long) after washing with 0.5 mL of saline

Fig. 3.

Biofilm formation by S. schleiferi inside the locking loop catheter of the SUB system. The locking loop catheter of the SUB system was observed via SEM. SEM images of inside: (a‒d) the kidney side, (e‒h) near the port, and (i‒l) the kidney side of the locking loop catheter. SEM images at: (a, e, and i) × 40, (b, f, and j) × 1000, (c, g, and k) × 5000, and (d, h, and l) × 10,000 magnifications. Scale bar = 2 mm, 100, 20 or 10 µm

Fig. 4.

Biofilm formation by S. schleiferi outside the subcutaneous port of the SUB system. The subcutaneous port of the SUB system was observed via SEM. SEM images of the: (a‒c) septum and (d‒f) metal parts. SEM images at: (a and d) × 1000, (b and e) × 5000, and (c and f) × 10,000 magnifications. Scale bar = 100, 20 or 10 µm

Furthermore, we evaluated the biofilm-forming ability of S. schleiferi strain 2404K2 in vitro. Growth of planktonic cells of the S. schleiferi strain 2404K2 was almost the same as that of other Staphylococcus spp. strains (Fig. 5a). However, adhesion of the biofilm of S. schleiferi strain 2404K2 was lower than that of S. pseudintermedius strain 2306K1, and the amount of precipitable biofilm of S. schleiferi strain 2404K2 was less than that of other Staphylococcus spp. strains (Fig. 5b). These results suggested that S. schleiferi strain 2404K2 had a low biofilm-forming ability but caused biofilm-related UTIs in the cat with the SUB system. Additionally, the isolate was negative in the coagulase test, consistent with identification as S. schleiferi subsp. schleiferi.

Fig. 5.

Evaluation of biofilm-forming ability of S. schleiferi strain 2404K2. S. aureus strains (ATCC 29213 and 25,923), S. pseudintermedius strains (2306K1 and 2406K1), and a S. schleiferi strain (2404K2) were cultured on a 96-well microtitre plate with a 96-peg lid (DOJINDO LABORATORIES, Kumamoto, Japan) in Luria–Bertani broth for 24 h at 39 °C. S. pseudintermedius strains (2306K1 and 2406K2) were isolated from the same cat and were used as comparable strains for biofilm formation. S. aureus strains (ATCC 29213 and 25,923) were used as control strains for biofilm formation. a Growth of planktonic cells determined by optical density at 595 nm (OD₅₉₅). b Biofilm formation determined by absorbance at 590 nm (Abs₅₉₀) after samples was stained with crystal violet. In these experiments, bacteria were cultured at 39 °C because the average normal cat body temperature is approximately 37.7–39.5 °C [11]. Adhesive refers to biofilm formation on a 96-peg lid. Precipitable refers to biofilm formation on a 96-well plate

Discussion and conclusion

The SUB system has revolutionised the treatment of upper urinary tract urolithiasis in cats [7]. However, chronic bacteriuria is a common complication of SUB placement [8, 9]. In the present study, we report the case of a cat with a UTI caused by S. schleiferi associated with the SUB system. After the initial implantation of the SUB system, the cat had no significant medical history and no UTI for 4 years, indicating that the first period of the SUB system in-use was uneventful. However, after replacement with the new subcutaneous port of the SUB system, S. schleiferi formed biofilms in the SUB system within 8 months, suggesting that the second period of the SUB system in-use was inappropriate. There are two possible routes through which S. schleiferi may have adhered to the SUB system. One possibility is that the cat habitually groomed the skin in contact with the subcutaneous port of the SUB system, allowing S. schleiferi from the oral cavity to adhere to the SUB system via damaged skin. Another possibility is that cleaning frequency of the SUB system was reduced from once a month to once every two months. The SUB system was cleaned once every month with alternating saline and saline with 2% tetra-EDTA during the first period, and once every 2 months with saline with 2% tetra-EDTA during the second period. A recent study reported that prophylactic use of 2% tetra-EDTA for SUB flushing may be helpful in reducing the frequency of long-term SUB device complications and could be considered part of the long-term management plan [12]. Although saline with 2% tetra-EDTA was used in the present study, biofilms formed in the SUB system. The threshold for UTI associated with the SUB system is presumed to be the frequency of SUB system flushing. A previous study reported that the incidence of bacteriuria in catheterised human patients is directly related to the duration of catheterisation; the daily rate of bacteriuria acquisition is approximately 3–10% [13]. Therefore, in cats with prolonged SUB system catheterisation, the frequency of the SUB system flushing may be an important factor in preventing UTIs. In the present case, the route by which S. schleiferi adhered to the SUB system remains unclear. As a strategy, a high flushing frequency may play an important factor in keeping the SUB system clean. However, risk of bacterial infection from contaminated septum must always be considered. Further studies are needed to develop strategies to prevent infection in cats with SUB system.

Deprey et al. reported that bacteriuria was resolved with appropriate antibiotic treatment in > 50% of cats, although the risk factors identified for positive urine cultures were longer hospitalisation duration and decreased body condition score [9]. However, in the present case, doxycycline administration was not effective against the UTI. Previous studies have reported that biofilm cells resist antibiotics in vitro [10, 14, 15], suggesting that biofilm-related chronic bacterial infections can be maintained in vivo despite antibiotic treatment. Currently, biofilm-forming bacteria cannot be detected using general bacterial tests. Thus, management of the SUB system is important for preventing bacterial biofilm formation.

As an example of pathogenicity, Staphylococcus aureus and the coagulase-variable species S. schleiferi are less frequently associated with superficial bacterial folliculitis in dogs than S. pseudintermedius [16, 17], suggesting that S. schleiferi has lower pathogenicity than S. pseudintermedius. Notably, S. schleiferi comprises two subspecies: S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans, which are coagulase-negative and coagulase-positive, respectively [18]. A recent study reported a case of catheter-related bloodstream infection caused by S. schleiferi subsp. coagulans in a human patient, thereby providing evidence that this subspecies has pathogenic potential in humans [19], suggesting that S. schleiferi subsp. coagulans may exhibit increasing pathogenic. However, the pathogenicity of S. schleiferi subsp. schleiferi remains uncertain. In the present case, although S. schleiferi subsp. schleiferi demonstrated low biofilm-forming ability in vitro, it nevertheless formed biofilms on the SUB system. While it is difficult to establish an experimental system that accurately mimics the host environment, the present case provides valuable insights into biofilm-related infections. Of course, further studies are necessary to elucidate the pathogenicity of S. schleiferi in companion animals.

In conclusion, bacterial infections in cats with SUB systems should be carefully monitored, and further research is warranted to develop strategies to prevent biofilm formation. One of the preventive measures is the development of more effective flushing solutions. Additionally, the pathogenicity of S. schleiferi remains unclear, and further studies are needed to clarify the virulence of S. schleiferi in companion animals and develop eradication methods against bacterial biofilms.

Abbreviations

UTI

Urinary tract infection

SUB

Subcutaneous ureteral bypass

EDTA

Ethylenediaminetetraacetic acid

BW

Body weight

SID

Semel in die

SEM

Scanning electron microscopy

OD₅₉₅

Optical density at 595 nm

Abs₅₉₀

Absorbance at 590 nm

Authors’ contributions

Y. Sato and Y. Suzuki contributed to analysis and interpretation of patient data regarding case management. N.H. was a major contributor to bacteria isolation and in vitro studies. Y. Sato and N.H. validated the in vitro study. Y. Sato wrote the manuscript. N.H., Y. Suzuki, N.Y., and Y.Y. revised the manuscript. All authors contributed to the discussion.

Funding

This research was supported by JSPS KAKENHI (grant number 24K11641) and ACRO Incubation Grants from Teikyo University (grant numbers 22–72 and 23–93).

Data availability

The datasets analysed in the present study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.