550171 research-article2014
JFM0010.1177/1098612X14550171Journal of Feline Medicine and SurgeryLund et al
Original Article
Antimicrobial susceptibility in bacterial isolates from Norwegian cats with lower urinary tract disease
Journal of Feline Medicine and Surgery 2015, Vol. 17(6) 507–515 © ISFM and AAFP 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1098612X14550171 jfms.com
Heidi Sjetne Lund, Gaute Skogtun, Henning Sørum and Anna Vigdís Eggertsdóttir
Abstract
Studies of feline lower urinary tract disease (FLUTD) among Norwegian cats have shown higher prevalences of bacterial cystitis than most previously published reports. The aims of the present study were to identify bacterial isolates obtained from the urine of Norwegian cats with FLUTD and their susceptibility to antimicrobial agents. Eighty-two bacterial isolates from 72 urine cultures obtained from 71 different cats were included. Escherichia coli, Staphylococcus species, Enterococcus species and Streptococcus species were the most frequently detected. The percentages of isolates susceptible to the included antimicrobial agents were as follows: enrofloxacin – 92%; trimethoprim/sulfonamide – 91%; nitrofurantoin – 89%; tetracycline – 78%; ampicillin – 73%; amoxicillin/clavulanic acid – 72%; trimethoprim – 68%; amoxicillin – 58%; cephalexin – 51%; spiramycin – 39%; penicillin – 34%; fucidic acid – 34%; lincomycin – 27%. Although several tendencies towards increasing antimicrobial resistance were detected among the isolates included, the species of bacteria isolated and their patterns of antimicrobial resistance were, in general, in concurrence with the existing literature. Thus, the results do not fully explain the higher prevalence of bacterial cystitis found in Norwegian cats. Moreover, additional explanatory factors beside the inclusion of primary accession cases rather than referred cases were not found. Accepted: 14 August 2014
Introduction While feline lower urinary tract disease (FLUTD) is common in small animal veterinary practice,1–3 bacterial cystitis in cats is reported to be relatively rare (11 years.11 The age groups constituted 19%, 42%, 29% and 11% of the material, respectively.11 In addition to aspects related to the sampling and handling of urine samples, factors of potential influence on the prevalence of bacterial cystitis may be the type of bacteria and their susceptibility to antimicrobial agents, or aspects related to the cat population itself, including genetic predisposition to bacterial cystitis in certain breeds or families of cats.
Norwegian University of Life Sciences, Oslo, Norway Corresponding author: Heidi S Lund DVM, Norwegian University of Life Sciences, PO Box 8146 Dep, Oslo, 0033, Norway Email: [email protected]
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508 The aims of the present study were to identify bacterial isolates obtained from urine from Norwegian cats with FLUTD and their susceptibility to antimicrobial agents.
Materials and methods Study sample The bacterial cultures presented stem from a larger study of FLUTD in Norwegian cats. The study population consisted of client-owned cats from Oslo and surrounding areas, presented at the clinics of the Small Animal Section, Norwegian School of Veterinary Science from 2003 until 2009. If informed consent was obtained from the owners, cats with clinical signs (dysuria, stranguria, haematuria, pollakiuria and/or periuria) and a final diagnosis consistent with FLUTD were eligible for inclusion as cases in the study. Exclusion criteria were treatment that could interfere with the diagnostics (antimicrobial medication within the last 3 months, hormones, or medication altering blood pressure, urine production and/or composition) and known concurrent diseases likely to be of influence on the urinary findings, such as chronic kidney disease, diabetes mellitus or hyperthyroidism. Only cats aged 7 months or older were included; no sex or breed restrictions were made. Methods A standardised questionnaire was used to register age, breed, sex, reproductive status, body weight, prior health history and information concerning housing conditions and feeding regime for each cat. A veterinarian performed a clinical examination and, in addition to urine samples, blood samples for routine haematology and clinical chemistry were collected. Abdominal radiography or ultrasound was included as part of the diagnostic investigation. If no indication of concurrent disease was found in the history, physical examination, laboratory results or radiological examination, concurrent disease was considered unlikely. Follow-up consultations were scheduled if concurrent disease non-detectable at the initial diagnostic investigation was suspected. Urine samples were obtained either by cystocentesis, aseptic catheterisation or collection of urine voided into sterile containers by gentle bladder compression on anaesthetised cats. Standard urinalysis was performed, including commercial urine dipstick analysis (Krulab; Kruuse), urine specific gravity measured with refractometer (URC-Ne; Atago) and microscopic examination of the urine sediment (native wet samples and wet samples stained with Sternheimer–Malbins staining solution). All urine samples were cultured on the day of collection. Quantitative bacteriology was performed by streaking 1 µl of urine onto blood agar (Blood Agar Base II; Difco), and qualitative bacteriology by cultivation on blood agar and selective bromo-thymol blue agar (Difco)
from sediments after centrifugation, incubated at 37oC aerobically and in a 5 % CO2-enriched atmosphere. Susceptibility to antimicrobial agents was determined in accordance with the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).12 Semi-confluent growth of the isolates on Mueller–Hinton agar (Difco) with antimicrobial discs (NeoSensitabs; Rosco) were incubated at 35oC. The selection of antimicrobial agents tested included penicillin, ampicillin, amoxicillin with and without clavulanic acid, cephalexin, trimethoprim, trimethoprim/sulfonamide, tetracycline, fucidic acid, enrofloxacin, spiramycin, lincomycin and nitrofurantoin. In this study, all cultures with ⩾103 cfu of two or fewer types of bacteria were included, independent of the method of urine sampling or whether the cats were diagnosed as having bacterial cystitis or not. However, in the total study population of cats from which the bacterial cultures in the present study stem from, a diagnosis of bacterial cystitis was only made after evaluating the method of urine collection in connection with the type and amount of bacteria detected.9,10,13 Cats with uroliths or urethral plugs in combination with positive bacterial cultures were categorised as having uroliths and urethral plugs, respectively. Further, samples obtained by unknown methodology were considered as voided samples in order not to overestimate the importance of the bacteria detected. Not all the information from the questionnaire and clinical investigation was used in the present study; other aspects of the obtained data have been previously published for subsets of the cats included in the present study.9–11,13,14 Statistical analyses The results are presented as frequencies of occurrence, expressed in percentages. Statistical analyses were performed in JMP 8 (SAS Institute).
Results Study sample A total of 82 bacterial isolates from 72 urine cultures from 71 different cats were included. Two urine samples were cultured from the same cat, but during separate episodes of clinical signs of FLUTD. Sixty-two of the cultures resulted in a single bacterial isolate, while in the remaining 10 cultures (all voided samples) two different isolates were detected. Descriptive statistics for the cats, including sex and reproductive status, age, breed, method of urine sampling and obstructed/non-obstructed form of FLUTD, are listed in Table 1. Among the purebred cats included in the study there were six Persians, five Norwegian Forest cats, two British Shorthairs, one Abyssinian and one Maine Coon.
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Table 1 Descriptive statistics of 71 cats with lower urinary tract disease and positive urine cultures Variable
Category
n (%)
Sex and reproductive status
Male Male neutered Female Female neutered 0–2 3–6 7–10 >11 Pure breed Domestic shorthair Voiding by manual compression Catheterisation Cystocentesis Unknown Non-obstructed Obstructed
8 (11) 39 (55) 9 (13) 15 (21) 23 (32) 22 (31) 15 (21) 11 (16) 15 (21) 56 (79) 42 (59) 8 (11) 19 (27) 2 (3) 53 (75) 18 (25)
Age (years)
Breed Method of urine sampling Obstruction
Bacterial isolates Escherichia coli was the most common bacteria, found as a single isolate in 38.8% of the cultures, and in combination with another bacterium in an additional 11.1% of the cultures. Other frequently detected bacteria were Staphylococcus species, Enterococcus species, Pasteurella species and Streptococcus species. An overview of the isolated bacteria is given in Table 2. Susceptibility to antimicrobial agents The proportions of isolates that were susceptible, intermediate-resistant and resistant to the antimicrobial agents tested are illustrated in Figure 1. Penicillin, fucidic acid, lincomycin and spiramycin had the highest proportion of resistant isolates (>50%). However, >80% of the isolates were susceptible to enrofloxacin, nitrofurantoin and the trimethoprim/sulfonamide combination. The pattern of antimicrobial susceptibility of E coli, Staphylococcus species, Enterococcus species, Pasteurella
Table 2 Results of urine cultures (n = 72) from 71 cats with lower urinary tract disease >103 cfu
Bacterial isolates, n (%) Escherichia coli Staphylococcus species Enterococcus species Pasteurella species Streptococcus species Enterobacter species Haemophilus species Proteus species Corynebacterium species Pseudomonas species Citrobacter species Escherichia coli/Staphylococcus species Escherichia coli/Clostridium species Escherichia coli/Enterococcus species Escherichia coli/Streptococcus species Haemophilus species/Pasteurella species Klebsiella species/Streptococcus species Total
7 4 3 3
1
>104 cfu
>105 cfu
Total
15
6 7 2 1 2 1
28 (38.8) 11 (15.3) 7 (9.7) 4 (5.5) 3 (4.2) 2 (2.8) 2 (2.8) 2 (2.8) 1 (1.4) 1 (1.4) 1 (1.4) 5 (6.9) 1 (1.4) 1 (1.4) 1 (1.4) 1 (1.4) 1 (1.4) 72 (100)
2 1 1 1
2 1 1 1 1* 1*
4* 1*
1* 1* 1* 24
26
22
*Refers to the lowest number of colony forming units (cfu) detected if different cfu were observed for the two different bacterial isolates
species and Streptococcus species included in the study is illustrated in Figure 2. Of the less frequently detected bacteria, the two Proteus species isolates were resistant to penicillin, fucidic acid and spiramycin, and intermediate-resistant to nitrofurantoin. One of the isolates was also intermediateresistant to amoxicillin and cephalexin. Enterobacter species (n = 2) were resistant to penicillin, ampicillin,
amoxicillin with and without clavulanic acid, fucidic acid, cephalexin, spiramycin and lincomycin. The only Pseudomonas species isolate was multidrug resistant, with intermediate resistance to enrofloxacin and tetracycline and otherwise resistant to all types of antimicrobial agents tested, except the combination of trimethoprim/sulfonamide, to which it tested susceptible.
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510
100% 90% 80% 70% 60%
Resistant
50%
Intermediate
40%
Suscep ble
30% 20% 10% 0%
Figure 1 Patterns of antimicrobial susceptibility of bacterial isolates from urine cultures from cats with lower urinary tract disease, presented by type of antibiotics, all isolates (n = 82)
(a)
(c)
(b)
Escherichia species (n = 36)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Intermediate
Suscep ble
Enterococcus species (n = 8)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Suscep ble
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Staphylococcus species (n = 16)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
(d)
Intermediate
(e)
The Citrobacter species isolate was multidrug resistant. Resistance was found to gentamicin, tobramycin, erythromycin and ciprofloxacin in addition to the antimicrobial agents routinely included in the testing. However, it was found to be susceptible to colistin and only intermediateresistant to neomycin and streptomycin. The Clostridium species isolate was resistant to trimethoprim/sulfonamide, fucidic acid, spiramycin and lincomycin, and intermediate-resistant to nitrofurantoin, while the Corynebacterium species isolate was susceptible to all the included antimicrobial agents. Patterns of antimicrobial susceptibility dependent on Gram staining and shape of bacteria (rods or cocci) are presented in Figure 3.
Resistant
Intermediate
Suscep ble
Streptococcus species (n = 5)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Intermediate
Suscep ble
Pasteurella species (n = 5)
Resistant
Intermediate
Suscep ble
Figure 2 Susceptibility to antimicrobials among bacterial isolates from cats with lower urinary tract disease. (a) Escherichia coli; (b) Staphylococcus species; (c) Enterococcus species; (d) Streptococcus species and (e) Pasteurella species
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Gram-nega ve rods
(b)
Gram-posi ve rods
100% 90% 80% 70% 60% 50% Resistant 40% Intermediate 30% 20% Suscep ble 10% 0%
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
(c)
Resistant
Intermediate
Suscep ble
Gram-posi ve cocci
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Intermediate
Suscep ble
Figure 3 Patterns of antimicrobial susceptibility of 82 bacterial isolates from urine cultures from cats with lower urinary tract disease, presented as (a) Gram-negative rods (n = 52), (b) Gram-positive rods (n = 2) and (c) Gram-positive cocci (n = 28)
Discussion Study population In contrast to most of the studies published on FLUTD, which commonly are based on referral cases,7,8 the bacterial isolates included in this study were collected from a population of primary accession cases, providing an opportunity to examine untreated cases and to include a variety of cases mirroring the situation in first opinion veterinary practice.9–11,13,14 This may explain, in part, the difference in the number of cats with bacterial cystitis previously detected, as cats easily treated with antibiotics in primary care practice may not represent a large proportion of the cats referred to larger veterinary clinics or university teaching hospitals. Previously, a predisposition to FLUTD has been described for purebred cats.15–17 In the present study, approximately 80% were mixed-breed cats (domestic short- or longhair cats), and none of the breeds dominated clearly among the purebred cats included. Further, 38% of the females and 17% of the males were intact, which constitutes a larger proportion of intact cats than recorded in most previous studies.8,15,18,19 Sexually intact females are previously described as having a decreased risk for bacterial cystitis.17 While prostatic diseases and chronic prostatitis as predisposing factors to bacterial cystitis are well recognised in dogs, the literature on cats is somewhat sparse.
However, some reports of prostatic carcinomas in both intact and neutered cats exist,20,21 as well as single reports of hyperplasia, cysts and fibroadenoma.20 Interestingly, there is also a recent report of prostatitis in an intact cat.22 Thus, the potential influence of a larger proportion of intact cats of both sexes on the results in the present study is uncertain. Bacterial isolates The pattern of bacterial isolates detected in this study was generally in accordance with the existing literature, with E coli, followed by Staphylococcus species and Enterococcus species, the most frequently isolated bacteria.5,6,8,18,19 Although not commonly reported, there are also recent publications of Corynebacterium species isolated from cats with signs of FLUTD.23,24 Susceptibility to antimicrobial agents Antimicrobial treatment of cats per os may represent a challenge. In addition to therapeutic efficacy, there are factors like palatability and owner convenience that have a major impact on the treatment outcome.25 In Norway, the assortment of antimicrobial formulations licensed for use in cats is limited, and, until recently, no central register of drugs prescribed to companion animals existed. However, anecdotal evidence indicates that amoxicillin and amoxicillin with clavulanic acid are
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512 most commonly used.26 This is in concurrence with national official therapeutic guidelines, which recommend broad-spectrum penicillin such as ampicillin or amoxicillin in cases of bacterial cystitis in cats pending microbiological results.27,28 Of the antimicrobial agents tested in the present study, enrofloxacin and trimethoprim/sulfonamide had the highest proportions of susceptible bacterial isolates, likely to be effective against >90% of the tested isolates. Also, nitrofurantoin (>80%) and ampicillin, amoxicillin/clavulanic acid and tetracycline (>70%) were likely to eradicate a larger percentage of the infections. These results are in line with the existing literature.5,18,29 The sensitivity of the E coli isolates to amoxicillin/clavulanic acid was lower than that published in several previous studies, which have reported a sensitivity ranging between 82% and 89%.5,18,29,30 Some of these studies allocated the isolates presenting as intermediateresistant to the susceptible group owing to the fact that susceptibility testing is based on attainable antibiotic concentrations in plasma or serum.30 When the active antimicrobial component is excreted unchanged in the urine, the actual concentration in urine may reach much higher levels.30,31 However, even when including the E coli isolates presenting as intermediate-resistant to amoxicillin/clavulanic acid in this study, the percentage of sensitive isolates is still lower – approximately 72%. Recent studies of multidrug-resistant E coli isolates from feline urine report resistance to β-lactams and tetracycline as most common.32,33 In the present study, almost 60% of the E coli isolates were intermediate-resistant to cephalexin. However, several years of susceptibility testing of E coli from different sources indicate that this is to be expected with the break points set by EUCAST (data not shown).12 Thus, the proportion of E coli isolates considered clinically susceptible to cephalosporins is probably higher than indicated by the in vitro susceptibility testing.34 The percentages of isolates resistant to tetracycline and trimethoprim with/without a sulfonamide should also be noted. Regional variations in bacterial prevalence and susceptibility patterns have been previously described.6,32 In studies of uropathogenic E coli isolates from dogs in other Nordic countries, the levels of resistance reported are generally lower than in the present study.35 Although not obtained from the same species, comparison is of interest owing to geographical similarities. Treatment of uropathogenic E coli may represent a growing challenge in veterinary medicine. Recent research has revealed the ability of E coli to invade and replicate within uroepithelial cells, and to form biofilmlike pods on the surface of the uroepithelium.36–38 Further, an association between increased risk of multidrug-resistant E coli urinary tract infection (UTI) and the number of different antimicrobial agents used within the last 3 months has been reported.33 In the light
of these results and the findings from the present study, it seems reasonable to monitor the resistance pattern among E coli isolates from urine samples in the Norwegian cat population. In this study, neither the Staphylococcus species isolates nor the Streptococcus species isolates represent a challenge with regard to choice of antibiotics. While, for years, the levels of antimicrobial resistance has been denoted as favourable in Norway compared with many other countries, a tendency towards increasing resistance against, for instance, narrow-spectrum penicillin is noted among the Staphylococcus species isolates in the present study.39–41 Also, the numbers of isolates resistant to trimethoprim with or without a sulfonamide component should be noted. Increasing resistance among Staphylococcus species isolates to antimicrobial agents, in general, and trimethoprim/sulfonamide, in particular, is previously published.40,42 Pasteurella species isolates were found to be susceptible to a broad range of antibiotics, which is in agreement with previous studies.5,18,29,31 Enterococcus species are often found to be resistant to several antibiotics.5,18,30,43 This was also the situation in this study, although nitrofurantoin, trimethoprim/sulfonamide and ampicillin presented as relatively effective in vitro. A large number of isolates resistant to cephalexin was noted, which is in keeping with previous results as intrinsic resistance against cephalosporins is expected in this group of bacteria.30,44 Also, resistance to trimethoprim and sulfonamides is previously described among Enterococcus species, and these drugs are commonly used in combination, although the actual synergistic effect has been questioned.43,45 In this study, the percentage of Enterococcus species isolates sensitive to trimethoprim alone was 90% of the infections with Gram-positive
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cocci would be successfully treated with amoxicillin, while the outcome of treating Gram-negative infections with amoxicillin/clavulanic acid would be more dubious. The latter is in contrast to the results from the studies included in the above-mentioned review, where amoxicillin with clavulanic acid was recommended for the treatment of Gram-negative bacteria.6,29,30 The main reason for this difference is probably a lower fraction of E coli isolates sensitive to amoxicillin with clavulanic acid found in the present study compared with previous studies, as E coli constitutes the dominant Gram-negative bacteria in all these studies.5,6,29,30 Further, it should be noted that narrow-spectrum penicillin would be sufficient to treat the UTIs caused by Streptococcus species, Pasteurella species and a large proportion of the UTIs caused by Staphylococcus species. In the present study, initiating therapy with broad-spectrum penicillins such as amoxicillin would thus be superfluous in these cases. Recently, the degree of exposure to different antibiotics as a risk factor for development of resistance and uncertainty about the reliability of results from microscopic examination of urinary sediment have been reported.11,18,19,31,33,47 These findings, in combination with the results from this study, demonstrate the importance of awaiting susceptibility testing before initiating antimicrobial treatment, not only to ensure effective treatment of urinary tract infections with as few unwanted medical and environmental side effects as possible, but also to prevent development of antimicrobial resistance. In this study, samples obtained by catheterisation and samples voided into sterile containers were included, as well as samples obtained by cystocentesis. This approach was chosen in order to increase the number of bacterial isolates and, subsequently, the number of susceptibility tests in order to get a broader view of the bacteria likely to colonise the urinary tract of Norwegian cats and their patterns of antimicrobial susceptibility. While cystocentesis is the method of choice in the diagnostic approach to bacterial cystitis in cats, the prevalence of significant bacterial growth and bacterial cystitis was already determined for the population of cats from which the bacterial isolates in the present study were collected.11 Thus, the method of urine sampling was considered to be of less importance than the number of isolates included. Although the risk of culturing contaminants is reduced by the fact that all samples were cultured at the day of collection without delay, the fact that the 10 samples producing two different bacteria were all voided samples needs attention. Further, the inclusion of samples obtained by methods other than cystocentesis may reduce the comparability of the present study.
Conclusions The species of bacteria isolated from the cats in the present study are in agreement with the existing literature.
Although several tendencies towards increasing antimicrobial resistance were detected among the isolates included, the patterns of antimicrobial susceptibility in the present study are, in general, in accordance with the existing literature and therefore do not fully explain the higher prevalence of bacterial cystitis previously detected in Norwegian cats. Moreover, additional explanatory factors beside the inclusion of primary accession cases rather than referred cases were not found. Comparable geographic trends or comparable results from other species have so far not been found. As literature on antimicrobial resistance in companion animals in Europe and especially the Nordic region is scarce, more research would be welcome. Acknowledgements We would like to thank the veterinarians and veterinary nurses at the Department of Companion Animal Clinical Sciences and the laboratory technicians at the Department of Food Safety and Infection Biology for their help and assistance in collecting the material.
Conflict of interest The authors do not have any potential conflicts of interest to declare.
Funding This work was supported by two not-for-profit foundations (Astri and Birger Torsted’s Foundation, and Veterinary Smidt’s Foundation).
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43 Sköld O. Resistance to trimethoprim and sulfonamides. Vet Res 2001; 32: 261–273. 44 Moellering RC. Emergence of Enterococcus as a significant pathogen. Clin Infect Dis 1992; 14: 1173–1178. 45 Huovinen P, Sundtröm L, Swedberg G, et al. Trimethoprim and sulfonamide resistance. Antimicrob Agents Chemother 1995; 39: 279–289.
46 Puskar M, Lemons C, Papich MG, et al. Antibiotic-resistant Corynebacterium jeikeium urinary tract infection in a cat. J Am Anim Hosp Assoc 2007; 43: 61–64. 47 Kataoka Y, Ito C, Kawashima A, et al. Identification and antimicrobial susceptibility of enterococci isolated from dogs and cats subjected to differing antibiotic pressures. J Vet Med Sci 2013; 75: 249–753.
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JFM0010.1177/1098612X14550171Journal of Feline Medicine and SurgeryLund et al
Original Article
Antimicrobial susceptibility in bacterial isolates from Norwegian cats with lower urinary tract disease
Journal of Feline Medicine and Surgery 2015, Vol. 17(6) 507–515 © ISFM and AAFP 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1098612X14550171 jfms.com
Heidi Sjetne Lund, Gaute Skogtun, Henning Sørum and Anna Vigdís Eggertsdóttir
Abstract
Studies of feline lower urinary tract disease (FLUTD) among Norwegian cats have shown higher prevalences of bacterial cystitis than most previously published reports. The aims of the present study were to identify bacterial isolates obtained from the urine of Norwegian cats with FLUTD and their susceptibility to antimicrobial agents. Eighty-two bacterial isolates from 72 urine cultures obtained from 71 different cats were included. Escherichia coli, Staphylococcus species, Enterococcus species and Streptococcus species were the most frequently detected. The percentages of isolates susceptible to the included antimicrobial agents were as follows: enrofloxacin – 92%; trimethoprim/sulfonamide – 91%; nitrofurantoin – 89%; tetracycline – 78%; ampicillin – 73%; amoxicillin/clavulanic acid – 72%; trimethoprim – 68%; amoxicillin – 58%; cephalexin – 51%; spiramycin – 39%; penicillin – 34%; fucidic acid – 34%; lincomycin – 27%. Although several tendencies towards increasing antimicrobial resistance were detected among the isolates included, the species of bacteria isolated and their patterns of antimicrobial resistance were, in general, in concurrence with the existing literature. Thus, the results do not fully explain the higher prevalence of bacterial cystitis found in Norwegian cats. Moreover, additional explanatory factors beside the inclusion of primary accession cases rather than referred cases were not found. Accepted: 14 August 2014
Introduction While feline lower urinary tract disease (FLUTD) is common in small animal veterinary practice,1–3 bacterial cystitis in cats is reported to be relatively rare (11 years.11 The age groups constituted 19%, 42%, 29% and 11% of the material, respectively.11 In addition to aspects related to the sampling and handling of urine samples, factors of potential influence on the prevalence of bacterial cystitis may be the type of bacteria and their susceptibility to antimicrobial agents, or aspects related to the cat population itself, including genetic predisposition to bacterial cystitis in certain breeds or families of cats.
Norwegian University of Life Sciences, Oslo, Norway Corresponding author: Heidi S Lund DVM, Norwegian University of Life Sciences, PO Box 8146 Dep, Oslo, 0033, Norway Email: [email protected]
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508 The aims of the present study were to identify bacterial isolates obtained from urine from Norwegian cats with FLUTD and their susceptibility to antimicrobial agents.
Materials and methods Study sample The bacterial cultures presented stem from a larger study of FLUTD in Norwegian cats. The study population consisted of client-owned cats from Oslo and surrounding areas, presented at the clinics of the Small Animal Section, Norwegian School of Veterinary Science from 2003 until 2009. If informed consent was obtained from the owners, cats with clinical signs (dysuria, stranguria, haematuria, pollakiuria and/or periuria) and a final diagnosis consistent with FLUTD were eligible for inclusion as cases in the study. Exclusion criteria were treatment that could interfere with the diagnostics (antimicrobial medication within the last 3 months, hormones, or medication altering blood pressure, urine production and/or composition) and known concurrent diseases likely to be of influence on the urinary findings, such as chronic kidney disease, diabetes mellitus or hyperthyroidism. Only cats aged 7 months or older were included; no sex or breed restrictions were made. Methods A standardised questionnaire was used to register age, breed, sex, reproductive status, body weight, prior health history and information concerning housing conditions and feeding regime for each cat. A veterinarian performed a clinical examination and, in addition to urine samples, blood samples for routine haematology and clinical chemistry were collected. Abdominal radiography or ultrasound was included as part of the diagnostic investigation. If no indication of concurrent disease was found in the history, physical examination, laboratory results or radiological examination, concurrent disease was considered unlikely. Follow-up consultations were scheduled if concurrent disease non-detectable at the initial diagnostic investigation was suspected. Urine samples were obtained either by cystocentesis, aseptic catheterisation or collection of urine voided into sterile containers by gentle bladder compression on anaesthetised cats. Standard urinalysis was performed, including commercial urine dipstick analysis (Krulab; Kruuse), urine specific gravity measured with refractometer (URC-Ne; Atago) and microscopic examination of the urine sediment (native wet samples and wet samples stained with Sternheimer–Malbins staining solution). All urine samples were cultured on the day of collection. Quantitative bacteriology was performed by streaking 1 µl of urine onto blood agar (Blood Agar Base II; Difco), and qualitative bacteriology by cultivation on blood agar and selective bromo-thymol blue agar (Difco)
from sediments after centrifugation, incubated at 37oC aerobically and in a 5 % CO2-enriched atmosphere. Susceptibility to antimicrobial agents was determined in accordance with the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).12 Semi-confluent growth of the isolates on Mueller–Hinton agar (Difco) with antimicrobial discs (NeoSensitabs; Rosco) were incubated at 35oC. The selection of antimicrobial agents tested included penicillin, ampicillin, amoxicillin with and without clavulanic acid, cephalexin, trimethoprim, trimethoprim/sulfonamide, tetracycline, fucidic acid, enrofloxacin, spiramycin, lincomycin and nitrofurantoin. In this study, all cultures with ⩾103 cfu of two or fewer types of bacteria were included, independent of the method of urine sampling or whether the cats were diagnosed as having bacterial cystitis or not. However, in the total study population of cats from which the bacterial cultures in the present study stem from, a diagnosis of bacterial cystitis was only made after evaluating the method of urine collection in connection with the type and amount of bacteria detected.9,10,13 Cats with uroliths or urethral plugs in combination with positive bacterial cultures were categorised as having uroliths and urethral plugs, respectively. Further, samples obtained by unknown methodology were considered as voided samples in order not to overestimate the importance of the bacteria detected. Not all the information from the questionnaire and clinical investigation was used in the present study; other aspects of the obtained data have been previously published for subsets of the cats included in the present study.9–11,13,14 Statistical analyses The results are presented as frequencies of occurrence, expressed in percentages. Statistical analyses were performed in JMP 8 (SAS Institute).
Results Study sample A total of 82 bacterial isolates from 72 urine cultures from 71 different cats were included. Two urine samples were cultured from the same cat, but during separate episodes of clinical signs of FLUTD. Sixty-two of the cultures resulted in a single bacterial isolate, while in the remaining 10 cultures (all voided samples) two different isolates were detected. Descriptive statistics for the cats, including sex and reproductive status, age, breed, method of urine sampling and obstructed/non-obstructed form of FLUTD, are listed in Table 1. Among the purebred cats included in the study there were six Persians, five Norwegian Forest cats, two British Shorthairs, one Abyssinian and one Maine Coon.
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Table 1 Descriptive statistics of 71 cats with lower urinary tract disease and positive urine cultures Variable
Category
n (%)
Sex and reproductive status
Male Male neutered Female Female neutered 0–2 3–6 7–10 >11 Pure breed Domestic shorthair Voiding by manual compression Catheterisation Cystocentesis Unknown Non-obstructed Obstructed
8 (11) 39 (55) 9 (13) 15 (21) 23 (32) 22 (31) 15 (21) 11 (16) 15 (21) 56 (79) 42 (59) 8 (11) 19 (27) 2 (3) 53 (75) 18 (25)
Age (years)
Breed Method of urine sampling Obstruction
Bacterial isolates Escherichia coli was the most common bacteria, found as a single isolate in 38.8% of the cultures, and in combination with another bacterium in an additional 11.1% of the cultures. Other frequently detected bacteria were Staphylococcus species, Enterococcus species, Pasteurella species and Streptococcus species. An overview of the isolated bacteria is given in Table 2. Susceptibility to antimicrobial agents The proportions of isolates that were susceptible, intermediate-resistant and resistant to the antimicrobial agents tested are illustrated in Figure 1. Penicillin, fucidic acid, lincomycin and spiramycin had the highest proportion of resistant isolates (>50%). However, >80% of the isolates were susceptible to enrofloxacin, nitrofurantoin and the trimethoprim/sulfonamide combination. The pattern of antimicrobial susceptibility of E coli, Staphylococcus species, Enterococcus species, Pasteurella
Table 2 Results of urine cultures (n = 72) from 71 cats with lower urinary tract disease >103 cfu
Bacterial isolates, n (%) Escherichia coli Staphylococcus species Enterococcus species Pasteurella species Streptococcus species Enterobacter species Haemophilus species Proteus species Corynebacterium species Pseudomonas species Citrobacter species Escherichia coli/Staphylococcus species Escherichia coli/Clostridium species Escherichia coli/Enterococcus species Escherichia coli/Streptococcus species Haemophilus species/Pasteurella species Klebsiella species/Streptococcus species Total
7 4 3 3
1
>104 cfu
>105 cfu
Total
15
6 7 2 1 2 1
28 (38.8) 11 (15.3) 7 (9.7) 4 (5.5) 3 (4.2) 2 (2.8) 2 (2.8) 2 (2.8) 1 (1.4) 1 (1.4) 1 (1.4) 5 (6.9) 1 (1.4) 1 (1.4) 1 (1.4) 1 (1.4) 1 (1.4) 72 (100)
2 1 1 1
2 1 1 1 1* 1*
4* 1*
1* 1* 1* 24
26
22
*Refers to the lowest number of colony forming units (cfu) detected if different cfu were observed for the two different bacterial isolates
species and Streptococcus species included in the study is illustrated in Figure 2. Of the less frequently detected bacteria, the two Proteus species isolates were resistant to penicillin, fucidic acid and spiramycin, and intermediate-resistant to nitrofurantoin. One of the isolates was also intermediateresistant to amoxicillin and cephalexin. Enterobacter species (n = 2) were resistant to penicillin, ampicillin,
amoxicillin with and without clavulanic acid, fucidic acid, cephalexin, spiramycin and lincomycin. The only Pseudomonas species isolate was multidrug resistant, with intermediate resistance to enrofloxacin and tetracycline and otherwise resistant to all types of antimicrobial agents tested, except the combination of trimethoprim/sulfonamide, to which it tested susceptible.
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510
100% 90% 80% 70% 60%
Resistant
50%
Intermediate
40%
Suscep ble
30% 20% 10% 0%
Figure 1 Patterns of antimicrobial susceptibility of bacterial isolates from urine cultures from cats with lower urinary tract disease, presented by type of antibiotics, all isolates (n = 82)
(a)
(c)
(b)
Escherichia species (n = 36)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Intermediate
Suscep ble
Enterococcus species (n = 8)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Suscep ble
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Staphylococcus species (n = 16)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
(d)
Intermediate
(e)
The Citrobacter species isolate was multidrug resistant. Resistance was found to gentamicin, tobramycin, erythromycin and ciprofloxacin in addition to the antimicrobial agents routinely included in the testing. However, it was found to be susceptible to colistin and only intermediateresistant to neomycin and streptomycin. The Clostridium species isolate was resistant to trimethoprim/sulfonamide, fucidic acid, spiramycin and lincomycin, and intermediate-resistant to nitrofurantoin, while the Corynebacterium species isolate was susceptible to all the included antimicrobial agents. Patterns of antimicrobial susceptibility dependent on Gram staining and shape of bacteria (rods or cocci) are presented in Figure 3.
Resistant
Intermediate
Suscep ble
Streptococcus species (n = 5)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Intermediate
Suscep ble
Pasteurella species (n = 5)
Resistant
Intermediate
Suscep ble
Figure 2 Susceptibility to antimicrobials among bacterial isolates from cats with lower urinary tract disease. (a) Escherichia coli; (b) Staphylococcus species; (c) Enterococcus species; (d) Streptococcus species and (e) Pasteurella species
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Gram-nega ve rods
(b)
Gram-posi ve rods
100% 90% 80% 70% 60% 50% Resistant 40% Intermediate 30% 20% Suscep ble 10% 0%
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
(c)
Resistant
Intermediate
Suscep ble
Gram-posi ve cocci
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Resistant
Intermediate
Suscep ble
Figure 3 Patterns of antimicrobial susceptibility of 82 bacterial isolates from urine cultures from cats with lower urinary tract disease, presented as (a) Gram-negative rods (n = 52), (b) Gram-positive rods (n = 2) and (c) Gram-positive cocci (n = 28)
Discussion Study population In contrast to most of the studies published on FLUTD, which commonly are based on referral cases,7,8 the bacterial isolates included in this study were collected from a population of primary accession cases, providing an opportunity to examine untreated cases and to include a variety of cases mirroring the situation in first opinion veterinary practice.9–11,13,14 This may explain, in part, the difference in the number of cats with bacterial cystitis previously detected, as cats easily treated with antibiotics in primary care practice may not represent a large proportion of the cats referred to larger veterinary clinics or university teaching hospitals. Previously, a predisposition to FLUTD has been described for purebred cats.15–17 In the present study, approximately 80% were mixed-breed cats (domestic short- or longhair cats), and none of the breeds dominated clearly among the purebred cats included. Further, 38% of the females and 17% of the males were intact, which constitutes a larger proportion of intact cats than recorded in most previous studies.8,15,18,19 Sexually intact females are previously described as having a decreased risk for bacterial cystitis.17 While prostatic diseases and chronic prostatitis as predisposing factors to bacterial cystitis are well recognised in dogs, the literature on cats is somewhat sparse.
However, some reports of prostatic carcinomas in both intact and neutered cats exist,20,21 as well as single reports of hyperplasia, cysts and fibroadenoma.20 Interestingly, there is also a recent report of prostatitis in an intact cat.22 Thus, the potential influence of a larger proportion of intact cats of both sexes on the results in the present study is uncertain. Bacterial isolates The pattern of bacterial isolates detected in this study was generally in accordance with the existing literature, with E coli, followed by Staphylococcus species and Enterococcus species, the most frequently isolated bacteria.5,6,8,18,19 Although not commonly reported, there are also recent publications of Corynebacterium species isolated from cats with signs of FLUTD.23,24 Susceptibility to antimicrobial agents Antimicrobial treatment of cats per os may represent a challenge. In addition to therapeutic efficacy, there are factors like palatability and owner convenience that have a major impact on the treatment outcome.25 In Norway, the assortment of antimicrobial formulations licensed for use in cats is limited, and, until recently, no central register of drugs prescribed to companion animals existed. However, anecdotal evidence indicates that amoxicillin and amoxicillin with clavulanic acid are
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512 most commonly used.26 This is in concurrence with national official therapeutic guidelines, which recommend broad-spectrum penicillin such as ampicillin or amoxicillin in cases of bacterial cystitis in cats pending microbiological results.27,28 Of the antimicrobial agents tested in the present study, enrofloxacin and trimethoprim/sulfonamide had the highest proportions of susceptible bacterial isolates, likely to be effective against >90% of the tested isolates. Also, nitrofurantoin (>80%) and ampicillin, amoxicillin/clavulanic acid and tetracycline (>70%) were likely to eradicate a larger percentage of the infections. These results are in line with the existing literature.5,18,29 The sensitivity of the E coli isolates to amoxicillin/clavulanic acid was lower than that published in several previous studies, which have reported a sensitivity ranging between 82% and 89%.5,18,29,30 Some of these studies allocated the isolates presenting as intermediateresistant to the susceptible group owing to the fact that susceptibility testing is based on attainable antibiotic concentrations in plasma or serum.30 When the active antimicrobial component is excreted unchanged in the urine, the actual concentration in urine may reach much higher levels.30,31 However, even when including the E coli isolates presenting as intermediate-resistant to amoxicillin/clavulanic acid in this study, the percentage of sensitive isolates is still lower – approximately 72%. Recent studies of multidrug-resistant E coli isolates from feline urine report resistance to β-lactams and tetracycline as most common.32,33 In the present study, almost 60% of the E coli isolates were intermediate-resistant to cephalexin. However, several years of susceptibility testing of E coli from different sources indicate that this is to be expected with the break points set by EUCAST (data not shown).12 Thus, the proportion of E coli isolates considered clinically susceptible to cephalosporins is probably higher than indicated by the in vitro susceptibility testing.34 The percentages of isolates resistant to tetracycline and trimethoprim with/without a sulfonamide should also be noted. Regional variations in bacterial prevalence and susceptibility patterns have been previously described.6,32 In studies of uropathogenic E coli isolates from dogs in other Nordic countries, the levels of resistance reported are generally lower than in the present study.35 Although not obtained from the same species, comparison is of interest owing to geographical similarities. Treatment of uropathogenic E coli may represent a growing challenge in veterinary medicine. Recent research has revealed the ability of E coli to invade and replicate within uroepithelial cells, and to form biofilmlike pods on the surface of the uroepithelium.36–38 Further, an association between increased risk of multidrug-resistant E coli urinary tract infection (UTI) and the number of different antimicrobial agents used within the last 3 months has been reported.33 In the light
of these results and the findings from the present study, it seems reasonable to monitor the resistance pattern among E coli isolates from urine samples in the Norwegian cat population. In this study, neither the Staphylococcus species isolates nor the Streptococcus species isolates represent a challenge with regard to choice of antibiotics. While, for years, the levels of antimicrobial resistance has been denoted as favourable in Norway compared with many other countries, a tendency towards increasing resistance against, for instance, narrow-spectrum penicillin is noted among the Staphylococcus species isolates in the present study.39–41 Also, the numbers of isolates resistant to trimethoprim with or without a sulfonamide component should be noted. Increasing resistance among Staphylococcus species isolates to antimicrobial agents, in general, and trimethoprim/sulfonamide, in particular, is previously published.40,42 Pasteurella species isolates were found to be susceptible to a broad range of antibiotics, which is in agreement with previous studies.5,18,29,31 Enterococcus species are often found to be resistant to several antibiotics.5,18,30,43 This was also the situation in this study, although nitrofurantoin, trimethoprim/sulfonamide and ampicillin presented as relatively effective in vitro. A large number of isolates resistant to cephalexin was noted, which is in keeping with previous results as intrinsic resistance against cephalosporins is expected in this group of bacteria.30,44 Also, resistance to trimethoprim and sulfonamides is previously described among Enterococcus species, and these drugs are commonly used in combination, although the actual synergistic effect has been questioned.43,45 In this study, the percentage of Enterococcus species isolates sensitive to trimethoprim alone was 90% of the infections with Gram-positive
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cocci would be successfully treated with amoxicillin, while the outcome of treating Gram-negative infections with amoxicillin/clavulanic acid would be more dubious. The latter is in contrast to the results from the studies included in the above-mentioned review, where amoxicillin with clavulanic acid was recommended for the treatment of Gram-negative bacteria.6,29,30 The main reason for this difference is probably a lower fraction of E coli isolates sensitive to amoxicillin with clavulanic acid found in the present study compared with previous studies, as E coli constitutes the dominant Gram-negative bacteria in all these studies.5,6,29,30 Further, it should be noted that narrow-spectrum penicillin would be sufficient to treat the UTIs caused by Streptococcus species, Pasteurella species and a large proportion of the UTIs caused by Staphylococcus species. In the present study, initiating therapy with broad-spectrum penicillins such as amoxicillin would thus be superfluous in these cases. Recently, the degree of exposure to different antibiotics as a risk factor for development of resistance and uncertainty about the reliability of results from microscopic examination of urinary sediment have been reported.11,18,19,31,33,47 These findings, in combination with the results from this study, demonstrate the importance of awaiting susceptibility testing before initiating antimicrobial treatment, not only to ensure effective treatment of urinary tract infections with as few unwanted medical and environmental side effects as possible, but also to prevent development of antimicrobial resistance. In this study, samples obtained by catheterisation and samples voided into sterile containers were included, as well as samples obtained by cystocentesis. This approach was chosen in order to increase the number of bacterial isolates and, subsequently, the number of susceptibility tests in order to get a broader view of the bacteria likely to colonise the urinary tract of Norwegian cats and their patterns of antimicrobial susceptibility. While cystocentesis is the method of choice in the diagnostic approach to bacterial cystitis in cats, the prevalence of significant bacterial growth and bacterial cystitis was already determined for the population of cats from which the bacterial isolates in the present study were collected.11 Thus, the method of urine sampling was considered to be of less importance than the number of isolates included. Although the risk of culturing contaminants is reduced by the fact that all samples were cultured at the day of collection without delay, the fact that the 10 samples producing two different bacteria were all voided samples needs attention. Further, the inclusion of samples obtained by methods other than cystocentesis may reduce the comparability of the present study.
Conclusions The species of bacteria isolated from the cats in the present study are in agreement with the existing literature.
Although several tendencies towards increasing antimicrobial resistance were detected among the isolates included, the patterns of antimicrobial susceptibility in the present study are, in general, in accordance with the existing literature and therefore do not fully explain the higher prevalence of bacterial cystitis previously detected in Norwegian cats. Moreover, additional explanatory factors beside the inclusion of primary accession cases rather than referred cases were not found. Comparable geographic trends or comparable results from other species have so far not been found. As literature on antimicrobial resistance in companion animals in Europe and especially the Nordic region is scarce, more research would be welcome. Acknowledgements We would like to thank the veterinarians and veterinary nurses at the Department of Companion Animal Clinical Sciences and the laboratory technicians at the Department of Food Safety and Infection Biology for their help and assistance in collecting the material.
Conflict of interest The authors do not have any potential conflicts of interest to declare.
Funding This work was supported by two not-for-profit foundations (Astri and Birger Torsted’s Foundation, and Veterinary Smidt’s Foundation).
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