Ribotype diversity of porcine Pasteurella multocida from Australia

OBJECTIVE: To use the technique of ribotyping to investigate the genetic diversity of Australian isolates of Pasteurella multocida associated with outbreaks of clinical disease in Australian pigs. DESIGN: One hundred and seven porcine P multocida isolates were analysed by ribotyping using the restriction enzymes HpaII and HindIII. The genetic population structure of the Australian porcine P multocida isolates was determined through statistical analysis of the joint ribotype patterns, and this was then compared with biochemical and epidemiological data available for the population. RESULTS: A total of 25 combined ribotypes were recognised, which were grouped into five ribotype clusters. Despite the deliberate selection of diverse isolates, the study revealed only a limited degree of genetic diversity. Fourteen of the ribotypes contained multiple isolates, and 12 of these ribotypes were present on more than one farm. Three of the seven biovars analysed in the study showed very limited diversity. All fifteen biovar 2 isolates (subsp multocida) were found in a single cluster (III), while all four biovar 8 isolates, which correspond to P multocida subsp gallicida, were allocated by themselves to a single cluster (IV). All nine of the biovar 12 isolates (lactose-positive subsp multocida) were assigned to a single cluster (I), together with the single biovar 14 isolate, which was the only other lactose-positive isolate in the population (ODC-negative). CONCLUSION: A limited number of ribotypes of P multocida are associated with Australian pigs. The majority of these ribotypes are widely distributed across multiple farms, and across multiple states. Individual farms can possess multiple ribotypes of P multocida. Some of the unusual biochemical variants of P multocida present in Australian pigs have a very limited genetic diversity. The nature of pig production in Australia, primarily involving continuous flow systems with few closed herds, has possibly contributed to the widespread distribution of a limited number ribotypes.     

Ribotyping of Indian Isolates of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.     

Characterization of Staphylococcus simulans strains isolated from cases of bovine mastitis

This study was conducted to characterize Staphylococcus simulans isolated from cases of bovine mastitis. A total of 134 isolates of S. simulans selected from 80 quarters from 61 cows or heifers in 37 different herds were characterized by EcoRI ribotyping. From 22 quarters two to seven consecutive isolates taken at weekly intervals were selected. Furthermore, three isolates from clinical infections in humans and two reference strains were included. A total of 16 different ribotypes were found, however, two types predominated. In most herds more than one type was found. From the 22 different quarters, where 76 paired or multiple isolates were at disposal, the same ribotype was constantly found in the same quarter. This study showed that S. simulans causing bovine mastitis could be divided into relatively large number of different types, but that two types predominated. More than one type could be found in the same herd and within different quarters of the same cow, but ribotyping confirmed that S. simulans could be the cause of persistent and stable infections.     

Strain discrimination of a novel serotype of Salmonella from harbour porpoises (Phocoena phocoena) by molecular techniques

The relatedness of 41 isolates of Salmonella of a novel serotype (antigenic formula 4,12:a:-) of serogroup B, obtained from harbour porpoises (Phocoena phocoena) stranded at various sites around the coastline of Scotland, was assessed by two molecular typing methods. Ribotyping showed that these isolates belonged to seven EcoRI (E) ribotypes and 11 PstI (P) ribotypes that were, in each case, distinct but closely related. Combined ribotyping data identified 15 different E/P ribotypes, the most common of which, E1/P1, was represented by 15 isolates from 14 animals stranded on both east and west coastlines. Strain discrimination achieved by E/P ribotyping was high (D=0.84). IS200 profiling revealed only three different fingerprints and strain discrimination by this method alone was poor (D=0.39). When E/P ribotyping and IS200 profiling were used together, they revealed the existence of 17 different types among the 41 isolates which formed two distinct, but related, groups of Salmonella serotype 4,12:a:-. This information should prove helpful in future studies examining the mode of transmission of this novel salmonella serotype and its association with disease in harbour porpoises.     

Epidemiology of Clostridium difficile on a veal farm: prevalence, molecular characterization and tetracycline resistance

Concern has been raised about the potential for Clostridium difficile to be a bovine and foodborne pathogen, yet limited study has been performed in cattle, and none in veal calves. This study evaluated the epidemiology and microbiology of C. difficile on one veal farm. Rectal swabs were obtained from calves within 48 h of arrival and at one, 17 and 21 weeks later. Selective culture for C. difficile was performed. Isolates were characterized by PCR ribotyping and PCR for tcdA, tcdB and cdtA. Tetracycline resistance and resistance genes were investigated. Multivariable logistic regression models were constructed to determine the relationship between shedding of the bacterium and specific ribotypes and the independent variables: time of sampling and area of housing. Calves were twice more likely to test positive 1 week after arrival (51%) when compared to initial results (32%). Shedding at 17 and 21 weeks was significantly lower (2% at both samplings). Ribotype 078 was the most common. Twelve different ribotypes were present initially with only three ribotypes found subsequently. Seventy-six percent (40/53) of isolates initially recovered were tetracycline resistant compared to 93% (81/87) from 2nd sampling. Tetracycline resistance genes were detected in 24% (13/53) of isolates during 1st and in 55% (50/91) during 2nd sampling. The high prevalence of pathogenic C. difficile in veal calves could be of zoonotic concern. The low prevalence before slaughter may be of importance for the evaluation of foodborne risks. Oxytetracycline administration to calves may have an impact on prevalence of C. difficile colonization.     

Automated ribotyping, a rapid typing method for analysis of Erysipelothrix spp. strains

Automated ribotyping classified 70 Erysipelothrix species strains, previously classified into 14 RAPD patterns and into 63 PFGE patterns, into 27 ribogroups. Twenty-three strains of the 70 analyzed and classified into 13 ribogroups were previously classified into six ribotypes by the traditional ribotyping method. Moreover, automated ribotyping differentiated seven strains that were not differentiated by PFGE. Therefore, automated ribotyping was more sensitive than RAPD and traditional ribotyping, and it might be a useful method for a rapid screening in epidemiological study of strains of this genus, and more accurate results can be obtained when this method is used together with PFGE.     

Bacterial isolates from blood and their susceptibility patterns in critically ill foals: 543 cases (1991-1998)

OBJECTIVE: To assess microorganisms isolated from blood specimens obtained from critically ill neonatal foals and to evaluate their antimicrobial susceptibility patterns. DESIGN: Retrospective study. ANIMALS: 543 neonatal foals. PROCEDURE: Medical records of foals that were < 1 month old and were admitted to a referral neonatal intensive care unit were reviewed for results of bacteriologic culture of blood and antimicrobial susceptibility patterns. RESULTS: At least 1 microorganism was isolated from 155 of 543 (28.5%) foals. Escherichia coli was the most commonly isolated bacterium. A single gram-positive organism was detected in 49 foals. Although 90% of the E coli isolates were susceptible to amikacin, some gram-negative and gram-positive organisms had resistance against multiple antimicrobials. CONCLUSIONS AND CLINICAL RELEVANCE: Gram-negative bacteria remain the most common isolates from blood of neonatal foals; however, gram-positive organisms were also found, and with greater prevalence than reported elsewhere. Susceptibility patterns may vary, and resistance to multiple antimicrobials may develop. This is especially true for organisms such as Enterobacter spp and Enterococcus spp. Prudent empirical treatment for neonatal sepsis should include broad-spectrum antimicrobials.     

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.     

Longitudinal study of Clostridium difficile and antimicrobial susceptibility of Escherichia coli in healthy horses in a community setting

Point prevalence studies have reported carriage rates of enteric pathogens in healthy horses, but longitudinal data are lacking. Commensal E. coli is an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data for horses. The objectives of this study were to investigate and molecularly characterize isolates of Clostridium difficile, Clostridium perfringens and Salmonella, collected sequentially over a one year period, and to determine the antibiotic susceptibility profile for E. coli. Fecal samples were collected monthly from 25 adult horses for one year. Selective cultures were performed for all above bacteria. C. difficile isolates were characterized via PCR toxin gene profiling and ribotyping. Broth microdilution was performed to assess antimicrobial susceptibility profiles of E. coli. Toxigenic Clostridium difficile was isolated from 15/275 (5.45%) samples from 10/25 (40%) horses. Four horses were positive at multiple sampling times but different ribotypes were found in three. Ribotypes included 078 (n=6), 001 (n=6) and C (n=3). C. perfringens was not isolated, nor was Salmonella. E. coli was isolated from 232/300 (77%) fecal samples. Resistance to ≥1 and ≥ 3 antimicrobials was present in 31/232 (13.4%) and 6/232 (2.6%) respectively. Only two horses shed the same strain of toxigenic C. difficile for more than one month, indicating that shedding is transient. The high number of ribotype 078 is consistent with recent emergence of this strain in the local horse population. The low prevalence of antibiotic resistance in commensal E. coli suggests that healthy horses are not likely a major reservoir of resistance for enteric bacteria.     

Antimicrobial Susceptibility and rRNA Gene Restriction Patterns among Staphylococcus intermedins from Healthy Dogs and from Dogs Suffering from Pyoderma or Otitis Externa

A total of 60 Staphylococcus intermedins strains from dogs were investigated by their sensitivity to various antibiotics (50 strains) and by their rRNA gene restriction patterns (ribotyping) (60 strains). Fifteen isolates were from healthy dogs, 9 with otitis externa, and 36 with pyoderma, including 10 strains from a previous study. Sixty per cent of the 50 strains tested for antibiotic susceptibility demonstrated resistance to penicillin, 24% to spiramycin, 20% to tetracycline, 16% to chloramphenicol, and 2% to fucidic acid. All isolates were susceptible to amoxycillin with clavulanic acid, enrofloxacin, and sulphonamides with trimethoprim. There were no significant differences in antimicrobial susceptibility patterns observed among isolates from pyoderma, otitis externa or healthy dogs. Among the 60 strains studied by ribotyping, 10 different ribotypes were identified: 6 different ribotypes among isolates from otitis externa, 8 among isolates from pyoderma, and 5 among isolates from healthy dogs. One ribotype (profile C) was dominant among the isolates from healthy dogs while another ribotype (profile A) was dominant among strains from dogs suffering from pyoderma. This profile was not demonstrated in any of the strains from healthy dogs. From 5 different dogs suffering from pyoderma, 2 different clones were demonstrated based on their plasmid profile and antibiogram. In these dogs 1 of the clones always belonged to ribotype A. The results concerning strains of S. intermedins isolated from furunculosis suggest the existence of distinct subpopulations with different pathogenicity to dogs.     

Occurrence and molecular analysis of Campylobacter in wildlife on livestock farms

Wildlife harbor a variety of Campylobacter spp. and may play a significant role in the transmission of Campylobacter to livestock. Although studies have been conducted on wildlife-associated Campylobacter isolates from farms in other countries, there are little data available for livestock farms in the United States. In addition, the critical questions of whether wildlife harbor Campylobacter that is pathogenic to ruminants and/or antibiotic-resistant Campylobacter have yet to be addressed. We captured wild small mammals (n=142) and small birds (n=188) at livestock farms in central Iowa and sampled them for thermophilic Campylobacter during autumn 2009, spring 2010, and autumn 2010. Overall prevalence was 4.79%, with isolates found only in wild birds. Molecular typing revealed four multilocus sequence types (STs), three of which are novel. The remaining ST (ST-806) was found in two house sparrows and is an ST previously associated with ruminant abortion cases. Further analysis of ST-806 wild bird and ruminant abortion isolates by pulsed-field gel electrophoresis, resistance gene location, and antibiotic susceptibility tests indicated that the isolates are nearly identical. This is the first account of isolation of Campylobacter types from wild birds that are known to be pathogenic to ruminants. Furthermore, these same two wild bird isolates are resistant to the antibiotic fluoroquinolone. Our results indicate there is an overall low prevalence of Campylobacter in selected wildlife in Iowa, but suggest that wildlife may play a role in the epidemiology of pathogenic Campylobacter for domestic livestock, and may also serve as a reservoir for antibiotic-resistant Campylobacter.     

Antimicrobial Susceptibility Patterns of Clostridium difficile Isolates from Family Dairy Farms

A significant risk factor for developing Clostridium difficile infection (CDI) in humans and animals is associated with the antimicrobial use. It has often been hypothesized that farm animals could be the source for human infection with Clostridium difficile (CD). In the European Union, family‐run dairy farms are the predominant farming model, which are more interlinked within the community compared to large‐scale intensive dairy or beef farms. Therefore, it is important to investigate antimicrobial susceptibility patterns of CD in such environment. A total of 159 CD isolates from 20 family dairy farms were tested with a customized broth microdilution plate for their antimicrobial resistance. Seventeen antimicrobials were selected (amoxicillin, ceftriaxone, clindamycin, daptomycin, erythromycin, fusidic acid, imipenem, levofloxacin, linezolid, metronidazole, moxifloxacin, oxacillin, rifampicin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin), which are commonly used for treatment of CDI in veterinary and human medicine, or were previously applied in CD epidemiological studies. Antimicrobials, which are used for treatment of CDI in humans (metronidazole, vancomycin, fusidic acid, tigecycline, linezolid) inhibited CD growth in vitro. Most CD isolates were resistant to erythromycin (93.1%), daptomycin (69.2%) and clindamycin (46.5%). High multiple‐resistance was found in CD ribotype 012 (n = 5, 100%), some CD SLO 060 (n = 4, 25%) and one CD 033 (n = 1, 1.1%). High multiple‐resistance in this study was linked with CD ribotypes and not with the origin of CD. The low prevalence of these ribotypes (6.3%; 10/159) indicates that family‐run dairy farms are an unlikely source of CD with multiple‐resistance to antimicrobials.     

Prevalence and Genotypic Characterization of Clostridium difficile From Ruminants in Switzerland

Clostridium difficile is an emerging enteric pathogen of humans and animals with a known main reservoir in the intestinal tract of various warm‐blooded animals. This study was carried out to evaluate the prevalence of C. difficile in 150 rectal swab samples collected from cattle and goats in Switzerland. The overall prevalence of C. difficile was 6.6%. The isolates belonged to the PCR ribotypes 033, 066, 070, 003, 001 and 137. In addition, the occurrence of C. difficile in faecal samples collected from farm ground was also evaluated and C. difficile was detected in 21% (7/30) of these samples. These isolates belonged to ribotypes 033, 066, 014 and 137. Several isolates pertained to ribotypes known to cause Clostridium difficile infections in humans.     

Ribotyping of Streptococcus uberis from a dairy's environment, bovine feces and milk

Streptococcus uberis is a major cause of bovine mastitis and infections commonly result from environmental exposure to the pathogen. To identify specific sources of mastitis-causing S. uberis strains, samples were collected monthly from the environment and feces of dry cows in a grazing herd. Environmental and fecal strains of S. uberis were compared to those found in milk. S. uberis was detected in 63% of 94 environmental samples, including water, soil, plant matter, bedding material, flies, and hay, in 23% of 107 fecal samples, and in 4% of 787 milk samples. Automated PvuII ribotyping revealed 48 ribotypes among 266 isolates. Per sample, up to five ribotypes were detected. The distribution of ribotypes did not differ significantly among environmental, fecal and milk samples. Specific environmental sources or strains of udder-pathogenic S. uberis were not identified. Fecal shedding was not persistent and did not differ between dry-off and calving. The proportion of fecal samples containing S. uberis was highest during the summer grazing season. S. uberis was common in farm soil (31 of 35 samples) but not in non-farm soil (0 of 11 samples). We hypothesize that fecal shedding of S. uberis may play a role in maintenance of S. uberis populations in the dairy ecosystem.     

Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark

The aim of the present study was to evaluate capsular-typing, plasmid-profiling, phage-typing and ribotyping for epidemiological studies of toxin-producing Pasteurella multocida ssp. multocida in Denmark. The evaluation of methods was based on 68 strains from nasal swabs and 14 strains from pneumonic lungs. Strains from lungs were all of capsular Type A, whereas strains from nasal swabs were of both capsular Types A and D. Only 9% of the strains contained plasmids, which could not be associated with antibiotic resistance. Phage-typing divided 61% of strains into 10 groups, while 39% were non-typable. CfoI ribotyping divided strains into four groups of which one type contained 94% of isolates. HindIII ribotyping divided strains into 18 types. A total of 18 strains from The Netherlands, UK and USA were subjected to HindIII ribotyping, resulting in 13 types of which six were identical to ribotypes of Danish strains. Phage-typing of isolates from an outbreak of atrophic rhinitis involving six herds in 1985 showed the existence of an epidemic strain. This type was recognised in the herd suspected of being the source of the infections and in four of the five infected herds. These findings were supported by HindIII ribotyping, as 85% of isolates from all herds were assigned to one ribotype. In conclusion, HindIII ribotyping seems to represent a useful tool for epidemiological studies of toxigenic P. multocida ssp. multocida.     

Evaluation and application of ribotyping for epidemiological studies of Actinobacillus pleuropneumoniae in Denmark

The aim of the present study was to evaluate ribotyping as an epidemiological tool for Actinobacillus pleuropneumoniae and apply the method in studies of A. pleuropneumoniae infections in Danish pig herds. The evaluation of ribotyping was based on the 13 international reference strains and 106 epidemiologically unrelated Danish field strains representing the nine serotypes of biotype 1 (1, 2, 5A/B, 6, 7, 8, 10, 12, and K2:O7) and one serotype 14 of biotype 2. Enzymes CfoI and HindIII were chosen for generation of ribotype patterns. Ribotyping of the reference strains resulted in 10 CfoI types and 11 HindIII types. Ribotyping of the Danish strains resulted in 17 different CfoI ribotypes and 24 different HindIII ribotypes. Combining HindIII- and CfoI-ribotyping divided the Danish strains into 26 different types. The stability, reproducibility and typability of ribotype patterns were good, and the discriminatory power was between 0.85–0.89. The relatively low discriminatory power was caused by four predominant types, containing 61% of the isolates. The typing system was applied in studies of routes of infection of specific pathogen-free (SPF) pig herds and included 112 strains of A. pleuropneumoniae. Airborne transmission from neighboring conventional pig farms was investigated in 12 cases of infected SPF herds. Transmission via vehicles transporting pigs between SPF herds was investigated in nine cases while transmission by trading of pigs between SPF herds was investigated in two cases. Serotype 2 was isolated from all SPF herds included in this study, except one, emphasizing the high prevalence of this serotype in Denmark. By ribotyping, airborne transmission was indicated in five of 12 cases, transmission via pig transporting vehicle was indicated in six of nine cases, and transmission via trading was indicated in one of two cases. In many cases findings of predominant ribotypes made interpretations of suspected routes of transmission difficult. The relationship of strains based on ribotypes was calculated using Dices coefficient and clustered by UPGMA. HindIII ribotypes of serotype 2 strains were closely related, though only showing 43% similarity to HindIII ribotypes of remaining serotypes.     

Usage of antimicrobials and occurrence of antimicrobial resistance among bacteria from mink

The usage of antimicrobials for treatment of mink and the occurrence of antimicrobial resistance among the most important bacterial pathogens in mink was investigated. The aim of the study was to provide data, which may serve as a basis for the formulation of recommendations for prudent use of antimicrobials for mink. A total of 164 haemolytic staphylococci, 49 haemolytic streptococci, 39 Pseudomonas aeruginosa, 13 Pasteurella multocida, and 1093 Escherichia coli isolates from Danish mink were included in the study. A high frequency of resistance among S. intermedius was found for tetracyclines (54.7%), followed by penicillin (21.7%), lincosamides (20.4%), macrolides (19.1%), and spectinomycin (18.5%). Very low frequencies of resistance were recorded for other antimicrobials. The highest frequency among the E. coli isolates was recorded for ampicillin, streptomycin, sulphonamides, and tetracyclines, whereas resistance to other antimicrobials was rare. All P. aeruginosa were sensitive to gentamicin and colistin and sensitive or intermediate to enrofloxacin, whereas most isolates were resistant to all other antimicrobials. All P. multocida and haemolytic streptococci were sensitive to penicillin. There was a steady increase in the use of antimicrobials during the period 2001-2006, the majority of the prescribed amount being extended spectrum penicillins followed by aminoglycosides, sulphonamides with trimethoprim, and macrolides.     

Identification of Pasteurella multocida Serogroup F isolates in rabbits

A total of 24 Pasteurella multocida rabbit isolates obtained from 24 rabbit flocks in the Czech Republic during the period of between 2001 and 2004 were analysed by capsular PCR typing. Apart from isolates identified as serogroups A (n = 14, 58.4%) and D (n = 2, 8.3%), eight isolates (33.3%) were identified as members of serogroup F. This serogroup had been predominantly associated with poultry infections so far. The rabbit serogroup F isolates were characterized in detail by ribotyping with restriction to endonuclease MspI revealing two distinct ribotypes. Seven serogroup F isolates were assigned to ribotype 1 and one isolate was assigned to ribotype 2.     

Clostridium difficile shedding by healthy dogs in Nigeria and Malawi

The objectives of this study were to determine the prevalence and characteristics of Clostridium difficile shedding in owned dogs in Nigeria and Malawi. Clostridium difficile was isolated from 31/120 (26%) dogs in Nigeria and 11/92 (12%) dogs in Malawi (p = 0.012). Overall, 22/42 (52%) isolates were toxigenic; 17/31 (55%) from Nigeria and 5/11 (45%) from Malawi. All toxigenic isolates possessing tcdA and tcdB, and only one also possessed cdtA/B. Sixteen different ribotypes were found, ten (63%) of which were non‐toxigenic. Most isolates corresponded to ribotypes that have been previously identified in humans or livestock. The role of dogs in transmission of C. difficile and the clinical implications of C. difficile shedding in dogs remain unclear. These data indicate that dogs could act as a source of C. difficile for exposure of other species, including humans; however, the true risk is unknown. Further study of the ecology of C. difficile and the role of dogs in disease of humans and other domestic animals is indicated.     

In vitro fluoroquinolone susceptibility of Pseudomonas aeruginosa isolates from dogs with ulcerative keratitis

OBJECTIVE: To determine the in vitro fluoroquinolone susceptibility profiles of Pseudomonas aeruginosa isolates from dogs with ulcerative keratitis. Animals-27 dogs with P. aeruginosa-associated ulcerative keratitis. PROCEDURES: P. aeruginosa isolates from dogs with ulcerative keratitis were collected during a 3-year period. Isolates were tested by use of the disk diffusion method for their susceptibility to 7 fluoroquinolones that are available as commercial ophthalmic preparations. The antimicrobials included second- (ciprofloxacin, ofloxacin, norfloxacin, and lomefloxacin), third- (levofloxacin), and fourth-generation (gatifloxacin and moxifloxacin) fluoroquinolones. Isolates were designated as susceptible, intermediate, or resistant to the various antimicrobials. The percentage of susceptible isolates was compared among individual fluoroquinolones and among fluoroquinolone generations. RESULTS: None of the dogs had received topical or systemic fluoroquinolone treatment prior to referral. Twenty-seven P. aeruginosa isolates were collected during the study period. In vitro, bacterial resistance to the tested fluoroquinolones was infrequently identified (24/ 27 isolates were susceptible to all fluoroquinolones evaluated); susceptibility percentages ranged from 88.9% to 100% for individual antimicrobials. There were no significant differences among isolate susceptibilities to the individual antimicrobials or among generations of fluoroquinolones. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of these in vitro data, none of the 7 evaluated fluoroquinolones (individually or collectively by generation) appeared to offer a clinically important advantage in the treatment of P. aeruginosa-associated ulcerative keratitis in dogs. Among the P. aeruginosa isolates collected from dogs with ulcerative keratitis in this study, the likelihood of susceptibility to the fluoroquinolones evaluated was high.