In vitro antimicrobial activity of a commercial ear antiseptic containing chlorhexidine and Tris–EDTA

Minimum bactericidal concentrations (MBCs) of a commercial ear antiseptic containing chlorhexidine 0.15% and Tris–EDTA (Otodine^®) were determined by broth microdilution for 150 isolates representing the most common pathogens associated with canine otitis. The microorganisms were classified into three groups according to their levels of susceptibility. The most susceptible group included Staphylococcus pseudintermedius, Malassezia pachydermatis, Streptococcus canis and Corynebacterium auriscanis, which were generally killed by 1 : 64 dilution of the antiseptic product (MBC = 23/0.8 μg/mL of chlorhexidine/Tris–EDTA). The most resistant organism was Proteus mirabilis, which survived up to 1 : 8 dilution of the product (MBC = 375/12 μg/mL). Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus displayed intermediate MBCs ranging between 188/6 and 47/1.5 μg/mL. Interestingly, S. pseudintermedius was more susceptible than S. aureus, and no significant difference was observed between meticillin‐resistant and meticillin‐susceptible isolates within each species, indicating that antiseptic use is unlikely to co‐select for meticillin resistance. Although the concentrations required for killing (MBCs) varied considerably with microorganism type, the combination of chlorhexidine 0.15% and Tris–EDTA was active against all the pathogens most commonly involved in canine otitis.     

In vitro evaluation of topical biocide and antimicrobial susceptibility of Staphylococcus pseudintermedius from dogs

Background – Staphylococcus pseudintermedius is an important canine pathogen, and the emergence and widespread dissemination of meticillin‐resistant strains (MRSP) is of significant concern. Multidrug‐resistant infections may require alternative approaches, such as the use of topical therapy. There is minimal information about the in vitro susceptibility of meticillin‐susceptible S. pseudintermedius (MSSP) and MRSP to biocides and topical antimicrobials. Hypothesis/Objectives – The hypothesis was that clinical isolates of MSSP and MRSP would not have universal susceptibility to topical biocides and antimicrobials. The goal of this study was to assess the susceptibility of a collection of S. pseudintermedius isolates to selected antimicrobials and biocides. Animals – The study was performed on clinical isolates of MSSP and MRSP from dogs with skin and soft tissue infections collected throughout North America between 2006 and 2008. Methods – The minimal inhibitory concentrations (MICs) of chlorhexidine digluconate, benzalkonium chloride, triclosan, accelerated hydrogen peroxide, geranium oil, tea tree oil and grapefruit seed extract were tested for 25 MRSP and 25 MSSP isolates from dogs using the agar dilution method. The MICs of fusidic acid, bacitracin and mupirocin were determined using Etests. Results – Triclosan demonstrated excellent activity against all bacterial isolates, with no growth at the lowest concentration evaluated (MIC ≤ 0.5 μg/mL). Conversely, grapefruit seed extract did not inhibit growth at the highest concentration tested (MIC > 3.84 μg/mL). All isolates were susceptible to mupirocin, fusidic acid and bacitracin. There were no significant differences noted in the range, MIC₅₀ or MIC₉₀ between MSSP and MRSP isolates. Conclusions and clinical importance – While isolates were susceptible to most of the tested compounds, universal susceptibility to all compounds with potential antimicrobial activity cannot be assumed, and specific testing is required.     

Treatment outcome of dogs with meticillin-resistant and meticillin-susceptible Staphylococcus pseudintermedius pyoderma

Background – The prevalence of meticillin‐ and multidrug‐resistant Staphylococcus pseudintermedius (MRSP) in canine pyoderma has been increasing in recent years; thus, treatment of these cases has become challenging. Hypothesis/Objectives – To compare treatment outcome (clinical resolution and treatment duration), adverse effects of medication, and concurrent diseases and medications in dogs with meticillin‐susceptible S. pseudintermedius (MSSP) and MRSP pyoderma. Animals/Methods – Medical records were reviewed retrospectively, and 123 MSSP and 93 MRSP clinical cases between January 2008 and April 2010 were included. Results – In MSSP infections, cefalexin and cefpodoxime were the most commonly prescribed antimicrobials, accounting for 43.2 and 34.4% of cases, respectively. In MRSP infections, chloramphenicol and doxycycline were most commonly prescribed, accounting for 52.6 and 14.4% of cases, respectively. Adverse effects were reported in seven MSSP and 31 MRSP cases. The most commonly reported adverse effects were gastrointestinal, prompting antibiotic discontinuation in three MSSP and 20 MRSP cases. Chloramphenicol was associated with the highest incidence of adverse reactions (27 of 51 cases). Of 164 cases with follow up, 43 of 88 MSSP infections and 29 of 76 MRSP infections achieved complete clinical resolution at the first recheck examination. Three MSSP and seven MRSP cases failed to improve or resolve at subsequent visits assessed at 3–4 week intervals. Conclusions and clinical importance – Results from this study showed that the majority of pyodermas resolved regardless of meticillin susceptibility. Although some cases of MRSP pyoderma took longer to treat, this is likely to be because of chronicity and not the organism. In addition, adverse effects were frequently associated with chloramphenicol administration.     

Comparative in vitro efficacy of antimicrobial shampoos: a pilot study

This study compared the antimicrobial efficacy of shampoos against meticillin‐sensitive Staphylococcus pseudintermedius (MSSP), meticillin‐resistant S. pseudintermedius (MRSP), antibiotic‐sensitive Pseudomonas aeruginosa (PA), multidrug‐resistant P. aeruginosa (MDR‐PA) and Malassezia pachydermatis. Three isolates were incubated for 10, 30 and 60 min with each shampoo diluted in phosphate‐buffered saline. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud’s dextrose agar (Malassezia). The minimal bactericidal concentrations (MBCs) for chlorhexidine products (Malaseb^®, Pyoderm^®/Microbex^® and Hibiscrub^®) were 1:1,024–1:2,048 for MSSP and MRSP, 1:512–1:1,024 for PA and MDR‐PA, and 1:2,048–1:5,096 for Malassezia at all time points. The MBCs for benzoyl peroxide (Paxcutol^®) for MSSP and MRSP were 1:2–1:8 at 10 min, and 1:256 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and 1:512–1:1,024 dilutions were effective against Malassezia at all time points. The MBCs for ethyl lactate (Etiderm^®) for MSSP and MRSP were 1:2 at 10 min, and 1:2–1:16 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and a 1:512 dilution was effective against Malassezia at all time points. Chloroxylenol (Coatex^®) and acetic acid–boric acid (Malacetic^®) were not effective against MSSP, MRSP or Pseudomonas. Both were effective against Malassezia at 1:8–1:16 dilution at 10 min, and at 1:8–1:32 dilution after 30 and 60 min. In conclusion, chlorhexidine appeared to be the most effective topical biocide, and MRSP and MDR‐PA were no less susceptible than antibiotic‐sensitive organisms. These results should, however, be confirmed with larger numbers of isolates.     

Prevalence of genes for enterotoxins,toxic shock syndrome toxin 1 and exfoliative toxin among clinical isolates of Staphylococcus pseudintermedius from canine origin

A total of 74 Staphylococcus pseudintermedius strains were isolated from the 99 clinical cases of canine pyoderma or chronic otitis in our veterinary teaching hospital during May 2006–February 2008. In this study, we examined the genetic distribution of staphylococcal pyogenic toxins such as staphylococcal enterotoxins A (sea), B (seb), C (sec), D (sed), E (see), and toxic shock syndrome toxin 1 (tst) as well as the previously characterized S. intermedius exfoliative toxin (siet) among those isolates. The polymerase chain reaction analyses with the toxin gene‐specific primers revealed that 18 (24.3%) of 74 S. pseudintermedius isolates carried the sec genes, but none of the sea, seb, sed, see and tst genes. Further DNA sequencing analysis of the amplified sec genes revealed that they all belonged to the canine type C staphylococcal enterotoxin (SEC_canine) whose superantigenic activity has been demonstrated. In addition to the sec_canine genes, our polymerase chain reaction results showed that all the 74 isolates carried the siet gene. Since both SEC_canine and SIET toxins are known to be biologically active, it would be interesting to investigate how those toxins are involved in the pathogenesis of the canine diseases by S. pseudintermedius such as pyoderma or chronic otitis.     

Clonal Spread of Salmonella enterica Serovar Infantis in Serbia: Acquisition of Mutations in the Topoisomerase Genes gyrA and parC Leads to Increased Resistance to Fluoroquinolones

Quinolone‐resistant Salmonella Infantis (n = 64) isolated from human stool samples, food and poultry during the years 2006–2011 were analysed for their resistance phenotypes, macrorestriction patterns and molecular mechanisms of decreased susceptibility to fluoroquinolones. Minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were determined by the agar dilution procedure, and the susceptibility to additional antimicrobial agents was determined by the disc diffusion method. To assess the influence of enhanced efflux activity, MICs were determined in the presence and absence of the inhibitor PAβN. The results of pulsed‐field gel electrophoresis (PFGE) typing revealed that quinolone‐resistant S. Infantis in Serbia had similar or indistinguishable PFGE profiles, suggesting a clonal spread. All S. Infantis showed combined resistance to NAL and tetracycline, whereas multiple drug resistance to three or more antibiotic classes was rare (2 isolates of human origin). The MICs ranged between 512 and 1024 μg/mL for NAL and 0.125–2 μg/mL for CIP. A single‐point mutation in the gene gyrA leading to a Ser83→Tyr exchange was detected in all isolates, and a second exchange (Ser80→Arg) in the gene parC was only present in eight S. Infantis isolates exhibiting slightly higher MICs of CIP (2 μg/mL). The inhibitor PAβN decreased the MIC values of CIP by two dilution steps and of NAL by at minimum 3–6 dilution steps, indicating that enhanced efflux plays an important role in quinolone resistance in these isolates. The plasmid‐mediated genes qnr, aac(6′)‐lb‐cr and qepA were not detected by PCR assays.     

Heterogeneity of Staphylococcus pseudintermedius isolates from atopic and healthy dogs

Staphylococcus pseudintermedius is part of the normal canine flora but frequently causes pyoderma in canine atopic dermatitis (AD). This study aimed to determine whether particular S. pseudintermedius strains were associated with AD and/or pyoderma. Ninety‐six S. pseudintermedius isolates from the ear, nares, perineum and lesions of 21 atopic and 16 healthy dogs were lysed with proteinase K and digested with 40 U SmaI. Restriction products were separated using pulsed‐field gel electrophoresis (PFGE) with an Oxford S. aureus control and lambda‐ladder DNA concatomer markers. A dendrogram was constructed by the unweighted pair group method. All isolates showed a ≥56% similarity coefficient. Nine distinct PFGE clusters were identified, as follows: five from both atopic and healthy dogs; three from atopic dogs only; and one from healthy dogs only. Nine clusters were isolated from the nares, eight from the perineum, five from the ears and six from pyoderma lesions. There were no significant differences in the frequency of isolation from atopic or healthy skin, body sites or infected lesions for any of the clusters. Two of six healthy dogs and 18 of 20 atopic dogs with multiple isolates had closely related isolates (less than three band differences) at more than one sampling site. Isolates from pyoderma lesions were closely related to at least one mucosal isolate in 11 of 16 dogs. Staphylococcus pseudintermedius isolates appear to be heterogeneous, and colonization or infection of atopic skin was not associated with any particular strain or cluster of strains.     

Antimicrobial resistance of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius, Staphylococcus intermedius and Staphylococcus delphini together comprise the S. intermedius group (SIG). Within the SIG, S. pseudintermedius represents the major pathogenic species and is involved in a wide variety of infections, mainly in dogs, but to a lesser degree also in other animal species and humans. Antimicrobial agents are commonly applied to control S. pseudintermedius infections; however, during recent years S. pseudintermedius isolates have been identified that are meticillin‐resistant and have also proved to be resistant to most of the antimicrobial agents approved for veterinary applications. This review deals with the genetic basis of antimicrobial resistance properties in S. pseudintermedius and other SIG members. A summary of the known resistance genes and their association with mobile genetic elements is given, as well as an update of the known resistance‐mediating mutations. These data show that, in contrast to other staphylococcal species, S. pseudintermedius seems to prefer transposon‐borne resistance genes, which are then incorporated into the chromosomal DNA, over plasmid‐located resistance genes.     

In vitro miconazole susceptibility of meticillin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus

Background – The emergence and dissemination of meticillin‐resistant staphylococci has created significant treatment challenges in veterinary medicine and increased interest in topical therapy for superficial infections. Concern has been expressed regarding the use of some topical antimicrobials in animals because of the potential for emergence of resistance, and additional options are required. Miconazole has limited antibacterial properties that include antistaphylococcal activity. Hypothesis/Objectives – The objective of this study was to assess the in vitro susceptibility of Staphylococcus pseudintermedius and Staphylococcus aureus to miconazole. Methods – In vitro susceptibility of 112 meticillin‐resistant S. pseudintermedius (MRSP), 53 meticillin‐resistant S. aureus (MRSA) and 37 meticillin‐susceptible S. pseudintermedius (MSSP) to miconazole was assessed using agar dilution. Results – The minimal inhibitory concentration (MIC) range, MIC₅₀ and MIC₉₀ for MRSP were 1–8, 2 and 4 μg/mL, respectively. Corresponding results for MRSA were 1–8, 2 and 6 μg/mL, and for MSSP 1–4, 2 and 2 μg/mL. The MIC for MSSP was a significantly lower MIC than that for both MRSP (P = 0.006) and MRSA (P < 0.001), while the MIC for MRSP was significantly lower than that for MRSA (P = 0.001). Conclusions and clinical importance – These in vitro data suggest that miconazole could be a useful therapeutic option for superficial infections caused by meticillin‐susceptible and meticillin‐resistant staphylococci, but proper clinical investigation is required.     

Prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) from skin and carriage sites of dogs after treatment of their meticillin-resistant or meticillin-sensitive staphylococcal pyoderma

Background – Meticillin‐resistant staphylococci are significant pathogens in veterinary dermatology, yet longitudinal studies of the impact of routine antimicrobial therapy on emergence or resolution of resistance are lacking. Objectives – To determine the prevalence of meticillin‐resistant staphylococci on skin and carriage sites in dogs with bacterial pyoderma and evaluate the prevalence of meticillin‐resistant Staphylococcus pseudintermedius (MRSP) colonization after successful treatment of pyoderma. Animals – One hundred and seventy‐three dogs that presented to a dermatology referral service with pyoderma and 41 healthy control dogs. Methods – Skin, nasal and rectal swabs for bacterial culture were collected at the time of referral and after clinical resolution of the pyoderma. Meticillin resistance was confirmed by demonstration of penicillin binding protein 2a antigen. Results – Initially, skin cultures yielded MRSP in 70 (40.5%) dogs, meticillin‐resistant Staphylococcus aureus (MRSA) in three (1.7%) and meticillin‐resistant Staphylococcus schleiferi ssp. coagulans (MRSScoag) in five (2.9%). Samples collected from the nose and rectum (carriage sites) yielded MRSP in 59 (34.1%) dogs, MRSA in 11 (6.4%) and MRSScoag in seven (4.0%). One hundred and two dogs were available for follow‐up cultures after clinical cure. Of 42 dogs initially diagnosed with MRSP pyoderma, MRSP was isolated at follow‐up from skin in 19 (45.2%) and carriage sites in 20 (47.6%). Of 60 dogs that did not have MRSP pyoderma initially, MRSP was isolated post‐treatment from the skin in 17 (28.3%), and MRSP from carriage sites increased from 7.8% (initially) to 26.7% (P = 0.0022). Conclusions and clinical importance – Colonization by MRSP often persists after resolution of MRSP pyoderma. Acquisition of MRSP during treatment appears to be common.     

Prevalence of and risk factors for isolation of meticillin‐resistant Staphylococcus spp. from dogs with pyoderma in northern California,USA

Background – Canine pyodermas associated with meticillin‐resistant Staphylococcus spp. (MRS) have increased in prevalence over the past decade. Hypothesis/Objectives – To compare the prevalence of MRS isolation from dogs with superficial pyoderma at a primary care clinic (PCC) and those at a tertiary care facility (VMTH) in California, USA, and identify associated risk factors. Animals – Client‐owned dogs from the VMTH (80 dogs) and the PCC (30 dogs). Methods – Aerobic bacterial culture and antibiotic susceptibility were performed on swab specimens collected from dogs, and meticillin resistance was determined using microdilution methods according to Clinical and Laboratory Standards Institute guidelines. A mecA gene PCR assay was used to confirm meticillin resistance when possible. Results – Of 89 staphylococcal isolates from the VMTH, 34 (38.2%) were meticillin resistant. In 31 dogs, pyoderma persisted, and one or more follow‐up isolates were obtained. The species isolated and drug susceptibility changed unpredictably during treatment. Of 33 PCC isolates, nine (27.3%) were meticillin resistant. Multiple drug resistance was identified in 41 of 53 (77.3%) MRS isolates from the VMTH and five of nine from the PCC. The sensitivity and specificity of PCR for the detection of meticillin resistance was 34 of 39 (87%) and 86 of 87 (99%), respectively. Risk factors for meticillin resistance for both sites were antibiotic treatment within the last year (P = 0.001), and for VMTH, hospitalization of dogs within the last year (P = 0.001). Conclusions and clinical importance – The prevalence of meticillin resistance was not different between VMTH and PCC isolates (P = 0.29). Previous antimicrobial therapy was an important risk factor for the isolation of MRS at both sites.     

In vitro assessment of chloramphenicol and florfenicol as second‐line antimicrobial agents in dogs

The aim of this study was to evaluate the potential of chloramphenicol and florfenicol as second‐line antimicrobial agents for treatment of infections caused by methicillin‐resistant Staphyococcus pseudintermedius (MRSP) and extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli in dogs, through a systematic in vitro assessment of the pharmacodynamic properties of the two drugs. Minimum inhibitory concentrations (MIC) and phenicol resistance genes were determined for 169 S. pseudintermedius and 167 E. coli isolates. Minimum bactericidal concentrations (MBC), time‐killing kinetics, and postantibiotic effect (PAE) of both agents against wild‐type isolates of each species were assessed. For S. pseudintermedius, the chloramphenicol MIC₉₀ was 32 μg/mL. No florfenicol resistance was detected in this species (MIC_90 = 4 μg/mL). The MIC₉₀ of both agents against E. coli was 8 μg/mL. Resistance genes found were cat_pC221 in S. pseudintermedius and catA1 and/or floR in E. coli. The phenicols displayed a time‐dependent, mainly, bacteriostatic effect on both species. Prolonged PAEs were observed for S. pseudintermedius, and no PAEs were detected for E. coli. More research into determination of PK/PD targets of efficacy is needed to further assess the clinical use of chloramphenicol and florfenicol as second‐line agents in dogs, optimize dosage regimens, and set up species‐specific clinical break points.     

Usefulness of cefovecin disk‐diffusion test for predicting mecA gene‐containing strains of Staphylococcus pseudintermedius and clinical efficacy of cefovecin in dogs with superficial pyoderma

Background –  Cefovecin has been widely used to treat skin infections in dogs. The relationship of the cefovecin disk‐diffusion test results to the presence of the mecA gene and the clinical efficacy of cefovecin have not been fully evaluated. Hypothesis/Objectives –  To determine the usefulness of an in vitro cefovecin disk‐diffusion test in predicting the presence of the mecA gene in Staphylococcus pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma. Methods –  Twenty‐six S. pseudintermedius strains isolated from 22 dogs with pyoderma were used. In vitro disk‐diffusion test results of cefovecin were compared with agar‐dilution test results, the presence of the mecA gene, and the improvement in clinical scores of dogs with superficial pyoderma at 14 days post treatment. Results –  There was a significant linear correlation (r = ?0.83) between the diameter of the obvious zone of inhibition by disk diffusion and the minimal inhibitory concentration for cefovecin (P < 0.0001). Receiver operating characteristic analysis revealed that zone diameters between 25 and 27 mm exhibited better sensitivity (92.9%) and specificity (100.0%) for detection of strains carrying the mecA gene. The mean improvement in clinical scores in dogs carrying cefovecin‐resistant strains was significantly lower than in dogs carrying cefovecin‐susceptible strains (P < 0.01). Conclusions and clinical importance –  The cefovecin disk‐diffusion test with a cut‐off value estimated in this study was valuable for predicting mecA gene carriage in S. pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius.     

Susceptibility of canine and feline bacterial pathogens to pradofloxacin and comparison with other fluoroquinolones approved for companion animals

In this study, 908 bacterial pathogens from defined infections of dogs and cats were tested for their susceptibility to the novel fluoroquinolone pradofloxacin, which was approved in 2011 for use in cats and dogs. Most of the bacteria tested (Staphylococcus aureus, Staphylococcus pseudintermedius, Escherichia coli, β-haemolytic streptococci, Pasteurella multocida and Bordetella bronchiseptica) exhibited low pradofloxacin MIC₉₀ values of ≤0.25 μg/ml. Solely Proteus spp. and Pseudomonas aeruginosa had higher MIC₉₀ values of ≥4 μg/ml. Only six (3.4%) of 177 S. pseudintermedius and 12 (5.3%) of 227 E. coli isolates showed pradofloxacin MICs of ≥2 μg/ml. Analysis of the quinolone resistance determining regions of the target genes identified double mutations in GyrA that resulted in amino acid exchanges S83L + D87N or S83L + D87Y and single or double mutations in ParC that resulted in amino acid exchanges S80I or S80I + E84G in all 12 E. coli isolates. The six S. pseudintermedius isolates exhibited amino acid exchanges S84L or E88K in GyrA and S80I in GrlA. Comparative analysis of the MICs of pradofloxacin and the MICs determined for enrofloxacin and its main metabolite ciprofloxacin, but also marbofloxacin, orbifloxacin, difloxacin and ibafloxacin was conducted for the target pathogens S. pseudintermedius, E. coli and P. multocida. This comparison confirmed that pradofloxacin MICs were significantly lower than those of the other tested fluoroquinolones.     

Antimicrobial Resistance Profiles of Campylobacter spp. Isolated from Broiler Chicken Meat of Estonian,Latvian and Lithuanian Origin at Estonian Retail Level and from Patients with Severe Enteric Infections in Estonia

The resistance patterns of Campylobacter spp. isolated from retail broiler chicken meat originating either from Estonia, Lithuania or Latvia collected in Estonia were determined. Additionally, in collaboration with the laboratories of several Estonian hospitals, antimicrobial susceptibility patterns were determined for Campylobacter isolates from patients with severe Campylobacter enteric infections. The isolates were identified at the species level by the PCR method. Respectively, 88.8% of the isolates were C. jejuni, and 11.2% were C. coli. In total, 126 Campylobacter isolates of broiler chicken meat and human origin were tested for minimal inhibitory concentrations (MICs) with the broth microdilution VetMIC^TH method (National Veterinary Institute; Uppsala, Sweden) for a total of six antimicrobials. Resistance to one or more antimicrobials was detected in 62 (63.3%) of Campylobacter broiler chicken meat isolates and in 20 (71.4%) of human‐origin isolates. Large proportions of the broiler chicken meat isolates were resistant to ciprofloxacin (60.2%). Multidrug resistance (i.e. to three or more unrelated antimicrobials) was detected in five (5.1%) C. jejuni isolates. Among the human isolates, 20 (71.4%) were resistant to fluoroquinolones, and two (7.1%) C. jejuni isolates exhibited multidrug resistance. The chicken meat isolates of Estonian origin were the most susceptible. However, a high proportion of fluoroquinolone‐resistant C. jejuni isolates were found in Latvian and Lithuanian products. The results of this study indicate that the problems caused by the inappropriate use of antimicrobials extend beyond the country in which a food originates; therefore, both domestic and international interventions and agreements are required to implement common policies on antimicrobial usage and to minimize the emergence of Campylobacter drug resistance.     

Staphylococcus pseudintermedius expresses surface proteins that closely resemble those from Staphylococcus aureus

Staphylococcus pseudintermedius is a commensal of dogs that is implicated in the pathogenesis of canine pyoderma. This study aimed to determine if S. pseudintermedius expresses surface proteins resembling those from Staphylococcus aureus and to characterise them. S. pseudintermedius strain 326 was shown to adhere strongly to purified fibrinogen, fibronectin and cytokeratin 10. It adhered to the α-chain of fibrinogen which, along with binding to cytokeratin 10, is the hallmark of clumping factor B of S. aureus, a surface protein that is in part responsible for colonisation of the human nares. Ligand-affinity blotting with cell-wall extracts demonstrated that S. pseudintermedius 326 expressed a cell-wall anchored fibronectin binding protein which recognised the N-terminal 29 kDa fragment. The ability to bind fibronectin is an important attribute of pathogenic S. aureus and is associated with the ability of S. aureus to colonise skin of human atopic dermatitis patients. S. pseudintermedius genomic DNA was probed with labelled DNA amplified from the serine-aspartate repeat encoding region of clfA of S. aureus. This probe hybridised to a single SpeI fragment of S. pseudintermedius DNA. In the cell-wall extract of S. pseudintermedius 326, a 180 kDa protein was discovered which bound to fibrinogen by ligand-affinity blotting and reacted in a Western blot with antibodies raised against the serine-aspartate repeat region of ClfA and the B-repeats of SdrD of S. aureus. It is proposed that this is an Sdr protein with B-repeats that has an A domain that binds to fibrinogen. Whether it is the same protein that binds cytokeratin 10 is not clear.     

Fluoroquinolone Resistance Mechanism of Clinical Isolates and Selected Mutants of Pasteurella multocida from Bovine Respiratory Disease in China

The minimum inhibitory concentrations (MICs), mutation prevention concentrations (MPCs) and contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (ciprofloxacin, enrofloxacin and orbifloxacin) susceptibility in 23 Pasteurella multocida (Pm) isolates were investigated. Fluoroquinolone-susceptible isolates (MICs ≤0.25 µg/ml, 9 isolates) had no QRDR mutations, and their respective MPCs were low. Fluoroquinolone-intermediate isolates (MICs=0.5 µg/ml, 14 isolates) had QRDR mutations (Asp87 to Asn or Ala84 to Pro in gyrA), and their respective MPCs were high (4–32 µg/ml). First-step mutants (n=5) and laboratory-derived highly resistant fluoroquinolone mutants (n=5) also had QRDR mutations. The MICs of fluoroquinolones for mutant-derived strains were decreased in the presence of efflux inhibitors. The results indicated that the fluoroquinolone resistance of Pm is mainly due to multiple target gene mutations in gyrA and parC and the overexpression of efflux pump genes.     

Identification of a multidrug efflux pump in Flavobacterium johnsoniae

In this study, the mechanism conferring multiple drug resistance in several strains of flavobacteria isolated from the ovarian fluids of hatchery reared 3-year old brook trout Salvelinus fontinalis was investigated. Metabolic fingerprinting and 16S rRNA gene sequences identified the isolates as Flavobacterium johnsoniae. The isolates exhibited multiple resistances to a wide range of antimicrobial classes including penicillin, cephem, monobactam, aminoglycoside, and phenicol. Although plasmids and other transposable elements containing antimicrobial resistance genes were not detected, the isolates did contain a genomic sequence for a chloramphenicol-inducible resistance-nodulation-division family multidrug efflux pump system. Efflux pumps are non-specific multidrug efflux systems. They are also a component of cell-cell communication systems, and respond specifically to cell membrane stressors such as oxidative or nitrosative stress. Understanding of efflux pump mediated antibiotic resistances will affect efficacy of clinical treatments of fishes associated with F. johnsoniae epizootics.     

Biofilm production by pathogens associated with canine otitis externa,and the antibiofilm activity of ionophores and antimicrobial adjuvants

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N‐acetylcysteine (NAC), Tris‐EDTA and disodium EDTA) were investigated spectrophotometrically (OD_570nm) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000–10,000 µg/ml and from 20,000–80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris‐EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16‐fold and eightfold higher than the MIC/MBC of NAC and Tris‐EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris‐EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm‐associated OE in dogs.