Pharmacokinetic–pharmacodynamic integration and modelling of oxytetracycline for the calf pathogens Mannheimia haemolytica and Pasteurella multocida

A calf tissue cage model was used to study the pharmacokinetics (PK) and pharmacodynamics (PD) of oxytetracycline in serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids. After intramuscular administration, the PK was characterized by a long mean residence time of 28.3 hr. Based on minimum inhibitory concentrations (MICs) for six isolates each of Mannheimia haemolytica and Pasteurella multocida, measured in serum, integration of in vivo PK and in vitro PD data established area under serum concentration–time curve (AUC_0–∞)/MIC ratios of 30.0 and 24.3 hr for M. haemolytica and P. multocida, respectively. Corresponding AUC_0–∞/MIC ratios based on MICs in broth were 656 and 745 hr, respectively. PK‐PD modelling of in vitro bacterial time–kill curves for oxytetracycline in serum established mean AUC_0–24 hr/MIC ratios for 3log₁₀ decrease in bacterial count of 27.5 hr (M. haemolytica) and 60.9 hr (P. multocida). Monte Carlo simulations predicted target attainment rate (TAR) dosages. Based on the potency of oxytetracycline in serum, the predicted 50% TAR single doses required to achieve a bacteriostatic action covering 48‐hr periods were 197 mg/kg (M. haemolytica) and 314 mg/kg (P. multocida), respectively, against susceptible populations. Dosages based on the potency of oxytetracycline in broth were 25‐ and 27‐fold lower (7.8 and 11.5 mg/kg) for M. haemolytica and P. multocida, respectively.     

Mannheimia haemolytica in Feedlot Cattle: Prevalence of Recovery and Associations with Antimicrobial Use,Resistance, and Health Outcomes

Background

Mannheimia haemolytica is an important etiological agent in bovine respiratory disease.

Objectives

Explore risk factors for recovery of susceptible and resistant M. haemolytica in feedlot cattle and explore associations with health outcomes.

Animals

Cattle (n = 5,498) from 4 feedlots sampled at arrival and later in feeding period.

Methods

Susceptibility of M. haemolytica isolates tested for 21 antimicrobials. Records of antimicrobial use and health events analyzed using multivariable regression.

Results

M. haemolytica recovered from 29% of cattle (1,596/5,498), 13.1% at arrival (95% CI, 12.3–14.1%), and 19.8% at second sampling (95% CI, 18.7–20.9%). Nearly half of study cattle received antimicrobial drugs (AMDs) parenterally, mostly as metaphylactic treatment at arrival. Individual parenteral AMD exposures were associated with decreased recovery of M. haemolytica (OR, 0.2; 95% CI, 0.02–1.2), whereas exposure in penmates was associated with increased recovery (OR, 1.5; 95% CI, 1.05–2.2). Most isolates were pan‐susceptible (87.8%; 95% CI, 87.0–89.4%). AMD exposures were not associated with resistance to any single drug. Multiply‐resistant isolates were rare (5.9%; 95% CI, 5.1–6.9%), but AMD exposures in pen mates were associated with increased odds of recovering multiply‐resistant M. haemolytica (OR, 23.9; 95% CI, 8.4–68.3). Cattle positive for M. haemolytica on arrival were more likely to become ill within 10 days (OR, 1.7; 95% CI, 1.1–2.4).

Conclusions and Clinical Importance

Resistance generally was rare in M. haemolytica. Antimicrobial drug exposures in penmates increased the risk of isolating susceptible and multiply‐resistant M. haemolytica, a finding that could be explained by contagious spread.     

Invasive Pasteurella multocida Infections – Report of Five Cases at a Minnesota Hospital, 2014

During October 2014, the Minnesota Department of Health was notified of five Hospital A patients with Pasteurella multocida bacteraemia; three had died. Human soft tissue infection with P. multocida typically results from cat or dog bites or scratches. Invasive infection, defined as a P. multocida isolate from a usually sterile site, is rare. We evaluated P. multocida isolations at Hospital A, compared with other Minnesota hospitals to understand invasive infection trends. A case was defined as clinically confirmed P. multocida in a Minnesota resident during 2012–2014. All hospital laboratories were queried; Fisher's exact test was used for comparison. Medical charts were reviewed for 2014 Hospital A patients with P. multocida infections. The Minnesota clinical laboratories survey response rate was 79% (63/80). At Hospital A, proportion of P. multocida isolates from usually sterile sites increased from 0% (0/2) during 2012 to 11% (1/9) during 2013, and to 86% (5/6) during 2014. The proportion of patients with P. multocida isolated from sterile sites was 35% (6/17) at Hospital A compared with 10% (58/583) statewide during 2012–2014 combined (P < 0.05). Among 2014 Hospital A patients with invasive P. multocida infection, all five were men; median age was 70 (range: 44–78) years. Four were temporally clustered within a 33‐day period; three of those had bacteraemia on admission, making hospital acquisition possible in only one. Among five bacteraemia patients, four had cirrhosis and/or skin ulcerations, and three died. The proportion of invasive P. multocida cases was substantially higher at Hospital A during 2014. No epidemiologic links between patients were found. Three had known pet exposure. Collaborative educational efforts of chronically ill pet owners by physicians and veterinarians can acknowledge the health benefits of pet ownership, while minimizing risk for serious invasive zoonotic infections, including those caused by P. multocida.     

牛溶血性曼氏杆菌及牛荚膜A型多杀性巴氏杆菌灭活疫苗对小鼠的保护性研究

牛溶血性曼氏杆菌及牛荚膜A型多杀性巴氏杆菌是导致牛呼吸道疾病（bovine respiratory disease,BRD）的重要细菌性病原,每年给养牛业带来巨大的经济损失,目前对其疫苗研究仍显不足。本研究选用牛溶血性曼氏杆菌（Mannheimia haemolytica,Mh） Mh422株和牛荚膜A型多杀性巴氏杆菌（Pasteurella multocida,Pm） PmCQ2株作为疫苗菌株,分别制备了2种菌体浓度的Mh和Pm单价灭活菌苗及3种菌量配比（1∶1、2∶1和3∶1）的Mh-Pm二联灭活苗,以小鼠为模型,皮下多点免疫（0.2 mL）,加强免疫2次,免疫剂量均为首免的一半。首免后第7天及其后每隔5 d,小鼠尾静脉采血分离血清,ELISA方法检测抗体效价,三免后第20天,分别以Mh422或PmCQ2进行腹腔攻毒测定免疫保护效果。结果显示,所有小鼠接种疫苗均无不良反应,二免后第10天抗体达较高水平,三免后抗体水平持续升高,第15天到达高峰,其后25 d维持高水平,后缓慢下降。Mh单菌苗的2种免疫剂量对Mh422株攻毒的免疫保护率均为0,而Pm单菌苗的2种免疫剂量对PmCQ2株攻毒的免疫保护率全为100%;Mh和Pm间无交叉免疫保护作用;3种菌量配比的Mh-Pm二联疫苗对Mh422株和PmCQ2株攻毒的各自免疫保护率分别为53%～71%和100%。该研究结果表明,所制备的Mh422单菌苗对同型攻毒无免疫保护作用,在诱导机体抗体产生方面,Mh和Pm间无相互抑制作用,PmCQ2株具有促进Mh422株灭活疫苗对Mh422的免疫保护作用,这为牛溶血性曼氏杆菌和牛多杀性巴氏杆菌二联疫苗的进一步研究提供了理论基础。     

Pharmacokinetic/pharmacodynamic integration and modelling of amoxicillin for the calf pathogens Mannheimia haemolytica and Pasteurella multocida

The antimicrobial properties of amoxicillin were determined for the bovine respiratory tract pathogens, Mannheima haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time‐kill curves were established. Pharmacokinetic (PK)/pharmacodynamic (PD) modelling of the time‐kill data, based on the sigmoidal E_max equation, generated parameters for three levels of efficacy, namely bacteriostatic, bactericidal (3log₁₀ reduction) and 4log₁₀ reduction in bacterial counts. For these levels, mean AUC_(0–24 h)/MIC serum values for M. haemolytica were 29.1, 57.3 and 71.5 h, respectively, and corresponding values for P. multocida were 28.1, 44.9 and 59.5 h. Amoxicillin PK was determined in calf serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids, after intramuscular administration of a depot formulation at a dosage of 15 mg/kg. Mean residence times were 16.5 (serum), 29.6 (exudate) and 29.0 h (transudate). Based on serum MICs, integration of in vivo PK and in vitro PD data established maximum concentration (C_max)/MIC ratios of 13.9:1 and 25.2:1, area under concentration–time curve (AUC_0–∞)/MIC ratios of 179 and 325 h and T>MIC of 40.3 and 57.6 h for P. multocida and M. haemolytica, respectively. Monte Carlo simulations for a 90% target attainment rate predicted single dose to achieve bacteriostatic and bactericidal actions over 48 h of 17.7 and 28.3 mg/kg (M. haemolytica) and 17.7 and 34.9 mg/kg (P. multocida).     

Comparative pharmacokinetics of florfenicol in healthy and Pasteurella multocida‐infected Gaoyou ducks

Pasteurella multocida is the causative agent of fowl cholera, and florfenicol (FF) has potent antibacterial activity against P. multocida and is widely used in the poultry industry. In this study, we established a P. multocida infection model in ducks and studied the pharmacokinetics of FF in serum and lung tissues after oral administration of 30 mg/kg bodyweight. The maximum concentrations reached (C_max) were lower in infected ducks (13.88 ± 2.70 μg/ml) vs. healthy control animals (17.86 ± 1.57 μg/ml). In contrast, the mean residence time (MRT: 2.35 ± 0.13 vs. 2.27 ± 0.18 hr) and elimination half‐life (T_½β: 1.63 ± 0.08 vs. 1.57 ± 0.12 hr) were similar for healthy and diseased animals, respectively. As a result, the area under the concentration curve for 0–12 hr (AUC_0–12 hr) for FF in healthy ducks was significantly greater than that in infected ducks (49.47 ± 5.31 vs. 34.52 ± 8.29 μg hr/ml). The pharmacokinetic differences of FF in lung tissues between the two groups correlated with the serum pharmacokinetic differences. The C_max and AUC_0–12 hr values of lung tissue in healthy ducks were higher than those in diseased ducks. The concentration of FF in lung tissues was approximately 1.2‐fold higher than that in serum both in infected and healthy ducks indicating that FF is effective in treating respiratory tract infections in ducks.     

Comparing the minimum inhibitory and mutant prevention concentrations of selected antibiotics against animal isolates of Pasteurella multocida and Salmonella typhimurium

Historically, the use of antibiotics was not well regulated in veterinary medicine. The emergence of antibiotic resistance (ABR) in pathogenic bacteria in human and veterinary medicine has driven the need for greater antibiotic stewardship. The preservation of certain antibiotic classes for use exclusively in humans, especially in cases of multidrug resistance, has highlighted the need for veterinarians to reduce its use and redefine dosage regimens of antibiotics to ensure efficacy and guard against the development of ABR pathogens. The minimum inhibitory concentration (MIC), the lowest concentration of an antibiotic drug that will prevent the growth of a bacterium, is recognised as a method to assist in antibiotic dosage determination. Minimum inhibitory concentrations sometimes fail to deal with first-step mutants in bacterial populations; therefore dosing regimens based solely on MIC can lead to the development of ABR. The mutant prevention concentration (MPC) is the minimum inhibitory antibiotic concentration of the most resistant first-step mutant. Mutant prevention concentration determination as a complementary and sometimes preferable alternative to MIC determination for veterinarians when managing bacterial pathogens. The results of this study focused on livestock pathogens and antibiotics used to treat them, which had a MIC value of 0.25 µg/mL for enrofloxacin against all 27 isolates of Salmonella typhimurium. The MPC values were 0.50 µg/mL, with the exception of five isolates that had MPC values of 4.00 µg/mL. The MPC test yielded 65.52% (18 isolates) Salmonella isolates with florfenicol MICs in the sensitive range, while 11 isolates were in the resistant range. Seventeen isolates (58.62%) of Pasteurella multocida had MIC values in the susceptible range and 41.38% (12 isolates) had an intermediate MIC value. Mutant prevention concentration determinations as done in this study is effective for the antibiotic treatment of bacterial infections and minimising the development of resistance. The MPC method can be used to better control to prevent the development of antibiotic drug resistance used in animals.     

Comparative pharmacokinetics of marbofloxacin in healthy and Mannheimia haemolytica infected calves

The pharmacokinetics of marbofloxacin were investigated in healthy (n=8) and Mannheimia haemolytica naturally infected (n=8) Simmental ruminant calves following intravenous (i.v.) and intramuscular (i.m.) administration of 2 mg kg(-1) body weight. The concentration of marbofloxacin in plasma was measured using high performance liquid chromatography with ultraviolet detection. Following i.v. administration of the drug, the elimination half-life (t(1/2 beta)) and mean residence time (MRT) were significantly longer in diseased calves (8.2h; 11.13 h) than in healthy ones (4.6 h; 6.1 h), respectively. The value of total body clearance (CL(B)) was larger in healthy calves (3 ml min(-1) kg(-1)) than in diseased ones (1.3 ml min(-1) kg(-1)). After single intramuscular (i.m.) administration of the drug, the elimination half-life, mean residence time (MRT) and maximum plasma concentration (C(max)) were higher in diseased calves (8.0, 12 h, 2.32 microg ml(-1)) than in healthy ones (4.7, 7.4 h, 1.4 microg ml(-1)), respectively. The plasma concentrations and AUC following administration of the drug by both routes were significantly higher in diseased calves than in healthy ones. Protein binding of Marbofloxacin was not significantly different in healthy and diseased calves. The mean value for MIC of marbofloxacin for M. haemolytica was 0.1+/-0.06 microg ml(-1). The C(max)/MIC and AUC(24)/MIC ratios were significantly higher in diseased calves (13.0-64.4 and 125-618 h) than in healthy calves (8-38.33 and 66.34-328 h). The obtained results for surrogate markers of antimicrobial activity (C(max)/MIC, AUC/MIC and T > or = MIC) indicate the excellent pharmacodynamic characteristics of the drug in diseased calves with M. haemolytica, which can be expected to optimize the clinical efficacy and minimize the development of resistance.     

Serological and molecular comparison of Mannheimia haemolytica and Pasteurella trehalosi strains isolated from wild and domestic ruminants in the French Alps

Over a period of 17 years, 84 bacterial isolates identified as Mannheimia haemolytica or M. glucosida, and 52 isolates identified as Pasteurella trehalosi were detected in the lungs of domestic and wild ruminants in the French Alps. The isolates were serotyped according to their surface capsular antigens, and those sharing common antigens were further characterized by pulsed field gel electrophoresis. The results showed that the bacterial isolates included in the study clustered according to the host species from which they were isolated. These findings indicate that the transmission of serotypes of M. haemolytica, M. glucosida or P. trehalosi from an animal host in which they are common to another species sharing the same geographical space may be a rare epidemiological event.     

Pharmacokinetic/pharmacodynamic integration of cefquinome against Pasteurella Multocida in a piglet tissue cage model

To explore the in vivo antimicrobial activity of cefquinome against Pasteurella multocida in piglets, a piglet tissue cage infection model was used in this study. After the population of P. multocida reached 10⁷ CFU/mL in a tissue cage, piglets received an intramuscular administration of cefquinome at 0.2, 0.4, 0.8, 1, 2, and 4 mg/kg once daily for 3 days. To assess the tissue cage pharmacokinetics (PKTCF) of cefquinome, tissue cage fluid was collected for cefquinome analysis at 1, 3, 6, 9, 12, and 24 hr after each of the 3 daily drug administrations. Bacteria were counted every 24 hr after drug administration and at 48 and 72 hr after the last administration. Evaluation of the relationship between pharmacokinetic/pharmacodynamic (PK/PD) parameters and the antibacterial effect showed that the surrogate of %T > minimum inhibitory concentration (MIC) (R² = 0.981) was the best PK/PD index that correlated with effectiveness of cefquinome against P. multocida. The respective values of %T > MIC required for continuous 1/3‐log, 1/2‐log, and 1‐log reductions were 14.23, 34.45, and 73.44%, respectively, during each 24‐hr treatment period. In conclusion, cefquinome exhibited a potent antibacterial effect against P. multocida. When %T > MIC reached 73.44%, cefquinome exhibited a bactericidal effect against P. multocida after three successive daily administrations.     

Diversity of Mannheimia haemolytica and Pasteurella trehalosi Serotypes from Apparently Healthy Sheep and Abattoir Specimens in the Highlands of Wollo,North East Ethiopia

The prevalence and serotypic diversity of Mannheimia [Pasteurella] haemolytica and Pasteurella trehalosi from nasal swabs, sera and abattoir specimens from sheep in the highlands of Wollo, North East Ethiopia was investigated. Prevalence rates of 83% and 75% of these microorganisms were found in the serum samples and nasal swabs, respectively, from apparently healthy sheep. In a local abattoir, 205 lungs were investigated, 34% of which showed pneumonia, from which samples were collected from 51 lungs and the same number of corresponding tonsils. Mannheimia and Pasteurella species were isolated from 59% of these pneumonic lungs and 69% of the respective tonsils. M. haemolytica serotypes accounted for 41 (59%) and P. trehalosi for 11 (32%) of the isolates from the abattoir specimens. The majority (67%) of isolates from nasal swabs were P. trehalosi, M. haemolytica being isolated f rom 4 (13%) of the swabs. M. glucosida was isolated only from the tonsils. The predominant serotypes of the isolates from both the nasal swabs and the abattoir specimens were M. haemolytica A1 (17%) and P. trehalosi T4 (16%) and T3 (13%). P. trehalosi T15 was less commonly encountered, while M. haemolytica A9 and A13 were not isolated. Studies on sera from 100 sheep indicated that antibodies against M. haemolytica serotype A1 (14%) were most common, followed by A5 and A8 (each 10%) and A9 and P. trehalosi T3 (each 9%) and T4 (8%). Antibodies against M. glucosida or serotype A11 occurred in 2% of the sera. Multiple serotypes were common in all types of samples. The importance of including in vaccines the most prevalent serotypes involved in the pneumonia of sheep in the area is discussed.     

Differential haptoglobin responsiveness to a Mannheimia haemolytica challenge altered immunologic,physiologic, and behavior responses in beef steers

Indicator traits associated with disease resiliency would be useful to improve the health and welfare of feedlot cattle. A post hoc analysis of data collected previously (Kayser et al., 2019a) was conducted to investigate differences in immunologic, physiologic, and behavioral responses of steers (N = 36, initial BW = 386 ± 24 kg) that had differential haptoglobin (HPT) responses to an experimentally induced challenge with Mannheimia haemolytica (MH). Rumen temperature, DMI, and feeding behavior data were collected continuously, and serial blood samples were collected following the MH challenge. Retrospectively, it was determined that 9 of the 18 MH-challenged steers mounted a minimal HPT response, despite having similar leukocyte and temperature responses to other MH-challenged steers with a greater HPT response. Our objective was to examine differences in behavioral and physiological responses between MH-challenged HPT responsive (RES; n = 9), MH-challenged HPT nonresponsive (NON; n = 9), and phosphate-buffered saline-inoculated controls (CON; n = 18). Additionally, ¹H NMR analysis was conducted to determine whether the HPT-responsive phenotype affected serum metabolite profiles. The RES steers had lesser (P < 0.05) cortisol concentrations than NON and CON steers. The magnitude of the increases in neutrophil concentrations and rumen temperature, and the reduction in DMI following the MH challenge were greatest (P < 0.05) in RES steers. Univariate analysis of serum metabolites indicated differences between RES, NON, and CON steers following the MH challenge; however, multivariate analysis revealed no difference between HPT-responsive phenotypes. Prior to the MH challenge, RES steers had longer (P < 0.05) head down and bunk visit durations, slower eating rates (P < 0.01) and greater (P < 0.05) daily variances in bunk visit frequency and head down duration compared with NON steers, suggesting that feeding behavior patterns were associated with the HPT-responsive phenotype. During the 28-d postchallenge period, RES steers had decreased (P < 0.05) final BW, tended (P = 0.06) to have lesser DMI, and had greater (P < 0.05) daily variances in head down and bunk visit durations compared with NON steers, which may have been attributed to their greater acute-phase protein response to the MH challenge. These results indicate that the HPT-responsive phenotype affected feeding behavior patterns and may be associated with disease resiliency in beef cattle.     

In vitro study of Pasteurella multocida adhesion to trachea, lung and aorta of rabbits

In vitro experiments were undertaken to study the adhesion and colonization to tracheal mucosa, lung and aorta explants from freshly killed rabbits of two different strains of Pasteurella multocida. Serotype A:3 (capsulated, fimbriae +, haemagglutination -, dermonecrotic toxin -) isolated from a rabbit with rhinitis, and serotype D:1 (non-capsulated, fimbriae +, haemagglutination +, dermonecrotic toxin +) isolated from a dead rabbit with septicaemia, were used. When the explants were observed under the scanning electron microscope, the type D strain was highly adherent to trachea and aorta explants compared to the type A strain. Adhesion to lung explants was best achieved by the type A strain after 45 min incubation, but after 2 h incubation no significant difference was observed between the strains. Our data indicate that the presence of fimbriae and the absence of capsule seem to enhance the adherence of P. multocida type D strain to tracheal tissue. The capsular material of P. multocida type A strain and the toxin of the type D strain seem to influence the adherence to lung tissue in rabbit. Adhesion of strain D to aorta may indicate the expression of receptors on the endothelium to that strain and may also explain the ability of certain strains to cause septicaemia.     

Pharmacokinetic–pharmacodynamic relationship of marbofloxacin for Escherichia coli and Pasturella multocida following repeated intramuscular administration in goats

The pharmacokinetics (PK) and pharmacodynamics (PD) of marbofloxacin (MBF) were determined in six healthy female goats of age 1.00–1.25 years after repeated administration of MBF. The MBF was administered intramuscularly (IM) at 2 mg kg^?1 day^?1 for 5 days. Plasma concentrations of MBF were determined by high‐performance liquid chromatography, and PK parameters were obtained using noncompartmental analysis. The MBF concentrations peaked at 1 hr, and peak concentration (C_max) was 1.760 µg/ml on day 1 and 1.817 µg/ml on day 5. Repeated dosing of MBF caused no significant change in PK parameters except area under curve (AUC) between day 1 (AUC_0–∞D1 = 7.67 ± 0.719 µg × hr/ml) and day 5 (AUC_0‐∞D5 = 8.70 ± 0.857 µg × hr/ml). A slight difference in mean residence time between 1st and 5th day of administration and accumulation index (AI = 1.13 ± 0.017) suggested lack of drug accumulation following repeated IM administration up to 5 days. Minimum inhibitory concentration (MIC) demonstrated that Escherichia coli (MIC = 0.04 µg/ml) and Pasturella multocida (MIC = 0.05 µg/ml) were highly sensitive to MBF. Time‐kill kinetics demonstrated rapid and concentration‐dependent activity of MBF against these pathogens. PK/PD integration of data for E. coli and P. multocida, using efficacy indices: C_max/MIC and AUC_0–24hr/MIC, suggested that IM administration of MBF at a dose of 2 mg kg^?1 day^?1 is appropriate to treat infections caused by E. coli. However, a dose of 5 mg kg^?1 day^?1 is recommended to treat pneumonia caused by P. multocida in goats. The study indicated that MBF can be used repeatedly at dosage of 2 mg/kg in goats without risk of drug accumulation up to 5 days.     

Synergistic inhibition of the growth in vitro of Microsporum canis by miconazole and chlorhexidine

An agar dilution technique was used to assess the minimum inhibitory concentrations (MIC) of miconazole, chlorhexidine and a 1:1 combination of both agents for 10 isolates of Microsporum canis. For nine of 10 of the isolates, a combination of miconazole and chlorhexidine was more effective than either agent alone; fractional inhibitory concentration indices indicated a synergistic effect for five isolates and an additive effect for four. These results illustrate the potent antimycotic effect of miconazole and chlorhexidine against M. canis and are in accordance with previous clinical studies that showed the value of using miconazole and chlorhexidine shampoo in association with oral griseofulvin in the treatment of feline dermatophytosis caused by M. canis.     

Epidemiology and genetic diversity of zoonotic pathogens in urban rats (Rattus spp.) from a subtropical city,Guangzhou, southern China

Commensal rats (Rattus spp.), which are globally distributed, harbour many pathogens responsible for significant human diseases. Despite this, we have a poor understanding of the epidemiology and genetic diversity of some recently neglected zoonotic pathogens, such as Leptospira spp., Bartonella spp. and hepatitis E virus (HEV), which constitute a major public health threat. Thus, we surveyed the occurrences, co‐infection and genetic diversity of these pathogens in 129 urban rats from China. For Rattus tanezumi, the prevalences of Leptospira spp., Bartonella spp. and HEV infection were 6.67%, 0% and 46.67%, respectively. The prevalences of Leptospira spp., Bartonella spp. and HEV infection were 57.89%, 9.65% and 57.89% for Rattus norvegicus respectively. Leptospira spp. and HEV infections were more likely to occur in mature R. norvegicus. Phylogenetic analyses showed that pathogenic Leptospira interrogans and Leptospira borgpetersenii might exist. We also found that Bartonella spp. showed high similarity to Bartonella elizabethae, Bartonella rochalimae and Bartonella tribocorum, which are implicated in human disease. Dual and triple infections were both detected. Moreover, dual infections with Leptospira spp. and HEV represented the most frequent co‐infection, and there was a significantly positive association between them. High genetic diversity was observed in genes segments from Leptospira, Bartonella and HEV. Our results first discover the occurrence of multiple co‐infections and genetic diversity of Leptospira, Bartonella and HEV in commensal rats from China. Altogether, the present study provides an insight into evaluating the risk of rat‐borne zoonoses in urban China.     

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.