Correction: Alhamami et al. First Emergence of Resistance to Macrolides and Tetracycline Identified in Mannheimia haemolytica and Pasteurella multocida Isolates from Beef Feedlots in Australia. Microorganisms 2021, 9, 1322

The authors wish to make the following corrections to this paper [1]: There are metadata errors in Supplementary Figure S2 and Table 8, which state that isolate 17BRD-035 was isolated from a feedlot in NSW when in fact it came from Queensland. Figure S2 in Supplementary Materials was changed accordingly and was included with a separate document: https://www.mdpi.com/article/10.3390/microorganisms9061322/s1 In Table 8, the ST column for strain P. m 17BRD-035 was changed from NSW to QLD, the correct Table 8 is as follows: Table 8. Resistance profile, RAPD pattern and presence of antimicrobial resistance genes among isolates of Pasteurella multocida (P. m) (n = 28) and Mannheimia haemolytica (M. h) (n = 1) +, present, −, absent. Table (see supplement) The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated

In vitro Antimicrobial Activity of Robenidine, Ethylenediaminetetraacetic Acid and Polymyxin B Nonapeptide Against Important Human and Veterinary Pathogens

The emergence and global spread of antimicrobial resistance among bacterial pathogens demand alternative strategies to treat life-threatening infections. Combination drugs and repurposing of old compounds with known safety profiles that are not currently used in human medicine can address the problem of multidrug-resistant infections and promote antimicrobial stewardship in veterinary medicine. In this study, the antimicrobial activity of robenidine alone or in combination with ethylenediaminetetraacetic acid (EDTA) or polymyxin B nonapeptide (PMBN) against Gram-negative bacterial pathogens, including those associated with canine otitis externa and human skin and soft tissue infection, was evaluated in vitro using microdilution susceptibility testing and the checkerboard method. Fractional inhibitory concentration indices (FICIs) and dose reduction indices (DRI) of the combinations against tested isolates were determined. Robenidine alone was bactericidal against Acinetobacter baumannii [minimum inhibitory concentrations (MIC) mode = 8 μg/ml] and Acinetobacter calcoaceticus (MIC mode = 2 μg/ml). Against Acinetobacter spp., an additivity/indifference of the combination of robenidine/EDTA (0.53 > FICIs > 1.06) and a synergistic effect of the combination of robenidine/PMBN (0.5 < FICI) were obtained. DRIs of robenidine were significantly increased in the presence of both EDTA and PMBN from 2- to 2048-fold. Robenidine exhibited antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, in the presence of sub-inhibitory concentrations of either EDTA or PMBN. Robenidine also demonstrated potent antibacterial activity against multidrug-resistant Gram-positive pathogens and all Gram-negative pathogens isolated from cases of canine otitis externa in the presence of EDTA. Robenidine did not demonstrate antibiofilm activity against Gram-positive and Gram-negative bacteria. EDTA facilitated biofilm biomass degradation for both Gram-positives and Gram-negatives. The addition of robenidine to EDTA was not associated with any change in the effect on biofilm biomass degradation. The combination of robenidine with EDTA or PMBN has potential for further exploration and pharmaceutical development, such as incorporation into topical and otic formulations for animal and human use

The Eimeria-host cell interaction in broiler chickens

Coccidiosis is an enteric infection of chickens caused by protozoan parasites of the genus Eimeria. Coccidiosis is a worldwide disease with an economic impact on broiler chicken production. An outbreak of disease can reduce weight gain and feed digestion in the entire flock, reducing the production of processed meat for market. The major characteristics of Eimeria species are the invasion of specific sites in the intestine of chickens and specificity of the immune response. To date, prophylaxis and vaccination are used to control coccidiosis. However, the continuous use of chemotherapeutics has led to increased drug resistance by Eimeria. In the case of vaccination, immunity against Eimeria is species-specific, hence, there is a need to vaccinate chickens against all species of Eimeria for complete protection. The Eimeria-host cell interaction is the first stage in the reproductive cycle in chickens that produces the damage in the chicken intestine. A more complete understanding of the enviromnental factors within the intestinal tract that influence this interaction will be useful to control the disease. The lack of a suitable method to study the interaction between Eimeria and host cells derived from different areas of intestine has hampered our understanding of the disease. The cell type of interest for this study was the chicken enterocyte. A layer of mucus is secreted by goblet cells in the intestinal epithelial to protect the enterocytes. Eimeria sporozoites have to cross the mucus layer in order to invade the epithelial cells. It is reasonable to assume that this mucus may have some involvement in Eimeriaenterocyte attachment. The objectives of this study were to investigate the roles of the enterocyte and intestinal mucus in the attachment process and the subsequent penetration of host cells by Eimeria sporozoites. Newly hatched, and 3-week-old chickens, were killed and intestinal segments were collected for developing an in vitro method ex vivo ( organ culture system, isolated enterocytes and a frozen section method) to study the Eimeria interaction with intestinal epithelial cells. Eimeria sporozoites were .extracted from oocysts and then labelled with a fluorescent dye (PKH-67). The frozen section model was found to be superior to the ~se of isolated enterocytes and organ culture systems, and was used for subsequent experiments in this project. This method was used to investigate the Eimeria-enterocyte attachment at preferred and non-preferred sites on the surface of enterocyte membranes. Indeed, the use of this method demonstrated that D-galactose on the surface of sporozoites had an important role in the attachment of E. tenella sporozoites to caecal enterocytes, with caecal and duodenal mucus both functioning as a physical barrier to E. tenella. In addition, two other major developments resulted from this project, these being; the development of a PCR protocol that can specifically identify different Eimeria species in a mixed sample containing at least 0.05 ng/μl of Eimeria DNA and a propidium iodide method that is a suitable indicator tool to assess the viability of oocysts and sporocysts. Finally, the inclusion of MgCh in the extraction buffer increases the hatchability of sporozoites from sporocysts. In conclusion, this study led to development of a frozen section method which can be used ex vivo to investigate further the role of mucus from vaccinated and non-vaccinated chickens, diets with different compositions, anticoccidial drugs, and the identification of the specific receptors in different areas of the chicken intestine. Finally, the propidium iodide method in combination with the PCR protocols can be used as a quality assurance tool in the production of Eimeria vaccines.Thesis (Ph.D.) -- University of Adelaide, Dept. of Agriculture and Animal Science, 200

Evaluation of effects of Mycoplasma mastitis on milk composition in dairy cattle from South Australia

Abstract Background Mycoplasma mastitis is increasingly posing significant impact on dairy industry. Although the effects of major conventional mastitis pathogens on milk components has been widely addressed in the literature, limited data on the effects of different Mycoplasma and Acholeplasma spp. on milk quality and quantity is available. The aim of this study was to determine the casual relationship of Mycoplasma spp. and A. laidlawii to mastitis and compare them to subclinical mastitis caused by conventional mastitis pathogens from a single dairy herd in South Australia; Mycoplasma spp. and A. laidlawii were detected using PCR applied directly to milk samples. The herd had mastitis problem with high somatic cell count and low response rate to conventional antimicrobial therapy. A total of 288 cow-level milk samples were collected aseptically and used in this study. Results Conventional culture showed a predominance of coagulase-negative staphylococci, followed by coagulase-positive staphylococci, Streptococcus spp., Enterococcus spp., E. coli, and Klebsiella spp. PCR results showed a high prevalence of mycoplasmas (76.7%), including A. laidlawii (10.8%), M. bovis (6.2%), M. bovirhinis (5.6%), M. arginini (2%), and (52.1%) of cows were co-infected with two or more Mycoplasma and Acholeplasma species. Mycoplasma co-infection significantly increased somatic cell counts (SCC) similar to conventional mastitis pathogens and compared to non-infected cows with 389.3, 550.3 and 67.3 respectively; and decreased the milk yield with 29.0, 29.9 and 34.4 l, respectively. Mycoplasma co-infection caused significant increase in protein percentage, and significant decrease in fat percentage and total milk solids, similar to other conventional mastitis pathogens. In contrast, changes in milk composition and yield caused by various individual Mycoplasma species were non-significant. Conclusions Mycoplasma mastitis had on-farm economic consequences similar to common conventional mastitis pathogens. Results of our study indicate that co-infection Mycoplasma mastitis caused similar effect on milk composition to other mastitis pathogens and we hope these findings raise the awareness of the importance of their detection on routine diagnostic panels

Invitro efficacy of cefovecin against anaerobic bacteria isolated from subgingival plaque of dogs and cats with periodontal disease

Periodontal disease is a common disease of dogs and cats often requiring antimicrobial treatment as an adjunct to mechanical debridement. However, correct compliance with oral antimicrobial therapy in companion animals is often difficult. Cefovecin is a recently introduced veterinary cephalosporin that has demonstrated prolonged concentrations in extracellular fluid, allowing for dosing intervals of up to 14 days. Subgingival samples were collected from the oral cavity of 29 dogs and eight cats exhibiting grade 2 or grade 3 periodontal disease. Samples were cultivated on Wilkin Chalgrens agar and incubated in an anaerobic chamber for seven days. Selected anaerobic bacteria were isolated and identified to species level using 16S rRNA gene sequence analysis. Minimum inhibitory concentrations were determined for cefovecin and six additional antimicrobials using the agar dilution methodology recommended by the Clinical and Laboratory Standards Institute. The 65 clinical isolates were identified as Porphyromonas gulae (n=45), Porphyromonas crevioricanis (n=12), Porphyromonas macacae (n=1), Porphyromonas cangingivalis (n=1) Fusobacterium nucleatum (n=2), Fusobacterium russii (n=1) and Solobacterium moorei (n=3). This is the first report of S.moorei being isolated from companion animals with periodontal disease. All isolates were highly susceptible to cefovecin, with a MIC of ≤0.125μg/ml. Conversely, different resistance rates to ampicillin, amoxicillin and erythromycin between isolates were detected. Cefovecin is thus shown to be effective invitro against anaerobic bacteria isolated from dogs and cats with periodontal disease

Evaluation of three cryoprotectants used with bovine milk affected with Mycoplasma bovis in different freezing conditions

Abstract Objectives Currently, there is no consensus protocols regarding the combination of glycerol (GLY), gelatin or foetal bovine serum (FBS) with dimethyl sulphoxide (DMSO) as cryoprotectants for Mycoplasma bovis in bovine milk samples. This study aimed to compare different cryopreservation compounds and storage temperatures for M. bovis. Results There were significant differences in the survival of M. bovis on different media. Differences were also observed between different storage conditions. All additives improved the survival of M. bovis in comparison to control (CON). The combination of GLY and DMSO was shown to be significantly different to CON with 57.1% (95% CI = 21.43–133.34) and 19.1% (95% CI = 11.73–60.27), respectively at week 16, and its use should be encouraged as a cryoprotectant for M. bovis at − 20 and − 80 °C. GEL/DMSO showed the highest survival rate for M. bovis with 57.14% (95% CI = 21.43–133.34) at 4 °C in comparison with CON 14.29% (95% CI = 9.60–50.39). FBS/DMSO showed the highest survival rate for the short-term preservation similarly to other additives. The evaluated cryopreservative compounds would improve survivability of M. bovis in milk for both transport and long-term storage. Hence, it is recommended to use the mentioned methods for routine transportation or storage purposes for suspicious M. bovis milk samples

Decontamination of aerosolised bacteria from a pig farm environment using a pH neutral electrochemically activated solution (Ecas4 anolyte).

An electrochemically activated solution (ECAS), generated by electrolysis of a dilute sodium chloride solution in a four-chamber electrolytic cell (Ecas4), was tested as a sanitising aerosol in eliminating bacteria from the environment of a weaning room vacated 24-48h earlier, at a continuous flow pig farm. An ultrasonic humidifier was used to fill the environment with a fog (droplets with diameters of 1-5 μm) containing 0.25 ppm of hypochlorous acid. The weaning room was fogged for 3 min at 30 min intervals during five hours of aerosol disinfection. An innovative sample treatment with propidium monoazide dye in conjunction with cyclonic air sampling was optimised and adapted for discerning live/dead bacteria in subsequent molecular quantification steps. Without fogging, total bacterial load ranged from 5.06 ± 0.04 to 5.75 ± 0.04 Log10 CFU/m3. After the first hour of fogging, a 78% total bacterial reduction was observed, which further increased to > 97% after the second hour, > 99.4% after the third and 99.8% after the fourth hour, finally resulting in a 99.99% reduction from the farm environment over five hours. Unlike the current formaldehyde spray disinfection protocol, which requires a long empty period because of its hazardous properties, this economically viable and environmentally friendly disinfection protocol may significantly lower downtime. Moreover, ECAS fogging can be easily adapted to a variety of applications, including the elimination of pathogens from livestock farm air environment for disease prevention, as well as decontamination after disease outbreaks

Characterisation of methicillin-resistant and methicillin-susceptible isolates of Staphylococcus pseudintermedius from cases of canine pyoderma in Australia

Meticillin-resistant Staphylococcus pseudintermedius (MRSP) has recently emerged as a worldwide cause of canine pyoderma. In this study, we characterized 22 S. pseudintermedius isolates cultured from 19 dogs with pyoderma that attended a veterinary dermatology referral clinic in Australia in 2011 and 2012. Twelve isolates were identified as MRSP by mecA real-time PCR and phenotypic resistance to oxacillin. In addition to β-lactam resistance, MRSP isolates were resistant to erythromycin (91.6 %), gentamicin (83.3 %), ciprofloxacin (83.3 %), chloramphenicol (75 %), clindamycin (66 %), oxytetracycline (66 %) and tetracycline (50 %), as shown by disc-diffusion susceptibility testing. Meticillin-susceptible S. pseudintermedius isolates only showed resistance to penicillin/ampicillin (90 %) and tetracycline (10 %). PFGE using the SmaI restriction enzyme was unable to type nine of the 12 MRSP isolates. However the nine isolates provided the same PFGE pulsotype using the Cfr91 restriction enzyme. Application of the mec-associated direct repeat unit (dru) typing method identified the nine SmaI PFGE-untypable isolates as dt11cb, a dru type that has only previously been associated with MRSP sequence type (ST)45 isolates that possess a unique SCCmec element. The dt11cb isolates shared a similar multidrug-resistant antibiogram phenotype profile, whereas the other MRSP isolates, dt11a, dt11af (dt11a-associated) and dt10h, were resistant to fewer antibiotic classes and had distinct PFGE profiles. This is the first report of MRSP causing pyoderma in dogs from Australia. The rapid intercontinental emergence and spread of multidrug-resistant MRSP strains confirms the urgent need for new treatment modalities for recurrent canine pyoderma in veterinary practice

Bioluminescent murine models of bacterial sepsis and scald wound infections for antimicrobial efficacy testing.

There are very few articles in the literature describing continuous models of bacterial infections that mimic disease pathogenesis in humans and animals without using separate cohorts of animals at each stage of disease. In this work, we developed bioluminescent mouse models of partial-thickness scald wound infection and sepsis that mimic disease pathogenesis in humans and animals using a recombinant luciferase-expressing Staphylococcus aureus strain (Xen29). Two days post-scald wound infection, mice were treated twice daily with a 2% topical mupirocin ointment for 7 days. For sepsis experiments, mice were treated intraperitoneally with 6 mg/kg daptomycin 2 h and 6 h post-infection and time to moribund monitored for 72 h. Consistent bacterial burden data were obtained from individual mice by regular photon intensity quantification on a Xenogen IVIS Lumina XRMS Series III biophotonic imaging system, with concomitant significant reduction in photon intensities in drug-treated mice. Post-mortem histopathological examination of wounds and bacterial counts in blood correlated closely with disease severity and total flux obtained from Xen29. The bioluminescent murine models provide a refinement to existing techniques of multiple bacterial enumeration during disease pathogenesis and promote animal usage reduction. The models also provide an efficient and information-rich platform for preclinical efficacy evaluation of new drug classes for treating acute and chronic human and animal bacterial infections