Hui San Allison

The increasing resistance of bacteria to antimicrobials is a major threat to public health. This study investigates the prevalence of antimicrobial resistance, both phenotypic and genotypic, among Campylobacter isolates from Australian meat chickens in 2022, as a follow up to investigate trends since the last national surveillance undertaken in 2016. Isolates (n = 186) were obtained at slaughter from 200 pooled cecal samples taken from 1,000 meat chickens. The majority of C. jejuni (68.7%) and C. coli (88.9%) isolates were susceptible to all the antibiotics that were tested, and no multi-drug resistance was found. Resistance to ciprofloxacin (fluoroquinolone) was detected in 24.4% of the C. jejuni and 3.2% of the C. coli isolates. Whole genome sequencing revealed a diverse range of sequence types (STs). These included 32 previously reported STs for C. jejuni and 13 for C. coli, as well as four and seven previously undescribed STs for each species, respectively. The STs containing fluoroquinolone-resistant isolates were ST2083, ST10130, ST2895, ST7323, ST2398, and ST1078 for C. jejuni, and ST860 and ST894 for C. coli. Although fluoroquinolones are not used in animal production in Australia, resistance amongst C. jejuni isolates was high (24.4%). This finding emphasizes the need for enhanced surveillance and regular sampling along the food chain to understand the source of the isolates and to mitigate risks of antimicrobial resistance to protect public health.

Controlling the use of the most critically important antimicrobials (CIAs) in food animals has been identified as one of the key measures required to curb the transmission of antimicrobial resistant bacteria from animals to humans. Expanding the evidence demonstrating the effectiveness of restricting CIA usage for preventing the emergence of resistance to key drugs amongst commensal organisms in animal production would do much to strengthen international efforts to control antimicrobial resistance (AMR). As Australia has strict controls on antimicrobial use in layer hens, and internationally comparatively low levels of poultry disease due to strict national biosecurity measures, we investigated whether these circumstances have resulted in curtailing development of critical forms of AMR. The work comprised a cross-sectional national survey of 62 commercial layer farms with each assessed for AMR in Escherichia coli isolates recovered from faeces. Minimum inhibitory concentration analysis using a panel of 13 antimicrobials was performed on 296 isolates, with those exhibiting phenotypic resistance to fluoroquinolones (a CIA) or multi-class drug resistance (MCR) subjected to whole genome sequencing. Overall, 53.0% of isolates were susceptible to all antimicrobials tested, and all isolates were susceptible to cefoxitin, ceftiofur, ceftriaxone, chloramphenicol and colistin. Resistance was observed for amoxicillin-clavulanate (9.1%), ampicillin (16.2%), ciprofloxacin (2.7%), florfenicol (2.4%), gentamicin (1.0%), streptomycin (4.7%), tetracycline (37.8%) and trimethoprim/sulfamethoxazole (9.5%). MCR was observed in 21 isolates (7.0%), with two isolates exhibiting resistance to four antimicrobial classes. Whole genome sequencing revealed that ciprofloxacin-resistant (fluoroquinolone) isolates were devoid of both known chromosomal mutations in the quinolone resistance determinant regions and plasmid-mediated quinolone resistance genes (qnr)—other than in one isolate (ST155) which carried the qnrS gene. Two MCR E. coli isolates with ciprofloxacin-resistance were found to be carrying known resistance genes including aadA1, dfrA1, strA, strB, sul1, sul2, tet(A), blaTEM-1B, qnrS1 and tet(A). Overall, this study found that E. coli from layer hens in Australia have low rates of AMR, likely due to strict control on antimicrobial usage achieved by the sum of regulation and voluntary measures.

This study aimed to determine the frequency and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) from Australian animals and whether animal-derived MRSA was similar to that from Australian veterinarians. A total of 1,080 clinical coagulase positive Staphylococcus isolates from Australian animals were collected during 2013. Sixteen (4%) of 360 S. aureus isolates were MRSA. Most MRSA came from companion animals, while none came from livestock. MRSA isolates were characterized using whole genome sequencing. ST22-IV (EMRSA-15) was the most common clone in dogs and cats. Clonal complex (CC) 8 was most common in horses. Most ST22-IV isolates were resistant to ciprofloxacin. Animal-derived MRSA genomes were interrogated for the presence of host-specific genetic markers (staphylokinase gene [scn], chemotaxis-inhibiting proteins gene [chp], staphylococcal complement inhibitor gene [sak], enterotoxin A gene [sea], and Von Willebrand Factor binding protein gene [vwb]). A subset of MRSA genomes previously collected from Australian veterinarians was also interrogated. There was no clear pattern in the distribution of host-specific markers among animal and veterinarian isolates. Animal- and veterinarian-derived MRSA were intermingled in the phylogenetic tree. The absence of MRSA in Australian livestock is in stark contrast with its presence in livestock from other countries. Possible explanations include Australia's geographic isolation, the absence of live animal importation into Australia, and most notably, the restrictions placed on the use of antimicrobials of critical importance in Australian livestock.

Increasing reports of multidrug-resistant bacterial infections in animals has created a need for novel antimicrobial agents that do not promote cross-resistance to critically important antimicrobial classes used in human medicine. In response to the recent emergence of antimicrobial resistance in several bovine mastitis pathogens, in vitro antimicrobial susceptibility was determined for four polyether ionophores (lasalocid, monensin, narasin and salinomycin) against Staphylococcus spp. and Streptococcus spp. isolated from clinical cases. In addition, erythrocyte haemolysis and WST-1 cell proliferation assays were used to assess in vitro mammalian cell cytotoxicity and biofilm susceptibility testing was performed using the minimum biofilm eradication concentration (MBEC) biofilm assay. Lasalocid, monensin, narasin and salinomycin exhibited bacteriostatic antimicrobial activity against all pathogens tested, including methicillin-resistant staphylococci, with MIC90 values <16g/ml. Narasin and monensin displayed the least toxicity against mammalian cell lines and all compounds significantly reduced viable cell numbers in a Staphylococcus aureus biofilm. Based on in vitro characterization, all four ionophores offer potentially novel treatments against bovine mastitis but in vivo studies will be essential to determine whether acceptable safety and efficacy is present following intramammary administration.

Methicillin-resistant coagulase-positive staphylococci (CoPS) have become increasingly recognised as opportunistic pathogens that limit therapeutic options in companion animals. The frequency of methicillin resistance amongst clinical isolates on an Australia-wide level is unknown. This study determined antimicrobial susceptibility patterns for CoPS isolated from clinical infections in companion animals (dogs, cats and horses) as part of the first nation-wide survey on antimicrobial resistance in animal pathogens in Australia for a oneyear period (January 2013 to January 2014). Clinical Staphylococcus spp. isolates (n = 888) obtained from 22 veterinary diagnostic laboratories were identified by MALDI-TOF mass spectrometry and subjected to antimicrobial susceptibility testing for 16 antimicrobials, representing 12 antimicrobial classes. Potential risk factors associated with methicillin resistance in Staphylococcus pseudintermedius isolates from dogs were analysed based on demographic factors and clinical history, including gender, age, previous antimicrobial treatment, chronic and/or recurrent diseases and site of infections. The most commonly identified CoPS were S. pseudintermedius (70.8%; dogs n = 616, cats n = 13) and S. aureus (13.2%, horses n = 53, dogs n = 47 and cats n = 17). Overall, the frequency of methicillin resistance among S. pseudintermedius (MRSP) and S. aureus (MRSA) was 11.8% and 12.8%, respectively. MRSP isolates were strongly associated with resistance to fluoroquinolones (OR 287; 95%CI 91.2±1144.8) and clindamycin (OR 105.2, 95%CI 48.5±231.9). MRSA isolates from dogs and cats were also more likely to be resistant to fluoroquinolones (OR 5.4, 95%CI 0.6±252.1), whereas MRSA from horses were more likely to be resistant to rifampicin. In multivariate analysis, MRSP-positive status was significantly associated with particular infection sites, including surgical (OR 8.8; 95%CI 3.74±20.7), and skin and soft tissue (OR 3.9; 95%CI 1.97±7.51). S. pseudintermedius isolated from dogs with surgical site infections were three times more likely to be methicillin-resistant if cases had received prior antimicrobial treatment. Whilst the survey results indicate the proportion of CoPS obtained from Australian companion animals that are methicillin-resistant is currently moderate, the identified risk factors suggest that it could rapidly increase without adequate biosecurity and infection control procedures in veterinary practice.

Multidrug-resistant (MDR) Escherichia coli have become a major public health concern to both humans and animal health. While the frequency of antimicrobial resistance (AMR) in clinical E. coli is monitored regularly in human medicine, current frequency of AMR in companion animals remains unknown in Australia. In this study we conducted antimicrobial susceptibility testing (AST) and where possible, determined potential risk factors for MDR infection among 883 clinical Escherichia coli isolated from dogs (n = 514), cats (n = 341) and horses (n = 28). AST was undertaken for 15 antimicrobial agents according to the Clinical Laboratory Standards Institute (CLSI) guidelines and interpreted using epidemiological cut-off values (ECOFFs) as well as CLSI veterinary and human clinical breakpoints. The AST revealed complete absence of resistance to carbapenems while resistance to amikacin was observed at a low level in isolates from dogs (1.6%) and cats (1.5%) compared to horses (10.7%). Among dog isolates, resistance to fluoroquinolones ranged from 9.1%–9.3% whereas among cat isolates, it ranged from 3.2%–5%. Among dog isolates, the proportion showing a 3rd generation cephalosporin (3GC) non-wild type phenotype was significantly higher (P < 0.05) in skin and soft tissue infection (SSTI, n = 122) isolates (17.2%–20.5%) compared to urinary tract infection (UTI, n = 392) isolates (9.9%–10.2%). The frequency of multidrug resistance was 18.1%, 11.7% and 42.9% in dog, cat and horse isolates, respectively. Risk factor analysis revealed that MDR E. coli isolated from UTI were positively associated with chronicity of infection and previous antimicrobial treatment. Dogs and cats with chronic UTI that had been previously treated with antimicrobials were eight times and six times more likely to be infected with MDR E. coli compared to dogs and cats with non-chronic UTI, and no history of antimicrobial treatment, respectively. This study revealed that pre-existing disease condition and prior antimicrobial use were the major risks associated with UTI with MDR E. coli in companion animals.

Robenidine, 1 (2,2′-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC’s of 8.1 and 4.7 μM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), and 4-C(CH3)3 (27) (23.7–71 μM) and with 3-Cl (3), 4-CH3 (21), and 4-CH(CH3)2 (26) (8.1–13.0 μM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH2OH moiety (75; 2,2′-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2′-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2′-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC’s of 4.2–21.6 μM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T1/2 > 247 min, CLint < 7 μL/min/mg protein, and EH < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75.

This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n = 324) from Australian food-producing animals [cattle (n = 169), porcine (n = 114), poultry (n = 32) and sheep (n = 9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0-88.6%), ampicillin (9.4-71.1%), trimethoprim/sulfamethoxazole (11.1-67.5%) and streptomycin (21.9-69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of blaCMY-2. This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages.

Background
Anecdotal reports suggest that rapid staining solutions can become contaminated with micro-organisms, especially Pseudomonas aeruginosa.

Hypothesis/Objectives
To determine whether inoculation of rapid Romanowsky-type stains with P. aeruginosa results in viable bacterial contamination, which could lead to cross-contamination of slides during cytological staining.

Methods
Pseudomonas aeruginosa was inoculated into clean and organically contaminated staining solutions (fixative, eosin and methylene blue) and positive (broth) and negative (bleach) control solutions. Subsequent viability and survival were detected by measuring colony-forming units per millilitre at various time points up to 2 weeks. Each sample was stained and microscopically examined to determine whether bacteria were visible.

Results
No bacteria could be cultured at any time point from the bleach or fixative solution. In clean eosin and methylene blue staining solutions, viable bacteria were recovered for up to 1 h, but by 24 h all bacteria were dead. In staining solutions contaminated with hair and dead skin cells, bacteria survived in methylene blue for up to 1 h, and viable bacteria persisted in the eosin stain for 2 weeks. In solutions containing viable organisms, the bacteria could be observed by microscopic examination; no bacteria were visible when the solutions contained no viable organisms.

Conclusions and clinical importance
Pseudomonas aeruginosa can survive in commonly used staining solutions for variable periods of time, but is unable to proliferate. Although theoretically this might complicate cytological interpretation and subsequent diagnosis, the likelihood of this in clinical practice appears remote when the correct staining technique is used.