A review - intestinal spirochaetal infections of pigs: An overview with an Australian perspective

Intestinal spirochaetes have become recognized over the last 25 years as an important group of enteric pathogens. These bacteria cause disease in a variety of animal species, especially pigs, poultry, dogs and human beings (Hampson and Stanton, 1996). In pigs, the bacteria cause two well-recognized conditions, swine dysentery (SD), and intestinal spirochaetosis (IS) (Taylor et al., 1980; Hampson, 1991). A third condition, referred to here as spirochaetal colitis (SC), is less clearly defined, but is associated with certain weakly beta-haemolytic spirochaetes other than those causing IS. Swine dysentery is one of the most significant production-limiting diseases of pigs and is a common problem throughout the world. The significance of IS and SC in reducing production is less clear; certainly, clinical manifestations of the conditions are much less severe than with SD. The prevalence of the diseases is not known, but the authors' observations suggest that IS occurs commonly in pigs in Australia and North America, whilst cases of IS and SC also have been reported in Europe (Taylor, 1992)

Genetic and antigenic studies on Haemophilus parasuis

Haemophilus parasuis, an organism dependent upon nicotinamide adenine dinucleotide (NAD) or V-factor for in-vitro growth, is the causative agent of porcine polyserositis and arthritis (Glasser's disease) (Nicolet, 1992). The principal lesions associated with this disease are fibrinous or serofibrinous meningitis, serositis, pleuritis, pericarditis, peritonitis and arthritis that can occur in various combinations or occasionally singly (Nicolet, 1992)

Qac genes and biocide tolerance in clinical veterinary methicillin-resistant and methicillin-susceptible Staphylococcus aureus and Staphylococcus pseudintermedius

Qac genes are associated with increased tolerance to quaternary ammonium compounds and other cationic biocides such as chlorhexidine. This study aimed to determine whether qac genes and increased biocide tolerance were present in 125 clinical methicillin-resistant and susceptible veterinary staphylococci. A total of 125 methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant and -susceptible Staphylococcus pseudintermedius (MRSP and MSSP) from three archived Australian veterinary staphylococci collections underwent whole genome sequencing, multilocus sequence typing and qac gene screening. Two MRSA isolates (12%) harboured qacA/B genes; both isolates were ST8 from horses. QacJ, qacG and smr genes were identified in 28/90 (31%) MRSP and 1/18 (6%) MSSP isolates. ST71 MRSP was significantly more likely to harbour qac genes than other MRSP clones (p < 0.05). A random subset of 31 isolates underwent minimum bactericidal concentration (MBC) testing against F10SCTM (benzalkonium chloride and biguanide), and HexaconTM (chlorhexidine gluconate), with and without the addition of bovine serum albumin (BSA) as an in vitro substitute for organic matter contamination. Qac genes were not associated with increased phenotypic biocide tolerance but biocide efficacy was significantly affected by the presence of BSA. In the absence of BSA, all MBC values were well below the recommended usage concentration. When BSA was present, regardless of qac gene presence, 50% of MRSA and 43% of MRSP had an F10SCTM MBC above the recommended concentration for general disinfection. Qac genes did not confer increased in vitro biocide tolerance to veterinary staphylococci. Organic matter contamination must be minimized to ensure the efficacy of biocides against MRSA and MRSP

Relative performance of antimicrobial susceptibility assays on clinical Escherichia coli isolates from animals

The assessment of antimicrobial resistance in bacteria derived from animals is often performed using the disc diffusion assay. However broth-microdilution is the preferred assay for national antimicrobial resistance surveillance programs. This study aimed to evaluate the accuracy of disc diffusion relative to broth-microdilution across a panel of 12 antimicrobials using data from a collection of 994 clinical Escherichia coli isolates from animals. Disc diffusion performance was evaluated by diagnostic sensitivity, specificity, likelihood ratio pairs and receive-operating characteristic (ROC) analysis. Data was dichotomised using CLSI susceptible and resistant clinical breakpoints. In addition, disc diffusion breakpoints produced using diffusion Breakpoint Estimation Testing Software (dBETS) were evaluated. Analysis revealed considerable variability in performance estimates for disc diffusion susceptible and resistant breakpoints (AUC ranges: 0.78–0.99 and 0.92–1.0, respectively) across the panel of antimicrobials. Ciprofloxacin, tetracycline, and ampicillin estimates were robust across both breakpoints, whereas estimates for several antimicrobials including amoxicillin-clavulanic acid, cefoxitin and gentamicin were less favourable using susceptible breakpoints. Overall performance estimates were moderately improved when dBETS susceptible breakpoints were applied. For most antimicrobials, disc diffusion was accurate at predicting resistance of clinical E. coli from animals that could otherwise be determined by broth-microdilution. While disc diffusion is suboptimal for assessing the proportion of fully susceptible isolates for some drugs, sensitivity and specificity estimates provided here allow for the use of standard formula to correct this. For this reason, disc diffusion has applicability in national surveillance provided the performance of the assay is taken into account

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

Spray and aerosolised pH-neutral electrochemically activated solution reduces Salmonella Enteritidis and total bacterial load on egg surface

Published: 13 January 2021The effectiveness of sprayed and aerosolised pH-neutral electrochemically activated solutions (ECAS) containing 150 mg/L of free available chlorine in reducing total bacteria load and artificially inoculated Salmonella enterica serotype Enteritidis 11RX on eggs surfaces was investigated. Treatment groups included untreated control, sodium hypochlorite (positive control), sprayed and aerosolised water and sprayed and aerosolised ECAS. Sprayed ECAS (150 mg/L, 45 s) showed a significant reduction in total bacterial load (2.2 log reduction, p < 0.0001) and S. Enteritidis (5.4 log reduction, p < 0.0001) when compared with the untreated control. Aerosolised ECAS (120 s) was effective in reducing both the total bacterial load (1.4 log reduction, p < 0.01) and S. Enteritidis (4.2 log reduction, p = 0.0022). However, aerosolised ECAS (60 s) only significantly reduced S. Enteritidis counts (2.8 log reduction, p < 0.0008), indicating that a longer time for bacterial reduction during fogging sanitisation is needed. Tests performed with one egg per oscillating tray were more effective in reducing both the total bacterial load and the S. Enteritidis counts than those with three eggs per oscillating tray. Sprayed ECAS (45 s) and aerosolised ECAS (120 s) did not deteriorate the egg cuticle integrity (DEab *), which was evaluated using Cuticle Blue dye solution and colour intensity measurement. Overall, both the reduction in total bacteria counts and S. Enteritidis from the egg surface and retention of cuticle integrity suggest that sprayed and aerosolised ECAS could be used as alternative sanitising approaches to improve the food safety aspect of table eggs.Sangay Tenzin 1, Sergio Ferro 2 , Samiullah Khan 3 , Permal Deo 4,* and Darren J. Trot

Companion animals are spillover hosts of the Multidrug-resistant human extraintestinal escherichia coli pandemic Clones ST131 and ST1193

Escherichia coli sequence types 131 (ST131) and 1193 are multidrug-resistant extraintestinal pathogens that have recently spread epidemically among humans and are occasionally isolated from companion animals. This study characterized a nationwide collection of fluoroquinolone-resistant (FQR) E. coli isolates from extraintestinal infections in Australian cats and dogs. For this, 59 cat and dog FQR clinical E. coli isolates (representing 6.9% of an 855-isolate collection) underwent PCR-based phylotyping and whole-genome sequencing (WGS). Isolates from commensal-associated phylogenetic groups A (14/59, 24%) and B1 (18/59, 31%) were dominant, with ST224 (10/59, 17%), and ST744 (8/59, 14%) predominating. Less prevalent were phylogenetic groups D (12/59, 20%), with ST38 (8/59, 14%) predominating, and virulence-associated phylogenetic group B2 (7/59, 12%), with ST131 predominating (6/7, 86%) and no ST1193 isolates identified. In a WGS-based comparison of 20 cat and dog-source ST131 isolates with 188 reference human and animal ST131 isolates, the cat and dog-source isolates were phylogenetically diverse. Although cat and dog-source ST131 isolates exhibited some minor sub-clustering, most were closely related to human-source ST131 strains. Furthermore, the prevalence of ST131 as a cause of FQR infections in Australian companion animals was relatively constant between this study and the 5-year-earlier study of Platell et al. (2010) (9/125 isolates, 7.2%). Thus, although the high degree of clonal commonality among FQR clinical isolates from humans vs. companion animals suggests the possibility of bi-directional between-species transmission, the much higher reported prevalence of ST131 and ST1193 among FQR clinical isolates from humans as compared to companion animals suggests that companion animals are spillover hosts rather than being a primary reservoir for these lineages

Characterization of staphylococcal cassette chromosome mec elements from methicillin-resistant Staphylococcus pseudintermedius infections in Australian animals

We examined the oxacillin resistance phenotype and genomic structure of staphylococcal cassette chromosome mec (SCCmec) elements from 77 veterinary methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates. Isolates were characterized by oxacillin broth microdilution, whole-genome sequencing, and bioformatics analysis. Five previously described SCCmec elements, and a sixth novel element, were identified: SCCmec III (also known as II-III), ΨSCCmec57395, and SCCmecNA45 (a SCCmec VII variant), all previously described in MRSP, and SCCmec IVg and SCCmec VT, previously described in both methicillin-resistant Staphylococcus aureus (MRSA) and MRSP. The sixth element was novel and found among nine geographically clustered isolates. This novel pseudostaphylococcal cassette chromosome (ΨSCCmecKW21) contained a class A mec gene complex but lacked ccr genes. It also harbored heavy metal (cadmium) resistance determinants. The median oxacillin MIC values among ΨSCCmecKW21, SCCmec III, and SCCmec VT isolates were significantly higher than those determined for the SCCmecNA45 VII variant isolates and ΨSCCmec57395 and SCCmec IVg isolates. ΨSCCmecKW21 was found exclusively in sequence type 497 (ST497), an MRSP clone that is locally successful in Victoria, Australia. Future studies are necessary to determine if this clone has disseminated further afield and if ΨSCCmecKW21 has moved into other MRSP lineages or staphylococcal species. IMPORTANCE Staphylococcus pseudintermedius is a significant veterinary pathogen and occasional cause of infections in humans. β-Lactams are an important group of antimicrobials used to treat staphylococcal infections in humans and animals. However, when staphylococci become methicillin resistant via the acquisition of a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec), they become resistant to all β-lactams. This study detected a novel SCCmec element among a cluster of methicillin-resistant S. pseudintermedius isolates from animals in Australia. It also detected SCCmec elements in S. pseudintermedius that had high similarity to those identified in methicillin-resistant Staphylococcus aureus, demonstrating how human and animal pathogens can share the same resistance determinants

Robotic Antimicrobial Susceptibility Platform (RASP): A next-generation approach to One Health surveillance of antimicrobial resistance

Background Surveillance of antimicrobial resistance (AMR) is critical to reducing its wide-reaching impact. Its reliance on sample size invites solutions to longstanding constraints regarding scalability. A robotic platform (RASP) was developed for high-throughput AMR surveillance in accordance with internationally recognized standards (CLSI and ISO 20776-1:2019) and validated through a series of experiments. Methods Experiment A compared RASP’s ability to achieve consistent MICs with that of a human technician across eight replicates for four Escherichia coli isolates. Experiment B assessed RASP’s agreement with human-performed MICs across 91 E. coli isolates with a diverse range of AMR profiles. Additionally, to demonstrate its real-world applicability, the RASP workflow was then applied to five faecal samples where a minimum of 47 E. coli per animal (239 total) were evaluated using an AMR indexing framework. Results For each drug–rater–isolate combination in Experiment A, there was a clear consensus of the MIC and deviation from the consensus remained within one doubling dilution (the exception being gentamicin at two dilutions). Experiment B revealed a concordance correlation coefficient of 0.9670 (95% CI: 0.9670–0.9670) between the robot- and human-performed MICs. RASP’s application to the five faecal samples highlighted the intra-animal diversity of gut commensal E. coli, identifying between five and nine unique isolate AMR phenotypes per sample. Conclusions While adhering to internationally accepted guidelines, RASP was superior in throughput, cost and data resolution when compared with an experienced human technician. Integration of robotics platforms in the microbiology laboratory is a necessary advancement for future One Health AMR endeavours

Dominance of Escherichia coli sequence types ST73, ST95, ST127 and ST131 in Australian urine isolates: a genomic analysis of antimicrobial resistance and virulence linked to F plasmids

Extraintestinal pathogenic Escherichia coli (ExPEC) are the most frequent cause of urinary tract infections (UTIs) globally. Most studies of clinical E. coli isolates are selected based on their antimicrobial resistance (AMR) phenotypes; however, this selection bias may not provide an accurate portrayal of which sequence types (STs) cause the most disease. Here, whole genome sequencing (WGS) was performed on 320 E. coli isolates from urine samples sourced from a regional hospital in Australia in 2006. Most isolates (91%) were sourced from patients with UTIs and were not selected based on any AMR phenotypes. No significant differences were observed in AMR and virulence genes profiles across age sex, and uro-clinical syndromes. While 88 STs were identified, ST73, ST95, ST127 and ST131 dominated. F virulence plasmids carrying senB-cjrABC (126/231; 55%) virulence genes were a feature of this collection. These senB-cjrABC+ plasmids were split into two categories: pUTI89-like (F29:A- :B10 and/or >95% identity to pUTI89) (n=73) and non-pUTI89-like (n=53). Compared to all other plasmid replicons, isolates with pUTI89-like plasmids carried fewer antibiotic resistance genes (ARGs), whilst isolates with senB-cjrABC+/non-pUTI89 plasmids had a significantly higher load of ARGs and class 1 integrons. F plasmids were not detected in 89 genomes, predominantly ST73. Our phylogenomic analyses identified closely related isolates from the same patient associated with different pathologies and evidence of strain-sharing events involving isolates sourced from companion and wild animals.Dmitriy Li, Paarthiphan Elankumaran, Timothy Kudinha, Amanda K. Kidsley, Darren J. Trott, Veronica Maria Jarocki, and Steven Philip Djordjevi