Antimicrobial susceptibility, plasmid replicon typing, phylogenetic grouping, and virulence potential of avian pathogenic and faecal Escherichia coli isolated from meat chickens in Australia.

Globally, avian colibacillosis is a leading cause of morbidity and mortality in poultry, associated with economic losses and welfare problems. Here, clinical avian pathogenic E. coli isolates (CEC; n = 50) and faecal E. coli isolates from healthy (FEC; n = 187) Australian meat chickens collected between 2006 and 2014 were subjected to antimicrobial susceptibility testing, phylogenetic grouping, plasmid replicon (PR) typing, multilocus sequence typing, and virulence gene (VG) profiling. Extended-spectrum cephalosporin (ESC)- and fluoroquinolone (FQ)-resistant E. coli isolates underwent further genetic characterization. Significant proportions of CEC and FEC were, respectively, susceptible (13/50; 48/187) or MDR (9/50; 26/187) to 20 tested antimicrobials. Phylogenetic groups A and C, and PR types IncFIB and IncFrep were most represented. Five tested CEC-associated VGs were more prevalent in CEC (≥ 90%) than FEC (≤ 58%). Some isolates (CEC n = 3; FEC n = 7) were resistant to ESCs and/or FQs and possessed signature mutations in chromosomal FQ target genes and plasmid-mediated qnrS, blaCMY-2, and blaDHA-1 genes. Sequence type 354 (n = 4), associated with extraintestinal infections in a broad range of hosts, was prevalent among ESC- and/or FQ-resistant FEC. This study confirmed existence of a small reservoir of ESC- and FQ-resistant E. coli in Australian commercial meat chickens despite absence of use in the industry of these drugs. Otherwise, diversity of VGs and PR types in both FEC and CEC populations was identified. We hypothesize that the source of ESC- and FQ-resistant E. coli is external to poultry production facilities.RESEARCH HIGHLIGHTSLow-level resistance to older and newer generation antimicrobial drugs detected.The most common sequence type (ST) associated with FQ resistance was ST354 (4/10).A small proportion of CEC (n = 3) and FEC (n = 7) were resistant to ESCs and/or FQs.

Inhibitors of second messenger pathways and Ca(2+)-induced exposure of phosphatidylserine in red blood cells of patients with sickle cell disease.

The present work investigates the contribution of various second messenger systems to Ca(2+)-induced phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients. The Ca(2+) dependence of PS exposure was confirmed using the Ca(2+) ionophore bromo-A23187 to clamp intracellular Ca(2+) over 4 orders of magnitude in high or low potassium-containing (HK or LK) saline. The percentage of RBCs showing PS exposure was significantly increased in LK over HK saline. This effect was reduced by the Gardos channel inhibitors, clotrimazole and charybdotoxin. Nevertheless, although Ca(2+) loading in the presence of an outwardly directed electrochemical gradient for K(+) stimulated PS exposure, substantial exposure still occurred in HK saline. Under the conditions used inhibitors of other second messenger systems (ABT491, quinacrine, acetylsalicylic acid, 3,4-dichloroisocoumarin, GW4869 and zVAD-fmk) did not inhibit the relationship between [Ca(2+)] and PS exposure. Inhibitors of phospholipase A2, cyclooxygenase, platelet-activating factor, sphingomyelinase and caspases, therefore, were without effect on Ca(2+)-induced PS exposure in RBCs, incubated in either HK or LK saline.

Effects of o-vanillin on K⁺ transport of red blood cells from patients with sickle cell disease.

Aromatic aldehydes like o-vanillin were designed to reduce the complications of sickle cell disease (SCD) by interaction with HbS, to reduce polymerisation and RBC sickling. Present results show that o-vanillin also directly affects RBC membrane permeability. Both the K(+)-Cl(-) cotransporter (KCC) and the Ca(2+)-activated K(+) channel (or Gardos channel) were inhibited with IC50 of about 0.3 and 1 mM, respectively, with activities almost completely abolished by 5 mM. Similar effects were observed in RBCs treated with the thiol reacting reagent N-ethylmaleimide or with the Ca(2+) ionophore A23187, to circumvent any action via HbS polymerisation. The deoxygenation-induced cation conductance (sometimes termed P(sickle)) was partially inhibited, whilst deoxygenation-induced exposure of phosphatidylserine was completely abrogated. Na(+)/K(+) pump activity was also reduced. Notwithstanding, o-vanillin stimulated K(+) efflux through an unidentified pathway and resulted in reduction in cell volume (as measured by wet weight-dry weight). These actions are relevant to understanding how aromatic aldehydes may affect RBC membrane permeability per se as well as HbS polymerisation and thereby inform design of compounds most efficacious in ameliorating the complications of SCD.

Antimicrobial susceptibility patterns of aerobic bacteria isolated from canine urinary samples in South East Queensland, 2013 to 2018.

Urinary tract infections are a common diagnosis in dogs presenting to veterinary practice. Veterinarians often treat suspected infections empirically, either in the absence of culture and susceptibility testing results or whilst waiting for them. This study aimed to identify the bacteria most frequently isolated from canine urinary samples and their antimicrobial susceptibility patterns in South East Queensland (SEQ) to help guide responsible empirical antimicrobial prescription by the veterinary community in this geographical location. Cumulative antibiograms were generated from the results of 1284 culture-positive urinary samples in SEQ, obtained from a commercial veterinary laboratory over a 5-year period. Escherichia coli was the most commonly isolated bacterial species (43%), followed by Staphylococcus spp. (23%), Proteus spp. (21%) and Enterococcus spp. (10%). Of the six most common isolates, 97% had susceptibility to at least one low-importance antimicrobial. Susceptibility to the low-importance and first-line antimicrobial recommendation, amoxicillin, was 81% for E. coli and 24% for Staphylococcus spp. Susceptibility of both E. coli and Staphylococcus spp. to medium-importance and commonly recommended empirical antimicrobials, trimethoprim sulphonamides and amoxicillin-clavulanic acid was ≥85% and >92% for high-importance antimicrobials enrofloxacin and ceftiofur. Of the E. coli and Staphylococcus spp. isolates, 8.8% and 4%, respectively, were considered multidrug resistant. There was no increase in resistance to antimicrobials detected over the study period. Susceptibilities suggest low- and medium-importance antimicrobials remain acceptable first-line empirical treatments. However, this should be continually assessed and updated using local surveillance data.

Understanding barriers to reducing antimicrobials on Australian dairy farms: A qualitative analysis.

INTRODUCTION: Reducing antibiotic use in production animal systems is one strategy which may help to limit the development of antimicrobial resistance. To reduce antimicrobial use in food-producing animals, it is important to first understand how antibiotics are used on farm and what barriers exist to decreasing their use. In dairy production systems, mastitis is one of the most common reasons for administering antimicrobials. Therefore, it is important to understand the motivations and behaviours of dairy farmers in relation to the diagnosis, treatment and prevention of mastitis. MATERIALS AND METHODS: In this study, we interviewed a sample of dairy farmers and dairy industry professionals from the major dairying regions of eastern Australia regarding their current practices used to diagnose, treat, and control subclinical and clinical mastitis. Inductive thematic analysis was used to code interview transcripts and identify the recurrent themes. RESULTS: Four overarching themes were identified: (1) the challenges associated with the detection and diagnosis of clinical mastitis, including with laboratory culture, (2) the motivations behind treatment decisions for different cases, (3) decisions around dry cow therapy and the role of herd recording, and (4) concerns regarding the development of antimicrobial resistance. DISCUSSION: This study identifies several challenges which may limit the ability of Australian dairy farmers to reduce antimicrobial use on farm, such as the need for rapid and reliable diagnostic tests capable of identifying the pathogenic causes of mastitis and the difficulties associated with conducting herd recording for the implementation of selective dry cow therapy. Industry professionals were concerned that farmers were not using individual cow records to aid in treatment decisions, which could result in unnecessary antimicrobial use. The results of this study can act as the basis for future research aimed at assessing these issues across the broader Australian dairy industry.

A non-electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease.

Abstract Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

Identification of the Ca²âº entry pathway involved in deoxygenation-induced phosphatidylserine exposure in red blood cells from patients with sickle cell disease.

Phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients is increased compared to levels in normal individuals and may participate in the anaemic and ischaemic complications of SCD. Exposure is increased by deoxygenation and occurs with elevation of intracellular Ca²âº to low micromolar levels. The Ca²âº entry step has not been defined but a role for the deoxygenation-induced pathway, Psickle, is postulated. Partial Psickle inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), 4,4'-dithiocyano-2,2'-stilbene-disulphonic acid (DIDS) and dipyridamole inhibited deoxygenation-induced PS exposure (DIDS IC50, 118 nM). Inhibitors and activators of other pathways (including these stimulated by depolarisation, benzodiazepines, glutamate and stretch) were without effect. Zn²âº and Gd³âº stimulated PS exposure to high levels. In the case of Zn²âº, this effect was independent of oxygen (and hence HbS polymerisation and RBC sickling) but required extracellular Ca²âº. The effect was completely abolished when Zn²âº (100 µM) was added to RBCs suspended in autologous plasma, implying a requirement of high levels of free Zn²âº.

The conductance of red blood cells from sickle cell patients: ion selectivity and inhibitors.

The abnormally high cation permeability in red blood cells (RBCs) from patients with sickle cell disease (SCD) occupies a central role in pathogenesis. Sickle RBC properties are notably heterogeneous, however, thus limiting conventional flux techniques that necessarily average out the behaviour of millions of cells. Here we use the whole-cell patch configuration to characterise the permeability of single RBCs from patients with SCD in more detail. A non-specific cation conductance was reversibly induced upon deoxygenation and was permeable to both univalent (Na+, K+, Rb+) and also divalent (Ca2+, Mg2+) cations. It was sensitive to the tarantula spider toxin GsMTx-4. Mn2+ caused partial, reversible inhibition. The aromatic aldehyde o-vanillin also irreversibly inhibited the deoxygenation-induced conductance, partially at 1mM and almost completely at 5mM. Nifedipine, amiloride and ethylisopropylamiloride were ineffective. In oxygenated RBCs, the current was pH sensitive showing a marked increase as pH fell from 7.4 to 6, with no change apparent when pH was raised from 7.4 to 8. The effects of acidification and deoxygenation together were not additive. Many features of this deoxygenation-induced conductance (non-specificity for cations, permeability toCa2+ andMg2+, pH sensitivity, reversibility, partial inhibition by DIDS and Mn2+) are shared with the flux pathway sometimes referred to as Psickle. Sensitivity to GsMTx-4 indicates its possible identity as a stretch-activated channel. Sensitivity to o-vanillin implies that activation requires HbS polymerisation but since the conductance was observed in whole-cell patches, results suggest that bulk intracellular Hb is not involved; rather a membrane-bound subfraction is responsible for channel activation. The ability to record P(sickle)-like activity in single RBCs will facilitate further studies and eventual molecular identification of the pathway involved.

Successful treatment of a chronic oroantral fistula infected with extensively drug resistant bacteria using long-term oesophageal tube feeding and several non-conventional treatments in a horse.

BACKGROUND: Chronic oroantral fistulae (OAF) with secondary sinusitis can occur following repulsion of cheek teeth in horses. CASE REPORT: An 8-year-old Andalusian cross gelding presented with an iatrogenic clinical crown fracture of tooth 209, which underwent repulsion of its apical portion (day 0). The horse was treated with intramuscular penicillin and intravenous gentamicin (5 days), followed by oral trimethoprim-sulphonamide (10 days) and then oral doxycycline (14 days). The acute iatrogenic OAF created during the initial repulsion persisted; a chronic OAF was identified on day 24. On day 48, septic sinusitis with multidrug-resistant (MDR) Escherichia coli was confirmed. Although susceptible to enrofloxacin in vitro, 30 days of therapy was unsuccessful. Subsequent serial cultures grew multiple MDR and extensively drug-resistant (XDR) gram-negative microorganisms. Whole-genome sequencing (WGS) revealed multiple sequence types of E. coli, with a range of resistance and virulence genes. The orientation of the OAF, regional osteomyelitis and septic sinusitis were confirmed with computed tomography on day 70. On day 74, enteral nutrition was provided through a cervical oesophagostomy tube for 3 months for prevention of oral feed contamination. The OAF was treated with various alternative therapeutics, including apple cider vinegar, propolis and amikacin impregnated products, until resolution on day 116. CONCLUSION: These non-conventional therapeutics, antimicrobials and long-term oesophagostomy contributed to the successful treatment of a complicated OAF. In the future, WGS may be useful to inform antimicrobial selection when MDR or XDR organisms are identified.

Risk factors for multidrug-resistant Escherichia coli rectal colonization of dogs on admission to a veterinary hospital.

This study aimed to identify risk factors for intestinal colonization with multidrug-resistant (MDR) E. coli in dogs on admission to a veterinary teaching hospital. Exposures to potential risk factors, including prior treatments, hospitalizations and interventions during the 42 days prior to admission were assessed for 82 case admissions and 82 time-matched controls in a retrospective prevalence-based case-control study of 20 months duration. On multivariable analyses, risk of MDR E. coli colonization on admission was increased with prior hospitalization for 4-7 days and >7 days relative to shorter periods, and in dogs that had prior diagnostic imaging techniques. Univariable analyses indicated that risk was increased following prior treatment with several antimicrobial agents. However, on multivariable analysis, administration of fluoroquinolones was associated with increased risk but risk did not appear to increase following administration of other antimicrobials. These results can inform management of canine patients and infection control procedures to mitigate the risk of clinical disease due to MDR bacteria in hospitalized dogs.

Risk factors for dogs becoming rectal carriers of multidrug-resistant Escherichia coli during hospitalization.

This study aimed to identify risk factors for dogs becoming rectal carriers of multidrug-resistant (MDR) Escherichia coli while hospitalized in a veterinary teaching hospital. Exposures to potential risk factors, including treatments, hospitalization, and interventions during a 42-day pre-admission period and hospitalization variables, were assessed for 90 cases and 93 controls in a retrospective, risk-based, case-control study. On multivariable analyses, hospitalization for >6 days [odds ratio (OR) 2·91-8·00], treatment with cephalosporins prior to admission (OR 5·04, 95% CI 1·25-20·27), treatment with cephalosporins for >1 day (OR 5·18, 95% CI 1·86-14·41), and treatment with metronidazole (OR 7·17, 95% CI 1·01-50·79) while hospitalized were associated with increased risk of rectal carriage of MDR E. coli during hospitalization. The majority of rectal isolates obtained during the study period conformed to MDR E. coli clonal groups previously obtained from extraintestinal infections. These results can assist the development of improved infection control guidelines for the management of dogs in veterinary hospitals to prevent the occurrence of nosocomial clinical infections.

Nanoscale adhesion profiling and membrane characterisation in sickle cell disease using hybrid atomic force microscopy-IR spectroscopy.

Sickle cell disease (SCD) presents a significant global health problem. At present there is no effective treatment, with most being supportive for its associated complications such as the vaso-occlusive crises that result from increased cell adhesion. Hypoxic sickle cells have previously shown greater phosphatidylserine (PS) exposure and oxidative damage, as well as being notably "stickier" suggesting that increased cell cohesion and adhesion to the blood vessel endothelium is a possible mechanism for vaso-occlusion. The present work uses the hybrid technique of atomic force microscopy nano-infrared spectroscopy (AFM-IR) to probe changes to the coefficient of friction and C-O IR intensity in SCD on a nanoscale for dried red blood cells (RBCs) fixed under conditions of hypoxia and correlates these observations with adhesive interactions at the membrane. Using functionalised AFM tips, it has been possible to probe adhesive interactions between hydrophilic and hydrophobic moieties exposed at the surface of the dried RBCs fixed under different oxygenation states and for different cell genotypes. The results are consistent with greater PS-exposure and oxidative damage in hypoxic sickle cells, as previously proposed, and also show strong correlation between localised oxidative damage and increased adhesion. A mechanistic explanation involving significant lipid tail disruption as a result of oxidative action, in combination with differing concentrations of externalised PS lipids, is proposed to explain the observed adhesion behaviour of each type of cell.

Treatment of moderate grade dog bite wounds using amoxicillin-clavulanic acid with and without enrofloxacin: a randomised non-inferiority trial.

BACKGROUND: Dog-to-dog bite wounds are a common veterinary emergency presentation: despite this, there is insufficient information to guide veterinarians on appropriate empirical antimicrobial management. OBJECTIVES: Investigate the effectiveness of amoxicillin-clavulanic acid with and without enrofloxacin in the treatment of moderate grade dog bite wounds (DBW). To describe common pathogens and their antimicrobial susceptibility patterns. MATERIALS AND METHODS: In a single-centre parallel group pragmatic trial, 50 dogs presenting with moderate grade DBW were prospectively randomised to receive amoxicillin-clavulanic acid (group A) or amoxicillin-clavulanic acid and enrofloxacin (group B). Swabs were taken for culture and susceptibility testing at admission. Stabilisation, wound care and surgical debridement were performed at the discretion of admitting clinicians. The primary outcome was complication due to infection at 10 days, with Bayesian inference used to estimate the difference in proportions between treatment groups. RESULTS: Of the 24 dogs in treatment group A, 1 required the addition of enrofloxacin at re-examination. None of the 26 dogs in group B required alteration of antimicrobial coverage. The difference in complication rate due to infection between treatment groups was 4.2%. Twenty-one different organisms were identified: Staphylococcus pseudintermedius, Neisseria spp., Pasteurella multocida and P. canis were the most common. Over 90% of gram-negative and gram-positive isolates were susceptible to amoxicillin-clavulanic acid. Ninety-six percent of gram-negative and 86% of gram-positive isolates were susceptible to enrofloxacin. CONCLUSION AND CLINICAL SIGNIFICANCE: Amoxicillin-clavulanic acid is an appropriate empirical antimicrobial choice for moderate DBW in South East Queensland. Reduced empirical enrofloxacin use will promote antimicrobial stewardship and potentially antimicrobial resistance.

Prevalence and spatial distribution of Coxiella burnetii seropositivity in northern Australian beef cattle adjusted for diagnostic test uncertainty.

Q fever is a zoonotic disease caused by infection with Coxiella burnetii transmitted from animals including, but not limited to, cattle, sheep and goats. The infection in cattle is typically sub-clinical with some evidence suggesting associated reproductive loss. There is currently limited data on the true prevalence and distribution of coxiellosis in beef cattle across northern Australia. During this study, 2,012 sera samples from beef cattle managed on commercial farms located in Queensland and the Northern Territory were tested using an indirect immunofluorescent assay (IFA) for serological evidence of IgG antibodies against C. burnetii. Bayesian latent class models were used to estimate the true prevalence, adjusted for diagnostic test sensitivity and specificity and incorporating the hierarchical structure of the cattle within farms and regions. In this study, cattle in the Northern Territory had lower estimated true prevalence than cattle within most regions of Queensland with the exception of south-east Queensland. Results from this study have described the geographic distribution and estimated the true prevalence of antibodies to C. burnetii in a sample of extensively managed beef cattle located across the tropical grazing regions of northern Australia.

Novel permeability characteristics of red blood cells from sickle cell patients heterozygous for HbS and HbC (HbSC genotype).

Individuals heterozygous for HbS and HbC (HbSC) represent about 1/3(rd) of sickle cell disease (SCD) patients. Whilst HbSC disease is generally milder, there is considerable overlap in symptoms with HbSS disease. HbSC patients, as well as HbSS ones, present with the chronic anaemia and panoply of acute vaso-occlusive complications that characterize SCD. However, there are important clinical and haematological differences. Certain complications occur with greater frequency in HbSC patients (like proliferative retinopathy and osteonecrosis) whilst intravascular haemolysis is reduced. Patients with HbSC disease can be considered as a discrete subset of SCD cases. Although much work has been carried out on understanding the pathogenesis of SCD in HbSS homozygotes, including the contribution of altered red blood cell permeability, relatively little pertains directly to HbSC individuals. Results reported in the literature suggest that HbSC cells, and particularly certain subpopulations, present with similar permeability to HbSS cells but there are also important differences - these have not been well characterized. We hypothesise that their unique cell transport properties accounts for the different pattern of disease in HbSC patients and represents a potential chemotherapeutic target not shared in red blood cells from HbSS patients. The distinct pattern of clinical haematology in HbSC disease is emphasised here. We analyse some of the electrophysiological properties of single red blood cells from HbSC patients, comparing them with those from HbSS patients and normal HbAA individuals. We also use the isosmotic haemolysis technique to investigate the behaviour of total red blood cell populations. Whilst both HbSS and HbSC cells show increased monovalent and divalent (Ca(2+)) cation conductance further elevated upon deoxygenation, the distribution of current magnitudes differs, and outward rectification is greatest for HbSC cells. In addition, although Gd(3+) largely abolishes the cation conductance of both HbSS and HbSC cells, only in HbSS ones are currents inhibited by the aminoglycosides like streptomycin. This distinction is retained in isosmotic lysis experiments where both HbSS and HbSC cells undergo haemolysis in sucrose solutions but streptomycin significantly inhibits lysis only in HbSS cells. These findings emphasise similarities but also differences in the permeability properties of HbSS and HbSC cells, which may be important in pathogenesis.

Yoda1 and phosphatidylserine exposure in red cells from patients with sickle cell anaemia.

Phosphatidylserine (PS) exposure is increased in red cells from sickle cell anaemia (SCA) patients. Externalised PS is prothrombotic and attractive to phagocytes and activated endothelial cells and thus contributes to the anaemic and ischaemic complications of SCA. The mechanism of PS exposure remains uncertain but it can follow increased intracellular Ca2+ concentration ([Ca2+]i). Normally, [Ca2+]i is maintained at very low levels but in sickle cells, Ca2+ permeability is increased, especially following deoxygenation and sickling, mediated by a pathway sometimes called Psickle. The molecular identity of Psickle is also unclear but recent work has implicated the mechanosensitive channel, PIEZO1. We used Yoda1, an PIEZO1 agonist, to investigate its role in sickle cells. Yoda1 caused an increase in [Ca2+]i and PS exposure, which was inhibited by its antagonist Dooku1 and the PIEZO1 inhibitor GsMTx4, consistent with functional PIEZO1. However, PS exposure did not necessitate an increase in [Ca2+]i. Two PKC inhibitors were also tested, chelerytherine chloride and calphostin C. Both reduced PS exposure whilst chelerytherine chloride also reduced Yoda1-induced increases in [Ca2+]i. Findings are therefore consistent with the presence of PIEZO1 in sickle cells, able to mediate Ca2+ entry but that PKC was also involved in both Ca2+ entry and PS exposure.

Non-electrolyte permeability of deoxygenated sickle cells compared.

The passive permeability pathways of red cells are poorly defined, with the exception of the Gardos channel. Several cation and anion pathways can be induced by a variety of manoeuvres, however, including treatment with oxidants, low ionic strength (LIS), shrinkage, swelling and also infection with the intra-erythrocytic malaria parasite. Several of these stimuli (malaria, swelling, LIS), in addition, also activate a non-electrolyte this permeability. Sickle cells uniquely show a deoxygenation-induced pathway, which is termed P(sickle) and is usually considered to be a conductive cationic pathway. In this report, we explore further the extent to which this permeability pathway of deoxygenated sickle cells is available for non-electrolyte transport. We show that a number of solutes are permeable, with greater permeability to sugars (notably lactose and maltose) and smaller molecules, and less to charged or zwitterionic species. Red cells from heterozygous HbSC patients also showed deoxygenation-induced haemolysis in isosmotic sucrose solution, though to a slightly lesser extent than for red cells from homozygous sickle cell patients. In contrast to sickle cells, red cells from beta-thalassaemic patients did not show haemolysis in isosmotic sucrose solutions, regardless of the O(2) tension. Of the secondary cellular changes resulting from incubation in non-electrolyte solutions (which include imposition of a highly positive membrane potential, marked intracellular alkalinisation and cell shrinkage), none appear to correlate with activation of the non-electrolyte permeability. Rather, findings indicate that it is low ionic strength per se that is responsible. Normal red cells also show changes in ionic and non-electrolyte permeability in low ionic strength media, and these permeabilities are compared to those found in deoxygenated sickle cells. The extent to which these different permeabilities in normal and sickle red cells can be ascribed to one or more common pathways remains to be determined.

Multidrug-resistant E. coli and enterobacter extraintestinal infection in 37 dogs.

BACKGROUND: Extraintestinal infections caused by multidrug-resistant (MDR) Escherichia coli and Enterobacter are becoming more common in veterinary medicine. OBJECTIVE: To generate hypotheses for risk factors for dogs acquiring extraintestinal infection caused by MDR E. coli and Enterobacter, describe antimicrobial resistance profiles and analyze treatment and clinical outcomes. ANIMALS: Thirty-seven dogs diagnosed with extraintestinal infection caused by MDR E. coli and Enterobacter spp. between October 1999 and June 2006. METHODS: Retrospective case series assembled from hospital records data, including clinical history before 1st MDR isolation and treatment outcome. Identity and antimicrobial susceptibility profiles were confirmed by standard microbiological techniques for 57 isolates. RESULTS: Most dogs had an underlying disease condition (97%), received prior antimicrobial treatment (87%), were hospitalized for >or =3 days (82%), and had a surgical intervention (57%). The urinary tract was the most common infection site (62%), and urinary catheterization, bladder stasis, or both were common among dogs (24%). Some dogs were treated with high doses of co-amoxyclavulanate (n = 14) and subsequently recovered even though the isolates showed in vitro resistance to this antimicrobial. Other dogs were successfully treated with chloramphenicol (n = 11) and imipenem (n = 2). CONCLUSION AND CLINICAL IMPORTANCE: Predisposing disease condition, any prior antimicrobial use rather than a specific class of antimicrobial, duration of hospitalization, and type of surgical procedure might be risk factors for acquiring MDR extraintestinal infections. Whereas culture and sensitivity results can indicate use of last-resort antimicrobials such as imipenem for MDR infections, some affected dogs can recover after administration of high doses of co-amoxyclavulanate.

Mycoplasma bovis and bovine respiratory disease: A risk factor study in Australian feeder cattle.

Mycoplasma bovis can be a bacterial inhabitant of the upper respiratory tract of healthy bovines. In body regions other that the upper respiratory tract however, M. bovis is associated with a number of clinical syndromes such as bovine respiratory disease (BRD). This study used two enzyme-linked immunosorbent assays to assess the sero-status of M. bovis-specific antibodies in Australian feeder cattle at the time of feedlot induction and at approximately 42 days on feed (follow-up). The apparent sero-prevalence of M. bovis-specific antibody at induction was estimated to be 3.5% (95% confidence interval [CI] 2.0-5.0%, 47/1354) and 25.3% (95% CI 21.9-28.8%, 343/1354) at follow-up. Exposure to M. bovis between induction and follow-up as demonstrated by an increase in serum antibodies was estimated to be 19.4% (95% CI 16.2-22.6%, 261/1349). Risk factors associated with sero-positivity at feedlot induction included the region where animals were 28 days prior to induction and saleyard exposure at least 27 days prior to induction. Risk factors associated with a sero-increase between induction and follow-up included breed, source region and access to water shared with an adjoining pen of animals. Of these, shared pen water was considered the most important (odds ratio [OR] 3.3, 95% CI 1.5-7.4, p = 0.003). Animals exposed to M. bovis between induction and follow-up were at a substantially increased risk of BRD (OR 2.2, 95% CI 1.4-3.4, p = 0.001). This is the first Australian study that has identified risk factors for M. bovis sero-positivity and sero-increase and shown an association between sero-increase and the risk of BRD in the feeder cattle population. These findings suggest that M. bovis is a significant pathogen in the Australian feeder cattle population. In addition, identification of defined risk factors associated with an increased risk of exposure to M. bovis can assist in the development of targeted control measures to reduce the economic impact of M. bovis associated disease and BRD in feeder cattle.

Antimicrobial resistance genes in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs.

OBJECTIVE: To identify genes associated with the observed antimicrobial resistance in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs. DESIGN: Isolates with known phenotypic resistance to ß-lactams, macrolides and tetracycline were screened for the presence of antimicrobial resistance genes. PROCEDURE: A total of 68 A. pleuropneumoniae, 62 H. parasuis and 20 P. multocida isolates exhibiting phenotypic antimicrobial resistance (A. pleuropneumoniae and P. multocida) or elevated minimal inhibitory concentrations (MICs) (H. parasuis) to any of the following antimicrobial agents - ampicillin, erythromycin, penicillin, tetracycline, tilmicosin and tulathromycin - were screened for a total of 19 associated antimicrobial resistance genes (ARGs) by PCR. RESULTS: The gene bla ROB-1 was found in all ampicillin- and penicillin-resistant isolates, but none harboured the bla TEM-1 gene. The tetB gene was found in 76% (74/97) of tetracycline-resistant isolates, 49/53 A. pleuropneumoniae, 17/30 H. parasuis and 8/14 P. multocida. One A. pleuropneumoniae isolate harboured the tetH gene, but none of the 97 isolates had tetA, tetC, tetD, tetE, tetL, tetM or tetO. A total of 92 isolates were screened for the presence of macrolide resistance genes. None was found to have ermA, ermB, ermC, erm42, mphE, mefA, msrA or msrE. CONCLUSION: The current study has provided a genetic explanation for the resistance or elevated MIC of the majority of isolates of Australian porcine respiratory pathogens to ampicillin, penicillin and tetracycline. However, the macrolide resistance observed by phenotypic testing remains genetically unexplained and further studies are required.