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.

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.

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.

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.

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.

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.

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.

Phenotypic and genotypic profiling of antimicrobial resistance in enteric Escherichia coli communities isolated from finisher pigs in Australia.

OBJECTIVE: To assess herd-to-herd variation in antimicrobial resistance phenotypes and associated antimicrobial resistance genes (ARGs) in faecal commensal Escherichia coli communities isolated from Australian slaughter-age pigs. METHODS: Hydrophobic grid-membrane filtration (HGMF) was used to screen populations of E. coli isolated from faecal samples obtained from pigs prior to or at slaughter. Multiplex PCRs were applied to the pooled DNA extracted from the samples to identify specific ARGs. METHODS: Pooled faecal samples from 30 finishers, from 72 different Australian pig farms, produced 5003 isolates for screening. HGMF techniques and image analysis were used to confirm E. coli resistance phenotypes to four antimicrobial agents (ampicillin, gentamicin, florfenicol and ceftiofur) using selective agars. Multiplex PCRs were performed on DNA from pooled samples for 35 ARGs associated with seven chemical classes. RESULTS: The prevalence of E. coli isolates showing no resistance to any of the drugs was 50.2% (95% confidence interval (CI) 41.8-58.6%). Ceftiofur resistance was very low (1.8%; CI 0.8-3.9%) and no ARGs associated with 3rd-generation cephalosporin resistance were detected. By contrast, ampicillin (29.4%, CI 22.8-37.0%), florfenicol (24.3%, CI 17.8-32.3%) and gentamicin (CI 17.5%, 10.7-27.2%) resistance prevalence varied greatly between farms and associated ARGs were common. The most common combined resistance phenotype was ampicillin-florfenicol. CONCLUSION: The use of registered antimicrobials in Australian pigs leads to the enteric commensal populations acquiring associated ARGs. However, despite a high intensity of sampling, ARGs imparting resistance to the critically important 3rd-generation cephalosporins were not detected.

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.

Isolation of Nocardia mexicana from focal proliferative tenosynovitis and arthritis in a steer.

CASE REPORT: An 18-month-old Charolais steer was presented with lameness and fluctuant swelling of the right stifle joint, which yielded neutrophils on fine-needle aspiration. A diagnosis of bacterial proliferative tenosynovitis and arthritis was made on postmortem and histological examination. Culture and 16S rRNA sequencing identified a Nocardia sp. with 99% homology with the corresponding DNA fragment of N. mexicana DSM 44952. Antimicrobial susceptibility testing revealed the isolate was susceptible to co-trimoxazole and third-generation cephalosporins. CONCLUSION: We report the first case, both in Australia and internationally, of proliferative tenosynovitis and arthritis caused by Nocardia spp. infection in a bovine and the first report of pathology attributed to N. mexicana in a veterinary patient. Given the limited susceptibility of the bacteria, the poor antimicrobial penetration that would be expected and the morphological changes that had taken place in the joint; the steer would have required protracted antimicrobial treatment in addition to invasive debridement of the lesion. This case emphasises the importance of routinely performing cytology and extended incubation of cultures in cases of arthritis in order to make ethical and economically viable treatment decisions.

Cation Homeostasis in Red Cells From Patients With Sickle Cell Disease Heterologous for HbS and HbC (HbSC Genotype).

Sickle cell disease (SCD) in patients of HbSC genotype is considered similar, albeit milder, to that in homozygous HbSS individuals--but with little justification. In SCD, elevated red cell cation permeability is critical as increased solute loss causes dehydration and encourages sickling. Recently, we showed that the KCl cotransporter (KCC) activity in red cells from HbSC patients correlated significantly with disease severity, but that in HbSS patients did not. Two transporters involved in red cell dehydration, the conductive channels Psickle and the Gardos channel, behaved similarly in red cells from the two genotypes, but were significantly less active in HbSC patients. By contrast, KCC activity was quantitatively greater in HbSC red cells. Results suggest that KCC is likely to have greater involvement in red cell dehydration in HbSC patients, which could explain its association with disease severity in this genotype. This work supports the hypothesis that SCD in HbSC patients is a distinct disease entity to that in HbSS patients. Results suggest the possibility of designing specific treatments of particular benefit to HbSC patients and a rationale for the development of prognostic markers, to inform early treatment of children likely to develop more severe complications of the disease.

Is Mycoplasma bovis a missing component of the bovine respiratory disease complex in Australia?

BACKGROUND: Bovine respiratory disease complex (BRDC) is a multi-factorial disease in which numerous factors, such as animal management, pathogen exposure and environmental conditions, contribute to the development of acute respiratory illness in feedlot cattle. The role of specific pathogens in the development of BRDC has been difficult to define because of the complex nature of the disease and the presence of implicated bacterial pathogens in the upper respiratory tract of healthy animals. Mycoplasma bovis is an important pathogen of cattle and recognised as a major contributor to cases of mastitis, caseonecrotic bronchopneumonia, arthritis and otitis media. To date, the role of M. bovis in the development of BRDC of Australian feeder cattle has not been investigated. METHODS: In this review, the current literature pertaining to the role of M. bovis in BRDC is evaluated. In addition, preliminary data are presented that identify M. bovis as a potential contributor to BRDC in Australian feedlots, which has not been considered previously. RESULTS AND CONCLUSION: The preliminary results demonstrate detection of M. bovis in samples from all feedlots studied. When considered in the context of the reviewed literature, they support the inclusion of M. bovis on the list of pathogens to be considered during investigations into BRDC in Australia.

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.

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.

Comparison of antimicrobial resistance phenotypes and genotypes in enterotoxigenic Escherichia coli isolated from Australian and Vietnamese pigs.

This study aimed to compare the antibiogram phenotype and carriage of antimicrobial resistance genes (ARGs) of 97 porcine multidrug-resistant (MDR) enterotoxigenic Escherichia coli (ETEC) isolates obtained from Vietnam and 117 porcine MDR-ETEC obtained from Australia, two countries with different antimicrobial regulation systems. An antimicrobial resistance index (ARI) was calculated to quantify their potential significance to public health. Both Vietnamese and Australian isolates had moderate to high levels of resistance to commonly used antibiotics (ampicillin, tetracycline and sulphonamides). None of the Australian isolates were resistant to fluoroquinolones or third-generation cephalosporins and none possessed associated plasmid-mediated ARGs. However, 23.1% of Australian isolates were resistant to gentamicin owing to ARGs associated with apramycin or neomycin resistance [e.g. aac(3)-IV] that impart cross-resistance to gentamicin. Whilst Vietnamese isolates carried aminoglycoside ARGs, 44.4% of commercial pig isolates were resistant to gentamicin in comparison with 0% of village pig isolates. The plasmid-mediated fluoroquinolone ARG qnrB was commonly detected in Vietnamese isolates (52.3% commercial, 44.1% village), but phenotypic resistance was low (3.2% and 11.8%, respectively). The mean ARI for Vietnamese isolates (26.0) was significantly different (P<0.001) from the mean ARI for Australian isolates (19.8), primarily reflecting fluoroquinolone resistance in the former collection. This comparison suggests the effectiveness of regulations that slow the dissemination of 'critical' resistance by restricting the availability of important classes of antimicrobials.

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.

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.