Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients.

Essentials Neutrophil extracellular traps (NETs) might play a role in cancer-related coagulopathy. We determined NET biomarkers and followed cancer patients for venous thromboembolism (VTE). We found a constant association with VTE for citrullinated histone H3. Biomarkers of NET formation could reflect a novel pathomechanism of cancer-related VTE. SUMMARY: Background Neutrophil extracellular traps (NETs) are decondensed chromatin fibers that might play a role in the prothrombotic state of cancer patients. Objectives To investigate whether the levels of citrullinated histone H3 (H3Cit), a biomarker for NET formation, cell-free DNA (cfDNA) and nucleosomes predict venous thromboembolism (VTE) in cancer patients. Patients/Methods Nine-hundred and forty-six patients with newly diagnosed cancer or progression after remission were enrolled in this prospective observational cohort study. H3Cit, cfDNA and nucleosome levels were determined at study inclusion, and patients were followed for 2 years. VTE occurred in 89 patients; the cumulative 3-month, 6-month, 12-month and 24-month incidence rates of VTE were 3.7%, 6.0%, 8.1%, and 10.0%, respectively. Results Patients with elevated H3Cit levels (> 75th percentile of its distribution, n = 236) experienced a higher cumulative incidence of VTE (2-year risk of 14.5%) than patients with levels below this cut-off (2-year risk of 8.5%, n = 710). In a competing-risk regression analysis, a 100 ng mL-1 increase in H3Cit level was associated with a 13% relative increase in VTE risk (subdistribution hazard ratio [SHR] 1.13, 95% confidence interval [CI] 1.04-1.22). This association remained after adjustment for high VTE risk and very high VTE risk tumor sites, D-dimer level, and soluble P-selectin level (SHR 1.13, 95% CI 1.04-1.22). The association of elevated nucleosome and cfDNA levels with VTE risk was time-dependent, with associations with a higher risk of VTE only during the first 3-6 months. Conclusion These data suggest that biomarkers of NET formation are associated with the occurrence of VTE in cancer patients, indicating a role of NETs in the pathogenesis of cancer-associated thrombosis.

OC-16 - Neutrophil extracellular traps and tissue factor-bearing microvesicles: a liaison dangereuse causing overt DIC in cancer patients?

INTRODUCTION: Overt disseminated intravascular coagulation (DIC) is a systemic process characterized by excessive coagulation activation and fibrinolysis that may occur in cancer patients. The underlying pathomechanisms are still poorly understood. Recent experimental studies found an important role for the interaction between procoagulant neutrophil extracellular traps (NETs) and tissue factor (TF) in the pathogenesis of thrombosis. AIM: To investigate whether NETs and TF-bearing microvesicles (MVs) play a central role in cancer-related overt DIC. MATERIALS AND METHODS: Twenty-eight cancer patients with overt DIC (ISTH score ≥5, 14 females, median age: 62 years [range: 21-80], 13 with solid tumors, 15 with acute leukemia) and 28 matched healthy controls were included. NET formation parameters (plasma DNA and nucleosomes), MVassociated TF activity, and routine coagulation parameters were determined at study inclusion. In 11 patients with acute myeloid leukemia (AML), follow-up measurements were also performed. RESULTS: Plasma DNA, nucleosomes, and MV-TF activity were highly elevated in patients with cancer-related DIC compared to healthy individuals (all p-values<0.001). Strong correlations were found between plasma DNA and nucleosomes (Spearman correlation-coefficient: r=0.68), nucleosomes and MV-TF activity (r=0.62), and DNA and MV-TF activity (r=0.57). In multivariate regression, altered routine coagulation parameters were highly associated with NET parameters and MV-TF activity. In detail, a doubling in plasma DNA was associated with a 7.6% decrease in fibrinogen (p=0.012), a 15.3% decrease in platelet count (p=0.002), a 3.9% decrease in prothrombin time (p=0.014), and a 41.0% increase in D-dimer (p<0.001). A 10% increase in nucleosomes was associated with a 3.1% decrease in fibrinogen (p<0.001), a 5.0% decrease in platelet count (p<0.001), a 1.0% decrease in prothrombin time (p<0.009), and a 112.7% increase in D-dimer (p<0.001). A 10% increase in MV-TF activity was associated with a 4.9% decrease in fibrinogen (p<0.001), a 7.1% decrease in platelet count (p<0.001), a 1.3% decrease in prothrombin time (p<0.001), and a 15.5% increase in D-dimer (p<0.001). After initiation of chemotherapy in AML patients, NET parameters and MV-TF activity decreased significantly (nucleosomes: 3.3-fold decrease and normalization after 1 week; DNA: 1.2-fold decrease after 1 week and 1.5-fold decrease after 1 month; MV-TF activity: 10-fold decease after 1 week and normalization after 1 month) (Figure 1), and routine coagulation parameters improved. CONCLUSIONS: Our results add to experimental studies that have investigated the interaction between NETs and TF. Taken together, evidence indicates the presence of a liaison dangereuse between NETs and TF-bearing MVs, which could be the underlying cause of cancer-related overt DIC.

Neutrophil elastase-deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis.

UNLABELLED: ESSENTIALS: Neutrophil elastase (NE) plays a role in extracellular trap formation (NETosis) triggered by microbes. The contribution of NE was evaluated in mouse NETosis models of sterile inflammation and thrombosis. NE is not required for mouse neutrophil NET production in vitro with non-infectious stimuli. NE deficiency had no significant effect on thrombosis in the inferior vena cava stenosis model. BACKGROUND: Neutrophil serine proteases have been implicated in coagulation and neutrophil extracellular trap (NET) formation. In human neutrophils, neutrophil elastase (NE) translocates to the nucleus during NETosis and cleaves histones, thus aiding in chromatin decondensation. NE(-/-) mice were shown not to release NETs in response to microbes. However, mouse studies evaluating the role of NE in NET formation in sterile inflammation and thrombosis are lacking. OBJECTIVE: We wished to establish if neutrophils from NE(-/-) mice have a defect in NETosis, similar to peptidylarginine deiminase 4 (PAD4(-/-)) mice, and how this might have an impact on venous thrombosis, a model where NETs are produced and are crucial to thrombus development. METHODS: We performed in vitro NET assays using neutrophils from wild-type (WT), NE(-/-), SerpinB1 (SB1)(-/-) and NE(-/-) SB1(-/-) mice. We compared WT and NE(-/-) animals using the inferior vena cava stenosis model of deep vein thrombosis (DVT). RESULTS: Neutrophil elastase deficiency resulted in a small reduction in ionomycin-induced NET formation in vitro without affecting histone citrullination. However, NET production in response to phorbol 12-myristate 13-acetate or platelet activating factor was normal in neutrophils from two independent NE-deficient mouse lines, and in NE(-/-) SB1(-/-) as compared with SB1(-/-) neutrophils. NE deficiency or inhibition did not prevent NETosis in vivo or DVT outcome. CONCLUSIONS: Neutrophil elastase is not required for NET formation in mice. NE(-/-) mice, which form pathological venous thrombi containing NETs, do not phenocopy PAD4(-/-) mice in in vitro NETosis assays or experimental venous thrombosis. Our study suggests that NET-targeted therapies need to be highly effective to have an impact on DVT.

Tissue factor expressed by circulating cancer cell-derived microparticles drastically increases the incidence of deep vein thrombosis in mice.

BACKGROUND: The risk of thrombotic complications such as deep vein thrombosis (DVT) during tumor development is well known. Tumors release into the circulation procoagulant microparticles (MPs) that can participate in thrombus formation following vessel injury. The importance of this MP tissue factor (TF) in the initiation of cancer-associated DVT remains uncertain. OBJECTIVE: To investigate how pancreatic cancer MPs promote DVT in vivo. METHODS: We combined a DVT mouse model in which thrombosis is induced by flow restriction in the inferior vena cava with one of subcutaneous pancreatic cancer in C57BL/6J mice. We infused high-TF and low-TF tumor MPs to determine the importance of TF in experimental cancer-associated DVT. RESULTS: Both tumor-bearing mice and mice infused with tumor MPs subjected to 3 h of partial flow restriction developed an occlusive thrombus; fewer than one-third of the control mice did. We observed that MPs adhered to neutrophil extracellular traps (NETs), which are functionally important players during DVT, whereas neither P-selectin nor glycoprotein Ib were required for MP recruitment in DVT. The thrombotic phenotype induced by MP infusion was suppressed by hirudin, suggesting the importance of thrombin generation. TF carried by tumor MPs was essential to promote DVT, as mice infused with low-TF tumor MPs had less thrombosis than mice infused with high-TF tumor MPs. CONCLUSIONS: TF expressed on tumor MPs contributes to the increased incidence of cancer-associated venous thrombosis in mice in vivo. These MPs may adhere to NETs formed at the site of thrombosis.

Neutrophil extracellular traps form predominantly during the organizing stage of human venous thromboembolism development.

BACKGROUND: A growing health problem, venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), requires refined diagnostic and therapeutic approaches. Neutrophils contribute to thrombus initiation and development in experimental DVT. Recent animal studies recognized neutrophil extracellular traps (NETs) as an important scaffold supporting thrombus stability. However, the hypothesis that human venous thrombi involve NETs has not undergone rigorous testing. OBJECTIVE: To explore the cellular composition and the presence of NETs within human venous thrombi at different stages of development. PATIENTS AND METHODS: We examined 16 thrombi obtained from 11 patients during surgery or at autopsy using histomorphological, immunohistochemical and immunofluorescence analyses. RESULTS: We classified thrombus regions as unorganized, organizing and organized according to their morphological characteristics. We then evaluated them, focusing on neutrophil and platelet deposition as well as micro-vascularization of the thrombus body. We observed evidence of NET accumulation, including the presence of citrullinated histone H3 (H3Cit)-positive cells. NETs, defined as extracellular diffuse H3Cit areas associated with myeloperoxidase and DNA, localized predominantly during the phase of organization in human venous thrombi. CONCLUSIONS: NETs are present in organizing thrombi in patients with VTE. They are associated with thrombus maturation in humans. Dissolution of NETs might thus facilitate thrombolysis. This finding provides new insights into the clinical development and pathology of thrombosis and provides new perspectives for therapeutic advances.

Plasma DNA is Elevated in Patients with Deep Vein Thrombosis.

OBJECTIVE: To investigate if plasma DNA is elevated in patients with deep vein thrombosis (DVT) and to determine whether there is a correlation with other biomarkers of DVT. BACKGROUND: Leukocytes release DNA to form extracellular traps (ETs), which have recently been linked to experimental DVT. In baboons and mice, extracellular DNA co-localized with von Willebrand factor (VWF) in the thrombus and DNA appeared in circulation at the time of thrombus formation. ETs have not been associated with clinical DVT. SETTING: From December 2008 to August 2010, patients were screened through the University of Michigan Diagnostic Vascular Unit and were divided into three distinct groups: 1) the DVT positive group, consisting of patients who were symptomatic for DVT, which was confirmed by compression duplex ultrasound (n=47); 2) the DVT negative group, consisting of patients that present with swelling and leg pain but had a negative compression duplex ultrasound, (n=28); and 3) a control group of healthy non-pregnant volunteers without signs or symptoms of active or previous DVT (n=19). Patients were excluded if they were less than 18 years of age, unwillingness to consent, pregnant, on an anticoagulant therapy, or diagnosed with isolated calf vein thrombosis. METHODS: Blood was collected for circulating DNA, CRP, D-dimer, VWF activity, myeloperoxidase (MPO), ADAMTS13 and VWF. The Wells score for a patient's risk of DVT was assessed. The Receiver Operating Characteristic (ROC) curve was generated to determine the strength of the relationship between circulating DNA levels and the presence of DVT. A Spearman correlation was performed to determine the relationship between the DNA levels and the biomarkers and the Wells score. Additionally the ratio of ADAMTS13/VWF was assessed. RESULTS: Our results showed that circulating DNA (a surrogate marker for NETs) was significantly elevated in DVT patients, compared to both DVT negative patients (57.7±6.3 vs. 17.9±3.5ng/mL, P<.01) and controls (57.7±6.3 vs. 23.9±2.1ng/mL, P<.01). There was a strong positive correlation with CRP (P<.01), D-dimer (P<.01), VWF (P<.01), Wells score (P<.01) and myeloperoxidase (MPO) (P<.01), along with a strong negative correlation with ADAMTS13 (P<.01) and the ADAMTS13/VWF ratio. The logistic regression model showed a strong association between plasma DNA and the presence of DVT (ROC curve was determined to be 0.814). CONCLUSIONS: Plasma DNA is elevated in patients with deep vein thrombosis and correlates with biomarkers of DVT. A strong correlation between circulating DNA and MPO suggests that neutrophils may be a source of plasma DNA in patients with DVT.

How platelets safeguard vascular integrity.

The haemostatic role of platelets was established in the 1880s by Bizzozero who observed their ability to adhere and aggregate at sites of vascular injury. It was only some 80 years later that the function of platelets in maintaining the structural integrity of intact blood vessels was reported by Danielli. Danielli noted that platelets help preserve the barrier function of endothelium during organ perfusion. Subsequent studies have demonstrated further that platelets are continuously needed to support intact mature blood vessels. More recently, platelets were shown to safeguard developing vessels, lymphatics, as well as the microvasculature at sites of leukocyte infiltration, including inflamed organs and tumours. Interestingly, from a mechanistic point of view, the supporting role of platelets in these various vessels does not necessarily involve the well-understood process of platelet plug formation but, rather, may rely on secretion of the various platelet granules and their many active components. The present review focuses on these nonconventional aspects of platelet biology and function by presenting situations in which platelets intervene to maintain vascular integrity and discusses possible mechanisms of their actions. We propose that modulating these newly described platelet functions may help treat haemorrhage as well as treat cancer by increasing the efficacy of drug delivery to tumours.

Differential stimulation of monocytic cells results in distinct populations of microparticles.

BACKGROUND: Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. METHODS: We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. RESULTS: Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera. CONCLUSION: We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

Serotonin stimulates platelet receptor shedding by tumor necrosis factor-alpha-converting enzyme (ADAM17).

BACKGROUND: Peripheral serotonin (5-hydroxytryptamine, 5-HT) is transported by platelets and released upon stimulation. In the platelet cytoplasm, 5-HT is transamidated to small GTPases, promoting alpha-granule release and primary hemostasis. OBJECTIVE: We hypothesized that 5-HT could also stimulate platelet receptor shedding after binding to the membrane 5-HT receptor (5-HT2AR). METHODS: Western blot and flow cytometry were used to determine levels of the adhesion receptor glycoprotein (GP)Ibalpha on platelets or its shed fragment glycocalicin in plasma and serum from wild-type mice, Tph1(-/-) mice lacking peripheral 5-HT, and mice lacking functional tumor necrosis factor-alpha-converting enzyme (TACE, ADAM17). Flow chamber experiments and intravital microscopy were used to examine the adhesive properties of platelets after stimulation of 5-HT2AR. RESULTS: Glycocalicin was significantly reduced in Tph1(-/-) plasma and serum. In isolated platelets, 5-HT induced shedding of GPIbalpha, which was increased to 60% when 5-HT uptake was inhibited by the selective serotonin reuptake inhibitor fluoxetine. Specific 5-HT2AR agonism and antagonism suggested activation of this receptor. The shedding could not be induced in TACE(DeltaZn/DeltaZn) platelets, suggesting that activated TACE mediated the shedding of GPIbalpha. Intracellular signaling involved phosphorylation of p38 mitogen-activated protein kinase rather than G-protein signaling. 5-HT2AR stimulation decreased platelet adhesion to collagen-bound von Willebrand factor under arterial shear (1500 s(-1)) and incorporation into FeCl3-induced thrombi in mesenteric arterioles. CONCLUSIONS: Stimulation of 5-HT2AR on platelets induces TACE-mediated shedding of GPIbalpha, the key adhesion molecule under high shear conditions. Our observations demonstrate a new pathway through which 5-HT could modulate cardiovascular disease.

Inhibition of von Willebrand factor-mediated platelet activation and thrombosis by the anti-von Willebrand factor A1-domain aptamer ARC1779.

BACKGROUND: von Willebrand factor (VWF) has a role in both hemostasis and thrombosis. Platelets adhere to damaged arteries by interactions between the VWF A1-domain and glycoprotein Ib receptors under conditions of high shear. This initial platelet binding event stimulates platelet activation, recruitment, and activation of the clotting cascade, promoting thrombus formation. OBJECTIVE: To characterize the inhibitory activity of a VWF inhibitory aptamer. METHODS: Using in vitro selection, aptamer stabilization, and conjugation to a 20-kDa poly(ethylene glycol), we generated a nuclease-resistant aptamer, ARC1779, that binds to the VWF A1-domain with high affinity (K(D) approximately 2 nM). The aptamer was assessed for inhibition of VWF-induced platelet aggregation. In vitro inhibition of platelet adhesion was assessed on collagen-coated slides and injured pig aortic segments. In vivo activity was assessed in a cynomolgus monkey carotid electrical injury thrombosis model. RESULTS AND CONCLUSION: ARC1779 inhibited botrocetin-induced platelet aggregation (IC90 approximately 300 nM) and shear force-induced platelet aggregation (IC95 approximately 400 nM). It reduced adhesion of platelets to collagen-coated matrices and formation of platelet thrombi on denuded porcine arteries. ARC1779 also inhibited the formation of occlusive thrombi in cynomolgus monkeys. We have discovered a novel anti-VWF aptamer that could have therapeutic use as an anti-VWF agent in the setting of VWF-mediated thrombosis.

Memory and the medial temporal lobe: hemispheric specialization reconsidered.

The role of the medial temporal lobe in learning and memory has been well established in research on humans and other animals. In humans, clinical and neuroimaging studies typically suggest material-specific lateralization in which the left and right temporal lobes are associated with verbal and nonverbal memory, respectively. It is often assumed that the temporal lobes are functionally alike, differing only in terms of the content to be learned. Here we present data that challenge this notion, showing that the type of material used during a memory task can influence fMRI activation patterns beyond the expected left-verbal/right-nonverbal dichotomy. Our results also suggest some degree of functional asymmetry in the medial temporal lobe that is independent of material type, pointing to underlying processing differences between the left and right temporal lobes.

Formation of platelet strings and microthrombi in the presence of ADAMTS-13 inhibitor does not require P-selectin or beta3 integrin.

BACKGROUND: Ultra-large von Willebrand factor (ULVWF) and the receptor P-selectin are released from endothelial Weibel-Palade bodies during injury or inflammation. VWF mediates platelet adhesion and P-selectin promotes leukocyte rolling. ADAMTS-13 limits the duration of platelet adhesion by cleaving the ULVWF. In the absence of ADAMTS-13, long VWF filaments decorated with platelets form. Recent in vitro studies suggested that P-selectin might anchor these platelet strings to endothelium, but whether the same mechanism exists in vivo remains to be elucidated. METHODS: We address the role of P-selectin and beta(3) integrin in platelet string formation in vivo using intravital microscopy by infusing inhibitory ADAMTS-13 antibody in P-selectin-/- and beta(3)-deficient mice and activating the endothelium by injecting histamine. RESULTS: We show that inhibition of ADAMTS-13 combined with endothelial activation leads to similar extents of platelet string formation in wild-type, P-selectin- and integrin beta(3)-deficient mice. Further, in venules the platelet strings can coalesce into VWF-platelet aggregates. This process utilizes neither the platelet beta(3) integrin nor P-selectin. We also show in vitro that platelets can act as a bridge between the VWF fibers and that VWF can self-associate even in areas devoid of platelets. CONCLUSIONS: The formation or retention of the platelet strings does not require P-selectin or the endothelial VWF receptor alpha(v)beta(3). Furthermore, in the presence of low ADAMTS-13 activity, VWF-dependent and alpha(IIb)beta(3)-independent platelet clustering occurs in veins, as has been shown at high arterial shear rates. Our study further supports the importance of regulation of VWF multimer size upon secretion from Weibel-Palade bodies.

Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo.

BACKGROUND: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg-deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF(-/-)). METHODS AND RESULTS: Here we studied Fg/VWF-independent platelet aggregation in vitro and found no aggregation in citrated platelet-rich plasma of Fg/VWF(-/-) mice. Surprisingly, in Fg/VWF(-/-) plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF(-/-) platelets but not of beta(3)-integrin-deficient (beta(3) (-/-)) platelets. In addition, beta(3) (-/-) platelets did not significantly incorporate into thrombi in Fg/VWF(-/-) mice. This Fg/VWF-independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF-NH(2)) induced aggregation of gel-filtered Fg/VWF(-/-) platelets in 1 mm Ca(2+) PIPES buffer. Notably, aggregation in PIPES buffer was only 50-60% of that observed in Fg/VWF(-/-) plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF(-/-) mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca(2+)in vitro. CONCLUSIONS: beta(3) integrin, thrombin, and Ca(2+) play critical roles in this Fg/VWF-independent aggregation, and both plasma and platelet granule proteins contribute to this process.

The P-selectin, tissue factor, coagulation triad.

The primary importance of tissue factor (TF) in blood coagulation and thrombus propagation has been recognized for many years. Nevertheless, our view about the origin of TF activity, necessary for normal hemostasis and found in pathologic conditions, needs to be revised in the light of recent observations. Pioneering work by Yale Nemerson's group showed that circulating TF on microparticles (MPs), could promote thrombus growth. The origin and characteristics of this 'blood-borne' TF are targets of intense research as well as intense debate. Surprising observations now implicate the adhesion receptor P-selectin (P-sel), known for its role in inflammation, in these MPs' generation. P-sel, translocated from granules to the cell surfaces of activated platelets and endothelial cells, was recently found to play multiple roles in hemostasis. Expressed on endothelium, it can mediate platelet rolling. Signaling by P-sel through its receptor on leukocytes, P-selectin glycoprotein ligand 1 (PSGL-1), induces the generation of TF-positive, highly procoagulant MPs. In addition, P-sel on activated platelets helps to recruit these MPs specifically to thrombi. In this review, we discuss the roles of P-sel and TF-positive MPs and highlight strategies to modulate hemostasis by modulating the P-sel, TF, coagulation triad.

Antimicrobial susceptibility and genetic relatedness of Salmonella serovars isolated from animal-derived dog treats in the USA.

OBJECTIVES: The objectives of this study were to determine the potential risk of dog treats in transmitting Salmonella to humans in the USA, and to characterize genetic relatedness and antimicrobial resistance among the isolates. METHODS: A total of 158 dog treats derived from pig ears and other animal parts were randomly collected nationwide and assayed for the presence of Salmonella. The Salmonella isolates were characterized using serotyping, pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. RESULTS: Forty-one percent (65/158) of samples were positive for Salmonella. Eighty-four Salmonella isolates, comprising 24 serotypes, were recovered from the 65 positive samples. Fourteen samples were contaminated with more than one Salmonella serotype. PFGE analysis of 78 Salmonella isolates yielded 64 patterns. S. Infantis with PFGE patterns indistinguishable from those of strains identified in Canadian outbreaks in 1999 were recovered in several dog treat products. The majority of Salmonella isolates were susceptible to the antimicrobials tested; however, resistance was observed to tetracycline (26%), streptomycin (23%), sulfamethoxazole (19%), chloramphenicol (8%) and ampicillin (8%). Twenty-eight (36%) Salmonella isolates were resistant to at least one antimicrobial and 10 (13%) isolates displayed resistance to four or more antimicrobials. Two isolates were identified as S. Typhimurium DT104 with the characteristic penta-resistance phenotype (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline). One S. Brandenburg isolate was resistant to eight antimicrobials. Seven Salmonella isolates also contained class I integrons encoding resistance genes to aminoglycosides, beta-lactam and streptothricin antimicrobials. CONCLUSIONS: The study indicates that animal-derived dog treats in the USA could be a potential source of animal and human infections with Salmonella, including multidrug-resistant Salmonella strains.

Prevalence of streptogramin resistance genes among Enterococcus isolates recovered from retail meats in the Greater Washington DC area.

The prevalence of streptogramin resistance genes in enterococci recovered from retail poultry in the Greater Washington DC area was examined. Forty-three chicken and 32 turkey retail samples were analysed. Thirty-one non-Enterococcus faecalis enterococcal strains were isolated that displayed MICs of quinupristin-dalfopristin and virginiamycin of > or = 4 mg/L. These included Enterococcus faecium (turkey n = 4, chicken n = 23), Enterococcus gallinarum (turkey n = 2, chicken n = 1) and Enterococcus hirae (chicken n = 1). The presence of streptogramin resistance genes was examined by PCR in all non-E. faecalis isolates. The vat(E) gene was detected in 10/23 chicken E. faecium and from 2/4 turkey E. faecium. No other streptogramin resistance genes were detected by PCR. In addition, erm(B) was detected in all the E. faecium and E. gallinarum found in turkeys and in 7/23 E. faecium found in chickens. The vat(E) gene was transferable by conjugation from only two of the 12 E. faecium isolates (one from chicken and one from turkey). This study suggests that there is a high prevalence of low-level streptogramin resistance among enterococci found in retail poultry and that other, yet to be identified, mechanisms operate in these isolates that confer streptogramin resistance in enterococci.

Characterization of Tn1546 in vancomycin-resistant Enterococcus faecium isolated from canine urinary tract infections: evidence of gene exchange between human and animal enterococci.

Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included Enterococcus faecium (n = 13), Enterococcus faecalis (n = 7), Enterococcus gallinarum (n = 11), and Enterococcus casseliflavus (n = 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One E. faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 micro g/ml) and gentamicin (MIC > 2,048 micro g/ml). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance, and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from a human vancomycin-resistant E. faecium. Transposons Tn5281 and Tn1546 were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had an 889-bp deletion and an IS1216V insertion at the 5' end and an IS1251 insertion between vanS and vanH. To date, this particular form of Tn1546 has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant E. faecium isolated from a companion animal in the United States.

Identification of vat(E) in Enterococcus faecalis isolates from retail poultry and its transferability to Enterococcus faecium.

Sixteen isolates of Enterococcus faecalis were recovered from retail poultry samples (seven chickens and nine turkeys) purchased from grocery stores in the greater Washington, D.C., area. PCR for known streptogramin resistance genes identified vat(E) in five E. faecalis isolates (three isolates from chickens and two isolates from turkeys). The vat(E) gene was transmissible on a ca. 70-kb plasmid, along with resistance to erythromycin, tetracycline, and streptomycin, by conjugation to E. faecalis and Enterococcus faecium recipient strains. DNA sequencing showed little variation between E. faecalis vat(E) genes from the chicken samples; however, one E. faecalis vat(E) gene from a turkey sample possessed 5 nucleotide changes that resulted in four amino acid substitutions. None of these substitutions in the vat(E) allele have previously been described. This is the first report of vat(E) in E. faecalis and its transferability to E. faecium, which indicates that E. faecalis can act as a reservoir for the dissemination of vat(E)-mediated streptogramin resistance to E. faecium.

The food safety perspective of antibiotic resistance.

Bacterial antimicrobial resistance in both the medical and agricultural fields has become a serious problem worldwide. Antibiotic resistant strains of bacteria are an increasing threat to animal and human health, with resistance mechanisms having been identified and described for all known antimicrobials currently available for clinical use. There is currently increased public and scientific interest regarding the administration of therapeutic and sub-therapeutic antimicrobials to animals, due primarily to the emergence and dissemination of multiple antibiotic resistant zoonotic bacterial pathogens. This issue has been the subject of heated debates for many years, however, there is still no complete consensus on the significance of antimicrobial use in animals, or resistance in bacterial isolates from animals, on the development and dissemination of antibiotic resistance among human bacterial pathogens. In fact, the debate regarding antimicrobial use in animals and subsequent human health implications has been going on for over 30 years, beginning with the release of the Swann report in the United Kingdom. The latest report released by the National Research Council (1998) confirmed that there were substantial information gaps that contribute to the difficulty of assessing potential detrimental effects of antimicrobials in food animals on human health. Regardless of the controversy, bacterial pathogens of animal and human origin are becoming increasingly resistant to most frontline antimicrobials, including expanded-spectrum cephalosporins, aminoglycosides, and even fluoroquinolones. The lion's share of these antimicrobial resistant phenotypes is gained from extra-chromosomal genes that may impart resistance to an entire antimicrobial class. In recent years, a number of these resistance genes have been associated with large, transferable, extra-chromosomal DNA elements, called plasmids, on which may be other DNA mobile elements, such as transposons and integrons. These DNA mobile elements have been shown to transmit genetic determinants for several different antimicrobial resistance mechanisms and may account for the rapid dissemination of resistance genes among different bacteria. The increasing incidence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. Although much scientific information is available on this subject, many aspects of the development of antimicrobial resistance still remain uncertain. The emergence and dissemination of bacterial antimicrobial resistance is the result of numerous complex interactions among antimicrobials, microorganisms, and the surrounding environments. Although research has linked the use of antibiotics in agriculture to the emergence of antibiotic-resistant foodborne pathogens, debate still continues whether this role is significant enough to merit further regulation or restriction.