Molecular mechanisms for anthelmintic resistance in strongyle nematode parasites of veterinary importance.

Veterinarians rely on a relatively limited spectrum of anthelmintic agents to control nematode parasites in domestic animals. Unfortunately, anthelmintic resistance has been an emerging problem in veterinary medicine. In particular, resistance has emerged among the strongyles, a group of gastrointestinal nematodes that infect a variety of hosts that range from large herbivores to small companion animals. Over the last several decades, a great deal of research effort has been directed toward developing an understanding of the mechanisms conferring resistance against the three major groups of anthelmintics: macrocyclic lactones, benzimidazoles, and nicotinic agonists. Our understanding of anthelmintic resistance has been largely formed by determining the mechanism of action for each drug class and then evaluating drug-resistant nematode isolates for mutations or differences in expression of target genes. More recently, drug efflux pumps have been recognized for their potential contribution to anthelmintic resistance. In this mini-review, we summarize the evidence for mechanisms of resistance in strongyle nematodes.

Inhibition of the virulence, antibiotic resistance, and fecal shedding of multiple antibiotic-resistant Salmonella Typhimurium in broilers fed Original XPC™.

Salmonella carriage is an insidious problem for the poultry industry. While most Salmonella serotypes are avirulent in poultry, these bacteria can contaminate chicken meat during processing, leading to one of the most important food safety hazards. In this study, we examined the anti-Salmonella effects of Diamond V Original XPC™ (XPC) included in the finisher diet fed to commercial broilers. On 3 occasions between day one (D1) and D20, broilers were experimentally infected with multiple antibiotic-resistant Salmonella Typhimurium. After confirming that the chicks were shedding Salmonella in the feces on D21, broiler chicks were fed a diet containing XPC (n = 57 birds; 1.25 kg/MT) or an XPC-free control diet (CON) (n = 57 birds) to D49. Fecal samples were obtained weekly and subjected to selective culture for enumerating and determining the antibiotic resistance of the Salmonella Salmonella isolates were then subjected to an in vitro virulence assay, which predicts the ability of Salmonella to cause illness in a mammalian host. Broilers were euthanized on D49 and a segment of the large intestine was removed and subjected to the same assays used for the fecal samples. When compared to the birds fed the CON diet, Salmonella fecal shedding, virulence (invasion and invasion gene expression), and antibiotic resistance were significantly decreased in birds fed XPC (5-fold, 7.5-fold, 6-fold, and 5.3-fold decreases, respectively). Birds fed XPC exhibited heavier body weight (BW) and greater BW gains than those fed the CON diet. The decrease in virulence was associated with a decreased expression of a genetic regulator of Salmonella invasion into cells (hilA), while the decrease in antibiotic resistance was due to a loss of an integron (SGI1) from the input strain. This study revealed that Original XPC™ inhibits the shedding, downstream virulence, and antibiotic resistance of Salmonella residing in broilers.

Non-typhoidal Salmonella encephalopathy involving lipopolysaccharide in cattle.

This study assessed the involvement of lipopolysaccharide (LPS) in the non-typhoidal Salmonella encephalopathy (NTSE) caused by a unique isolate of Salmonella enterica serovar Saint-paul (SstpNPG). NTSE was prevented by genetic (deletion of murE) or pharmacologic (polymyxin) disruption of LPS on SstpNPG although the disruption of LPS did not deter brain penetration of the strain. This is the first study to demonstrate that LPS is involved in the manifestations of NTSE.

Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena.

In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs) as transducers of Salmonella engulfment by the model protozoan Tetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena) engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates by Tetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymena was assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule.

Experimental reproduction of bovine Salmonella encephalopathy using a norepinephrine-based stress model.

Neurological disease represents a sporadic but serious manifestation of bovine salmonellosis that is thought to be related to systemic infection. Salmonella enterica serovar Dublin (S. Dublin) is the serovar most associated with systemic infection in cattle, although reports of neurological disease associated with S. Dublin or any other serovar are rare and usually anecdotal. This study reports the involvement of three strains of S. enterica, serovars Saintpaul, Montevideo, and Enteritidis, in Salmonella encephalopathies. Encephalopathies were reproduced in calves using a norepinephrine-based stress model. Neurological signs were not observed in calves infected with control strains of S. enterica, including S. Dublin, or in calves infected with clinical strains in the absence of norepinephrine. Therefore, norepinephrine may play a role in Salmonella encephalopathies.

Klebsiella to Salmonella gene transfer within rumen protozoa: implications for antibiotic resistance and rumen defaunation.

The rumen has long been thought to be a site of gene transfer for microorganisms. Rumen protozoa (RPz) are active predators of bacteria that can harbor antibiotic resistance genes. In this study, RPz were assessed as sites of gene transfer between two bacterial species, Salmonella and Klebsiella. One Klebsiella isolate carried a plasmid bearing bla(CMY-2), encoding an extended-spectrum beta-lactamase conferring ceftriaxone resistance, while the Salmonella was susceptible to ceftriaxone yet capable of thriving within protozoa. In vitro studies revealed that ceftriaxone-resistant Salmonella could be isolated following co-incubation of Salmonella and Klebsiella with RPz obtained from adult cattle and goats. Ceftriaxone-resistant Salmonella were not recovered in the presence of an inhibitor of protozoa engulfment or when a protozoa-sensitive Salmonella was part of the co-incubation. This transfer event was additionally observed in vitro for protozoa-independent stressors although at a significantly lower frequency. The gene transfer event was related to bacterial conjugation since a conjugation inhibitor, nalidixic acid, perturbed the phenomenon. Ceftriaxone-resistant Salmonella were recovered from calves, sheep, and goats co-challenged with ceftriaxone-resistant Klebsiella and ceftriaxone-sensitive Salmonella. However, the transfer event was not observed in calves and sheep that were defaunated prior to the co-challenge. Moreover, Salmonella transconjugants were isolated from separate bovine in vivo studies involving a Klebsiella donor carrying a plasmid conferring colicin activity while no such transconjugants were obtained from defaunated calves. These results provide an important basis for evaluating and preventing the spread of antibiotic resistance and other selective advantages for pathogens present in ruminants.

Early epithelial invasion by Salmonella enterica serovar Typhimurium DT104 in the swine ileum.

Salmonella enterica serovar Typhimurium is an important intestinal pathogen in swine. This study was performed to document the early cellular invasion of Salmonella serovar Typhimurium in swine ileum. Ileal gut-loops were surgically prepared in ten 4- to 5-week-old mixed-breed pigs and inoculated for 0-60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy (SEM and TEM, respectively). Preferential bacterial adherence to microfold cells (M cells) was seen within 5 minutes, and by 10 minutes bacterial invasion of the apical membrane was seen in M cells, goblet cells, and enterocytes. This multicellular invasion was observed throughout the course of infection. In addition, SEM revealed a specific affinity of Salmonella serovar Typhimurium to sites of cell extrusion. Using TEM, bacteria in these areas were focused in the crevices formed by the extruding cell and the adjacent cells and in the cytoplasm immediately beneath the extruding cell. Our results suggest that early cellular invasion by Salmonella serovar Typhimurium is nonspecific and rapid in swine. Furthermore, the combination of SEM and TEM data suggests that Salmonella serovar Typhimurium may use sites of cell extrusion as an additional mechanism for early invasion.

Abomasitis associated with multiple antibiotic resistant Salmonella enterica serotype Typhimurium phagetype DT104.

Salmonella enterica serotype Typhimurium phagetype DT104 is a multiple antibiotic resistant pathogen that has been purported to be more pathogenic than other Salmonella. In this study, we evaluated the possibility that DT104 is the causative agent of veal calf abomasitis observed in four independent outbreaks of salmonellosis. This study was undertaken to determine if the outbreaks might be due to hypervirulent S. enterica serotype Typhimurium phagetype DT104 (DT104) since Salmonella does not usually cause abomasitis. Tissues and fluids from these calves were subjected to bacteriologic culture. Pure Salmonella cultures were then used in bovine challenge experiments. DT104 was identified as the causative agent of abomasitis in calves. Thus, abomasitis is a potential indicator of infection with multiple antibiotic resistant DT104 and adds credence to the apparent hypervirulence of this pathogen.

Secretion of a putative cytotoxin in multiple antibiotic resistant Salmonella enterica serotype Typhimurium phagetype DT104.

Salmonella enterica serotype Typhimurium phagetype DT104 (DT104) is a multiple antibiotic-resistant pathogen. DT104 infections have been reported in a multitude of hosts including humans, companion animals, livestock and wildlife. Recently, several isolates of DT104 were recovered from veal calves exhibiting abomasitis, a finding that is inconsistent with classic salmonellosis. One of these isolates was used in murine ligated loop experiments where it was observed that multiresistant DT104 can elaborate a putative cytotoxin. Thus it appears that DT104 has the ability to evade pharmacologic interventions, via antibiotic resistance, and elaborate a toxin that can damage cells.

Antibiotic resistance in Salmonella enterica serovar Typhimurium exposed to microcin-producing Escherichia coli.

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.

Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype typhimurium phage type DT104.

PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 422 veterinary isolates of Salmonella enterica serotype Typhimurium. The identities of extra-integron genes encoding resistance to streptomycin, gentamicin, kanamycin, and apramycin were evaluated. Gentamicin resistance was conferred by the aadB gene. Kanamycin resistance was encoded by either the aphA1-Iab gene or the Kn gene. Apramycin resistance was determined by the aacC4 gene. Analysis of gene distribution did not reveal significant differences with regard to phage type, host species, or region except for the Kn gene, which was found mostly in nonclinical isolates. The data from this study indicate that pentaresistant DT104 does not acquire extra-integron genes in species- or geography-related foci, which supports the hypothesis that clonal expansion is the method of spread of this organism.

Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture.

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in

Evaluation of invasion-conferring genotypes and antibiotic-induced hyperinvasive phenotypes in multiple antibiotic resistant Salmonella typhimurium DT104.

Antibiotic resistance in pathogenic bacteria is a problem in both industrialized and developing countries. This is especially evident in Salmonella typhimurium, a foodborne pathogen that causes gastrointestinal and systemic disease throughout the world. S. typhimurium DT104 further poses a major health concern due to its apparent enhanced ability to acquire multiple antibiotic resistance genes and its putative hypervirulent phenotype. Recently, we demonstrated that multiresistant S. typhimurium do not appear to be more invasive than non-resistant cohorts. In the present study, we evaluated the presence of Salmonella pathogenicity island 1 (SPI1) flanking and internal sequences in over 400 isolates of multiresistant S. typhimurium. With these same isolates, we also used a tissue culture invasion assay to evaluate a potential relationship between antibiotic exposure and a hyperinvasive phenotype. Our studies revealed that SPI1 flanking sequences are similar in multiresistant and non-resistant S. typhimurium. Furthermore, we failed to identify any isolates that were hyperinvasive in the presence of any of the 14 antibiotics evaluated. These results further indicate that the putative hypervirulence of multiresistant S. typhimurium is not likely to occur at the level of invasion.

Augmentation of antibiotic resistance in Salmonella typhimurium DT104 following exposure to penicillin derivatives.

Antibiotic resistance in pathogenic bacteria has been a problem in both developed and developing countries. This problem is especially evident in Salmonella typhimurium, one of the most prevalent foodborne pathogens. While performing in vitro gentamicin protection-based invasion assays, we found that certain isolates of multiresistant S. typhimurium can be 'induced' to exhibit new resistance profiles. That is, bacteria become resistant to a wider range of antibiotics and they also exhibit quantitative increases in MIC values for antibiotics that were part of their pre-induction antibiograms. This 'induction' process involves growing the bacteria to stationary phase in the presence of antibiotics such as ampicillin, amoxicillin or ticarcillin. Since the isolates studied exhibited resistance to ampicillin, amoxicillin and ticarcillin prior to exposing the bacteria to these antibiotics, the observed phenomenon suggests that resistant Salmonella not only have a selective advantage over non-resistant Salmonella but their resistance phenotypes can be accentuated when an inappropriate antibiotic is used therapeutically.

Identification of diminished tissue culture invasiveness among multiple antibiotic resistant Salmonella typhimurium DT104.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium further poses a major health concern due to its apparent enhanced ability to acquire multiple antibiotic resistance genes. Currently it is unclear if multiresistant S. typhimurium are more or less pathogenic than non-resistant counterparts. Using an in vitro invasion assay, we evaluated the relative pathogenicity of over 400 multiresistant S. typhimurium isolates. Our studies failed to identify any <<<>>> isolates. However, we identified 12 isolates exhibiting invasive phenotypes that were constrained relative to controls. These <<<>>> strains were found in a variety of phagetypes all possessing at least a hexaresistant profile. Further studies revealed that the alterations in invasion were not due to changes in adherence. Limited studies exploring in vivo virulence revealed a mildly decreased ability to cause murine lethality for the hypoinvasive strain examined. These results indicate that the ability to cause disease is not increased but is rather mildly attenuated for certain isolates of multiresistant S. typhimurium.

Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant S. typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistant S. typhimurium DT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in S. typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments from S. typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistant Salmonella such as S. typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant S. typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected in only 3-4 h.

Inhibition of Salmonella typhimurium invasion by host cell expression of secreted bacterial invasion proteins.

Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability of Salmonella typhimurium to enter mammalian cells that express portions of the Salmonella invasion proteins SipB and SipC. Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor. Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blocked Salmonella invasion, whereas expression of their chimeric counterparts had no effect on invasion. The effect on invasion was specific for Salmonella since the perturbation of uptake was not extended to other invasive bacterial species. These results suggest that Salmonella invasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC. In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.

Use of the alveolar-arterial oxygen gradient in the assessment of acute pulmonary embolism.

To evaluate the utility of the alveolar-arterial (A-a) oxygen gradient in the diagnosis of acute pulmonary embolism (PE), a retrospective analysis was done of consecutive emergency department patients who underwent pulmonary angiography for the presumed diagnosis of acute PE. Patients were categorized into two groups depending on the presence or absence of prior cardiopulmonary disease. Arterial blood gas samples were chosen for analysis only if obtained when the PE first was suspected clinically (before lung scans or angiograms) and the patient was breathing room air. A total of 152 patients met all study criteria; 59 patients (39%) had angiographically documented emboli. In comparison with the study patients in whom PE was excluded, there was no significant difference in mean PaO2 (64 v 67 mm Hg) or A-a gradient (39 v 36 mm Hg). Various combinations of the A-a gradient and blood gas levels failed to exclude PE in more than 35% of patients with no prior cardiovascular disease and in 25% of patients with prior cardiovascular disease. The A-a gradient did show a linear correlation with the severity of the PE, as assessed by the PaO2 (r = -0.87) and pulmonary artery mean pressure (r = 0.63). These results indicate that the A-a oxygen gradient, in combination with blood gas levels, may contribute to the formulation of a clinical assessment. However, these laboratory parameters are of insufficient discriminant value to permit exclusion of the diagnosis of PE.