Kristi L. Anderson

Effects of subtherapeutic concentrations of antimicrobials on gene acquisition events in Yersinia, Proteus, Shigella, and Salmonella recipient organisms in isolated ligated intestinal loops of swine

OBJECTIVE To assess antimicrobial resistance and transfer of virulence genes facilitated by subtherapeutic concentrations of antimicrobials in swine intestines. ANIMALS 20 anesthetized pigs experimentally inoculated with donor and recipient bacteria. PROCEDURES 4 recipient pathogenic bacteria (Salmonella enterica serotype Typhimurium, Yersinia enterocolitica, Shigella flexneri, or Proteus mirabilis) were incubated with donor bacteria in the presence of subinhibitory concentrations of 1 of 16 antimicrobials in isolated ligated intestinal loops in swine. Donor Escherichia coli contained transferrable antimicrobial resistance or virulence genes. After coincubations, intestinal contents were removed and assessed for pathogens that acquired new antimicrobial resistance or virulence genes following exposure to the subtherapeutic concentrations of antimicrobials. RESULTS 3 antimicrobials (apramycin, lincomycin, and neomycin) enhanced transfer of an antimicrobial resistance plasmid from commensal E coli organisms to Yersinia and Proteus organisms, whereas 7 antimicrobials (florfenicol, hygromycin, penicillin G, roxarsone, sulfamethazine, tetracycline, and tylosin) exacerbated transfer of an integron (Salmonella genomic island 1) from Salmonella organisms to Yersinia organisms. Sulfamethazine induced the transfer of Salmonella pathogenicity island 1 from pathogenic to nonpathogenic Salmonella organisms. Six antimicrobials (bacitracin, carbadox, erythromycin, sulfathiazole, tiamulin, and virginiamycin) did not mediate any transfer events. Sulfamethazine was the only antimicrobial implicated in 2 types of transfer events. CONCLUSIONS AND CLINICAL RELEVANCE 10 of 16 antimicrobials at subinhibitory or subtherapeutic concentrations augmented specific antimicrobial resistance or transfer of virulence genes into pathogenic bacteria in isolated intestinal loops in swine. Use of subtherapeutic antimicrobials in animal feed may be associated with unwanted collateral effects.

Comparisons of Salmonella conjugation and virulence gene hyperexpression mediated by rumen protozoa from domestic and exotic ruminants

Recent studies have identified a phenomenon in which ciliated protozoa engulf Salmonella and the intra-protozoal environment hyperactivates virulence gene expression and provides a venue for conjugal transfer of antibiotic resistance plasmids. The former observation is relegated to Salmonella bearing the SGI1 multiresistance integron while the latter phenomenon appears to be a more generalized event for recipient Salmonella. Our previous studies have assessed virulence gene hyperexpression only with protozoa from the bovine rumen while conjugal transfer has been demonstrated in rumen protozoa from cattle and goats. The present study examined virulence gene hyperexpression for Salmonella exposed to rumen protozoa obtained from cattle, sheep, goats, or two African ruminants (giraffe and bongo). Conjugal transfer was also assessed in these protozoa using Salmonella as the recipient. Virulence gene hyperexpression was only observed following exposure to the rumen protozoa from cattle and sheep while elevated virulence was also observed in these animals. Conjugal transfer events were, however, observed in all protozoa evaluated. It therefore appears that the protozoa-based hypervirulence is not universal to all ruminants while conjugal transfer is more ubiquitous.

Beta-lactam antibiotics prevent Salmonella-mediated bovine encephalopathy regardless of the β-lactam resistance status of the bacteria

This study assessed the capacity of β-lactam antibiotics to prevent salmonella-mediated encephalopathy in calves given the putative neuroprotective effects of these drugs of increasing glutamate export from the brain. Both ampicillin and ceftiofur prevented the development of encephalopathy despite resistance of the inoculated Salmonella enterica serovar Saint-Paul isolate to both drugs. A glutamate receptor antagonist also prevented this salmonella-mediated encephalopathy. Glutamate exporters were hyper-expressed in the presence of β-lactam antibiotics while a glutamate export inhibitor obviated the effects of these antibiotics, demonstrating a neuroprotective effect through glutamate export from the brain. The findings indicate that β-lactam antibiotics remain an important treatment option for this atypical form of bovine salmonellosis.

Abrogation of Salmonella and E. coli O157:H7 in Feedlot Cattle Fed a Proprietary Saccharomyces cerevisiae Fermentation Prototype

Salmonella and E. coli O157:H7 are insidious problems for the beef industry. Asymptomatic fecal shedding of these pathogens contaminates the hide and carcass. Furthermore, Salmonella are unique in their ability to infiltrate lymph nodes leading to the post-harvest contamination of ground beef. These contaminations yield the two most important food safety hazards associated with the consumption of beef. Herein, we report the anti-Salmonella and anti-E. coli O157:H7 effects of a novel Saccharomyces cerevisiae fermentation prototype (PRT; NaturSafeTM) fed to feedlot cattle. Cattle fed PRT were compared to those fed a combination of monensin, tylosin, and a direct-fed microbial- a standard conventional practice in the U.S. beef industry. In this investigator-blinded study, 1,495 feedlot heifers (300-400 kg) were fed PRT (n=747 heifers) or the standard industry diet (PC; n=748 heifers) without PRT for 125-146 days prior to slaughter. At the abattoir, fecal swabs were obtained from 400 animals (n=200/group) and subjected to selective culture for enumerating Salmonella and E. coli O157:H7. Additionally, subiliac lymph nodes were obtained from 400 carcasses for enumeration of Salmonella spp. Salmonella isolated from the feces and lymph nodes were subjected to a virulence assay and some antibiotic susceptibility and Salmonella serovar testing. When compared to cattle that received PC, Salmonella fecal shedding, lymph node infiltration, virulence, and antibiotic resistances were significantly decreased in cattle fed PRT. Additionally, PRT-fed cattle had a lower prevalence of certain Salmonella serovars (Newport, Typhimurium, and Dublin) and shed fewer E. coli O157:H7. The decrease in Salmonella virulence was associated with a decreased expression of hilA, a genetic regulator of Salmonella invasion into eukaryotic cells. This study revealed that a proprietary Saccharomyces cerevisiae fermentation prototype inhibits the shedding, lymph node carriage, downstream virulence, and antibiotic resistance of Salmonella residing in cattle beyond the standard conventional practice that includes monensin, tylosin, and a direct-fed microbial.

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.

Doxycycline as an inhibitor of p-glycoprotein in the alpaca for the purpose of maintaining avermectins in the CNS during treatment for parelaphostrongylosis.

Meningeal worms (Parelaphostrongylus tenuis) are a common malady of alpacas, often refractory to conventional treatments. Ivermectin is a very effective anthelmintic used against a variety of parasites but this drug is not consistently effective against alpaca meningeal worms once the parasite has gained access to the CNS, even if used in a protracted treatment protocol. Ivermectin is not effective against clinical cases of P. tenuis, raising the possibility that the drug is not sustained at therapeutic concentrations in the central nervous system (CNS). A specific protein (designated as p-glycoprotein (PGP)) effluxes ivermectin from the brain at the blood-brain barrier, thus hampering the maintenance of therapeutic concentrations of the drug in the CNS. Minocycline is a synthetic tetracycline antibiotic with an excellent safety profile in all animals tested to date. Minocycline has three unique characteristics that could be useful for treating meningeal worms in conjunction with ivermectin. First, minocycline is an inhibitor of PGP at the blood-brain barrier and this inhibition could maintain effective concentrations of ivermectin in the brain and meninges. Second, minocycline protects neurons in vivo through a number of different mechanisms and this neuroprotection could alleviate the potential untoward neurologic effects of meningeal worms. Third, minocycline is a highly lipid-soluble drug, thus facilitating efficient brain penetration. We thus hypothesized that minocycline will maintain ivermectin, or a related avermectin approved in ruminants (abamectin, doramectin, or eprinomectin), in the alpaca CNS. To test this hypothesis, we cloned the gene encoding the alpaca PGP, expressed the alpaca PGP in a heterologous expression system involving MDCK cells, and measured the ability of minocycline to inhibit the efflux of avermectins from the MDCK cells; doxycycline was used as a putative negative control (based on studies in other species). Our in vitro studies surprisingly revealed that doxycycline was effective at inhibiting the efflux of ivermectin and doramectin (minocycline had no effect). These two avermectins, in combination with doxycycline, should be considered when treating meningeal worms in alpacas.