Characterization of antibiotic and disinfectant susceptibility profiles among Pseudomonas aeruginosa veterinary isolates recovered during 1994-2003.

AIMS: To evaluate susceptibility of Pseudomonas aeruginosa veterinary isolates to antibiotics and disinfectants. METHODS AND RESULTS: Pseudomonas aeruginosa isolates collected from dogs (n = 155) and other animals (n = 20) from sixteen states during 1994-2003 were tested for susceptibility. Most isolates were resistant to twenty-one antimicrobials tested, and the highest prevalence of resistance was to ß-lactams (93.8%) and sulphonamides (93.5%). Fluoroquinolone resistance did not increase from 1994 to 2003. Ciprofloxacin and enrofloxacin had a 5 and 16% prevalence of resistance, respectively, while sarafloxacin and nalidixic acid had a prevalence of resistance of 97 and 98%, respectively. Strains were pan-resistant to triclosan and chlorhexidine, were highly resistant to benzalkonium chloride and demonstrated high susceptibility to other disinfectants. Didecyldimethylammonium chloride was the most active ammonium chloride. Inducible resistance was observed to cetyl ammonium halides, chlorhexidine and benzyl ammonium chlorides, which formulate disinfectants used in veterinary clinics and dairies. Organic acid inhibition was associated with the dissociated acid species. CONCLUSIONS: Dissociated organic acids appear able to inhibit Ps. aeruginosa, and rates of fluoroquinolone resistance merit sustained companion animal isolate surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of Ps. aeruginosa susceptibility to 24 disinfectants and illustrates the high resistance of Ps. aeruginosa to both antibiotics and disinfectants.

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated from canine infections.

AIMS: To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. METHODS AND RESULTS: Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed-field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the ß-lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4-aadA1, bla(OXA-31) -aadA2, aadA1-arr-3-catB3 and cmlA5-cmlA-aadA1). CONCLUSIONS: A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti-pseudomonal agents were observed in these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene bla(OXA-31) in a canine Ps. aeruginosa isolate.

Salmonella enterica isolates from pasture-raised poultry exhibit antimicrobial resistance and class I integrons.

AIMS: While considerable foodborne pathogen research has been conducted on conventionally produced broilers and turkeys, few studies have focused on free-range (organic) or pastured poultry. The current surveillance study was designed to isolate, identify and genetically characterize Salmonella from pastured poultry farm environment and from retail samples. METHODS AND RESULTS: In this study, 59 isolates were collected from two pastured poultry farms (n = 164; pens, feed, water and insect traps) and retail carcasses (n = 36) from a local natural foods store and a local processing plant. All isolates were serotyped and analysed phenotypically (antimicrobial resistance profiles) and genotypically (DNA fingerprints, plasmid profiles and integron analysis). Salmonella enterica was detected using standard microbiological methods. Salmonella Kentucky was the most prevalent serotype detected from the sampled sources (53%), followed by Salmonella Enteritidis (24%), Bareilly (10%), Mbandaka (7%), Montevideo (5%) or Newport (2%). All isolates were resistant to sulfisoxazole and novobiocin, and the majority (40/59) possessed class I integrons shown by PCR detection. Each Salmonella serotype elicited a distinct pulsed-field gel electrophoresis fingerprint profile, and unique differences were observed among the serotypes. CONCLUSIONS: The findings of this study show that Salmonella serotypes isolated from pasture-raised poultry exhibit antimicrobial resistance and class I integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that despite the cessation of antibiotic usage in poultry production, antibiotic resistant Salmonella may still be recovered from the environment and poultry products.

Salmonella challenges: prevalence in swine and poultry and potential pathogenicity of such isolates.

Salmonellosis is the second leading cause of bacterial foodborne illness in the United States, and the great majority of these infections are associated with the consumption of products such as meat, poultry, eggs, milk, seafood, and fresh produce contaminated with Salmonella. The per capita consumption of meat and poultry in United States has increased significantly over the past century. This increase is especially evident with poultry products, where there has been a nearly 6-fold increase in chicken consumption and 17-fold increase in turkey consumption since 1909. The per capita consumption of pork has also increased over this time from 18.7 to 21.7 kg/yr. With this increase in meat and poultry consumption, the dynamics of animal production and consumer exposure have changed leading to new challenges in limiting salmonellosis. To meet the demands of consumers, more intensive agricultural practices have been adopted, which has likely changed the population characteristics of Salmonella present among poultry flocks and swine populations. In Salmonella isolated from swine in the United States, S. Typhimurium has replaced S. Choleraesuis as the predominant serovar in recent years. Among isolates from turkeys collected in 2004, serovars S. Senftenberg and S. Hadar were most common overall; however, S. Heidelberg was most common from clinical diagnostic sources, potentially indicating increased virulence. Salmonella Heidelberg was also the most commonly detected serovar among chicken isolates from clinically ill birds and Salmonella surveillance samples. Overall among the 10 serovars most commonly associated with human infections, 6 are also found in the top serovars of swine and poultry. These include S. Typhimurium, S. Enteritidis, S. Heidelberg, S. Montevideo, S. Saintpaul, and S. I 4,[5],12:i:-.

Food animal-associated Salmonella challenges: pathogenicity and antimicrobial resistance.

Salmonellosis is a worldwide health problem; Salmonella infections are the second leading cause of bacterial foodborne illness in the United States. Approximately 95% of cases of human salmonellosis are associated with the consumption of contaminated products such as meat, poultry, eggs, milk, seafood, and fresh produce. Salmonella can cause a number of different disease syndromes including gastroenteritis, bacteremia, and typhoid fever, with the most common being gastroenteritis, which is often characterized by abdominal pain, nausea, vomiting, diarrhea, and headache. Typically the disease is self-limiting; however, with more severe manifestations such as bacteremia, antimicrobial therapy is often administered to treat the infection. Currently, there are over 2,500 identified serotypes of Salmonella. A smaller number of these serotypes are significantly associated with animal and human disease including Typhimurium, Enteritidis, Newport, Heidelberg, and Montevideo. Increasingly, isolates from these serotypes are being detected that demonstrate resistance to multiple antimicrobial agents, including third-generation cephalosporins, which are recommended for the treatment of severe infections. Many of the genes that encode resistance are located on transmissible elements such as plasmids that allow for potential transfer of resistance among strains. Plasmids are also known to harbor virulence factors that contribute to Salmonella pathogenicity. Several serotypes of medical importance, including Typhimurium, Enteritidis, Newport, Dublin, and Choleraesuis, are known to harbor virulence plasmids containing genes that code for fimbriae, serum resistance, and other factors. Additionally, many Salmonella contain pathogenicity islands scattered throughout their genomes that encode factors essential for bacterial adhesion, invasion, and infection. Salmonella have evolved several virulence and antimicrobial resistance mechanisms that allow for continued challenges to our public health infrastructure.

Characterization of Salmonella Typhimurium of animal origin obtained from the National Antimicrobial Resistance Monitoring System.

Salmonella Typhimurium remains one of the most common causes of salmonellosis in animals and humans in the United States. The emergence of multi-drug resistant Salmonella reduces the therapeutic options in cases of invasive infections, and has been shown to be associated with an increased burden of illness. In this study, 588 S. Typhimurium (including var. Copenhagen) isolates obtained from either animal diagnostic specimens (n = 199) or food animals after slaughter/processing (n = 389) were examined for antimicrobial susceptibility, presence of class-1 integrons, and characterized using pulsed-field gel electrophoresis and phage typing. Seventy-six percent (448/588) of isolates were resistant to at least one antimicrobial. Salmonella isolates displayed resistance most often to streptomycin (63%), tetracycline (61%), ampicillin (61%), and to a lesser extent, chloramphenicol (36%), ceftiofur (15%), gentamicin (9%), and nalidixic acid (4%), with more resistance observed among diagnostic isolates. Salmonella recovered from turkeys (n = 38) exhibited the highest rates of resistance, with 92% of isolates resistant to least one antimicrobial, and 58% resistant to > or =10 antimicrobials. Class 1 integrons were present in 51% of all isolates. Five integron associated resistance genes (aadA, aadB, pse-1, oxa-2 and dhfr) were identified. A total of 311 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained 146 isolates, including DT104 isolates obtained from all seven animal species. Results demonstrated a varied spectrum of antimicrobial resistance, including several multidrug resistant clonal groups, among S. Typhimurium and S. Typhimurium var. Copenhagen isolates recovered from both diagnostic and slaughter/processing samples.

Characterization of Salmonella enterica serotype newport isolated from humans and food animals.

Salmonella enterica serotype Newport isolates resistant to at least nine antimicrobials (including extended-spectrum cephalosporins), known as serotype Newport MDR-AmpC isolates, have been rapidly emerging as pathogens in both animals and humans throughout the United States. Resistance to extended-spectrum cephalosporins is associated with clinical failures, including death, in patients with systemic infections. In this study, 87 Salmonella serotype Newport strains were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing and examined for the presence of class 1 integrons and bla(CMY) genes. Thirty-five PFGE patterns were observed with XbaI, and three of these patterns were indistinguishable among isolates from humans and animals. Fifty-three (60%) Salmonella serotype Newport isolates were identified as serotype Newport MDR-AmpC, including 16 (53%) of 30 human isolates, 27 (93%) of 29 cattle isolates, 7 (70%) of 10 swine isolates, and 3 (30%) of 10 chicken isolates. However, 28 (32%) Salmonella serotype Newport isolates were susceptible to all 16 antimicrobials tested. The bla(CMY) gene was present in all serotype Newport MDR-AmpC isolates. Furthermore, the plasmid-mediated bla(CMY) gene was transferable via conjugation to an Escherichia coli strain. The transconjugant showed the MDR-AmpC resistance profile. Thirty-five (40%) of the isolates possessed class 1 integrons. Sequence analyses of the integrons showed that they contained aadA, which confers resistance to streptomycin, or aadA and dhfr, which confer resistance to trimethoprim-sulfamethoxazole. One integron from a swine isolate contained the sat-1 gene, which encodes resistance to streptothricin, an antimicrobial agent that has never been approved for use in the United States. In conclusion, Salmonella serotype Newport MDR-AmpC was commonly identified among Salmonella serotype Newport isolates recovered from humans and food animals. These findings support the possibility of transmission of this organism to humans through the food chain.

Resistance to serum complement, iss, and virulence of avian Escherichia coli.

Control of avian colibacillosis is hampered by lack of easily identifiable markers for virulent Escherichia coli. Resistance to serum complement appears to be a widespread trait of virulent avian E. coil, suggesting that bacterial factors promoting survival in serum may be useful in discriminating between virulent and avirulent isolates. Such distinguishing factors may prove useful in diagnostic protocols or as targets in future colibacillosis control protocols. Interestingly, the factors responsible for resistance to complement differ in the E. coli isolated from mammalian and avian hosts, which may reflect differences in the nature of avian and mammalian colibacillosis. In some cases, genetic determinants for serum complement resistance in avian E. coli are found on aerobactin- or Colicin V-encoding plasmids. One such gene, iss, first described for its role in the serum resistance associated with a ColV plasmid from a human E. coli isolate, occurs much more frequently in isolates from birds with colibacillosis than in faecal isolates from healthy birds. Efforts to identify the genomic location of iss in a single, virulent avian E. coli isolate have revealed that it occurs in association with several purported virulence genes, all linked to a large conjugative R plasmid. At this time, it is not known whether iss merely marks the presence of a larger pathogenicity unit or is itself a contributor to virulence. Nevertheless, the presence of the complement-resistance determinant, iss, may be a marker of virulent avian E. coli exploitable in controlling avian colibacillosis.

Virulence factors of Escherichia cofi from cellulitis or colisepticemia lesions in chickens.

This study was designed to compare virulence factors of cellulitis-derived Escherichia coli to colisepticemic E. coli in order to clarify whether E. coli associated with cellulitis comprise a unique subset of pathogenic E. coli. Isolates were tested for serotype, capsule, aerobactin production, colicin production, the presence of the iss gene, and serum resistance. Untypable isolates made up the greatest percentage of each group. Serotypes O2 and O78 were the most commonly identified among both groups of isolates. No statistical differences in the distribution of aerobactin or colicin production, capsule, or iss gene were observed between groups. Cluster analysis showed that 90% of the E. coli isolates had greater than 42% livability in serum-resistance tests. No separation of colisepticemic vs. cellulitis E. coli isolates was observed on the basis of SR. Colicin production by E. coli was highly correlated with serum resistance (P = 0.0029). These data suggest that cellulitis E. coli have virulence traits similar to those of colisepticemic E. coli.

Iss from a virulent avian Escherichia coli.

No single characteristic of virulent avian Escherichia coli has been identified that can be exploited in colibacillosis detection protocols. Research in our lab suggests a strong association between the presence of an iss DNA sequence with an isolate's disease-causing ability. The study presented here focuses on the techniques used in the expression, purification, and characterization of avian E. coli Iss protein. In brief, iss was cloned into an expression vector, the construct was transformed into a protease-deficient E. coli, and expression was induced. The protein was expressed as a glutathione-S-transferase (GST) fusion and purified by affinity chromatography. The GST portion was cleaved from Iss, Iss was harvested by affinity chromatography, and the identity of Iss was confirmed by N-terminal sequencing. Currently, purified Iss is being used to prepare hybridomas for production of monoclonal antibodies with the goal of evaluating anti-Iss as a reagent for the detection of virulent avian E. coli.