Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH, from a fowl cholera isolate of P. multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3 sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3 and 2-6 sialyl lactose, while NanH demonstrated activity only on 2-3 sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8 sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.Pasteurella multocida is a gram-negative coccobacillus of the family Pasteurellaceae and is a normal inhabitant of the upper respiratory system of many animals (24). The organism has a broad host range and is commonly a secondary pathogen in upper respiratory infections. Serotype D virulent isolates are toxigenic, but all serotypes produce capsules which confer serum resistance and resistance to phagocytosis (42). However, it is unusual to isolate a P. multocida strain that does not produce sialidase activity (40). Sialidases (neuraminidases; EC 3.2.1.18) are enzymes that liberate sialic acid from sialylconjugated glycoproteins, glycolipids, or colominic acids by cleaving alphaketosidic linkages. It is hypothesized that sialidase contributes to the virulence of some pathogenic organisms, especially those that inhabit and invade mucosal surfaces (7). Drzeniek (14) found sialidase activity in bacterial isolates that belong to the orders Pseudomonadales and Eubacteriales, and sialidases have been cloned from Clostridium species (35,36,37), Vibrio cholerae (48), Streptococcus pneumoniae (4, 5), Micromonospora viridifaciens (38), and Salmonella enterica serotype Typhimurium (21). Many of these bacterial sialidases have about 20% similarity at the amino acid level (21).Sialidases have been implicated as bacterial virulence factors (7, 34). It has been shown that a sialidase-deficient mutant of S. pneumoniae was less able to colonize and persist on mucosal surfaces than the wild type (46). In addition, a Bacteroides fragilis sialidase-deficient mutant was attenuated in the rat abscess model (18). The role of sialidase in ...

Multidrug-resistant opportunistic pathogens have become endemic to the veterinary hospital environment. Escherichia coli isolates resistant to 12 antibiotics were isolated from two dogs that were housed in the intensive care unit at The University of Georgia Veterinary Teaching Hospital within 48 h of each other. Review of 21 retrospective and prospective hospital-acquired E. coli infections revealed that the isolates had similar antibiotic resistance profiles, characterized by resistance to most cephalosporins, ␤-lactams, and the ␤-lactamase inhibitor clavulanic acid as well as resistance to tetracycline, spectinomycin, sulfonamides, chloramphenicol, and gentamicin. E. coli isolates with similar resistance profiles were also isolated from the environment in the intensive care unit and surgery wards. Multiple E. coli genetic types were endemic to the hospital environment, with the pulsed-field gel electrophoresis fingerprint identified among E. coli isolates from diseased animals and the hospital environment matching. The extended-spectrum cephalosporin resistance in these nosocomial E. coli isolates was attributed to the cephamycinase-encoding gene, bla CMY2 . Chloramphenicol resistance was due in part to the dissemination of the florfenicol resistance gene, flo, among these isolates. Resistance encoded by both genes was self-transmissible. Although bla CMY2 and flo were common to the polyclonal, nosocomial E. coli isolates, there was considerable diversity in the genetic compositions of class 1 integrons, especially among isolates belonging to the same genetic type. Two or more integrons were generally present in these isolates. The gene cassettes present within each integron ranged in size from 0.6 to 2.4 kb, although a 1.7-kb gene cassette was the most prevalent. The 1.7-kb gene cassette contained spectinomycin resistance gene aadA5 and trimethoprim resistance gene dfrA17.

Escherichia coli is a common inhabitant of the gastrointestinal tract of most animals. Like most pathogenic E. coli, avian isolates cannot be distinguished biochemically from the normal commensals inhabiting the gastrointestinal tract of birds. Using a molecular approach, we were able to identify genetic differences among avian E. coli isolates by restriction fragment length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) by the polymerase chain reaction (PCR). Several different RFLPs were observed among avian E. coli isolates using DNA probes for 16S ribosomal RNA genes (rrn) and insertion sequence elements (IS2). We were also able to observe differences in DNA banding patterns generated by RAPD analysis. Similarities and differences among avian E. coli were discernible using RFLPs and RAPD analysis, whereas conventional bacteriological methods failed to differentiate these isolates. Based on RAPD patterns, avian E. coli appear to be genetically diverse. Of 16 different RAPD types (RT) encountered, 84% of E. coli fell into seven major RTs. One RT was present in clinical isolates but absent from the commensals isolated in this study. Many of these different E. coli RTs were not geographically restricted to northern Georgia but were also observed in other southern states in the United States. Resistance to various antibiotics was randomly associated with different E. coli RTs. Sarafloxacin resistance was present among different E. coli RTs, suggesting that antibiotic usage is not selecting for a clonal population in avian E. coli. RAPD provides a rapid and powerful tool to study the epidemiology of avian E. coli.

No abstract