Florfenicol, a veterinary fluorinated analog of thiamphenicol, is approved for treatment of bovine respiratory pathogens in the United States. However, florfenicol resistance has recently emerged among veterinary Escherichia coli isolates incriminated in bovine diarrhea. The flo gene, which confers resistance to florfenicol and chloramphenicol, has previously been identified inPhotobacterium piscicida and Salmonella enterica serovar Typhimurium DT104. The flo gene product is closely related to the CmlA protein identified inPseudomonas aeruginosa. The cmlA gene confers nonenzymatic chloramphenicol resistance via an efflux mechanism. Forty-eight E. coli isolates recovered from calves with diarrhea, including 41 that were both chloramphenicol and florfenicol resistant, were assayed for the presence of both flo andcmlA genes. Forty-two of the 44 isolates for which florfenicol MICs were ≥16 μg/ml were positive via PCR for theflo gene. All E. coli isolates for which florfenicol MICs were ≤8 μg/ml were negative for the flogene (n = 4). Twelve E. coli isolates were positive for cmlA, and chloramphenicol MICs for all 12 were ≥32 μg/ml. Additionally, eight isolates were positive for bothflo and cmlA, and both florfenicol and chloramphenicol MICs for these isolates were ≥64 μg/ml. DNA sequence analysis of the E. coli flo gene demonstrated 98% identity to the published GenBank sequences of both serovar TyphimuriumfloSt and P. piscicida pp-flo. Theflo gene was identified on high-molecular-weight plasmids of approximately 225 kb among the majority of florfenicol-resistantE. coli isolates. However, not all of the florfenicol-resistant E. coli isolates tested contained the large flo-positive plasmids. This suggests that several of the E. coli isolates may possess a chromosomalflo gene. The E. coli flo gene specifies nonenzymatic cross-resistance to both florfenicol and chloramphenicol, and its presence among bovine E. coli isolates of diverse genetic backgrounds indicates a distribution much wider than previously thought.

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Salmonella enterica serotype typhimurium(S. typhimurium) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 was determined by producing antibiotic-resistant Escherichia coli host strain clones from DT104 DNA. DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes floS. typhimurium (floSt ),int, invA, and spvC were produced for colony blot hybridizations. One hundred nine Salmonellaisolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The genefloSt , reported in this study, confers chloramphenicol and florfenicol resistance on S. typhimurium DT104. Florfenicol resistance is unique to S. typhimurium DT104 and multidrug-resistant S. typhimurium isolates with the same drug resistance profile among all isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the floSt -positive genotype. Resistances to florfenicol and chloramphenicol are conferred by the genefloSt , described in this paper. Presumptive identification of S. typhimurium DT104 can be made rapidly based on the presence of the floSt gene or its resulting phenotype.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within eachrfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe toEscherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coliO157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.

We have cloned fragments of DNA (up to 13 kb), from Pseudomonas putida AJ, that code for two stereospecific haloalkanoate dehalogenases. These enzymes are highly specific for D and L substrates. The two genes, designated hadD and hadL, have been isolated and independently expressed in Escherichia coli and P. putida hosts by using broad-host-range vectors. Pseudomonas putida AJ was isolated from soil samples exposed to 2-chloropropionate and shown to contain both Dand L-stereospecific 2-haloalkanoate dehalogenases (13). The purification and characterization of the D-specific enzyme have been described recently (25). It is highly isomer specific, converting D-chloropropionate with isomeric inversion to lactate. We have used this strain to clone both the Dand L-specific dehalogenase genes (hadD and hadL) separately and together into Escherichia coli.To facilitate the transfer of these genes between various bacterial species, we used the broad-host-range IncQ vectors that we have developed in recent years as cloning vectors. DNA sequence data for the D-specific gene and an upstream open reading frame are presented. These sequences and their deduced oligopeptides appear to have no significant homologies with previously described sequences.The hadL sequence is presented elsewhere (15).

The objective of this study was to define combinations of pH, salt, and moisture that produce growth, stasis, or inactivation of Listeria monocytogenes in Mexican-style cheese. A soft, directly acidified, rennet-coagulated, fresh cheese similar to Mexican-style cheese was produced. The cheese was subsequently altered in composition as required by the experimental protocol. A factorial design with four moisture contents (42, 50, 55, and 60%), four salt concentrations (2.0, 4.0, 6.0, and 8.0% wt/wt), six pH levels (5.0, 5.25, 5.50, 5.75, 6.0, and 6.5), and three replications was used. Observations of growth, stasis, or death were obtained for each combination after 21 and 42 days of incubation at 10 degrees C. Binary logistic regression was used to develop an equation to determine the probability of growth or no growth for any combination within the range of the data set. In addition, ordinal logistic regression was used to calculate proportional odds ratios for growth, stasis, and death for each treatment combination. Ordinal logistic regression was also used to develop equations to determine the probability of growth, stasis, and death for formulations within the range of the data set. Models were validated with independently produced data. Of 60 samples formulated to have a 5% probability of Listeria growth (pH, 5.0 to 6.0; brine concentration, 8.17 to 16.00%), none supported growth. Of 30 samples formulated to have 50% probability of growth using the binary model (pH, 5.50 to 6.50; brine concentration, 3.23 to 12.50%), 20 supported growth. Of 30 samples formulated to have a 50% probability of growth according to the ordinal model (pH, 5.50 to 6.50; brine concentration, 3.37 to 10.90%), 16 supported growth. These data indicate that the logistic regression models presented accurately predict the behavior of L. monocytogenes in Mexican-style cheese.

L .F . B O LT ON A ND J. F . F RA N K. 1999. A simple, novel method for determining stressadaptive response of Listeria monocytogenes in food systems is presented. The method involves plating samples on Listeria-selective agar (LSA) acidified to pH 5·25 with incubation at 36°C for 60 h to detect acid adaptation and plating on LSA with 70 gl −1 NaCl and incubation at 7°C for 7 d to detect cold-osmotic adaptation. Adapted cells produced larger colonies (×1 mm) under these conditions than unadapted cells. Scot A (97%) and Brie-1 (100%) cells incubated in milk at pH 5 for 3 h manifested the acid-adapted colony type compared with 6% and 21% of viable cells in the unstressed control population. After a 5-d adaptation period at 4°C in milk with 80 gl −1 salt, 29% of Scot A and 91% of Brie-1 viable cells exhibited the adapted colony type compared with ³1% of the unstressed control population. Stress-adapted L. monocytogenes were isolated from soft cheese held for 42 d at 10 C.

In 2006, DuPont Qualicon introduced the BAX<sup/> system Q7 instrument for use with its assays. To demonstrate the equivalence of the new and old instruments, a validation study was conducted using the BAX system PCR Assay for Salmonella, AOAC Official MethodSM 2003.09, on three food types. The foods were simultaneously analyzed with the BAX system Q7 instrument and either the U.S. Food and Drug Administration Bacteriological Analytical Manual or the U.S. Department of AgricultureFood Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Comparable performance between the BAX system and the reference methods was observed. Of the 75 paired samples analyzed, 39 samples were positive by both the BAX system and reference methods, and 36 samples were negative by both the BAX system and reference methods, demonstrating 100 correlation. Inclusivity and exclusivity for the BAX system Q7 instrument were also established by testing 50 Salmonella strains and 20 non-Salmonella isolates. All Salmonella strains returned positive results, and all non-Salmonella isolates returned a negative response.