Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n ‫؍‬ 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC > 16 g/ml) and extended-spectrum-␤-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the bla CTX-M-1 -group ␤-lactamase genes in addition to one or more of the following ␤-lactamase genes: bla TEM , bla SHV-3 , bla CMY-2 , bla CTX-M-14-like , and bla OXA-1. Different bla TEM sequence variants were detected in some isolates (n ‫؍‬ 40). Three isolates harbored a bla TEM-181 gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases ؊28 and ؊29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of ␤-lactamase genes increased the MICs (>16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators.

There are an estimated 8 million users of smokeless tobacco products (STPs) in the United States, and yet limited data on microbial populations within these products exist. To better understand the potential microbiological risks associated with STP use, a study was conducted to provide a baseline microbiological profile of STPs. A total of 90 samples, representing 15 common STPs, were purchased in metropolitan areas in Little Rock, AR, and Washington, DC, in November 2012, March 2013, and July 2013. Bacterial populations were evaluated using culture, pyrosequencing, and denaturing gradient gel electrophoresis (DGGE). Moist-snuff products exhibited higher levels of bacteria (average of 1.05 ؋ 10 6 CFU/g STP) and diversity of bacterial populations than snus (average of 8.33 ؋ 10 1 CFU/g STP) and some chewing tobacco products (average of 2.54 ؋ 10 5 CFU/g STP). The most common species identified by culturing were Bacillus pumilus, B. licheniformis, B. safensis, and B. subtilis, followed by members of the genera Oceanobacillus, Staphylococcus, and Tetragenococcus. Pyrosequencing analyses of the 16S rRNA genes identified the genera Tetragenococcus, Carnobacterium, Lactobacillus, Geobacillus, Bacillus, and Staphylococcus as the predominant taxa. Several species identified are of possible concern due to their potential to cause opportunistic infections and reported abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N=-nitrosamines. This report provides a microbiological baseline to help fill knowledge gaps associated with microbiological risks of STPs and to inform potential regulations regarding manufacture and testing of STPs. IMPORTANCEIt is estimated that there 8 million users of smokeless tobacco products (STPs) in the United States; however, there are limited data on microbial populations that exist within these products. The current study was undertaken to better understand the potential microbiological risks associated with STP use and provide a baseline microbiological profile of STPs. Several bacterial species were identified that are of possible concern due to their potential to cause opportunistic infections. In addition, some species have abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobaccospecific N=-nitrosamines. Overall, this report provides a microbiological baseline to help fill knowledge gaps related to the microbiological risks of STPs and to inform potential regulations regarding the manufacture and testing of STPs.

Seventy-eight Salmonella enterica serovar Heidelberg isolates from humans were tested for antimicrobial susceptibility, resistance genes, and plasmids and genotyped by pulsed-field gel electrophoresis (PFGE). Most (88%) contained plasmids, and 47% were resistant to antimicrobials. The overall results were compared to those of previous S. Heidelberg studies of food-and animal-related sources, and multiple similarities were observed.

The degradation of phenanthrene and pyrene was investigated by using five different wood-decaying fungi. After 63 days of incubation in liquid culture, 13.8 and 4.3% of the [ring U-14 C]phenanthrene and 2.4 and 1.4% of the [4,5,9,10-14 C]pyrene were mineralized by Trametes versicolor and Kuehneromyces mutabilis, respectively. No 14 CO 2 evolution was detected in either [ 14 C]phenanthrene or [ 14 C]pyrene liquid cultures of Flammulina velutipes, Laetiporus sulphureus, and Agrocybe aegerita. Cultivation in straw cultures demonstrated that, in addition to T. versicolor (15.5%) and K. mutabilis (5.0%), L. sulphureus (10.7%) and A. aegerita (3.7%) were also capable of mineralizing phenanthrene in a period of 63 days. Additionally, K. mutabilis (6.7%), L. sulphureus (4.3%), and A. aegerita (3.3%) mineralized [ 14 C]pyrene in straw cultures. The highest mineralization of [ 14 C] pyrene was detected in straw cultures of T. versicolor (34.1%), which suggested that mineralization of both compounds by fungi may be independent of the number of aromatic rings. Phenanthrene and pyrene metabolites were purified by high-performance liquid chromatography and identified by UV absorption, mass, and 1 H nuclear magnetic resonance spectrometry. Fungi capable of mineralizing phenanthrene and pyrene in liquid culture produced enriched metabolites substituted in the K region (C-9,10 position of phenanthrene and C-4,5 position of pyrene), whereas all other fungi investigated produced metabolites substituted in the C-1,2, C-3,4, and C-9,10 positions of phenanthrene and the C-1 position of pyrene.

Three filamentous fungi were examined for the ability to biotransform phenanthrene to oxidative (phase I) and conjugative (phase II) metabolites. Phenanthrene metabolites were purified by high-performance liquid chromatography (HPLC) and identified by UV/visible absorption, mass, and 1H NMR spectra. Aspergillus niger ATCC 6275, Syncephalastrum racemosum UT-70, and Cunninghamella elegans ATCC 9245 initially transformed [9-(14)C]phenanthrene to produce metabolites at the 9,10-, 1,2-, and 3,4-positions. Subsequently, sulfate conjugates of phase I metabolites were formed by A. niger, S. racemosum, and C. elegans. Minor glucuronide conjugates of 9-phenanthrol and phenanthrene trans-9, 10-dihydrodiol were formed by S. racemosum and A. niger, respectively. In addition, C. elegans produced the glucose conjugates 1-phenanthryl beta-D-glucopyranoside and 2-hydroxy-1-phenanthryl beta-D-glucopyranoside, a novel metabolite. [9-(14)C]Phenanthrene metabolites were not detected in organic extracts from biotransformation experiments with the yeasts, Candida lipolytica 37-1, Candida tropicalis ATCC 32113, and Candida maltosa R-42.

Because fluoroquinolone antimicrobial agents may be released into the environment, the potential for environmental bacteria to biotransform these drugs was investigated. Eight Mycobacterium sp. cultures in a sorbitol-yeast extract medium were dosed with 100 g ml ؊1 of norfloxacin and incubated for 7 days. The MICs of norfloxacin for these strains, tested by an agar dilution method, were 1.6 to 25 g ml ؊1 . Cultures were extracted with ethyl acetate, and potential metabolites in the extracts were purified by high-performance liquid chromatography. The metabolites were identified using mass spectrometry and nuclear magnetic resonance spectroscopy. N-Acetylnorfloxacin (5 to 50% of the total absorbance at 280 nm) was produced by the eight Mycobacterium strains. N-Nitrosonorfloxacin (5 to 30% of the total absorbance) was also produced by Mycobacterium sp. strain PYR100 and Mycobacterium gilvum PYR-GCK. The MICs of N-nitrosonorfloxacin and N-acetylnorfloxacin were 2-to 38-and 4-to 1,000-fold higher, respectively, than those of norfloxacin for several different bacteria, including the two strains that produced both metabolites. Although N-nitrosonorfloxacin had less antibacterial activity, nitrosamines are potentially carcinogenic. The biotransformation of fluoroquinolones by mycobacteria may serve as a resistance mechanism.Environmental residues of fluoroquinolone antibacterial agents, such as ciprofloxacin and norfloxacin (11), are of concern because they may select for resistant strains of potentially pathogenic bacteria (12). Because fluoroquinolones are excreted largely unchanged (30), they may be released into the environment (28). Several fluoroquinolones are metabolized by soil fungi, but the role of bacteria in fluoroquinolone metabolism has been less studied (8,25,37).Norfloxacin is a fluoroquinolone used in the treatment of several bacterial diseases, including urinary tract infections in humans (17), enteritis in dogs (2), and chronic respiratory disease in chickens (34). A dextran-linked prodrug has been developed from norfloxacin for treatment of Mycobacterium bovis infections (10). Metabolism of norfloxacin via N acetylation, oxidation, and breakdown of the piperazine ring has been reported for humans (26) and fungi (25).Several Mycobacterium spp. biotransform fluoroquinolones, polycyclic aromatic hydrocarbons (PAHs), and other ring compounds (8, 13). Chen et al. (8) demonstrated by chromatographic techniques and radiolabeling methods that several soil microorganisms, including two strains of mycobacteria, biotransform the fluoroquinolone danofloxacin to N-desmethyldanofloxacin, 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, and other metabolites that may also include CO 2 . However, nothing is known about the bacterial transformation of other fluoroquinolones that may persist in the environment (11, 28).Based on this existing knowledge and on the potential of mycobacteria to degrade high-priority pollutants, such as PAHs (5, 9, 13), we screened extracts from dosed cultures...

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