An amoxicillin-resistant (Amox r ) strain of Helicobacter pylori was selected for by culturing an amoxicillinsensitive (Amox s ) strain in increasingly higher concentrations of amoxicillin, resulting in a 133-fold increase in MIC, from 0.03 to 0.06 g/ml to 4 to 8 g/ml. This resistance was stable upon freezing for at least 6 months and conferred cross-resistance to seven other ␤-lactam antibiotics. ␤-Lactamase activity was not detected in this Amox r strain; however, analysis of the penicillin-binding protein (PBP) profiles generated from isolated bacterial membranes of the Amox s parental strain and the Amox r strain revealed a significant decrease in labeling of PBP 1 by biotinylated amoxicillin (bio-Amox) in the Amox r strain. Comparative binding studies of PBP 1 for several ␤-lactams demonstrated that PBP 1 in the Amox r strain had decreased affinity for mezlocillin but not significantly decreased affinity for penicillin G. In addition, PBP profiles prepared from whole bacterial cells showed decreased labeling of PBP 1 and PBP 2 in the Amox r strain at all bio-Amox concentrations tested, suggesting a diffusional barrier to bio-Amox or a possible antibiotic efflux mechanism. Uptake analysis of 14 C-labeled penicillin G showed a significant decrease in uptake of the labeled antibiotic by the Amox r strain compared to the Amox s strain, which was not affected by pretreatment with carbonyl cyanide m-chlorophenylhydrazone, eliminating the possibility of an efflux mechanism in the resistant strain. These results demonstrate that alterations in PBP 1 and in the uptake of ␤-lactam antibiotics in H. pylori can be selected for by prolonged exposure to amoxicillin, resulting in increased resistance to this antibiotic.Helicobacter pylori is the most common cause of gastric and duodenal ulcers and is strongly associated with the development of gastric adenocarcinoma (see references 14 and 32 for reviews). It is estimated that at least a third of the world's population is infected with H. pylori, making it one of the most common infections in humans (14). Successful treatment of H. pylori infections most often employs the use of two or more antibiotics and the addition of either bismuth or a proton pump inhibitor (14,17,18). However, H. pylori resistance to many of the commonly used antibiotics in this triple regimen is rising (19), including resistance to metronidazole (1, 27, 36), clarithromycin (1, 6, 9, 27), rifampin or rifabutin (24), and, recently, amoxicillin (11, 12, 13, 22, 38).Resistance to ␤-lactam antibiotics by gram-negative bacteria is most commonly due to the production of ␤-lactamase, either chromosomally encoded or, more often, plasmid mediated (see reference 30 for a review). Other important mechanisms of resistance include alterations in penicillin-binding proteins (PBPs), decreased permeation of the antibiotic into the bacterial cell, or combinations of these resistance strategies (see reference 28 for a review). Active efflux pumps in gramnegative bacteria which excrete drugs, including multidrug effl...

Vibrio fischeri, a luminescent marine bacterium, specifically colonizes the light organ of its symbiotic partner, the Hawaiian squid Euprymna scolopes. In a screen for V. fischeri colonization mutants, we identified a strain that exhibited on average a 10-fold decrease in colonization levels relative to that achieved by wild-type V. fischeri. Further characterization revealed that this defect did not result from reduced luminescence or motility, two processes required for normal colonization. We determined that the transposon in this mutant disrupted a gene with high sequence identity to the pgm (phosphoglucomutase) gene of Escherichia coli, which encodes an enzyme that functions in both galactose metabolism and the synthesis of UDP-glucose. The V. fischeri mutant grew poorly with galactose as a sole carbon source and was defective for phosphoglucomutase activity, suggesting functional identity between E. coli Pgm and the product of the V. fischeri gene, which was therefore designated pgm. In addition, lipopolysaccharide profiles of the mutant were distinct from that of the parent strain and the mutant exhibited increased sensitivity to various cationic agents and detergents. Chromosomal complementation with the wild-type pgm allele restored the colonization ability to the mutant and also complemented the other noted defects. Unlike the pgm mutant, a galactose-utilization mutant (galK) of V. fischeri colonized juvenile squid to wild-type levels, indicating that the symbiotic defect of the pgm mutant is not due to an inability to catabolize galactose. Thus, pgm represents a new gene required for promoting colonization of E. scolopes by V. fischeri.

The penicillin-binding proteins (PBPs) of helical (log-phase)Helicobacter pylori ATCC 43579 were identified by using biotinylated ampicillin. The major PBPs had apparent molecular masses of 47, 60, 63, and 66 kDa; an additional minor PBP of 95 to 100 kDa was also detected. The relative affinities of various β-lactams for these PBPs were tested by competitive-binding assays. Only PBP63 appeared to be significantly bound to each of the competing antibiotics, whereas PBP66 strongly bound mezlocillin, oxacillin, amoxicillin, and ceftriaxone. Whereas most of the β-lactams significantly bound two or more PBPs, aztreonam specifically targeted PBP63. The influence of sub-MICs of these β-lactams on the morphologies of log-phase H. pylori was observed at both the phase-contrast and transmission electron microscopy levels. Each of the eight β-lactams examined induced blebbing and sphere formation, whereas aztreonam was the only antibiotic studied which induced pronounced filamentation in H. pylori. Finally, studies comparing the PBPs of helical (log-phase) cultures with those of coccoid (7-, 14-, and 21-day-old) cultures of H. pylori revealed that the major PBPs at 60 and 63 kDa seen in the helical form were almost undetectable in the coccoid forms, whereas PBP66 remained the major PBP in the coccoid forms, although somewhat reduced in level compared to the helical form. PBP47 was present in both forms at approximately equal concentrations. These studies thus identified the major PBPs in both helical and coccoid forms of H. pylori and compared the relative affinities of seven different β-lactams for the PBPs in the helical forms and their effects on bacterial morphology.

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