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Acute Helicobacter pylori infection produces hypochlorhydria. The decrease in acid facilitates survival of the bacterium and its colonization of the stomach. The present study was designed to identify the pathways in oxyntic mucosa by which acute H. pylori infection inhibits acid secretion. In rat fundic sheets in an Ussing chamber, perfusion of the luminal surface with H. pylori in spent broth (103–108cfu/ml) or spent broth alone (1:105to 1:100final dilution) caused a concentration-dependent increase in somatostatin (SST; maximal: 200 ± 20 and 194 ± 9% above basal; P < 0.001) and decrease in histamine secretion (maximal: 45 ± 5 and 48 ± 2% below basal; P < 0.001); the latter was abolished by SST antibody, implying that changes in histamine secretion reflected changes in SST secretion. Both responses were abolished by the axonal blocker tetrodotoxin (TTX), the sensory neurotoxin capsaicin, or the CGRP antagonist CGRP8-37, implying that the reciprocal changes in SST and histamine secretion were due to release of CGRP from sensory neurons. In isolated rabbit oxyntic glands, H. pylori inhibited basal and histamine-stimulated acid secretion in a concentration-dependent manner; the responses were not affected by TTX or SST antibody, implying that H. pylori can directly inhibit parietal cell function. In conclusion, acute administration of H. pylori is capable of inhibiting acid secretion directly as well as indirectly by activating intramural CGRP sensory neurons coupled to stimulation of SST and inhibition of histamine secretion. Activation of neural pathways provides one explanation as to how initial patchy colonization of the superficial gastric mucosa by H. pylori can acutely inhibit acid secretion.

The mechanism of bismuth's bactericidal activity against Helicobacter pylori was investigated using transmission electron microscopy (TEM) and analytical electron microscopy (AEM); time-kill kinetic methods evaluated the effect of excess divalent cations. TEM analysis of untreated H. pylori revealed a normal morphology. In contrast, H. pylori exposed to bismuth salts had swollen, distorted cells with membrane-cell wall blebbing and a cytoplasm containing electron-dense, sometimes crystalline aggregates. By AEM, swollen cells contained bismuth at the cell periphery, whereas bacillary forms contained cytoplasmic bismuth localizations. Time-kill studies showed that the bactericidal activity of bismuth could be prevented by pretreatment with divalent cations. The effects of bismuth salts on the glycocalyces-cell walls of H. pylori with reversal of bactericidal activity by divalent cations are identical to those produced by other polycationic agents on various gram-negative bacilli. We conclude that disruption of the glycocalyces-cell walls of H. pylori is one mechanism of action for bismuth salts.

Vibrio furnissii in the blood is rarely reported, which may explain why clinical features of bloodstream infections with this organism have not been described. We describe a patient who developed skin lesions and V. furnissii bacteremia and was successfully treated with fluoroquinolones. V. furnissii may be a serious pathogen in patients with underlying comorbidities who are exposed to seafood.

Non-beta-lactam inhibitor-based methods were evaluated for detecting plasmid-mediated AmpC β-lactamases in Klebsiella spp., Escherichia coli , and Proteus mirabilis . Using CLSI methodology and disks containing cefotetan alone and in combination with 400 μg of boronic acid, 9 of 10 positive control strains and 54 of 55 AmpC-PCR-positive clinical isolates were detected. Importantly 71% and 40% of these clinical isolates were susceptible by routine testing to ceftriaxone and ceftazidime, respectively. Boronic acid disks also enhanced detection of expanded-spectrum β-lactamases in AmpC producers.

AmpC beta-lactamases are cephalosporinases that confer resistance to a wide variety of β-lactam drugs and that may thereby create serious therapeutic problems. Although reported with increasing frequency, the true rate of occurrence of AmpC beta-lactamases in Escherichia coli , Klebsiella pneumoniae , and Proteus mirabilis remains unknown. We tested a total of 1,286 consecutive, nonrepeat isolates of these three species and found that, overall, 45 (3.5%) yielded a cefoxitin zone diameter less than 18 mm (screen positive) and that 16 (1.2%) demonstrated AmpC bands by isoelectric focusing. Based on the species, of 683 E. coli , 371 K. pneumoniae , and 232 P. mirabilis isolates tested, 13 (1.9%), 28 (7.6%), and 4 (1.7%), respectively, demonstrated decreased zone diameters and 11 (1.6%), 4 (1.1%), and 1 (0.4%), respectively, demonstrated AmpC bands. Cefoxitin resistance was transferred for all but 8 ( E. coli ) of the 16 AmpC producers. We also describe a three-dimensional extract test, which was used to detect phenotypically isolates that harbor AmpC beta-lactamase. Of the 45 cefoxitin-resistant isolates, the three-dimensional extract test accurately identified all 16 AmpC producers and 28 of 29 (97%) isolates as non-AmpC producers. Interestingly, most (86%) isolates in the latter group were K. pneumoniae isolates. These data confirm that, at our institution, E. coli , K. pneumoniae , and P. mirabilis harbor plasmid-mediated AmpC enzymes.

An organism resembling Bacillus alvei was isolated from the lung and pleural fluid of an immunocompetent patient. The isolate differed from the type strain of B. alvei in its ability to reduce nitrate and its inability to produce dihydroxyacetone and acetylmethylcarbinol. The isolate was resistant to ciprofloxacin and showed intermediate susceptibility to vancomycin.

We evaluated the in vitro bactericidal effect of clarithromycin versus ampicillin alone and in combination against clarithromycin-sensitive and clarithromycin-resistant strains of Helicobacter pylori. No combination containing clarithromycin achieved complete bactericidal effect against clarithromycin-resistant strains. Complete bactericidal effect was achieved for all strains by triple-agent combinations that contained bismuth, omeprazole, and relatively high concentrations of ampicillin. These in vitro results demonstrate the additive bactericidal activity provided by a combination of agents for the eradication of H. pylori. Bismuth may play a particularly important role in the bactericidal effect.

Widespread antibiotic use has been associated with increases in both bacterial resistance and nosocomial infection.To characterize the impact of hospital-wide clindamycin restriction on the incidence of Clostridium difficile-associated diarrhea and on antimicrobial prescribing practices.Prospective, observational cohort study.University-affiliated Veterans Affairs Medical Center.Hospitalized patients with symptomatic diarrhea.Clinical data on individual patients and data on antibiotic use were obtained from hospital pharmacy records. Hospital-wide use of antimicrobial agents was monitored. Isolates of C. difficile underwent antimicrobial susceptibility testing and molecular typing.An outbreak of C. difficile-associated diarrhea was caused by a clonal isolate of clindamycin-resistant C. difficile and was associated with increased use of clindamycin. Hospital-wide requirement of approval by an infectious disease consultant of clindamycin use led to an overall reduction in clindamycin use, a sustained reduction in the mean number of cases of C. difficile-associated diarrhea (11.5 cases/month compared with 3.33 cases/month; P0.001), and an increase in clindamycin susceptibility among C. difficile isolates (9% compared with 61%; P0.001). A parallel increase was noted in the use of and costs associated with other antibiotics with antianaerobic activity, including cefotetan, ticarcillin-clavulanate, and imipenem-cilastin. The hospital realized overall cost savings as a result of the decreased incidence of C. difficile-associated diarrhea.Hospital formulary restriction of clindamycin is an effective way to decrease the number of infections due to C. difficile. It can also lead to a return in clindamycin susceptibility among isolates and can effect cost savings to the hospital.

Resistance to expanded-spectrum cephalosporins commonly develops in Enterobacter aerogenes during therapy due to selection of mutants producing high levels of the chromosomal Bush group 1 β-lactamase. Recently, resistant strains producing plasmid-mediated extended-spectrum β-lactamases (ESBLs) have been isolated as well. A study was designed to investigate ESBL production among 31 clinical isolates of E. aerogenes from Richmond, Va., with decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk potentiation test. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. β-Lactamases were investigated by an isoelectric focusing overlay technique which simultaneously determined isoelectric points (pIs) and substrate or inhibitor profiles. Decreased susceptibility to cefotaxime, ceftazidime, and aztreonam (MIC range, 1 to 64 μg/ml) was detected and associated with resistance to gentamicin and trimethoprim-sulfamethoxazole. All strains produced an inducible Bush group 1 β-lactamase (pI 8.3). Twenty-nine of the 31 isolates also produced an enzyme similar to SHV-4 (pI 7.8), while 1 isolate each produced an enzyme similar to SHV-3 (pI 6.9) and to SHV-5 (pI 8.2). The three different SHV-derived ESBLs were transferred by transconjugation to Escherichia coli C600N and amplified by PCR. Plasmid profiles of the clinical isolates showed a variety of different large plasmids. Because of the linkage of resistance to aminoglycosides and trimethoprim-sulfamethoxazole with ESBL production, it is possible that the usage of these drugs was responsible for selecting plasmid-mediated resistance to extended-spectrum cephalosporins in E. aerogenes . Furthermore, it is important that strains such as these be recognized, because they can be responsible for institutional spread of resistance genes.

A total of 907 consecutive isolates of members of the family Enterobacteriaceae recovered during a 20-week period were tested for production of extended-spectrum beta-lactamases (ESBLs) by the double-disk (DD) potentiation method. Of 84 DD-positive isolates, 83 (9.2%) produced ESBLs based on isoelectric focusing. SHV-derived ESBLs and several TEM-derived ESBLs were present in nine species, including the first isolate of Citrobacter koserii and Morganella morganii known to harbor an SHV-derived ESBL. Results of testing 58 nonrepeat isolates for ESBL production by several recommended methods were as follows (percent detected in parentheses): DD method with aztreonam (95), ceftazidime (79), ceftriaxone (88), or cefpodoxime (90); broth microdilution method with ceftazidime (86) or cefotaxime (91) alone or in combination with clavulanate; and the standard disk diffusion method with new breakpoints and standard concentrations of aztreonam (78), ceftazidime (79), ceftriaxone (83), or cefpodoxime (98) or a novel concentration (5 microg) of ceftazidime (88). In three instances during an extended part of the study, an ESBL-producing isolate and a non-ESBL-producing isolate of the same species were recovered from a single blood culture bottle. These data indicate that ESBLs occur in several species of Enterobacteriaceae and at a relatively high incidence at our institution and that the standard disk diffusion method with cefpodoxime and the DD method with several beta-lactams are practical and cost-effective methods for detecting ESBL-producing isolates of Enterobacteriaceae.

Optimal therapy for Helicobacter pylori infection to date, consists of metronidazole, bismuth, and tetracycline. This combination, however, is less effective against metronidazole-resistant organisms. We used a time-kill kinetic methodology to assess the bactericidal effects of selected agents, alone and in combination, to a metronidazole-susceptible and a metronidazole-resistant strain of H. pylori. single, double, and triple agents showed increasing bactericidal activity. The combination of metronidazole, bismuth, and tetracycline showed maximal killing effect (no detectable regrowth) against the susceptible strain, but against the resistant strain this combination showed less killing. The time-kill methodology may therefore offer an in vitro approach to the initial selection of agents to be evaluated for the treatment of H. pylori infections.

Assessment of in vitro susceptibility testing of Helicobacter pylori is difficult because of the fastidious, slowly growing nature of this microorganism. The high rate of relapse observed clinically and a possible subpopulation of cells that are not actively replicating suggest the potential need for bactericidal therapy in order to eradicate H. pylori. We used modified time-kill kinetic methodology in order to evaluate the bactericidal activities of ampicillin and bismuth, alone and in combination, against three strains of H. pylori in both a stationary (slow) growth phase and a logarithmic (rapid) growth phase. We found that ampicillin produced a decrease in CFU per milliliter (2 to 4 log10 units) for three strains of H. pylori when tested in logarithmic growth phases but was less inhibitory (< 1-log10-unit decrease in CFU per milliliter) when tested in a stationary growth phase. In contrast, bismuth, when tested in a logarithmic growth phase, produced little inhibitory effect, as the CFU for all strains tested increased above the inoculum. However, when tested in a stationary growth phase, bismuth produced a decrease in CFU per milliliter of < 1 to > 3 log10 units). The activities of these two agents when combined mimicked the activity of the most active drug alone for that growth phase. We conclude that the clinical use of ampicillin combined with bismuth has been more effective than that of either agent used alone because ampicillin targets replicating cells, whereas bismuth targets cells that are not actively replicating.

Beta-Lactamase-producing, aminoglycoside-resistant strains of Enterococcus faecalis have been isolated from different geographic areas and are endemic at our institution. We report the isolation of a beta-lactamase-producing, aminoglycoside-resistant strain of E. faecium. The beta-lactamase was plasmid mediated and transferable with high frequency into a plasmid-free E. faecalis recipient strain. MICs suggested that the E. faecium strain also contained intrinsic (chromosomal) resistance to penicillins.

• Recent clinical studies have emphasized the importance of diphtheroids, previously regarded as nonpathogenic bacteria or contaminants, as causes of ocular disease. We encountered two patients with endophthalmitis following cataract extraction and anterior chamber intraocular lens implantation. Both patients had previously been treated with subconjunctival and/or oral corticosteroids for presumed sterile endophthalmitis. Vitreous cultures in each case yielded pure growth of a diphtheroid that was subsequently identified as coryneform group A-4. The clinical response to standard intraocular therapy with gentamicin and cefazolin was delayed, although both patients eventually had restoration of functional vision. A comparison of the antibiotic minimum inhibitory and minimum bactericidal concentrations of the isolates may help to explain the delayed response to therapy seen in these two patients.

We treated a farmer who had Listeria monocytogenes bacterial keratitis. Therapy with topical antibiotics was unsuccessful; it was necessary to treat the patient with topical and systemic penicillin and gentamicin. To elucidate the pathogenesis of this infection, we developed a rabbit model. Using the patient's strain of L. monocytogenes , we determined that the severity of the rabbit infection was dose-related. If we used an inoculum of more than 10 7 organisms, many of the features of the human Listeria keratitis were mimicked. We also found that treatment with either penicillin or gentamicin did not control the infection as well as using both antibiotics simultaneously, a combination which resulted in relatively rapid resolution of infection and no corneal scarring. The human and animal data indicate that L. monocytogenes can be a virulent corneal pathogen. Listeria corneal infections must be treated aggressively with both penicillin and gentamicin to prevent permanent visual loss.

To investigate the risk factors, clinical features, molecular epidemiology, and treatment outcomes associated with an outbreak of infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis.Case-control and molecular genetics study.Tertiary care Veterans Affairs hospital.Sixty-five patients infected or colonized with beta-lactamase-producing high-level gentamicin-resistant E. faecalis (case patients) were matched and compared with 65 randomly selected patients infected or colonized with beta-lactamase-negative, gentamicin-susceptible E. faecalis.During the 20-month study period, 124 of 1506 isolates (8.2%) of E. faecalis from 70 patients were found to produce beta-lactamase. Univariate analysis showed older age, higher APACHE II score, nosocomial acquisition, recent surgical procedure, total parenteral nutrition, and antibiotic treatment to be significantly associated with the acquisition of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis. A multivariate analysis, done using stepwise multiple logistic regressions, showed that only two variables remained significant: an APACHE II score greater than 6 (odds ratio, 9.5; 95% CI, 4.4 to 20.3) and antibiotic treatment (odds ratio, 10.2; CI, 4.5 to 23.2). The mortality rate for case patients was 7.7% (5 of 65 patients; CI, 1.2% to 14.2%); no control patient died. Of 23 infections occurring in case patients, 13 were treated with "inappropriate" antibiotics (regimens that included a beta-lactamase-unstable antibiotic); 2 patients improved and 11 had complete resolution of disease. "Appropriate" treatments (regimens that included a beta-lactamase-stable antibiotic) were used in 10 patients; 5 of 10 infections were fatal. Restriction enzyme digests of total chromosomal DNA showed nearly identical patterns for selected isolates of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis, suggesting dissemination through the hospital of a single strain of E. faecalis.Fatal infections were observed despite treatment with beta-lactamase-stable antibiotics. The risk for infection or colonization with beta-lactamase-producing, high-level gentamicin-resistant E. faecalis was strongly associated with severe underlying disease (acute physiology and chronic health evaluation [APACHE] II score, greater than 6) and previous antibiotic treatment. These results may be useful in targeting high-risk patients for infection-control interventions.

Because of the immunosuppressive therapy received by patients undergoing cardiac transplantation, disseminated infections, including disseminated fungal infections, often develop. Disseminated coccidioidomycosis developed in a 23-year-old man soon after undergoing orthotopic cardiac transplantation. Clinical manifestations included an unusual rash, severe myositis and arthropathy, a rapid downhill course, and pathologic evidence of widespread fungal invasion, including invasion of the cardiac graft. Detailed travel and geographic histories, and perhaps skin testing and antibody determinations for geographic-specific pathogens, should be part of the preoperative evaluation of all transplant candidates.

The conventional cell culture method for detection of Chlamydia trachomatis requires two to six days and is technically difficult to perform. The authors evaluated a new, relatively simple, non-culture method (MicroTrak™, Syva Co., Palo Alto, CA) that requires less than one hour to complete. Two hundred fifty-one cervical and 209 male urethral specimens from three Richmond health clinics were read by direct immunofluorescence staining and compared with cell culture technics using iodine staining. Patient specimens were applied directly onto microscope slides (8 mm well) and stained with a fluorescein-labeled monoclonal antibody. Slides were examined for 10-15 minutes at ×1,000 using an AO epifluorescent microscope and were considered positive if five or more typical elementary bodies were seen. The sensitivity, specificity, positive and negative predictive values for the direct smear were 89%, 97%, 85%, and 98% for males, and 93%, 96%, 85%, and 98% for females, respectively. The rapid direct specimen test appears to be a satisfactory method for detecting chlamydia in male and female genital specimens.

Stomatococcus mucilaginosus was isolated from the blood of a patient with endocarditis and a past history of drug abuse and aortic valve replacement. At autopsy, Gram stain of the aortic valve revealed gram-positive cocci. Our isolate was atypical for S. mucilaginosus in that colonies were nonmucoid and nonadherent to agar surfaces. Cellular capsules were demonstrated by light and electron microscopy. Phenotypic characteristics identified by conventional methods as well as profile numbers obtained by using two commercial identification systems for staphylococci, the API Staph-Ident and the dms Staph Trac, are presented. Practical tests that differentiate S. mucilaginosus from the genera Micrococcus and Staphylococcus include growth on nutrient agar containing salt and lysostaphin susceptibility. Additional tests that helped differentiate our isolate from group D streptococci included hydrolysis of L-pyrrolidonyl-beta-naphthylamide and streptococcal serogrouping.

The peroxisomal acyl-CoA oxidase has been purified from extracts of the yeast Candida tropicalis grown with alkanes as the principal energy source. The enzyme has a molecular weight of 552,000 and a subunit molecular weight of 72,100. Using an experimentally determined molar extinction coefficient for the enzyme-bound flavin, a minimum molecular weight of 146,700 was determined. Based on these data, the oxidase contains eight perhaps identical subunits and four equivalents of FAD. No other β-oxidation enzyme activities are detected in purified preparations of the oxidase. The oxidase flavin does not react with sulfite to form an N(5) flavin-sulfite complex. Photochemical reduction of the oxidase flavin yields a red semiquinone; however, the yield of semiquinone is strongly pH dependent. The yield of semiquinone is significantly reduced below pH 7.5. The flavin semiquinone can be further reduced to the hydroquinone. The behavior of the oxidase flavin during photoreduction and its reactivity toward sulfite are interpreted to reflect the interaction in the N(1)-C(2)O region of the flavin with a group on the protein which acts as a hydrogen-bond acceptor. Like the acyl-CoA dehydrogenases which catalyze the same transformation of acyl-CoA substrates, the oxidase is inactivated by the acetylenic substrate analog, 3-octynoyl-CoA, which acts as an active site-directed inhibitor.