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PubMed2002

Constitutive high expression of chromosomal beta-lactamase in Pseudomonas aeruginosa caused by a new insertion sequence (IS1669) located in ampD.

The expression of chromosomal AmpC beta-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutations in ampD to find evidence for the genetic changes leading to high-level expression of chromosomal beta-lactamase. A new insertion sequence, IS1669, was found located in the ampD genes of two clinical P. aeruginosa isolates and several biofilm-isolated variants. The presence of IS1669 in ampD resulted in the expression of high levels of AmpC beta-lactamase. Complementation of these isolates with ampD from the reference P. aeruginosa strain PAO1 caused a dramatic decrease in the expression of AmpC beta-lactamase and a parallel decrease of the MIC of ceftazidime to a level comparable to that of PAO1. One highly resistant, constitutive beta-lactamase-producing variant contained no mutations in ampD, but a point mutation was observed in ampR, resulting in an Asp-135-->Asn change. An identical mutation of AmpR in Enterobacter cloacae has been reported to cause a 450-fold higher AmpC expression. However, in many of the isolates expressing high levels of chromosomal beta-lactamase, no changes were found in either ampD, ampR, or in the promoter region of ampD, ampR, or ampC. Our results suggest that multiple pathways may exist leading to increased antimicrobial resistance due to chromosomal beta-lactamase.

Amino Acid Sequence↗
PubMed2004

tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus.

The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus. By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.

Anti-Bacterial Agents↗
PubMed2004

De novo design of potent antimicrobial peptides.

Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic beta-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed.

Anti-Bacterial Agents↗
PubMed2005

Combination therapy with intravenous colistin for management of infections due to multidrug-resistant Gram-negative bacteria in patients without cystic fibrosis.

Colistin, an antibiotic almost abandoned for intravenous administration for many years due to its reported toxicity, has been recently reintroduced in clinical practice due to the emergence of multidrug-resistant gram-negative bacteria and the lack of development of new antibiotics to combat them. To assess the safety and effectiveness of intravenous colistin, in combination with other antimicrobial agents, in the treatment of serious infections in patients without cystic fibrosis, a retrospective cohort study in a 450-bed tertiary-care hospital in Athens, Greece, was performed. Patients who were hospitalized from 1 October 2000 to 31 January 2004 and received intravenous colistin for more than 72 h were further analyzed. The primary outcome measure was the in-hospital mortality; secondary end points were the clinical outcome of the infections and the occurrence of colistin toxicity. Fifty patients received intravenous colistin with a median (mean) daily dose of 3 (4.5) million IU for 16.5 (21.3) days for the management of 54 episodes of infections due to multidrug-resistant gram-negative bacteria. The predominant infections were pneumonia (33.3%), bacteremia (27.8%), urinary tract infection (11.1%), and intra-abdominal infection (11.1%). The responsible pathogens were Acinetobacter baumannii (51.9%), Pseudomonas aeruginosa (42.6%), and Klebsiella pneumoniae (3.7%) strains (no pathogen was isolated from one case). In-hospital mortality was 24% (12/50 patients). Clinical response (cure or improvement) of the infection was observed in 66.7% of episodes (36/54). In the studied group, serum creatinine levels were decreased, at the end of colistin treatment, by an average of 0.2 +/- 1.3 mg/dl compared to baseline levels. Deterioration of renal function during colistin therapy was observed in 4/50 patients (8%). Coadministration of other antimicrobial agents with spectrum against gram-negative microorganisms and the absence of a control group constitute the major limitations of this study. The use of intravenous colistin for the treatment of infections due to multidrug-resistant gram-negative bacteria appears to be safe and effective.

Adult↗
PubMed1998

Identification of the rrmA gene encoding the 23S rRNA m1G745 methyltransferase in Escherichia coli and characterization of an m1G745-deficient mutant.

An Escherichia coli mutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83-100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. The rrmA gene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on the E. coli chromosome and identified to be identical to the previously sequenced gene yebH.

Chromatography, High Pressure Liquid↗
PubMed1995

Characterization of Vibrio cgolerae non-O1 serogroups obtained from an outbreak of diarrhea in Lima, Peru.

In February 1994, an outbreak of diarrhea caused by non-O1 Vibrio cholerae occurred among volunteers in a vaccine trial study area in Lima, Peru. Clinically, 95% of the patients presented with liquid diarrhea with either no or mild dehydration. Serogrouping of 58 isolates recovered from diarrheal patients affected in the outbreak revealed seven different serogroups, with serogroups O10 (21%) and O12 (65%) being predominant. Most of these isolates were susceptible to a variety of antimicrobial agents. None of the 58 isolates hybridized with a DNA probe previously used to detect the gene encoding the heat-stable enterotoxin NAG-ST or produced cholera toxin as assessed by GM1 ganglioside enzyme-linked immunosorbent assay. Ribotyping exhibited 10 different BglI ribotype patterns among the 58 V. cholera non-O1 strains studied. However, ribotyping showed that all isolates belonging to serogroup O12 exhibited identical ribotypes and that 83% of the serogroup O10 isolates belonged to another identical ribotype, thus showing excellent correlation between ribotypes and serogroups. Among a group of O10 and O12 isolates selected for virulence studies, none produced enterotoxin whereas the majority produced a cytotoxin, as assessed in Y1 and HeLa cells. These isolates were also negative for the gene encoding zonula occludens toxin (Zot) as assessed by a PCR assay. The isolates tested showed strong adherence and some degree of invasion in the HEp-2 cell assay, whereas none of the isolates was positive in the PCR assay for the gene encoding the toxin coregulated pilus subunit A antigen (tcpA). In the removable intestinal tie adult rabbit diarrhea model, O10 and O12 serogroup isolates produced severe diarrhea and occasionally death when rabbits were challenged with 10(10) bacterial cells. Fluid accumulation was shown in the rabbit intestinal loop test when whole cultures were injected. No significant difference in virulence was shown between serogroup O10 and O12 isolates. This study provides further evidence that V. chlorae non-O1 non-O139 strains have diarrhegenic potential for humans through a yet-undefined mechanism(s) and that such strains can cause outbreaks.

Animals↗
PubMedDate not supplied

Inhibitory effect of antibacterial resin composite against Streptococcus mutans.

Dental resin composites with antibacterial activity may be useful for preventing the secondary caries frequently seen around restorations. Three types of silver-supported antibacterial materials (Novaron, Amenitop and AIS) inhibited the growth of the major oral pathogen Streptococcus mutans. Minimum inhibitory amounts in suspensions of Novaron, Amenitop and AIS against S. mutans were 40, 30 and 400 microg/ml, respectively. These antibacterial materials were incorporated into TEGDMA-UDMA-based light-activated resin composite, and the antibacterial activities of these composites were examined. Composites incorporating 5 wt% (N-5) or more of Novaron and 7 wt% (AM-7) or more of Amenitop inhibited the growth of S. mutans, whereas composites incorporating up to 10 wt% of AIS did not. No significant difference in either compressive or flexural strength was observed between the control and N-5 composites after 1 day and 6 months of storage in water. However, for AM-5 composite, there was a significant difference in either strength parameter between the two immersion periods. There was no or extremely little release of silver ions from the N-5 and AM-5 composites after 1 day or 6 months of immersion in water. These results indicated that a light-activated resin composite incorporating silver-supported antibacterial material such as Novaron may be clinically useful due to its inhibitory effect against S. mutans and favorable mechanical properties.

Analysis of Variance↗
PubMed2006

Antimicrobial activity of Arctium lappa constituents against microorganisms commonly found in endodontic infections.

This study evaluated in vitro the antimicrobial activity of rough extracts from leaves of Arctium lappa and their phases. The following microorganisms, commonly found in the oral cavity, specifically in endodontic infections, were used: Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans. The agar-diffusion method allowed detection of the hexanic phase as an inhibitor of microbial growth. Bioautographic assays identified antimicrobial substances in the extract. The results showed the existence, in the rough hexanic phase and in its fractions, of constituents that have retention factors (Rf) in three distinct zones, thereby suggesting the presence of active constituents with chemical structures of different polarities that exhibited specificity against the target microorganisms. It may be concluded that the Arctium lappa constituents exhibited a great microbial inhibition potential against the tested endodontic pathogens.

Acetates↗
PubMed1981

Epidemiologic features of urinary infections due to enterobacteriaceae resistant to nalidixic acid and trimethoprim.

For a period of one year we identified all urinary isolates of Enterobacteriaceae resistant to either nalidixic acid (NA) or trimethoprim (TMP). Host and organism characteristics associated with the occurrence of 68 NA and 61 TMP-resistant isolates were compared with 61 matched antimicrobial-susceptible controls. Minimum inhibitory concentrations to NA and TMP were carried out on all isolates, Escherichia coli isolates were biotyped and TMP-sulfamethoxazole (SMX) synergy studies were performed on TMP-resistant isolates. Study patients were reviewed with regard to age, renal function, presence of structural urinary tract abnormality, history of antimicrobial treatment and persistence of study strains within the urinary tract. Resistance to both drugs was associated with prior treatment with the relevant antimicrobial and with underlying urinary tract abnormality. No association was noted between resistance and patient age or renal function. Once resistant organisms infected an abnormal urinary tract, they were able to persist almost indefinitely. Patients with persistent urinary infections associated with structural urinary tract abnormality account for most NA or TMP-resistant infections in our hospital and constitute an identifiable group in whom the therapeutic usefulness of these drugs is limited.

Adult↗
PubMedDate not supplied

Evaluation of antibacterial activity of three dentin primers using an in vitro tooth model.

This study compared the antibacterial activities of three dentin primers and investigated a newly designed experimental system using a bovine tooth model method for evaluating antibacterial activity by comparing this method with a conventional disk diffusion method. The antibacterial activities of SA primer in Clearfil Liner Bond, LB primer in Clearfil Liner Bond II, and ED primer in Panavia 21 were evaluated using the conventional disk diffusion method. The 50 microliters aliquot of each primer was applied to three sterilized paper disks, then placed onto Tryptic Soy agar plates already inoculated with Streptococcus mutans. After anaerobic incubation for 48 hours, the diffusion of antibacterial components was determined using the inhibition zone produced around the paper disk. The diameter of the inhibition zones was measured and the average calculated. Standardized cavities (diameter 5.0 mm, depth 3.0 mm) were prepared on the labial surfaces of bovine teeth and inoculated with S mutans (10(6) CFU/microliter) following sterilization by 60Co gamma rays (50 KGy). The teeth were divided into four groups: SA primer, LB primer, ED primer, and a control group. Except for the control teeth, the cavity preparations were treated with the respective dentin primers, and then firmly sealed with a temporary sealing material. The teeth were placed in bottles containing melted Tryptic Soy agar. Five ml of Tryptic Soy broth was then added to the surface of the hardened Tryptic Soy agar. After 1 week's incubation of the teeth in the bottles at 37 degrees C, the number of bacteria remaining in each cavity was counted, except for eight specimens, which were used for SEM observation. The ED primer showed the widest inhibition zone in the disk diffusion test, which was significantly different from the other primers. Using the bovine tooth model, all dentin primers showed antibacterial activity, with significant differences found among the four groups. The results indicated that ED primer had the strongest antibacterial effect among the three primers.

Aminosalicylic Acids↗
PubMed2001

Isolation and characterization of a chromosomally encoded disulphide oxidoreductase from Salmonella enterica serovar Typhimurium.

In this study, the chromosomally encoded disulphide oxidoreductase dsbA from Salmonella typhimurium was cloned and characterized. A survey of a number of serovars of Salmonella subspecies I showed that dsbA is highly conserved in most, but not all members of this subclass of Salmonella species. Using motility, beta-galactosidase, and alkaline phosphatase assays as indirect indicators of disulphide oxidoreductase activity, we demonstrated that DsbA from S. typhimurium LT2 can only partially complement an Escherichia coli dsbA-null strain. This is surprising considering the high degree of conservation between these two DsbA proteins (87% amino acid identity). To determine the contribution of DsbA to the proper folding and assembly of proteins of S. typhimurium, deletion mutants were created in the avirulent strain LT2 and in the virulent strain SL1344. These null alleles were constructed by partial deletion of the dsbA-coding region and then insertion of an antibiotic resistance marker in the gene. Mutants no longer expressing a functional disulphide oxidoreductase exhibit pleitropic effects, including an increase in colony mucoidy, a dramatic decrease in motility, and an increased susceptibility to the cationic peptide protamine sulphate. The disruption of disulphide bond formation was also shown to specifically affect the stability of several proteins secreted into the extracellular environment.

Amino Acid Sequence↗
PubMed1989

Marine invertebrate mucus--agglutinating and antibacterial activity, with emphasis on Metridium senile.

The agglutinating and antibacterial activities of mucus from marine invertebrates are described. Mucus from eleven species was tested for the presence of haemagglutinins using a range of vertebrate erythrocytes. Mucus from Metridium senile, Ophiocomina nigra and Branchiostoma lanceolatum yielded positive results and was investigated further. The haemagglutinin of M. senile was purified, characterized and shown to be mannose-specific. Mucus from this animal was subsequently assayed for antibacterial activity using seawater isolates and verified marine and terrestrial cultures. Only two test organisms were inhibited, while the majority showed significantly enhanced growth. Possible functions of the mucus barrier in marine invertebrates are discussed.

Agglutinins↗