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Characterization of emeA, a NorA homolog and multidrug resistance efflux pump, in Enterococcus faecalis.

We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).

Amino Acid Sequence↗

In vitro selection and characterization of Bacillus anthracis mutants with high-level resistance to ciprofloxacin.

Mutants of attenuated Bacillus anthracis with high-level ciprofloxacin resistance were isolated using a three-step in vitro selection. Ciprofloxacin MICs were 0.5 micro g/ml for first-step mutants, which had one of two gyrA quinolone resistance-determining region (QRDR) mutations. Ciprofloxacin MICs were 8 and 16 microg/ml for second-step mutants, which had one of three parC QRDR mutations. Ciprofloxacin MICs for third-step mutants were 32 and 64 microg/ml. Mutants for which MICs were 64 microg/ml had one of two additional mutations within the gyrA QRDR or one of two mutations within the gyrB QRDR. Mutants for which MICs were 32 microg/ml had no additional target modifications but showed evidence of enhanced ciprofloxacin efflux.

Anti-Infective Agents↗

Resistance to antimicrobial peptides and stress response in Mycoplasma pulmonis.

Antimicrobial peptides are widely distributed in nature, and in vertebrates, they play a key function in the innate immune defense system. It is generally agreed that these molecules may provide new antibiotics with therapeutic value. However, there are still many unsolved questions regarding the mechanisms underlying their antimicrobial activity as well as the mechanisms of resistance evolved by microorganisms against these molecules. The second point was addressed in this study. After determining the activity of 10 antimicrobial peptides against Mycoplasma pulmonis, a murine respiratory pathogen, the development of resistance was investigated. Following in vitro selection using subinhibitory concentrations of peptides, clones of this bacterium showing increased resistance to melittin or gramicidin D were obtained. For some of the clones, a cross-resistance was observed between these two peptides, in spite of their deep structural differences, and also with tetracycline. A proteomic analysis suggested that the stress response in these clones was constitutively activated, and this was confirmed by finding mutations in the hrcA gene; in mycoplasmas, bacteria which lack alternative sigma factors, the HrcA protein is supposed to play a key role as a negative regulator of heat shock proteins. By complementation of the hrcA mutants with the wild-type gene, the initial MICs of melittin and gramicidin D decreased to values close to the initial ones. This indicates that the resistance of M. pulmonis to these two antimicrobial peptides could result from a stress response involving HrcA-regulated genes.

Anti-Infective Agents↗

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.

Acriflavine↗

Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump.

AcrAB-TolC is the major, constitutively expressed efflux protein complex that provides resistance to a variety of antimicrobial agents in Escherichia coli. Previous studies showed that AcrA, a periplasmic protein of the membrane fusion protein family, could function with at least two other resistance-nodulation-division family pumps, AcrD and AcrF, in addition to its cognate partner, AcrB. We found that, among other E. coli resistance-nodulation-division pumps, YhiV, but not MdtB or MdtC, could also function with AcrA. When AcrB was assessed for the capacity to function with AcrA homologs, only AcrE, but not YhiU or MdtA, could complement an AcrA deficiency. Since AcrA could, but YhiU could not, function with AcrB, we engineered a series of chimeric mutants of these proteins in order to determine the domain(s) of AcrA that is required for its support of AcrB function. The 290-residue N-terminal segment of the 398-residue protein AcrA could be replaced with a sequence coding for the corresponding region of YhiU, but replacement of the region between residues 290 and 357 produced a protein incapable of functioning with AcrB. In contrast, the replacement of residues 357 through 397 of AcrA still produced a functional protein. We conclude that a small region of AcrA close to, but not at, its C terminus is involved in the interaction with its cognate pump protein, AcrB.

Carrier Proteins↗

Clinical impact of rapid in vitro susceptibility testing and bacterial identification.

During the past decade, a variety of instrument-assisted bacterial identification and antimicrobial susceptibility test systems have been developed which permit provision of test results in a matter of hours rather than days, as has been the case with traditional overnight procedures. These newer rapid techniques are much more expensive than older methods. It has been presumed but not proven that the clinical benefits of rapid testing to patients with infection offset the added cost. The intent of this study was to objectively define the clinical impact of rapid bacterial identification and antimicrobial susceptibility testing. A 1-year study was performed in which infected, hospitalized patients in a tertiary-care, teaching, medical center were randomly assigned to one of two groups: patients for whom identification and susceptibility testing was performed by using a semi-automated, rapid, same-day procedure and those for whom testing was accomplished by using traditional overnight techniques. The two groups were compared with respect to numerous demographic descriptors, and then patients were monitored prospectively through the end of their hospitalization with the aim of determining whether there existed objectively defineable differences in management and outcome between the two groups. The mean lengths of time to provision of susceptibility and identification test results in the rapid test group were 11.3 and 9.6 h, respectively. In the overnight test group, these values were 19.6 and 25.9 h, respectively (P < 0.0005). There were 273 evaluable patients in the first group and 300 in the second group. Other than the length of time required to provide susceptibility and identification test results, no significant differences were noted between the two groups with respect to > 100 demographic descriptors. With regard to measures of outcome, the mean lengths of hospitalization were also the same in both groups. Mortality rates were however, lower in the rapid test group (i.e., 8.8% versus 15.3%). Similarly, statistically significantly fewer laboratory studies, imaging procedures, days of intubation, and days in an intensive or intermediate-care area were observed with patients in the rapid test group. Rapid testing was also associated with significantly shortened lengths of elapsed time prior to alterations in antimicrobial therapy. Lastly, patient costs for hospitalization were significantly lower in the rapid test group. The results of this study indicate the rapid same-day bacterial identification and susceptibility testing in the microbiology laboratory can have a major impact on the care and outcome of hospitalized patients with infection.

Adolescent↗

Serotypes of group B meningococci.

Strains of group B meningococci isolated from cases of sporadic infection, epidemic infection, and from patients unassociated with clinical meningococcal infection have been examined by a serological typing technique. The majority of group B meningococci from clinical infections in the UK have serotype 2 antigen. Such strains were relatively uncommon among carriers who had no association with meningococcal disease.

Agglutination Tests↗

Sensitivity index of antimicrobial agents as a simple solution for multidrug resistance in Salmonella Typhi.

BACKGROUND & OBJECTIVE: Multi drug-resistant (MDR) typhoid in India is an escalating problem. MDR isolates of Salmonella Typhi are on rise and are becoming a challenge for timely and appropriate treatment. Occurrence of per cent sensitive (%S), per cent resistant (%R) and per cent intermediate (%I) isolates may vary geographically and treatment decided on the basis of only one of these three subpopulations may lead to selection of inappropriate drug for treatment and thus treatment failure. Determination of sensitivity index (SI) of antimicrobial agents, instead of %S or %R subpopulations, may give clearer insight regarding selection of appropriate antimicrobial for treatment of typhoid. In present work, the data of sensitivity testing were analysed and interpreted both in terms of SI as well as %S, %I and %R. METHODS: A total of 205 isolates of Salmonella Typhi were collected during June 2000 and August 2002 from a network of five institutes- Lady Hardinge Medical College (LHMC, N=110), Ram Manohar Lohia Hospital (RML, N=14), Majeedia Hospital (MH, N=48), Lal's Pathology Lab (LAL, N=28) and All India Institute of Medical Sciences (AIIMS, N=5) on nutrient agar slopes. Of these, 142 isolates were subjected to phage typing and biotyping at National Salmonella Phage Typing Centre, New Delhi. Five isolates resistant to 3-7 and one isolate susceptible to all of total 12 antimicrobial tested were subjected to plasmid analysis. SI for various antimicrobials was determined as the ratio of %S and %R values derived form %RIS analysis using WHONET5. RESULTS: 18 (8.7%) isolates were susceptible to all tested antimicrobials and 124 (60%) were MDR. Of the 142 isolates, 103 were phage type E1 and biotype I. SI of antimicrobials rather than individual %S or %R or %I population presents a better criterion for interpretation of sensitivity testing data as well as selection of the most appropriate antimicrobial for timely treatment. Presence of 140, 48 and 23 Kb size plasmids in all 5 MDR isolates and none in susceptible isolate was observed. INTERPRETATION & CONCLUSION: Re-emergence of chloramphenicol sensitivity in Salmonella typhi was observed in the present study. Interpretation in terms of SI criteria warrants that reintroduction of chloramphenicol at present for treatment of typhoid may rebound resistance. Current empiric therapy used for treatment of typhoid may soon become ineffective. SI being a ratio will not only eliminate geographical variation of %RIS data but also its interpretation. SI can provide guidelines for clinicians in remote areas where facilities for sensitivity testing are not available.

Drug Resistance, Multiple, Bacterial↗

[Diffusion of fosfomycin in the presence of glucose-6-phosphate from discs for antibiotic sensitivity testing].

A disc containing 50 microgram fosfomycin and 25 microgram glucose-6-phosphate (G-6-P) has been developed for fosfomycin susceptibility testing. The present study was designed to examine the influence of G-6-P on the diffusion gradient of fosfomycin in Mueller-Hinton agar. Discs containing 50 microgram 3H-fosfomycin and/or 25 microgram 14C-G-6-P were placed in the center of agar plates. Radioactivity of agar cylinders removed along the radius of the Petri plate at different times from 30 sec to 24 hours was measured. Results showed that the fosfomycin/G-6-P ratio throughout diffusion remained close to the initial ratio in the disc, suggesting that loads of both substances borne by the discs are adequate for antibiotic sensitivity testing.

Agar↗