S P Cohen

Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction.

Chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, selected on agar containing low concentrations of tetracycline or chloramphenicol, were 6- to 18-fold less susceptible to the fluoroquinolones than were their wild-type E. coli K-12 or E. coli C parental strains. The frequency of emergence of such mutants was at least 1,000-fold higher than that of those selected by the fluoroquinolone norfloxacin directly. When Mar mutants, but not wild-type cells, were plated on norfloxacin, mutants resistant to high levels of norfloxacin (2 micrograms/ml) appeared at a relatively high (approximately 10(-7] frequency. In addition to decreased amounts of OmpF, Mar mutants had other outer membrane protein changes and were four- to eightfold less susceptible to fluoroquinolones than was an ompF::Tn5 mutant lacking only OmpF. Accumulation of [3H]norfloxacin was more than threefold lower in the Mar mutants than in wild-type cells and twofold lower than in the OmpF-deficient derivative. These differences were not attributable to a change in the endogenous active efflux system for norfloxacin in E. coli. Norfloxacin-induced inhibition of DNA synthesis was threefold lower in intact cells of a Mar mutant than in susceptible cells, but this difference was not seen in toluene-permeabilized cells. Insertion of Tn5 into marA (min 34.05 on the chromosome) led to a return of the wild-type patterns of norfloxacin accumulation, fluoroquinolone and other antimicrobial agent susceptibilities, and outer membrane protein profile, including partial restoration of OmpF. These findings together suggest that marA-dependent fluoroquinolone resistance is linked to decreased cell permeability, only part of which can be accounted for by the reduction in OmpF. Once mutated to marA, cells can achieve high levels of quinolone resistance at a relatively high frequency. Images

Endogenous active efflux of norfloxacin in susceptible Escherichia coli.

Escherichia coli was shown to have an energy-dependent reduced uptake of the fluoroquinolone antimicrobial agent norfloxacin. Studies of everted inner membrane vesicles suggested that this reduced accumulation involved a carrier-mediated norfloxacin active efflux generated by proton motive force with an apparent Km of 0.2 mM and a Vmax of 3 nmol min-1 mg of protein-1. Other hydrophilic, but not hydrophobic, quinolones competed with norfloxacin for transport. Porin (OmpF)-deficient E. coli cells were twofold less susceptible to norfloxacin and showed twice as much energy-dependent reduction in drug uptake. However, active efflux assayed in everted vesicles from the OmpF strain was unchanged compared with that in the parental strain. These findings suggest that in the OmpF mutant decreased outer membrane permeability, combined with active efflux across the inner membrane, in some manner results in decreased steady-state uptake of norfloxacin and lowered drug susceptibility.

Identification of Cpgp40/15 Type Ib as the Predominant Allele in Isolates of Cryptosporidium spp. from a Waterborne Outbreak of Gastroenteritis in South Burgundy, France

ABSTRACT Cryptosporidium sp. isolates from a waterborne outbreak of diarrhea in France were analyzed by PCR-restriction fragment length polymorphism analysis and sequencing of the Cpgp40/15 locus. Ninety-one percent of the isolates were Cryptosporidium hominis type Ib. The results of this study and those of studies of other outbreaks suggest that the type Ib allele is the predominant allele associated with waterborne cryptosporidiosis.

An Early Intestinal Mucosal Source of Gamma Interferon Is Associated with Resistance to and Control of Cryptosporidium parvum Infection in Mice

ABSTRACT Resistance to and control of Cryptosporidium parvum infection in mice in the absence of adaptive immunity appears to be gamma interferon (IFN-γ) dependent. Using an IFN-γ-neutralizing antibody in a murine model, we demonstrated increased susceptibility to infection within 24 h. We correlated this early resistance and control with increased mucosal expression of IFN-γ and demonstrate that CD8+ T-cell receptor αβ intestinal intraepithelial lymphocytes express and secrete this cytokine shortly after infection. The rapid kinetics of IFN-γ expression and secretion by naive CD8+ T cells in response to a protozoan pathogen have not previously been demonstrated.

Mycoplasma genitalium Prevalence, Coinfection, and Macrolide Antibiotic Resistance Frequency in a Multicenter Clinical Study Cohort in the United States

ABSTRACT The prevalence rates of Mycoplasma genitalium infections and coinfections with other sexually transmitted organisms and the frequency of a macrolide antibiotic resistance phenotype were determined in urogenital specimens collected from female and male subjects enrolled in a multicenter clinical study in the United States. Specimens from 946 subjects seeking care from seven geographically diverse clinical sites were tested for M. genitalium and for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. Sequencing was used to assess macrolide antibiotic resistance among M. genitalium-positive subjects. M. genitalium prevalence rates were 16.1% for females and 17.2% for males. Significant risk factors for M. genitalium infections were black race, younger age, non-Hispanic ethnicity, and female symptomatic status. Female M. genitalium infections were significantly more prevalent than C. trachomatis and N. gonorrhoeae infections, while the M. genitalium infection rate in males was significantly higher than the N. gonorrhoeae and T. vaginalis infection rates. The macrolide-resistant phenotype was found in 50.8% of females and 42% of males. These results show a high prevalence of M. genitalium single infections, a lower prevalence of coinfections with other sexually transmitted organisms, and high rates of macrolide antibiotic resistance in a diverse sample of subjects seeking care across a wide geographic area of the United States.

Seasonality of Cryptosporidium oocyst detection in surface waters of Meru, Kenya as determined by two isolation methods followed by PCR

Meru, Kenya has watersheds which are shared by wildlife, humans and domesticated animals. These surface waters can be contaminated by the waterborne pathogen Cryptosporidium. To quantify the seasonality and prevalence of Cryptosporidium in Meru regional surface waters, we used a calcium carbonate flocculation (CCF) and sucrose floatation method, and a filtration and immunomagnetic bead separation method, each of which used PCR for Cryptosporidium detection and genotyping. Monthly water samples were collected from January through June in 2003 and 2004, bracketing two April-May rainy seasons. We detected significant seasonality with 8 of 9 positive samples from May and June (p<0.0014), which followed peak rainy season precipitation and includes some of the subsequent dry season. Six of 9 positive samples revealed C. parvum, and 3 contained C. andersoni. None contained C. hominis. Our results indicate that Meru surface waters are Cryptosporidium-contaminated at the end of rainy seasons, consistent with the timing of human infections reported by others from East Africa and contrasting with the onset of rainy season peak incidence reported from West Africa.

Untranslated sequence upstream of MarA in the multiple antibiotic resistance locus ofEscherichia coli is related to the effector-binding domain of the XylS transcriptional activator

MarA, the 129-amino-acid (aa) protein which plays a crucial role in the multiple antibiotic resistance (Mar) phenotype inEscherichia coli, shows homology to members of the XylS/AraC family of transcriptional regulators. Although these proteins vary in size from around 100 to 350 aa they all contain a DNA-binding domain with a helix-turn-helix motif. The larger ones, e.g., XylS, AraC, and Rob, contain an additional domain either at their amino- or at their carboxy-terminus. This domain is important for effector-binding or dimerization or of unknown function. MarA consists only of the DNA-binding component. Nevertheless, a sequence with a coding potential of 141 aa upstream of its ATG start-codon showed 20.5–26.9% aa identity with the corresponding section within the effector-binding domain of XylS from the TOL plasmid ofPseudomonas putida when translated in the same reading frame as MarA. However, the reading frame was interrupted by 11 translational stops. In another frame, this upstream sequence actually codes for a real protein, MarR, that is completely unrelated to XylS. Implications for the putative evolution of regulatory proteins through translocation of domains followed by adaptation are discussed.