European Journal of Clinical Microbiology and Infectious Diseases

The value of the chromogenic limulus assay for detection of gram-negative bacteria in urine was determined. The assay was performed in microtiter plates at room temperature. In 311 consecutively collected urine samples from patients with suspected urinary tract infection, the assay was positive in all 35 samples containing ⩾ 105 bacteria/ml. No false positive or false negative results were obtained. Four of six urine specimens from patients with gonococcal infection were positive in the assay, whereas samples from patients with chlamydial infection did not yield a positive result. The assay is a rapid and reliable method for detection of urinary tract infection caused by gram-negative bacteria when ⩾ 105 bacteria/ml are present.

 The in vitro activity of ranalexin alone and in combination with other cationic peptides, macrolides, rifampin, and rifabutin was investigated against a clinical isolate of Cryptosporidium parvum. Susceptibility tests were performed by inoculation of the isolate onto cell monolayers and determining the parasite count after 48 h of incubation at 37  °C. Antibiotic-free cultures were used as controls in the study. Ranalexin showed low anticryptosporidial activity: it suppressed the growth of parasites by ≥40% at 50 μM. Ranalexin showed enhanced activity when it was combined with noninhibitory concentrations of other compounds: a 74.4–94.1% reduction in the number of parasites was observed when ranalexin 50 μM was combined with magainin II 50 μM, indolicidin 50 μM, clarithromycin 8 mg/l, azithromycin 8 mg/l, rifampin 8 mg/l, and rifabutin 8 mg/l. The results suggest that ranalexin may be effective in inhibiting Cryptosporidium parvum growth in vitro upon combination with other peptides and hydrophobic antibiotics.

The objectives of this study were to determine rates, patterns, and mechanisms of antibiotic resistance, and to assess connections between chicken commensal, human commensal, and pathogenic ciprofloxacin-resistant Escherichia coli isolates. All E. coli isolates collected from chickens, their farmers, and patients in the Constantine region (North-east Algeria) were analyzed for bla and plasmid-mediated quinolone resistance (PMQR) gene contents, phylogroups, Rep-PCR profiles, and multilocus sequence types. A high prevalence of resistance to fluoroquinolones (51.4 % to ciprofloxacin) was recorded in avian isolates. Of these, 22.2 % carried the aac(6’)-Ib-cr gene, whereas lower resistance levels to these antibiotics were recorded in chicken farmers’ isolates. None of the commensal isolates harbored the qnr, qepA, or oqxAB genes. One human pathogenic isolate was ertapenem-resistant and harbored the bla OXA-48 gene, 84 showed an extended-spectrum β-lactamase phenotype, with bla CTX-M-15 gene prevalent in 87.2 % of them. Seventy isolates were resistant to fluoroquinolones, with aac(6’)-Ib-cr present in 72.8 %, qnrB in 5.7 %, and qnrS in 10 %. Three Rep-PCR profiles were common to chicken commensal and human pathogenic isolates (phylogroups D and B1; ST21, ST48, and ST471 respectively); one was found in both chicken and chicken-farmer commensal strains (D; ST108), while another profile was identified in a chicken-farmer commensal strain and a human pathogenic one (B1; ST19). These findings suggest clonal and epidemiologic links between chicken and human ciprofloxacin-resistant E. coli isolates and the important role that poultry may play in the epidemiology of human E. coli infections in the Constantine region.

Colistin is an old antibiotic, which is abandoned decades ago because of high nephrotoxicity rates. However, it is reintroduced to clinical medicine due to lack of newly discovered antibiotics and is still widely used for the treatment of resistant gram-negative infections. Discovering mechanisms to reduce nephrotoxicity risk is of significant importance since exposed patients may have many other factors that alter kidney functions. Several agents were evaluated in animal models of colistin nephrotoxicity as a means to prevent kidney injury. Considerable heterogeneity exists in terms of reporting colistin dosing and experimental designs. This issue leads clinicians to face difficulties in designing studies and sometimes may lead to report dosing strategies inadequately. Here, we present a review according to animal models of colistin nephrotoxicity using data gathered from previous experiments to draw attention on possible complexities that researchers may encounter.

Bone allografts retrieved from multi-organ donors can be decontaminated with minimally aggressive methods. Therefore, we evaluated the efficacy of antibiotics and antiseptics in the decontamination of bone fragments actively contaminated with coagulase-negative staphylococci. Gentamicin (512/1,024 μg/mL), rifampicin (400/1,000 μg/mL), chlorhexidine in alcohol and chlorhexidine soap were tested with different contact times and temperatures and a delay in starting decontamination. Gentamicin-susceptible strains dried on bone could be removed by gentamicin 512 μg/mL after 19 h of contact, while strains not dried on bone could be eliminated by soaking bone for 60 min in gentamicin 512 μg/mL. Rifampicin-susceptible strains could be eliminated by soaking bone for 60 min in rifampicin 1,000 μg/mL. In none of the experimental conditions could gentamicin/rifampicin-resistant staphylococci be eliminated. Antiseptics could not eliminate staphylococci from bone. Different antibiotics need different protocols in order to decontaminate bone allografts.

The in vitro post-antibiotic effect (PAE) of cefepime, cefotaxime, ceftazidime and imipenem on reference strains ofEscherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa andSerratia marcescens were evaluated by bioluminescence assay of bacterial ATP. In parallel with the PAE determination, initial killing and morphology studies were performed. Imipenem produced>1 h PAE on all strains tested, cefepime and cefotaxime on four strains and ceftazidime only on one of the strains tested. The length of the PAE on different strains did not correlate in the same way to MIC. Imipenem induced>1 h PAE at 1/4-2 MIC while the cephalosporins caused>1 h PAE at 4−256 × MIC. A PAE exceeding 1.2 h was seen concomitantly with spheroplasts but there was not necessarily strong (≥99 %) initial killing at the same time. The PAE duration at≥99 % initial killing varied between 2.0 h and 5.0 h. When the cephalosporins produced<1 h PAEs, this was seen concomitantly with production of filaments and weak initial killing. The bioluminescence method was not jeopardized by filament formation and no negative PAE was found in contrast to the viable count method. The study showed that neither a certain multiple of MIC, the presence of spheroplasts nor strong initial killing can predict the length of PAE for β-lactam antibiotics on gram-negative bacteria.

Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) identification of mycobacteria requires a standard acetonitrile/formic acid pre-MALDI-TOF-MS. We prospectively compared this standard protocol with direct deposit with matrix for the identification of mycobacteria cultured on solid media. We first verified that Mycobacterium tuberculosis was killed after it was mixed with matrix. Then, 111 Mycobacterium isolates previously identified by partial rpoB gene sequencing were tested in parallel by the two protocols. An identification score >1.7 was obtained in 86/111 (77.5 %) isolates after protein extraction versus 97/111 (87.4 %) isolates after direct deposit (p = 0.039, Chi-squared test). In a third step, we determined that direct deposit achieved identification for as few as 2.104 M. tuberculosis organisms. In a fourth step, we evaluated direct deposit of one colony for 116 solid medium-cultured clinical isolates finally identified as representative of 12 species (63.8 % M. tuberculosis). For 114/116 (98.3 %) isolates with an identification score >1.2, the MALDI-TOF-MS identification was in complete agreement with the reference rpoB gene sequencing identification. One isolate with a MALDI-TOF-MS identification score of 1.22 for M. fortuitum was identified as M. avium by partial rpoB gene sequencing. One other isolate with a MALDI-TOF-MS identification score of 1.22 for M. tuberculosis was identified as M. tuberculosis by genotyping. All the original MALDI-TOF-MS spectra reported here have been deposited in a public database. Direct deposit of one colony on a MALDI-TOF-MS plate allows for an accurate identification of mycobacteria for an identification score >1.3.