Macrophage adaptation leads to parallel evolution of genetically diverseEscherichia colismall-colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.

OCCURRENCE AND ANTIBIOTIC SENSIBILITY PROFILE OF Salmonella spp. ISOLATED FROM PORK MEAT CUTS COMMERCIALIZED IN THE OPEN MARKETS IN PELOTAS, RS (BRAZIL)

The objective of the present study was to evaluate the occurrence of Salmonella spp. in 15 samples of pork meat cuts (T-bone, shank, sausage and ribs) commercialized in open markets of Pelotas (RS, Brazil) and verify the prevalent serovars, and test the isolates profile of sensitivity to several antibiotics of importance in medicine (nalidixic acid, ampicillin, aztreonam, kanamycin, carbenicillin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, chloramphenicol, gentamicin, sulfonamide, tetracycline and trimetoprina). Twelve samples (80%) were contaminated by Salmonella enterica, serovars Infantis, Derby, Panama and Typhimurium. All isolates were susceptible to trimetoprin, aztreonam, ciprofloxacin, ceftriaxone and cefoxitin. For the other antibiotics, the pattern of sensitivity varied as serovar. In addition, 39.1% of isolates showed up to be multiresistant.

Trial-of-antibiotic algorithm for the diagnosis of tuberculosis in a district hospital in a developing country with high HIV prevalence

OBJECTIVE: To evaluate a diagnostic algorithm for pulmonary tuberculosis based on smear microscopy and objective response to trial of antibiotics. SETTING: Adult medical wards, Hlabisa Hospital, South Africa, 1996-1997. METHODS: Adults with chronic chest symptoms and abnormal chest X-ray had sputum examined for Ziehl-Neelsen stained acid-fast bacilli by light microscopy. Those with negative smears were treated with amoxycillin for 5 days and assessed. Those who had not improved were treated with erythromycin for 5 days and reassessed. Response was compared with mycobacterial culture. RESULTS: Of 280 suspects who completed the diagnostic pathway, 160 (57%) had a positive smear, 46 (17%) responded to amoxycillin, 34 (12%) responded to erythromycin and 40 (14%) were treated as smear-negative tuberculosis. The sensitivity (89%) and specificity (84%) of the full algorithm for culture-positive tuberculosis were high. However, 11 patients (positive predictive value [PPV] 95%) were incorrectly diagnosed with tuberculosis, and 24 cases of tuberculosis (negative predictive value [NPV] 70%) were not identified. NPV improved to 75% when anaemia was included as a predictor. Algorithm performance was independent of human immunodeficiency virus status. CONCLUSION: Sputum smear microscopy plus trial of antibiotic algorithm among a selected group of tuberculosis suspects may increase diagnostic accuracy in district hospitals in developing countries.

Molecularly-imprinted materials for potentiometric transduction: application to the antibiotic enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mVΔlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL 1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ±0.7%), fast response, good sensitivity (47 mV/Dlog([ENR]/ M), wide linear range (1.0×10-5–1.0×10-3 M), low detection limit (0.9 µg mL-1), and a stable baseline for a 5×10-2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.

Molecularly-Imprinted Materials for Potentiometric Transduction: Application to the Antibiotic Enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mV/Δlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL−1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ± 0.7%), fast response, good sensitivity (47 mV/Δlog([ENR]/M), wide linear range (1.0 × 10−5–1.0 × 10−3 M), low detection limit (0.9 µg mL−1), and a stable baseline for a 5 × 10−2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.

Biomimetic Sensor Potentiometric System for Doxycycline Antibiotic Using a Molecularly Imprinted Polymer as an Artificial Recognition Element

Molecular imprinting is a useful technique for the preparation of functional materials with molecular recognition properties. A Biomimetic Sensor Potentiometric System was developed for assessment of doxycycline (DOX) antibiotic. The molecularly imprinted polymer (MIP) was synthesized by using doxycycline as a template molecule, methacrylic acid (MAA) and/or acrylamide (AA) as a functional monomer and ethylene glycol dimethacrylat (EGDMA) as a cross-linking agent. The sensing elements were fabricated by the inclusion of DOX imprinted polymers in polyvinyl chloride (PVC) matrix. The sensors showed a high selectivity and a sensitive response to the template in aqueous system. Electrochemical evaluation of these sensors under static (batch) mode of operation reveals near-Nernstian response. MIP/MAA membrane sensor was incorporated in flow-through cells and used as detectors for flow injection analysis (FIA) of DOX. The method has the requisite accuracy, sensitivity and precision to assay DOX in tablets and biological fluids.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Antibiotic susceptibility of Waddlia chondrophila in Acanthamoeba castellanii amoebae.

Waddlia chondrophila is an emerging cause of miscarriage in bovines and humans. Given the strict intracellular growth of this Chlamydia-like organism, its antibiotic susceptibility was tested by amoebal coculture, cell culture, and real-time PCR. W. chondrophila was susceptible to doxycycline and azithromycin but resistant to beta-lactams and fluoroquinolones.

Antibiotic resistant bacteria/genes dissemination in lacustrine sediments highly increased following cultural eutrophication of Lake Geneva (Switzerland).

This study investigates faecal indicator bacteria (FIB), multiple antibiotic resistant (MAR), and antibiotic resistance genes (ARGs), of sediment profiles from different parts of Lake Geneva (Switzerland) over the last decades. MARs consist to expose culturable Escherichia coli (EC) and Enterococcus (ENT) to mixed five antibiotics including Ampicillin, Tetracycline, Amoxicillin, Chloramphenicol and Erythromycin. Culture-independent is performed to assess the distribution of ARGs responsible for, β-lactams (blaTEM; Amoxicillin/Ampicillin), Streptomycin/Spectinomycin (aadA), Tetracycline (tet) Chloramphenicol (cmlA) and Vancomycin (van). Bacterial cultures reveal that in the sediments deposited following eutrophication of Lake Geneva in the 1970s, the percentage of MARs to five antibiotics varied from 0.12% to 4.6% and 0.016% to 11.6% of total culturable EC and ENT, respectively. In these organic-rich bacteria-contaminated sediments, the blaTEM resistant of FIB varied from 22% to 48% and 16% to 37% for EC and ENT respectively, whereas the positive PCR assays responsible for tested ARGs were observed for EC, ENT, and total DNA from all samples. The aadA resistance gene was amplified for all the sediment samples, including those not influenced by WWTP effluent water. Our results demonstrate that bacteria MARs and ARGs highly increased in the sediments contaminated with WWTP effluent following the cultural eutrophication of Lake Geneva. Hence, the human-induced changing limnological conditions highly enhanced the sediment microbial activity, and therein the spreading of antibiotic resistant bacteria and genes in this aquatic environment used to supply drinking water in a highly populated area. Furthermore, the presence of the antibiotic resistance gene aadA in all the studied samples points out a regional dissemination of this emerging contaminant in freshwater sediments since at least the late nineteenth century.

Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.

Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.

Résistance aux antibiotiques chez les pneumocoques [Pneumococcal antibiotic resistance].

In 1875, 7 years prior to the description of the Koch bacillus, Klebs visualized the first Streptococcus pneumoniae in a pleural fluid. Since then, this organism has played a determinant role in biomedical science. From a biological point of view, it was largely implicated in the development of passive and active immunization by serotherapy and vaccination, respectively. Genetic transformation was also first observed in S. pneumoniae, leading to the discovery of DNA. From a clinical point of view, S. pneumoniae is still today a prime cause of otitis media in children and of pneumonia in all age groups, as well as a predominant cause of meningitis and bacteremia. In adults, bacteremia is still entailed with a mortality of over 25%. Although S. pneumoniae remained very sensitive to penicillin for many years, penicillin-resistance has emerged and increased dramatically over the last 15 years. During this period of time, the frequency of penicillin-resistant isolates has increased from < or = 1% to frequencies varying from 20 to 60% in geographic areas as diverse as South Africa, Spain, France, Hungary, Iceland, Alaska, and numerous regions of the United States and South America. In Switzerland, the current frequency of penicillin-resistant pneumococci ranges between 5 and > or = 10%. The increase in penicillin-resistant pneumococci correlates with the intensive use of beta-lactam antibiotics. The mechanism of resistance is not due to bacterial production of penicillinase, but to an alteration of the bacterial target of penicillin, the so-called penicillin-binding proteins. Resistance is subdivided into (i) inter mediate level resistance (minimal inhibitory concentration [MIC] of penicillin of 0.1-1 mg/L) and (ii) high level resistance (MCI > or = 2 mg/L). The clinical significance of intermediate resistance remains poorly defined. On the other hand, highly resistant strains were responsible for numerous therapeutical failures, especially in cases of meningitis. Antibiotics recommended against penicillin-resistant pneumococci include cefotaxime, ceftriaxone, imipenem and in some instances vancomycin. However, penicillin-resistant pneumococci tend to present cross-resistances to all the antibotics of the beta-lactam family and could even become resistant to the last resort drugs mentioned above. Thus, in conclusion, the explosion of resistance to penicillin in pneumococci is a ubiquitous phenomenon which must be fought against by (i) a strict utilization of antibiotics, (ii) the practice of microbiological sampling of infected foci before treatment, (iii) the systematic surveillance of resistance profiles of pneumococci against antibiotics and (iv) the adequate vaccination of populations at risk.

Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHA0 with enhanced antibiotic production

Introduction of the recombinant cosmid pME3090 into Pseudomonas fluorescens strain CHAO, a good biocontrol agent of various diseases caused by soilborne pathogens, increased three- to five-fold the production of the antibiotic metabolites pyoluteorin (Pit) and 2,4-diacetylphlorogIucinol (Phi) in vitro. Strain CHAO/pME3090 also overproduced Pit and Phi in the rhizosphere of wheat infected or not infected with Pythium ultimum. The biocontrol activity of the wild-type and recombinant Straitis was compared using various plant pathogen-host combinations in a gnotobiotic system. Antibiotic overproduction affected neither the protection of wheat against P. ultimum and Gaeumannomyces graminis var. tritici nor the growth of wheat plants. In contrast, strain CHA0/pME3090 showed an increased capacity to protect cucumber against Fusarium oxysporum f. sp. cucumerinum and Phomopsis sclerotioides, compared with the wild-type strain CHAO, The antibiotic overproducing strain protected tobacco roots significantly better against Thielaviopsis basicola than the wild-type strain but drastically reduced the growth of tobacco plants and was also toxic to the growth of sweet com. On King's B agar and on malt agar, the recombinant strain CHA0/pME3090 inhibited all pathogens more than did the parental strain CHAO. Synthetic Pit and Phi were toxic to all fungi tested. Tobacco and sweet com were more sensitive to synthetic Pit and Phi than were cucumber and wheat. There was no correlation between the sensitivity of the pathogens to the synthetic antibiotics and the degree of disease suppression by strain CHAO pME3090. However, there was a correlation between the sensitivity of the plants and the toxicity of the recombinant strain. We conclude that the plant species rather than the pathogen determines whether cosmid pME3090 in P. fluorescens strain CHAO leads to improved disease suppression.

A simple blood-culture bacterial pellet preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing with the VITEK 2 system.

An ammonium chloride procedure was used to prepare a bacterial pellet from positive blood cultures, which was used for direct inoculation of VITEK 2 cards. Correct identification reached 99% for Enterobacteriaceae and 74% for staphylococci. For antibiotic susceptibility testing, very major and major errors were 0.1 and 0.3% for Enterobacteriaceae, and 0.7 and 0.1% for staphylococci, respectively. Thus, bacterial pellets prepared with ammonium chloride allow direct inoculation of VITEK cards with excellent accuracy for Enterobacteriaceae and a lower accuracy for staphylococci.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Rapid analysis of multiclass antibiotic residues and some of their metabolites in hospital, urban wastewater and river water by ultra-highperformance liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry

The present work describes the development of a fast and robust analytical method for the determination of 53 antibiotic residues, covering various chemical groups and some of their metabolites, in environmental matrices that are considered important sources of antibiotic pollution, namely hospital and urban wastewaters, as well as in river waters. The method is based on automated off-line solid phase extraction (SPE) followed by ultra-high-performance liquid chromatography coupled to quadrupole linear ion trap tandem mass spectrometry (UHPLC–QqLIT). For unequivocal identification and confirmation, and in order to fulfill EU guidelines, two selected reaction monitoring (SRM) transitions per compound are monitored (the most intense one is used for quantification and the second one for confirmation). Quantification of target antibiotics is performed by the internal standard approach, using one isotopically labeled compound for each chemical group, in order to correct matrix effects. The main advantages of the method are automation and speed-up of sample preparation, by the reduction of extraction volumes for all matrices, the fast separation of a wide spectrum of antibiotics by using ultra-high-performance liquid chromatography, its sensitivity (limits of detection in the low ng/L range) and selectivity (due to the use of tandem mass spectrometry) The inclusion of β-lactam antibiotics (penicillins and cephalosporins), which are compounds difficult to analyze in multi-residue methods due to their instability in water matrices, and some antibiotics metabolites are other important benefits of the method developed. As part of the validation procedure, the method developed was applied to the analysis of antibiotics residues in hospital, urban influent and effluent wastewaters as well as in river water samples