Concentration of Airborne Staphylococcus aureus (MRSA and MSSA), Total Bacteria, and Endotoxins in Pig Farms.

Pigs are very often colonized by Staphylococcus aureus and transmission of such pig-associated S. aureus to humans can cause serious medical, hygiene, and economic problems. The transmission route of zoonotic pathogens colonizing farm animals to humans is not well established and bioaerosols could play an important role. The aim of this study was to assess the potential occupational risk of working with S. aureus-colonized pigs in Switzerland. We estimated the airborne contamination by S. aureus in 37 pig farms (20 nursery and 17 fattening units; 25 in summer, 12 in winter). Quantification of total airborne bacterial DNA, airborne Staphylococcus sp. DNA, fungi, and airborne endotoxins was also performed. In this experiment, the presence of cultivable airborne methicillin-resistant S. aureus (MRSA) CC398 in a pig farm in Switzerland was reported for the first time. Airborne methicillin-sensitive S. aureus (MSSA) was found in ~30% of farms. The average airborne concentration of DNA copy number of total bacteria and Staphylococcus sp. measured by quantitative polymerase chain reaction was very high, respectively reaching values of 75 (± 28) × 10(7) and 35 (± 9.8) × 10(5) copy numbers m(-3) in summer and 96 (± 19) × 10(8) and 40 (± 12) × 10(6) copy numbers m(-3) in winter. Total mean airborne concentrations of endotoxins (1298 units of endotoxin m(-3)) and fungi (5707 colony-forming units m(-3)) exceeded the Swiss recommended values and were higher in winter than in summer. In conclusion, Swiss pig farmers will have to tackle a new emerging occupational risk, which could also have a strong impact on public health. The need to inform pig farmers about biological occupational risks is therefore crucial.

Transmission of MRSA and MSSA between humans and farm animals

Human nares are the main niche of Staphylococcus aureus, but farm animals can be also infected (cows) or colonized (pigs) constituting significant reservoir of this pathogen. Previous studies indicated that human and animal strains are quite distinct but the extent of cross-species specialization and transmission remains largely unknown. However, recent reports from several European countries as well as USA and Canada have indicated that employment in farming is an emerging risk factor for MRSA carriage. Pigs were found to be frequently colonized with MRSA, usually with a strain belonging to CC398. It is not known whether animal-human transmission was specific to this particular MRSA strain. S. aureus isolates from cow mastitis and pig colonization isolates were collected in parallel to nasal swab isolates from the animals' caretakers. The isolates were genotyped by AFLP, spatyping, and when appropriate by MLST. The isolates from cow mastitis were genetically uniform in comparison with human isolates. They were quite distinct from farmers\' carriage isolates, indicating pronounced hostspecialization. However, several cases where an infected cow and a colonized farmer had the same strain were detected, including one farm where two farmers were colonized and two cows were infected with MRSA belonging to CC398. Pig isolates were genetically more diverse than cow isolates. They were different from both human and cow isolates with one notable exception. Large fraction of pigs (20%) and pig caretakers (50%) were colonized with isolates belonging to CC398, majority of which were MSSA (2 cases of MRSA). These results indicate that host specialization in S. aureus is quite pronounced. Transmission between humans and farm animals was consequently quite rare. Both MSSA and MRSA strains belonging to otherwise pig-specific CC398 had increased capacity to colonize humans. Study of the genetic factors responsible for host specialization is underway.

Improvement of the antibacterial activity of daptomycin-loaded polymeric microparticles by Eudragit RL 100: An assessment by isothermal microcalorimetry.

The aim of the present study was to develop novel daptomycin-loaded acrylic microparticles with improved release profiles and antibacterial activity against two clinically relevant methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains (MSSA and MRSA, respectively). Daptomycin was encapsulated into poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (EUD) microparticles by a double emulsion-solvent evaporation method. For comparison purposes similar formulations were prepared with vancomycin. Particle morphology, size distribution, encapsulation efficiency, surface charge, physicochemical properties, in vitro release and biocompatibility were assessed. Particles exhibited a micrometer size and a spherical morphology. The addition of EUD to the formulation caused a shift in the surface charge of the particles from negative zeta potential values (100% PMMA formulations) to strongly positive. It also improved daptomycin encapsulation efficiency and release, whereas vancomycin encapsulation and release were strongly hindered. Plain and antibiotic-loaded particles presented comparable biocompatibility profiles. The antibacterial activity of the particles was assessed by isothermal microcalorimetry against both MSSA and MRSA. Daptomycin-loaded PMMA-EUD particles presented the highest antibacterial activity against both strains. The addition of 30% EUD to the daptomycin-loaded PMMA particles caused a 40- and 20-fold decrease in the minimum inhibitory (MIC) and bactericidal concentration (MBC) values, respectively, when compared to the 100% PMMA formulations. On the other hand, vancomycin-loaded microparticles presented the highest antibacterial activity in PMMA particles. Unlike conventional methods, isothermal microcalorimetry proved to be a real-time, sensitive and accurate method for assessment of antibacterial activity of antibiotic-loaded polymeric microparticles. Finally, the addition of EUD to formulations proved to be a powerful strategy to improve daptomycin encapsulation efficiency and release, and consequently improving the microparticles activity against two relevant S. aureus strains.

Blood culture-based diagnosis of bacteraemia: state of the art.

Blood culture remains the best approach to identify the incriminating microorganisms when a bloodstream infection is suspected, and to guarantee that the antimicrobial treatment is adequate. Major improvements have been made in the last years to increase the sensitivity and specificity and to reduce the time to identification of microorganisms recovered from blood cultures. Among other factors, the introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology revolutionized the identification of microorganisms whereas the introduction of nucleic-acid-based methods, such as DNA hybridization or rapid PCR-based test, significantly reduce the time to results. Together with traditional antimicrobial susceptibility testing, new rapid methods for the detection of resistance mechanisms respond to major epidemiological concerns such as methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase or carbapenemases. This review presents and discusses the recent developments in microbial diagnosis of bloodstream infections based on blood cultures.

In vivo effect of flucloxacillin in experimental endocarditis caused by mecC-positive staphylococcus aureus showing temperature-dependent susceptibility in vitro.

Methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene (mecC-MRSA) exhibited at 37°C MICs of oxacillin close to those of methicillin-susceptible S. aureus (MSSA). We investigated whether at this temperature, mecC-MRSA strains respond to flucloxacillin treatment like MSSA strains, using a rat model of endocarditis. Flucloxacillin (human-like kinetics of 2 g intravenously every 6 h) cured 80 to 100% of aortic vegetations infected with five different mecC-MRSA strains. These results suggest that mecC-MRSA infections may successfully respond to treatment with β-lactams.

Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was observed. Finally, all formulations proved to be biocompatible with both ISO compliant L929 fibroblasts and human MG63 osteoblast-like cells.

Estimation de l'impact économique de l'introduction d'un test diagnostique rapide pour le dépistage des MRSA au CHUV

Contexte : Parmi les infections nosocomiales, le Staphylocoque méticilline résistant (MRSA) est le germe pathogène le plus couramment identifié dans les hôpitaux du monde entier. La stratégie de contrôle des MRSA au CHUV implique le dépistage des patients à risque. Avec la méthode de dépistage par culture, le temps d'attente est de plusieurs jours. Ceci occasionne des problèmes dans la gestion des flux des patients, principalement à cause des mesures d'isolement. Pour réduire le temps d'attente, l'hôpital envisage d'utiliser une méthode de diagnostic rapide par "polymerase chain reaction" (PCR). Méthodologie : Les données concernant les dépistages réalisés, dans trois services durant l'année 2007, ont été utilisées. Le nombre de jours d'isolement a d'abord été déterminé par patient et par service. Ensuite une analyse des coûts a été effectuée afin d'évaluer la différence des coûts entre les deux méthodes pour chaque service. Résultats : Le principal impact économique de la méthode par PCR dépend principalement du nombre de jours d'isolements évités par rapport à la méthode de culture. Aux services de soins, l'analyse a été menée sur 192 dépistages. Quand la différence de jours d'isolement est de deux jours, le coût des dépistages diminue de plus de 12kCHF et le nombre de jours d'isolement diminue de 384 jours. Au centre interdisciplinaire des urgences, sur 96 dépistages, le gain potentiel avec la méthode PCR est de 6kCHF avec une diminution de 192 jours d'isolement. Aux soins intensifs adultes, la méthode de dépistage par PCR est la méthode la plus rentable avec une diminution des coûts entre 4KCHF et 20K CHF et une diminution des jours d'isolement entre 170 et 310. Pour les trois services analysés, les résultats montrent un rapport coût-efficacité favorable pour la méthode PCR lorsque la diminution des jours d'isolement est supérieure à 1.3 jour. Quand la différence de jours d'isolement est inférieure à 1.3, il faut tenir compte d'autres paramètres, comme le coût de matériel qui doit être supérieur à 45.5 CHF, et du nombre d'analyses par dépistage, qui doit être inférieur à 3, pour que la PCR reste l'alternative la plus intéressante. Conclusions : La méthode par PCR montre des avantages potentiels importants, tant économiques qu'organisationnels qui limitent ou diminuent les contraintes liées à la stratégie de contrôle des MRSA au CHUV. [Auteure, p. 3]

Analytical methods for vancomycin determination in biological fluids and in pharmaceuticals

Vancomycin is a glycopeptide antibiotic employed in the treatment of infections caused by certain methicillin-resistant staphylococci. It is indicated also for patients allergic to penicillin or when there is no response to penicillins or cephalosporins. The adequate vancomycin concentration levels in blood serum lies between 5 and 10 mg/L. Higher values are toxic, causing mainly nephrotoxicity and ototoxicity. Various analytical methods are described in the literature: spectrophotometric, immunologic, biologic and chromatographic methods. This paper reviews the main analytical methods for vancomycin determination in biological fluids and in pharmaceutical preparations.

Atividade antibacteriana de floroglucinóis e do extrato hexânico de Hypericum brasiliense Choysi

Three phloroglucinols were obtained from Hypericum brasiliense: japonicine A (1), isouliginosin B (2) and uliginosin B (3). Bioautography and disk diffusion methods were used to determine antibacterial activity of the hexanic extract. Strains of the Coagulase Negative Staphylococcus and American Methicillin Resistant Staphylococcus aureus clones showed a growth inhibition zone ranging from 10 to 12 mm and 7 to 15 mm, respectively. Minimal inhibitory concentration (MIC) values were used to measure antistaphylococcal activity for all phloroglucinols. Isouliginosin B and uliginosin B presented MIC values of 1.5 and 3.0 µg/mL, respectively, while japonicine A displayed MIC value of 50.0 µg/mL.

PCR-based ribotyping of Staphylococcus aureus

Genotyping techniques are valuable tools for the epidemiologic study of Staphylococcus aureus infections in the hospital setting. Pulsed-field gel electrophoresis (PFGE) is the current method of choice for S. aureus strain typing. However, the method is laborious and requires expensive equipment. In the present study, we evaluated the natural polymorphism of the genomic 16S-23S rRNA region for genotyping purpose, by PCR-based ribotyping. Three primer pairs were tested to determine the size of amplicons produced and to obtain better discrimination with agar gel electrophoresis and ethidium bromide staining. The resolution of the typing system was determined using sets of bacteria obtained from clinical specimens from a large tertiary care hospital. These included DNA from three samples obtained from a bacteremic patient, six strains with known and diverse PGFE patterns, and 88 strains collected over a 3-month period in the same hospital. Amplification patterns obtained from samples from the same patient were identical, and PFGE from samples known to be different produced three genotypes. Amplification of DNA from 61 methicillin-resistant isolates produced only one pattern. Methicillin-sensitive strains yielded a diversity of patterns, pointing to a true polyclonal distribution throughout the hospital (22 unique patterns from 27 strains). Computer-based software can be used to differentiate among identifiable strains, given the low number of bands and good characterization of PCR products. PCR-based ribotyping can be a useful technique for genotyping methicillin-sensitive S. aureus strains, but is of limited value for methicillin-resistant strains.

In vitro antimicrobial activity of a new series of 1,4-naphthoquinones

The antibacterial activity of a series of 1,4-naphthoquinones was demonstrated. Disk diffusion tests were carried out against several Gram-positive and Gram-negative bacteria. The compound 5-amino-8-hydroxy-1,4-naphthoquinone was the most effective, presenting inhibition zones measuring 20 mm against staphylococci, streptococci and bacilli at 50 µg/ml. Methicillin-resistant Staphylococcus aureus and several clinical isolates of this bacterium were also inhibited. Naphthazarin, 5-acetamido-8-hydroxy-1,4-naphthoquinone, and 2,3-diamino-1,4-naphthoquinone were the next most active compounds. The minimal inhibitory concentration of the active compounds was determined against S. aureus, ranging from 30 to 125 µg/ml. All compounds presented a minimal bactericidal concentration higher than 500 µg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50% of maximal effect, was 8 µg/ml for 5-amino-8-hydroxy-1,4-naphthoquinone against S. aureus, S. intermedius, and S. epidermidis. These results indicate an effective in vitro activity of 5-amino-8-hydroxy-1,4-naphthoquinone and encourage further studies for its application in antibiotic therapy.

New multilocus sequence typing of MRSA in São Paulo, Brazil

An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE) profile and molecular sequence typing (MLST) genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5), showing a new yqiL allele gene, resulting in a new sequence typing (ST) (1176). Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.

Clinical impact of Achromobacter xylosoxidans colonization/infection in patients with cystic fibrosis

The rate of diagnosis of colonization/infection of the airways with Achromobacter xylosoxidans has increased in cystic fibrosis patients, but its clinical significance is still controversial. This retrospective, case-control study aimed to evaluate the clinical impact of A. xylosoxidans colonization/infection in cystic fibrosis patients. Individuals who were chronically colonized/infected (n=10), intermittently colonized/infected (n=15), and never colonized/infected with A. xylosoxidans (n=18) were retrospectively evaluated during two periods that were 2 years apart. Demographic characteristics, clinical data, lung function, and chronic bacterial co-colonization data were evaluated. Of the total study population, 87% were pediatric patients and 65.1% were female. Individuals chronically colonized/infected with A. xylosoxidans had decreased forced expiratory volume in 1 s (51.7% in the chronic colonization/infection group vs 82.7% in the intermittent colonization/infection group vs 76% in the never colonized/infected group). Compared with the other two groups, the rate of co-colonization with methicillin-resistant Staphylococcus aureus was higher in individuals chronically colonized/infected with A. xylosoxidans (P=0.002). Changes in lung function over 2 years in the three groups were not significant, although a trend toward a greater decrease in lung function was observed in the chronically colonized/infected group. Compared with the other two groups, there was a greater number of annual hospitalizations in patients chronically colonized/infected with A. xylosoxidans (P=0.033). In cystic fibrosis patients, there was an increased frequency of A. xylosoxidans colonization/infection in children, and lung function was reduced in patients who were chronically colonized/infected with A. xylosoxidans. Additionally, there were no differences in clinical outcomes during the 2-year period, except for an increased number of hospitalizations in patients with A. xylosoxidans.

Étude sur le Staphylococcus aureus résistant à la méthicilline chez le porc à l'abattoir au Québec, Canada

Le Staphylococcus aureus résistant à la méthicilline (SARM) est un pathogène important qui a été identifié comme agent d‟infection chez les animaux d‟élevage et les travailleurs exposés à ces animaux. Au Canada, très peu d‟informations sont disponibles concernant les SARMs d‟origine porcine. L‟objectif de cette étude était de déterminer la prévalence des SARMs provenant de porcs à l‟abattoir, de caractériser leur résistance aux antibiotiques ainsi que d‟évaluer le niveau de séroconversion des porcs envers le S. aureus chez les animaux porteurs ou non du SARM. Un total de 107 isolats ont été identifiés positifs aux SARMs sur 660 échantillons. La prévalence de SARMs à l‟abattoir A était de 30,8% et de 23,8% à l‟abattoir B. La susceptibilité aux antibiotiques a été déterminée en utilisant la méthode de micro-dilution de Sensititre. Tous les isolats ont démontré une sensibilité envers la ciprofloxacine, la gatifloxacine, la gentamicine, la lévofloxacine, le linézolide, la quinupristine/dalfopristine, la rifampicine, la streptomycine, le triméthoprime/sulfaméthoxazole et la vancomycine. De la résistance a été observée envers la daptomycine (0,93%), l‟érythromycine (29%), la clindamycine (29%), la tétracycline (98,1%). De plus, 30% des SARMs isolés étaient résistants à plus de deux antibiotiques autres que les β-lactamines. Par typage, deux clones prédominants ont été obtenus ainsi que deux types de SCCmec (type V et possiblement un nouveau type comprenant les cassettes III et IVb). 15 clones ont été identifiés par typage MLVA, comprenant les clones prédominants VI (40.1%; 43/107) et XI (17.7%; 19/107). Deux souches de SARMs ont été caractérisées par biopuce à ADN et des gènes d‟antibiorésistance, de typage (SCCmec et MLST) et de virulence ont été identifiés. Sans considération pour le site de colonisation, les porcs SA-/MRSA- (n=34) et les porcs SA+ (n=194) montrent, respectivement, des taux de séroconversion de 20.6% et 32.5%. Les porcs colonisés par un SARM à un site de iv prélèvement et non colonisés par un SA à l‟autre site (n=18) montrent une séroconversion (5.6%) significativement (P < 0.05) plus faible comparativement aux porcs colonisés par SA à un ou deux sites de prélèvement et n‟ayant pas de SARM. Nos résultats démontrent que les porcs provenant d‟abattoir peuvent être colonisés par des SARMs multi-résistants aux antibiotiques. De plus, ces SARMs sont possiblement capable de coloniser leurs hôtes sans stimuler la production d‟anticorps et ce par l‟atténuation de la réponse immunitaire ou par la colonisation de porcs qui sont moins immunocompétents.

Étude clinique sur l’impact du Staphylococcus aureus résistant à la méthicilline sur l’évolution des patients atteints de fibrose kystique

La prévalence du Staphylococcus aureus résistant à la méthicilline (SARM) a augmenté de façon dramatique dans les dernières années chez les patients atteints de fibrose kystique. Quoique le rôle du SARM dans la pathogénèse de l’atteinte respiratoire de la fibrose kystique ne soit pas clairement déterminé, certaines études récentes ont suggéré une association entre la persistance du SARM et le déclin accéléré de la fonction respiratoire. Cependant, l’importance clinique des diverses souches qui colonisent les patients atteints de fibrose kystique n’a pas encore été élucidée. Les objectifs de ce mémoire étaient de déterminer les effets d’une colonisation persistante par un SARM sur le statut clinique et respiratoire des enfants atteints de fibrose kystique. Également, nous tenions à étudier les caractéristiques des différentes souches de SARM dans cette population. Nous avons réalisé une étude rétrospective en analysant les données cliniques ainsi que les mesures de la fonction respiratoire chez les enfants atteints de fibrose kystique suivis à la Clinique de fibrose kystique du CHU Sainte-Justine entre 1996 et 2008 et ayant une colonisation persistante par un SARM. Afin de déterminer les souches qui colonisent cette population, nous avons effectué une caractérisation moléculaire des isolats du SARM. Nous avons identifié 22 patients avec une colonisation persistante par un SARM. Les résultats n’ont démontré aucun changement significatif en ce qui concernait le taux de déclin de la fonction respiratoire avant ou après l’acquisition du SARM. Cependant, la colonisation persistante par un SARM était associée à une augmentation du nombre d’hospitalisations pour une exacerbation pulmonaire. La plupart de nos patients étaient colonisés par le CMRSA-2, une souche épidémique au Canada. La présente étude suggère que la souche épidémique CMRSA-2 pouvait affecter l’évolution clinique des enfants atteints de fibrose kystique.