Fosfomycin susceptibility in carbapenem-resistant Enterobacteriaceae from Germany.

Due to the increase in multidrug-resistant Enterobacteriaceae, the interest in older antimicrobial agents, like fosfomycin, has increased. In this study, we used agar dilution for testing susceptibilities to fosfomycin in a collection of 107 carbapenem-nonsusceptible Enterobacteriaceae isolates, of which 80 produced various types of carbapenemases, including KPC, VIM, NDM, and OXA-48. Overall, 78% of the strains had fosfomycin MICs of ≤ 32 mg/liter and were thus considered to be susceptible according to the current EUCAST breakpoint. The MIC50 and MIC90 were 8 mg/liter and 512 mg/liter, respectively. Escherichia coli strains had significantly lower fosfomycin MICs than the Klebsiella pneumoniae and Enterobacter cloacae strains. Furthermore, comparisons of the susceptibility testing methods, like Etest and disk diffusion, were performed against agar dilution as the reference method. Essential agreement between Etest and agar dilution was 78.9%, and categorical agreement between the two methods was 92.5%, with 20% very major errors and 2.6% major errors. Disk diffusion was studied with 50-µg and 200-µg fosfomycin disks, but no inhibition zone breakpoint that reduced very major and major errors to an acceptable level was found. Etest and disk diffusion showed poor agreement with fosfomycin agar dilution.

Description of the metallo-ß-lactamase GIM-1 in Acinetobacter pittii.

OBJECTIVES: To characterize the mechanisms involved in the reduced carbapenem susceptibility of five Acinetobacter pittii strains isolated from different regions of Germany. METHODS: The strains were analysed by susceptibility testing, phenotypic tests for metallo-ß-lactamase production, sequencing of the integron structure and strain typing by PFGE, as well as multilocus sequence typing (MLST) and plasmid analysis by S1 restriction and hybridization. RESULTS: Despite GIM-1 production, the MICs of imipenem were only 4 mg/L for four strains and some methods of phenotypic MBL detection failed. According to PFGE and MLST, the strains belonged to four different clones, but blaGIM-1 was present in identical integron structures in all strains and carried on plasmids of ∼60 kb. CONCLUSIONS: For the first time, GIM-1 has been demonstrated in A. pittii. This resistance mechanism has previously been reported only in Enterobacteriaceae and Pseudomonas aeruginosa. As GIM-1 was found in strains with diverse clonal backgrounds, but encoded on plasmids of a similar size, further spread among Acinetobacter spp. seems possible. The detection of GIM-1 production might be challenging in some strains due to the low MICs of carbapenems.

Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR.

A commercial multiplex PCR (hyplex SuperBug ID) was tested with a collection of 132 clinical Enterobacteriaceae strains producing different carbapenemases. The sensitivity for the detection of KPC-, VIM-, NDM-, and OXA-48-encoding genes was 100%, whereas two IMP variants were missed.

First description of Escherichia coli producing CTX-M-15- extended spectrum beta lactamase (ESBL) in out-patients from south eastern Nigeria.

We studied the presence of extended spectrum beta lactamases (ESBLs) in 44 clinical isolates of Escherichia coli collected from out-patients in two university teaching hospitals in South-Eastern Nigeria. Species identification was performed by standard microbiology methods and re-confirmed by MALDI-TOF technology. Phenotypic characterization of ESBL enzymes was done by double disc synergy test and presence of ESBL genes was determined by specific PCR followed by sequencing. Transfer of plasmid DNA was carried out by transformation using E. coli DH5 as recipient strain. Phenotypic characterization identified all isolates to be ESBL positive. 77% of strains were from urine, 13.6% from vaginal swabs and 9.0% from wound swabs. 63.6% were from female patients, 68% were from outpatients and 95.5% from patients younger than 30 years. All ESBL producers were positive in a PCR for bla(CTX-M-1) cluster, in exemplary strains bla(CTX-M-15) was found by sequencing. In all strains ISEcp1 was found upstream and ORF477 downstream of bla(CTX-M). PCR for bla(TEM) and bla(OXA-1) was positive in 93.1% of strains, whereas bla(SHV) was not detected, aac(6')-Ib-cr was found in 97.7% of strains. RAPD analysis revealed seven different clonal groups named A through G with the majority of the strains (65.9%) belonging to clone A. Transfer of an ESBL plasmid with co-resistance to gentamicin, kanamycin, tobramycin, doxycycline and trimethropim-sulfamethoxazole was successful in 19 (43.2%) strains. This study showed a high rate of CTX-M-1 cluster - ESBLs in South-Eastern Nigeria and further confirms the worldwide spread of CTX-M ESBL in clinical isolates.

The matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)-based protein peaks of 4448 and 5302 Da are not associated with the presence of Panton-Valentine leukocidin.

The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus plays an important role in the pathogenesis of necrotizing pneumonia and recurrent skin and soft tissue infections. The gene encoding for PVL, lukS/F-PV, is distributed by prophages and can thus spread between isolates. Molecular methods have normally been used to identify lukS/F-PV-positive strains. Recently, however, a matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)-based method has been described to identify PVL-positive S. aureus strains. The aim of this study was thus to investigate the association of distinct MALDI-TOF MS peaks to the toxin profile in molecularly characterized methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA) strains harbouring lukS/F-PV. In contrast to the previous results, the MALDI-TOF MS peaks were detected in all 104 recently described molecularly divergent MRSA irrespective of the presence of PVL. Our result indicates that these described peaks seem to be independent of the presence of PVL.

Performance of MicroScan WalkAway and Vitek 2 for detection of oxacillin resistance in a set of methicillin-resistant Staphylococcus aureus isolates with diverse genetic backgrounds.

Of 104 genotypically diverse methicillin-resistant Staphylococcus aureus (MRSA) isolates tested with the MicroScan WalkAway (Pos MIC 24 panel) and Vitek 2 (AST-P549 card) systems, 7 and 6 isolates, respectively, showed an oxacillin MIC of < or =2mg/liter. Most of these MRSA isolates were community acquired. However, if the cefoxitin screen of AST-P549 was also considered, MRSA detection failed for only one isolate.

Staphylococcus saprophyticus ATCC 15305 is internalized into human urinary bladder carcinoma cell line 5637.

Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.

Effects of a Cognitive Training With and Without Additional Physical Activity in Healthy Older Adults: A Follow-Up 1 Year After a Randomized Controlled Trial.

Background: Combining cognitive training (CT) with physical activity (CPT) has been suggested to be most effective in maintaining cognition in healthy older adults, but data are scarce and inconsistent regarding long-term effects (follow-up; FU) and predictors of success. Objective: To investigate the 1-year FU effects of CPT versus CT and CPT plus counseling (CPT+C), and to identify predictors for CPT success at FU. Setting and Participants: We included 55 healthy older participants in the data analyses; 18 participants (CPT group) were used for the predictor analysis. Interventions: In a randomized controlled trial, participants conducted a CT, CPT, or CPT+C for 7 weeks. Outcome Measures: Overall cognition, verbal, figural, and working memory, verbal fluency, attention, planning, and visuo-construction. Results: While within-group comparisons showed cognitive improvements for all types of training, only one significant interaction Group × Time favoring CPT in comparison to CPT+C was found for overall cognition and verbal long-term memory. The most consistent predictor for CPT success (in verbal short-term memory, verbal fluency, attention) was an initial low baseline performance. Lower education predicted working memory gains. Higher levels of insulin-like growth factor 1 (IGF-1) and lower levels of brain-derived neurotrophic factor at baseline (BDNF) predicted alternating letter verbal fluency gains. Discussion: Within-group comparisons indicate that all used training types are helpful to maintain cognition. The fact that cognitive and sociodemographic data as well as nerve growth factors predict long-term benefits of CPT contributes to the understanding of the mechanisms underlying training success and may ultimately help to adapt training to individual profiles. Clinical Trial Registration: WHO ICTRP (http://apps.who.int/trialsearch/), identifier DRKS00005194.

Influence of cardiac resynchronisation therapy on different types of sleep disordered breathing.

AIMS: This study investigates the influence of cardiac resynchronisation therapy (CRT) on sleep disordered breathing (SDB) in patients with severe heart failure (HF). METHODS AND RESULTS: Seventy-seven patients with HF (19 females; 62.6+/-10 years) eligible for CRT were screened for presence, type, and severity of SDB before and after CRT initiation (5.3+/-3 months) using cardiorespiratory polygraphy. NYHA class, frequency of nycturia, cardiopulmonary exercise, 6-minute walking test results, and echocardiography parameters were obtained at baseline and follow-up. Central sleep apnoea (CSA) was documented in 36 (47%), obstructive sleep apnoea (OSA) in 26 (34%), and no SDB in 15 (19%) patients. CRT improved clinical and haemodynamic parameters. SDB parameters improved in CSA patients only (apnoea hypopnoea index: 31.2+/-15.5 to 17.3+/-13.7/h, p<0.001; SaO2min: 81.8+/-6.6 to 84.8+/-3.3%, p=0.02, desaturation: 6.5+/-2.3 to 5.5+/-0.8%, p=0.004). Daytime capillary pCO2 was significantly lower in CSA patients compared to those without SDB with a trend towards increase with CRT (35.5+/-4.2 to 37.9+/-5.7 mm Hg, ns). After classifying short term clinical and haemodynamic CRT effects, improved SDB parameters in CSA occurred in responders only. CONCLUSIONS: In patients with severe HF eligible for CRT, CSA is common and can be influenced by CRT, this improvement depends on good clinical and haemodynamic response to CRT.

Cognitive training with and without additional physical activity in healthy older adults: cognitive effects, neurobiological mechanisms, and prediction of training success.

Data is inconsistent concerning the question whether cognitive-physical training (CPT) yields stronger cognitive gains than cognitive training (CT). Effects of additional counseling, neurobiological mechanisms, and predictors have scarcely been studied. Healthy older adults were trained with CT (n = 20), CPT (n = 25), or CPT with counseling (CPT+C; n = 23). Cognition, physical fitness, BDNF, IGF-1, and VEGF were assessed at pre- and post-test. No interaction effects were found except for one effect showing that CPT+C led to stronger gains in verbal fluency than CPT (p = 0.03). However, this superiority could not be assigned to additional physical training gains. Low baseline cognitive performance and BDNF, not carrying apoE4, gains in physical fitness and the moderation of gains in physical fitness × gains in BDNF predicted training success. Although all types of interventions seem successful to enhance cognition, our data do not support the hypotheses that CPT shows superior CT gains compared to CT or that CPT+C adds merit to CPT. However, as CPT leads to additional gains in physical fitness which in turn is known to have positive impact on cognition in the long-term, CPT seems more beneficial. Training success can partly be predicted by neuropsychological, neurobiological, and genetic parameters. Unique Identifier: WHO ICTRP (http://www.who.int/ictrp); ID: DRKS00005194.

Multiresistant strains are as susceptible to photodynamic inactivation as their naïve counterparts: protoporphyrin IX-mediated photoinactivation reveals differences between methicillin-resistant and methicillin-sensitive Staphylococcus aureus strains.

OBJECTIVE: The current study was aimed at the investigation of differences in response to photoinactivation between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates. Moreover, we aimed to elucidate if the observed variation resulted from antimicrobial resistance mechanisms and strains' susceptibility to antibiotic therapy. BACKGROUND DATA: Because of the emergence of multidrug resistance, the development of alternative antimicrobial strategies seems to be required. The concept of photodynamic inactivation (PDI) involves cell exposure to appropriate wavelength light that leads to the excitation of photosensitizer molecules, resulting in the production of reactive oxygen species responsible for cell inactivation and death. Recently, we have demonstrated a strain-dependent response of S. aureus to photoinactivation, and observed elevated resistance to PDI among MRSA strains. Nevertheless, the mechanism underlying this phenomenon remains unexplained. METHODS: S. aureus response to protoporphyrin IX (PPIX)-mediated photoinactivation was studied for 424 MRSA/MSSA isolates. VITEK 2 Advanced Expert System was used to detect antimicrobial resistance mechanisms and strains' susceptibility to antibiotictherapy. RESULTS: Data obtained demonstrated that MRSA are significantly more resistant to photoinactivation than MSSA strains; however, the difference observed did not result from antimicrobial susceptibility or resistance mechanisms. Furthermore, regardless of the strains' origin, a similar effectiveness of PDI could be achieved. Moreover, it was determined that the ability to form biofilms in vitro, and the presence of mec element, does not explain the observed differences between MRSA and MSSA strains. CONCLUSIONS: PDI could be highly effective against multidrug resistant pathogens as well as their naïve counterparts. Nevertheless, regardless of the antimicrobial resistance mechanism, the difference in response to PDI between MRSA and MSSA exists.

The major autolysin of Staphylococcus lugdunensis, AtlL, is involved in cell separation, stress-induced autolysis and contributes to bacterial pathogenesis.

Staphylococcus lugdunensis is a human skin commensal organism, but it is considered as a virulent Staphylococcus species. In a previous study, we described the first S. lugdunensis autolysin, AtlL. This enzyme displays two enzymatic domains and generates two peptidoglycan hydrolases, an N-acetylmuramoyl-l-alanine amidase and an N-acetylglucosaminidase. In this study, to further investigate the functions of this autolysin, a ΔatlL mutant was constructed. The microscopic examination of the mutant showed cell aggregates and revealed a rough outer cell surface demonstrating, respectively, the roles of AtlL in cell separation and peptidoglycan turnover. This ΔatlL mutant exhibited a lower susceptibility to Triton X-100-induced autolysis assays and appears to be more resistant to cell wall antibiotic-induced lysis and death compared with its parental strain. The atlL mutation affected the biofilm formation capacity of S. lugdunensis. Furthermore, the ΔatlL mutant showed trends toward reduced virulence using the Caenorhabditis elegans model. Overall, AtlL appears as a major cell wall autolysin of S. lugdunensis implicated in cell separation, in stress-induced autolysis and in bacterial pathogenesis.

Staphylococcus saprophyticus surface-associated protein (Ssp) is associated with lifespan reduction in Caenorhabditis elegans.

Staphylococcal lipases have been proposed as pathogenicity factors. In Staphylococcus saprophyticus the surface-associated protein (Ssp) has been previously characterized as a cell wall-associated true lipase. A S. saprophyticus Δssp::ermB mutant has been described as less virulent in an in vivo model of urinary tract infection compared with its wild-type. This is the first report showing that S. saprophyticus induced a lifespan reduction in Caenorhabditis elegans similar to that of S. aureus RN4220. In two S. saprophyticus Δssp::ermB mutants lifespan reduction in C. elegans was partly abolished. In order to attribute virulence to the lipase activity itself and distinguish this phenomenon from the presence of the Ssp-protein, the conserved active site of the lipase was modified by site-directed ligase-independent mutagenesis and lipase activity-deficient mutants were constructed. These results indicate that the Ssp is associated with pathogenicity in C. elegans and one could speculate that the lipase activity itself is responsible for this virulence.

Identical MALDI TOF MS-derived peak profiles in a pair of isogenic SCCmec-harboring and SCCmec-lacking strains of Staphylococcus aureus.

MALDI-TOF MS-based peak differences in oxacillin-resistant Staphylococcus aureus and oxacillin-susceptible S. aureus isolates have been described previously. Unfortunately, these isolates were not isogenic with respect to their mecA gene. Ours is the first to use a SCCmec-harboring parent and a SCCmec-lacking daughter strain, with the same genetic background, to unequivocally rule out strain-specific protein peaks. We could not show differences in the peak profiles within the preset Biotyper settings used for MALDI-TOF-based identification in this pair of SCCmec-harboring parent and SCCmec-lacking daughter strains.

Evaluation of species-specific score cut-off values for various Staphylococcus species using a MALDI Biotyper-based identification.

Although previous studies investigating the MALDI Biotyper database (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification) have proven its high accuracy for bacterial identification, the studies differed in sample preparation, number of replicates, quantity of shots and target types used. In particular, the score cut-off values of special importance for reliable species identification varied. The aim of the present study was to identify species-specific differences in the mean score values for staphylococci. Cut-off values recommended by the manufacturer were adapted using the 20th percentile to rule out unknown score-modifying factors, even though the specificity was high and the lowest cut-off values would also yield an accurate result. Whilst correct species diagnosis was obtained in 97.32 % of samples (1382/1420), only 220 of all duplicates (15.49 %) revealed a score of ≥2.3, whilst 968 (68.17 %) had a score between 2.0 and 2.299, and 194 (13.66 %) had a score of <2.0. Ten of 21 species had a calculated 20th percentile of <2.0 and one species of <1.7. In conclusion, the use of species-specific cut-off values improves the relative sensitivity of species identification in staphylococci.

Construction and characterization of three knockout mutants of the fbl gene in Staphylococcus lugdunensis.

Staphylococcus lugdunensis is an important human pathogen that causes infectious diseases similar to those caused by Staphylococcus aureus. In contrast to S. aureus, only a very few pathogenicity factors of S. lugdunensis have been characterized. Notably, a genetic manipulation of S. lugdunensis has not yet been described. Ours is the first report where transformation of three different plasmids (pBT2, pRB473, and pT181) into S. lugdunensis and a directed genetic manipulation of S. lugdunensis are described. We constructed fbl knockout mutants from three different strains of S. lugdunensis to show that at least in these strains, the fibrinogen binding is exclusively mediated by Fbl.

Fbl is not involved in the invasion of eukaryotic epithelial and endothelial cells by Staphylococcus lugdunensis.

For several Staphylococci, such as Staphylococcus aureus, Staphylococcus saprophyticus, and Staphylococcus epidermidis, invasion of eukaryotic cells has been described and this mechanism has been considered an important part of the infection process. The fibrinogen-binding protein (Fbl) of Staphylococcus lugdunensis, a homolog of the clumping factor A of S. aureus, has been described as fibrinogen-binding adhesin and might promote invasion of cells. We therefore characterized several clinical strains of S. lugdunensis in terms of whole cell fibrinogen and fibronectin binding and correlated these results with the invasion of epithelial and endothelial cells by S. lugdunensis. We described for the first time invasion of cells by S. lugdunensis. As invasion of cells by S. lugdunensis was only partly inhibited by cytochalasin D in contrast to a complete inhibition of invasion of cells by S. aureus, further invasion mechanisms are likely to be present in S. lugdunensis. In addition, the Fbl of S. lugdunensis is not involved in the invasion of cells as ruled out by an isogenic fbl mutant.

Late Periprosthetic Joint Infection due to Staphylococcus lugdunensis Identified by Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry: A Case Report and Review of the Literature.

Staphylococcus lugdunensis, member to the group of coagulase-negative staphylococci, is previously thought to be rarely isolated. Recently other staphylococci have been described, which were supposedly related to S. lugdunensis, such as Staphylococcus pseudolugdunensis and Staphylococcus pettenkoferi. To decrease the rate misidentifications, an accurate identification method, such as matrix-assisted laser desorption ionization time of flight mass spectrometry or molecular methods, should be used. S. lugdunensis is usually associated with severe infections similar to those caused by S. aureus. Moreover, it has been described that skin infections due to S. lugdunensis are severely underreported and could be also underreported in periprosthetic joint infections. Ours is the first case of a late periprosthetic infection of the hip due to S. lugdunensis, identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. A periprosthetic infection due to S. lugdunensis should be treated according to protocols of S. aureus periprosthetic infections, and therefore an accurate species identification is desirable.

Occurrence of genes of putative fibrinogen binding proteins and hemolysins, as well as of their phenotypic correlates in isolates of S. lugdunensis of different origins.

BACKGROUND: Staphylococcus lugdunensis is an important human pathogen that causes potentially fatal endocarditis, osteomyelitis and skin and soft tissue infections similar to diseases caused by Staphylococcus aureus. Nevertheless, in contrast to S. aureus, data on pathogenicity factors of S. lugdunensis is scarce. Two adhesins, a fibrinogen and a von Willebrand factor binding protein, and a S. lugdunensis synergistic hemolysin (SLUSH) have been previously described. Moreover, the newly sequenced genome of S. lugdunensis revealed genes of other putative fibrinogen binding adhesins and hemolysins. The aim of this study was to gain more insight into the occurrence of genes likely coding for fibrinogen binding adhesins and hemolysins using clinical strains of S. lugdunensis. FINDINGS: Most of the putative adhesin genes and hemolysin genes investigated in this study were highly prevalent, except for the SLUSH gene cluster. In contrast to previous reports, binding to fibrinogen was detected in 29.3% of the S. lugdunensis strains. In most strains, hemolysis on blood agar plates was weak after 24 h and distinct after 48 h of incubation. The fibrinogen binding and hemolysis phenotypes were also independent of the type of clinical specimen, from which the isolates were obtained. CONCLUSION: In this study we described a pyrrolidonyl arylamidase negative S. lugdunensis isolate. Our data indicate that a matrix-assisted laser desorption ionisation time-of-flight MS-based identification of S. lugdunensis or species-specific PCR's should be performed in favour of pyrrolidonyl arylamidase testing. In contrast to the high occurrence of putative fibrinogen binding protein genes, 29.3% of the S. lugdunensis strains bound to fibrinogen. Putative hemolysin genes were also prevalent in most of the S. lugdunensis strains, irrespective of their hemolysis activity on Columbia blood agar plates. Similar to a previous report, hemolysis after 48 h of incubation is also indicative for S. lugdunensis. The SLUSH gene cluster was detected in an estimated 50% of the strains, indicating that this locus is different or non-prevalent in many strains.

Identification of molecularly defined Staphylococcus aureus strains using matrix-assisted laser desorption/ionization time of flight mass spectrometry and the Biotyper 2.0 database.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced for bacterial identification. To our knowledge, this is the first study where the Biotyper 2.0 database (Bruker Daltonics) has been applied for bacterial identification in a local strain collection of molecularly defined Staphylococcus aureus. We showed that the accuracy of the Biotyper 2.0-based identification for 602 molecularly defined strains of S. aureus, irrespective of meticillin resistance, was equivalent to that of the molecularly defined reference even at a score cut-off value of 2. Also, 412 isolates of 20 different species of non-S. aureus staphylococci were all correctly identified to species level compared to the molecularly defined reference. Moreover, the MALDI-TOF MS-based S. aureus identification approach was clearly faster than more time-consuming methods such as a molecular identification approach.