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1.  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 
Photomedicine and Laser Surgery  2014;32(3):121-129.
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.
PMCID: PMC3952657  PMID: 24527879
3.  Fosfomycin Susceptibility in Carbapenem-Resistant Enterobacteriaceae from Germany 
Journal of Clinical Microbiology  2014;52(6):1893-1897.
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.
PMCID: PMC4042753  PMID: 24648559
4.  Staphylococcus saprophyticus surface-associated protein (Ssp) is associated with lifespan reduction in Caenorhabditis elegans 
Virulence  2013;4(7):604-611.
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.
PMCID: PMC3906294  PMID: 23959029
Staphylococcus saprophyticus; Ssp; lipase; C. elegans; lifespan reduction
5.  Detection of Carbapenemases in Enterobacteriaceae by a Commercial Multiplex PCR 
Journal of Clinical Microbiology  2012;50(9):3115-3118.
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.
PMCID: PMC3421779  PMID: 22785190
6.  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 blaCTX-M-1 cluster, in exemplary strains blaCTX-M-15 was found by sequencing. In all strains ISEcp1 was found upstream and ORF477 downstream of blaCTX-M. PCR for blaTEM and blaOXA-1 was positive in 93.1% of strains, whereas blaSHV 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.
PMCID: PMC3473344  PMID: 22824236
Outpatients; ESBL; CTX-M; Escherichia coli
7.  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 
Case Reports in Medicine  2011;2011:608919.
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.
PMCID: PMC3138060  PMID: 21776276
8.  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 
BMC Research Notes  2011;4:113.
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.
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.
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.
PMCID: PMC3089787  PMID: 21477287
9.  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 ▿  
Journal of Clinical Microbiology  2009;47(8):2623-2625.
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 ≤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.
PMCID: PMC2725681  PMID: 19515835
10.  Tropheryma whipplei Infection of an Acellular Porcine Heart Valve Bioprosthesis in a Patient Who Did Not Have Intestinal Whipple's Disease 
Journal of Clinical Microbiology  2004;42(10):4487-4493.
Rare cases of culture-negative infective endocarditis are caused by Tropheryma whipplei, the uncommon bacterium of Whipple's disease. We evaluated an 80-year-old woman with valvular heart disease but without intestinal Whipple's disease. The diagnosis of aortic valve xenograft culture-negative infection with T. whipplei was established by multiple molecular assays and by electron microscopy. First, a PCR with broad-range primers identified the complete 16S ribosomal DNA of T. whipplei in bioprosthesis tissue. Novel real-time reverse transcription-PCR assays were developed to detect mRNAs encoding recently identified proteins determined from the T. whipplei genome, specifically Whipplei surface protein (TW113) and a DNA polymerase III subunit (TW727). The positive detection of mRNAs indicated the presence of metabolically active bacteria and suggested the viability of T. whipplei. The quantification of T. whipplei genome equivalents by real-time PCR indicated a high-density bacterial colonization of the valve tissue. Additionally, an ultrastructural examination revealed numerous rod-shaped bacteria consistent in size with T. whipplei in the extracellular collagen matrix of the bioprosthesis. We conclude that extracellular growth of T. whipplei can occur in the microenvironment of biological prosthetic valve tissue and that T. whipplei endocarditis can occur in the absence of intestinal Whipple's disease.
PMCID: PMC522317  PMID: 15472298

Results 1-10 (10)