Rapid Detection of Vancomycin-Intermediate Staphylococcus aureus by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Vancomycin is the standard of care for the treatment of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections. Infections with vancomycin-nonsusceptible MRSA, including vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA), are clinically challenging and are associated with poor patient outcomes. The identification of VISA in the clinical laboratory depends on standard susceptibility testing, which takes at least 24 h to complete after isolate subculture, whereas hVISA is not routinely detected in clinical labs. We therefore sought to determine whether VISA and hVISA can be differentiated from vancomycin-susceptible S. aureus (VSSA) using the spectra produced by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Strains of MRSA were characterized for vancomycin susceptibility phenotype by broth microdilution and modified population analysis. We tested 21 VISA, 21 hVISA, and 38 VSSA isolates by MALDI-TOF MS. Susceptibility phenotypes were separated by using a support vector machine (SVM) machine learning algorithm. The resulting model was validated by leave-one-out cross validation. Models were developed and validated by using spectral profiles generated under various subculture conditions, as well as with and without hVISA strains. Using SVM, we correctly identified 100% of the VISA and 97% of the VSSA isolates with an overall classification accuracy of 98%. Addition of hVISA to the model resulted in 76% hVISA identification, 100% VISA identification, and 89% VSSA identification, for an overall classification accuracy of 89%. We conclude that VISA/hVISA and VSSA isolates are separable by MALDI-TOF MS with SVM analysis.     

In vivo development of daptomycin resistance in vancomycin-susceptible methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> severe infections previously treated with glycopeptides

Our aim was to describe the clinical and microbiological features of four cases of severe vancomycin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) infections in which the vancomycin non-susceptibility development and daptomycin resistance occurred under therapy with teicoplanin (three cases) and daptomycin switched to vancomycin (one case). Clinical data were retrospectively reviewed. On nine clinical epidemiologically unrelated daptomycin-susceptible (DAP-S) and daptomycin-resistant (DAP-R) MRSA, we performed: (i) DAP-VAN-TEC-CFX-RIF minimum inhibitory concentrations (MICs); (ii) glycopeptide resistance detection (GRD) by δ-hemolysis; (iii) glycopeptide population analysis; (iv) molecular characterization by PFGE-MLST-SCCmec-agr-typing; (v) rpoB and mprF single nucleotide polymorphisms (SNPs); (vi) dltA-mprF-atl-sceD expression by real-time quantitative polymerase chain reaction (qPCR). Three out of the four patients did not survive despite salvage treatment; two died with active MRSA infection and one died because of Stenotrophomonas maltophilia sepsis. The fourth patient, in which a reversion to a DAP-S phenotype occurred, survived with daptomycin plus trimethoprim/sulfamethoxazole and oxacillin treatment, and endovascular device removal. Daptomycin resistance development was preceded by a stable heterogeneous vancomycin-intermediate S. aureus (hVISA) or VISA phenotype acquisition, while in one case, daptomycin resistance was preceded by an unstable daptomycin heteroresistance (hDAP) behavior reverting to DAP-S during vancomycin plus rifampin therapy followed by high doses of daptomycin. All DAP-R strains showed hVISA or DAP-R traits, including mutations and/or up-regulation of genes involved in cell wall turnover and cell membrane perturbation. In our study, daptomycin resistance arose during glycopeptide therapy. The emergence of DAP-R isolates was preceded by a stable VISA or hVISA phenotype or by instability reverting to a DAP-S heteroresistant phenotype. Daptomycin, as first-line therapy for the treatment of severe MRSA infections, should be used at optimal dosage combined with other agents such as beta-lactams, to prevent daptomycin resistance occurrence.     

Survivability and Fitness Cost of Heterogeneous Vancomycin-intermediate Staphylococcus aureus

The aim of this study was to observe the survivability and fitness cost of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) isolates. Survivability study was performed on dry cotton swab, and fitness cost was evaluated by estimating growth kinetics and generation time constant in BACTEC automated system. Total mean maximum time of recovery on primary culture was 4.1 and 7.1 weeks (P = 0.0001) for hVISA and vancomycin-sensitive S. aureus (VSSA), respectively, in dry starved condition. No significant difference between the mean value of lag phase duration (P = 0.89) was noted between hVISA and VSSA isolate in growth kinetics. However, we observed lesser generation time of hVISA isolates compared to S. aureus ATCC 29213 (P = 0.0076). This study concluded that a significant difference in generation time between VSSA and hVISA and suggests that hVISA have fitness cost compared to VSSA. However, further studies with more cases are required.     

Update of the activity of telavancin against a global collection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> causing bacteremia,including endocarditis (2011–2014)

The efficacy and safety of telavancin is under evaluation for the treatment of subjects with complicated Staphylococcus aureus bacteremia and S. aureus right-sided infective endocarditis. This study evaluated the telavancin activity against a global collection of S. aureus causing bloodstream infections (BSI), including endocarditis, to support the development of bacteremia/endocarditis clinical indications. This study included a total of 4191?S. aureus [1490 methicillin-resistant S. aureus (MRSA)], which were unique (one per patient) clinical isolates recovered from blood samples collected during 2011–2014 in a global network of hospitals. All isolates were deemed responsible for BSI, including endocarditis, by local guidelines. Isolates were tested for susceptibility by broth microdilution. Telavancin (MIC_50/90, 0.03/0.06 μg/ml) inhibited all S. aureus at ≤0.12 μg/ml, the breakpoint for susceptibility. Equivalent minimum inhibitory concentration (MIC) values (MIC_50/90, 0.03/0.06 μg/ml) were obtained for telavancin against methicillin-susceptible S. aureus (MSSA) and MRSA isolates, as well as MRSA from community and healthcare origins. Similar telavancin activities (MIC₅₀, 0.03 μg/ml) were observed against MRSA subsets from North America and Europe, while isolates from the Asia-Pacific (APAC) and Latin America regions had MIC₅₀ values of 0.06 μg/ml. MRSA with vancomycin MIC values of 2–4 μg/ml and the multidrug resistance (MDR) subset had telavancin MIC₅₀ results of 0.06 μg/ml, although the MIC₁₀₀ result obtained against these subsets remained identical to those of MSSA (MIC₁₀₀, 0.12 μg/ml, respectively). This study updates the telavancin in vitro activity, which continues to demonstrate great potency against invasive S. aureus, regardless of the susceptibility phenotype or demographic characteristics (100.0% susceptible), and supports the sought-after subsequent indications.     

In vitro activity of tedizolid against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> collected in 2013 and 2014 from sites in Latin American countries,Australia, New Zealand,and China

Tedizolid is an oxazolidinone with an antimicrobial in vitro potency advantage against Gram-positive bacterial pathogens compared to other currently marketed drugs in this class, including linezolid. Tedizolid was compared to linezolid when tested against Staphylococcus aureus and Streptococcus pneumoniae isolates collected from countries in Latin America and the Asia-Pacific. Isolates were tested by broth microdilution susceptibility methods against tedizolid, linezolid, and non-class comparators in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The activity of tedizolid against S. aureus was potent and consistent in Latin America (MIC₉₀, 0.5 mg/L), Australia and New Zealand (MIC₉₀, 0.25 mg/L), and China (MIC₉₀, 0.5 mg/L). Based on MIC₉₀ results, tedizolid was four- to eight-fold more active than linezolid against S. aureus, including both methicillin-susceptible and -resistant isolates. Only two tedizolid non-susceptible strains were observed; both had intermediate minimum inhibitory concentration (MIC) values of 1 mg/L, for which the MICs of linezolid was higher (≥2 mg/L). Tedizolid (MIC₉₀, 0.25 mg/L) was four-fold more potent than linezolid (MIC₉₀, 1 mg/L) against S. pneumoniae in all countries that provided isolates. The findings from this study support the global clinical development of tedizolid for Gram-positive infections.     

In vitro antibacterial effects of statins against bacterial pathogens causing skin infections

With financial considerations impeding research and development of new antibiotics, drug repurposing (finding new indications for old drugs) emerges as a feasible alternative. Statins are extensively prescribed around the world to lower cholesterol, but they also possess inherent antimicrobial properties. This study identifies statins with the greatest potential to be repurposed as topical antibiotics and postulates a mechanism of action for statins’ antibacterial activity. Using broth microdilution, the direct antibacterial effects of all seven parent statins currently registered for human use and three selected statin metabolites were tested against bacterial skin pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens. Simvastatin and pitavastatin lactone exerted the greatest antibacterial effects (minimum inhibitory concentrations of 64 and 128 μg/mL, respectively) against S. aureus. None of the statins tested were effective against E. coli, P. aeruginosa, or S. marcescens, but simvastatin hydroxy acid acid might be active against S. aureus, E. coli, and S. marcescens at drug concentrations >?256 μg/mL. It was found that S. aureus may exhibit a paradoxical growth effect when exposed to simvastatin; thus, treatment failure at high drug concentrations is theoretically probable. Through structure-activity relationship analysis, we postulate that statins’ antibacterial action may involve disrupting the teichoic acid structures or decreasing the number of alanine residues present on Gram-positive bacterial cell surfaces, which could reduce biofilm formation, diminish bacterial adhesion to environmental surfaces, or impede S. aureus cell division.     

Phenotypic changes of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> during vancomycin therapy for persistent bacteraemia and related clinical outcome

Persistent bacteraemia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) that fails to respond to glycopeptide therapy is a well-documented clinical problem. There are limited data on changes in agr functionality, vancomycin susceptibility and heteroresistance during MRSA PB. Thus, the frequency of these changes and their clinical significance remain unclear. Only patients with MRSA PB (≥7 days) from a prospective cohort of S. aureus bacteraemia were included. We collected isogenic paired strains and compared vancomycin MIC, vancomycin heteroresistance, and agr functionality between initial and final blood isolates. We also assessed the clinical outcome. A total of 49 patients had MRSA PB over 22 months. Bacteraemia persisted for a median of 13 days and most patients (98%) received glycopeptide as initial therapy. Among 49 isogenic pairs, only one pair showed a vancomycin MIC increase ≥2-fold by broth microdilution method, and only seven (14%) by E-test. Significant portions of initial isolates had vancomycin heteroresistance (49%) and agr dysfunction (76%). Development of vancomycin heteroresistance during PB occurred in four (16%) among 25 initial vancomycin-susceptible isolates, and acquisition of agr dysfunction occurred in two (16%) among 12 initial agr-functional isolates. Changes in the opposite direction occasionally occurred. These phenotypic changes during PB were not associated with mortality, whereas agr dysfunction of the initial isolates was significantly associated with mortality. During MRSA PB, phenotypic changes of MRSA isolates occurred occasionally under prolonged vancomycin exposure but were not significantly associated with clinical outcome. In contrast, initial agr dysfunction could be a predictor for mortality in MRSA PB.     

Antimicrobial susceptibility testing of Gram-positive and -negative bacterial isolates directly from spiked blood culture media with Raman spectroscopy

Patients suffering from bacterial bloodstream infections have an increased risk of developing systematic inflammatory response syndrome (SIRS), which can result in rapid deterioration of the patients’ health. Diagnostic methods for bacterial identification and antimicrobial susceptibility tests are time-consuming. The aim of this study was to investigate whether Raman spectroscopy would be able to rapidly provide an antimicrobial susceptibility profile from bacteria isolated directly from positive blood cultures. First, bacterial strains (n?=?133) were inoculated in tryptic soy broth and incubated in the presence or absence of antibiotics for 5 h. Antimicrobial susceptibility profiles were analyzed by Raman spectroscopy. Subsequently, a selection of strains was isolated from blood cultures and analyzed similarly. VITEK®2 technology and broth dilution were used as the reference methods. Raman spectra from 67 antibiotic-susceptible strains showed discriminatory spectra in the absence or at low concentrations of antibiotics as compared to high antibiotic concentrations. For 66 antibiotic-resistant strains, no antimicrobial effect was observed on the bacterial Raman spectra. Full concordance with VITEK®2 data and broth dilution was obtained for the antibiotic-susceptible strains, 68 % and 98 %, respectively, for the resistant strains. Discriminative antimicrobial susceptibility testing (AST) profiles were obtained for all bacterial strains isolated from blood cultures, resulting in full concordance with the VITEK®2 data. It can be concluded that Raman spectroscopy is able to detect the antimicrobial susceptibility of bacterial species isolated from a positive blood culture bottle within 5 h. Although Raman spectroscopy is cheap and rapid, further optimization is required, to fulfill a great promise for future AST profiling technology development.     

Mass spectrometric identification of phenol-soluble modulins in the ATCC® 43300 standard strain of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> harboring two distinct phenotypes

Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 43300?) is a representative methicillin-resistant S. aureus (MRSA) strain that is used as a quality control strain for testing the methicillin susceptibility of clinical isolates. It has been consistently observed that colonies with two different colors (golden yellow and white) grow from the ATCC stock on blood agar plates. In this study, staphylococcal peptide and protein profiling was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. From the extract of the potentially hemolytic 43300 strain, we identified a single significant peak at an observed mass-to-charge (m/z) value of 2306.9, which correlates well with the predicted mass of formylated phenol-soluble modulin α2, a major staphylococcal virulence factor. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the hemolytic 43300 cells predominantly produced various phenol-soluble modulins (PSMs) and their export proteins, including four α type PSM peptides, PSMβ1, PSM-mec, PmtC and PmtD. However, none of the PSM peptides was detected in the presumably non-hemolytic 43300 strain, but the export proteins PmtC and PmtD. We found that this MRSA standard strain expresses two distinct phenotypes and that their phenotypic features are more likely associated with PSM toxin production. Therefore, careful attention is needed when MRSA reference strains are utilized for the diagnosis and susceptibility testing of MRSA in clinical settings.     

The ArlRS two-component system is a regulator of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>-induced endothelial cell damage

Staphylococcus aureus endovascular infections retain a high morbidity and mortality despite antibiotics and supportive care. The destruction of endothelial cells (ECs) is a critical step in the pathogenesis of S. aureus endovascular infections. In order to better understand S. aureus-induced EC damage, we systematically screened a collection of two-component regulatory system mutants of methicillin-resistant S. aureus (MRSA) USA300 strain JE2 for damage induction in human umbilical vein ECs (HUVECs). This screen revealed that the two-component regulatory system ArlRS is required for maximum damage: arlRS inactivation leads to a > 70% reduction in damage. In a different genetic S. aureus background (RN6390, MSSA strain) arlRS inactivation had a smaller but also significant effect on EC damage. In both strains, the reduction in EC damage was accompanied by a significant reduction in internalization. In conclusion, we determined a novel role of ArlRS in S. aureus-induced EC damage, which will help to better understand the pathogenesis of S. aureus endovascular infection.     

Reduced Vancomycin Susceptibility in Staphylococcus aureus,Including Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Strains: Resistance Mechanisms,Laboratory Detection,and Clinical Implications

Summary: The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) over the past decade has provided a challenge to diagnostic microbiologists to detect these strains, clinicians treating patients with infections due to these strains, and researchers attempting to understand the resistance mechanisms. Recent data show that these strains have been detected globally and in many cases are associated with glycopeptide treatment failure; however, more rigorous clinical studies are required to clearly define the contribution of hVISA to glycopeptide treatment outcomes. It is now becoming clear that sequential point mutations in key global regulatory genes contribute to the hVISA and VISA phenotypes, which are associated predominately with cell wall thickening and restricted vancomycin access to its site of activity in the division septum; however, the phenotypic features of these strains can vary because the mutations leading to resistance can vary. Interestingly, changes in the staphylococcal surface and expression of agr are likely to impact host-pathogen interactions in hVISA and VISA infections. Given the subtleties of vancomycin susceptibility testing against S. aureus, it is imperative that diagnostic laboratories use well-standardized methods and have a framework for detecting reduced vancomycin susceptibility in S. aureus.     

Biofilm Forming Ability and <Emphasis Type="Italic">Spa</Emphasis> Gene Polymorphism in Methicillin Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Clinical Isolates in North of Iran

Spa typing has been shown to function as a genetic marker for Staphylococcus aureus outbreak investigations and epidemiological studies. This study was aimed to investigate biofilm formation capacity and spa gene polymorphism in methicillin resistant S. aureus (MRSA) strains isolated from clinical samples. A total of 102 S. aureus isolated during 2016, were analyzed for methicillin resistance and biofilm formation using phenotypic assays and PCR-based detection of associated genes. The polymorphic region of the spa gene was amplified by PCR using specific primers and subsequently in MRSA strains the amplified products were sequenced and spa types determined by using the spa database website. Out of 102 S. aureus, 41 isolates (40.2%) recognized as MRSA in phenotypic and genotypic investigations. In phenotypic assay, biofilm forming ability was detected in 71 isolates. The frequency of icaA and fnbA in test isolate were 53.9 and 65.7% respectively. Amplification of polymorphic region of the spa gene in all 102 tested isolates resulted in eight size fragments ranged between 168–336 bp. In MRSA strains thirteen distinct spa types with 5–12 repeats were observed. The most frequent types of spa were t030, t037, t325, t421, t937, t1814 and t084. spa types t2421, t1814, t359 and t2617 identified for the first time in Iran. The present results showed high biofilm formation capacity and great diversity of variable region of spa gene in MRSA strains and confirmed that, spa typing provides valuable information on the epidemiologic features and discrimination of this bacterium.     

Preliminary antimycobacterial study on selected Turkish plants (Lamiaceae) against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and search for some phenolic constituents

Background

The global resurgence of tuberculosis is a significant threat. Lamiaceae members have been used in folk remedies for centuries. This study was designed to assess the in-vitro antimycobacterial activity of eighteen crude extracts from six plants (Lamiaceae) and to characterize their phenolic and flavonoid compounds.

Methods

Six Turkish medicinal plants of the family Lamiaceae (Stachys tmolea Boiss., Stachys thirkei C. Koch, Ballota acetabulosa (L.) Benth., Thymus sipthorpii Benth., Satureja aintabensis P.H. Davis, and Micromeria juliana (L.) Benth. ex Reich.) were collected in 2009 – 2010. Dried and crushed plant samples were subjected to sequential extraction with petroleum ether, ethyl acetate, and methanol in order of increasing polarity. A broth microdilution method was employed to screen extracts against four mycobacterial strains of Mycobacterium tuberculosis. Phenolic and flavonoid compounds were characterized by liquid chromatography–mass spectrometry.

Results

S. aintabensis, T. sibthorpii, and M. juliana were found to develop considerable activity against the four strains of M. tuberculosis with the minimal inhibitory concentrations value of 12.5-100 μg/ml. S. aintabensis and T. sibthorpii extracts killed M. tuberculosis with the minimum bactericidal concentration value of 50–800 μg/ml. On the basis of these prominent antimycobacterial activity, we suggest that they could be a source of natural anti-tuberculosis agents.

Conclusion

S. aintabensis and T. sibthorpii showed activity by killing Mycobacteria strains. The major phenolic compound was rosmarinic for T. sibthorpii and S. aintabensis. Flavonoids might be “a modal” for the drug design.

     

DNA microarray analysis of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> causing bloodstream infection: bacterial genes associated with mortality?

Providing evidence for microbial genetic determinants’ impact on outcome in Staphylococcus aureus bloodstream infections (SABSI) is challenging due to the complex and dynamic microbe–host interaction. Our recent population-based prospective study reported an association between the S. aureus clonal complex (CC) 30 genotype and mortality in SABSI patients. This follow-up investigation aimed to examine the genetic profiles of the SABSI isolates and test the hypothesis that specific genetic characteristics in S. aureus are associated with mortality. SABSI isolates (n?=?305) and S. aureus CC30 isolates from asymptomatic nasal carriers (n?=?38) were characterised by DNA microarray analysis and spa typing. Fisher’s exact test, least absolute shrinkage and selection operator (LASSO) and elastic net regressions were performed to discern within four groups defined by patient outcome and characteristics. No specific S. aureus genetic determinants were found to be associated with mortality in SABSI patients. By applying LASSO and elastic net regressions, we found evidence suggesting that agrIII and cna were positively and setC (=selX) and seh were negatively associated with S. aureus CC30 versus non-CC30 isolates. The genes chp and sak, encoding immune evasion molecules, were found in higher frequencies in CC30 SABSI isolates compared to CC30 carrier isolates, indicating a higher virulence potential. In conclusion, no specific S. aureus genes were found to be associated with mortality by DNA microarray analysis and state-of-the-art statistical analyses. The next natural step is to test the hypothesis in larger samples with higher resolution methods, like whole genome sequencing.     

Microbiology of chronic rhinosinusitis

Most sinus infections are viral and only a small percentage develop bacterial infection. Rhino-, influenza, and para-influenza viruses are the most frequent viral causes of sinusitis. The most common bacterial isolates from children and adult patients with community-acquired acute bacterial sinusitis are Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes. Staphylococcus aureus and anaerobic organisms (Prevotella and Porphyromonas, Fusobacterium, and Peptostreptococcus spp.) are the commonest isolates in chronic rhinosinusitis (CRS). Aerobic and anaerobic beta lactamase-producing bacteria (BLPB) were recovered from over a third of these patients. Methicillin-resistant S. aureus (MRSA) accounted for over 60 % of S. aureus isolates. Pseudomonas aeruginosa and other aerobic and facultative Gram-negative rods are frequently recovered in nosocomial sinusitis, the immunocompromised host, individuals with human immunodeficiency virus infection, and in cystic fibrosis. The CRS infection evolves the formation of a biofilm that might play a significant role in the pathogenesis and persistence of CRS. The microbiology of sinusitis is influenced by previous antimicrobial therapy, vaccinations, and the presence of normal flora capable of interfering with the growth of pathogens. Recognition of the unique microbiology of CRS and their antimicrobial susceptibility is of great importance when selecting antimicrobial therapy.     

Development of EUCAST zone diameter breakpoints and quality control criteria for ceftazidime-avibactam 10-4 μg

Ceftazidime-avibactam disk studies were performed for disk mass selection and for establishing EUCAST quality control ranges and zone diameter breakpoints. The disk mass study included disk diffusion testing with ceftazidime-avibactam 10-4 and 10-6 μg disks and broth microdilution MIC testing for challenge set of 94 Enterobacteriaceae and 45 Pseudomonas aeruginosa. EUCAST SOP 9.0-based QC and MIC-disk correlations studies were followed for development of ceftazidime-avibactam 10-4 μg ranges for Escherichia coli ATCC 25922, P. aeruginosa ATCC 27583, and Klebsiella pneumoniae ATCC 700603 and for zone diameter breakpoint determination. The ceftazidime-avibactam 10-4 and 10-6 μg disks performed similar in comparison to broth microdilution, with zones ≤?14 mm for all resistant strains. The 10-4 μg disk was selected and used in QC and breakpoint studies. There was minimal variation of ceftazidime-avibactam 10-4 μg QC study results between disks, media, and sites. The QC ranges were within 7 mm for all strains. The zone diameter breakpoint study demonstrated good correlation of MIC and disk results. The established zone diameter breakpoints resulted in false susceptible rates of 1.6 and 4.0% for Enterobacteriaceae and P. aeruginosa. EUCAST selected the ceftazidime-avibactam 10-4 μg disk and established QC ranges for E. coli 25922 of 24–30 mm, P. aeruginosa ATCC 27853 of 21–27 mm, and K. pneumoniae ATCC 700603 of 18–24 mm. The zone diameter breakpoints that correlated best with the MIC breakpoints of susceptible ≤?8 mg/L and resistant >?8 mg/L were Enterobacteriaceae (S?≥?13, R?<?13 mm) and P. aeruginosa (S?≥?17, R?<?17 mm).     

Synergistic effect of vancomycin combined with cefotaxime,imipenem, or meropenem against Staphylococcus aureus with reduced susceptibility to vancomycin

Background/aim We investigated the synergistic effect between vancomycin and β-lactams against vancomycin-susceptible (VSSA) and nonsusceptible MRSA isolates [heterogeneous vancomycin-intermediate S. aureus (hVISA) and VISA].Materials and methods A total of 29 MRSA, including 6 VISA, 14 hVISA, and 9 VSSA isolates, were subjected to a microbroth dilution-minimum inhibitory concentration (MIC) checkerboard using vancomycin combined with cefotaxime, imipenem, or meropenem. To confirm synergistic activity, the representative strains of VISA, hVISA, and VSSA were then selected for the time-kill curve method.Results The combination of vancomycin with imipenem, meropenem, or cefotaxime exhibited synergistic effects against 17 (2 VISA, 9 hVISA, and 6 VSSA), 14 (3 VISA, 9 hVISA and 2 VSSA), and 5 (3 VISA and 2 hVISA) isolates, respectively. Additive and indifferent effects were found in the remaining isolates, but no antagonistic effect was observed. Using time-kill assay, the vancomycin combined with either imipenem or cefotaxime demonstrated synergism against both VISA and hVISA isolates, while the synergistic effect with meropenem was obtained only in the VISA isolates.Conclusion This study demonstrated in vitro enhanced antibacterial activity of vancomycin plus β-lactams against clinical hVISA or VISA isolates. These combinations may be an alternative treatment for MRSA infections in clinical practice.     

Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures

The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla _CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.     

Spa type distribution in MRSA and MSSA bacteremias and association of spa clonal complexes with the clinical characteristics of bacteremia

The genetic distribution of invasive methicillin-susceptible (MSSA) and resistant S. aureus (MRSA) strains has to be addressed in order to target infection control strategies. A large MRSA epidemic caused by a certain MRSA strain (spa type 067) broke out in 2001 in our health district. We wanted to investigate the current spa type distribution in MRSA and MSSA bacteremias and assess the potential association of spa clonal complexes (spaCC) with the clinical characteristics of S. aureus bacteremia. One hundred nine invasive MRSA isolates and 353 invasive MSSA isolates were spa typed and grouped into clonal complexes (spaCC). Spa type distribution was compared to that of colonizing MRSA strains. Spa type and spaCC data linked to clinical information on the course of bacteremic cases was used to search for differences in virulence between strains. Spa type distribution in MRSA is less heterogenic than in MSSA. t067 dominates both in MRSA colonisations and in invasive findings. Among MSSA, no such dominating strains were found. Of spaCCs, mortality was the highest in spaCC 067 (25.6%). SpaCC 008 was more often associated with endocarditis than other CCs (22.7 vs 5.8%, p =?0.013), spaCC 2133 with skin infections (68.4 vs 36.4%, p =?0.007), and spaCC 012 with foreign body infections (25.0 vs 9.3%, p =?0.029) than other clonal complexes. A single successful strain can explain the major proportion of MRSA among S. aureus bacteremias. Certain spaCCs showed association with certain clinical characteristics. These findings suggest that S. aureus strains differ in their virulence and invasiveness.     

Low occurrence of the new species <Emphasis Type="Italic">Staphylococcus argenteus</Emphasis> in a <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> collection of human isolates from Belgium

Staphylococcus argenteus is a novel Staphylococcus species closely related to Staphylococcus aureus that has been recently described. In this study, we investigated the proportion and the characteristics of S. argenteus recovered from humans in Belgium. S. aureus. human isolates collected in Belgium from 2006 to 2015 (n?=?1,903) were retrospectively characterised via the presence of non-pigmented colonies on chocolate agar, spa typing and rpoB sequencing to determine if some of them were in fact S. argenteus. Out of 73 strains non-pigmented on chocolate plates, 3 isolates (0.16 %) showed rpoB sequences, in addition to spa and sequence types (ST2250/t5787, ST2250/t6675, ST3240/t6675), related to S. argenteus. Two of them were methicillin-resistant, harbouring a SCCmec type IV. The three S. argenteus isolates carried genes (sak, scn) of the immune evasion cluster. This first Belgian nationwide analysis showed a low occurrence of S. argenteus. Further studies should be conducted to identify the distribution range and the clinical impact of this new species.