Real-time PCR assay for detection of Staphylococcus aureus, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Rapid detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important concern for both treatment and implementation of infection control policies. The present study provides an \u2018in house\u2019 real-time PCR assay to detect directly nuc, pvl, and mecA genes. The assay is able to perform identification of MRSA, Methicillin-Sensitive S. aureus, Methicillin-Resistant Coagulase Negative Staphylococci and the Panton-Valentine leukocidin virulence gene from rectal and pharyngeal swab samples in a screening context. We found an analytical sensitivity of this current Triplex PCR assay of 514 CFU/mL. Analytical specificity was tested with different Gram-positive and Gram-negative species and yielded no false-positive PCR signal. The sensitivity and specificity of the Triplex Real Time PCR were both 100% for these targets when compared with the culture and conventional methods. This assay is readily adaptable for routine use in a microbiology laboratory, as it will enable the implementation of timely and properly guided therapy and infection control strategies

Intrinsic role of coagulase negative staphylococci norA-like efflux system in fluoroquinolones resistance

NorA is a Staphylococcus aureus multidrug transporter that exports structurally distinct compounds including fluoroquinolones. In this study norA-like genes of Staphylococcus epidermidis (norASEP) and Staphylococcus haemolyticus (norASHAE) were identified and sequenced. The nucleotide identity of norASEP and norASHAE with norA was 75.3 and 74.1%, respectively, and the amino acid identity 87.7 and 86%, respectively. Inactivation of norASEP increased the ciprofloxacin susceptibility of E. coli DH5\u3b1 carrying the pB SK 198 norASEP EZ cat norASEP plasmid

Effects of Mushroom and Chicory Extracts on the Physiology and Shape of Prevotella intermedia, a Periodontopathogenic Bacterium

Contrary to the common assumption that food has a negative impact on oral health, research has shown that several foods contain a number of components with antibacterial and antiplaque activity. These natural compounds may be useful for improving daily oral hygiene. In this study we evaluate the mode of antimicrobial action of fractions of mushroom and red chicory extracts on Prevotella intermedia, a periodontopathogenic bacterium. The minimal inhibitory concentration corresponded to 0.5x compared to the natural food concentration for both extracts. This concentration resulted in a bacteriostatic effect in mushroom extract and in a slightly bactericidal effect in chicory extract. Cell mass continued to increase even after division stopped. As regards macromolecular synthesis, DNA was almost totally inhibited upon addition of either mushroom or chicory extract, and RNA to a lesser extent, while protein synthesis continued. Cell elongation occurred after septum inhibition as documented by scanning electron microscopy and cell measurement. The morphogenetic effects are reminiscent of the mode of action of antibiotics such as quinolones or β-lactams. The discovery of an antibiotic-like mode of action suggests that these extracts can be advantageously employed for daily oral hygiene in formulations of cosmetic products such as mouthwashes and toothpastes

Cerebral Palsy:Early Markers of Clinical Phenotype and Functional Outcome

The Prechtl General Movement Assessment (GMA) has become a cornerstone assessment in early identification of cerebral palsy (CP), particularly during the fidgety movement period at 3-5 months of age. Additionally, assessment of motor repertoire, such as antigravity movements and postural patterns, which form the Motor Optimality Score (MOS), may provide insight into an infant's later motor function. This study aimed to identify early specific markers for ambulation, gross motor function (using the Gross Motor Function Classification System, GMFCS), topography (unilateral, bilateral), and type (spastic, dyskinetic, ataxic, and hypotonic) of CP in a large worldwide cohort of 468 infants. We found that 95% of children with CP did not have fidgety movements, with 100% having non-optimal MOS. GMFCS level was strongly correlated to MOS. An MOS > 14 was most likely associated with GMFCS outcomes I or II, whereas GMFCS outcomes IV or V were hardly ever associated with an MOS > 8. A number of different movement patterns were associated with more severe functional impairment (GMFCS III-V), including atypical arching and persistent cramped-synchronized movements. Asymmetrical segmental movements were strongly associated with unilateral CP. Circular arm movements were associated with dyskinetic CP. This study demonstrated that use of the MOS contributes to understanding later CP prognosis, including early markers for type and severity

Predicting the Need for Biopsy to Detect Clinically Significant Prostate Cancer in Patients with a Magnetic Resonance Imaging-detected Prostate Imaging Reporting and Data System/Likert ≥3 Lesion: Development and Multinational External Validation of the Imperial Rapid Access to Prostate Imaging and Diagnosis Risk Score

BACKGROUND: Although multiparametric magnetic resonance imaging (MRI) has high sensitivity, its lower specificity leads to a high prevalence of false-positive lesions requiring biopsy. OBJECTIVE: To develop and externally validate a scoring system for MRI-detected Prostate Imaging Reporting and Data System (PIRADS)/Likert ≥3 lesions containing clinically significant prostate cancer (csPCa). DESIGN, SETTING, AND PARTICIPANTS: The multicentre Rapid Access to Prostate Imaging and Diagnosis (RAPID) pathway included 1189 patients referred to urology due to elevated age-specific prostate-specific antigen (PSA) and/or abnormal digital rectal examination (DRE); April 27, 2017 to October 25, 2019. INTERVENTION: Visual-registration or image-fusion targeted and systematic transperineal biopsies for an MRI score of ≥4 or 3 + PSA density ≥0.12 ng/ml/ml. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Fourteen variables were used in multivariable logistic regression for Gleason ≥3 + 4 (primary) and Gleason ≥4 + 3, and PROMIS definition 1 (any ≥4 + 3 or ≥6 mm any grade; secondary). Nomograms were created and a decision curve analysis (DCA) was performed. Models with varying complexity were externally validated in 2374 patients from six international cohorts. RESULTS AND LIMITATIONS: The five-item Imperial RAPID risk score used age, PSA density, prior negative biopsy, prostate volume, and highest MRI score (corrected c-index for Gleason ≥3 + 4 of 0.82 and 0.80-0.86 externally). Incorporating family history, DRE, and Black ethnicity within the eight-item Imperial RAPID risk score provided similar outcomes. The DCA showed similar superiority of all models, with net benefit differences increasing in higher threshold probabilities. At 20%, 30%, and 40% of predicted Gleason ≥3 + 4 prostate cancer, the RAPID risk score was able to reduce, respectively, 11%, 21%, and 31% of biopsies against 1.8%, 6.2%, and 14% of missed csPCa (or 9.6%, 17%, and 26% of foregone biopsies, respectively). CONCLUSIONS: The Imperial RAPID risk score provides a standardised tool for the prediction of csPCa in patients with an MRI-detected PIRADS/Likert ≥3 lesion and can support the decision for prostate biopsy. PATIENT SUMMARY: In this multinational study, we developed a scoring system incorporating clinical and magnetic resonance imaging characteristics to predict which patients have prostate cancer requiring treatment and which patients can safely forego an invasive prostate biopsy. This model was validated in several other countries

Characterization of Escherichia coli strains resistant to carbapenems isolated from rectal swab in a multidrug-resistant strains screening programme

The aim of the study is to characterize 28 Escherichia coli carbapenem-resistant strains isolated from multi-resistant screening. All the strains were tested through CARBA NP test and PCR analysis for molecular characterization of carbapenemase. Plasmid characterization and phylogenetic study was performed. The molecular characterization revealed that 24 of 28 strains harbour carbapenemases. The most involved plasmids are FIA, FIB, FIIS and FrepB replicons that belong to the IncF group. The phylo-typing analysis revealed a greater presence of the B2 group. Carbapenem resistance in E. coli, should be constantly monitored to avoid the onset of new epidemic episodes

Multiple detection of hypermucoviscous and hypervirulent strains of Klebsiella pneumoniae: An emergent health care threat

This study focused on the characterization of 19 hypermucoviscous Klebsiella pneumoniae strains, that were identified from 26 hypermucosal strains. In order to identify hypermucoviscous strains of K. pneumoniae, the string test was applied. This phenotype is known in the literature as one of the virulence factors of this species together with the production of biofilm and other hypervirulence factor genes such as: rmpA, rmpA2, iucA, iroB, peg-344. We also investigated presence of magA gene that correlates with the hyper-production of capsule of K1 serotype. Of the strains under study, 13 out of 19 harboured at least one virulence factor.Sequence type (ST) was determined in order to identify known high-risk clones or new emerging high-risk clones and their variability in a single clinical setting. Important STs found among these strains were ST65 and ST29. Carbapenem resistance was also investigated and 4 out of 19 strains harboured at least a carbapenemase: one strain harboured a KPC enzyme alone, one strain carried a KPC and an OXA-48 like, one strain produced OXA-48-like alone, and the last strain harboured two metallo-β-lactamases (VIM-1 and NDM-5) plus OXA-48-like. In particular, this latter strain belongs to ST383, which was recently reported in Northern Italy as a hypervirulent and XDR strain.The global spread of hypervirulent K. pneumoniae is an important epidemiological issue that should be considered in diagnostic and therapeutic managements of patients with K. pneumoniae infections

Klebsiella pneumoniae KPC producer resistant to ceftazidime-avibactam due to a deletion in the blaKPC3 gene

Klebsiella pneumoniae producing KPC is a great health concern and therapy with ceftazidime-avibactam represent a choice for the treatment of infections supported by these strains. We report a ceftazidime-avibactam resistant strain by a deletion of 6 nucleotides in the blaKPC gene sequence

Ceftazidime/avibactam resistance is associated with different mechanisms in KPC-producing Klebsiella pneumoniae strains

This study focused on Klebsiella pneumoniae isolates that were resistant or had low susceptibility to a combination of ceftazidime/avibactam. We aimed to investigate the mechanisms underlying this resistance. A total of 24 multi-drug resistant isolates of K. pneumoniae were included in the study. The phenotypic determination of carbapenemase presence was based on the CARBA NP test. NG-Test CARBA 5 was also performed, and it showed KPC production in 22 out 24 strains. The molecular characterisation of blaKPC carbapenemase gene, ESBL genes (blaCTX-M, blaTEM, and blaSHV) and porin genes ompK35/36 was performed using the PCR. Finally, ILLUMINA sequencing was performed to determine the presence of genetic mutations.Various types of mutations in the KPC sequence, leading to ceftazidime/avibactam resistance, were detected in the analysed resistant strains. We observed that KPC-31 harboured the D179Y mutation, the deletion of the amino acids 167-168, and the mutation of T243M associated with ceftazidime/avibactam resistance. The isolates that did not present carbapenemase alterations were found to have other mechanisms such as mutations in the porins. The mutations both on the KPC-3 enzyme and in the porins confirmed, that diverse mechanisms confer resistance to ceftazidime/avibactam in K. pneumoniae

Different OXA-Carbapenemases in Genetically Unrelated Klebsiella pneumoniae Strains Isolated in a North Italian Hospital During Multidrug Resistance Screening

: Klebsiella pneumoniae is one of the main opportunistic pathogens that cause a broad spectrum of diseases with increasingly frequent acquisition of resistance to antibiotics, namely carbapenems. This study focused on the characterization of 23 OXA-48-like carbapenemase-producing K. pneumoniae isolates using phenotypic and molecular tests. Phenotypic determination of the presence of β-lactamases was performed using the extended-spectrum beta-lactamase (ESBL) NP test, and phenotypic determination of the presence of carbapenemase was based on the Carba NP test. Antimicrobial susceptibility tests were performed to assess the resistance against carbapenems. Molecular characterization of ESBL genes and carbapenemase genes (blaOXA-48, blaKPC, blaVIM, and blaNDM) was performed using polymerase chain reaction (PCR) techniques. In addition, K. pneumoniae strains were analyzed for their relatedness using multilocus sequence typing PCR analysis based on the Institut Pasteur protocol, which produces allelic profiles that contain their evolutionary and geographic pattern. Following further Sanger sequencing of the blaOXA-48 genes, no genetic mutations were found. Some OXA-48-producing K. pneumoniae isolates coharbored blaKPC, blaNDM, and blaVIM genes, which encode other carbapenemases that can hydrolyze carbapenem antibiotics. The final part of the study focused on the characterization of the plasmid profiles of all isolates to better understand the spreading of the IncL/M blaOXA-48 plasmid gene. The plasmid profile also revealed other incompatibility groups, suggesting that other plasmid genes are spreading in K. pneumoniae isolates, which can coharbor and spread different carbapenemases simultaneously