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Characterization of Escherichia coli strains resistant to carbapenems isolated from rectal swab in a multidrug-resistant strains screening programme
No full textThe 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
Real-time PCR assay for detection of Staphylococcus aureus, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples
Full text linkRapid 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 ‘in house’ 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
Klebsiella pneumoniae carbapenemase (KPC) producer resistant to ceftazidime–avibactam due to a deletion in the blaKPC3 gene
No full textKlebsiella 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
Multiple detection of hypermucoviscous and hypervirulent strains of Klebsiella pneumoniae: An emergent health care threat
No full textThis 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
Ceftazidime/avibactam resistance is associated with different mechanisms in KPC-producing Klebsiella pneumoniae strains
Full text linkThis 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
Intrinsic role of coagulase negative staphylococci norA-like efflux system in fluoroquinolones resistance
Full text linkNorA 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 DH5α carrying the pB SK 198 norASEP EZ cat norASEP plasmid
spa Typing and Molecular Characterization of Antimicrobial Resistance in Staphylococcus aureus Strains from Patients with Cystic Fibrosis
No full textWe characterized Staphylococcus aureus strains isolated from cystic fibrosis (CF) patients during screening for multidrug-resistant strains to determine mechanisms of antibiotic resistance and conduct spa typing. We investigated 53 S. aureus isolates collected from different CF patients, excluding multiple isolates from the same patient. Genotypic characterization was based on spa type (protein A); staphylococcal cassette chromosome mec (SCCmec) type for S. aureus resistant to methicillin (methicillin-resistant S. aureus [MRSA]); and resistance to the most common macrolides, lincosamides, and streptogramins b and fluoroquinolones. Most strains (78.41%) were resistant to one or more antibiotics; 16.96% were MRSA, whereas 69.81% showed resistance to erythromycin. MRSA strains revealed the acquisition and insertion of SCCmec of class I (n = 1) (hospital-acquired), IV (n = 5), and V (n = 1) (community-acquired), along with two cases that were not typeable. We detected 34 different spa types, with t571 being the most frequent. The spa minimum spanning tree of the tested strains showed evidence of strain relatedness
Different OXA-Carbapenemases in Genetically Unrelated Klebsiella pneumoniae Strains Isolated in a North Italian Hospital During Multidrug Resistance Screening
No full text: 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
Evaluation of rapid KPC carbapenemase detection method based on MALDI-TOF VITEK MS spectra analysis
No full textClinical microbiology laboratories in hospital settings need to be able to identify patients who carry carbapenemase-producing bacterial strains quickly in order to contain their spread and initiate proper pharmacological therapy. The aim of this study was to confirm the correlation between KPC production and a characteristic mass spectrometry (MS) peak (11 109 Da±8) to validate the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS as a rapid screening tool. With this aim, 176 selected clinical samples that were KPC-producing and 260 control samples that were carbapenem-susceptible or carbapenem-resistant through other resistance mechanisms, or were producing hydrolytic enzymes other than KPC, were analysed. The presence of the 11 109 Da peak in the spectra of 99.4 % (175/176) of the KPC-producing strains compared to the controls, which all lacked the peak, confirmed a strong correlation between KPC production and the presence of the 11 109 Da peak in the MALDI-TOF MS spectrum. The high sensitivity (98.7 %) and specificity (100 %) of the peak searching in the MALDI-TOF MS spectra mean that 11 109 Da peak searching is a suitable screening tool in KPC-endemic regions
Molecular detection of bacterial RNA in atheromatous plaques of patients with stroke
No full textAim: Infections and chronic inflammatory conditions such as periodontitis, have been associated with the development and progression of atherosclerosic process and susceptibility to plaque rupture. The chronicity of periodontal disease provides a rich source of subgingival bacteria, mostly gram negative, which are frequently spread in the blood as a result of the periodontal lesion. DNA from periodontal pathogens has been detected in atherosclerotic lesions, but viable bacteria have not yet been isolated.
This study was carried out to detect, by molecular techniques, the presence of RNA of bacteria in atheromatous plaques collected from both symptomatic and asymptomatic carotid stenosis patients.
Methods: Total RNA extracts, derived from eighty-one atheromatous plaques collected during carotid endartrectomy (36 from symptomatic patients and 45 from asymptomatic patients) underwent reverse transcription to cDNA using random primers. All cDNA samples obtained were tested by PCR using universal primers for global bacterial detection (16S rRNA).
Results: Of eighty-one sample tested, seven were found positive (8.6%), six out of the thirty-six (16.6%) of symptomatic patients and only one out forty-five (2.2%) from the asymptomatic patients group.
Conclusion: This pilot study suggests the preferential presence of bacterial RNA in recently symptomatic atherosclerotic plaques. These results are of interest because the presence of viable bacteria may be presumed into the atheromatous plaques and, therefore, indicates a possible bacterial role in activation of several inflammatory factors and in atherosclerotic plaque vulnerability
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