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 ß-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.

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-ß-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.

spa Typing and Molecular Characterization of Antimicrobial Resistance in Staphylococcus aureus Strains from Patients with Cystic Fibrosis.

We 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.

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.

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.

Dynamics of SARS-CoV2 Infection and Multi-Drug Resistant Bacteria Superinfection in Patients With Assisted Mechanical Ventilation.

Objective: To investigate the presence of bacteria and fungi in bronchial aspirate (BA) samples from 43 mechanically ventilated patients with severe COVID-19 disease. Methods: Detection of SARS-CoV-2 was performed using Allplex 2019-nCoV assay kits. Isolation and characterisation of bacteria and fungi were carried out in BA specimens treated with 1X dithiothreitol 1% for 30 min at room temperature, using standard culture procedures. Results: Bacterial and/or fungal superinfection was detected in 25 out of 43 mechanically ventilated patients, generally after 7 days of hospitalisation in an intensive care unit (ICU). Microbial colonisation (colony forming units (CFU) <1000 colonies/ml) in BA samples was observed in 11 out of 43 patients, whereas only 7 patients did not show any signs of bacterial or fungal growth. Pseudomonas aeruginosa was identified in 17 patients. Interestingly, 11 out of these 17 isolates also showed carbapenem resistance. The molecular analysis demonstrated that resistance to carbapenems was primarily related to OprD mutation or deletion. Klebsiella pneumoniae was the second most isolated pathogen found in 13 samples, of which 8 were carbapenemase-producer strains. Conclusion: These data demonstrate the detection of bacterial superinfection and antimicrobial resistance in severe SARS-CoV-2-infected patients and suggest that bacteria may play an important role in COVID-19 evolution. A prospective study is needed to verify the incidence of bacterial and fungal infections and their influence on the health outcomes of COVID-19 patients.

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 '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.

Evaluation of rapid KPC carbapenemase detection method based on MALDI-TOF VITEK MS spectra analysis.

Clinical 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.

The anti-adhesive mode of action of a purified mushroom (Lentinus edodes) extract with anticaries and antigingivitis properties in two oral bacterial phatogens.

BACKGROUND: In previous works we have shown that a low-molecular-mass (LMM) fraction from mushroom (Lentinus edodes) homogenate interferes with binding of Streptococcus mutans to hydroxyapatite and Prevotella intermedia to gingival cells. Additionally, inhibition of biofilm formation of both odonto- and periodonto-pathogenic bacteria and detachment from preformed biofilms have been described for this compound. Further purification of mushroom extract has been recently achieved and a sub-fraction (i.e. # 5) has been identified as containing the majority of the mentioned biological activities. The aim of this study was to characterise the bacterial receptors for the purified mushroom sub-fraction #5 in order to better elucidate the mode of action of this compound when interfering with bacterial adhesion to host surfaces or with bacteria-bacteria interactions in the biofilm state. METHODS: Candidate bacterial molecules to act as target of this compound were bacterial surface molecules involved in cell adhesion and biofilm formation, and, thus, we have considered cell wall associated proteins (CWPs), teichoic acid (TA) and lipoteichoic acid (LTA) of S. mutans, and outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of P. intermedia. RESULTS: Fifteen S. mutans CWPs and TA were capable of binding sub-fraction #5, while LTA did not. As far as P. intermedia is concerned, we show that five OMPs interact with sub-fraction # 5. Capacity of binding to P. intermedia LPS was also studied but in this case negative results were obtained. CONCLUSIONS: Binding sub-fraction # 5 to surface molecules of S. mutans or P. intermedia may result in inactivation of their physiological functions. As a whole, these results indicate, at molecular level, the bacterial surface alterations affecting adhesion and biofim formation. For these antimicrobial properties, the compound may find use in daily oral hygiene.

Effects of mushroom and chicory extracts on the shape, physiology and proteome of the cariogenic bacterium Streptococcus mutans.

BACKGROUND: Dental caries is an infectious disease which results from the acidic demineralisation of the tooth enamel and dentine as a consequence of the dental plaque (a microbial biofilm) accumulation. Research showed that several foods contain some components with antibacterial and antiplaque activity. Previous studies indicated antimicrobial and antiplaque activities in a low-molecular-mass (LMM) fraction of extracts from either an edible mushroom (Lentinus edodes) or from Italian red chicory (Cichorium intybus). METHODS: We have evaluated the antimicrobial mode of action of these fractions on Streptococcus mutans, the etiological agent of human dental caries. The effects on shape, macromolecular syntheses and cell proteome were analysed. RESULTS: The best antimicrobial activity has been displayed by the LMM mushroom extract with a bacteriostatic effect. At the MIC of both extracts DNA synthesis was the main macromolecular synthesis inhibited, RNA synthesis was less inhibited than that of DNA and protein synthesis was inhibited only by roughly 50%. The partial inhibition of protein synthesis is compatible with the observed significant increase in cell mass. The increase in these parameters is linked to the morphological alteration with transition from cocci of the untreated control to elongated cells. Interestingly, these modifications were also observed at sub-MIC concentrations. Finally, membrane and cytosol proteome analysis was conducted under LMM mushroom extract treatment in comparison with untreated S. mutans cells. Significant changes were observed for 31 membrane proteins and 20 of the cytosol fractions. The possible role of the changed proteins is discussed. CONCLUSIONS: This report has shown an antibiotic-like mode of action of mushroom and chicory extracts as demonstrated by induced morphogenetic effects and inhibition of specific macromolecular synthesis. This feature as well as the safe use of this extract as result of its natural origin render the LMM both mushroom and chicory extracts suitable for the formulation into products for daily oral hygiene such as mouthwashes or toothpastes.

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 ß-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.