Correction: Morroni et al. Antimicrobial Activity of Aztreonam in Combination with Old and New ß-Lactamase Inhibitors against MBL and ESBL Co-Producing Gram-Negative Clinical Isolates: Possible Options for the Treatment of Complicated Infections. Antibiotics 2021, 10, 1341.

Results have been implemented with additional data, which have been commented in the following paragraphs [...].

Antimicrobial Activity of Aztreonam in Combination with Old and New ß-Lactamase Inhibitors against MBL and ESBL Co-Producing Gram-Negative Clinical Isolates: Possible Options for the Treatment of Complicated Infections.

Metallo-ß-lactamases (MBLs) are among the most challenging bacterial enzymes to overcome. Aztreonam (ATM) is the only ß-lactam not hydrolyzed by MBLs but is often inactivated by co-produced extended-spectrum ß-lactamases (ESBL). We assessed the activity of the combination of ATM with old and new ß-lactamases inhibitors (BLIs) against MBL and ESBL co-producing Gram-negative clinical isolates. Six Enterobacterales and three non-fermenting bacilli co-producing MBL and ESBL determinants were selected as difficult-to-treat pathogens. ESBLs and MBLs genes were characterized by PCR and sequencing. The activity of ATM in combination with seven different BLIs (clavulanate, sulbactam, tazobactam, vaborbactam, avibactam, relebactam, zidebactam) was assessed by microdilution assay and time-kill curve. ATM plus avibactam was the most effective combination, able to restore ATM susceptibility in four out of nine tested isolates, reaching in some cases a 128-fold reduction of the MIC of ATM. In addition, relebactam and zidebactam showed to be effective, but with lesser reduction of the MIC of ATM. E. meningoseptica and C. indologenes were not inhibited by any ATM-BLI combination. ATM-BLI combinations demonstrated to be promising against MBL and ESBL co-producers, hence providing multiple options for treatment of related infections. However, no effective combination was found for some non-fermentative bacilli, suggesting the presence of additional resistance mechanisms that complicate the choice of an active therapy.

Interplay of OpdP Porin and Chromosomal Carbapenemases in the Determination of Carbapenem Resistance/Susceptibility in Pseudomonas aeruginosa.

Carbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.

Zidovudine in synergistic combination with fosfomycin: an in vitro and in vivo evaluation against multidrug-resistant Enterobacterales.

Multidrug-resistant (MDR) Enterobacterales are a priority health issue with few treatment options. Recently, fosfomycin has been reconsidered for MDR bacterial infections. Zidovudine, licensed for the treatment of human immunodeficiency virus (HIV), has unexploited antibacterial properties and has been considered for drug repurposing. The aim of this study was to assess the effect of the combination of fosfomycin plus zidovudine against clinical MDR Enterobacterales isolates. Minimum inhibitory concentration (MIC) determination and checkerboard assays for 36 MDR Enterobacterales strains were performed. In addition, fosfomycin-resistant strains were evaluated using time-kill assay and in an in vivo Galleria mellonella infection model. Zidovudine and fosfomycin MICs ranged between 0.06 to >64 mg/L and 0.125 to >512 mg/L, respectively. A synergistic effect [fractional inhibitory concentration index (FICI) ≤0.5] was observed in 25 isolates and no antagonistic effect was observed in the remaining isolates. For 7 of 8 fosfomycin-resistant strains (MIC > 32 mg/L), zidovudine combination was able to restore fosfomycin susceptibility. These results were confirmed by time-kill assays. Fosfomycin + zidovudine presented greater larval survival (20-50%) than monotherapy. Synergistic activity was observed for fosfomycin + zidovudine in 69.4% of the tested strains. In vivo experiments confirmed the enhanced effectiveness of the combination. The zidovudine concentrations tested here can be reached in human serum using the actual licensed dosage, therefore this combination deserves further clinical investigation.

Synergistic activity of fosfomycin and chloramphenicol against vancomycin-resistant Enterococcus faecium (VREfm) isolates from bloodstream infections.

Vancomycin-resistant Enterococcus faecium (VREfm) infections are increasing. Current anti-VREfm options (linezolid and daptomycin) are suboptimal. Fosfomycin maintains good efficacy against VREfm and chloramphenicol is active against ≥ 90% of VREfm. We tested chloramphenicol + fosfomycin (CAF+FOS) against 10 VREfm isolated from blood. MICs were 64 to 512 µg/mL for fosfomycin and 8 to 16 µg/mL for chloramphenicol. The combination decreased both MICs, with a synergic effect in 50% of the isolates and an additive effect in the remaining 50%. Time-kill assays performed on fractional inhibitory concentration index ≤ 0.5 strains confirmed the synergism. The antibiotic combination at » of minimum inhibitory concentrations (MICs) caused a ≥ 2 log10 reduction compared to the two antibiotics alone. Finally, we provided a proof of concept of the in vitro efficacy of CAF+FOS in G. mellonella. The survival of G. mellonella larvae treated with the combination was significantly higher. The activity of fosfomycin and chloramphenicol against VREfm increases when they are used in combination.

Aspergillus fumigatus biofilm formation on different bone substitutes used in maxillary sinus augmentation: an in vitro analysis.

BACKGROUND: Fungus ball (FB) typically affects healthy adults, and Aspergillus fumigatus is the most frequent etiologic agent: iatrogenic factors represent an important issue in FB pathogenesis. Moreover, a recent study suggested a significant association between the use of anorganic bovine bone as sinus grafting material and subsequent development of FB. The aim of the present investigation is to evaluate in vitro eventual differences in the ability of Aspergillus fumigatus to colonize different bone grafting materials and grow on them as biofilm. FINDINGS: Five different bone substitutes (demineralized bone matrix, anorganic bovine bone, ß-tricalcium phosphate, synthetic nano-hydroxyapatite, and synthetic hydroxyapatite), commonly used in sinus floor augmentation procedures, were inoculated with conidia suspensions of A. fumigatus and incubated at 37 °C for 4 and 8 h, in standardized conditions. Biofilm bound to the different materials underwent quantitative and qualitative analysis by confocal and scanning electron microscopy. A. fumigatus proved to be able to adhere and form biofilm on all the tested bone substitutes. The surface plot representation of the samples displayed some differences in the density of the superficial layer, due to the physical characteristics of the biomaterials. Nevertheless, Kruskal-Wallis test showed no significant differences in biomass amount among the five bone substitutes (p = 0.236 and p = 0.55 after 4 and 8 h adhesion, respectively). CONCLUSIONS: All the bone substitutes normally used in sinus floor augmentation represent a favorable substrate for fungal growth, due to their physical and chemical characteristics. During sinus floor elevation procedures, Schneiderian membrane integrity should be maintained in order to avoid the exposure of the grafting material at the respiratory environment, with potential risks of fungal colonization.

Sub-MIC effects of a proline-rich antibacterial peptide on clinical isolates of Acinetobacter baumannii.

INTRODUCTION: Acinetobacter baumannii is one of the most important nosocomial pathogens, mainly due to its ability to accumulate antibiotic-resistances and to persist in the hospital environment - characteristics related to biofilm production. It is well-known that A. baumannii is inhibited by the proline-rich peptide Bac7(1-35), but its putative effects at sub-MICs were never considered. AIMS: We examined the sub-MIC effect of Bac7(1-35) on the growth rate, resistance induction and some A. baumannii features linked to virulence. METHODOLOGY: Growth kinetics in the presence of sub-MICs of Bac7(1-35) were evaluated spectrophotometrically. Peptide uptake was quantified by cytometric analysis. The ability of Bac7(1-35) to interfere with biofilm production was investigated by the crystal violet method and confocal microscopy. Bacterial motility was observed at the interphase between a layer of a semi-solid medium and the polystyrene bottom of a Petri dish. The induction of resistance was evaluated after serial passages with sub-MICs of the peptide. RESULTS: Although the MIC of Bac7(1-35) was between 2-4 µM for all tested strains, its effect on the growth rate at sub-MICs was strain-dependent and correlated with the amount of peptide internalized by each strain. Sub-MICs of Bac7(1-35) induced a strongly strain-dependent effect on biofilm formation and reduced motility in almost all strains, but interestingly the peptide did not induce resistance. CONCLUSION: Bac7(1-35) is internalized into A. baumannii and is able to inhibit biofilm formation and bacterial motility, without inducing resistance. This study stresses the importance of considering possible effects that antimicrobials could have at sub-MICs, mimicking a common condition during antibiotic treatment.

Spread of Vancomycin-Resistant Enterococcus faecium Isolates Despite Validated Infection Control Measures in an Italian Hospital: Antibiotic Resistance and Genotypic Characterization of the Endemic Strain.

An alarming increase of vancomycin-resistant Enterococcus faecium (VREfm) isolates was detected in an Italian referral hospital subjected to policies of infection control validated by the Joint Commission International. Analysis of the population structure of 122 consecutive, nonreplicate VREfm isolates collected over an 18-month period identified a single major clone that spread around the whole hospital, rapidly establishing an endemic state. It belonged to sequence type (ST) 17 and showed a highly multidrug-resistant phenotype, being resistant to all antimicrobial classes for the carriage of several resistance determinants. Furthermore, some strains with decreased susceptibility to daptomycin were detected. Eighteen out of the 122 isolates did not group in the major clone. They showed a low spreading potential inside the hospital wards, even if most of them displayed a multidrug-resistant phenotype and belonged to a hospital-adapted lineage. Causes that led to the VREfm endemic state have not been fully elucidated. However, it is conceivable that the increase in systemic antibiotic consumption and the use of selective digestive tract decontamination, including vancomycin in critically ill patients during the period before 2014, may have played a role in the ST17 clone dissemination, but additional traits conferring high fitness in hospital environment cannot be excluded.

Epidemic Dissemination of a Carbapenem-Resistant Acinetobacter baumannii Clone Carrying armA Two Years After Its First Isolation in an Italian Hospital.

This study describes the dissemination of a carbapenem-resistant Acinetobacter baumannii (CRAB) strain in a university hospital in Northeast Italy. Characterization of the outbreak strain was combined with a retrospective analysis of all CRAB isolates collected in the same hospital during the 5 years preceding the outbreak, with the aim of elucidating the origin of the epidemic spread. The outbreak strain was shown to belong to the International Clone II and carry the blaOXA-23 gene, flanked by two ISAba1 sequences in opposite orientation (Tn2006 arrangement). The epidemic clone harbored also the blaOXA-66 allele of the carbapenemase intrinsic to A. baumannii, the determinant of ArmA 16S rRNA methylase and a class 1 integron, with the aacA4, catB8, and aadA1 cassette array. Genotype analysis, performed by macrorestriction analysis and VRBA, revealed that isolates related to outbreak strain had been sporadically collected from inpatients in the 2 years preceding outbreak start. Carriage of blaOXA-66, armA, and the integron further supported relatedness of these isolates to the outbreak clone. Outbreak initially involved three medical wards, typically hosting elderly patients with a history of prolonged hospitalization. The study highlights the need to adopt strict infection control measures also when CRAB isolation appears to be a sporadic event.

pKBuS13, a KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 833, carrying Tn4401b inserted into an Xer site-specific recombination locus.

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.

Evaluation of a genotyping method based on the ospA gene to detect Borrelia burgdorferi sensu lato in multiple samples of lyme borreliosis patients.

In this study we have developed a new Restriction-Fragment-Length-Polymorphism (RFLP) genotyping method for rapid detection and identification of Borrelia genospecies present as unique species or as co-infection in multiple specimens obtained simultaneously from 29 individual patients affected by early or late Lyme borreliosis (LB). The target of the RFLP-genotyping was the heterogeneous plasmid located ospA gene, thus we developed a method able to detect and differentiate between six clinically relevant Borrelia genospecies circulating in Europe, B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, B. bissettii and B. spielmanii. In this study Borrelia DNA could be detected by PCR in at least one specimen of each patient, except in one case of neuroborreliosis (NB); blood samples gave the highest sensitivity in all patient groups. The genotyping indicated that B. afzelii was present in 8 patients with skin involvement, B. garinii in 2 cases of NB and 4 cases with skin involvement, B. burgdorferi sensu stricto was detected in one patient with skin involvement and another with Lyme arthritis. Different Borrelia species in distinct specimens were identified in one patient with EM. The RFLP analysis of 11 patients revealed mixed patterns, which suggested pluri-infection with different Borrelia species.

Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii.

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.