Functional features of KPC-109, a novel 270-loop KPC-3 mutant mediating resistance to avibactam-based ß-lactamase inhibitor combinations and cefiderocol.

OBJECTIVES: To investigate a ceftazidime/avibactam (CZA)-resistant Klebsiella pneumoniae (NE368), isolated from a patient exposed to CZA, expressing a novel K. pneumoniae carbapenemase (KPC)-3 variant (KPC-109). METHODS: Antimicrobial susceptibility testing was performed by reference broth microdilution. Whole-genome sequencing (WGS) analysis of NE368 was performed combining a short- and long-reads approach (Illumina and Oxford Nanopore Technologies). Functional characterization of KPC-109 was performed to investigate the impact of KPC-109 production on the ß-lactam resistance phenotype of various Escherichia coli and Klebsiella pneumoniae strains, including derivatives of K. pneumoniae with OmpK35 and OmpK36 porin alterations. Horizontal transfer of the KPC-109-encoding plasmid was investigated by conjugation and transformation experiments. RESULTS: K. pneumoniae NE368 was isolated from a patient after repeated CZA exposure, and showed resistance to CZA, fluoroquinolones, piperacillin/tazobactam, expanded-spectrum cephalosporins, amikacin, carbapenems and cefiderocol. WGS revealed the presence of a large chimeric plasmid of original structure (pKPN-NE368), encoding a novel 270-loop mutated KPC-3 variant (KPC-109; ins_270_KYNKDD). KPC-109 production mediated resistance/decreased susceptibility to avibactam-based combinations (with ceftazidime, cefepime and aztreonam) and cefiderocol, with a trade-off on carbapenem resistance. However, in the presence of porin alterations commonly encountered in high-risk clonal lineages of K. pneumoniae, KPC-109 was also able to confer clinical-level resistance to carbapenems. Resistance of NE368 to cefiderocol was likely contributed by KPC-109 production acting in concert with a mutated EnvZ sensor kinase. The KPC-109-encoding plasmid did not appear to be conjugative. CONCLUSIONS: These findings expand current knowledge about the diversity of emerging KPC enzyme variants with 270-loop alterations that can be encountered in the clinical setting.

T2Bacteria and T2Resistance Assays in Critically Ill Patients with Sepsis or Septic Shock: A Descriptive Experience.

The use of rapid molecular tests may anticipate the identification of causative agents and resistance determinants in the blood of critically ill patients with sepsis. From April to December 2021, all intensive care unit patients with sepsis or septic shock who were tested with the T2Bacteria and T2Resistance assays were included in a retrospective, single center study. The primary descriptive endpoints were results of rapid molecular tests and concomitant blood cultures. Overall, 38 combinations of T2Bacteria and T2Resistance tests were performed. One or more causative agent(s) were identified by the T2Bacteria assay in 26% of episodes (10/38), whereas negative and invalid results were obtained in 66% (25/38) and 8% (3/38) of episodes, respectively. The same pathogen detected by the T2Bacteria test grew from blood cultures in 30% of cases (3/10). One or more determinant(s) of resistance were identified by the T2Resistance assay in 11% of episodes (4/38). Changes in therapy based on T2Bacteria and/or T2Resistance results occurred in 21% of episodes (8/38). In conclusion, T2Bacteria/T2Resistance results can influence early treatment decisions in critically ill patients with sepsis or septic shock in real-life practice. Large, controlled studies remain necessary to confirm a favorable impact on patients' outcomes and antimicrobial stewardship interventions.

Candida auris Candidemia in Critically Ill, Colonized Patients: Cumulative Incidence and Risk Factors.

INTRODUCTION: Candida auris (C. auris) is an emerging nosocomial pathogen, and a sharp rise in cases of colonization and infection has been registered in intensive care units (ICUs) during the ongoing coronavirus disease 2019 (COVID-19) pandemic. The unfavorable resistance profile of C. auris and the potential high mortality of C. auris infections represent an important challenge for physicians. METHODS: We conducted a single-center retrospective study including all patients admitted to ICUs with isolation of C. auris in any non-sterile body site between February 20, 2020, and May 31, 2021. The primary aim of the study was to assess the cumulative incidence of C. auris candidemia in colonized patients. The secondary aim was to identify predictors of C. auris candidemia in the study population. RESULTS: During the study period, 157 patients admitted to ICUs in our hospital became colonized with C. auris; 59% of them were affected by COVID-19. Overall, 27 patients (17%) developed C. auris candidemia. The cumulative risk of developing C. auris candidemia was > 25% at 60 days after first detection of C. auris colonization. Seven patients with C. auris candidemia (26%) also developed a late recurrent episode. All C. auris blood isolates during the first occurring episode were resistant to fluconazole and susceptible to echinocandins, while 15 (56%) were resistant to amphotericin B. During late recurrent episodes, emergent resistance to caspofungin and amphotericin B occurred in one case each. In the final multivariable model, only multisite colonization retained an independent association with the development of C. auris candidemia. CONCLUSION: Candida auris candidemia may occur in up to one fourth of colonized critically ill patients, and multisite colonization is an independent risk factor for the development of candidemia. Implementing adequate infection control measures remains crucial to prevent colonization with C. auris and indirectly the subsequent development of infection.

Spread of Carbapenem-Resistant Gram-Negatives and Candida auris during the COVID-19 Pandemic in Critically Ill Patients: One Step Back in Antimicrobial Stewardship?

The possible negative impact of severe adult respiratory distress caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (COVID-19) on antimicrobial stewardship and infection control has been postulated, but few real-life data are available. The aim of this study was to report our experience with colonization/infection of carbapenem-resistant Pseudomonas aeruginosa (CRPA), carbapenem-resistant Klebsiella pneumoniae (CR-Kp) and Candida auris among critically ill COVID-19 patients admitted to the intensive care unit (ICU). All COVID-19 patients admitted to the ICUs at San Martino Policlinico Hospital-IRCCS in Genoa, Italy, were screened from 28 February to 31 May 2020. One-hundred and eighteen patients admitted to COVID-19 ICUs were included in the study. Among them, 12 (10.2%) became colonized/infected with CRPA, 6 (5.1%) with C. auris and 2 (1.6%) with CR-Kp. All patients with CRPA received prior treatment with meropenem, and in 11 (91.7%) infection was not preceded by colonization. Four patients (66.7%) developed C. auris candidemia. A significant spread of resistant pathogens was observed among critically ill COVID-19 patients. Dedicated strategies are warranted to prevent horizontal spread and maintain effective antimicrobial stewardship programs in the setting of COVID-19 care.

The natural plant compound carvacrol as an antimicrobial and anti-biofilm agent: mechanisms, synergies and bio-inspired anti-infective materials.

Carvacrol (5-isopropyl-2-methyl phenol) is a natural compound that occurs in the leaves of a number of plants and herbs including wild bergamot, thyme and pepperwort, but which is most abundant in oregano. The aim of this review is to analyse the scientific data from the last five years (2012-2017) on the antimicrobial and anti-biofilm activities of carvacrol, targeting different bacteria and fungi responsible for human infectious diseases. The antimicrobial and anti-biofilm mechanisms of carvacrol and its synergies with antibiotics are illustrated. The potential of carvacrol-loaded anti-infective nanomaterials is underlined. Carvacrol shows excellent antimicrobial and anti-biofilm activities, and is a very interesting bioactive compound against fungi and a wide range of Gram-positive and Gram-negative bacteria, and being active against both planktonic and sessile human pathogens. Moreover, carvacrol lends itself to being combined with nanomaterials, thus providing an opportunity for preventing biofilm-associated infections by new bio-inspired, anti-infective materials.

Hypoalbuminemia as a predictor of acute kidney injury during colistin treatment.

This study aimed to assess the predictors of acute kidney injury (AKI) during colistin therapy in a cohort of patients with bloodstream infections (BSI) due to colistin-susceptible Gram-negative bacteria, focusing on the role of serum albumin levels. The study consisted of two parts: (1) a multicentre retrospective clinical study to assess the predictors of AKI during colistin therapy, defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria; and (2) bioinformatic and biochemical characterization of the possible interaction between human serum albumin and colistin. Among the 170 patients included in the study, 71 (42%), 35 (21%), and 11 (6%) developed KDIGO stage 1 (K1-AKI), KDIGO stage 2 (K2-AKI), and KDIGO stage 3 (K3-AKI), respectively. In multivariable analyses, serum albumin <2.5 g/dL was independently associated with K1-AKI (subdistribution hazard ratio [sHR] 1.85, 95% confidence interval [CI] 1.17-2.93, p = 0.009) and K2-AKI (sHR 2.37, 95% CI 1.15-4.87, p = 0.019). Bioinformatic and biochemical analyses provided additional information nurturing the discussion on how hypoalbuminemia favors development of AKI during colistin therapy. In conclusion, severe hypoalbuminemia independently predicted AKI during colistin therapy in a large cohort of patients with BSI due to colistin-susceptible Gram-negative bacteria. Further study is needed to clarify the underlying causal pathways.

Pharmacokinetics of high-dose extended-infusion meropenem during pulmonary exacerbation in adult cystic fibrosis patients: a case series.

This case series explored the pharmacokinetic/pharmacodynamic (PK/PD) characteristics of meropenem (MEM) in adult cystic fibrosis (CF) patients hospitalized for a pulmonary exacerbation. From January 2015 to June 2016, all adult patients with cystic fibrosis (CF) and chronic pulmonary infection due to meropenem (MEM)-susceptible/intermediate Pseudomonas aeruginosa who received at least 48 h of MEM as an extended 3-hour infusion for treating a pulmonary exacerbation were enrolled. MEM plasma concentrations were determined by high-performance liquid chromatography. Six adult CF patients with a median age of 47 years were included in the study. MEM showed a high Vd (mean 45.98 L, standard deviation [SD] ±34.45). A minimal PK/PD target of 40% T > minimum inhibitory concentration (MIC) with respect to the MEM MIC of P. aeruginosa strains isolated from sputum during exacerbation was achieved in 5/6 patients (83%). MEM failed to achieve this target only in one patient, whose strain showed the highest MEM MIC in our cohort (8 mg/L). In all patients, MEM was well tolerated, and no adverse events were reported. In conclusion, high-dose, extended-infusion MEM during pulmonary exacerbation showed a high Vd in six adult CF patients with high median age, and was well tolerated.

Antimicrobial activity of eugenol and essential oils containing eugenol: A mechanistic viewpoint.

Eugenol is a hydroxyphenyl propene, naturally occurring in the essential oils of several plants belonging to the Lamiaceae, Lauraceae, Myrtaceae, and Myristicaceae families. It is one of the major constituents of clove (Syzygium aromaticum (L.) Merr. & L.M. Perry, Myrtaceae) oil and is largely used in both foods and cosmetics as a flavoring agent. A large body of recent scientific evidence supports claims from traditional medicine that eugenol exerts beneficial effects on human health. These effects are mainly associated with antioxidant and anti-inflammatory activities. Eugenol has also shown excellent antimicrobial activity in studies, being active against fungi and a wide range of gram-negative and gram-positive bacteria. The aim of this review is to analyze scientific data from the main published studies describing the antibacterial and antifungal activities of eugenol targeting different kind of microorganisms, such as those responsible for human infectious diseases, diseases of the oral cavity, and food-borne pathogens. This article also reports the effects of eugenol on multi-drug resistant microorganisms. On the basis of this collected data, eugenol represents a very interesting bioactive compound with broad spectrum antimicrobial activity against both planktonic and sessile cells belonging to food-decaying microorganisms and human pathogens.

Phytochemicals for human disease: An update on plant-derived compounds antibacterial activity.

In recent years, many studies have shown that phytochemicals exert their antibacterial activity through different mechanisms of action, such as damage to the bacterial membrane and suppression of virulence factors, including inhibition of the activity of enzymes and toxins, and bacterial biofilm formation. In this review, we summarise data from the available literature regarding the antibacterial effects of the main phytochemicals belonging to different chemical classes, alkaloids, sulfur-containing phytochemicals, terpenoids, and polyphenols. Some phytochemicals, besides having direct antimicrobial activity, showed an in vitro synergistic effect when tested in combination with conventional antibiotics, modifying antibiotic resistance. Review of the literature showed that phytochemicals represent a possible source of effective, cheap and safe antimicrobial agents, though much work must still be carried out, especially in in vivo conditions to ensure the selection of effective antimicrobial substances with low side and adverse effects.

The effect of sub-inhibitory concentrations of rifaximin on urease production and on other virulence factors expressed by Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus.

Rifaximin, a topical derivative of rifampin, inhibited urease production and other virulence factors at sub-MIC concentrations in strains involved in hepatic encephalopathy and the expression of methicillin resistance in Staphylococcus aureus. In particular, urease production was affected in all Proteus mirabilis and Klebsiella pneumoniae strains as well as in all tested Pseudomonas aeruginosa isolates. Other exotoxins, synthesized by P. aeruginosa, such as protease, gelatinase, lipase, lecithinase and DNAse were also not metabolized in the presence of rifaximin. This antibiotic inhibited pigment production in both P. aeruginosa and Chromobacterium violaceum, a biosensor control strain. Lastly, rifaximin affected haemolysin production in S. aureus and was able to restore cefoxitin susceptibility when the strain was cultured in the presence of sub-MICs of the drug. The present findings confirm and extend previous observations about the beneficial effects of rifaximin for the treatment of gastrointestinal diseases, since in this anatomic site, it reaches a large array of concentrations which prevents enterobacteria from thriving and/or producing their major virulence factors.

Meropenem for treating KPC-producing Klebsiella pneumoniae bloodstream infections: Should we get to the PK/PD root of the paradox?

The objective of this study was to assess the achievement of pharmacokinetic/pharmacodynamic (PK/PD) targets of meropenem (MEM) in critically-ill patients with bloodstream infections (BSI) due to Klebsiella pneumoniae-carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) with MEM minimum inhibitory concentrations (MICs) ≥16 mg/L. Nineteen critically-ill patients with KPC-Kp BSI were given combination therapy including MEM, tigecycline, plus colistin or gentamicin (according to susceptibility testing). MEM was administered as an extended 3-hour infusion of 2 g every 8 hours, or adjusted according to renal function. MEM plasma concentrations were determined by high-performance liquid chromatography. PK/PD targets for MEM were defined as T > 40% 1×MIC and T > 40% 4×MIC. Possible synergisms between MEM and coadministered agents were assessed by time-kill assays based on plasma levels for MEM and on fixed plasma concentrations for the other agents. In none of 19 patients MEM reached any PK/PD target. The actual MEM MICs were 256, 512, and 1024 mg/L in 1, 3, and 15 isolates, respectively. However, theoretically, the PK/PD target of T > 40% 1×MIC could have been achieved in 95%, 68%, 32% and 0% of the isolates for MIC equal to 8, 16, 32, and 64 mg/L, respectively. No synergisms were observed between MEM and coadministered agents. In conclusion, high-dose MEM failed to reach PK/PD targets in 19 patients with BSI due to KPC-Kp with very high MEM MICs. On a theoretical basis, our results suggest a possible usefulness of MEM against resistant blood isolates with MICs up to 32 mg/L.

Risk factors for infections due to carbapenem-resistant Klebsiella pneumoniae after open heart surgery.

OBJECTIVES: Patients undergoing major surgery are at increased risk of developing infections due to resistant organisms, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp). In this study, we assessed risk factors for CR-Kp infections after open heart surgery in a teaching hospital in northern Italy. METHODS: A retrospective study was conducted from January to December 2014. The primary outcome measure was postoperative CR-Kp infection, defined as a time-to-event end-point. The effect of potentially related variables was assessed by univariable and multivariable analyses. Secondary end-points were in-hospital mortality and 180-day postoperative mortality. RESULTS: Among 553 patients undergoing open heart surgery, 32 developed CR-Kp infections (6%). In the final multivariable model, CR-Kp colonization [hazard ratio (HR) 227.45, 95% confidence intervals (CI) 67.13-1225.20, P < 0.001], cardiopulmonary bypass time in minutes (HR 1.01, 95% CI 1.01-1.02, P < 0.001), chronic obstructive pulmonary disease (HR 3.99, 95% CI 1.61-9.45, P = 0.004), SOFA score (HR 1.29, 95% CI 1.08-1.53, P = 0.007), preoperative mechanical ventilation (HR 8.10, 95% CI 1.31-48.57, P = 0.026), prolonged mechanical ventilation (HR 2.48, 95% CI 1.08-6.15, P = 0.032) and female sex (HR 2.08, 95% CI 1.00-4.36, P = 0.049) were associated with the development of CR-Kp infection. Increased in-hospital mortality and 180-day mortality were observed in patients who developed CR-Kp infections in comparison with those who did not. CONCLUSIONS: In our cohort, CR-Kp colonization was an important predictor of CR-Kp infection after open heart surgery. CR-Kp infection after surgery significantly affected survival. Preventing colonization is conceivably the most effective current strategy to reduce the impact of CR-Kp.

Antistaphylococcal activity and metabolite profiling of manuka honey (Leptospermum scoparium L.) after in vitro simulated digestion.

The antistaphylococcal activity against methicillin-susceptible and -resistant Staphylococcus aureus and the metabolite profiling of manuka honey (MH) were investigated before and after in vitro simulated gastric (GD) and gastroduodenal (GDD) digestions. Undigested manuka honey showed antibacterial activity against all the tested strains, the GD sample showed no activity against S. aureus, and the GDD honey showed an antistaphylococcal activity, which was slightly reduced in comparison with the undigested sample. To explain these results, methylglyoxal (MGO), to which most of the antibacterial activity of MH is ascribed, was subjected to in vitro simulated GD and GDD. After digestion, MGO showed antibacterial activity at concentrations definitively higher than those registered in digested MH samples. These results showed that the antistaphylococcal activity registered after digestion cannot be ascribed to MGO. Thus metabolite analysis, carried out using an explorative untargeted NMR-based approach and a targeted RP-HPLC-PAD-ESI-MSn analysis focused on bio-active substances, was used to highlight the chemical modifications occurring from digestion. The results showed that (1) the level of MGO decreases and (2) the content of aromatic compounds, such as leptosin and methyl syringate, markers of manuka honey, was stable under gastric and gastroduodenal conditions, whereas (3) the levels of acetic and lactic acids increase in particular after gastroduodenal digestion, being 1.5 and 2.8 times higher in GDD-MH than in UND-MH, respectively. Overall, the results obtained from chemical analysis provide at least a partial explanation of the registered antibacterial activity observed after gastroduodenal digestion.

Chemical Characterization and in Vitro Antibacterial Activity of Myrcianthes hallii (O. Berg) McVaugh (Myrtaceae), a Traditional Plant Growing in Ecuador.

Myrcianthes hallii (O. Berg) McVaugh (Myrtaceae) is a plant native to Ecuador, traditionally used for its antiseptic properties. The composition of the hydro-methanolic extract of this plant was determined by submitting it to ultra-high performance liquid chromatography (UHPLC) hyphenated to heated-electrospray ionization mass spectrometry and UV detection. The presence of antimicrobial components prompted us to test the extract against methicillin-resistant and methicillin-susceptible Staphylococcus aureus, multidrug-resistant and susceptible Escherichia coli, Pseudomonas aeruginosa, Enterococcus spp. and Streptococcus pyogenes strains. The chromatographic analysis led to the identification of 38 compounds, including polyphenols and organic acids, and represents the first chemical characterization of this plant. The extract showed modest antibacterial activity against all tested bacteria, with the exception of E. coli which was found to be less sensitive. Whilst methicillin-resistant strains usually display resistance to several drugs, no relevant differences were observed between methicillin-susceptible and resistant strains. Considering its long-standing use in folk medicine, which suggests the relative safety of the plant, and the presence of many known antibacterial polyphenolic compounds responsible for its antibacterial activity, the results show that M. hallii extract could be used as a potential new antiseptic agent. Moreover, new anti-infective biomaterials and nanomaterials could be designed through the incorporation of M. hallii polyphenols. This prospective biomedical application is also discussed.

Assessment of the Antimicrobial Activity of Algae Extracts on Bacteria Responsible of External Otitis.

External otitis is a diffuse inflammation around the external auditory canal and auricle, which is often occurred by microbial infection. This disease is generally treated using antibiotics, but the frequent occurrence of antibiotic resistance requires the development of new antibiotic agents. In this context, unexplored bioactive natural candidates could be a chance for the production of targeted drugs provided with antimicrobial activity. In this paper, microbial pathogens were isolated from patients with external otitis using ear swabs for over one year, and the antimicrobial activity of the two methanol extracts from selected marine (Dunaliella salina) and freshwater (Pseudokirchneriella subcapitata) microalgae was tested on the isolated pathogens. Totally, 114 bacterial and 11 fungal strains were isolated, of which Staphylococcus spp. (28.8%) and Pseudomonas aeruginosa (P. aeruginosa) (24.8%) were the major pathogens. Only three Staphylococcus aureus (S. aureus) strains and 11 coagulase-negative Staphylococci showed resistance to methicillin. The two algal extracts showed interesting antimicrobial properties, which mostly inhibited the growth of isolated S. aureus, P. aeruginosa, Escherichia coli, and Klebsiella spp. with MICs range of 1.4 × 108 to 2.2 × 10(10) cells/mL. These results suggest that the two algae have potential as resources for the development of antimicrobial agents.

First report of chronic pulmonary infection by KPC-3-producing and colistin-resistant Klebsiella pneumoniae sequence type 258 (ST258) in an adult patient with cystic fibrosis.

The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae continues to increase, and the possible development of KPC-producing K. pneumoniae infections in cystic fibrosis (CF) patients is a matter of concern. Here, we describe the establishment of a chronic lung infection due to a colistin-resistant KPC-producing K. pneumoniae isolate in an Italian CF patient.

Emergence of a KPC-3-Producing Escherichia coli ST69 as a Cause of Bloodstream Infections in Italy.

The spread of carbapenem-resistant gram negatives is a global emergency, and surveillance of new resistant clones is critical from both public health and clinical standpoints. Herein, we describe the emergence of a KPC-3-producing Escherichia coli ST69 as a cause of bloodstream infection in two Italian patients.

Antibacterial activity of polyphenols.

Polyphenols are a widely distributed group of natural products found in fruits, vegetables, nuts, seeds, stems and flowers. Such compounds, especially dietary flavonoids and tannins, have been shown to exert antioxidant, antiinflammatory, anti-cancer and antibacterial effects and may have beneficial effects on human health. The antimicrobial activity of polyphenols has been widely studied and hundreds of publications reporting the antimicrobial activity of polyphenols have been recently published. In an era of increasing antibiotic resistance, the development of new strategies to fight bacteria is welcome. Further studies are needed to evaluate the therapeutic potential of polyphenols alone or in combination with currently available antibiotics.

Identification of a New Delhi metallo-ß-lactamase-4 (NDM-4)-producing Escherichia coli in Italy.

BACKGROUND: During June-July 2012, six imipenem-resistant Escherichia coli isolates were isolated from two patients hospitalized in a ward of one large tertiary-care hospital in Genoa, Italy. Genetic features associated with blaNDM-4 gene were investigated. RESULTS: The isolates exhibited the same PFGE profile and a multidrug-resistant (MDR) phenotype to aminoglycosides, fluoroquinolones, and ß-lactams. The strains produced the NDM-4 carbapenemase and the blaNDM-4 gene was part of the variable region of a class 1 integron. MLST analysis revealed that all isolates belonged to sequence type 405 (ST405). CONCLUSIONS: This is the first report on the emergence of an MDR strain of E.coli producing the NDM-4 MBL in Italy.