Molecular Characterization of Enterococcus faecium Clinical Isolates Harbouring erm (T) from an Italian Hospital.

The presence of erm(T) gene conferring resistance to macrolides, lincosamides and streptogramin B (MLSB), was screened in 296 enterococci collected from clinical samples in a central Italy hospital and seven Enterococcus faecium isolates resulted positive to erm(T) by PCR. All isolates were resistant to erythromycin, tetracycline, ciprofloxacin and ampicillin but susceptible to vancomycin and chloramphenicol. Whole Genome Sequencing analysis revealed that in five E. faecium isolates, all belonging to the sequence type ST80 included in the clonal complex CC17 responsible of nosocomial infections, erm (T) gene was chromosome-located, in different genetic contexts. In E. faecium 735,236, erm (T) was on a 4,159-bp region flanked by two IS1216 and inserted at the 3' end of the mp gene. In E. faecium 711,448 and 739,437, erm (T) was found in a 4,463-bp region identical to that detected in E. faecium 735,236 except for 319 bp. In E. faecium 713,729 and 757,415, erm (T) was on a 7,038-bp region flanked by IS1251 and ISEfm2 transposases and encompassed between the genes encoding a recombinase and three hypothetical proteins. erm(T)-carrying minicircles were detected in all isolates by inverse PCR assays demonstrating that erm(T) was included in mobile elements. However, in conjugation assays by filter mating, the erm(T) transferability was unsuccessful. Although macrolides are not used to treat enterococcal infections, the resistance is nonetheless widespread. These antibiotics are critically important in human medicine, but only few studies focused on erm (T)-harbouring clinical enterococci. The emergence of erm (T)-mediated erythromycin resistance among enterococci, potentially transferable to other nosocomial pathogens, should be constantly monitored.

Genetic analysis of vancomycin-variable Enterococcus faecium clinical isolates in Italy.

PURPOSE: To investigate the occurrence of vancomycin-variable enterococci (VVE) in a hospital in central Italy. METHODS: vanA positive but vancomycin-susceptible Enterococcus faecium isolates (VVE-S) were characterized by antibiotic susceptibility tests, molecular typing (PFGE and MLST), and WGS approach. The reversion of VVE-S to a resistant phenotype was assessed by exposure to increasing vancomycin concentrations, and the revertant isolates were used in filter mating experiments. qPCR was used to analyze the plasmid copy number. RESULTS: Eleven putative VVE-S were selected. WGS revealed two categories of vanA cluster plasmid located: the first type showed the lack of vanR, the deletion of vanS, and an intact vanH/vanA/vanX cluster; the second type was devoid of both vanR and vanS and showed a deletion of 544-bp at the 5'-end of the vanH. Strains (n = 7) carrying the first type of vanA cluster were considered VVE-S and were able to regain a resistance phenotype (VVE-R) in the presence of vancomycin, due to a 44-bp deletion in the promoter region of vanH/vanA/vanX, causing its constitutive expression. VVE-R strains were not able to transfer resistance by conjugation, and the resistance phenotype was unstable: after 11 days of growth without selective pressure, the revertants were still resistant but showed a lower vancomycin MIC. A higher plasmid copy number in the revertant strains was probably related to the resistance phenotype. CONCLUSION: We highlight the importance of VVE transition to VRE under vancomycin therapy resulting in a potential failure treatment. We also report the first-time identification of VVE-S isolates pstS-null belonging to ST1478.

Genomic Analysis of a Linezolid-Resistant Staphylococcus capitis Causing Bacteremia: Report from a University Hospital in Central Italy.

Although coagulase negative staphylococci are rarely associated with complicated diseases, in some cases they cause life-threatening infections. Here we described a clinical case of a bacteremia due to a methicillin- and linezolid-resistant Staphylococcus capitis in a patient previously treated with linezolid. Whole genome sequencing revealed the common mutation G2576T in all rDNA 23S alleles and several acquired resistance genes. Moreover, the isolate was epidemiologically distant from the NRCS-A clade, usually responsible for nosocomial infections in neonatal intensive care units. Our findings further confirm the ability of minor staphylococci to acquire antibiotic resistances and challenge the treatment of these infections.

Detecting Carbapenemases in Animal and Food Samples by Droplet Digital PCR.

BACKGROUND: The presence of carbapenemase-producing bacteria (CPB) in animal hosts and along the food chain may result in the development of reservoirs for human infections. Several CPB strains isolated from animals have been reported, suggesting that transmission and dissemination of the corresponding genes between humans and animals may occur. Animal and food samples have complex backgrounds that hinder the detection of CPB present in low concentrations by standard detection procedures. METHODS: We evaluated the possibility of detecting blaKPC, blaVIM, and blaOXA-48-like carbapenemases in 286 animal and food samples (faeces from farm and companion animals, raw meat, bivalve molluscs) by culture-based and standard molecular methods and by ddPCR. RESULTS: The proposed ddPCR managed to detect the target genes, also in samples resulting negative to standard methods. While the presence of blaKPC and blaVIM was detected in few samples (~3%), one third of the samples (n = 94/283) carried different variants of blaOXA-48-like genes. CONCLUSION: A specific and sensitive method such as ddPCR could be suitable to evaluate the current veterinarian and environmental situation and to assess the dynamic transmission and persistence of CPB between animals and humans and vice versa.

Clinical and microbiological features of ceftolozane/tazobactam-resistant Pseudomonas aeruginosa isolates in a university hospital in central Italy.

OBJECTIVES: Ceftolozane/tazobactam (C/T) is a novel cephalosporin and ß-lactamase inhibitor combination with great activity against Pseudomonas aeruginosa. To assess P. aeruginosa susceptibility to C/T, a surveillance study was conducted from October 2018 to March 2019 at the University Hospital 'Ospedali Riuniti' in Ancona, Italy. METHODS: Minimum inhibitory concentrations (MICs) to C/T were determined by Etest strip. Resistant isolates were characterized by phenotypic (broth microdilution antimicrobial susceptibility testing and modified Carbapenem Inactivation Method [mCIM]) and genotypic (Polymerase Chain Reaction [PCR], Pulsed Field Gel Electrophoresis [PFGE], and whole-genome sequencing [WGS]) methods. Clinical variables of patients infected by C/T-resistant P. aeruginosa were collected from medical records. RESULTS: Fifteen of 317 P. aeruginosa collected showed resistance to C/T (4.7%). Ten strains demonstrated carbapenemase activity by mCIM method, and PCR confirmed that eight strains harbored a blaVIM gene while the other two were positive for blaIMP. Additionally, three isolates carried acquired extended spectrum ß-lactamase genes (two isolates carried blaPER and one carried blaGES). Eight strains were strictly related by PFGE and WGS analysis confirmed that they belonged to sequence type (ST)111. The other STs found were ST175 (two isolates), ST235 (two isolates), ST70 (one isolate), ST621 (one isolate), and the new ST3354 (one isolate). Most patients had received previous antibiotic therapies, carried invasive devices, and experienced prolonged hospitalization. CONCLUSION: This study demonstrated the presence of C/T-resistant P. aeruginosa isolates in a regional hospital carrying a number of resistance mechanisms acquired by different high-risk clones.

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 [...].

Synthesis and Biological Evaluation of Novel Cinnamic Acid-Based Antimicrobials.

The main antimicrobial resistance (AMR) nosocomial strains (ESKAPE pathogens such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are the most widespread bacteria in cutaneous infections. In this work we report the synthesis, in silico skin permeability prediction, antimicrobial, antibiofilm, and wound healing properties of novel cinnamic acid-based antimicrobials (DM1-11) as novel antibacterial drugs for the treatment of ESKAPE-related skin infections. Antimicrobial and wound healing scratch assays were performed to evaluate the antibacterial properties of DM1-11. In silico skin permeability capabilities of DM1-11 were evaluated using Swiss-ADME online database. Cytotoxicity assays were performed on keratinocytes and fibroblasts. DM2, bearing a catechol group on the aromatic ring of the cinnamic portion of the molecule, possesses a significant antibacterial activity against S. aureus (MIC range 16-64 mg/L) and contrasts the biofilm-mediated S. epidermidis infection at low concentrations. Wound healing assays showed that wound closure in 48 h was observed in DM2-treated keratinocytes with a better healing pattern at all the used concentrations (0.1, 1.0, and 10 µM). A potential good skin permeation for DM2, that could guarantee its effectiveness at the target site, was also observed. Cytotoxicity studies revealed that DM2 may be a safe compound for topical use. Taking together all these data confirm that DM2 could represent a safe wound-healing topical agent for the treatment of skin wound infections caused by two of main Gram-positive bacteria belonging to ESKAPE microorganisms.

Linezolid-resistant Enterococcus gallinarum isolate of swine origin carrying cfr, optrA and poxtA genes.

OBJECTIVES: To characterize a linezolid-resistant Enterococcus gallinarum isolate of porcine origin co-carrying cfr, optrA and poxtA genes. METHODS: The genome was sequenced using the Illumina and Nanopore platforms. The presence of circular intermediates was examined by inverse PCR. Transferability of oxazolidinone resistance genes was investigated by transformation and conjugation. RESULTS: Two plasmids, the cfr- and optrA-carrying pEgFS4-1 (35 kb) and the poxtA-harbouring pEgFS4-2 (38 kb), were identified. pEgFS4-1 disclosed a distinctive mosaic structure with two cargo regions bounded by identical IS1216 elements interpolated into a backbone related to that of Enterococcus faecium vanA-containing pVEF2. The first cargo region included the cfr and optrA contexts, whereas the second one carried a Tn554 remnant and the lnu(A) gene. Both regions were able to excise in circular form as a unique translocable unit. pEgFS4-2 plasmid was 99% identical to a not fully described E. faecium pSBC1 plasmid. The poxtA environment, flanked by IS1216, was proved to be unstable. pEgFS4-2 also exhibited another cargo region containing the tet(M)-tet(L) genes arranged in tandem and its circular form was detected. Transformation and conjugation experiments failed to demonstrate the transferability of both plasmids to enterococcal recipients. Both plasmids persisted in the absence of selective pressure. CONCLUSIONS: To the best of our knowledge, this is the first description of a linezolid-resistant E. gallinarum isolate of swine origin carrying cfr, optrA and poxtA genes. The co-presence of three linezolid resistance determinants in an intrinsically vancomycin-resistant enterococcal species is cause of concern.

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.

Characterization and Clonal Diffusion of Ceftaroline Non-Susceptible MRSA in Two Hospitals in Central Italy.

BACKGROUND: Ceftaroline represents a novel fifth-generation cephalosporin to treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Ceftaroline susceptibility of 239 MRSA isolates was assessed by disk diffusion and a MIC test strip following both EUCAST and CLSI guidelines. Non-susceptible isolates were epidemiologically characterized by pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing, and further investigated by PCR and whole genome sequencing to detect penicillin-binding protein (PBP) mutations as well as antibiotic resistance and virulence genes. RESULTS: Fourteen isolates out of two hundred and thirty-nine (5.8%) were non-susceptible to ceftaroline (MIC > 1 mg/L), with differences between the EUCAST and CLSI interpretations. The characterized isolates belonged to seven different pulsotypes and three different clones (ST228/CC5-t041-SCCmecI, ST22/CC22-t18014-SCCmecIV, and ST22/CC22-t022-SCCmecIV), confirming a clonal diffusion of ceftaroline non-susceptible strains. Mutations in PBPs involved PBP2a for ST228-t041-SCCmecI strains and all the other PBPs for ST22-t18014-SCCmecIV and ST22-t022-SCCmecIV clones. All isolates harbored antibiotic resistance and virulence genes with a clonal distribution. CONCLUSION: Our study demonstrated that ceftaroline non-susceptibile isolates belonged not only to ST228 strains (the most widespread clone in Italy) but also to ST22, confirming the increasing role of these clones in hospital infections.

High prevalence of carbapenem-resistant Klebsiella pneumoniae ST307 recovered from fecal samples in an Italian hospital.

Aim: This study reports the characterization of carbapenem-resistant colonizing strains of K. pneumoniae. Methods: 650 stool samples were screened for carbapenem-resistant K. pneumoniae (CR-Kp). All strains were characterized for antibiotic susceptibility, typing features, main carbapenemases and extended-spectrum ß-lactamases. The carbapenemase transferability was assessed by interspecific conjugation. Results: Eighteen CR-Kp were multidrug resistant, five were KPC producing. A predominance of ST307 isolates, constituting the predominant cluster by PFGE analysis, was identified (50% were KPC-2 producers). Conjugation data showed the co-transfer of blaKPC-2, blaTEM-1, blaOXA-1, blaCTX-M-15 in a single large pKPN3-like plasmid. Conclusion: Our data pointed out the diversity of colonizing K. pneumoniae strains compared with clinical ones. The predominance of ST307 strains suggested an increased spreading, even in our area, of this high-risk clone.

Lay abstract Carbapenem-resistant Klebsiella pneumoniae represents a major antibiotic resistance threat worldwide. These microorganisms are associated with high mortality and difficult-to-treat infections. Of particular interest is the production of carbapenemase, enzymes capable of inactivating life-saving drugs such as carbapenems. In the interaction with humans, K. pneumoniae plays different roles: commensal, opportunistic pathogen or true pathogen. Our study aimed to analyze the population of K. pneumoniae obtained from a fecal screening, since gut-colonizing strains are considered the common source of K. pneumoniae nosocomial infections. There are many differences between clinical and colonizing isolates, but the latter are much less characterized. The careful characterization of colonizing strains is crucial, in order to better understand how K. pneumoniae may change its role from commensal to pathogen.

First IncHI2 Plasmid Carrying mcr-9.1, blaVIM-1, and Double Copies of blaKPC-3 in a Multidrug-Resistant Escherichia coli Human Isolate.

We report a novel IncHI2 plasmid coharboring blaVIM-1, two copies of blaKPC-3, and mcr-9.1 resistance genes in a human Escherichia coli isolate of the new serogroup O188. The blaVIM-1 gene was included in a class 1 integron, mcr-9.1 in a cassette bracketed by IS903 and ΔIS1R, and blaKPC-3 in two copies within a new composite Tn4401-like transposon. The emergence of carbapenem and colistin resistance genes in a single plasmid is of great concern for upcoming clinical therapies.

Synergistic effect of antimicrobial peptide LL-37 and colistin combination against multidrug-resistant Escherichia coli isolates.

Overview: The global spread of antibiotic resistance represents a serious threat for public health. Aim: We evaluated the efficacy of the antimicrobial peptide LL-37 as antimicrobial agent against multidrug-resistant Escherichia coli. Results: LL-37 showed good activity against mcr-1 carrying, extended spectrum ß-lactamase- and carbapenemase-producing E. coli (minimum inhibitory concentration, MIC, from 16 to 64 mg/l). Checkerboard assays demonstrated synergistic effect of LL-37/colistin combination against all tested strains, further confirmed by time-kill and post antibiotic effect assays. MIC and sub-MIC concentrations of LL-37 were able to reduce biofilm formation. Conclusion: Our preliminary data indicated that LL-37/colistin combination was effective against multidrug-resistant E. coli strains and suggested a new possible clinical application.

Testing Surgical Face Masks in an Emergency Context: The Experience of Italian Laboratories during the COVID-19 Pandemic Crisis.

The first wave of the COVID-19 pandemic brought about a broader use of masks by both professionals and the general population. This resulted in a severe worldwide shortage of devices and the need to increase import and activate production of safe and effective surgical masks at the national level. In order to support the demand for testing surgical masks in the Italian context, Universities provided their contribution by setting up laboratories for testing mask performance before releasing products into the national market. This paper reports the effort of seven Italian university laboratories who set up facilities for testing face masks during the emergency period of the COVID-19 pandemic. Measurement set-ups were built, adapting the methods specified in the EN 14683:2019+AC. Data on differential pressure (DP) and bacterial filtration efficiency (BFE) of 120 masks, including different materials and designs, were collected over three months. More than 60% of the masks satisfied requirements for DP and BFE set by the standard. Masks made of nonwoven polypropylene with at least three layers (spunbonded-meltblown-spunbonded) showed the best results, ensuring both good breathability and high filtration efficiency. The majority of the masks created with alternative materials and designs did not comply with both standard requirements, resulting in suitability only as community masks. The effective partnering between universities and industries to meet a public need in an emergency context represented a fruitful example of the so-called university "third-mission".

Human leptospirosis in the Marche region: Over 10 years of surveillance.

We conducted a 10 years' retrospective study in 347 symptomatic individuals to assess the regional distribution of leptospirosis. A total of 173 individuals were diagnosed positive (49.8%): 11.5% were found positive to Leptospira by microscopic agglutination test positive, whereas 38.3% were found positive by microscopy analysis. The maximum peak of leptospirosis was reached in 2017 (n = 32). The most common serovars were Icterohaemorrhagiae and Poi.

Increase and diversity of carbapenemase-producing Escherichia coli isolates, Italy.

Aim: This study reports on a surveillance in an Italian hospital focused on carbapenemase-producing Escherichia coli (CP-Ec). Materials & methods: Eighteen isolates (nine from clinical specimens and nine from rectal swab) were characterized for antibiotic susceptibilities, typing features, main carbapenemase, extended-spectrum ß-lactamases (ESBLs) and other bla genes, and their transferability by conjugation and transformation. Results: An increase in CP-Ec isolates was observed during 3-year surveillance period. Compared with the clinical isolates, all belonging to one sequence type (ST), ST131, those from rectal swab were very heterogeneous and belonged to eight STs. Transfer data confirmed the role of conjugative plasmids in the spreading of carbapenemase genes. Conclusion: The prevalence of CP-Ec in Italy has risen, with a substantial increase over the last year.