A novel KPC-166 in ceftazidime/avibactam resistant ST307 Klebsiella pneumoniae causing an outbreak in intensive care COVID Unit, Italy.

INTRODUCTION: Aim of the study was the molecular characterization of 21 ceftazidime/avibactam resistant (CZA-R) Klebsiella pneumoniae strains, collected in the period October 2021-March 2022 from an Intensive Care COVID Unit in a Northern Italian Hospital. METHODS: After growth on selective/chromogenic culture media and susceptibility tests assessment, resistance genes content was ascertained for all the isolates by the HybriSpot 12 multiplexing, PCR and Whole-Genome Sequencing (WGS). Clonality was assessed by PFGE and MLST according to the Pasteur scheme. A SNPs-based phylogenetic tree was obtained comparing representative isolates and global genomes. The blaKPC gene horizontal transmission was evaluated by conjugation experiments. blaKPC-166 was cloned in a pCR2.1 vector and transformed in chemically competent TOP10 cells. RESULTS: Sixteen inpatients resulted positive for colonization and/or infection by KPC-producing K. pneumoniae (KPC-Kp) strains. The 21 CZA-R KPC-Kp isolates obtained showed MDR phenotype; susceptibility to meropenem was always retained. All the CZA-R KPC-Kp presented a novel blaKPC variant, named blaKPC-166, showing a single nucleotide substitution (T811C) compared to the blaKPC-94; but related to blaKPC-2. TWO DIFFERENT PULSOTYPES WERE DETECTED: A in 18/21 and B in 1/21 cases, two strains from the same patient being untypable by PFGE. Interestingly, the outbreak was sustained by the high-risk clone ST307, although the ST22, ST6342, ST6418 and ST6811 have also been identified and associated to KPC-166. Worryingly, blaKPC-166 could be transferred horizontally and, after cloning, it conferred resistance to CZA. DISCUSSION: This novel variant confers CZA-resistance and carbapenems susceptibility restoration. As KPC-166 was found expressed by multiple Kp clones, greater efforts should be made to prevent the further dissemination of such strains in Italian clinical settings.

Enterobacter asburiae ST229: an emerging carbapenemases producer.

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. "Policlinico San Matteo" in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

Clinical isolates of ST131 blaOXA-244-positive Escherichia coli, Italy, December 2022 to July 2023.

The dissemination of carbapenemase-producing Escherichia coli, although still at low level, should be continuously monitored. OXA-244 is emerging in Europe, mainly in E. coli. In Italy, this carbapenemase was reported from an environmental river sample in 2019. We report clinical isolates of OXA-244-producing ST131 E. coli in four patients admitted to an acute care hospital in Pavia, Italy. The association of this difficult-to-detect determinant with a globally circulating high-risk clone, ST131 E. coli, is of clinical relevance.

Concomitant Resistance to Cefiderocol and Ceftazidime/Avibactam in Two Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Lung Transplant Patients.

In this study, we present two cases of Klebsiella pneumoniae, one KPC-33- and one NDM-1-producing, showing resistance to cefiderocol and ceftazidime/avibactam, collected in the intensive care unit of a hospital in Northern Italy from two patients who had recently undergone lung transplantation. Whole-genome sequencing was performed to investigate the molecular features of these strains.

Colonization of Residents and Staff of an Italian Long-Term Care Facility and an Adjacent Acute Care Hospital Geriatrics Unit by Multidrug-Resistant Bacteria.

In 2022, we undertook a point prevalence screening study for Enterobacterales with extended-spectrum ß-lactamases (ESBLs), high-level AmpC cephalosporinases and carbapenemases, and also methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) in a long-term care facility (LTCF) and the associated acute-care hospital Geriatrics unit in Bolzano, Northern Italy. Urine samples and rectal, inguinal, oropharyngeal, and nasal swabs were plated on selective agar plates. Metadata of the patients, including demographic data, were collected, and risk factors for colonization were determined. ESBL, AmpC, carbapenemase, and quinolone resistance genes were investigated by the HybriSpot 12 PCR AUTO System. The following colonization percentages by multidrug-resistant (MDR) bacteria have been found in LTCF residents: all MDR organisms, 59.5%; ESBL producers, 46.0% (mainly CTX-M-type enzymes); carbapenemase producers, 1.1% (one Klebsiella pneumoniae with KPC-type); MRSA, 4.5%; VRE, 6.7%. Colonization by MDR bacteria was 18.9% for LTCF staff and 45.0% for Geriatrics unit patients. Peripheral vascular disease, the presence of any medical device, cancer, and a Katz Index of 0 were significant risk factors for colonization of LTCF residents by MDR bacteria in univariate and/or multivariate regression analysis. To conclude, the ongoing widespread diffusion of MDR bacteria in the LTCF suggests that efforts should be strengthened on MDR screening, implementation of infection control strategies, and antibiotic stewardship programs targeting the unique aspects of LTCFs. ClinicalTrials.gov ID: 0530250-BZ Reg01 30/08/2022.

Bactericidal and Anti-Biofilm Activity of the FtsZ Inhibitor C109 against Acinetobacter baumannii.

In the last few years, Acinetobacter baumannii has ranked as a number one priority due to its Multi Drug Resistant phenotype. The different metabolic states, such as the one adopted when growing as biofilm, help the bacterium to resist a wide variety of compounds, placing the discovery of new molecules able to counteract this pathogen as a topic of utmost importance. In this context, bacterial cell division machinery and the conserved protein FtsZ are considered very interesting cellular targets. The benzothiadiazole compound C109 is able to inhibit bacterial growth and to block FtsZ GTPase and polymerization activities in Burkholderia cenocepacia, Pseudomonas aeruginosa, and Staphylococcus aureus. In this work, the activity of C109 was tested against a panel of antibiotic sensitive and resistant A. baumannii strains. Its ability to inhibit biofilm formation was explored, together with its activity against the A. baumannii FtsZ purified protein. Our results indicated that C109 has good MIC values against A. baumannii clinical isolates. Moreover, its antibiofilm activity makes it an interesting alternative treatment, effective against diverse metabolic states. Finally, its activity was confirmed against A. baumannii FtsZ.

The Role of Virulence Factors in Neonatal Sepsis Caused by Enterobacterales: A Systematic Review.

Neonatal sepsis is a life-threatening condition with high mortality. Virulence determinants relevant in causing Gram-negative (GN) neonatal sepsis are still poorly characterized. A better understanding of virulence factors (VFs) associated with GN neonatal sepsis could offer new targets for therapeutic interventions. The aim of this review was to assess the role of GN VFs in neonatal sepsis. We primarily aimed to investigate the main VFs leading to adverse outcome and second to evaluate VFs associated with increased invasiveness/pathogenicity in neonates. MEDLINE, Embase, and Cochrane Library were systematically searched for studies reporting data on the role of virulome/VFs in bloodstream infections caused by Enterobacterales among neonates and infants aged 0-90 days. Twenty studies fulfilled the inclusion criteria. Only 4 studies reported data on the association between pathogen virulence determinants and neonatal mortality, whereas 16 studies were included in the secondary analyses. The quality of reporting was suboptimal in the great majority of the published studies. No consistent association between virulence determinants and GN strains causing neonatal sepsis was identified. Considerable heterogeneity was found in terms of VFs analysed and reported, included population and microbiological methods, with the included studies often showing conflicting data. This variability hampered the comparison of the results. In conclusions, pathogens responsible for neonatal sepsis are widely heterogenous and can use different pathways to develop invasive disease. The recent genome-wide approach needs to include multicentre studies with larger sample sizes, analyses of VF gene profiles instead of single VF genes, alongside a comprehensive collection of clinical information. A better understanding of the roles of virulence genes in neonatal GN bacteraemia may offer new vaccine targets and new markers of highly virulent strains. This information can potentially be used for screening and preventive interventions as well as for new targets for anti-virulence antibiotic-sparing therapies.

Hypervariable-Locus Melting Typing: a Novel Approach for More Effective High-Resolution Melting-Based Typing, Suitable for Large Microbiological Surveillance Programs.

Pathogen typing is pivotal to detecting the emergence of high-risk clones in hospital settings and to limit their spread. Unfortunately, the most commonly used typing methods (i.e., pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and whole-genome sequencing [WGS]) are expensive or time-consuming, limiting their application to real-time surveillance. High-resolution melting (HRM) can be applied to perform cost-effective and fast pathogen typing, but developing highly discriminatory protocols is challenging. Here, we present hypervariable-locus melting typing (HLMT), a novel approach to HRM-based typing that enables the development of more effective and portable typing protocols. HLMT types the strains by assigning them to melting types (MTs) on the basis of a reference data set (HLMT-assignment) and/or by clustering them using melting temperatures (HLMT-clustering). We applied the HLMT protocol developed on the capsular gene wzi for Klebsiella pneumoniae on 134 strains collected during surveillance programs in four hospitals. Then, we compared the HLMT results to those obtained using wzi, MLST, WGS, and PFGE typing. HLMT distinguished most of the K. pneumoniae high-risk clones with a sensitivity comparable to that of PFGE and MLST+wzi. It also drew surveillance epidemiological curves comparable to those obtained using MLST+wzi, PFGE, and WGS typing. Furthermore, the results obtained using HLMT-assignment were consistent with those of wzi typing for 95% of the typed strains, with a Jaccard index value of 0.9. HLMT is a fast and scalable approach for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive, and thus, it is applicable for infection control programs in low- and middle-income countries. IMPORTANCE In this work, we describe hypervariable-locus melting typing (HLMT), a novel fast approach to pathogen typing using the high-resolution melting (HRM) assay. The method includes a novel approach for gene target selection, primer design, and HRM data analysis. We successfully applied this method to distinguish the high-risk clones of Klebsiella pneumoniae, one of the most important nosocomial pathogens worldwide. We also compared HLMT to typing using WGS, the capsular gene wzi, MLST, and PFGE. Our results show that HLMT is a typing method suitable for real-time epidemiological investigation. The application of HLMT to hospital microbiology surveillance can help to rapidly detect outbreak emergence, improving the effectiveness of infection control strategies.

In Vitro Activity of Sulbactam-Durlobactam against Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates: A Multicentre Report from Italy.

In the present study, the in vitro activity of the sulbactam-durlobactam (SUL-DUR) combination was evaluated against 141 carbapenem-resistant A. baumannii (CRAb) clinical strains collected from six Italian laboratories. Over half (54.6%) of these isolates were resistant to colistin. The SUL-DUR combination was active against these CRAb isolates with MIC50 and MIC90 values of 0.5 mg/L and 4 mg/L, respectively. Only eleven isolates were resistant to SUL-DUR with MIC values ranging from 8 to 128 mg/L. The SUL-DUR resistant A. baumannii exhibited several antimicrobial resistance genes (ARGs) such as blaOXA-20, blaOXA-58, blaOXA-66, blaADC-25, aac(6')-Ib3 and aac(6')-Ib-cr and mutations in gyrA (S81L) and parC (V104I, D105E). However, in these isolates, mutations Q488K and Y528H were found in PBP3. Different determinants were also identified in these CRAb isolates, including adeABC, adeFGH, adeIJK, abeS, abaQ and abaR, which encode multidrug efflux pumps associated with resistance to multiple antibacterial agents. This is the first report on the antimicrobial activity of SUL-DUR against carbapenem-resistant A. baumannii isolates selected from multiple regions in Italy.

OXA-244-Producing ST131 Escherichia coli From Surface and Groundwaters of Pavia Urban Area (Po Plain, Northern Italy).

The study aimed to investigate (i) the occurrence of third-generation cephalosporins and/or carbapenems non-sensitive Enterobacterales in Pavia surface and groundwaters, (ii) their resistance determinants, and (iii) the clonal features of the most relevant strains. During May 13 and 14, 2019, n = 18 water samples from n = 12 sampling sites in the urban/peri-urban area of Pavia (Po Plain, Northern Italy) have been evaluated. At first, hydrochemical analysis and bacterial plate counts were carried out on all the water samples. One milliliter of each water sample was then screened on both MacConkey agar (MC) added with cefotaxime (1 mg/L; 2 mg/L) and MC plus meropenem (0.25 mg/L; 4 mg/L). Species identification and antimicrobial susceptibilities were assessed by MicroScan autoSCAN-4. Double Disk Synergy (DD) test, CT103XL microarray, acc(6')-Ib-cr, qnrS, blaCTX-M-/MOX-/VEB-/OXA-type genes targeted PCR and sequencing, Pulsed-Field Gel Electrophoresis (PFGE), MultiLocus Sequence Typing (MLST), and Whole-Genome Sequencing on selected strains were performed. A total of n = 30 isolates grown on ß-lactams enriched MC: Escherichia coli (n = 21; 70%), Klebsiella spp. (n = 5; 16.6%), Citrobacter freundii (n = 2; 6.7%), and Kluyvera intermedia (n = 2; 6.7%). All E. coli and K. pneumoniae were ESßL-producers by DD. The 66.6, 38.0, and 19.0% of E. coli were ciprofloxacin/levofloxacin, trimethoprim-sulfamethoxazole, and gentamicin resistant (EUCAST 2019 breakpoints), respectively. A blaCTX-M-type determinant was identified in E. coli (n = 20/21; 95.2%) and K. pneumoniae (n = 2/3; 66.7%). The remaining E. coli was blaVEB-1 and blaMOX-2 genes positive. The aac(6')-Ib-cr determinant was found in n = 7 E. coli and n = 1 K. pneumoniae, while qnrS was found in n = 1 E. coli and n = 2 K. pneumoniae. PFGE showed clonal heterogeneity among ESßL-E. coli. Two out of four E. coli detected as blaOXA-244-positive, belonged to the pandemic ST131. One XDR K. pneumoniae from a stream sample, detected as blaKPC-2 positive, resulted of ST258. The epidemiological impact of blaOXA-244 ST131 E. coli and blaKPC-2 ST258 K. pneumoniae presence in surface waters of an urban area in Northern Italy must not be underestimated.

Wild Boars as an Indicator of Environmental Spread of ESßL-Producing Escherichia coli.

Antimicrobial resistance (AMR) represents an increasing issue worldwide, spreading not only in humans and farmed animals but also in wildlife. One of the most relevant problems is represented by Extended-Spectrum Beta-Lactamases (ESßLs) producing Escherichia coli because they are the cause of important infections in human. Wild boars (Sus scrofa) as a source of ESßLs attracted attention due to their increasing density and their habits that lead them to be at the human-livestock-wildlife interface. The aim of this study was to increase the knowledge about the ESßLs E. coli strains carried by wild boars living in a particularly high-density area of Northern Italy. The analysis of 60 animals allowed to isolate 16 ESßL-producing E. coli strains (prevalence 23.3%), which were characterised from a phenotypical and molecular point of view. The overall analysis revealed that the 16 isolates were all not only ESßL producers but also multidrug resistant and carried different types of plasmid replicons. The genome analysis performed on a subset of isolates confirmed the heterogeneity observed with pulsed-field gel electrophoresis (PFGE) and highlighted the presence of two pandemic sequence types, ST131 and ST10, with different collections of virulence factors. The genomic context of ESßL genes further evidenced that all of them were surrounded by transposons and insertion sequences, suggesting the possibility to exchange AMR genes. Overall, this study shows the worrying dissemination of ESßL-producing E. coli in wild boars in Northern Italy, suggesting the role of these animals as a spreader of AMR and their inclusion in surveillance programmes, to shed light on the "One Health" complex interactions.

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 [...].

Comparative analysis of SARS-CoV-2 quasispecies in the upper and lower respiratory tract shows an ongoing evolution in the spike cleavage site.

Studies are needed to better understand the genomic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe viral quasispecies population of upper and lower respiratory tract by next-generation sequencing in patients admitted to intensive care unit. A deep sequencing of the S gene of SARS-CoV-2 from 109 clinical specimens, sampled from the upper respiratory tract (URT) and lower respiratory tract (LRT) of 77 patients was performed. A higher incidence of non-synonymous mutations and indels was observed in the LRT among minority variants. This might be explained by the ability of the virus to invade cells without interacting with ACE2 (e.g. exploiting macrophage phagocytosis). Minority variants are highly concentrated around the gene portion encoding for the Spike cleavage site, with a higher incidence in the URT; four mutations are highly recurring among samples and were found associated with the URT. Interestingly, 55.8% of minority variants detected in this locus were T>G and G>T transversions. Results from this study evidenced the presence of selective pressure and suggest that an evolutionary process is still ongoing in one of the crucial sites of spike protein associated with the spillover to humans.

Isolation of a Colistin-Susceptible MDR Pantoea calida Harboring the mcr-9 Gene Suggests the Silent Spread of the Resistance Factor.

Pantoea spp. are bacteria that are often detected in the environment and as symbionts of arthropods. They sporadically cause infections in humans and recently extended spectrum beta-lactamases (ESBL)- and carbapenemase-producing strains have started to emerge. In this study, we report the isolation and the complete genome sequence of a strain of Pantoea calida encoding the colistin-resistance gene mcr-9. The strain was isolated from a preterm newborn in a neonatal pathology ward. On clinical examination, his vital signs were normal and blood culture was negative. Rectal swab screening for ESBL-producing Enterobacterales allowed to isolate the bacterium, and a complete genome was obtained using both short and long read sequencing. The mcr-9 gene was found to be encoded on a IncHI2 superplasmid, which confers resistance to six classes of antibiotics, including beta lactams (ESBL). Despite the presence of mcr-9, the isolate retains susceptibility to colistin, which could be explained by the absence of compatible regulatory genes (qseBC) from the genome. The presence of the resistance gene is undetectable with the routine clinical procedures, that is, phenotypic tests. This suggests that a silent spread might be ongoing in the ward. To our knowledge, this is the first description of an MDR P. calida and of a Pantoea spp. encoding any mobile colistin resistance gene.

Antimicrobial Susceptibility, Virulence, and Genomic Features of a Hypervirulent Serotype K2, ST65 Klebsiella pneumoniae Causing Meningitis in Italy.

The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including blaSHV-11, blaSHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage.

In Vitro Antimicrobial Activity of the Siderophore Cephalosporin Cefiderocol against Acinetobacter baumannii Strains Recovered from Clinical Samples.

BACKGROUND: Cefiderocol is a siderophore cephalosporin that exhibits antimicrobial activity against most multi-drug resistant Gram-negative bacteria, including Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia. METHODS: A total of 20 multidrug-resistant A. baumannii strains were isolated from 2020 to 2021, molecularly characterized and tested to assess the in vitro antibacterial activity of cefiderocol. Thirteen strains were carbapenem-hydrolysing oxacillinase OXA-23-like producers, while seven were non-OXA-23-like producers. Minimum inhibitory concentrations (MICs) were determined by broth microdilution, considered as the gold standard method. Disk diffusion test was also carried out using iron-depleted CAMHB plates for cefiderocol. RESULTS: Cefiderocol MICs ranged from 0.5 to 1 mg/L for OXA-23-like non-producing A. baumannii strains and from 0.25 to >32 mg/L for OXA-23-like producers, using the broth microdilution method. Cefiderocol MIC90 was 8 mg/L. Diameter of inhibition zone of cefiderocol ranged from 18 to 25 mm for OXA-23-like non-producers and from 15 to 36 mm for OXA-23-like producers, using the diffusion disk method. A large variability and a low reproducibility were observed during the determination of diameter inhibition zone. Molecular characterization showed that all isolates presented the ISAba1 genetic element upstream the blaOXA-51. Among OXA-23-like non-producers, four were blaOXA-58 positive and two were negative for all the resistance determinants analyzed. CONCLUSIONS: Cefiderocol showed in vitro antimicrobial activity against both carbapenem-susceptible and non-susceptible A. baumannii strains, although some OXA-23-like producers were resistant. Further clinical studies are needed to consolidate the role of cefiderocol as an antibiotic against MDR A. baumannii.

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.