Multidrug resistance (MDR) represents a serious global threat due to the rapid global spread and limited antimicrobial options for treatment of difficult-to-treat (DTR) infections sustained by MDR pathogens. Recently, novel β-lactams/β-lactamase inhibitor combinations (βL-βLICs) have been developed for the treatment of DTR infections due to MDR Gram-negative pathogens. Although novel βL-βLICs exhibited promising in vitro and in vivo activities against MDR pathogens, emerging resistances to these novel molecules have recently been reported. Resistance to novel βL-βLICs is due to several mechanisms including porin deficiencies, increasing carbapenemase expression and/or enzyme mutations. In this review, we summarized the main mechanisms related to the resistance to ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam in MDR Gram-negative micro-organisms. We focused on antimicrobial activities and resistance traits with particular regard to molecular mechanisms related to resistance to novel βL-βLICs. Lastly, we described and discussed the main detection methods for antimicrobial susceptibility testing of such molecules. With increasing reports of resistance to novel βL-βLICs, continuous attention should be maintained on the monitoring of the phenotypic traits of MDR pathogens, into the characterization of related mechanisms, and on the emergence of cross-resistance to these novel antimicrobials.

The SARS-CoV-2-associated COVID-19 pandemic has shaken the global healthcare system. Although the best-known symptoms are dry cough and pneumonia, viral RNA has been detected in the stool and about half of COVID-19 patients exhibit gastrointestinal upset. In this scenario, special attention is being paid to the possible role of the gut microbiota (GM). Fecal samples from 69 COVID-19 patients from three different hospitals of Bologna (Italy) were analyzed by 16S rRNA gene-based sequencing. The GM profile was compared with the publicly available one of healthy age- and gender-matched Italians, as well as with that of other critically ill non-COVID-19 patients. The GM of COVID-19 patients appeared severely dysbiotic, with reduced diversity, loss of health-associated microorganisms and enrichment of potential pathogens, particularly Enterococcus. This genus was far overrepresented in patients developing bloodstream infections (BSI) and admitted to the intensive care unit, while almost absent in other critically ill non-COVID-19 patients. Interestingly, the percentage of patients with BSI due to Enterococcus spp. was significantly higher during the COVID-19 pandemic than in the previous 3 years. Monitoring the GM of critically ill COVID-19 patients could help clinical management, by predicting the onset of medical complications such as difficult-to-treat secondary infections.

Combination therapy is recommended for the treatment of KPC-producing Klebsiella pneumoniae (KPC-Kp), but the optimal regimen for colistin-resistant strains is unknown. We compared the synergistic activity and post-antibiotic effect (PAE) of colistin in combination with other antimicrobials against colistin-susceptible and -resistant KPC-Kp bloodstream isolates. The genotypes of nine colistin-susceptible and eight colistin-resistant KPC-Kp bloodstream isolates were analysed using PCR and amplicon sequencing. Combinations of colistin, meropenem, tigecycline, rifampicin and teicoplanin were then screened using the Etest, a chequerboard assay and time–kill studies. Synergistic combinations were also analysed with respect to the PAE in time–kill curves and the PAE at clinically achievable concentrations. Insertional inactivation of the PhoQ/PhoB two-component regulatory system by mgrB-IS5 was identified in 6/8 (75%) colistin-resistant KPC-Kp. Colistin/rifampicin combinations resulted in no interactions [fractional inhibitory concentration (FIC) indices 1.5–2] for colistin-susceptible strains, but were uniformly synergistic (FIC indices 0.1–0.4) against colistin-resistant KPC-Kp. Time–kill kinetic analysis, at clinically achievable fixed concentrations of rifampicin and colistin, confirmed synergy and produced persistent growth inhibition (3 h) of colistin-resistant KPC-Kp strains exposed to colistin/rifampicin or colistin/rifampicin/tigecycline combinations. Combinations of colistin plus rifampicin, and less frequently tigecycline, exhibited synergistic activity in vitro against colistin-resistant KPC-Kp strains.

Meropenem/Vaborbactam (MEM-VAB) is a novel carbapenem- β-lactamase inhibitor active against KPC-producing Enterobacteria. Herein, we evaluate the incidence of meropenem/vaborbactam-resistance among KPC-producing K. pneumoniae (KPC-Kp) bloodstream infection in a large Italian hospital. Meropenem/vaborbactam-resistance was found in 8% (n = 5) KPC-Kp, while 5% (n = 3) strains exhibited cross-resistance to ceftazidime/avibactam (CAZ-AVI). Genomic analysis revealed that meropenem/vaborbactam-resistance was associated with truncated OmpK35 and insertion of glycine and aspartic acid within OmpK36 at position 134–135 (GD134–135). Notably, no specific mutation was associated to cross-resistance. No specific antimicrobial treatment was related to favorable clinical outcomes, while cross-resistance was not associated to higher clinical and/or microbiological failures. Our study indicated that resistance to meropenem/vaborbactam was due to porins mutations and is associated with reduced susceptibility to both ceftazidime/avibactam and carbapenems.

In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced. Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST). Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively. Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.

The new Xpert MTB/RIF Ultra assay (Ultra, Cepheid, Sunnyvale, USA) is a cartridge-based automated diagnostic test that can simultaneously identify Mycobacterium tuberculosis complex (MTB) and resistance to Rifampicin (RIF). With respect to the previous version Xpert MTB/RIF assay (Xpert), IS6110/IS1081 repetitive elements probes have been added allowing the detection of lower MTB load, defined by the new semi-quantitative category “trace” with indeterminate RIF resistance. The aim of this study was to evaluate performance of the new version Ultra on Xpert-negative, but TB culture-positive clinical samples. The de-identified frozen samples (-20 °C) collected over a 4-year period (February 2014-October 2017), which had previously resulted smear-negative, Xpert-negative but MTB culture-positive, were analyzed with Ultra. The de-frosted samples were loaded into the cartridge using the same process as the previous version, according to manufacturer’s instruction. During the study period 382 MTB culture-positive samples were archived: 314 resulted Xpert-positive and 68 Xpert-negative. Thirty-one of the 68 Xpert-negative samples resulted positive with Ultra, with an overall improvement in MTB detection of 45.6%. Out of 36 Xpert-negative respiratory samples, 18 resulted Ultra-positive with the following semi-quantitative loads: “low”(n = 1), “very low”(n = 11), “trace”(n = 6), with an improvement in MTB detection of 50%. The best performance was achieved on bronchoalveolar lavage specimens (53.8%). Out of 32 Xpert-negative non-respiratory samples, 13 resulted Ultra-positive with the following semi-quantitative loads: “very low”(n = 7), “trace”(n = 6), with an improvement in MTB detection of 40.6%. The best performance was achieved on biopsies (55.6%) and lymph nodes (50%). The new category “trace” detected 12 out of the 31 Ultra-positive MTB samples; in the remaining 19 samples RIF susceptibility was determined with 100% concordance with the phenotypic susceptibility test. The mean time to positivity of samples found negative by Ultra was significantly longer in comparison to positive samples in liquid culture. Our results are consistent with the few studies published so far and confirm the better performance of Ultra compared to the previous version in both respiratory and non-respiratory smear-negative samples, with an overall improvement of 45.6%.