ESKAPE bacteria are a major cause of multidrug-resistant infections, and new drugs are urgently needed to combat these pathogens. Given the importance of iron in bacterial physiology and pathogenicity, iron uptake and metabolism have become attractive targets for the development of new antibacterial drugs. In this scenario, the FDA-approved iron mimetic metal Gallium [Ga(III)] has been successfully repurposed as an antimicrobial drug. Ga(III) disrupts ferric iron-dependent metabolic pathways, thereby inhibiting microbial growth. This work provides the first comparative assessment of the antibacterial activity of Ga(NO₃)₃ (GaN), Ga(III)-maltolate (GaM), and Ga(III)-protoporphyrin IX (GaPPIX), belonging to the first-, second- and third-generation of Ga(III) formulations, respectively, on ESKAPE species, including reference strains and multidrug-resistant (MDR) clinical isolates. In addition to the standard culture medium Mueller Hinton broth (MHB), iron-depleted MHB (DMHB) and RPMI-1640 supplemented with 10% human serum (HS) (RPMI-HS) were also included in Ga(III)-susceptibility tests, because of their different nutrient and iron contents. All ESKAPE species were resistant to all Ga(III) compounds in MHB and DMHB (MIC > 32 μM), except Staphylococcus aureus and Acinetobacter baumannii, which were susceptible to GaPPIX. Conversely, the antibacterial activity of GaN and GaM was very evident in RPMI-HS, in which the low iron content and the presence of HS better mimic the in vivo environment. In RPMI-HS about 50% of the strains were sensitive (MIC < 32) to GaN and GaM, both compounds showing a similar spectrum of activity, although GaM was more effective than GaN. In contrast, GaPPIX lost its antibacterial activity in RPMI-HS likely due to the presence of albumin, which binds GaPPIX and counteracts its inhibitory effect. We also demonstrated that the presence of multiple heme-uptake systems strongly influences GaPPIX susceptibility in A. baumannii. Interestingly, GaN and GaM showed only a bacteriostatic effect, whereas GaPPIX exerted a bactericidal activity on susceptible strains. Altogether, our findings raise hope for the future development of Ga(III)-based compounds in the treatment of infections caused by multidrug-resistant ESKAPE pathogens.

Background: Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 398 is recognized as an occupational hazard for workers employed in intensive animal husbandry, especially in the swine-breeding chain. In this study, we compared the prevalence and epidemiological type of MRSA isolates from swine and farm workers in a large area of southern Italy. Methods: Between January and March 2018, 88 workers from 32 farms where we had previously performed a survey for MRSA colonization of farmed pigs, were sampled by nasal swabbing. A follow-up investigation was conducted on seven workers 1 year after primary screening. MRSA isolates were characterized by MLST, spa and SCCmec typing, and tested for susceptibility to 15 antimicrobials. Epidemiological correlations between human and swine MRSA isolates were supported by Rep-MP3 and RAPD PCR fingerprinting, and whole-genome sequencing. Results: The overall colonization rate of MRSA in swine farm workers was 21.6%, being significantly higher in intensive farms and in workers with direct animal contact. All human MRSA isolates were multi-drug resistant, belonged to the ST398 livestock clade, and did not carry Panton-Valentine leukocidin and enterotoxin genes. Notably, 94.1% of human MRSA isolates belonged to the same epidemiological type as swine MRSA isolates from the corresponding farm. Persistent MRSA carriage was documented in some workers 1 year after primary sampling. Conclusions: We report a high prevalence of MRSA among swine farm workers, with higher colonization rates associated with intensive breeding and animal exposure. Our findings suggest unidirectional animal-to-human transmission of LA-MRSA and denote the high zoonotic transmissibility of the ST398 livestock clade.

Colonization by livestock-associated MRSA (LA-MRSA) has increasingly been reported in the swine population worldwide. The aim of this study was to assess the prevalence of MRSA nasal carriage in healthy pigs, including the black (Calabrese) breed, from farms in the Calabria Region (Southern Italy). Between January and March 2018, a total of 475 healthy pigs reared in 32 farms were sampled by nasal swabbing. MRSA isolates were characterized by spa, MLST and SCCmec typing, and susceptibility testing to 17 antimicrobials. 22 of 32 (66.8%) pig farms resulted positive for MRSA. The prevalence of MRSA was 46.1% (219 MRSA culture-positive out of 475 samples). MRSA colonization was significantly higher in intensive farms and in pigs with a recent or ongoing antimicrobial treatment. All 219 MRSA isolates were assigned to ST398. The most common spa types were t011 (37.0%), t034 (22.4%) and t899 (15.1%). A novel spa type (t18290) was detected in one isolate. An insertion of IS256 in the ST398-specific A07 fragment of the SAPIG2195 gene was detected in 10 out of 81 t011 isolates. Nearly all isolates carried the SCCmec type V element, except 11 isolates that carried the SCCmec type IVc. None of the isolates was positive for the Panton-Valentine leukocidin. All isolates were resistant to tetracycline. High resistance rates were also found for clindamycin (93.1%), trimethoprim/sulfamethoxazole (68.4%), fluoroquinolones (47.9–65.3%) and erythromycin (46.1%). None of the isolates was resistant to vancomycin and fusidic acid. Overall, a multidrug resistant phenotype was observed in 88.6% of isolates. We report a high prevalence of MRSA among healthy swine in Southern Italy farms, with higher isolation frequency associated with intensive farming. The epidemiological types identified in our study reflect those reported in other European countries. Our findings underscore the importance of monitoring the evolution of LA-MRSA in pig farms in order to implement control measures and reduce the risk of spread in the animal population.

Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC ( n = 15 and 16, respectively) and 1/4 MIC ( n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes ( n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG , which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli , shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.