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The discovery, commercialization, and regular administration of antimicrobial agents have revolutionized the therapeutic paradigm, making it possible to treat previously untreatable and fatal infections. However, the excessive use of antibiotics has led to develop resistance soon after their use in clinical practice, to the point of becoming a global emergency. The mechanisms of bacterial resistance to antibiotics are manifold, including mechanisms of destruction or inactivation, target site modification, or active efflux, and represent the main examples of evolutionary adaptation for the survival of bacterial species. The acquirement of new resistance mechanisms is a consequence of the great genetic plasticity of bacteria, which triggers specific responses that result in mutational adaptation, acquisition of genetic material, or alteration of gene expression, virtually producing resistance to all currently available antibiotics. Understanding resistance processes is critical to the development of new antimicrobial agents to counteract drug-resistant microorganisms. In this review, both the mechanisms of action of antibiotic resistance (AMR) and the antibiotic resistance genes (ARGs) mainly found in clinical and environmental bacteria will be reviewed. Furthermore, the evolutionary background of multidrug-resistant bacteria will be examined, and some promising elements to control or reduce the emergence and spread of AMR will be proposed.

Background Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is an urgent public health issue in Italy. This pattern of resistance is due mainly to dissemination of carbapenemase genes. Molecular characterization of carbapenem-resistant Klebsiella pneumoniae (CR-KP) strains was performed over a three-year period. In-depth analysis was performed on a subset of emerging CR-KP ST101 and ST307 clones. Methods A prospective study was performed on 691 patients with CR-KP bloodstream infections hospitalized in 19 hospitals located in three large provinces in Southern Italy. Carbapenemase genes were identified via genotyping methods. Multi-locus sequence typing (MLST) and Whole Genome Sequencing (WGS) were carried out on ST101 and ST307 isolates. Results Among the CR-KP isolates, bla KPC was found in 95.6%, bla VIM was found in 3.5%, bla NDM was found in 0.1% and bla OXA-48 was found in 0.1%. The bla KPC-3 variant was identified in all 104 characterized KPC-KP isolates. MLST of 231 representative isolates revealed ST512 in 45.5%, ST101 in 20.3% and ST307 in 18.2% of the isolates. cgMLST of ST307 and ST101 isolates revealed presence of more than one beta-lactam resistance gene. Amino acid substitution in the chromosomal colistin-resistance gene pmrB was found in two ST101 isolates. Conclusions ST512 is widespread in Southern Italy, but ST101 and ST307 are emerging since they were found in a significant proportion of cases. Aggressive infection control measures and a continuous monitoring of these high-risk clones are necessary to avoid rapid spread of CR-KP, especially in hospital settings.

Background: The spread of ESBL-producing Enterobacteriaceae (ESBL-PE) strains in food poses a potential risk to human health. The aim of the study was to determine the occurrence of ESBL-PE and to investigate their distribution on foods. Methods: A total of 1000 food samples, including both raw and ready-to-eat products, was analyzed for the presence of ESBL-producing Enterobacteriaceae using chromogenic selective agar. Antibiotic resistance in the isolated strains was assessed using conventional methods, while whole-genome sequencing was employed to predict antimicrobial resistance and virulence genes. Results: The overall occurrence of ESBL-PE strains was 2.8%, with the highest contamination in raw meat samples (10%). A total of 31 multidrug-resistant (MDR) strains was isolated, mainly Escherichia coli, followed by Klebsiella pneumoniae, Salmonella enterica, and Enterobacter hormaechei. All strains exhibited high levels of resistance to at least four different β-lactam antibiotics, as well as to other antimicrobial classes including sulfonamides, tetracyclines, aminoglycosides, and quinolones. Whole-genome sequencing identified 63 antimicrobial resistance genes, with blaCTX-M being the most prevalent ESBL gene. Twenty-eight (90%) isolates carried Inc plasmids, known vectors of multiple antimicrobial resistance genes, including those associated with ESBLs. Furthermore, several virulence genes were identified. Conclusions: The contamination of food with ESBL-PE represents a potential public health risk, underscoring the importance of the implementation of genomic surveillance to monitor and control the spread of antimicrobial resistance.

Chryseobacterium spp. are Gram-negative, opportunistic pathogens antibiotic-resistant commonly found in the environment. The aim of this study was to investigate potential sources of Chryseobacterium infections in healthcare settings by comparing clinical and environmental isolates using phenotypic and genotypic analyses. Between June and July 2023, six cases of infection with Chryseobacterium spp. were identified in a hospital in Apulia, southern Italy. Environmental sampling (air, surfaces and water) was performed in parallel with routine clinical investigations. Isolates were subjected to antibiotic susceptibility testing and genotypic analysis using Sanger and Next-Generation Sequencing. Five cases of Chryseobacterium spp. infection were recorded in the Gastroenterology Department (Pavilion A) and one in the Vertebral Surgery Department (Pavilion B). C. indologenes was identified in blood and tracheal aspirate samples using MALDI-TOF MS. Environmental analysis carried out in the pavilions A and B isolated C. indologenes from sink tap in Pavilion B. Subsequently, genome sequencing revealed that Chryseobacterium strains misidentified as C. indologenes were more closely related to C. arthrosphaerae . Genetic analysis confirmed the cluster hypothesis involving four patients from the pavilion A, while no genetic link was found between the environmental and clinical strains. Innovative molecular methods in clinical and environmental investigations have allowed more accurate identification of the etiologic agent and possibly tracing the source of infection in the nosocomial setting. Control measures, such as patient isolation and room disinfection, have prevented the spread of infection.

The Bacillus cereus group (B. cereus group) comprises several closely related species that share high genetic similarity but display markedly different phenotypic traits and pathogenic potential. Reliable and rapid discrimination at the species level remains challenging using conventional microbiological and molecular methods. In this study, Fourier Transform Infrared (FTIR) spectroscopy was evaluated as a rapid phenotypic approach to differentiate seven members of the Bacillus cereus sensu stricto (B. cereus s.s.), Bacillus anthracis (B. anthracis), Bacillus thuringiensis (B. thuringensis), Bacillus mycoides (B. mycoides), Bacillus toyonensis (B. toyonensis), Bacillus wiedmannii (B. wiedmannii) and Bacillus weihenstephanensis (B. weihenstephanensis). A collection of 190 isolates previously characterized by whole genome sequencing was analyzed using the IR Biotyper system. Spectral data were processed through multivariate analyses, including principal component analysis and linear discriminant analysis, following a hierarchical classification strategy. FTIR spectroscopy enabled clear discrimination of B. anthracis from other members of the B. cereus group and allowed the separation of several additional species based on distinct spectral signatures. A further discrimination step permitted differentiation between B. cereus sensu stricto and B. thuringiensis, with minimal overlap. These findings demonstrate that FTIR spectroscopy represents a promising and rapid tool for species-level discrimination within the B. cereus group. While the results should be considered preliminary for species represented by a limited number of isolates, this approach shows strong potential as a complementary method to molecular techniques in routine diagnostics in food safety and veterinary microbiology.

Staphylococcus aureus and coagulase-negative staphylococci (CNS) are the main causative agents of mastitis in sheep. Their ability to form biofilms in vivo is considered an important virulence factor underlying mastitis outbreaks refractory to antibiotic treatments. Furthermore, pre- and postdipping immersion during milking in iodine substances could determine the presence of residues in milk and therefore represent a health risk factor for consumers. The aim of this study was to evaluate the antibacterial and biofilm inhibitory activity of Thymus vulgaris L. essential oil (TEO) against staphylococci strains isolated from ovine clinical mastitis. In particular, 3 reference strains (S. aureus 25923 and 11623 and S. epidermidis 12228) and 12 clinical isolates (6 S. aureus and 6 CNS) were used. TEO solutions, from a concentration of 1% (v/v) to 1.25% (v/v), corresponding to 9.28–2.32 mg/mL, were obtained after solubilization in 10% dimethyl sulfoxide (DMSO) and used to evaluate the bacterial time-kill compared to that of an iodine-based solution. Antibacterial efficacy was then assessed by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), while biofilm inhibition was assessed by minimum biofilm inhibitory concentration (MBIC) using a spectrophotometer at a wavelength of 570 nm. Additionally, biofilm-associated genes (icaA and icaD) were evaluated in all tested strains by PCR. The tested TEO concentrations were able to significantly and prominently reduce bacterial growth compared to controls, as demonstrated by bacterial time-kills. The MIC value was obtained at a concentration of 0.50% (v/v) for a single coagulation-positive isolate (S. aureus (f)) and at a concentration of 0.25% (v/v) for all other isolates. TEO showed effective bactericidal action with a 99.9% reduction in CFU/mL of all isolates in the MBC test at a concentration of 0.25% (v/v) for most of the tested strains. Furthermore, a marked inhibition in biofilm formation at all tested concentrations was observed, with MBIC value of 0.25%. All S. aureus tested were biofilm-producing strains and positive for icaA and icaD genes, while two CNS biofilm-producing strains were negative for both genes. These preliminary results suggest that TEO could be a promising alternative as an udder disinfectant during milking practices. Although in vivo studies are needed to confirm the efficacy and safety of TEO as an adjuvant in the prevention and treatment of udder infections, TEO could help counteract the emergence of antimicrobial resistance and reduce the potential risk of iodine residues in milk.

Bacillus cereus is isolated from a variety of foods where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature. In this study, we identified members of B. cereus groups in 65% of the ice cream samples analyzed, which were characterized based on multi locus variable number tandem repeats analysis (MLVA) and whole genome sequencing (WGS). The MLVA revealed that 36 strains showed different allelic profiles. Analyses of WGS data enabled the identification of three members of the B. cereus group: B. cereus sensu stricto, B. mosaicus and B. thuringiensis. Based on the multi locus sequence typing (MLST) scheme, the strains were classified in 27 sequence types (STs), including ST26 that causes food poisoning. Toxin genes’ detection revealed the presence of the genes encoding nonhemolytic enterotoxin (NHE), hemolysin BL (HBL), cytotoxin K (cytK) and cereulide (ces) in 100%, 44%, 42% and 8% of the strains, respectively. The identification of eleven antimicrobial resistance (AMR) genes predicted the resistance to five different antimicrobials, and the resistance to beta-lactam antibiotics was confirmed with a phenotypic antimicrobial test. Taken together, the results showed that the B. cereus strains isolated from ice cream were a potential hazard for consumer safety.

The emergence of colistin-resistant Enterobacteriaceae in food products is a growing concern due to the potential transfer of resistance to human pathogens. This study aimed to assess the prevalence of colistin-resistant Enterobacteriaceae in raw and ready-to-eat food samples collected from two regions of Italy (Apulia and Basilicata) and to evaluate their resistance phenotypes and genetic characteristics. A total of 1000 food samples were screened, with a prevalence of 4.4% of colistin-resistant Enterobacteriaceae. The majority of the isolates belonged to Enterobacter spp. (60%), followed by Moellerella wisconsensis, Atlantibacter hermannii, Klebsiella pneumoniae, and Escherichia coli, among others. Genomic sequencing and antimicrobial susceptibility testing revealed high levels of resistance to β-lactams, with most isolates exhibiting multidrug resistance (MDR). Notably, seven isolates harbored mcr genes (mcr-1, mcr-9, and mcr-10). Additionally, in four of them were predicted the IncHI2 plasmids, known to facilitate the spread of colistin resistance. Furthermore, 56 antimicrobial resistance genes were identified, suggesting the genetic mechanisms underlying resistance to several antibiotic classes. Virulence gene analysis showed that E. coli and other isolates carried genes linked to pathogenicity, increasing the potential risk to public health. This study emphasizes the role of food as a potential reservoir for colistin-resistant bacteria and the importance of monitoring the spread of AMR genes in foodborne pathogens.

The Bacillus cereus group includes species that act as food-borne pathogens causing diarrheal and emetic symptoms. They are widely distributed and can be found in various foods. In this study, out of 550 samples of milk and cheeses, 139 (25.3%) were found to be contaminated by B. cereus sensu lato (s.l.). One isolate per positive sample was characterized by Multilocus Sequence Typing (MLST) and for the presence of ten virulence genes. Based on MLST, all isolates were classified into 73 different sequence types (STs), of which 12 isolates were assigned to new STs. Virulence genes detection revealed that 90% and 61% of the isolates harboured the nheABC and the hblCDA gene cluster, respectively. Ninety-four percent of the isolates harboured the enterotoxin genes entS and entFM; 8% of the isolates possessed the ces gene. Thirty-eight different genetic profiles were identified, suggesting a high genetic diversity. Our study clearly shows the widespread diffusion of potentially toxigenic isolates of B. cereus s.l. in milk and cheeses in the Apulia region highlighting the need to adopt GMP and HACCP procedures along every step of the milk and cheese production chain in order to reduce the public health risk linked to the consumption of foods contaminated by B. cereus s.l.

In order to provide insights into the evolutionary and epidemiological viral dynamics during the current COVID-19 pandemic in South Eastern Italy, a total of 298 genomes of SARS-CoV-2 strains collected in the Apulia and Basilicata regions, between March 2020 and January 2021, were sequenced. The genomic analysis performed on the draft genomes allowed us to assign the genetic clades and lineages of belonging to each sample and provide an overview of the main circulating viral variants. Our data showed the spread in Apulia and Basilicata of SARS-CoV-2 variants which have emerged during the second wave of infections and are being currently monitored worldwide for their increased transmission rate and their possible impact on vaccines and therapies. These results emphasize the importance of genome sequencing for the epidemiological surveillance of the new SARS-CoV-2 variants’ spread.