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This article reports a rapid and unexpected spread of colonization cases of NDM-1 carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in a neonatal surgical unit (NSU) at Bambino Gesù Children’s Hospital in Rome, Italy. Between the 16th of November 2020 and the 18th of January 2021, a total of 20 NDM-1 carbapenemase-producing K. pneumoniae (n = 8) and E. coli (n = 12) were isolated from 17 out of 230 stool samples collected from neonates admitted in the aforementioned ward and time period by an active surveillance culture program routinely in place to monitor the prevalence of colonization/infection with multidrug-resistant Gram-negative microorganisms. All strains were characterized by antimicrobial susceptibility testing, detection of resistance determinants, PCR-based replicon typing (PBRT) and multilocus-sequence typing (MLST). All isolates were highly resistant to most of the tested antibiotics, and molecular characterization revealed that all of them harbored the blaNDM-1 gene. Overall, IncA/C was the most common Inc group (n = 20/20), followed by IncFIA (n = 17/20), IncFIIK (n = 14/20) and IncFII (n = 11/20). MLST analysis was performed on all 20 carbapenemase-producing Enterobacterales (CPE) strains, revealing three different Sequence Types (STs) among E. coli isolates, with the prevalence of ST131 (n = 10/12; 83%). Additionally, among the 8 K. pneumoniae strains we found 2 STs with the prevalence of ST37 (n = 7/8; 87.5%). Although patient results were positive for CPE colonization during their hospital stay, infection control interventions prevented their dissemination in the ward and no cases of infection were recorded in the same time period.

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e., "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment.

The spread of carbapenemase-producing Enterobacterales (CPE), especially Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), is a serious public health threat in pediatric hospitals. The associated risk in newborns is due to their underdeveloped immune system and limited treatment options. The aim was to estimate the prevalence and circulation of CPE among the neonatal intensive units of a major pediatric hospital in Italy and to investigate their molecular features. A total of 124 CPE were isolated from rectal swabs of 99 newborn patients at Bambino Gesù Children’s Hospital between July 2016 and December 2019. All strains were characterized by antimicrobial susceptibility testing, detection of resistance genes, and PCR-based replicon typing (PBRT). One strain for each PBRT profile of K. pneumoniae or E. coli was characterized by multilocus-sequence typing (MLST). Interestingly, the majority of strains were multidrug-resistant and carried the blaNDM gene. A large part was characterized by a multireplicon status, and FII, A/C, FIA (15%) was the predominant. Despite the limited size of collection, MLST analysis revealed a high number of Sequence Types (STs): 14 STs among 28 K. pneumoniae and 8 STs among 11 E. coli, with the prevalence of the well-known clones ST307 and ST131, respectively. This issue indicated that some strains shared the same circulating clone. We identified a novel, so far never described, ST named ST10555, found in one E. coli strain. Our investigation showed a high heterogeneity of CPE circulating among neonatal units, confirming the need to monitor their dissemination in the hospital also through molecular methods.

This study aimed at monitoring and characterize the Staphylococcus aureus carriage status of employees in a pasta company in order to evaluate the associated risk factors. Food handlers (n = 21) were sampled between 2013 and 2015 through nasal and hand swabs to determine the colonization status. Seven out of 21 employees (33%) were contaminated with S. aureus and the prevalence decreased to 9.5% over the last year. Only two persistent carriers were identified. Twenty-eight strains were isolated from both hand and nasal samples. Each of them was resistant to at least one class of antibiotics and the multidrug resistance strains were isolated from the nose. The highest resistance rate was observed towards penicillin G (79%) and to linezolid (64%) confirming the rapid spread of linezolid resistant strains recently described in Italy. The dominant toxin gene was sem (93%), which is usually not among the most prevalent, whereas the primary agr group was the agrIII (43%) and the most frequent spa type was t030 (39%). These results combined with the genomic macrorestriction analysis revealed high genetic diversity. The increased virulence, antibiotic resistance and molecular variability of isolates highlighted the importance of monitoring activity in food company to assess the potential associated risk of foodborne diseases.

The emerging spread of carbapenemase-producing Enterobacterales (CPE) strains, in particular, Klebsiella pneumoniae and Escherichia coli, has become a significant threat to hospitalized patients. Carbapenemase genes are frequently located on plasmids than can be exchanged among clonal strains, increasing the antibiotic resistance rate. The aim of this study was to determine the prevalence of CPE in patients upon their admission and to analyze selected associated factors. An investigation of the antibiotic resistance and genetic features of circulating CPE was carried out. Phenotypic tests and molecular typing were performed on 48 carbapenemase-producing strains of K. pneumoniae and E. coli collected from rectal swabs of adult patients. Carbapenem-resistance was confirmed by PCR detection of resistance genes. All strains were analyzed by PCR-based replicon typing (PBRT) and multilocus sequence typing (MLST) was performed on a representative isolate of each PBRT profile. More than 50% of the strains were found to be multidrug-resistant, and the blaKPC gene was detected in all the isolates with the exception of an E. coli strain. A multireplicon status was observed, and the most prevalent profile was FIIK, FIB KQ (33%). MLST analysis revealed the prevalence of sequence type 512 (ST512). This study highlights the importance of screening patients upon their admission to limit the spread of CRE in hospitals.

Nontyphoidal salmonellosis (NTS) is the second most commonly reported gastrointestinal infection in humans and an important cause of food-borne outbreaks in Europe. The use of antimicrobial agents for animals, plants, and food production contributes to the development of antibiotic-resistant Salmonella strains that are transmissible to humans through food. The aim of this study was to investigate the presence and the potential dissemination of multidrug-resistant (MDR) Salmonella strains isolated in the Marche Region (Central Italy) via the food chain. Strains were isolated from different sources: food, human, food animal/livestock, and the food-processing environment. Among them, we selected MDR strains to perform their further characterization in terms of resistance to tetracycline agent, carriage of tet genes, and plasmid profiles. Tetracycline resistance genes were detected by PCR and plasmid replicons by PCR-based replicon typing (PBRT). A total of 102 MDR Salmonella strains were selected among the most prevalent serovars: S. Infantis (n = 36/102), S. Derby (n = 20/102), S. Typhimurium (n = 18/102), and a monophasic variant of S. Typhimurium (MVST, n = 28/102). Resistance to sulfisoxazole (86%) and tetracycline (81%) were the most common, followed by ampicillin (76%). FIIS was the most predominant replicon (17%), followed by FII (11%) and FIB (11%) belonging to the IncF incompatibility group. Concerning the characterization of tet genes, tetB was the most frequently detected (27/89), followed by tetA (10/89), tetG (5/89), and tetM (1/89). This study showed the potential risk associated with the MDR Salmonella strains circulating along the food chain. Hence, epidemiological surveillance supported by molecular typing could be a very useful tool to prevent transmission of resistant Salmonella from food to humans, in line with the One Health approach.

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10−4–1 × 10−6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.

Purpose. The presence of Staphylococcus aureus in poultry and poultry products, including eggs, increases its potential to enter the food chain, resulting in foodborne diseases. In this context, eggshell colonization by staphylococci may represent a risk factor. This study aimed to investigate the contamination of rural eggshell by S. aureus and to characterize the key features of the isolated strains. Methodology. Antibiotic resistance was assessed by disc diffusion. Resistant isolates were analysed by PCR for the identification of associated genetic determinants of resistance. PCR was also used to screen for the presence of genes coding for toxins, namely, sea, sec, sei, sem, seo and tst. The genetic characterization was extended by means of agr locus typing and spa typing. Results. 34 S. aureus were isolated. Macrolide- and tetracycline-resistant strains were prevalent. All strains were susceptible to oxacillin, cefoxitin and trimethoprim-sulfamethoxazole. PCR screening for genes encoding enterotoxins detected several virulence patterns, which, together with s pa-typing and agr-locus typing, allowed cluster analysis and the description of novel clones. Conclusion. Continuous monitoring of staphylococci is needed also in rural or natural settings. Increasing the number of samples and expanding the geographical region will be needed to further extend the significance of the study.