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Background: VIM metallo-β-lactamases are enzymes characterized by the ability to hydrolyze all β-lactams. Usually, blaVIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM- 1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of blaVIM genes. Materials and methods: 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. Results: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the blaVIM-1 allele while six isolates carried the blaVIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The blaVIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the blaVIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two blaVIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. Conclusion: We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution. [ABSTRACT FROM AUTHOR]

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, qnr S, bla CTX-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 β-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 ESβL-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 bla CTX-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 bla VEB-1 and bla MOX-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 ESβL- E. coli. Two out of four E. coli detected as bla OXA-244-positive, belonged to the pandemic ST131. One XDR K. pneumoniae from a stream sample, detected as bla KPC-2 positive, resulted of ST258. The epidemiological impact of bla OXA-244 ST131 E. coli and bla KPC-2 ST258 K. pneumoniae presence in surface waters of an urban area in Northern Italy must not be underestimated. [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize four Enterobacterales co-producing NDM- and OXA-48-like carbapenemases from Czech patients with travel history or/and previous hospitalization abroad. Klebsiella pneumoniae isolates belonged to "high risk" clones ST147, ST11, and ST15, while the Escherichia coli isolate was assigned to ST167. All isolates expressed resistance against most β-lactams, including carbapenems, while retaining susceptibility to colistin. Furthermore, analysis of WGS data showed that all four isolates co-produced OXA-48- and NDM-type carbapenemases, in different combinations (Kpn47733: bla NDM–5 + bla OXA–181; Kpn50595: bla NDM–1 + bla OXA–181; Kpn51015: bla NDM–1 + bla OXA–244; Eco52418: bla NDM–5 + bla OXA–244). In Kpn51015, the bla OXA–244 was found on plasmid p51015_OXA-244, while the respective gene was localized in the chromosomal contig of E. coli Eco52418. On the other hand, bla OXA–181 was identified on a ColKP3 plasmid in isolate Kpn47733, while a bla OXA–181-carrying plasmid being an IncX3-ColKP3 fusion was identified in Kpn50595. The bla NDM–1 gene was found on two different plasmids, p51015_NDM-1 belonging to a novel IncH plasmid group and p51015_NDM-1 being an IncF K 1-FIB replicon. Furthermore, the bla NDM–5 was found in two IncFII plasmids exhibiting limited nucleotide similarity to each other. In both plasmids, the genetic environment of bla NDM–5 was identical. Finally, in all four carbapenemase-producing isolates, a diverse number of additional replicons, some of these associated with important resistance determinants, like bla CTX–M–15, arr-2 and ermB , were identified. In conclusion, this study reports the first description of OXA-244-producing Enterobacterales isolated from Czech hospitals. Additionally, our findings indicated the genetic plurality involved in the acquisition and dissemination of determinants encoding OXA/NDM carbapenemases. [ABSTRACT FROM AUTHOR]

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired β-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring bla TEM–1b, bla CTX–M–15, bla OXA–1, aph(6)-Ib , aac(6′)Ib-cr , aac(3)-Ila , aph(6)-Id , aadA1 , qnrB1 , sul2 , catA1 , catB3 , tetA , and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, bla DHA–1, qnrB4 , mph(A) , sul1 , and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A bla CMY–2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA , iss , astA , and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 bla CTX–M–15, bla CMY–2, bla DHA–1, and aac(6′)-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir. [ABSTRACT FROM AUTHOR]

KPC-producing Escherichia coli (KPC-Ec) remains uncommon, being mainly reported as the cause of sporadic episodes of infection rather than outbreak events. Here we retrospectively describe the dynamics of a large hospital outbreak sustained by KPC-Ec, involving 106 patients and 25 hospital wards, during a six-month period. Twenty-nine representative KPC-Ec isolates (8/29 from rectal swabs; 21/29 from other clinical specimens) have been investigated by Whole-Genome Sequencing (WGS). Outbreak isolates showed a multidrug-resistant profile and harbored several resistance determinants, including blaCTX-M-27, aadA5, dfrA17, sulI, gyrA1AB and parC1aAB. Phylogenomic analysis identified the ST131 cluster 1 (23/29 isolates), H30Rx clade C, as responsible for the epidemic event. A further two KPC-Ec ST131 clusters were identified: cluster 2 (n = 2/29) and cluster 3 (n = 1/29). The remaining KPC-Ec resulted in ST978 (n = 2/29) and ST1193 (n = 1/29), and were blaKPC-3 associated. The KPC-Ec ST131 cluster 1, originated in a previous KPC-Kp endemic context probably by plasmid transfer, and showed a clonal dissemination strategy. Transmission of the blaKPC gene to the globally disseminated high-risk ST131 clone represents a serious cause of concern. Application of WGS in outbreak investigations could be useful to better understand the evolution of epidemic events in order to address infection control and contrast interventions, especially when high-risk epidemic clones are involved. [ABSTRACT FROM AUTHOR]

Background: VIM (Verona Integron-encoded Metallo-beta-lactamase) is a member of the Metallo-Beta-Lactamases (MBLs), and is able to hydrolyze all beta-lactams antibiotics, except for monobactams, and including carbapenems. Here we characterize a VIM-producing IncA plasmid isolated from a clinical ST69 Escherichia coli strain from an Italian Long-Term Care Facility (LTCF) inpatient. Methods: An antimicrobial susceptibility test and conjugation assay were carried out, and the transferability of the blaVIM-type gene was confirmed in the transconjugant. Whole-genome sequencing (WGS) of the strain 550 was performed using the Sequel I platform. Genome assembly was performed using "Microbial Assembly". Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases. Results: Assembly resulted in three complete circular contigs: the chromosome (4,962,700 bp), an IncA plasmid (p550_IncA_VIM_1; 162,608 bp), harboring genes coding for aminoglycoside resistance (aac(6′)-Ib4, ant(3″)-Ia, aph(3″)-Ib, aph(3′)-XV, aph(6)-Id), beta-lactam resistance (blaSHV-12, blaVIM-1), macrolides resistance (mph(A)), phenicol resistance (catB2), quinolones resistance (qnrS1), sulphonamide resistance (sul1, sul2), and trimethoprim resistance (dfrA14), and an IncK/Z plasmid (p550_IncB_O_K_Z; 100,306 bp), free of antibiotic resistance genes. Conclusions: The increase in reports of IncA plasmids bearing different antimicrobial resistance genes highlights the overall important role of IncA plasmids in disseminating carbapenemase genes, with a preference for the blaVIM-1 gene in Italy. [ABSTRACT FROM AUTHOR]

Background: The spread of carbapenemase genes, such as blaNDM-1, in Proteus mirabilis poses a public health threat. The aim of the study was to characterize the genome and plasmids sequences of an NDM-1-positive strain (IBCRE14), which was isolated in 2019 from a catheterized patient hospitalized in Italy. Methods: Whole genome sequencing (WGS) of IBCRE14 was performed on extracted genomic DNA using Sequel I platform. Genome assembly was performed using "Microbial Assembly". Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases from the Center for Genomic Epidemiology. Results: IBCRE14 had a genome size of 4,018,329 bp and harboured genes coding for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ), and trimethoprim (dfrA1). A large plasmid (pIB_NDM_1) harboured antibiotic resistance genes against sulphonamide (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr-2), aminoglycosides (aadA1, aph3-VI), and beta-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) harboured a qnrD1 gene coding for quinolone resistance. Conclusion: The ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could both acquire more resistance genes and easily disseminate. To our knowledge, this is the first report on an untypable plasmid harbouring blaNDM-1 in P. mirabilis, in Italy. [ABSTRACT FROM AUTHOR]

We investigated an Italian OXA-181-producing Escherichia coli clinical isolate (ECS1_14) by whole-genome sequencing. The strain coharbored blaCTX-M-15, blaCMY-2, and qnrS1 genes; it belonged to ST410(Achtman)/ST692(Pasteur) and phylogroup A. The blaOXA-181 gene was harbored on a plasmid highly similar (99% identity) to the pOXA181_EC14828 plasmid, recently reported in China. [ABSTRACT FROM AUTHOR]

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales , but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii . The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3 , bla KPC-2, and bla VIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3 -harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales , affecting even low-virulence pathogens, as C. freundii ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Mattioni Marchetti, Venturelli, Cassetti, Meschiari, Migliavacca and Bitar.)

Fosfomycin-resistant FosA8-producing Enterobacterales are uncommon strains with extremely low incidence in Europe, based on only three reports in the literature. We detected FosA8-producing Escherichia coli ST131 in clinical isolates from two patients admitted in February 2023 to a rehabilitation unit in Italy. The occurrence of rare fosA -like genes in the high-risk clone ST131 is of clinical relevance. The dissemination of FosA-producing E. coli, although still at low levels, should be continuously monitored.

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.

Antimicrobial resistance is well-known to be a global health and development threat. Due to the decrease of effective antimicrobials, re-evaluation in clinical practice of old antibiotics, as fosfomycin (FOS), have been necessary. FOS is a phosphonic acid derivate that regained interest in clinical practice for the treatment of complicated infection by multi-drug resistant (MDR) bacteria. Globally, FOS resistant Gram-negative pathogens are raising, affecting the public health, and compromising the use of the antibiotic. In particular, the increased prevalence of FOS resistance (FOS R ) profiles among Enterobacterales family is concerning. Decrease in FOS effectiveness can be caused by i ) alteration of FOS influx inside bacterial cell or ii ) acquiring antimicrobial resistance genes. In this review, we investigate the main components implicated in FOS flow and report specific mutations that affect FOS influx inside bacterial cell and, thus, its effectiveness. FosA enzymes were identified in 1980 from Serratia marcescens but only in recent years the scientific community has started studying their spread. We summarize the global epidemiology of FosA/C2/L1-2 enzymes among Enterobacterales family. To date, 11 different variants of FosA have been reported globally. Among acquired mechanisms, FosA3 is the most spread variant in Enterobacterales , followed by FosA7 and FosA5. Based on recently published studies, we clarify and represent the molecular and genetic composition of fosA/C2 genes enviroment, analyzing the mechanisms by which such genes are slowly transmitting in emerging and high-risk clones, such as E. coli ST69 and ST131, and K. pneumoniae ST11. FOS is indicated as first line option against uncomplicated urinary tract infections and shows remarkable qualities in combination with other antibiotics. A rapid and accurate identification of FOS R type in Enterobacterales is difficult to achieve due to the lack of commercial phenotypic susceptibility tests and of rapid systems for MIC detection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mattioni Marchetti, Hrabak and Bitar.)

The aim of this study was to report the characterization of the first mcr -positive Enterobacterales isolated from Czech hospitals. In 2019, one Citrobacter freundii and four Enterobacter isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying mcr genes, the genomic DNAs of mcr -carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried bla VIM - and mcr -like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried mcr-9 -like alleles. Furthermore, the three sequence type 106 (ST106) Enterobacter hormaechei isolates harbored the bla VIM-1 gene, while the ST764 E. hormaechei and ST95 C. freundii included bla VIM-4 Analysis of plasmid sequences showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each mcr-9 -carrying IncHI2 plasmid. The bla VIM-4 gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, bla VIM-1 was localized on plasmids (∼55 kb) exhibiting repA -like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9 -positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the bla VIM-1 on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants. IMPORTANCE Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr , in different host species and geographic areas were published. Here, we report the first detection of Enterobacterales carrying mcr -9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored bla VIM-1 , while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included bla VIM-4 Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. bla VIM-4 was found in the MDR regions of IncHI2 plasmids, while bla VIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms., (Copyright © 2020 Bitar et al.)

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired β-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring bla TEM-1b , bla CTX-M-15 , bla OXA-1 , aph(6)-Ib , aac(6')Ib-cr , aac(3)-Ila , aph(6)-Id , aadA1 , qnrB1 , sul2 , catA1 , catB3 , tetA , and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, bla DHA-1 , qnrB4 , mph(A) , sul1 , and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A bla CMY-2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA , iss , astA , and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 bla CTX-M-15 , bla CMY-2 , bla DHA-1 , and aac(6')-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir., (Copyright © 2020 Mattioni Marchetti, Bitar, Mercato, Nucleo, Marchesini, Mancinelli, Prati, Scarsi, Hrabak, Pagani, Fabbi and Migliavacca.)

We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, bla KPC-2 was harbored on a novel variant of a Tn 4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, bla KPC-3 was on a Tn 4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying bla KPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria., (Copyright © 2019 American Society for Microbiology.)

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy.

In 2016, we undertook a point prevalence screening study for Enterobacteriaceae with extended-spectrum β-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 geriatric unit in Bolzano, Northern Italy. Urine samples and rectal, inguinal, oropharyngeal and nasal swabs were plated on selective agars. Demographic data were collected. ESBL and carbapenemase genes were sought by PCR. We found the following colonization percentages with multidrug-resistant (MDR) bacteria in 2016 in LTCF residents: all MDR organisms, 66.1%; ESBL producers, 53.0%; carbapenemase-producers, 1.7%; MRSA, 14.8%; VRE, 0.8%. Colonization by all MDR bacteria was 19.4% for LTCF staff and 26.0% for geriatric unit patients. PCR showed that 80.3% of Escherichia coli isolates from LTCF residents, all E. coli isolates from LTCF staff, 62.5% and 100% of Klebsiella pneumoniae from LTCF residents and geriatric unit patients, respectively, had a blaCTX-M-type gene. All carbapenemase-producing Enterobacteriaceae harboured a blaVIM-type gene. 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.