The study evaluated the degree of colonization with drug-resistant bacteria among residents of LTCFs located in three Northern Italian Provinces, finding high colonization of residents in Milan (75.0%), Piacenza (69.4%) and Bolzano (66.1%). Many residents had more than one target organism, underscoring the role of LTCFs as a reservoir for these isolates [2,3,4].
Colonization of LTCF residents with ESβL-producing enterobacteria was highly prevalent in all the surveyed LTCFs (60.5% in Milan, 66.1% in Piacenza and 53.0% in Bolzano), and group-1 CTX-M-type enzymes were highly predominant, especially in E. coli (80–97% of isolates). Notably, about 82% of K. pneumoniae and 32% of P. mirabilis isolates also harbored a blaCTX-M-type gene. In the same Bolzano LTCF, here screened for ESβL-producing enterobacteria, high colonization percentages, equal to 64.0 and 49.0%, were previously found in 2008  and 2012 , respectively; the latter survey also screened a second LTCF in the Province of Bolzano, showing a colonization prevalence of 56.0%. In an Italian study carried out in 2006, a colonization prevalence of 54.0% was found in LTCF residents bearing a urinary catheter , while a more recent multicenter study, performed in 2015 and involving 12 Italian LTCFs, reported a mean ESβL colonization of 57.3% (range: 32.8–81.5%) . In all these Italian studies, CTX-M enzymes were the predominantly produced ESβLs. The high ESβL colonization rates of > 50% in Italian LTCF residents are paralleled by high ESβL prevalence in invasive E. coli isolates . Generally, ESβL carriage in most European countries is strikingly lower than that found in Italy , with exceptions reported from Ireland [27, 28] and Portugal .
In our screening study, high-level AmpC-producing Enterobacteriaceae were rarely isolated in LTCF residents in Milan and Piacenza, but 24.3% of LTCF residents in Bolzano were colonized by M. morganii expressing a high-level DHA-AmpC phenotype; blaDHA-type genes in LTCF isolates have previously been found in a few E. coli and K. pneumoniae strains from Korea , but to our knowledge have not yet been reported in Italian LTCFs.
Carbapenemase-producing enterobacteria were not found in LTCF residents in Piacenza, rarely in Bolzano (1.6%) and more frequently in Milan (7.6%). As found in previous studies of carbapenemase-producing Enterobacteriaceae from Bolzano [22, 23, 31], the VIM-1-producing E. coli and C. amalonaticus isolates from residents in this study were also positive for blaSHV-12. In the present study, all carbapenemase producers from Milan, except an E. cloacae complex isolate expressing a blaVIM-1 gene, had KPC-type enzymes; similar results have been reported by other Italian studies in LTCF residents [25, 32, 33]. Carbapenemase-producing enterobacteria, especially KPC-producing K. pneumoniae, are epidemically spread in Italy  and the emergence of this MDR phenotype in LTCFs is worrying, expanding the reservoir of this health care threat. Nevertheless, as previously summarized , carbapenemase-producing Enterobacteriaceae are still rare in Italian LTCF residents; the reasons are probably multifactorial, comprising clinical characteristics of the enrolled residents  and the low carbapenem selective pressure in LTCFs. On average, only 1.1% of residents enrolled in our screening study received carbapenems within the previous 3 months (data not shown). Nevertheless, a carbapenemase-producing enterobacteria prevalence of 7.6% (mainly KPC-producing K. pneumoniae), reported here for the LTCF in Milan, gives rise to concern and has to be addressed by future hygiene and antibiotic stewardship measures.
This study shows the emergence of carbapenemase-producing P. aeruginosa in LTCF residents in Piacenza, identifying single isolates with blaVIM-type and blaGES-5 determinants. P. aeruginosa expressing blaVIM-type determinants is widely spread in Italy , and an outbreak of GES-5-producing P. aeruginosa was reported from a LTCF in Japan . Moreover, the ESβL genes blaGES-1 and blaBEL-like were found in two and one P. aeruginosa isolates, respectively; the latter rarely detected β-lactamase was previously recovered in P. aeruginosa strains from Belgium . A. baumannii producing OXA-23 carbapenemases have an epidemic diffusion in Italy , reflected in the present study by the isolation of this resistance type from LTCF residents in Milan (1.9%) and Piacenza (5.8%).
MRSA colonization prevalence here reported ranged widely in the surveyed LTCFs (5.7, 14.8 and 24.0% in Milan, Piacenza and Bolzano, respectively), similar to other Italian studies [25, 39, 40]. Varying MRSA colonization prevalence, ranging from close to zero up to levels higher than 37%, has been reported in European studies .
Colonization by VRE in the present study was highly variable, ranging from 0.8 to 20.2%. VRE-carriage in European LTCF residents was found to be low, ranging from 0.0–3% [28, 41, 42].
For Enterobacteriaceae significant differences in colonization frequencies of LTCF residents were found: i) for CTX-M-type ESβL-producing E. coli between Piacenza (highest prevalence) and Bolzano, ii) for high-level AmpC-producing M. morganii (highest prevalence in Bolzano), iii) for carbapenemase producers, with highest prevalence in Milan, iv) for carbapenemase-producing A. baumannii, showing highest prevalence in Piacenza, and v) for MRSA and VRE, most prevalent in Milan. Therefore, no clear picture of general colonization differences can be deduced from overall colonization prevalence data.
A variety of risk factors for MRSA and ESβL colonization have previously been reported ; many of these have also been analyzed in the present survey. Interestingly, male residents carried a more than double risk for MRSA carriage when compared with female residents, probably because of the higher frequencies of other risk factors in males (administration of any antibiotic within the previous 3 months, hospitalization within the previous 12 months and coma), predisposing men rather than women to MRSA acquisition. Moreover, in our study the trend for an inverse correlation (p = 0.09) between age > 85 years and MRSA prevalence was associated with a significantly lower percentage of male residents > 85 years, compared to females; similar results have been found by other authors . In the present survey, administration of cephalosporins during the previous 3 months resulted to be an independent risk factor for ESβL colonization; the LTCFs in Piacenza registered the highest consumption of cephalosporins, correlating with highest ESβL prevalence in LTCF residents from Piacenza. Other independent risk factors for ESβL colonization were physical disability, the presence of any invasive medical device and cancer. Whereas no significant differences were found between residents in the three Provinces for cancer as risk factor, physical disability and the presence of any medical device showed highest prevalence in the LTCF in Bolzano; nonetheless, LTCF residents in Bolzano had the lowest ESβL prevalence in the present screening study.
Therefore, further factors may have contributed to the observed differences, comprising staff/resident ratio and practiced hygiene and infection control measures . The LTCF in Bolzano showed the highest staff/resident ratio, and understaffing has been shown to be a risk factor for colonization of LTCF residents by MDR organisms . All of the surveyed LTCFs in the present study follow hygiene, infection prevention and control measures according to guidelines of The Society for Healthcare Epidemiology of America (SHEA) and The Association for Professionals in Infection Control and Epidemiology (APIC) . Nonetheless, the Bolzano LTCF had introduced enforced hygiene measures, according to the World Health Organization guidelines , after the 2008 screening study, showing an ESβL colonization prevalence of 64.0% in LTCF residents ; colonization frequency decreased significantly to 49.0% (p = 0.02) in 2012 , arriving at a slightly higher percentage of 53.0% in 2016, but other factors such as changed case mixes and risk factors may also have contributed to this decrease in ESβL prevalence .
Significant differences in antibiotic resistance epidemiology of blood culture isolates, used as a proxy for the general local antibiotic resistance epidemiology, were registered, as derived from European Antimicrobial Resistance Surveillance Network (EARS-Net) data for 2016 . Specifically, we found the following antibiotic resistance data referred to the geographic regions of Milan, Piacenza and Bolzano, respectively: E. coli third generation cephalosporin-resistant: 22.1% (29/131), 29.4% (71/259) and 17.8% (56/314); K. pneumoniae carbapenem-resistant: 29.2% (7/24), 13.5% (10/74) and 6.2% (4/64); A. baumannii carbapenem-resistant: 50.0% (1/2,) 100.0% (24/24) and 0.0% (0/2); MRSA: 36.0% (18/50), 49.7% (82/165) and 14.6% (20/137); E. faecalis VRE: 0.0% (0/20), 2.4% (2/83) and 0.0% (0/41); E. faecium VRE: 10.0% (1/10), 22.2% (6/27) and 8.0% (2/25). This data for blood culture isolates, compared with our LTCF screening data, correlates well for ESβL-producing E. coli, carbapenem-resistant K. pneumoniae and A. baumannii; on the other hand, no correlation for MRSA and VRE can be derived. Patient transfer between acute-care facilities and LTCFs contribute to the diffusion of MDR organisms in both settings; such bi-directional movement of MDR bacteria, related to acute systemic infections, might be more significant for Enterobacteriaceae and A. baumannii than for MRSA and VRE.
Moreover, the snapshot approach used in this study might lead to the sudden increase in prevalence of a specific resistance phenotype, as shown for high-level AmpC-producing M. morganii detected in 2016 from Bolzano LTCF residents , which could be a transient phenomenon. Similarly, the high prevalence of VRE in LTCF residents from Milan could be due to a transitory local epidemic event.
Finally, the local circulation of highly transmissible clones, for example ESβL-producing E. coli, KPC-producing K. pneumoniae and OXA-23-producing A. baumannii could contribute to the explanation of the here reported screening results [38, 47].
This study has some limitations. First, it has been done in only four LTCFs, located in three different Provinces in Northern Italy, and therefore data may not be extrapolated to other Italian LTCFs with differing characteristics. Second, the number of LTCF residents participating in the study was variable, ranging from 34% in Milan up to 100% in Bolzano. Third, we did not use an enrichment step during the laboratory analysis; this limitation is partially compensated by using 4–5 different specimen types for the screening of MDR bacteria. Fourth, different sample types, types of media and laboratory methodologies have been used in the three laboratories processing the samples from the different LTCFs. Fifth, molecular characterization and typing of isolates in the 2016 study was limited, not including pulsed-field gel electrophoresis (PFGE) and sequence typing (ST) of isolates and therefore not permitting the identification of epidemic clusters. Finally, screening of healthcare workers has been done only in one of the enrolled LTCFs , but not in the other surveyed facilities. Despite these limitations, the strength of our study is the comparison of colonization prevalence between LTCFs located in three different Provinces, comparing it also with differences in risk factors for colonization and in the local epidemiology of invasive isolates.