Risk factors for colonization with multidrug-resistant Gram-negative bacteria and Clostridioides difficile in Long Term Care Facilities (LTCFs) residents: the evidence from 27 facilities in a high endemic setting.

Introduction: Residency in LTCFs increases the likelihood of colonization with multidrug resistant Gram-negative bacteria (MDR-GNB). We assessed the prevalence and risk factors for enteric colonization by III-generation cephalosporins-resistant and carbapenem-resistant (CR) GNB in a large group of LTCFs in a high endemic setting. We also assessed the prevalence and risk factors for C. difficile colonization. Methods: A point prevalence survey with rectal screening (RS) was conducted in 27 LTCFs in north Italy. Epidemiological and clinical variables on the survey day, history of hospitalization and surgery within one year, and antibiotics within three months, were collected. The presence of III-generation cephalosporin resistant and CR GNB was assessed using a selective culture on chromogenic medium and PCR for carbapenemase detection. The presence of C. difficile was assessed using ELISA for GDH and RT-PCR to identify toxigenic strains. Multi-variable analyses were performed using two-level logistic regression models. Results: In the study period 1947 RSs were performed. The prevalence of colonization by at least one GNB resistant to III-generation cephalosporin was 51% (E. coli 65%, K. pneumoniae 14% of isolates). The prevalence of colonization by CR GNB was 6%. 6% of all isolates (1150 strains) resulted in a carbapenem-resistant K. pneumoniae, and 3% in a carbapenem-resistant E. coli. KPC was the most frequent carbapenemase (73%) identified by PCR, followed by VIM (23%). The prevalence of colonization by C. difficile was 11.7%. The presence of a medical device (OR 2.67) and previous antibiotic use (OR 1.48) were significantly associated with III-generation cephalosporin resistant GNB colonization. The presence of a medical device (OR 2.67) and previous hospitalization (OR 1.80) were significantly associated with CR GNB. The presence of a medical device (OR 2.30) was significantly associated with C. difficile colonization. Main previously used antibiotic classes were fluoroquinolones (32% of previously treated subjects), III-generation cephalosporins (21%), and penicillins (19%). Conclusion: Antimicrobial stewardship in LTCFs is a critical issue, being previous antibiotic treatment a risk factor for colonization by MDR-GNB. The prevalence of colonization by III-generation cephalosporin and CR GNB among LTCF residents also underlines the importance to adhere to hand hygiene indications, infection prevention and control measures, and environmental hygiene protocols, more achievable than rigorous contact precautions in this type of social setting.

Resistome and Virulome of Multi-Drug Resistant E. coli ST131 Isolated from Residents of Long-Term Care Facilities in the Northern Italian Region.

Long-term care facilities (LTCFs) are important reservoirs of antimicrobial-resistant (AMR) bacteria which colonize patients transferred from the hospital, or they may emerge in the facility as a result of mutation or gene transfer. In the present study, we characterized, from a molecular point of view, 43 E. coli strains collected from residents of LTCFs in Northern Italy. The most common lineage found was ST131, followed by sporadic presence of ST12, ST69, ST48, ST95, ST410 and ST1193. All strains were incubators of several virulence factors, with iss, sat, iha and senB being found in 84%, 72%, 63% and 51% of E. coli, respectively. Thirty of the ST131 analyzed were of the O25b:H4 serotype and H30 subclone. The ST131 isolates were found to be mainly associated with IncF plasmids, CTX-M-1, CTX-M-3, CTX-M-15, CTX-M-27 and gyrA/parC/parE mutations. Metallo-ß-lactamases were not found in ST131, whereas KPC-3 carbapenemase was found only in two ST131 and one ST1193. In conclusion, we confirmed the spread of extended-spectrum ß-lactamase genes in E. coli ST131 isolated from colonized residents living inside LTCFs. The ST131 represents an incubator of fluoroquinolones, aminoglycosides and other antibiotic resistance genes in addition to different virulence factors.

Whole-Genome Sequencing (WGS) of Carbapenem-Resistant K. pneumoniae Isolated in Long-Term Care Facilities in the Northern Italian Region.

K. pneumoniae (KPN) is one of the widest spread bacteria in which combined resistance to several antimicrobial groups is frequent. The most common ß-lactamases found in K. pneumoniae are class A carbapenemases, both chromosomal-encoded (i.e., NMCA, IMI-1) and plasmid-encoded (i.e., GES-enzymes, IMI-2), VIM, IMP, NDM, OXA-48, and extended-spectrum ß-lactamases (ESBLs) such as CTX-M enzymes. In the present study, a total of 68 carbapenem-resistant KPN were collected from twelve long-term care facilities (LTCFs) in the Northern Italian region. The whole-genome sequencing (WGS) of each KPN strain was determined using a MiSeq Illumina sequencing platform and analysed by a bacterial analysis pipeline (BAP) tool. The WGS analysis showed the prevalence of ST307, ST512, and ST37 as major lineages diffused among the twelve LTCFs. The other lineages found were: ST11, ST16, ST35, ST253, ST273, ST321, ST416, ST1519, ST2623, and ST3227. The blaKPC-2, blaKPC-3, blaKPC-9, blaSHV-11, blaSHV-28, blaCTX-M-15, blaOXA-1, blaOXA-9, blaOXA-23, qnrS1, qnrB19, qnrB66, aac(6')-Ib-cr, and fosA were the resistance genes widespread in most LTCFs. In this study, we demonstrated the spreading of thirteen KPN lineages among the LTCFs. Additionally, KPC carbapenemases are the most widespread ß-lactamase.

Molecular characterization of carbapenem-resistant Klebsiella pneumoniae ST14 and ST512 causing bloodstream infections in ICU and surgery wards of a tertiary university hospital of Verona (northern Italy): co-production of KPC-3, OXA-48, and CTX-M-15 ß-lactamases.

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae, isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-ß-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that blaKPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas blaCTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.

Outbreak of Saprochaete clavata Sepsis in Hematology Patients: Combined Use of MALDI-TOF and Sequencing Strategy to Identify and Correlate the Episodes.

INTRODUCTION: New fungal species are increasingly reported in immunocompromised patients. Saprochaete clavata (S. clavata), an ascomycetous fungus formerly called Geotrichum clavatum, is intrinsically resistant to echinocandins and is often misidentified. OBJECTIVE: We describe a cluster of seven S. clavata infections in hospitalized hematology patients who developed this rare fungemia within a span of 11 months. Three of the seven patients died. Identification of the isolates was determined only with the Saramis database of VitekMS system and sequencing of the internal transcribed spacer (ITS) region. Clonal relatedness of the isolates was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) analysis; clonal correlation between the strains was investigated by means of phylogenetic analysis, based on single-nucleotide variants (SNPs). Clinical presentation, 1-3 ß-D-glucan (BG) and galactomannan (GM) antigen results and analysis of possible sources of contamination are also described with a prospective case-control study of the outbreak. RESULTS: MALDI-TOF MS-Vitek (bioMerieux, Marcy l'Etoile, France) failed to identify the six isolates, while SARAMIS (bioMerieux, Marcy l'Etoile, France) identified the isolates as S. clavata. Initially, Vitek 2 identified the strains as Geotrichum capitatum in two of the seven cases. Molecular identification gave 99% homology with S. clavata. BG was positive in three out of six patients (range 159 to >523 pg/ml), GM results were always negative. All the isolates were resistant to echinocandins (anidulafungin, micafungin, and caspofungin) and Fluconazole, but susceptible to Flucytosine and Voriconazole. One isolate showed acquired resistance to Flucytosine and Amphotericin B during treatment. Both the correlation-based dendrograms obtained by MALDI-TOF MS (Bruker Daltonics) and MS-Vitek not only clustered six of the seven bloodstream infection (BSI) isolates in the same group, but also showed their strong relatedness. Phylogenetic analysis using SNPrelate showed that the seven samples recorded during the investigation period clustered together. We observed a split between one case and the remainder with a node supported by a z-score of 2.3 (p-value = 0.021) and 16 mutations unique to each branch. CONCLUSION: The use of proteomics for identification and evaluation of strain clonality in outbreaks of rare pathogens is a promising alternative to laborious and time-consuming molecular methods, even if molecular whole-genome sequencing (WGS) typing will still remain the reference method for rare emergent pathogens.