The changing epidemiology of carbapenemase-producing Klebsiella pneumoniae in Italy: toward polyclonal evolution with emergence of high-risk lineages.

BACKGROUND: Previous studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates. METHODS: From March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome. RESULTS: Twenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395. CONCLUSIONS: This WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.

Infections in liver and lung transplant recipients: a national prospective cohort.

Infections are a major complication of solid organ transplants (SOTs). This study aimed to describe recipients' characteristics, and the frequency and etiology of infections and transplant outcome in liver and lung SOTs, and to investigate exposures associated to infection and death in liver transplant recipients. The study population included recipients of SOTs performed in Italy during a 1-year period in ten Italian lung transplant units and eight liver transplant units. Data on comorbidities, infections, retransplantation, and death were prospectively collected using a web-based system, with a 6-month follow-up. The cumulative incidence of infection was 31.7% and 47.8% in liver and lung transplants, respectively, with most infections occurring within the first month after transplantation. Gram-negatives, which were primarily multidrug-resistant, were the most frequent cause of infection. Death rates were 0.42 per 1000 recipient-days in liver transplants and 1.41 per 1000 recipient-days in lung transplants. Infection after SOT in adult liver recipients is associated to an increased risk of death (OR = 13.25; p-value < 0.001). Given the frequency of infection caused by multidrug-resistant microorganisms in SOT recipients in Italy and the heavy impact of infections on the transplant outcome, the reinforcement of surveillance and control activities to prevent the transmission of multidrug-resistant microorganisms in SOT recipients represents a priority. The implementation of the study protocol in liver and lung transplant units and the sharing of results have increased the awareness about the threat due to antimicrobial resistance in the country.

Respiratory diphtheria due to Corynebacterium ulcerans transmitted by a companion dog, Italy 2014.

A serious respiratory tract infection due to Corynebacterium ulcerans was observed in a 70-year-old woman. Clinical presentation included pseudomembranes in the upper respiratory tract and lung involvement. C. ulcerans was recovered from the nose of the patient's dog. Both dog's and patient's isolates belonged to Sequence Type 331.

Detection of KPC-216, a Novel KPC-3 Variant, in a Clinical Isolate of Klebsiella pneumoniae ST101 Co-Resistant to Ceftazidime-Avibactam and Cefiderocol.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CP-KP) represents a global threat to public health, with limited antimicrobial therapeutic options. In this study, we analyzed a ceftazidime/avibactam (CAZ-AVI)-resistant K. pneumoniae isolate obtained from a patient previously exposed to CAZ-AVI expressing a novel K. pneumoniae carbapenemase (KPC)-3 variant. METHODS: Antimicrobial susceptibility testing was performed using reference broth microdilution. Whole-genome sequencing (WGS) was performed using Illumina and Nanopore Technologies. Short- and long-reads were combined with Unicycler. Assemblies were investigated for multilocus sequence typing (MLST), antimicrobial resistance genes, porins, and plasmids. RESULTS: The K. pneumoniae isolate (KP_RM_1) was resistant to CAZ-AVI, expanded-spectrum cephalosporins, amikacin, ertapenem, and cefiderocol (FDC) but was susceptible to tigecycline, colistin, trimethoprim/sulfamethoxazole, meropenem-vaborbactam, and imipenem-relebactam. WGS revealed that the KP_RM_1 genome is composed of a single chromosome of 5 Mbp and five circular plasmids. Further analysis showed the presence of novel blaKPC-216 located on a 72 kb plasmid. KPC-216 differs from KPC-3 by a Lysin (K) insertion at position 168 (+K168). CONCLUSIONS: We report the identification of a new KPC-3 variant associated with CAZ-AVI resistance. The KPC variants associated with CAZ-AVI resistance should be determined to promptly inform clinicians and start the appropriate antimicrobial therapy.

Whole-Genome Sequencing and Molecular Analysis of Ceftazidime-Avibactam-Resistant KPC-Producing Klebsiella pneumoniae from Intestinal Colonization in Elderly Patients.

Ceftazidime-avibactam (CAZ-AVI) is an active antibiotic combination of a ß-lactam-ß-lactamase inhibitor against carbapenemase-producing Enterobacterales. Reports of resistance to CAZ-AVI other than metallo-ß-lactamases have increased in recent years. The aim of this study was to analyze KPC-Klebsiella pneumoniae (KP) isolates resistant to CAZ-AVI from the intestinal carriage of hospitalized elderly patients in Italy, in February 2018-January 2020. Characterization of CAZ-AVI-resistant KP isolates, including MLST, resistome, virulome and plasmid content, was performed by WGS analysis. Out of six CAZ-AVI-resistant KP isolates, three belonged to ST101 and three to ST512; two isolates produced KPC-3 (both ST512), four had mutated KPC-3 (KPC-31, in ST101 and ST512, and KPC-46, both ST101). All CAZ-AVI-resistant KP isolates were multidrug-resistant and carried several resistance genes. The yersiniabactin ybt9 gene cluster was present in all ST101 isolates, while, in ST512 isolates, no virulence genes were detected. Several plasmids were detected: IncF was present in all isolates, as well as IncR and Col440 in ST101 and IncX3 in ST512 isolates. In conclusion, it is important to monitor the circulation of K. pneumoniae resistant to CAZ-AVI to prevent the spread of clones causing difficult-to-treat infections. The presence of mutated KPC-3 in high-risk K. pneumoniae clones resistant to CAZ-AVI in hospitalized patients deserves attention.

Multicenter study on the prevalence of colonization due to carbapenem-resistant Enterobacterales strains before and during the first year of COVID-19, Italy 2018-2020.

Background: Among multidrug-resistant (MDR) bacteria able to threaten human health, carbapenem-resistant Enterobacterales (CRE) have become a major public health threat globally. National and international guidelines point out the importance of active routine surveillance policies to prevent CRE transmission. Therefore, defining lines of intervention and strategies capable of containing and controlling the spread of CRE is considered determinant. CRE screening is one of the main actions to curb transmission and control outbreaks, outlining the presence and also the prevalence and types of carbapenemase enzymes circulating locally. Objective: The purpose of this study was to outline the epidemiology of CRE colonization in Italy, detecting CRE-colonized patients at admission and during hospitalization, before and during the first year of COVID-19. Materials and methods: A total of 11,063 patients admitted to seven different hospitals (Bologna, Catania, Florence, Genoa, Naples, Palermo, and Turin) in Intensive Care Units (ICU) and other wards (non-ICU) located in the North, Center, and South of Italy were enrolled and screened for CRE carriage at admission (T0) and during the first 3 weeks of hospitalization (T1-T3). The study spanned two periods, before (September 2018-Septemeber 2019, I observational period) and during the COVID-19 pandemic (October 2019-September 2020, II observational period). Results: Overall, the prevalence of CRE-colonized patients at admission in ICU or in other ward, ranged from 3.9 to 11.5%, while a percentage from 5.1 to 15.5% of patients acquired CRE during hospital stay. There were large differences between the I and II period of study according to the different geographical areas and enrolling centers. Overall, comparison of prevalence of CRE-positive patients showed a significant increased trend between I and II observational periods both in ICU and non-ICU wards, mostly in the Southern participating centers. KPC-producing Klebsiella pneumoniae was the most frequent CRE species-carbapenemase combination reported in this study. In particular, the presence of KPC-producing K. pneumoniae was reported in period I during hospitalization in all the CRE-positive patients enrolled in ICU in Turin (North Italy), while in period II at admission in all the CRE-positive patients enrolled in ICU in Catania and in 58.3% of non-ICU CRE-positive patients in Naples (both centers in South Italy). Conclusion: The prevalence of CRE in Italy highly increased during the COVID-19 pandemic, mostly in the Southern hospital centers. KPC-producing K. pneumoniae was the most frequent colonizing CRE species reported. The results of our study confirmed the crucial value of active surveillance as well as the importance of multicenter studies representing diverse geographical areas even in endemic countries. Differences in CRE colonization prevalence among centers suggest the need for diversified and center-specific interventions as well as for strengthening efforts in infection prevention and control practices and policies.

Dynamics of carbapenemase-producing Enterobacterales intestinal colonisation in the elderly population after hospital discharge, Italy, 2018-2020.

Carbapenemase-producing Enterobacterales (CPE) represent a serious threat to public health worldwide. Elderly patients are at increased risk of colonisation/infection with CPE. This study aimed to evaluate the persistence of CPE colonisation and the genotypic characteristics of persistent strains in elderly people discharged from Italian hospitals. A longitudinal study was conducted in two Italian cities (March 2018 to September 2020) enrolling 137 patients aged ≥65 years with CPE intestinal colonisation at hospital discharge. CPE colonisation was evaluated after 4, 8 and 12 months. Competing risk analysis was used to explore the association between baseline characteristics and persistence at 4 months. For all isolates, carbapenemase typing and multilocus sequence typing were performed. Persistent isolates underwent whole-genome sequencing. Of 137 patients, 91% carried carbapenemase-producing Klebsiella pneumoniae (CP-KP) and 8.8% carried carbapenemase-producing Escherichia coli. Although a large number of patients were lost to follow-up owing to death or withdrawal, 28/65 patients (43.1%) remained colonised at Month 4; 16/42 (38.1%) and 5/28 (17.9%) were found colonised up to Months 8 and 12, respectively. Colonisation persistence was more frequent in patients with bacteraemia or complicated urinary tract infection while in hospital and in those staying in long-term care facilities (LTCFs). Clonal characteristics of CP-KP isolates did not appear to influence persistence. Isolates obtained from each persistent carrier were identical or highly related by SNP phylogenetic analysis. Identification of patients at higher risk of persistent intestinal carriage after hospital discharge can prompt control measures to limit the transmission of CPE in the community, especially in LTCF settings.

Case Report: First Report of Fatal Legionella pneumophila and Klebsiella pneumoniae Coinfection in a Kidney Transplant Recipient.

A very rare case of pulmonary Klebsiella pneumoniae-Legionella pneumophila coinfection in a double kidney transplanted man affected by the chronic renal disease is described. Cases of Legionnaires' disease with an incubation period of 14 days have rarely been documented. Despite the long period of hospitalization, typing of clinical and environmental L. pneumophila strains demonstrated that the patient's home water distribution system was the source of infection, highlighting that Legionella house contamination can be a hidden risk, especially for immune-compromised people.

Whole Genome Sequencing and Molecular Analysis of Carbapenemase-Producing Escherichia coli from Intestinal Carriage in Elderly Inpatients.

The spread of carbapenemase-producing (CP) Enterobacterales is currently a worldwide concern, especially in the elderly. Twelve CP-E. coli isolated from rectal swabs of colonized inpatients aged ≥65 years from four hospitals in two Italian cities (Milan and Rome) were analyzed by whole genome sequencing (WGS) to obtain multi-locus sequence typing (MLST), identification of carbapenemase-encoding genes, resistome, plasmid content, and virulence genes. MLST analysis showed the presence of 10 unrelated lineages: ST410 (three isolates from three different hospitals in two cities) and ST12, ST38, ST69, ST95, ST131, ST189, ST648, ST1288, and ST1598 (one isolate each). Most isolates (9/12, 75%) contained a serine-ß-lactamase gene (5 blaKPC-3, 2 blaKPC-2, and 2 blaOXA-181), while three isolates harbored a metallo-ß-lactamase gene (two blaNDM-5 and one blaVIM-1). In most CP-E. coli, the presence of more than one plasmid was observed, with the predominance of IncF. Several virulence genes were detected. All isolates contained genes enhancing the bacterial fitness, such as gad and terC, and all isolates but one, fimH, encoding type 1 fimbriae. In conclusion, CP-E. coli clones colonizing elderly patients showed heterogeneous genetic backgrounds. We recommend strict surveillance to monitor and prevent the spread of successful, high-risk clones in healthcare settings.

Laboratory management for SARS-CoV-2 detection: a user-friendly combination of the heat treatment approach and rt-Real-time PCR testing.

The RNA purification is the gold standard for the detection of SARS-CoV-2 in swab samples, but it is dependent on the availability of chemical reagents. In this study, we evaluated the heat treatment method without RNA extraction as a reliable option to nucleic acid purification.

Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread.

Public health interventions to control the current epidemic of carbapenem-resistant Klebsiella pneumoniae rely on a comprehensive understanding of its emergence and spread over a wide range of geographical scales. We analysed the genome sequences and epidemiological data of >1,700 K. pneumoniae samples isolated from patients in 244 hospitals in 32 countries during the European Survey of Carbapenemase-Producing Enterobacteriaceae. We demonstrate that carbapenemase acquisition is the main cause of carbapenem resistance and that it occurred across diverse phylogenetic backgrounds. However, 477 of 682 (69.9%) carbapenemase-positive isolates are concentrated in four clonal lineages, sequence types 11, 15, 101, 258/512 and their derivatives. Combined analysis of the genetic and geographic distances between isolates with different ß-lactam resistance determinants suggests that the propensity of K. pneumoniae to spread in hospital environments correlates with the degree of resistance and that carbapenemase-positive isolates have the highest transmissibility. Indeed, we found that over half of the hospitals that contributed carbapenemase-positive isolates probably experienced within-hospital transmission, and interhospital spread is far more frequent within, rather than between, countries. Finally, we propose a value of 21 for the number of single nucleotide polymorphisms that optimizes the discrimination of hospital clusters and detail the international spread of the successful epidemic lineage, ST258/512.

Daptomycin Resistant Staphylococcus aureus Clinical Strain With Novel Non-synonymous Mutations in the mprF and vraS Genes: A New Insight Into Daptomycin Resistance.

Objectives: Daptomycin (DAP) resistance in Staphylococcus aureus is uncommon but there are increasing reports of the emergence of resistance during DAP therapy. Most clinical DAP-resistant S. aureus isolates investigated carried mutations in the mprF gene. The aim of this study was to identify mutations between a clinical pair of methicillin-susceptible S. aureus (MSSA) isolates (DAP-susceptible and DAP-resistant). Additionally, the activity of genes previously associated with DAP resistance was assessed. Materials and Methods: Two MSSA isolates from patient with left-sided endocarditis were analyzed by whole genome sequencing (WGS) and reverse transcription-quantitative real-time PCR (RT-qPCR). The first isolate, DAP-susceptible, was obtained before initiation of treatment and the second isolate, DAP-resistant, was recovered after 4 weeks of DAP therapy. Results: Comparison of complete genomes of DAP-susceptible and its DAP-resistant variant identified two non-synonymous and one synonymous mutations. The non-synonymous mutations consisted of a S829L substitution in mprF and a T331I substitution in vraS. The RT-qPCR experiments revealed an increased expression of vraS, dltA, mprF, and sceD genes in DAP-resistant variant. Strikingly, the expression of dltA and mprF genes was significantly downregulated by DAP. Conclusion: The mprF and vraS genes were previously associated with DAP resistance, however, none of the mutations described in this study had been previously identified and linked to DAP resistance. Moreover, we provide a new insight into the DAP action on S. aureus, in which the expression of key genes in DAP resistance is decreased by the antibiotic.

Emergence of NDM-5-producing Escherichia coli sequence type 167 clone in Italy.

The emergence of carbapenemase-producing Enterobacteriaceae (CPE) is a critical concern worldwide. In Italy, CPE isolates are very frequent, with the KPC enzyme types strongly predominant whereas the New Delhi metallo-ß-lactamase (NDM) enzymes are extremely rare. Here we report the first detection of NDM-5-producing Escherichia coli sequence type 167 (ST167) isolates from two patients with urinary tract infection (Ec001 and Ec002 from urines), including one with colonisation (Ec003 from faeces) admitted to the same hospital 2 months apart in 2017. Minimum inhibitory concentrations (MICs) were determined by broth microdilution. The carbapenemase type was identified both by phenotypic and genotypic methods. Isolate genotypes were investigated by phylogenetic typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Next-generation sequencing (NGS) was used to obtain complete sequences of plasmids. The three E. coli isolates carried the blaNDM-5 gene, shared the same resistance phenotype and belonged to ST167. By PFGE, isolates showed the same profile, suggesting that they were the same strain. NGS revealed that the blaNDM-5 gene was located on a 99-kb multireplicon plasmid (designed pNDM-5-IT) with a peculiar scaffold constituted by four replicons of the IncF type (FIA, FIB and two copies of the FII replicon). pNDM-5-IT plasmid harboured multiple resistance and virulence determinants, including the arginine deaminase (ADI) cluster never found associated with plasmids before. Since NDM-5-producing E. coli ST167 has been regarded as a successful epidemic clone in China, the emergence of such a clone carrying a plasmid associated both with multiresistance and virulence could be a public-health threat.