A multicentre evaluation of the NG-test DetecTool OXA-23 for the rapid detection of OXA-23 carbapenemase directly from blood cultures.

Objectives: A multicentre study evaluating NG-Test DetecTool OXA-23 for the detection of OXA-23 carbapenemase directly from positive blood cultures (PBCs). Methods: The NG-Test DetecTool OXA-23 is an immunoassay that integrates a sample preparation device. We evaluated NG-Test DetecTool OXA-23 on 189 spiked and 126 clinical PBCs. The clinical samples' standard-of-care procedure consisted of bacterial identification from the first day of positivity by MALDI-TOF MS, conventional culture and antimicrobial susceptibility testing. The immunoassay results were verified molecularly. The strains used for the spiked samples consisted of well-characterized Acinetobacter baumannii and Proteus mirabilis strains. Results: The NG-Test DetecTool OXA-23 was evaluated on 315 PBCs and revealed sensitivity of 100% (95% CI: 98.21%-100.00%) and specificity of 100% (95% CI: 96.73%-100.00%). It provided 204 true-positive results for OXA-23 in 196 bottles with carbapenem-resistant A. baumannii (CRAB) and 8 bottles with carbapenem-resistant P. mirabilis and also provided 111 true-negative results. There were no false-positive and no false-negative results. Among the 315 PBCs studied, 83 clinical blood cultures collected in the ICU of a Greek university hospital, which were tested prospectively, all yielded CRAB, and OXA-23 was correctly detected in all samples from the first day of positivity using the NG-Test DetecTool OXA-23. Conclusions: The NG-Test DetecTool OXA-23 has exhibited excellent sensitivity and specificity for OXA-23 detection in PBCs and can provide valuable information for appropriate selection of antibiotic therapy and early implementation of infection control measures.

Multicenter study to assess the use of BL-DetecTool for the detection of CTX-M-type ESBLs and carbapenemases directly from clinical specimens.

Antimicrobial resistance (AMR) is one of the major public health problems worldwide. Multiple strategies have been put in place to address this problem. One of them is the rapid detection of the mechanisms of resistance, such as extended-spectrum beta-lactamases (ESBLs) and/or carbapenemases. We conducted a multicenter study that included nine European centers for the assessment of prototypes of a novel lateral flow immunoassay-based device (BL-DetecTool) for a rapid detection of ESBL (NG-Test CTX-M-MULTI DetecTool) and/or carbapenemases (NG-Test CARBA 5 DetecTool) from Enterobacterales and Pseudomonas aeruginosa in positive urine, positive blood cultures, and rectal swabs. We performed a prospective analysis between January 2021 and June 2022, including overall 22,010 samples. Based on each hospital information, the sensitivity to detect CTX-M was 84%-100%, 90.9%-100%, and 75%-100% for urine, positive blood cultures, and enriched rectal swabs, respectively. On the other hand, the sensitivity to detect carbapenemases was 42.8%-100%, 75%-100%, and 66.6%-100% for urine, positive blood cultures, and enriched rectal swab, respectively. BL-DetecTool allows a rapid and reliable detection of ESBL and carbapenemases directly from urine, positive blood cultures, or enriched rectal swabs, being an easy technique to implement in the workflow of clinical microbiology laboratories. IMPORTANCE: The assessed rapid assay to detect CTX-M beta-lactamases and carbapenemases directly from clinical samples can favor in the rapid detection of these mechanisms of resistance and hence the administration of a more adequate antimicrobial treatment.

Citrobacter freundii resistant to novel ß-lactamase inhibitor combinations and cefiderocol, co-producing class A, B and D carbapenemases encoded by transferable plasmids.

OBJECTIVES: To characterize a carbapenem-resistant Citrobacter freundii (Cf-Emp) co-producing class A, B and D carbapenemases, resistant to novel ß-lactamase inhibitor combinations (BLICs) and cefiderocol. METHODS: Carbapenemase production was tested by an immunochromatography assay. Antibiotic susceptibility testing (AST) was performed by broth microdilution. WGS was performed using short- and long-read sequencing. Transfer of carbapenemase-encoding plasmids was assessed by conjugation experiments. RESULTS: Cf-Emp was isolated on selective medium for carbapenem-resistant Enterobacterales from the surveillance rectal swab taken at hospital admission from a patient of Moroccan origin. Cf-Emp produced three different carbapenemases, including KPC-2, OXA-181 and VIM-1, and was resistant to all ß-lactams including carbapenems, novel BLICs (ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam) and cefiderocol. MIC of aztreonam/avibactam was 0.25 mg/L. The strain belonged to ST22, one of the C. freundii lineages of global diffusion, known to be associated with carbapenemase production. Each carbapenemase gene was located aboard a different plasmid (named pCf-KPC, pCf-OXA and pCf-VIM, respectively), which also carried other clinically relevant resistance genes, such as armA (pCf-KPC), blaSHV-12 (pCf-VIM) and qnrS1 (pCf-OXA). Transferability to Escherichia coli J53 by conjugation was observed for all plasmids. CONCLUSIONS: The finding of enterobacterial strains carrying multiple carbapenemase genes on transferable plasmids is alarming, because similar strains could provide an important reservoir for disseminating these clinically relevant resistance determinants.

Seven Years of Culture Collection of Neisseria gonorrhoeae: Antimicrobial Resistance and Molecular Epidemiology.

The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobials, in particular to ceftriaxone monotherapy or ceftriaxone plus azithromycin, represents a global public health concern. This study aimed to analyze the trend of antimicrobial resistance in a 7-year isolate collection retrospective analysis in Italy. Molecular typing on a subsample of gonococci was also included. A total of 1,810 culture-positive gonorrhea cases, collected from 2013 to 2019, were investigated by antimicrobial susceptibility, using gradient diffusion method, and by the N. gonorrhoeae multiantigen sequence typing (NG-MAST). The majority of infections occurred among men with urogenital infections and 57.9% of male patients were men who have sex with men. Overall, the cefixime resistance remained stable during the time. An increase of azithromycin resistance was observed until 2018 (26.5%) with a slight decrease in the last year. In 2019, gonococci showing azithromycin minimum inhibitory concentration above the EUCAST epidemiological cutoff value (ECOFF) accounted for 9.9%. Ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae (PPNG) percentages increased reaching 79.1% and 18.7% in 2019, respectively. The most common sequence types identified were 5,441, 1,407, 6,360, and 5,624. The predominant genogroup (G) was the 1,407; moreover, a new genogroup G13070 was also detected. A variation in the antimicrobial resistance rates and high genetic variability were observed in this study. The main phenotypic and genotypic characteristics of N. gonorrhoeae isolates were described to monitor the spread of drug-resistant gonorrhea.

Virulence Determinants in Staphylococcus aureus Clones Causing Osteomyelitis in Italy.

Staphylococcus aureus is the most common pathogen causing osteomyelitis (OM). The aim of this study was to explore the clonal complex (CC) distribution and the pattern of virulence determinants of S. aureus isolates from OM in Italy. Whole-genome sequencing was performed on 83 S. aureus isolates from OM cases in six hospitals. Antibiotic susceptibility tests showed that 30.1% of the isolates were methicillin-resistant S. aureus (MRSA). The most frequent CCs detected were CC22, CC5, CC8, CC30, and CC15, which represent the most common lineages circulating in Italian hospitals. MRSA were limited in the number of lineages (CC22, CC5, CC8, and CC1). Phylogenetic analysis followed the sequence type-CC groupings and revealed a non-uniform distribution of the isolates from the different hospitals. No significant difference in the mean number of virulence genes carried by MRSA or MSSA isolates was observed. Some virulence genes, namely cna, fib, fnbA, coa, lukD, lukE, sak, and tst, were correlated with the CC. However, different categories of virulence factors, such as adhesins, exoenzymes, and toxins, were frequently detected and unevenly distributed among all lineages. Indeed, each lineage carried a variable combination of virulence genes, likely reflecting functional redundancy, and arguing for the importance of those traits for the pathogenicity in OM. In conclusion, no specific genetic trait in the most frequent lineages could explain their high prevalence among OM isolates. Our findings highlight that CCs detected in OM isolates follow the epidemiology of S. aureus infections in the country. It is conceivable that any of the most common S. aureus CC can cause a variety of infections, including OM.

Update on activity of dalbavancin and comparators against clinical isolates of Gram-positive pathogens from Europe and Russia (2017-2018), and on clonal distribution of MRSA.

BACKGROUND: Gram-positive pathogens remain a major cause of healthcare- and community-associated infections. In particular, the dissemination of methicillin-resistant staphylococci, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), have significantly reduced the therapeutic options, making the management of these infections even more challenging. Dalbavancin is a second-generation lipoglycopeptide approved for the treatment of moderate to severe acute bacterial skin and skin structure infections (ABSSSIs) caused by Gram-positive organisms, showing a bactericidal effect and a low propensity towards the selection of resistance over time. AIM: To evaluate the antimicrobial activity of dalbavancin and other comparators against recent clinical isolates of Gram-positive pathogens obtained from different sources and from several European countries, including countries of Southern and Eastern Europe, and Russia, where resistance rates are typically high. This study also aimed to describe the clonal relationship of MRSA strains circulating in Southern and Eastern Europe and Russia. RESULTS: In total, 1478 isolates were collected. Study results demonstrated the excellent and stable activity of dalbavancin against Gram-positive microorganisms, including MRSA. Interestingly, dalbavancin has retained unaltered minimum inhibitory concentration (MIC50 and MIC90) values over the years, and seems to have a low propensity towards the selection of resistance. CONCLUSIONS: These data support the potential efficacy of dalbavancin against Gram-positive bacteria and uncommon Gram-positive pathogens in patients with ABSSSIs. Of note, few coagulase-negative staphylococci (CoNS) isolates were resistant to dalbavancin and susceptible to vancomycin, highlighting the importance of testing for susceptibility to dalbavancin before its administration for CoNS infections.

Rapid diagnostic tests in the management of pneumonia.

INTRODUCTION: Pneumonia is one of the main causes of mortality associated with infectious diseases worldwide. Several challenges have been identified in the management of patients with pneumonia, ranging from accurate and cost-effective microbiological investigations, prompt and adequate therapeutic management, and optimal treatment duration. AREAS COVERED: In this review, an updated summary on the current management of pneumonia patients is provided and the epidemiological issues of infectious respiratory diseases, which in the current pandemic situation are of particular concern, are addressed. The clinical and microbiological approaches to pneumonia diagnosis are reviewed, including discussion about the new molecular assays pointing out both their strengths and limitations. Finally, the current recommendations about antibiotic treatment are examined and discussed depending on the epidemiological contexts, including those with high prevalence of multidrug-resistant bacteria. EXPERT OPINION: We claim that rapid diagnostic tests, if well-positioned in the diagnostic workflow and reserved for the subset of patients who could most benefit from these technologies, may represent an interesting and feasible tool to optimize timing of targeted treatments especially in terms of early de-escalation or discontinuation of antibiotic therapy.

Evaluation of Revogene® Carba C assay for the rapid detection of carbapenemase genes in gram-negative pathogens.

The rapid detection of carbapenemase-producing microorganisms is crucial for clinical and infection control purposes. Here we evaluated Revogene®Carba C assay on 154 carbapenemase-producing and -nonproducing Gram-negatives. Up to 8 samples per 70 minutes were processed by Revogene®Carba C assay which showed excellent performances with 100% sensitivity, 99.5% specificity,100% NPV, and 97.8% positive predictive value.

Inappropriate use of carbapenems in an internal medicine ward: Impact of a carbapenem-focused antimicrobial stewardship program.

PURPOSE: Carbapenem consumption is a major driver for selection and spread of carbapenem-resistant Enterobacterales (CRE). We assessed the impact of a carbapenem-focused multimodal antimicrobial stewardship program (ASP) in the internal medicine unit of a medium-size acute-care hospital. METHODS: We compared the percentage of inappropriate carbapenem prescriptions and the proportion of carbapenem treated patients registered in a 12-month pre-intervention and in a 24-month post-intervention period by using an interrupted time series analysis. The consumption of carbapenems, expressed in defined daily doses (DDD), was also assessed. As a secondary objective, the incidence of infections by carbapenem-resistant Klebsiella pneumoniae (CRKP) and the percentage of CRKP invasive isolates in the same time periods were compared. RESULTS: After the ASP intervention, the mean monthly percentage of inappropriate carbapenem prescriptions dropped from 59% to 25%, and the mean monthly proportion of carbapenem treated patients decreased from 3% to 1%. The interrupted time series analysis confirmed a significant decrease in the percentage of inappropriate carbapenem prescriptions (-41.6%, p = 0.0262) and in the proportion of carbapenem treated patients (-2.1%, p < 0.0001). Carbapenem consumption decreased from 5.2 to 1.6 DDD x 100 patient-days. The incidence of CRKP infections remained unchanged (29.1 × 100,000 patient-days vs 28.9 × 100,000 patient-days, p = 0.9864) and the percentage of CRKP invasive isolates decreased, though not significantly, from 36.4% to 13.3% (p = 0.3478). CONCLUSIONS: The implementation of a carbapenem-focused ASP was effective at limiting the inappropriate use of carbapenems and was associated with a significant decrease in carbapenem consumption. Such effects were sustained during a 24-month post-intervention period.

Genomic analysis of Neisseria meningitidis carriage isolates during an outbreak of serogroup C clonal complex 11, Tuscany, Italy.

BACKGROUND: In 2015-2016, a cross-sectional carriage survey was performed in Tuscany Region, Italy, during an outbreak of invasive meningococcal disease due to Neisseria meningitidis serogroup C clonal complex 11 (MenC:cc11). This study aims to evaluate the genomic profile of meningococcal carriage isolates collected during the survey. METHODS: Whole-genome sequencing (WGS) was performed using Illumina MiSeq on 85 cultivated meningococcal carriage isolates received at the Dept. of Infectious Disease, National Institute of Health (Istituto Superiore di Sanità, ISS), as National Reference Laboratory (NRL) for Invasive Meningococcal Disease (IMD). De novo assembled genomes were scanned by the BIGSdb platform to assign: the genotypic profiles, the cgMLST, the vaccine antigen variants and allele types of antimicrobial resistance associated genes, together with denitrification pathway loci. RESULTS: Capsule null and non-groupable meningococci accounted for 52.9% and 10.6%, respectively. Among the remaining carriage isolates, serogroup B was the predominant (71.0%). Serogroup C meningococci were culture negative and unavailable for WGS. Overall, 64 genotypic profiles were identified and, based on cgMLST, isolates clustered according to clonal complexes. Eight isolates (9.4%) harbored at least one gene encoding a 4CMenB vaccine antigen. Mutated penA alleles were found in more than 82%. Finally, complete aniA and norB coding sequences were detected among 71.8% of carriage isolates. CONCLUSIONS: Meningococcal carriage isolates collected during the MenC:cc11 outbreak were characterized by an extensive genetic diversity. The lack of outbreak-related isolates among carriage might be attributable to the high transmissibility with low duration of colonization of MenC:cc11 meningococci.

Carriage rates and risk factors during an outbreak of invasive meningococcal disease due to Neisseria meningitidis serogroup C ST-11 (cc11) in Tuscany, Italy: a cross-sectional study.

BACKGROUND: During 2015-2016 an outbreak of invasive meningococcal disease due to N. meningitidis serogroup C ST-11 (cc11) occurred in Tuscany, Italy. The outbreak affected mainly the age group 20-30 years, men who have sex with men, and the area located between the cities of Firenze, Prato and Empoli, with discos and gay-venues associated-clusters. A cross-sectional-survey was conducted to assess the prevalence and risk factors for meningococcal-carriage, in order to address public health interventions. METHODS: A convenience sample of people aged 11-45 years provided oropharyngeal swab specimens and completed questionnaires on risk factors for meningococcal carriage during a 3 months study-period, conducted either in the outbreak-area and in a control-area not affected by the outbreak (cities of Grosseto and Siena). Isolates were tested by culture plus polymerase chain reaction. Serogroup C meningococcal isolates were further characterized using multilocus sequence typing. Univariate and multivariate analyses were performed to estimate adjusted odds ratios (AORs) for meningococcal carriage. RESULTS: A total of 2285 oropharyngeal samples were collected. Overall, meningococcal carriage prevalence was 4.8% (n = 110), with nonencapsulated meningococci most prevalent (2.3%; n = 52). Among encapsulated meningococci, serogroup B was the most prevalent (1.8%; n = 41), followed by serogroup Y (0.5%; n = 11) and serogroup C (0.2%; n = 4); one carrier of serogroup E and one of serogroup Z, were also found (0.04%). Three individuals from the city of Empoli were found to carry the outbreak strain, C:ST-11 (cc11); this city also had the highest serogroup C carriage prevalence (0.5%). At the multivariate analyses, risk factors for meningococcal carriage were: illicit-drugs consumption (AOR 6.30; p < 0.01), active smoking (AOR 2.78; p = 0.01), disco/clubs/parties attendance (AOR 2.06; p = 0.04), being aged 20-30 years (AOR 3.08; p < 0.01), and have had same-sex intercourses (AOR 6.69; p < 0.01). CONCLUSIONS: A low prevalence of meningococcal serogroup C carriage in an area affected by an outbreak due to the hypervirulent N. meningitidis serogroup C ST-11 (cc11) strain was found. The city of Empoli had the highest attack-rate during the outbreak and also the highest meningococcal serogroup C carriage-prevalence due to the outbreak-strain. Multivariate analyses underlined a convergence of risk factors, which partially confirmed those observed among meningococcal outbreak-cases, and that should be considered in targeted immunization campaigns.

Evaluation of candidemia and antifungal consumption in a large tertiary care Italian hospital over a 12-year period.

PURPOSE: An early adequate antifungal therapy based on the knowledge of local epidemiology can reduce the candidemia-attributable mortality and the length of hospitalization. We performed a retrospective study to analyze the epidemiology of candidemia and the antifungal consumption in our hospital. METHODS: We analyzed Candida spp. isolated from the blood, and their susceptibility profile from 2005 to 2016 in Careggi University Hospital, Florence, Italy. We also performed a stratified analysis by clinical setting where Candida spp. were isolated (Medical Wards, Surgery, Intensive Care Unit-ICU). Then, we retrospectively reviewed the annual consumption of antifungal agents and calculated the defined daily dosing for 10,000 hospital days. RESULTS: The rate of candidemia was higher in ICU than other settings and Candida albicans was the first cause of candidemia (61.2%). After adjustment for hospital days, the rate of C. albicans showed a statistically significant parabolic trend (p < 0.001), with a peak of incidence in 2010. After 2010, we observed a reduction of candidemia due to both C. albicans and non-albicans species. Between 2005 and 2015, we reported an increasing increased use of echinocandins. As far as resistance profile is concerned, only one Candida glabrata isolate was resistant to caspofungin (1.9%) and 30% of C. glabrata were resistant to fluconazole. CONCLUSIONS: Our data describe C. albicans as the first cause of candidemia in all the studied settings and the low rate of echinocandin resistance, despite their increased use over the study period. ICU was confirmed as the setting with the highest incidence of candidemia.

Complete Genome Sequence of the First KPC-Type Carbapenemase-Positive Proteus mirabilis Strain from a Bloodstream Infection.

Sequencing of the blaKPC-positive strain Proteus mirabilis AOUC-001 was performed using both the MiSeq and PacBio RS II platforms and yielded a single molecule of 4,272,433 bp, representing the complete chromosome. Genome analysis showed the presence of several acquired resistance determinants, including two copies of blaKPC-2 carried on a fragment of a KPC-producing plasmid previously described in Klebsiella pneumoniae.

Characterization of KPC-encoding plasmids from two endemic settings, Greece and Italy.

OBJECTIVES: Global dissemination of KPC-type carbapenemases is mainly associated with the spread of high-risk clones of Klebsiella pneumoniae and of KPC-encoding plasmids. In this study, we explored the population structure of KPC-encoding plasmids from the recent epidemics of KPC-producing K. pneumoniae (KPC-Kp) in Greece and Italy, the two major European endemic settings. METHODS: Thirty-four non-replicate clinical strains of KPC-Kp representative of the early phases (2008-11) of the Greek (n = 22) and Italian (n = 12) epidemics were studied. Isolates were typed by MLST, and blaKPC-carrying plasmids were characterized by S1 profiling, PCR-based replicon typing and RFLP. Transfer experiments by conjugation or transformation were carried out with Escherichia coli recipients. Eleven plasmids, representative of all different restriction profiles, were completely sequenced. RESULTS: The representative Greek strains belonged to 14 sequence types (STs), with a predominance of ST258. The representative Italian strains belonged to three STs, with a predominance of clonal complex 258 (ST258, ST512). The 34 strains carried plasmids of variable size (78-166 kb), either with blaKPC-2 or blaKPC-3 gene embedded in a Tn4401a transposon. Plasmids from Greek strains were mostly of a single RFLP type (A) and resembled the archetypal pKpQIL KPC-encoding plasmid, while plasmids from Italian strains belonged to a more heterogeneous population, showing five RFLP profiles (A, C-F). Types A and C resembled pKpQIL or deletion derivatives thereof, while types D-F included plasmids with hybrid structures between pKpQIL, pKPN3 and pKPN101-IT. CONCLUSIONS: pKpQIL-like plasmids played a major role in the dissemination of blaKPC in Greece and Italy, but evolved with different dynamics in these endemic settings.