Performance evaluation of Bruker UMIC® microdilution panel and disc diffusion to determine cefiderocol susceptibility in Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and Burkolderia species.

PURPOSE: Cefiderocol susceptibility testing (AST) represents an open challenge for clinical microbiology. Herein, we evaluated the performance of the UMIC® Cefiderocol broth microdilution (BMD) test and disc diffusion on Gram-negative species. METHODS: UMIC® Cefiderocol BMD test, disc diffusion and reference BMD were in parallel performed on a collection of 256 clinical isolates. Categorical agreement (CA), essential agreement (EA), bias, major errors (MEs) and very major errors (VMEs) were calculated for both AST methods. RESULTS: The UMIC® Cefiderocol BMD strip exhibited an EA < 90% (85.5%), a CA higher than 90% (93.7%) and a low number of VMEs (n = 4, 4.2%) and MEs (n = 12, 7.4%). UMIC® Cefiderocol identified 96.2% of the resistant isolates [Enterobacterales, (39/40); P. aeruginosa (19/19); A. xylosoxidans (5/6); S. maltophilia (5/6); Burkholderia spp. (8/8)]. Disc diffusion showed a high CA (from 94.9 to 100%) regardless of disc manufacturer in Enterobacterales, P. aeuroginosa, A. baumannii and S. maltophilia. However, high rates of results falling in the area of technical uncertainty (ATU) were observed in Enterobacterales (34/90, 37.8%) and P. aeruginosa (16/40, 40%). Disc diffusion showed a poor performance in A. xylosoxidans and Burkholderia spp. if PK/PD breakpoint was used (overall, 5/9 VMEs; in contrast, the use of P. aeruginosa-specific breakpoints resulted in 100% of CA with 24.6% of results in the ATU). CONCLUSION: In conclusion, disc diffusion and UMIC® Cefiderocol are valid methods for the determination of cefiderocol susceptibility. Given the high number of results in the ATU by disc diffusion, a combined use of both AST methods may represent a solution to overcome the challenge of cefiderocol susceptibility testing in routine microbiology laboratories.

Whole Genome Sequencing of a Chlamydia trachomatis Strain Responsible for a Case of Rectal Lymphogranuloma Venereum in Italy.

Lymphogranuloma venereum (LGV) is a systemic sexually transmitted infection caused by Chlamydia trachomatis serovars L1 to L3. The current LGV cases in Europe are mainly characterized by an anorectal syndrome, spreading within men who have sex with men (MSM). Whole-genome sequencing of LGV strains is crucial to the study of bacterial genomic variants and to improve strategies for contact tracing and prevention. In this study, we described the whole genome of a C. trachomatis strain (LGV/17) responsible for a case of rectal LGV. LGV/17 strain was isolated in 2017 in Bologna (North of Italy) from a HIV-positive MSM, presenting a symptomatic proctitis. After the propagation in LLC-MK2 cells, the strain underwent whole-genome sequencing by means of two platforms. Sequence type was determined using the tool MLST 2.0, whereas the genovariant was characterized by an ompA sequence evaluation. A phylogenetic tree was generated by comparing the LGV/17 sequence with a series of L2 genomes, downloaded from the NCBI website. LGV/17 belonged to sequence type ST44 and to the genovariant L2f. Nine ORFs encoding for polymorphic membrane proteins A-I and eight encoding for glycoproteins Pgp1-8 were detected in the chromosome and in the plasmid, respectively. LGV/17 was closely related to other L2f strains, even in the light of a not-negligible variability. The LGV/17 strain showed a genomic structure similar to reference sequences and was phylogenetically related to isolates from disparate parts of the world, indicative of the long-distance dynamics of transmission.

Could an optimized joint pharmacokinetic/pharmacodynamic target attainment of continuous infusion ceftazidime-avibactam be a way to avoid the need for combo therapy in the targeted treatment of deep-seated DTR Gram-negative infections?

The objective of this study was to assess the relationship between joint pharmacokinetic/pharmacodynamic (PK/PD) target attainment of continuous infusion (CI) ceftazidime-avibactam and the microbiological outcome of documented difficult-to-treat resistant (DTR) Gram-negative infections. A 2-year retrospective cohort study was performed in patients receiving CI ceftazidime-avibactam mono- or combo therapy for documented DTR Gram-negative infections and undergoing therapeutic drug monitoring of both ceftazidime and avibactam. The free fractions of steady-state concentrations (fCss) of ceftazidime and avibactam were calculated. The joint PK/PD target was considered optimal when both the fCss/MIC ratio for ceftazidime ≥4 (equivalent to 100% fT>4xMIC) and the fCss/CT ratio for avibactam >1 (equivalent to 100% fT >CT of 4.0 mg/L) were simultaneously achieved (quasi-optimal if only one of the two and suboptimal if neither of the two was achieved). Multivariate logistic regression analysis was applied for testing potential variables associated with microbiological failure. Fifty-eight patients were treated with CI ceftazidime-avibactam mono- (36) or combo therapy (22) for documented DTR Gram-negative infections [74.2% for primary or secondary bloodstream infections (BSIs)]. Combo therapy was administered more frequently to intensive care unit (ICU) patients (P = 0.023) or for pneumonia (P = 0.001) and less frequently for intra-abdominal infections and BSIs (P = 0.04). Microbiological failure occurred in five cases (8.6%, three in mono- and two in combo therapy). In the multivariate analysis, the suboptimal/quasi-optimal joint PK/PD target emerged as the only independent predictor of microbiological failure (odds ratio [OR] 11.11; 95% confidence interval [CI] 1.31-93.98; P = 0.023), whereas monotherapy was not (P = 0.99). Optimized joint PK/PD target attainment of CI ceftazidime-avibactam monotherapy could represent a way forward for allowing microbiological eradication of DTR Gram-negative infections and could render unnecessary combo therapy.

Case report of Usutu virus infection in an immunocompromised patient in Italy, 2022.

Usutu virus (USUV) is an arthropod-borne flavivirus emerged in Africa in 1950s and in Eruope in 1990s causing a massive number of birds' deaths. The role of USUV as human pathogen has been only recently hypothesized and cases of USUV infection in humans remain limited and often related to immunocompromised subjects. Herein, we report a case of USUV meningoencephalitis infection in an immunocompromised patient with no history of previous flavivirus infection. The infection due to USUV evolved rapidly since hospital admission thus resulting fatal in few days after symptoms onset and, although not proven, a suspected bacteria co-infection has been hypothesized. Based on these findings, we suggested that when USUV meningoencephalitis is suspected in countries endemic, careful attention should be applied to neurological syndromes during summer months especially among immunocompromised patients.

Severe Escherichia coli infections in critical adult patients: two case reports and genomic analysis.

Pathogenic Escherichia coli strains can infect a variety of body sites due to the expression of virulence factors necessary to overcome the host defenses. Here, we present two cases of E. coli infection in adults and discuss the associated genomic features. Whole-genome sequencing was performed using both Illumina iSeq 100 and Oxford Nanopore MinION systems. Assembly was carried out with Unicycler using a hybrid approach. The genomes were annotated with RASTtk and scanned for genes involved in antimicrobial resistance, virulence and stress response with AMRFinderPlus. Sequence analysis was conducted using tools from the Center for Genomic Epidemiology (CGE) website. The two strains, named SO80 and SO81, carried a genome of 5,229,956 and 5,437,935 base pairs, respectively. SO80 belonged to ST70 and carried 13 virulence factors, 6 of which were located on a 170 Kb plasmid, while SO81 belonged to ST69 and carried 29 virulence factors, 5 of which were located on a 113 Kb plasmid. Our work highlights key factors which may have contributed to the complicated clinical status of these patients, and provides new in-depth data on E. coli infections with few precedents in the literature.

Increased blaKPC Copy Number and OmpK35 and OmpK36 Porins Disruption Mediated Resistance to Imipenem/Relebactam and Meropenem/Vaborbactam in a KPC-Producing Klebsiella pneumoniae Clinical Isolate.

Herein, we report the in vivo evolution of imipenem/relebactam-resistance in KPC-producing K. pneumoniae (KPC-Kp) isolated from a critically-ill patient treated with multiple combination therapies based on ceftazidime-avibactam or meropenem-vaborbactam. Imipenem/relebactam-resistance was associated to meropenem-vaborbactam cross-resistance and was due to truncated OmpK35 and OmpK36 porins and increased copy of blaKPC copy number. Genome analysis demonstrated that imipenem/relebactam-resistant KPC-Kp harbored a second copy of blaKPC-carrying Tn4401 in a ColRNAI plasmid as a consequence of a transposition event.

Dynamic evolution of imipenem/relebactam resistance in a KPC-producing Klebsiella pneumoniae from a single patient during ceftazidime/avibactam-based treatments.

OBJECTIVES: The novel carbapenem/ß-lactamase inhibitor combination imipenem/cilastatin/relebactam has been developed for the treatment of infections due to carbapenemase-producing Enterobacteriaceae (CPE). Herein, we describe the in vivo evolution of imipenem/cilastatin/relebactam resistance in longitudinal intra-patient Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) strains isolated from a patient following ceftazidime/avibactam-based treatments. METHODS: WGS analysis was performed on KPC-Kp strains isolated at different times and during antimicrobial treatments from the same patient. Genome assemblies were performed using a hybrid approach using Illumina iSeq 100 and Minion Oxford Nanopore platforms. Subpopulation analysis and allele frequency determination was performed by mapping Illumina reads to blaKPC. RESULTS: During antimicrobial treatment, resistance to ceftazidime/avibactam was observed following 16 days of antimicrobial therapy. WGS results showed that all KPC-Kp exhibited a low SNP rate of divergence, belonged to ST512 and shared similar antimicrobial resistance and porin gene patterns. Genetic analysis demonstrated that the first ceftazidime/avibactam-resistant KPC-Kp strain harboured a blaKPC-53 gene in a Tn4401 transposon moved from IncFII(K) to a 43 kb IncX3 plasmid, while a imipenem/cilastatin/relebactam-resistant strain exhibited two copies of the Tn4401 transposon in IncFII(K) and IncX3 plasmids, resulting in an increased blaKPC copy number. Of note, frequency analysis demonstrated that imipenem/cilastatin/relebactam-resistant KPC-Kp consisted of mixed subpopulations harbouring blaKPC-40 and blaKPC-53 alleles. CONCLUSIONS: Our results show the in vivo evolution of genetic rearrangement conferring resistance to imipenem/relebactam in a patient with KPC-Kp infection and treated with different ceftazidime/avibactam-based treatments. The rapid development of mutations and the high adaptability of its genome highlight the potential threat of KPC-Kp.

COVID-19 in patients with HIV-1 infection: a single-centre experience in northern Italy.

BACKGROUND: Since the end of February 2020, the Coronavirus Disease 2019 (COVID-19) outbreak rapidly spread throughout Italy and other European countries, but limited information has been available about its characteristics in HIV-infected patients. METHODS: We have described a case series of patients with HIV infection and COVID-19 diagnosed at the S.Orsola Hospital (Bologna, Italy) during March and April, 2020. RESULTS: We reported a case series of 26 HIV-infected patients with COVID-19. Nineteen subjects were men, the median age was 54 years, 73% of patients had one or more comorbidities. Only 5 patients with interstitial pneumonia were hospitalized, but there were no admissions to intensive care unit and no deaths. CONCLUSIONS: In our experience, COVID-19 associated with HIV infection had a clinical presentation comparable to the general population and was frequently associated with chronic comorbidities.

Comparative serum bactericidal activity of meropenem-based combination regimens against extended-spectrum beta-lactamase and KPC-producing Klebsiella pneumoniae.

Combination therapies are frequently used in the treatment of multidrug-resistant Klebsiella pneumoniae infection without consensus regarding which combination is the most effective. We compared bactericidal titres from sera collected from critically ill patients receiving meropenem plus tigecycline (n = 5), meropenem plus colistin (n = 5), or meropenem, colistin and tigecycline (n = 5) against K. pneumoniae isolates that included ESBL-producing (n = 7) and KPC-producing strains (n = 14) with varying sensitivity patterns to colistin and tigecycline. Meropenem concentrations (Cmin) were measured in all samples by LC-MS/MS, and indexed to respective pathogen MICs to explore differences in patterns of bactericidal activity for two versus three drug combination regimens. All combination regimens achieved higher SBTs against ESBL (median reciprocal titre 128, IQR 32-256) versus KPC (4, IQR 2-32) strains. Sera from patients treated with meropenem-colistin yielded higher median SBTs (256, IQR 64-512) than either meropenem-tigecycline (32, IQR 8-256; P < 0.001). The addition of tigecycline was associated with a lower probability of achieving a reciprocal SBT above 8 when meropenem concentrations were below the MIC (P = 0.04). Although the clinical significance is unknown, sera from patients receiving tigecycline-based combination regimens produce lower serum bactericidal titres against ESBL or KPC-producing K. pneumoniae. SBTs may represent a useful complimentary endpoint for comparing pharmacodynamics of combinations regimens for MDR Enterobacteriaceae.

In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment.

Objectives: KPC-producing Klebsiella pneumoniae (KPC-Kp) represent a serious problem worldwide. Herein, we describe the evolution of ceftazidime/avibactam resistance by sequencing longitudinal clinical isolates from a patient with KPC-Kp bloodstream infection undergoing ceftazidime/avibactam treatment. Methods: WGS was performed on one ceftazidime/avibactam-susceptible KPC-Kp (BOT-CA-S) and two phenotypically different ceftazidime/avibactam-resistant KPC-Kp with low (BOT-CA-R) and high (BOT-EMO) carbapenem MICs. The population diversity was assessed by the frequency of allele mutations and population analysis profiles (PAPs). Results: Phylogenetic analysis demonstrated clonal relatedness of the KPC-Kp isolates, all belonging to the clone ST1519. The D179Y mutation in blaKPC-3 was detected in both of the ceftazidime/avibactam-resistant KPC-Kp, whereas it was absent in the ceftazidime/avibactam-susceptible isolate. The mutation emerged independently in the two ceftazidime/avibactam-resistant isolates and was associated with a significant reduction in carbapenem MICs in BOT-CA-R, but not in BOT-EMO. WGS analysis revealed that the frequency of the D179Y mutation was 96.32% and 51.05% in BOT-CA-R and BOT-EMO, respectively. PAP results demonstrated that carbapenem resistance in BOT-EMO was due to the coexistence of mixed subpopulations harbouring WT and mutated blaKPC-3. A bacterial subpopulation with high ceftazidime/avibactam resistance for BOT-EMO KPC-Kp showed low carbapenem MICs, whereas a subpopulation with high meropenem resistance had a low MIC of ceftazidime/avibactam. Conclusions: Our analysis indicates that mixed subpopulations of ceftazidime/avibactam-resistant KPC-Kp emerge after ceftazidime/avibactam treatment. The evolution of different subpopulations that are highly resistant to ceftazidime/avibactam likely contributes to treatment failure, thereby highlighting the need for combination treatment strategies to limit selection of ceftazidime/avibactam-resistant KPC-Kp subpopulations.

Two cases of relapsed HIV-associated visceral leishmaniasis successfully treated with combination therapy.

BACKGROUND: The management of visceral leishmaniasis (VL) in HIV-infected patients is often complex with patients experiencing higher mortality rates, more toxic side effects and a higher possibility of treatment failure and relapse than HIV-negative individuals with VL. CASE PRESENTATION: We report on successful salvage therapy in two HIV-infected patients suffering with disseminated cutaneous and visceral leishmaniasis, recalcitrant to therapy with liposomal amphotericin B. After the employment of combination anti-leishmanial treatment, parasite genomes were not detectable up to the last follow up visit, 57 and 78 weeks after treatment onset, respectively. CD4+ lymphocyte counts fluctuated over time, but were generally higher than counts detected at treatment onset, which likely contributed to protection against VL relapse. CONCLUSIONS: Results achieved with the anti-leishmanial combination treatment were promising, but are based on only two patients. Future investigation is necessary to confirm the efficacy of this salvage therapy in sustaining the immunological response and control of VL.

Characterization of an IncL/M plasmid carrying blaOXA-48 in a Klebsiella pneumoniae strain from Italy.

Here we report the complete nucleotide sequence of a 49.257-bp IncL/M conjugative plasmid (pRAY) carrying the blaOXA-48 gene collected from a Klebsiella pneumoniae clinical strain isolated in Italy. The genetic environment of pRAY plasmid revealed that the blaOXA-48 gene was located within a Tn1999.2 transposon. The pRAY plasmid differed from blaOXA-48-harboring IncL/M plasmids by genetic context and size. Comparative analysis demonstrated that pRAY plasmid lacked a region of ~15 kb carrying genes encoding proteins involved in pilus assembly and plasmid conjugative apparatus.

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of KPC-Producing Klebsiella pneumoniae.

We evaluated a real-time single-peak (11.109-Da) detection assay based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae Our results demonstrated that the 11.109-Da peak was detected in 88.2% of the KPC producers. Analysis of blaKPC-producing K. pneumoniae showed that the gene encoding the 11.109-Da protein was commonly (97.8%) associated with the Tn4401a isoform.

Genomic epidemiology of Klebsiella pneumoniae in Italy and novel insights into the origin and global evolution of its resistance to carbapenem antibiotics.

Klebsiella pneumoniae is at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated with K. pneumoniae infections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniae strains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology of K. pneumoniae in Italy. Thirty-one strains were carbapenem-resistant K. pneumoniae carbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity of K. pneumoniae worldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼ 1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group of K. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding of K. pneumoniae evolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

Matrix-assisted laser desorption ionization-time of flight and comparative genomic analysis of M-18 group a Streptococcus strains associated with an acute rheumatic fever outbreak in northeast Italy in 2012 and 2013.

Acute rheumatic fever (ARF) is a postsuppurative sequela caused by Streptococcus pyogenes infections affecting school-age children. We describe here the occurrence of an ARF outbreak that occurred in Bologna province, northeastern Italy, between November 2012 and May 2013. Molecular analysis revealed that ARF-related group A Streptococcus (GAS) strains belonged to the M-18 serotype, including subtypes emm18.29 and emm18.32. All M-18 GAS strains shared the same antigenic profile, including SpeA, SpeB, SpeC, SpeL, SpeM, and SmeZ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed that M-18 GAS strains grouped separately from other serotypes, suggesting a different S. pyogenes lineage. Single nucleotide polymorphisms and phylogenetic analysis based on whole-genome sequencing showed that emm18.29 and emm18.32 GAS strains clustered in two distinct groups, highlighting genetic variations between these subtypes. Comparative analysis revealed a similar genome architecture between emm18.29 and emm18.32 strains that differed from noninvasive emm18.0 strains. The major sources of differences between M-18 genomes were attributable to the prophage elements. Prophage regions contained several virulence factors that could have contributed to the pathogenic potential of emm18.29 and emm18.32 strains. Notably, phage ΦSPBO.1 carried erythrogenic toxin A gene (speA1) in six ARF-related M-18 GAS strains but not in emm18.0 strains. In addition, a phage-encoded hyaluronidase gene (hylP.2) presented different variants among M-18 GAS strains by showing internal deletions located in the α-helical and TSßH regions. In conclusion, our study yielded insights into the genome structure of M-18 GAS strains responsible for the ARF outbreak in Italy, thus expanding our knowledge of this serotype.

Human infection with highly pathogenic A(H7N7) avian influenza virus, Italy, 2013.

During an influenza A(H7N7) virus outbreak among poultry in Italy during August-September 2013, infection with a highly pathogenic A(H7N7) avian influenza virus was diagnosed for 3 poultry workers with conjunctivitis. Genetic analyses revealed that the viruses from the humans were closely related to those from chickens on affected farms.

In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment.

Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB(L82R) in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB(L82R) mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

In vitro activity and post-antibiotic effects of colistin in combination with other antimicrobials against colistin-resistant KPC-producing Klebsiella pneumoniae bloodstream isolates.

OBJECTIVES: Combination therapy is recommended for the treatment of KPC-producing Klebsiella pneumoniae (KPC-Kp), but the optimal regimen for colistin-resistant strains is unknown. We compared the synergistic activity and post-antibiotic effect (PAE) of colistin in combination with other antimicrobials against colistin-susceptible and -resistant KPC-Kp bloodstream isolates. METHODS: The genotypes of nine colistin-susceptible and eight colistin-resistant KPC-Kp bloodstream isolates were analysed using PCR and amplicon sequencing. Combinations of colistin, meropenem, tigecycline, rifampicin and teicoplanin were then screened using the Etest, a chequerboard assay and time-kill studies. Synergistic combinations were also analysed with respect to the PAE in time-kill curves and the PAE at clinically achievable concentrations. RESULTS: Insertional inactivation of the PhoQ/PhoB two-component regulatory system by mgrB-IS5 was identified in 6/8 (75%) colistin-resistant KPC-Kp. Colistin/rifampicin combinations resulted in no interactions [fractional inhibitory concentration (FIC) indices 1.5-2] for colistin-susceptible strains, but were uniformly synergistic (FIC indices 0.1-0.4) against colistin-resistant KPC-Kp. Time-kill kinetic analysis, at clinically achievable fixed concentrations of rifampicin and colistin, confirmed synergy and produced persistent growth inhibition (3 h) of colistin-resistant KPC-Kp strains exposed to colistin/rifampicin or colistin/rifampicin/tigecycline combinations. CONCLUSIONS: Combinations of colistin plus rifampicin, and less frequently tigecycline, exhibited synergistic activity in vitro against colistin-resistant KPC-Kp strains.