Genome characterization of a Klebsiella pneumoniae co-producing OXA-181 and KPC-121 resistant to ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam and cefiderocol isolated from a critically ill patient

Objectives: : Carbapenemase-producing Enterobacterales (CPE) represent a public health concern. The limited antimicrobial options against CPE have led to the development of novel antimicrobial molecules. In the present study, we characterized the genetic determinants associated with the resistance to cef tazidime/avibactam (CAZ-AVI), meropenem/vaborbactam (MER-VAB), imipenem/relebactam (IMI-REL) and cefiderocol (CFD) in a carbapenemase-producing Klebsiella pneumoniae strain isolated from a critically ill patient. Methods: : Genomic DNA was sequenced using Illumina iSeq 100 and Minion Oxford Nanopore plat forms. Assemblies were performed with a de novo approach using short-read, hybrid and long-lead as sembly approaches. Final assembly was manually curated and carefully verified. Circular elements were screened for antimicrobial-resistance genes, porins, virulence factors and prophage regions. Results: : KPC-Kp (KPC-producing Klebsiella pneumoniae) BO743 was resistant to all novel β-lactams including CAZ-AVI, MER-VAB, IMI-REL and CFD. The genome of strain BO743 is composed of a single chromosome of 5 347 606 bp and three circular plasmids of 363 634 bp (pBO743-363Kb), 120 290 bp (pBO743-120Kb) and 54 339 bp (pBO743-54Kb). Sequence analysis demonstrated that KPC-Kp BO743 co harboured blaOXA-181 and novel blaKPC-121 located, respectively, on the pBO743-54Kb and pBO743-120Kb plasmids. KPC-121 differed by a serine insertion at position 181 than KPC-3. Conclusion: : The description of the genome of KPC-Kp cross-resistant to novel βL-βLICs and cefiderocol reveals the presence of numerous antimicrobial resistance genes including blaOXA-181 and novel variant blaKPC-121. The characterization of this multidrug-resistant phenotype provides evidence that needs further attention and monitoring of such MDR clinical isolate

Rectal screening of carbapenemase-producing Enterobacteriaceae: a proposed workflow

Abstract Objectives Active screening is a crucial element for the prevention of carbapenemase-producing Enterobacteriaceae (CPE) transmission in healthcare settings. Here, we proposed a culture-based protocol for rectal swab CPE screening that combines the detection of CPE and the identification of carbapenamase type. Methods The workflow integrates an automatic digital analysis of selective chromogenic media (WASPLab, Copan), with subsequent rapid tests for the confirmation of carbapenemase production (i.e. detection of Klebsiella pneumoniae KPC-specific peak by MALDI-TOF mass spectrometry; a multiplex immunochromatographic assay identifying the five commonest carbapenemase types). To in-depth evaluate the performance of this protocol, data about 21 162 rectal swabs submitted for CPE screening at the Microbiology of S. Orsola-Malpighi Hospital in Bologna were analyzed. Results Considering its ability to correctly segregate plates with/without Enterobacteriaceae, WASPLab Image Analysis Software showed globally a sensitivity and a specificity of 100% and 79.4%, respectively. Out of the plates with a bacterial growth (n = 901), 76.9% were found positive for CPE by MALDI-TOF (specific KPC-peak for K. pneumoniae) or by the immunochromatographic assay. Only 2.8% of KPC-positive K. pneumoniae strains were missed by the specific MALDI-TOF MS algorithm, being detected by the immunochromatographic assay. The mean turn-around-time needed from the sample arrival to the final report ranged between 18 to 24 hours, with a significant time saving compared to a manual reading. Conclusions This workflow proved to be fast and reliable, being particularly suitable for KPC-K. pneumoniae endemic areas and for high-throughput laboratories

Dengue and falciparum malaria co-infection in travelers returning from Burkina Faso: Report of two cases in Northeastern Italy

Rationale: Malaria and dengue are the most prevalent vector-borne diseases in tropical countries. Plasmodium parasite and dengue virus (DENV) concurrent infection is possible and often under-recognized in geographical areas where these infections are both endemic. Patients concern and diagnosis: We describe the first two cases of Plasmodium falciparum and DENV-3 co-infection in travelers returning to northeastern Italy from Burkina Faso during 2013-2014. Interventions: Malaria infection in both patients was treated with mefloquine. Due to the persistence of symptoms despite of the antimalaria treatment, dengue was also investigated; the treatment of dengue was symptomatic. Outcomes: The patients were discharged in good general condition. Lessons: The need for surveillance of potential malaria and dengue co-infection in travelers returning to Europe from endemic areas is highlighted, as infection with Plasmodium does not exclude arboviral co-infection

Valutazione di un metodo rapido per la diagnosi d'infezione da H. pylori attraverso ricerca di antigene fecale

n/

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

Successful Treatment of Bloodstream Infection due to a KPC-Producing Klebsiella Pneumoniae Resistant to Imipenem/Relebactam in a Hematological Patient

Novel carbapenem-β-lactamase inhibitor combination, imipenem/relebactam (IMI-REL), has been recently approved for treatment of infections with limited or no alternative treatment options. In this study, we described the emergence of the IMI-REL-resistance in a KPC-producing Klebsiella pneumoniae (KPC-Kp) strain collected from a hematological patient with no evidence of prior colonization. Interestingly, IMI-REL-resistance was associated with meropenem/vaborbactam (MER-VAB) cross-resistance but was not associated with cross-resistance to ceftazidime/avibactam (CAZ-AVI). Although treatment with CAZ-AVI and gentamicin completely eradicated the infection due KPC-Kp cross-resistance to IMI-REL and MER-VAB, the patient became colonized subsequently by KPC-Kp strains susceptible to IMI-REL and MER-VAB. Whole-genome sequencing performed by hybrid approach using Illumina and Oxford Nanopore platforms demonstrated that all KPC-Kp strains isolated from hematological patient belonged to the ST512 and were clonally related. Analysis of antimicrobial and porins genes demonstrated that cross-resistance to IMI-REL and MER-VAB was associated with increased blaKPC-3 copy number and truncated OmpK35 and OmpK36 with GD134-135 insertion. Phylogenetic analysis demonstrated that KPC-Kp cross-resistance to IMI-REL and MER-VAB was clonally related to a KPC-Kp resistant to IMI-REL as previously described, demonstrating the spread of this multidrug resistant clone in the hematological unit. In conclusion, the results presented in this study reported the emergence of cross-resistance to MER-VAB and IMI-REL in a KPC-Kp strain isolated from a hematological patient and highlight the potential development and diffusion of new multidrug resistance traits

Retrospective screening of serum and cerebrospinal fluid samples from patients with acute meningo-encephalitis does not reveal past Japanese encephalitis virus infection, Emilia Romagna, Italy, 2011.

P Gaibani1, A C Finarelli2, R Cagarelli2, A Pierro1, G Rossini1, M Calzolari3, M Dottori3, P Bonilauri3, M P Landini1, V Sambri ([email protected])1 1. Operative Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies, S. Orsola-Malpighi University Hospital, Bologna, Italy 2. Public Health Authority, Emilia Romagna Region, Bologna, Italy 3. Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna 'B. Ubertini' (IZSLER; Experimental veterinary institute of Lombardy and Emilia Romagna), Reggio Emilia, Ital

Comparative genomics of closely related strains of Klebsiella pneumoniae reveals genes possibly involved in colistin resistance

Strains of colistin-resistant Klebsiella pneumoniae are emerging worldwide, due to the increased use of this molecule in antibiotic-resistant nosocomial infections. Comparative genomics was performed on three closely related K. pneumoniae strains isolated from three patients in a single hospital in Bologna, Italy. Two of these isolates are colistin-resistant, while the third is sensitive to this antibiotic. The designed bioinformatic approach detected, among the three analyzed genomes, single nucleotide polymorphisms, insertions and deletions, specific patterns of gene presence and absence, in a total of 270 genes. These genes were analyzed by automatic and manual methods, to identify those potentially involved in colistin resistance, based on the data available in the literature and on the mechanism of action of colistin, the alteration of the outer membrane. Three of the identified genes (waaL, rfbA, vacJ), all presenting non-synonymous substitutions in the colistin resistant strains, resulted to be of special interest, due to the specific function of their protein products, involved in the biosynthesis of the outer bacterial membrane

In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator

Colistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniae producing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrB inactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrB deletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrB inactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrB gene, upregulated transcription of phoP, phoQ, and pmrK (which is part of the pmrHFIJKLM operon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniae and were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLM operon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target

Chikungunya: an emerging and spreading arthropod-borne viral disease

The infection caused by the virus Chikungunya is known since the last 50 years, but since the disease was mainly diffuse in geographical areas located in developing countries, a few research work have been made available until the appearance of an important epidemiological outbreak in 2005 in the island of La Reunion, that is part of metropolitan France even if located in the Southern Eastern part of the Indian Ocean. In 2007, a smaller outbreak of Chikungunya developed in the Northern Eastern part of Italy, where the local transmission has been made possible by the enormous population of Aedes albopictus and the presence of a viremic patient coming from the Indian Ocean area. Nowadays, Chikungunya is spreading in Southeast Asia countries, including Indonesia, Malaysia, Thailand and Singapore. This paper reviews different aspects of the disease caused by Chikungunya virus, including: history, epidemiology, biological and pathogenetic aspects, clinical pictures, diagnosis and treatment