Genomics of Klebsiella pneumoniae ST16 producing NDM-1, CTX-M-15, and OXA-232

OBJECTIVES: Genomic characterization of the internationally spread sequence type (ST) 16 carbapenem-resistant Klebsiella pneumoniae. METHODS: The complete genomes of three carbapenem producing ST16 K. pneumoniae from Italian patients were analysed by single-nucleotide polymorphism-based phylogeny, core genome multilocus sequence typing, resistance, plasmid, and virulence content and compared with ten genomes of ST16 strains isolated in other countries. Plasmids carrying blaNDM-1 or blaOXA-232 carbapenemase genes were assembled and sequences were analysed. RESULTS: The internationally spread ST16 K. pneumoniae clone showed variability in terms of distribution of NDM-1 and OXA-232 type carbapenemases. In some ST16 strains, up to six plasmids can be simultaneously present in the same cell, including ColE-like plasmids carrying blaOXA-232 and IncF plasmids carrying blaNDM-1. The differences observed in plasmid, resistance, and virulence content and core genome suggested that there is not a unique, highly conserved ST16 clone, but instead different variants of this lineage circulate worldwide. CONCLUSIONS: The ST16 K. pneumoniae clone has spread worldwide and may become a high-risk clone

OXA-244-Producing ST131 Escherichia coli From Surface and Groundwaters of Pavia Urban Area (Po Plain, Northern Italy)

The study aimed to investigate (i) the occurrence of third-generation cephalosporins and/or carbapenems non-sensitive Enterobacterales in Pavia surface and groundwaters, (ii) their resistance determinants, and (iii) the clonal features of the most relevant strains. During May 13 and 14, 2019, n = 18 water samples from n = 12 sampling sites in the urban/peri-urban area of Pavia (Po Plain, Northern Italy) have been evaluated. At first, hydrochemical analysis and bacterial plate counts were carried out on all the water samples. One milliliter of each water sample was then screened on both MacConkey agar (MC) added with cefotaxime (1 mg/L; 2 mg/L) and MC plus meropenem (0.25 mg/L; 4 mg/L). Species identification and antimicrobial susceptibilities were assessed by MicroScan autoSCAN-4. Double Disk Synergy (DD) test, CT103XL microarray, acc(6')-Ib-cr, qnrS, blaCTX-M-/MOX-/VEB-/OXA-type genes targeted PCR and sequencing, Pulsed-Field Gel Electrophoresis (PFGE), MultiLocus Sequence Typing (MLST), and Whole-Genome Sequencing on selected strains were performed. A total of n = 30 isolates grown on β-lactams enriched MC: Escherichia coli (n = 21; 70%), Klebsiella spp. (n = 5; 16.6%), Citrobacter freundii (n = 2; 6.7%), and Kluyvera intermedia (n = 2; 6.7%). All E. coli and K. pneumoniae were ESβL-producers by DD. The 66.6, 38.0, and 19.0% of E. coli were ciprofloxacin/levofloxacin, trimethoprim-sulfamethoxazole, and gentamicin resistant (EUCAST 2019 breakpoints), respectively. A blaCTX-M-type determinant was identified in E. coli (n = 20/21; 95.2%) and K. pneumoniae (n = 2/3; 66.7%). The remaining E. coli was blaVEB-1 and blaMOX-2 genes positive. The aac(6')-Ib-cr determinant was found in n = 7 E. coli and n = 1 K. pneumoniae, while qnrS was found in n = 1 E. coli and n = 2 K. pneumoniae. PFGE showed clonal heterogeneity among ESβL-E. coli. Two out of four E. coli detected as blaOXA-244-positive, belonged to the pandemic ST131. One XDR K. pneumoniae from a stream sample, detected as blaKPC-2 positive, resulted of ST258. The epidemiological impact of blaOXA-244 ST131 E. coli and blaKPC-2 ST258 K. pneumoniae presence in surface waters of an urban area in Northern Italy must not be underestimated

Detection of ST1702 Escherichia coli blaNDM-5 and blaCMY-42 genes positive isolates from a Northern Italian hospital

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy

Genomic Insight of VIM-harboring IncA Plasmid from a Clinical ST69 Escherichia coli Strain in Italy

Background: VIM (Verona Integron-encoded Metallo-beta-lactamase) is a member of the Metallo-Beta-Lactamases (MBLs), and is able to hydrolyze all beta-lactams antibiotics, except for monobactams, and including carbapenems. Here we characterize a VIM-producing IncA plasmid isolated from a clinical ST69 Escherichia coli strain from an Italian Long-Term Care Facility (LTCF) inpatient. Methods: An antimicrobial susceptibility test and conjugation assay were carried out, and the transferability of the blaVIM-type gene was confirmed in the transconjugant. Whole-genome sequencing (WGS) of the strain 550 was performed using the Sequel I platform. Genome assembly was performed using “Microbial Assembly”. Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases. Results: Assembly resulted in three complete circular contigs: the chromosome (4,962,700 bp), an IncA plasmid (p550_IncA_VIM_1; 162,608 bp), harboring genes coding for aminoglycoside resistance (aac(6′)-Ib4, ant(3″)-Ia, aph(3″)-Ib, aph(3′)-XV, aph(6)-Id), beta-lactam resistance (blaSHV-12, blaVIM-1), macrolides resistance (mph(A)), phenicol resistance (catB2), quinolones resistance (qnrS1), sulphonamide resistance (sul1, sul2), and trimethoprim resistance (dfrA14), and an IncK/Z plasmid (p550_IncB_O_K_Z; 100,306 bp), free of antibiotic resistance genes. Conclusions: The increase in reports of IncA plasmids bearing different antimicrobial resistance genes highlights the overall important role of IncA plasmids in disseminating carbapenemase genes, with a preference for the blaVIM-1 gene in Italy

Multidrug-resistant (MDR) and Extensively Drug-resistant (XDR) Gram-negative bacteria of clinical interest: laboratory diagnosis, clinical management and infection control

Antimicrobial resistance (AMR) represents one of the major 21st century public health tasks: its increasing has reduced the effective prevention and treatment strategies against infection events. The problem of AMR is especially urgent for Gram-negative bacteria, on which an over-/inappropriate use (incorrect choices, inadequate dosing, and poor adherence to treatment guidelines) of antibiotics have acted as a selective pressure, favouring the survival and multiplication of strains with acquired antibiotic-resistant traits. The aim of my PhD research were: i) to perform an evaluation and characterization of the antibiotic-resistance determinants circulating in Gram-negative bacteria collected from both human and veterinary settings; ii) to obtain, through NGS analysis, a complete picture of the structure of replicons and plasmids harbouring such resistance genes arrays, and iii) to get the own genetic characteristics of the most spread MDR successful clones/plasmids collected. The first project provides the characterization of two carbapenemase-producing Citrobacter freundii strains from two Italian patients without any apparent epidemiological link. The isolates were susceptible only to gentamycin, colistin, tigecycline and trimethoprim-sulfamethoxazole. Illumina sequencing revealed the presence of two different KPC variants: blaKPC-2 gene in a novel variant of Tn4401 trasposon, inserted in an IncN plasmid; blaKPC-3 gene inserted in an IncX3-IncA self-conjugative plasmid fusion. The two isolates resulted clonally unrelated belonging to different sequence types (ST46 and ST19). The second project was on two carbapenem-resistant E. coli strains collected in Italy from a three years-old outpatient affected by diarrhea, after a travel in India. The strains resulted clonally unreleated and showed an MDR profile, retaining susceptibility only to colistin, tigecycline and fosfomycin. WGS analysis identified an ST2659 and an ST405 E. coli. One strain contained a 130,562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) coding blaOXA-, blaNDM-5, blaTEM-1B, blaCTX-M-15, aac(6’)-lb-cr, aadA5, rmtB, sul1, dfrA17, qacEΔ1 and catB3. The other isolate carried four plasmids: a 153,866 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), a 41143 bp IncI plasmid (pISV_IncI_CMY-42), a 51,480 bp IncX3 plasmid (pISV_IncX3_OXA-181) and an 89,866 bp IncI1 plasmid. pISV_IncFII_NDM-5 carried genes coding blaOXA-1, blaNDM-5, blaTEM-1B, aac(6’)-lb-cr, aadA5, tet(B), catB3 and catA1. The third project describes a deadly infection events occurred in a bulldog breeding located in Central Italy. A total of five E. coli strains were collected from biopsy specimens of a two-weeks old dog, belonged to an eight-puppy litter. The entire litter contracted haemorrhagic enteritis with renal involvement and cerebral vessels congestion. All the strains were clonally releated, belonging to ST58. WGS analysis, performed on a representative strain, revealed the presence of: i) an IncHI2α replicon plasmid (pMBC_CTX-M-15, 302,597 bp) harboured dfrA14, catA1, sul2, aac(6’)-Ib-cr, qnrB1, tet(A), blaOXA-1, blaTEM-1b, blaCTX-M-15, aac(3)-Iia and aph(6)-Id resistance genes; ii) an IncI1 replicon plasmid (pMBC_CMY-2, 83,077 bp) harboured the blaCMY-2 determinat only; iii) an IncFII replicon plasmid (pMBC_DHA-1, 83,429 bp) harboured sul1, qnrB4 and blaDHA-1 resistance genes. The source of contamination and infection still remains unknown. The results show that a worldwide spread of antibiotic-resistance genes is occurring in different Gram-negative microorganisms, through MDR hyper-epidemic plasmids or successful clone’s dissemination. The molecular characterization of the antibiotic-resistance genes represents an essential support to explain the epidemiology, modes and high speed rate ability to spread of certain bacterial clones or their resistance determinants.Antimicrobial resistance (AMR) represents one of the major 21st century public health tasks: its increasing has reduced the effective prevention and treatment strategies against infection events. The problem of AMR is especially urgent for Gram-negative bacteria, on which an over-/inappropriate use (incorrect choices, inadequate dosing, and poor adherence to treatment guidelines) of antibiotics have acted as a selective pressure, favouring the survival and multiplication of strains with acquired antibiotic-resistant traits. The aim of my PhD research were: i) to perform an evaluation and characterization of the antibiotic-resistance determinants circulating in Gram-negative bacteria collected from both human and veterinary settings; ii) to obtain, through NGS analysis, a complete picture of the structure of replicons and plasmids harbouring such resistance genes arrays, and iii) to get the own genetic characteristics of the most spread MDR successful clones/plasmids collected. The first project provides the characterization of two carbapenemase-producing Citrobacter freundii strains from two Italian patients without any apparent epidemiological link. The isolates were susceptible only to gentamycin, colistin, tigecycline and trimethoprim-sulfamethoxazole. Illumina sequencing revealed the presence of two different KPC variants: blaKPC-2 gene in a novel variant of Tn4401 trasposon, inserted in an IncN plasmid; blaKPC-3 gene inserted in an IncX3-IncA self-conjugative plasmid fusion. The two isolates resulted clonally unrelated belonging to different sequence types (ST46 and ST19). The second project was on two carbapenem-resistant E. coli strains collected in Italy from a three years-old outpatient affected by diarrhea, after a travel in India. The strains resulted clonally unreleated and showed an MDR profile, retaining susceptibility only to colistin, tigecycline and fosfomycin. WGS analysis identified an ST2659 and an ST405 E. coli. One strain contained a 130,562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) coding blaOXA-, blaNDM-5, blaTEM-1B, blaCTX-M-15, aac(6’)-lb-cr, aadA5, rmtB, sul1, dfrA17, qacEΔ1 and catB3. The other isolate carried four plasmids: a 153,866 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), a 41143 bp IncI plasmid (pISV_IncI_CMY-42), a 51,480 bp IncX3 plasmid (pISV_IncX3_OXA-181) and an 89,866 bp IncI1 plasmid. pISV_IncFII_NDM-5 carried genes coding blaOXA-1, blaNDM-5, blaTEM-1B, aac(6’)-lb-cr, aadA5, tet(B), catB3 and catA1. The third project describes a deadly infection events occurred in a bulldog breeding located in Central Italy. A total of five E. coli strains were collected from biopsy specimens of a two-weeks old dog, belonged to an eight-puppy litter. The entire litter contracted haemorrhagic enteritis with renal involvement and cerebral vessels congestion. All the strains were clonally releated, belonging to ST58. WGS analysis, performed on a representative strain, revealed the presence of: i) an IncHI2α replicon plasmid (pMBC_CTX-M-15, 302,597 bp) harboured dfrA14, catA1, sul2, aac(6’)-Ib-cr, qnrB1, tet(A), blaOXA-1, blaTEM-1b, blaCTX-M-15, aac(3)-Iia and aph(6)-Id resistance genes; ii) an IncI1 replicon plasmid (pMBC_CMY-2, 83,077 bp) harboured the blaCMY-2 determinat only; iii) an IncFII replicon plasmid (pMBC_DHA-1, 83,429 bp) harboured sul1, qnrB4 and blaDHA-1 resistance genes. The source of contamination and infection still remains unknown. The results show that a worldwide spread of antibiotic-resistance genes is occurring in different Gram-negative microorganisms, through MDR hyper-epidemic plasmids or successful clone’s dissemination. The molecular characterization of the antibiotic-resistance genes represents an essential support to explain the epidemiology, modes and high speed rate ability to spread of certain bacterial clones or their resistance determinants

Fosfomycin resistance mechanisms in Enterobacterales: an increasing threat

Antimicrobial resistance is well-known to be a global health and development threat. Due to the decrease of effective antimicrobials, re-evaluation in clinical practice of old antibiotics, as fosfomycin (FOS), have been necessary. FOS is a phosphonic acid derivate that regained interest in clinical practice for the treatment of complicated infection by multi-drug resistant (MDR) bacteria. Globally, FOS resistant Gram-negative pathogens are raising, affecting the public health, and compromising the use of the antibiotic. In particular, the increased prevalence of FOS resistance (FOSR) profiles among Enterobacterales family is concerning. Decrease in FOS effectiveness can be caused by i) alteration of FOS influx inside bacterial cell or ii) acquiring antimicrobial resistance genes. In this review, we investigate the main components implicated in FOS flow and report specific mutations that affect FOS influx inside bacterial cell and, thus, its effectiveness. FosA enzymes were identified in 1980 from Serratia marcescens but only in recent years the scientific community has started studying their spread. We summarize the global epidemiology of FosA/ C2/L1-2 enzymes among Enterobacterales family. To date, 11 different variants of FosA have been reported globally. Among acquired mechanisms, FosA3 is the most spread variant in Enterobacterales, followed by FosA7 and FosA5. Based on recently published studies, we clarify and represent the molecular and genetic composition of fosA/C2 genes enviroment, analyzing the mechanisms by which such genes are slowly transmitting in emerging and high-risk clones, such as E. coli ST69 and ST131, and K. pneumoniae ST11. FOS is indicated as first line option against uncomplicated urinary tract infections and shows remarkable qualities in combination with other antibiotics. A rapid and accurate identification of FOSR type in Enterobacterales is difficult to achieve due to the lack of commercial phenotypic susceptibility tests and of rapid systems for MIC detection

OXA-48 and NDM-1 Klebsiella pneumoniae of Sequence Type 101 from blood in a patient with travel history abroad, Italy

Klebsiella pneumoniae (KP) is an important pathogen involved in serious nosocomial infections all over the world. Here, we describe the first report on a blood-stream infection caused by an OXA-48/NDM-1 ST101-KP, in Italy. The patient was an Italian woman, transferred from Cairo Hospital to a Neurosurgery ward in Cuneo (IT). The detection described here enhances the need for an effective National infection control strategy in Italy

Genomic Insight of VIM-harboring IncA Plasmid from a Clinical ST69 Escherichia coli Strain in Italy

Background: VIM (Verona Integron-encoded Metallo-beta-lactamase) is a member of the Metallo-Beta-Lactamases (MBLs), and is able to hydrolyze all beta-lactams antibiotics, except for monobactams, and including carbapenems. Here we characterize a VIM-producing IncA plasmid isolated from a clinical ST69 Escherichia coli strain from an Italian Long-Term Care Facility (LTCF) inpatient. Methods: An antimicrobial susceptibility test and conjugation assay were carried out, and the transferability of the blaVIM-type gene was confirmed in the transconjugant. Whole-genome sequencing (WGS) of the strain 550 was performed using the Sequel I platform. Genome assembly was performed using "Microbial Assembly". Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases. Results: Assembly resulted in three complete circular contigs: the chromosome (4,962,700 bp), an IncA plasmid (p550_IncA_VIM_1; 162,608 bp), harboring genes coding for aminoglycoside resistance (aac(6')-Ib4, ant(3″)-Ia, aph(3″)-Ib, aph(3')-XV, aph(6)-Id), beta-lactam resistance (blaSHV-12, blaVIM-1), macrolides resistance (mph(A)), phenicol resistance (catB2), quinolones resistance (qnrS1), sulphonamide resistance (sul1, sul2), and trimethoprim resistance (dfrA14), and an IncK/Z plasmid (p550_IncB_O_K_Z; 100,306 bp), free of antibiotic resistance genes. Conclusions: The increase in reports of IncA plasmids bearing different antimicrobial resistance genes highlights the overall important role of IncA plasmids in disseminating carbapenemase genes, with a preference for the blaVIM-1 gene in Italy

Genomic Characterization of VIM and MCR Co-Producers: The First Two Clinical Cases, in Italy

Background: the co-production of carbapenemases and mcr-genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy. Methods: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the bla(VIM-) and mcr- type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio). Results: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the bla(VIM-1) determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and bla(VIM-1) on a non-conjugative IncA plasmid. Conclusions: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy