Evidence review and recommendations for the implementation of genomics for antimicrobial resistance surveillance: reports from an international expert group.

Nearly a century after the beginning of the antibiotic era, which has been associated with unparalleled improvements in human health and reductions in mortality associated with infection, the dwindling pipeline for new antibiotic classes coupled with the inevitable spread of antimicrobial resistance (AMR) poses a major global challenge. Historically, surveillance of bacteria with AMR typically relied on phenotypic analysis of isolates taken from infected individuals, which provides only a low-resolution view of the epidemiology behind an individual infection or wider outbreak. Recent years have seen increasing adoption of powerful new genomic technologies with the potential to revolutionise AMR surveillance by providing a high-resolution picture of the AMR profile of the bacteria causing infections and providing real-time actionable information for treating and preventing infection. However, many barriers remain to be overcome before genomic technologies can be adopted as a standard part of routine AMR surveillance around the world. Accordingly, the Surveillance and Epidemiology of Drug-resistant Infections Consortium convened an expert working group to assess the benefits and challenges of using genomics for AMR surveillance. In this Series, we detail these discussions and provide recommendations from the working group that can help to realise the massive potential benefits for genomics in surveillance of AMR.

Genotypic Evolution of Klebsiella pneumoniae Sequence Type 512 during Ceftazidime/Avibactam, Meropenem/Vaborbactam, and Cefiderocol Treatment, Italy.

In February 2022, a critically ill patient colonized with a carbapenem-resistant K. pneumoniae producing KPC-3 and VIM-1 carbapenemases was hospitalized for SARS-CoV-2 in the intensive care unit of Policlinico Umberto I hospital in Rome, Italy. During 95 days of hospitalization, ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol were administered consecutively to treat 3 respiratory tract infections sustained by different bacterial agents. Those therapies altered the resistome of K. pneumoniae sequence type 512 colonizing or infecting the patient during the hospitalization period. In vivo evolution of the K. pneumoniae sequence type 512 resistome occurred through plasmid loss, outer membrane porin alteration, and a nonsense mutation in the cirA siderophore gene, resulting in high levels of cefiderocol resistance. Cross-selection can occur between K. pneumoniae and treatments prescribed for other infective agents. K. pneumoniae can stably colonize a patient, and antimicrobial-selective pressure can promote progressive K. pneumoniae resistome evolution, indicating a substantial public health threat.

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

Isolation and Characterisation of Human-Derived blaKPC-3-Producing Salmonella enterica Serovar Rissen in 2018.

In this study, we describe a Salmonella enterica serovar (S.) Rissen strain with a reduced susceptibility to meropenem, isolated from a urinary infection in an 89-year-old woman in 2018 during activity surveillance in Italy (Enter-Net Italia). The genomic characteristics, pathogenicity, and antimicrobial resistance mechanisms were investigated via a genomic approach. Antimicrobial susceptibility testing revealed a "susceptible, increased exposure" phenotype to meropenem in the S. Rissen strain (4_29_19). Whole-genome sequencing (WGS) was performed using both the NovaSeq 6000 S4 PE150 XP platform (Illumina, San Diego, CA, USA) and MinION (Oxford Nanopore). The S. Rissen 4_29_19 strain harboured two plasmids: a pKpQIL-like plasmid carrying the blaKPC-3 resistance gene in a Tn4401a transposon (pKPC_4_29_19), and a ColE-like plasmid (p4_4_29_19) without resistance genes, highly prevalent among Enterobacterales. Comparative analysis revealed that the pKPC_4_29_19 plasmid was highly related to the pKpQIL reference plasmid (GU595196), with 57% coverage and 99.96% identity, but lacking a region of about 30 kb, involving the FIIK2 replicon region and the entire transfer locus, causing the loss of its ability to conjugate. To our knowledge, this is the first time that a pKpQIL-like plasmid, carrying blaKPC-3, highly diffused in Klebsiella pneumoniae strains, has been identified in a Salmonella strain in our country. The acquisition of blaKPC genes by Salmonella spp. is extremely rare, and is reported only sporadically. In zoonotic bacteria isolated from humans, the presence of a carbapenem resistance gene carried by mobile genetic elements, usually described in healthcare-associated infection bacteria, represents an important concern for public health.

Multiplicity of blaKPC Genes and pKpQIL Plasmid Plasticity in the Development of Ceftazidime-Avibactam and Meropenem Coresistance in Klebsiella pneumoniae Sequence Type 307.

In 2021, Klebsiella pneumoniae sequence type 307 (ST307) strains causing pulmonary and bloodstream infections identified in a hospital in Rome, Italy, reached high levels of resistance to ceftazidime-avibactam (CZA). One of these strains reached high levels of resistance to both CZA and carbapenems and carried two copies of blaKPC-3 and one copy of blaKPC-31 located on plasmid pKpQIL. The genomes and plasmids of CZA-resistant ST307 strains were analyzed to identify the molecular mechanisms leading to the evolution of resistance and compared with ST307 genomes at local and global levels. A complex pattern of multiple plasmids in rearranged configurations, coresident within the CZA-carbapenem-resistant K. pneumoniae strain, was observed. Characterization of these plasmids revealed recombination and segregation events explaining why K. pneumoniae isolates from the same patient had different antibiotic resistance profiles. This study illustrates the intense genetic plasticity occurring in ST307, one of the most worldwide-diffused K. pneumoniae high-risk clones.

Genome-Based Retrospective Analysis of a Providencia stuartii Outbreak in Rome, Italy: Broad Spectrum IncC Plasmids Spread the NDM Carbapenemase within the Hospital.

Providencia stuartii is a member of the Morganellaceae family, notorious for its intrinsic resistance to several antibiotics, including last-resort drugs such as colistin and tigecycline. Between February and March 2022, a four-patient outbreak sustained by P. stuartii occurred in a hospital in Rome. Phenotypic analyses defined these strains as eXtensively Drug-Resistant (XDR). Whole-genome sequencing was performed on the representative P. stuartii strains and resulted in fully closed genomes and plasmids. The genomes were highly related phylogenetically and encoded various virulence factors, including fimbrial clusters. The XDR phenotype was primarily driven by the presence of the blaNDM-1 metallo-ß-lactamase alongside the rmtC 16S rRNA methyltransferase, conferring resistance to most ß-lactams and every aminoglycoside, respectively. These genes were found on an IncC plasmid that was highly related to an NDM-IncC plasmid retrieved from a ST15 Klebsiella pneumoniae strain circulating in the same hospital two years earlier. Given its ability to acquire resistance plasmids and its intrinsic resistance mechanisms, P. stuartii is a formidable pathogen. The emergence of XDR P. stuartii strains poses a significant public health threat. It is essential to monitor the spread of these strains and develop new strategies for their control and treatment.

Ceftazidime-avibactam resistance in Klebsiella pneumoniae sequence type 37: a decade of persistence and concealed evolution.

The first reports of carbapenem-resistant Enterobacterales in our hospital date back to 2006. In that period, few ertapenem-resistant but meropenem-susceptible Klebsiella pneumoniae isolates belonging to sequence type (ST) 37 were retrieved from clinical samples. These strains produced the CTX-M-15 extended spectrum ß-lactamase, OmpK35 was depleted due to a nonsense mutation, and a novel OmpK36 variant was identified. Yet, starting from 2010, Klebsiella pneumoniae carbapenemase (KPC)-producing ST512 isolates started prevailing and ST37 vanished from sight. Since 2018 the clinical use of the combination of ceftazidime-avibactam (CZA) has been introduced in clinical practice for the treatment of bacteria producing serine-ß-lactamases, but KPC-producing, CZA-resistant K. pneumoniae are emerging. In 2021, four CZA-resistant ST37 isolates producing KPC variants were isolated from the same number of patients. blaKPC gene cloning in Escherichia coli was used to define the role of those KPC variants on CZA resistance, and whole genome sequencing was performed on these isolates and on three ST37 historical isolates from 2011. CZA resistance was due to mutations in the blaKPC genes carried on related pKpQIL-type plasmids, and three variants of the KPC enzyme have been identified in the four ST37 strains. The four ST37 isolates were closely related to each other and to the historical isolates, suggesting that ST37 survived without notice in our hospital for 10 years, waiting to re-emerge as a CZA-resistant K. pneumoniae clone. The ancestor of these contemporary isolates derives from ST37 wild-type porin strains, with no other mutations in chromosomal genes involved in conferring antibiotic resistance (parC, gyrA, ramR, mgrB, pmrB).

Global spread and evolutionary convergence of multidrug-resistant and hypervirulent Klebsiella pneumoniae high-risk clones.

For people living in developed countries life span is growing at a faster pace than ever. One of the main reasons for such success is attributable to the introduction and extensive use in the clinical practice of antibiotics over the course of the last seven decades. In hospital settings, Klebsiella pneumoniae represents a well-known and commonly described opportunistic pathogen, typically characterized by resistance to several antibiotic classes. On the other hand, the broad wedge of population living in Low and/or Middle Income Countries is increasing rapidly, allowing the spread of several commensal bacteria which are transmitted via human contact. Community transmission has been the original milieu of K. pneumoniae isolates characterized by an outstanding virulence (hypervirulent). These two characteristics, also defined as "pathotypes", originally emerged as different pathways in the evolutionary history of K. pneumoniae. For a long time, the Sequence Type (ST), which is defined by the combination of alleles of the 7 housekeeping genes of the Multi-Locus Sequence Typing, has been a reliable marker of the pathotype: multidrug-resistant clones (e.g. ST258, ST147, ST101) in the Western world and hypervirulent clones (e.g. ST23, ST65, ST86) in the Eastern. Currently, the boundaries separating the two pathotypes are fading away due to several factors, and we are witnessing a worrisome convergence in certain high-risk clones. Here we review the evidence available on confluence of multidrug-resistance and hypervirulence in specific K. pneumoniae clones.

An outbreak sustained by ST15 Klebsiella pneumoniae carrying 16S rRNA methyltransferases and blaNDM: evaluation of the global dissemination of these resistance determinants.

The spread of extremely-drug-resistant Klebsiella pneumoniae has become a major health threat worldwide. This is largely mediated by certain lineages, recognized as high-risk clones dispersed throughout the world. Analysis of an outbreak of nine ST15, NDM-1 metallo-ß-lactamase-producing K. pneumoniae was performed. An IncC plasmid carrying the blaNDM-1 gene also carried the rare rmtC gene, encoding for 16S rRNA methyltransferases (16RMTases), conferring resistance to all aminoglycosides. The global spread of New Delhi metallo (NDM) variants and their association with the 16RMTases among K. pneumoniae complete genomes available in GenBank was studied, and a complete overview of the association of 16RMTases and NDM in K. pneumoniae genomics was produced. NDM is often associated with16RMTases, and both are spreading in K. pneumoniae, conferring resistance to all beta-lactams and aminoglycosides. This analysis suggests that aminoglycosides have a limited future as a second-line treatment against NDM-producing K. pneumoniae.

Virulence plasmid pINV as a genetic signature for Shigella flexneri phylogeny.

Shigella flexneri is a major health burden in low- and middle-income countries, where it is a leading cause of mortality associated with diarrhoea in children, and shows an increasing incidence among travellers and men having sex with men. Like all Shigella spp., S. flexneri has evolved from commensal Escherichia coli following the acquisition of a large plasmid pINV, which contains genes essential for virulence. Current sequence typing schemes of Shigella are based on combinations of chromosomal genetic loci, since pINV-encoded virulence genes are often lost during growth in the laboratory, making these elements inappropriate for sequence typing. By performing comparative analysis of pINVs from S. flexneri strains isolated from different geographical regions and belonging to different serotypes, we found that in contrast to plasmid-encoded virulence genes, plasmid maintenance genes are highly stable pINV-encoded elements. For the first time, to our knowledge, we have developed a S. flexneri plasmid multilocus sequence typing (pMLST) method based on different combinations of alleles of the vapBC and yacAB toxin-antitoxin (TA) systems, and the parAB partitioning system. This enables typing of S. flexneri pINV plasmids into distinct 'virulence sequence types' (vSTs). Furthermore, the phylogenies of vST alleles and bacterial host core genomes suggests an intimate co-evolution of pINV with the chromosome of its bacterial host, consistent with previous findings. This work demonstrates the potential of plasmid maintenance loci as genetic characteristics to study as well as to trace the molecular phylogenesis of S. flexneri pINV and the phylogenetic relationship of this plasmid with its bacterial host.

Consensus on ß-Lactamase Nomenclature.

Assigning names to ß-lactamase variants has been inconsistent and has led to confusion in the published literature. The common availability of whole genome sequencing has resulted in an exponential growth in the number of new ß-lactamase genes. In November 2021 an international group of ß-lactamase experts met virtually to develop a consensus for the way naturally-occurring ß-lactamase genes should be named. This document formalizes the process for naming novel ß-lactamases, followed by their subsequent publication.

Colistin Resistance Mechanisms in Human Salmonella enterica Strains Isolated by the National Surveillance Enter-Net Italia (2016-2018).

BACKGROUND: A collection of human-epidemiologically unrelated S. enterica strains collected over a 3-year period (2016 to 2018) in Italy by the national surveillance Enter-Net Italia was analysed. METHODS: Antimicrobial susceptibility tests, including the determination of minimal inhibitory concentrations (MICs) for colistin, were performed. Colistin resistant strains were analysed by PCR to detect mobile colistin resistance (mcr) genes. In mcr-negative S. enterica serovar Enteritidis strains, chromosomal mutations potentially involved in colistin resistance were identified by a genomic approach. RESULTS: The prevalence of colistin-resistant S. enterica strains was 7.7%, the majority (87.5%) were S. Enteritidis. mcr genes were identified only in one strain, a S. Typhimurium monophasic variant, positive for both mcr-1.1 and mcr-5.1 genes in an IncHI2 ST4 plasmid. Several chromosomal mutations were identified in the colistin-resistant mcr-negative S. Enteritidis strains in proteins involved in lipopolysaccharide and outer membrane synthesis and modification (RfbN, LolB, ZraR) and in a component of a multidrug efflux pump (MdsC). These mutated proteins were defined as possible candidates for colistin resistance in mcr-negative S. Enteritidis of our collection. CONCLUSIONS: The colistin national surveillance in Salmonella spp. in humans, implemented with genomic-based surveillance, permitted to monitor colistin resistance, determining the prevalence of mcr determinants and the study of new candidate mechanisms for colistin resistance.

Interplay between Klebsiella pneumoniae producing KPC-31 and KPC-3 under treatment with high dosage meropenem: a case report.

The objective was to study ceftazidime-avibactam resistant and susceptible Klebsiella pneumoniae isolated from a patient admitted to the Policlinico Umberto I of Rome for SARS-CoV2. Data on the evolution of patient's conditions, antimicrobial therapies, and microbiological data were collected. Whole-genome sequencing performed by Illumina and Nanopore sequencing methods were used to type the strains. During the hospitalization, a SARS-CoV2-infected patient was colonized by a KPC-producing K. pneumoniae strain and empirically treated with ceftazidime-avibactam (CZA) when presenting spiking fever symptoms. Successively, ST2502 CZA-resistant strain producing the KPC-31 variant gave a pulmonary infection to the patient. The infection was treated with high doses of meropenem. The KPC-31-producing strain disappeared but the patient remained colonized by a KPC-3-producing K. pneumoniae strain. An interplay between highly conserved KPC-31- and KPC-3-producing ST2502 strains occurred in the SARS-CoV2 patient during the hospitalization, selected by CZA and carbapenem treatments, respectively.

Spread of hypervirulent multidrug-resistant ST147 Klebsiella pneumoniae in patients with severe COVID-19: an observational study from Italy, 2020-21.

OBJECTIVES: To report an outbreak of hypervirulent Klebsiella pneumoniae (hvKp) in COVID-19 patients. METHODS: Prospective, observational study including consecutive COVID-19 patients with hvKp infections admitted to the University Hospital of Pisa (Italy). Clinical data and outcome of patients were collected. All patients were followed-up to 30 days from the diagnosis of infection. Mortality within 30 days of the diagnosis of hvKp infection was reported. The hypermucoviscous phenotype was determined by the 'string test'. Molecular typing was performed on three strains collected during different periods of the outbreak. The strains underwent whole genome sequencing using the Illumina MiSeq instrument. The complete circular assemblies were also obtained for the chromosome and a large plasmid using the Unicycler tool. RESULTS: From November 2020 to March 2021, hvKp has been isolated from 36 COVID-19 patients: 29/36 (80.6%) had infections (15 bloodstream infections, 8 ventilator-associated pneumonias and 6 complicated urinary tract infections), while 7/36 (19.4%) had colonization (3 urine, 2 rectal and 2 skin). The isolates belonged to ST147 and their plasmid carried three replicons of the IncFIB (Mar), IncR and IncHI1B types and several resistance genes, including the rmpADC genes encoding enhancers of capsular synthesis. The hvKp isolates displayed an ESBL phenotype, with resistance to piperacillin/tazobactam and ceftolozane/tazobactam and susceptibility only to meropenem and ceftazidime/avibactam. The majority of patients were treated with meropenem alone or in combination with fosfomycin. Thirty-day mortality was 48.3% (14/29). CONCLUSIONS: ST147 ESBL-producing hvKp is associated with high mortality in COVID-19 patients. Strict microbiological surveillance and infection control measures are needed in this population.

Evolutionary Trajectories toward Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Clinical Isolates.

From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA)-resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the sequence type 512 (ST512), ST101, and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among the nine, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69, and KPC-70 showed amino acid substitutions, insertions, and deletions in the Ω loop of the protein. KPC-29 has a duplication, while the novel KPC-67 has a triplication, of the KDD triplet in the 270-loop, a secondary loop of the KPC-3 protein. Genomics performed on contemporary resistant and susceptible clones underlined that these novel mutations emerged in blaKPC-3 genes located on conserved plasmids: in ST512, all blaKPC-3 mutant genes were located in pKpQIL plasmids, while the three novel blaKPC-3 mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy-number pTOPO vector, the Ω loop mutated variants showed the CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.

Meropenem-Vaborbactam as Salvage Therapy for Ceftazidime-Avibactam-, Cefiderocol-Resistant ST-512 Klebsiella pneumoniae-Producing KPC-31, a D179Y Variant of KPC-3.

A 68-year-old man had recurrent bacteremia by Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae resistant to ceftazidime-avibactam and cefiderocol. The sequencing of a target region showed that it harbored a KPC-3 variant enzyme (D179Y; KPC-31), which confers resistance to ceftazidime-avibactam and restores meropenem susceptibility. The patient was successfully treated with meropenem-vaborbactam.

Molecular epidemiology of NDM-5-producing Escherichia coli high-risk clones identified in two Italian hospitals in 2017-2019.

Between November 2018 and October 2019, carbapenem-resistant Enterobacterales carrying New Delhi Metallo-ß-lactamase (NDM) caused one of the largest and persistent outbreaks occurred in Italy and intensified surveillance measures have been taken in all Italian hospitals. In this study we analyzed NDM-5- producing Escherichia coli identified in 2 hospitals of the Lazio region in Italy. Epidemiological and microbiological data demonstrated that in 2018-2019 the NDM-5-producing high-risk E. coli ST167 clone circulated in patients from both hospitals. In 2019, another NDM-5-producing E. coli clone, identified by MLST as ST617 was introduced in one of the 2 hospitals and caused an outbreak. This study describes an application of genomics as a useful method to discern endemic and outbreak clones when applied to strains of the same species (E. coli) with the same resistance determinant (NDM-5) and the relevance of screening patients admitted in critical units for carbapenemase producers to prevent outbreaks.

First evidence of blaNDM-1 and blaOXA-23 carbapenemase genes in human body lice infesting a second-hand T-shirt in a street market in Italy.

BACKGROUND: The spread of carbapenems resistance is a public health concern. The main group of carbapenemases encoding the ß-lactamases activity (bla genes) is the Metallo-ß-lactamases (MBLs). METHODS: The presence of carbapenemase blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, blaOXA-58-like, and blaNDM-1 genes was screened by real time PCR in 26 Pediculus humanus insects identified from second-hand clothes in a local market in Central Italy. Bacteria diversity was also characterized through shotgun metagenomic amplification for a deep sequencing of the host-associated bacterial microbiomes. RESULTS: The blaOXA-23 and blaNDM-1 carbapenemases genes were found and metagenomic analysis showed a great presence of Acinetobacter species. CONCLUSIONS: These results suggest a new potential transmission path for carbapenemase gene spread through bacteria ingested by insects infesting humans.