The ICU environment contributes to the endemicity of the "Serratia marcescens complex" in the hospital setting.

Serratia marcescens is an opportunistic pathogen historically associated with sudden outbreaks in intensive care units (ICUs) and the spread of carbapenem-resistant genes. However, the ecology of S. marcescens populations in the hospital ecosystem remains largely unknown. We combined epidemiological information of 1,432 Serratia spp. isolates collected from sinks of a large ICU that underwent demographic and operational changes (2019-2021) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003-2019) with 165 genomic data. These genomes were grouped into clades (1-4) and subclades (A and B) associated with distinct species: Serratia nematodiphila (1A), S. marcescens (1B), Serratia bockelmannii (2A), Serratia ureilytica (2B), S. marcescens/Serratia nevei (3), and S. nevei (4A and 4B). They may be classified into an S. marcescens complex (SMC) due to the similarity between/within subclades (average nucleotide identity >95%-98%), with clades 3 and 4 predominating in our study and publicly available databases. Chromosomal AmpC ß-lactamase with unusual basal-like expression and prodigiosin-lacking species contrasted classical features of Serratia. We found persistent and coexisting clones in sinks of subclades 4A (ST92 and ST490) and 4B (ST424), clonally related to outbreak isolates carrying blaVIM-1 or blaOXA-48 on prevalent IncL/pB77-CPsm plasmids from our hospital since 2017. The distribution of SMC populations in ICU sinks and patients reflects how Serratia species acquire, maintain, and enable plasmid evolution in both "source" (permanent, sinks) and "sink" (transient, patients) hospital patches. The results contribute to understanding how water sinks serve as reservoirs of Enterobacterales clones and plasmids that enable the persistence of carbapenemase genes in healthcare settings, potentially leading to outbreaks and/or hospital-acquired infections.IMPORTANCEThe "hospital environment," including sinks and surfaces, is increasingly recognized as a reservoir for bacterial species, clones, and plasmids of high epidemiological concern. Available studies on Serratia epidemiology have focused mainly on outbreaks of multidrug-resistant species, overlooking local longitudinal analyses necessary for understanding the dynamics of opportunistic pathogens and antibiotic-resistant genes within the hospital setting. This long-term genomic comparative analysis of Serratia isolated from the ICU environment with isolates causing nosocomial infections and/or outbreaks within the same hospital revealed the coexistence and persistence of Serratia populations in water reservoirs. Moreover, predominant sink strains may acquire highly conserved and widely distributed plasmids carrying carbapenemase genes, such as the prevalent IncL-pB77-CPsm (pOXA48), persisting in ICU sinks for years. The work highlights the relevance of ICU environmental reservoirs in the endemicity of certain opportunistic pathogens and resistance mechanisms mainly confined to hospitals.

Enterobacter asburiae ST229: an emerging carbapenemases producer.

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. "Policlinico San Matteo" in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.

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.

First Case of VIM-1-like-Producing Pseudomonas putida Bacteremia in an Oncohematological Pediatric Patient in Italy.

Bacterial infections caused by multidrug-resistant (MDR) Gram-negatives are of great concern worldwide, as they are frequently associated with high mortality and morbidity rates. To date, two cases of VIM-2 metallo-ß-lactamase (MBL)-producing Pseudomonas putida bacteremia have been ever reported in France and Spain between 2004 and 2010. Here, we present the first case of VIM-1-like-producing P. putida isolated in blood culture collected from an oncohematological pediatric patient admitted to Bambino Gesù Children's Hospital (IRCCS) in Rome, Italy.

Antimicrobial Activity of Cefiderocol against the Carbapenemase-Producing Enterobacter cloacae Complex and Characterization of Reduced Susceptibility Associated with Metallo-ß-Lactamase VIM-1.

Emergence of cefiderocol resistance among carbapenemase-producing Enterobacterales, particularly those in the Enterobacter cloacae complex (ECC), is becoming of alarming concern; however, the mechanistic basis of this phenomenon remains poorly understood. We describe the acquisition of VIM-1-mediated reduced cefiderocol susceptibility (MICs 0.5 to 4 mg/L) in a collection of 54 carbapenemase-producing isolates belonging to the ECC. MICs were determined by reference methodologies. Antimicrobial resistance genomic analysis was performed through hybrid WGS. The impact of VIM-1 production on cefiderocol resistance in the ECC background was examined at microbiological, molecular, biochemical, and atomic levels. Antimicrobial susceptibility testing yielded 83.3% susceptible isolates and MIC50/90 values of 1/4 mg/L. Decreased susceptibility to cefiderocol was mainly associated with isolates producing VIM-1, with cefiderocol MICs 2- to 4-fold higher than for isolates carrying other types of carbapenemases. E. cloacae and Escherichia coli VIM-1 transformants displayed significantly enhanced cefiderocol MICs. Biochemical assays with purified VIM-1 protein revealed low but detectable cefiderocol hydrolysis. Simulation studies revealed how cefiderocol is anchored to the VIM-1 active site. Additional molecular assays and WGS data analysis highlighted the implication of SHV-12 coproduction and suggested the inactivation of the FcuA-like siderophore receptor as further contributors to the higher cefiderocol MICs. Our findings warn of the potential of the VIM-1 carbapenemase to at least partly limit the activity of cefiderocol in the ECC. This effect is probably enhanced due to combination with additional mechanisms, such as ESBL production and siderophore inactivation, and indicates the need for active surveillance to extend the life span of this promising cephalosporin.

The Arabidopsis APOLO and human UPAT sequence-unrelated long noncoding RNAs can modulate DNA and histone methylation machineries in plants.

BACKGROUND: RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. RESULTS: Here, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner. CONCLUSIONS: Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.

Exploring the Global Spread of Klebsiella grimontii Isolates Possessing blaVIM-1 and mcr-9.

The spread of plasmid-mediated carbapenemases within Klebsiella oxytoca is well-documented. In contrast, data concerning the closely related species Klebsiella grimontii are scarce. In fact, despite the recent report of the first blaKPC-2-producing K. grimontii, nothing is known about its clonality and antibiotic resistance patterns. In a retrospective search in our collection, we identified 2 blaVIM-positive K. oxytoca strains. Whole-genome sequencing with both Illumina and Nanopore indicated that our strains actually belonged to K. grimontii and were of sequence type 172 (ST172) and ST189. Moreover, the two strains were associated with 297-kb IncHI2/HI2A-pST1 and 90.6-kb IncFII(Yp) plasmids carrying blaVIM-1 together with mcr-9 and blaVIM-1, respectively. In the IncHI2/HI2A plasmid, blaVIM-1 was located in a class 1 integron (In110), while mcr-9 was associated with the qseC-qseB-like regulatory elements. Overall, this plasmid was shown to be very similar to those carried by other Enterobacterales isolated from food and animal sources (e.g., Salmonella and Enterobacter spp. detected in Germany and Egypt). The IncFII(Yp) plasmid was unique, and its blaVIM-1 region was associated with a rare integron (In1373). Mapping of In1373 indicated a possible origin in Austria from an Enterobacter hormaechei carrying a highly similar plasmid. Core-genome phylogenies indicated that the ST172 K. grimontii belonged to a clone of identical Swedish and Swiss strains (≤15 single nucleotide variants [SNVs] to each other), whereas the ST189 strain was sporadic. Surveillance of carbapenemase-producing K. oxytoca strains should be reinforced to detect and prevent the dissemination of new species belonging to the Klebsiella genus.

Combination of aztreonam, ceftazidime-avibactam and amikacin in the treatment of VIM-1 Pseudomonas aeruginosa ST235 osteomyelitis.

We describe a challenging case of patient with metallo-beta-lactamase-producing Pseudomonas aeruginosa sternal osteomyelitis following aortic valve replacement with biological prosthesis. The strain exhibited a multidrug-resistance phenotype carrying the blaVIM-1 gene and belonged to the high-risk clone sequence type ST235. The patient was successfully treated with surgical debridement plus antibiotic therapy with ceftazidime/avibactam, aztreonam, and amikacin. Time-kill curves showed that this triple antibiotic combination at 1 × MIC was strongly synergic after 8 h, achieving 99.9% killing and maintaining this until 48 h.

Emerging peptide antibiotics with therapeutic potential.

This review covers some of the recent progress in the field of peptide antibiotics with a focus on compounds with novel or established mode of action and with demonstrated efficacy in animal infection models. Novel drug discovery approaches, linear and macrocyclic peptide antibiotics, lipopeptides like the polymyxins as well as peptides addressing targets located in the plasma membrane or in the outer membrane of bacterial cells are discussed.

Characterization of 16S rRNA methylase genes in Enterobacterales and Pseudomonas aeruginosa in Athens Metropolitan area, 2015-2016.

The aim of this study was to characterize the 16S rRNA methylase (RMT) genes in aminoglycoside-resistant Enterobacterales and Pseudomonas aeruginosa isolates in 2015-2016 in hospitals in Athens, Greece. Single-patient, Gram-negative clinical isolates resistant to both amikacin and gentamicin (n = 292) were consecutively collected during a two-year period (2015-2016) in five tertiary care hospitals in Athens. RMT genes were detected by PCR. In all RMT-producing isolates, ESBL and carbapenemase production was confirmed by PCR, and the clonal relatedness and the plasmid contents were also characterized. None of the 138 P. aeruginosa isolates harbored any of the RMT genes surveyed although some were highly resistant to aminoglycosides (MICs > = 512 mg/L). Among 154 Enterobacterales, 31 Providencia stuartii (93.9%), 42 Klebsiella pneumoniae (37.8%), six Proteus mirabilis (75%), and two Escherichia coli (100%) isolates were confirmed as highly resistant to amikacin, gentamicin, and tobramycin with MICs ≥ 512 mg/L, harboring mainly the rmtB (98.8%). All were carbapenemase producers. P. stuartii, P. mirabilis, and E. coli produced VIM-type carbapenemases. K. pneumoniae produced KPC- (n = 34, 81.0%), OXA-48 (n = 4, 9.5%), KPC- and VIM- (n = 3, 7.1%), or only VIM-type (n = 1, 2.4%) enzymes. Two groups of similar IncC plasmids were detected one harboring rmtB1, blaVEB-1, blaOXA-10, and blaTEM-1, and the other additionally blaVIM-1 and blaSHV-5. Among RMT-producing Enterobacterales, rmtB1 predominated and was associated with carbapenemase-encoding gene(s). Similar IncC plasmids carrying a multiresistant region, including ESBL genes, and in the case of VIM-producing isolates, the blaVIM-1, were responsible for this dissemination. The co-dissemination of these genes poses a public health threat.

Emergence of Enterobacter cloacae Complex Co-Producing IMP-10 and CTX-M, and Klebsiella pneumoniae Producing VIM-1 in Clinical Isolates in Japan.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging threat in healthcare settings worldwide. OBJECTIVES: We evaluated the presence of carbapenemase genes in CPE in a tertiary care university hospital in Tokyo, Japan. METHODS: Carbapenem-resistant clinical isolates were collected in 2018 at Teikyo University Hospital (Tokyo, Japan). Bacterial species were identified using MALDI-TOF MS. Carbapenemase production was evaluated using a carbapenemase inactivation method. The presence of carbapenemase genes was confirmed by multiplex PCR and DNA sequencing. RESULTS: Four CPE isolates were identified: two Enterobacter cloacae complex strains and Klebsiella oxytoca and Klebsiella pneumoniae strains. Three of the isolates (E. cloacae complex and K. oxytoca) were IMP-1-type producers, including IMP-10 in their produced metallo-ß-lactamase, and are epidemic in East Japan. The IMP-10-producing E. cloacae complex strain also produced CTX-M ESBL. The other CPE isolate (K. pneumoniae) is a VIM-1 producer. VIM-1-producing K. pneumoniae is epidemic in Europe, especially in Greece. Accordingly, the VIM-1 producer was isolated from a patient with a medical history in Greece. CONCLUSIONS: This study revealed the emergence of E. cloacae complex co-producing IMP-1-type carbapenemase and CTX-M ESBL, and K. pneumoniae producing VIM-1 carbapenemase in clinical isolates in Japan. Metallo-ß-lactamase was the most prevalent type of carbapenemase at Teikyo University Hospital, especially IMP-1-type carbapenemase. The detection of VIM-1-producing K. pneumoniae suggests that epidemic CPE from overseas can spread to countries with low CPE prevalence, such as Japan, highlighting the need for active surveillance.

First Genomic Characterization of blaVIM-1 and mcr-9-Coharbouring Enterobacter hormaechei Isolated from Food of Animal Origin.

We describe here the complete genome sequence of an Enterobacter hormaechei ST279 coharbouring blaVIM-1 and mcr-9 recovered from uncooked beef patty in June 2017, Egypt. The tested isolate was resistant to carbapenem but susceptible to colistin (minimum inhibitory concentration (MIC), 0.5 µg/mL). The antimicrobial susceptibility profile and conjugation experiments were performed. The entire genome was sequenced by the Illumina MiniSeq and Oxford Nanopore methods. The blaVIM-1 and mcr-9 genes are carried on the same IncHI2/pMLST1 plasmid, pMS37a (Size of 270.9 kb). The mcr-9 gene was located within the physical boundaries demarcated by two insertion elements IS903 (upstream) and IS1 (downstream) but did not possess the downstream regulatory genes (qseC/qseB) which regulate the expression of mcr-9. Therefore, the mcr-9 might be silently disseminated among carbapenem-resistant Enterobacterales. In addition to blaVIM-1 and mcr-9, plasmid pMS37a harbored various antibiotic resistance genes including aac(6')-Il, ΔaadA22, aac(6')-Ib-cr, sul1, dfrA1 and tetA. To the best of our knowledge, this is the first report of a blaVIM-1 and mcr-9-coharbouring E. hormaechei isolate of food origin worldwide. The identification of a multidrug-resistant VIM-1 and mcr-9 positive Enterobacter hormaechei isolate from food is worrisome as retail meat and meat products could serve as a vehicle for these MDR bacteria, which could be transferred between animals and humans through the food chain. It further highlights that Enterobacterales co-producing MCR and carbapenemases being found in the food chain indeed correspond to a One-Health issue, highlighting the need for serious steps to prevent their further dissemination.

Early OXA-48-Producing Enterobacterales Isolates Recovered in a Spanish Hospital Reveal a Complex Introduction Dominated by Sequence Type 11 (ST11) and ST405 Klebsiella pneumoniae Clones.

Carbapenemase-producing Enterobacterales (CPE) have become an important public health concern. In our hospital, VIM enzymes were first detected in 2005, Klebsiella pneumoniae carbapenemase (KPC) enzymes in 2009, and OXA-48 enzymes in 2012. We assess the population biology of the first OXA-48-producing Enterobacterales isolates recovered in our hospital (2012 to 2013) where infections by other carbapenemases had been endemic for several years. Over a 21-month period, 71 isolates (61 Klebsiella pneumoniae, 5 Escherichia coli, 2 Klebsiella aerogenes, and 1 each of Enterobacter cloacae, Klebsiella oxytoca, and Citrobacter amalonaticus) recovered from clinical and surveillance specimens from 57 patients (22.8% nonhospitalized) were investigated for OXA-48-like-producing enzymes. Analyses for characterization and determination of the location of the blaOXA-48 gene, plasmid transferability, sequence, and clonal relatedness were performed. Most of the isolates also coproduced CTX-M-15 (57/71, 80.3%) and/or VIM-1 (7/71, 9.8%). K. pneumoniae was predominantly identified as sequence type 11 (ST11) (63.4%) and ST405 (9.8%) and E. coli as ST540, ST1406, ST3163, and ST4301. The blaOXA-48 gene was part of Tn1999.2 located at the tir gene of plasmids (ca. ≥50 kb) of the IncL/M group, also carrying blaVIM-1 and blaCTX-M-15 genes. We selected one ST11 K. pneumoniae isolate for whole-genome sequencing in which we studied the plasmid containing the blaOXA-48 gene. This plasmid was compared with indexed plasmids existing in NCBI database by the use of BRIG and MAUVE. Our data suggest a rapid spread of blaOXA-48 genes between commonly isolated high-risk clones of Enterobacterales species, frequently associated with antibiotic resistance. Moreover, the emergence of the multiresistant ST11 K. pneumoniae clone among nonhospitalized patients emphasizes the difficulty of preventing its dissemination into the community.IMPORTANCE We present results of microbiological analysis of the first Enterobacterales isolates that were isolated in 2012 in our institution expressing OXA-48 carbapenemase. This enzyme confers resistance to carbapenems, an important group of antibiotics widely used in the hospitals. OXA-48 carbapenemase is currently present in many parts of the world, but it is found particularly frequently in the Mediterranean area. It was disseminated at the Ramón y Cajal Hospital and found to be associated with a particular Klebsiella pneumoniae strain, so-called high-risk clone ST11, which was previously found in our institution in association with other enzymes such as CTX-M-15, VIM-1, and KPC-3. This clone might have acquired a plasmid harboring the blaOXA-48 gene. Our results point out the importance of local epidemiology in the dissemination and maintenance of multidrug-resistant bacteria.

Genomic characterization of Citrobacter freundii strains coproducing OXA-48 and VIM-1 carbapenemase enzymes isolated in leukemic patient in Spain.

Background: The emergence of carbapenemase-producing (CP) Citrobacter freundii poses a significant threat to public health, especially in high-risk populations. In this study, whole genome sequencing was used to characterize the carbapenem resistance mechanism of three C. freundii clinical isolates recovered from fecal samples of patients with acute leukemia (AL) from Spain. Materials and methods: Twelve fecal samples, collected between 2013 and 2015 from 9 patients with AL, were screened for the presence of CP strains by selecting them on MacConkey agar supplemented with ertapenem (0.5 mg/L). Bacteria were identified by MALDI-TOF mass spectrometry and were phenotypically characterized. Whole genome sequencing of C. freundii isolates was performed using the MinION and MiSeq Illumina sequencers. Bioinformatic analysis was performed in order to identify the molecular support of carbapenem resistance and to study the genetic environment of carbapenem resistance encoding genes. Results: Three carbapenem-resistant C. freundii strains (imipenem MIC≥32 mg/L) corresponding to three different AL patients were isolated. Positive modified Carba NP test results suggested carbapenemase production. The genomes of each C. freundii tested were assembled into a single chromosomal contig and plasmids contig. In all the strains, the carbapenem resistance was due to the coproduction of OXA-48 and VIM-1 enzymes encoded by genes located on chromosome and on an IncHI2 plasmid, respectively. According to the MLST and the SNPs analysis, all strains belonged to the same clone ST169. Conclusion: We report in our study, the intestinal carrying of C. freundii clone ST169 coproducing OXA-48 and VIM-1 identified in leukemic patients.

Characterization of VIM-1-Producing E. coli Isolated From a German Fattening Pig Farm by an Improved Isolation Procedure.

A few reports indicate that livestock might represent a new reservoir for carbapenemase-producing Enterobacteriaceae (CPE). In 2015, VIM-1-producing Escherichia coli were detected at slaughter in colon contents of animals from a German fattening pig farm within the national monitoring on ESBL-producing E. coli. In this study, pooled faces samples from pigs, as well as samples from the barn surrounding environment of this fattening farm were taken, to evaluate the dissemination of CPEs. Several modifications of the culture-dependent detection procedure were investigated for their potential to improve the sensitivity of the CPE isolation method. The current reference procedure was adapted by adding a real-time PCR pre-screening and additional enrichment steps. It was possible to isolate 32 VIM-1-producing E. coli from four fecal samples of three different barns using two serial enrichment steps in combination with real-time PCR and selective agar plates. By genetic typing, we confirmed the presence of two E. coli clonal lineages circulating on this particular farm: one was harboring the bla VIM- 1 on an IncHI2 plasmid while the second lineage carried the gene on the chromosome. Despite its different locations, the bla VIM- 1 gene was harbored on a class 1 integron in both clonal lineages. Whole-genome sequencing revealed that the VIM-1-carrying plasmids exhibited only slight variability in its compositions and sizes. We assume that the prevalence of CPEs in animal production in Germany and other European countries might be underestimated and there is a concern of further spread of VIM-1-producing bacteria in German livestock and food.

Increase and diversity of carbapenemase-producing Escherichia coli isolates, Italy.

Aim: This study reports on a surveillance in an Italian hospital focused on carbapenemase-producing Escherichia coli (CP-Ec). Materials & methods: Eighteen isolates (nine from clinical specimens and nine from rectal swab) were characterized for antibiotic susceptibilities, typing features, main carbapenemase, extended-spectrum ß-lactamases (ESBLs) and other bla genes, and their transferability by conjugation and transformation. Results: An increase in CP-Ec isolates was observed during 3-year surveillance period. Compared with the clinical isolates, all belonging to one sequence type (ST), ST131, those from rectal swab were very heterogeneous and belonged to eight STs. Transfer data confirmed the role of conjugative plasmids in the spreading of carbapenemase genes. Conclusion: The prevalence of CP-Ec in Italy has risen, with a substantial increase over the last year.

First Report of NDM and VIM Coproducing Klebsiella pneumoniae in Tunisia and Emergence of Novel Clones.

Metallo-ß-lactamase (MBL) producing bacteria constitute nowadays a serious global concern worldwide. The purpose of our present study was to characterize molecular features of MBL producing bacteria and to identify the existing clones in our area. Thirteen MBL-producing-Klebsiella pneumoniae were detected in clinical samples from patients hospitalized in the Military hospital of Tunisia during 2017. The molecular research by polymerase chain reaction and sequencing of gene encoding MBL enzymes showed that only two types were identified in our study: blaNDM-1 and blaVIM-1 genes detected, respectively, in eight and six isolates. An association between these two MBL genes (blaNDM-1+blaVIM-1) has been observed in one of our isolates. Other ß-lactamase types (CTXM-15/4 isolates; SHV/2 isolates; OXA-48/3 isolates) were detected in association with New Delhi metallo-beta-lactamase (NDM) and/or Verona Integron-Mediated Metallo-ß-lactamase (VIM) enzymes. Furthermore, these isolates were resistant to other antimicrobial agents, including gentamicin [aac(3)-II/11 isolates], tetracycline (tetB or tetA/2 isolates), chloramphenicol (cmlA and/or floR/3 isolates), streptomycin (aadA/5 isolates), and sulfonamides (sul1 or sul2 or sul3/4isolates). The Multilocus Sequence Typing revealed 10 different Sequence types (ST) of which 7 novel ST: ST147 (3 isolates), ST101 (1 isolate), ST630 (1 isolate), ST3485 (1 isolate), ST3486 (1 isolate), ST3487 (1 isolate), ST3488 (1 isolate), ST3489 (1 isolate), ST3490 (1 isolate), ST3491 (2 isolates). Our study provides new data about MBL producing K. pneumoniae in Tunisia. Thus, we report for the first time the coexpression of blaNDM-1 and blaVIM-1 in our country and also we describe seven novel ST of MBL producing K. pneumoniae in the world.

Prevalence and Diversity of Antibiotic Resistance Genes in Swedish Aquatic Environments Impacted by Household and Hospital Wastewater.

Antibiotic-resistant Enterobacteriaceae and non-lactose fermenting Gram-negative bacteria are a major cause of nosocomial infections. Antibiotic misuse has fueled the worldwide spread of resistant bacteria and the genes responsible for antibiotic resistance (ARGs). There is evidence that ARGs are ubiquitous in non-clinical environments, especially those affected by anthropogenic activity. However, the emergence and primary sources of ARGs in the environment of countries with strict regulations for antibiotics usage are not fully explored. The aim of the present study was to evaluate the repertoire of ARGs of culturable Gram-negative bacteria from directionally connected sites from the hospital to the wastewater treatment plant (WWTP), and downstream aquatic environments in central Sweden. The ARGs were detected from genomic DNA isolated from a population of selectively cultured coliform and Gram-negative bacteria using qPCR. The results show that hospital wastewater was a reservoir of several class B ß-lactamase genes such as bla IMP-1 , bla IMP-2, and bla OXA-23, however, most of these genes were not observed in downstream locations. Moreover, ß-lactamase genes such as bla OXA-48, bla CTX-M-8, and bla SFC-1, bla V IM-1, and bla V IM-13 were detected in downstream river water but not in the WWTP. The results indicate that the WWTP and hospital wastewaters were reservoirs of most ARGs and contribute to the diversity of ARGs in associated natural environments. However, this study suggests that other factors may also have minor contributions to the prevalence and diversity of ARGs in natural environments.

Increased Resistance to Carbapenems in Proteus mirabilis Mediated by Amplification of the blaVIM-1-Carrying and IS26-Associated Class 1 Integron.

Objective: The aim of the study was to decipher the mechanisms and associated genetic determinants responsible for increased carbapenem resistance among Proteus mirabilis clinical isolates. Methods: The entire genetic structure surrounding the ß-lactam resistance genes was characterized by PCR, gene walking, and DNA sequencing. Results: A series of clinical P. mirabilis isolates were consecutively recovered from different patients at the Military hospital of Sofia, Bulgaria. They showed variable levels of resistance to carbapenems. All isolates produced the same carbapenemase VIM-1 that was chromosomally encoded. We showed that increased resistance to carbapenems was related to an increased number of blaVIM-1 gene copies. Conclusion: We showed here that increased carbapenem resistance in P. mirabilis may result from increased expression of the blaVIM-1 carbapenemase gene through multiplication of its copy number.

Retrospective Analysis of Bacterial Cultures Sampled in German Chicken-Fattening Farms During the Years 2011-2012 Revealed Additional VIM-1 Carbapenemase-Producing Escherichia coli and a Serologically Rough Salmonella enterica Serovar Infantis.

Carbapenems are last-resort antibiotics used in human medicine. The increased detection of carbapenem-resistant Enterobacteriaceae (CRE) is therefore worrying. In 2011 we reported the first livestock-associated VIM-1-producing Salmonella (S.) enterica serovar Infantis (R3) isolate from dust, sampled in a German chicken fattening farm. Due to this observation we retrospectively investigated more than 536 stored bacterial cultures, isolated from 45 chicken fattening farms during the years 2011 and 2012. After a non-selective overnight incubation, the bacteria were transferred to selective media. Escherichia (E.) coli and Salmonella growing on these media were further investigated, including antibiotic susceptibility testing, carbapenemase gene screening and whole genome sequencing (WGS). In total, four CRE were found in three out of 45 investigated farms: Besides R3, one additional Salmonella (G-336-1a) as well as two E. coli isolates (G-336-2, G-268-2). All but G-268-2 harbored the blaVIM-1 gene. Salmonella isolates R3 and G-336-1 were closely related although derived from two different farms. All three blaVIM-1-encoding isolates possessed identical plasmids and the blaVIM-1- containing transposon showed mobility at least in vitro. In isolate G-268-2, the AmpC beta-lactamase gene blaCMY-2 but no known carbapenemase gene was identified. However, a transfer of the phenotypic resistance was possible. Furthermore, G-268-2 contained the mcr-1 gene, combining phenotypical carbapenem- as well as colistin resistance in one isolate. Carbapenem-resistant Enterobacteriaceae have been found in three out of 45 investigated chicken flocks. This finding is alarming and emphasizes the importance of intervention strategies to contain the environmental spread of resistant bacteria in animals and humans.