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Abstract 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.

Antimicrobial resistance leads to the ineffectiveness of antimicrobials, hampering the ability to cure infections. Zinc-dependent metallo ß-lactamases (MBLs) producing bacteria represent a threat to public health due to limited therapeutic options. Zinc chelators are able to inhibit MBLs and have the potential to restore carbapenem susceptibility. Recently, polypyridine ligands named Bispicen (N,N’-Bis(2-pyridymethyl)-ethylenediamine), Trispicen (N,N,N’-Tris(2-pyridylmethyl)-ethylenediamine), TrispicenA (N,N,N’-tris([2-pyridylmethyl)-ethylenediamine-N’-acetic acid) and TrispicenDALA (N,N,N’-tris([2-pyridylmethyl)-ethylenediamine-N’-acetyl-D-alanyl-D-alanyl-D-alanine methyl ester) were evaluated for their ability to interfere with the Zn-ion network by interactions in the binding site of MBLs. We report on the inhibitory activity of these ligands towards three subclass B1 MBLs (NDM-1, VIM-1 and IMP-1) by kinetic analysis, demonstrating that all compounds, except TrispicenDALA, acted as competitive inhibitors towards NDM-1 and VIM-1. Bispicen was more active against VIM-1 with Ki value of 0.13 μM while NDM-1 was highly inhibited by Trispicen with Ki value of 0.08 μM. The IMP-1 enzyme was resistant to these compounds showing Ki and IC50 values higher than 500 μM. Docking study reveals that all compounds interact with residues of the MBL active site mainly by the formation of pi-interactions and chelation of both Zn ions. Polypyridine ligands deserve attention for their inhibitory potential towards NDM-1 and VIM-1.

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.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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 β-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 bla VIM-1 or bla OXA-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., Competing Interests: The authors declare no conflict of interest.

ABSTRACT 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.

The non-hospital environment particularly poultry farms and abattoirs are fast becoming reservoir channels for the transmission of antibiotic resistant bacteria including those that produce metallo beta-lactamases (MBLs). Foodproducing animal’s habouring multidrug resistant bacteria including those that produce metallo-beta-lactamases (MBLs) poses health risks to the human population. This study investigated the prevalence of blaIMP-1 and blaVIM-1 MBL genes in Pseudomonas aeruginosa isolates from food-producing animals by multiplex PCR technique. Anal swab samples (n=120) were bacteriologically analyzed on cetrimide selective agar for the selective isolation of P. aeruginosa isolates. Antibiogram was carried out as per the Clinical and Laboratory Standard Institute (CLSI) criteria. The production of MBLs was detected phenotypically and genotypically using the modified Hodges test method and multiplex PCR technique respectively. DNA products were run on 1.5 % agarose gel, and visualized using a UV transilluminator at 260 nm. Data was analyzed statistically using SPSS version 23.0. Out of the 120 anal swab samples, a total of 43 (35.8 %) isolates of P. aeruginosa was bacteriologically recovered. The P. aeruginosa isolates were found to be resistant to ampicillin (88.4 %), cefotaxime (81.4 %), gentamicin (79.1 %), sulphamethoxazole-trimethoprim (72.1 %), oxacillin (76.7 %), nitrofurantoin (76.7 %), meropenem (62.8 %), ofloxacin (67.4 %), imipenem (65.1 %) and cloxacillin (69.8 %). MBL was phenotypically detected in 15 (34.9 %) isolates of P. aeruginosa. However, the multiplex PCR technique significantly confirmed MBL production in only 12 (27.9) isolates of P. aeruginosa (p

• Metallo-beta-lactamase-producing Pseudomonas aeruginosa osteomyelitis is hard to treat. • Aztreonam/ceftazidime–avibactam combined with amikacin was strongly synergic. • Time-kill curves showed that triple combination achieved 99.9% killing until 48 h. • Antipseudomonal synergistic agents plus debridement resolved the infection. 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 bla VIM-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. [ABSTRACT FROM AUTHOR]

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 blaVIM–1 on an IncHI2 plasmid while the second lineage carried the gene on the chromosome. Despite its different locations, the blaVIM–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.

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 β-lactamase genes such as blaIMP-1, blaIMP-2, and blaOXA-23, however, most of these genes were not observed in downstream locations. Moreover, β-lactamase genes such as blaOXA-48, blaCTX-M-8, and blaSFC-1, blaV IM-1, and blaV 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.

Metallo-β-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 β-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-β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

Abstract The monobactam antibiotic, aztreonam, can be hydrolyzed by some β-lactamases (NDM-1), whereas other β-lactamases, including VIM-1, have no hydrolytic activity on aztreonam. NDM-1 and VIM-1 are both metallo-β-lactamases (MBLs), but they catalyze hydrolysis at different active site residues, which is reflected in their obvious activity difference with aztreonam. In this work, to explore the catalytic hydrolysis mechanism of MBLs at the atomic level, molecular dynamic simulations were performed for NDM-1-aztreonam and VIM-1-aztreonam complexes based on the molecular docking analysis. Molecular modeling revealed the binding of aztreonam to the active regions of NDM-1 and VIM-1. Residues Met67, Phe70, Asp124, Thr190, and His250 play key roles in the binding of aztreonam with NDM-1. His116, Asp118, Cys198, His201, and His240 are the critical residues for the binding of aztreonam with VIM-1. Interestingly, Asp124 displayed the strongest binding energy (ΔE total = −11.28 kcal/mol), which was nearly 10 times higher than that of the other residues in the NDM-1-aztreonam complex. A similar result was found for the binding of aztreonam with VIM-1, with Asp118 displaying the strongest binding energy (ΔE total = −2.95 kcal/mol). These results implicated aspartic acid as the critical active site in the catalytic hydrolysis pocket of NDM-1 and VIM-1. However, in the VIM-1-aztreonam complex, because of the strong π-π interaction between the thiazol ring group of aztreonam and the imidazole ring groups of His201 and His240, the binding energy obtained from Asp118 become significantly weaker than that of aztreonam with Asp124 of NDM-1. Analysis of the simulation trajectory indicated that the thiazol ring plane of aztreonam is almost parallel to the imidazole ring planes of His201 and His240, implying that they can form a strong π-π interaction, which is consistent with the above results. On the basis of the computational biology results, it was confirmed that aspartic acid in the active pocket of NDM-1 and VIM-1 can effectively promote substrate hydrolysis, while histidine on the other side of the active pocket can block the binding of the substrate with aspartic acid, leading to the loss of hydrolysis. Graphical abstract Unlabelled Image Highlights • The interaction of aztreonam towards the NDM-1 and VIM-1 complexes were explored. • The binding sites of aztreonam in the complexes were identified. • Aspartic acid in the active pocket of NDM-1 and VIM-1 promote substrate hydrolysis. • Histidine in the active pocket block the hydrolysis of the substrate. [ABSTRACT FROM AUTHOR]

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 β-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, β-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. [ABSTRACT FROM AUTHOR]

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-β-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-β-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.

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.

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.

The non-hospital environment particularly poultry farms and abattoirs are fast becoming reservoir channels for the transmission of antibiotic resistant bacteria including those that produce metallo beta-lactamases (MBLs). Foodproducing animal's habouring multidrug resistant bacteria including those that produce metallo-beta-lactamases (MBLs) poses health risks to the human population. This study investigated the prevalence of blaIMP-1 and blaVIM-1 MBL genes in Pseudomonas aeruginosa isolates from food-producing animals by multiplex PCR technique. Anal swab samples (n=120) were bacteriologically analyzed on cetrimide selective agar for the selective isolation of P. aeruginosa isolates. Antibiogram was carried out as per the Clinical and Laboratory Standard Institute (CLSI) criteria. The production of MBLs was detected phenotypically and genotypically using the modified Hodges test method and multiplex PCR technique respectively. DNA products were run on 1.5 % agarose gel, and visualized using a UV transilluminator at 260 nm. Data was analyzed statistically using SPSS version 23.0. Out of the 120 anal swab samples, a total of 43 (35.8 %) isolates of P. aeruginosa was bacteriologically recovered. The P. aeruginosa isolates were found to be resistant to ampicillin (88.4 %), cefotaxime (81.4 %), gentamicin (79.1 %), sulphamethoxazole-trimethoprim (72.1 %), oxacillin (76.7 %), nitrofurantoin (76.7 %), meropenem (62.8 %), ofloxacin (67.4 %), imipenem (65.1 %) and cloxacillin (69.8 %). MBL was phenotypically detected in 15 (34.9 %) isolates of P. aeruginosa. However, the multiplex PCR technique significantly confirmed MBL production in only 12 (27.9) isolates of P. aeruginosa (p<0.001) that harboured the blaIMP-1 MBL genes. The blaVIM-1 MBL genes were not detected in the MBL positive P. aeruginosa phenotypes. The P. aeruginosa isolates that harboured the blaIMP-1 MBL genes were found to be multiply resistant. This study reported for the first time the prevalence of blaIMP-1 MBL genes from P. aeruginosa isolates from anal swab samples of food-producing animals in Abakaliki, Nigeria. The long-term exposure of food-producing animals to antibiotics could cause accumulation of antibiotic resistance determinants in the gut microbiota of these animals. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas aeruginosa is an opportunistic human pathogen which causes serious problems, especially in people who have immunodeficiency. Metallo beta-lactamase (MBL) resistance in this bacterium has led some difficulties in treating bacterial infections. MBLs are being reported with increasing frequency worldwide. The aim of the present systematic review and meta-analysis was to collect data about the relative frequency (RF) of VIM-1-imipenem resistant P. aeruginosa (VIM-1-IRPA) in different regions of Iran and report an overall prevalence if possible. Materials and Methods: PubMed, ISI web of science, Scopus and Google Scholar were searched using following key terms: "P. aeruginosa," "imipenem," "VIM-1" and "Iran" were. Articles/abstracts, which used clinical specimens and had done polymerase chain reaction to detect the VIM-1 gene of MBL genes, were included in this review. STATA SE version 11.2 (StataCorp, College Station, TX, USA) was used for statistical analysis. Results: Out of 5457 results found, 10 articles were eligible to be included in our systematic review and meta-analysis. These studies were carried out in Tehran, Isfahan, Kurdistan, Ahvaz, Markazi and Northwest of Iran (Orumieh and Tabriz). Pooled estimation of 1972 P. aeruginosa samples showed that 13% (95% confidence interval = 10.5-16.5%]) of strains were VIM-1 positive. VIM-1-IRPA RF in different studies varied from 0% to 19.5% in Isfahan and Markazi provinces, respectively. We found a moderate heterogeneity (Chochran Q-test, P = 0.032, I-squared = 50.7%) of VIM-1-IRPA RF among studies. Conclusion: According to the results of this study VIM-1-IRPA RF in Iran is in low-level Prevention strategies to reduce the prevalence rates of VIM-1 positive strains in Iran are needed.

Carbapenemase producing Citrobacter freundii (CPCF) infections are still uncommon in European countries. Here we report a molecular study conducted in a tertiary care facility in southern Madrid, Spain, from 2009 to 2014 to investigate the epidemiology of CPCF. The bla IMP-1, bla IMP-2, bla KPC, bla NDM, bla OXA-48, bla VIM-1 and bla VIM-2 genes were screened by PCR. Molecular typing was carried out by Pulsed-field gel electrophoresis analysis (PFGE) and multilocus sequence typing (MLST). Whole genome sequencing (WGS) was performed to characterize the resistome and the mobile genetic elements associated with the carbapenems resistance of CPCF. A total of 11/521 (2.1%) isolates had reduced susceptibility to carbapenems. PCR amplification revealed the presence of bla VIM-1 in 10 isolates and bla KPC-2 in 2 isolates. One C. freundii isolate co-harbored bla VIM-1 and bla KPC-2 genes. PFGE and MLST assigned 10 different clonal, 4 previously reported (ST11, ST18, ST22 and ST64) and 6 new STs (ST89, ST90, ST91, ST92, ST92 and ST94). The bla VIM-1 gene was part of In624 ( intI1-bla VIM-1 - aacA4 - dfrB1-aadA1 - catB2 - qacEΔ1 / sul1 ). In 3 of these isolates, plasmid-mediated quinolone resistance genes ( qnrA1 and qnrB4 ) were present in its downstream region, taking part of a complex class 1 integron ([In624:ISCR1: qnrB4 - bla DHA-1 ] and [In624:ISCR1: qnrA1 ]). On the other hand, the bla KPC-2 gene was associated with a Tn3-based transposon. The dissemination of the bla VIM-1 gene among various clones suggests a successful horizontal transfer of integron carrying elements that play a dominant role in the development of multidrug resistance in Enterobacteriaceae . [ABSTRACT FROM AUTHOR]

Worldwide increase in carbapenem resistance and transferable carbapenemases are significant challenges in treatment of Pseudomonas aeruginosa infections. In this study, investigation of carbapenemase production in carbapenem-resistant P. aeruginosa isolates recovered from clinical specimens in a tertiary hospital was aimed. A total of 84 carbapenem-resistant P. aeruginosa isolates were examined. 'Carbapenem inactivation method' (CIM) was used for phenotypic detection of carbapenemase production. The existence of blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48, and blaGES genes was investigated by polymerase chain reaction (PCR). Subtypes of the detected genes were identified by sequence analysis. Arbitrarily primed PCR (AP-PCR) was performed to evaluate the clonal relationship among the isolates. The presence of high-risk clones in carbapenemase producers was investigated by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Three isolates (3.5%) were identified as carbapenemase producers by CIM tests, while PCR tests demonstrated three isolates carrying carbapenemase genes as well. blaVIM gene was found in two isolates and blaGES gene was found in one isolate. Sequence analysis demonstrated that the carbapenemases were VIM-1, VIM-2, and GES-5. AP-PCR yielded high clonal diversity among the isolates. According to MALDI-TOF MS analysis, none of the carbapenemase-producing strains belonged to the high-risk clones. In conclusion, the presence of VIM-1, VIM-2, and GES-5 type carbapenemases in P. aeruginosa isolates was demonstrated for the first time in our hospital, GES-5 being reported for the second time in Turkey. Our results will lead strategies for controlling the spread of carbapenemases and contribute to epidemiological data from Turkey. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Enterobacteriaceae are not any more isolated only from human settings, but also from livestock. We reported for the first time the presence of VIM-1 carbapenemases in a livestock farm in Germany. The VIM-1 resistance gene found in these farms was located on IncHI2 plasmids. In order to be able to analyse these plasmids in more detail, two different plasmids from a single farm (pRH-R27 from Salmonella enterica and pRH-R178 from Escherichia coli ) were completely sequenced and analysed for the presence of antibiotic and heavy metal resistances. The plasmids showed to harbour bla VIM-1 , aacA4 , aadA1 , sul1 , qacEΔ (encoded in an In 110 class 1 integron), as well as bla ACC-1 , strA / strB , and catA1 genes together with resistance to heavy metals ( ter -, mer -, sil -, ars -, rcn -, and pco ). Comparison with other IncHI2 plasmid revealed that while pRH-R27 is a mosaic IncHI2 plasmid with both high homology to the plasmid pSTm-A54650 and R478, both isolated from humans, pRH-R178 is a deletion derivative of pRH-R27, presumably caused by several IS-mediated deletions indicating genetic evolution of plasmids in this environment. [ABSTRACT FROM AUTHOR]

"n 800x600 Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 st1":*{behavior:url(#ieooui) } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif";} Background: Pseudomonas aeruginosa is an important opportunistic pathogen causes clinical infections among burn patients. Metallo-β-lactamases (MBLs) are important mechanisms of Carbapenem (drug of choice) resistance among Pseudomonas aeruginosa isolates. The aims of this study were to determine the antibiotic susceptibility pattern and to detect the prevalence of MBLs among Pseudomonas aeruginosa"n"nMethods: Initially, the antibiotic resistance patterns of 170 clinical strains isolated from burn patients in Motahari Hospital in Tehran, Iran were determined by Kirby-Bauer disc diffusion method. All of the clinical isolates using two phenotypic and genotypic methods. Pseudomonas aeruginosa isolates resistant to Imipenem were screened for production of MBL by E test with Imipenem / Imipenem plus EDTA (E test MBL). PCR assay was performed for detection of blaVIM genes."n"nResults: Based on the study results, the percentage of resistance was as below: Imipenem (10 μg) 52.9%, Amikacin (30 μg) 81.7%, Carbenicilin (100 μg) 74.7%, Polymixine B (300 unit) 10%, Ticarcilin (75 μg) 84.7%, Tobramycin (10 μg) 88.2%, Colisitin (10 μg) 34.1, Colisitin (25 μg) 28.3%. Of 90 Carbapenem resistant isolates, 10(11/1%) isolates were positive by E test, all were sensitive to Colisitin and Polymixine B. All of the Imipenem resistant Pseudomonas aeruginosa isolates were examined by PCR for the presence of the blaVIM genes. All MBL-producing isolates carried blaVIM-1 genes."n"nConclusion: Considering the high prevalence and clinical importance of MBL-producing isolates, rapid identification of them and use of the appropriate infection control measures are necessary to prevent further spread of infections by these organisms.

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 MIC 50/90 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., Competing Interests: The authors declare no conflict of interest.

Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin β-lactamases, class B metallo-β-lactamases (MBL) or class D OXA-48-like β-lactamases. The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia. In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST. Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 β-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae , Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively. Three different types of carbapenemases were identified showing the complexity of CRE in Croatia. [ABSTRACT FROM AUTHOR]

The distribution of carbapenemase genes in Escherichia coli strains isolated between September 2009 and May 2013 in Germany was investigated. Out of 192 isolates with carbapenemase production OXA-48 was found in 44.8%, VIM-1 in 18.8%, NDM-1 in 11.5% and KPC-2 in 6.8%. Patients with VIM-1 producing E. coli ( n = 36) differed from patients with OXA-48 by an older age, less frequent mention of travel history and an increased proportion of clinical over screening specimens. These data might indicate that introduction from abroad is of minor importance for VIM-1 producing E. coli compared to other carbapenemases. Multilocus sequence typing revealed that E. coli with VIM-1 were mostly multiclonal, emphasizing the role of horizontal gene transfer in its spread. Susceptibility testing of VIM-1 producing E. coli demonstrated aztreonam susceptibility in 55.6%. Among non-β-lactams susceptibility rates of >90% were observed for amikacin, tigecycline, colistin, fosfomycin and nitrofurantoin. [ABSTRACT FROM AUTHOR]

In 2004 and 2005, 5 metallo-β-lactamase (MBL)-positive Acinetobacter baumannii isolates were found in 2 Greek hospitals. Isolates were unrelated and carried blaVIM-1 in a class 1 integron; blaOXA-51- and blaOXA-58-like carbapenemase genes were also detected. VIM-1 MBL in Acinetobacter spp. causes concern, given the increasing resistance of this species.

Sequence type 11 Klebsiella pneumoniae , coproducing NDM-1 and VIM-1 metallo-β-lactamases, were isolated in a Greek hospital. bla NDM-1 was part of a Tn 125 derivative, located on an ~90-kb plasmid similar to the NDM-1-encoding plasmid pB-3002cz. bla VIM-1 was located in an In-e541-like integron, carried on a multireplicon (IncA/C and IncR) plasmid of ~180 kb. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas aeruginosa is an opportunistic human pathogen which causes serious problems, especially in people who have immunodeficiency. Metallo beta-lactamase (MBL) resistance in this bacterium has led some difficulties in treating bacterial infections. MBLs are being reported with increasing frequency worldwide. The aim of the present systematic review and meta-analysis was to collect data about the relative frequency (RF) of VIM-1-imipenem resistant P. aeruginosa (VIM-1-IRPA) in different regions of Iran and report an overall prevalence if possible. Materials and Methods: PubMed, ISI web of science, Scopus and Google Scholar were searched using following key terms: “P. aeruginosa,” “imipenem,” “VIM-1” and “Iran” were. Articles/abstracts, which used clinical specimens and had done polymerase chain reaction to detect the VIM-1 gene of MBL genes, were included in this review. STATA SE version 11.2 (StataCorp, College Station, TX, USA) was used for statistical analysis. Results: Out of 5457 results found, 10 articles were eligible to be included in our systematic review and meta-analysis. These studies were carried out in Tehran, Isfahan, Kurdistan, Ahvaz, Markazi and Northwest of Iran (Orumieh and Tabriz). Pooled estimation of 1972 P. aeruginosa samples showed that 13% (95% confidence interval = 10.5-16.5%]) of strains were VIM-1 positive. VIM-1-IRPA RF in different studies varied from 0% to 19.5% in Isfahan and Markazi provinces, respectively. We found a moderate heterogeneity (Chochran Q-test, P = 0.032, I-squared = 50.7%) of VIM-1-IRPA RF among studies. Conclusion: According to the results of this study VIM-1-IRPA RF in Iran is in low-level Prevention strategies to reduce the prevalence rates of VIM-1 positive strains in Iran are needed. [ABSTRACT FROM AUTHOR]

Abstract: Over a 6-year period (2007–2012), the emergence of Enterobacter cloacae isolates resistant to β-lactams and with reduced susceptibility to carbapenems was observed in Hospital Universitario 12 de Octubre (Madrid, Spain). To determine the possible role of metallo-β-lactamases (MBLs) in the resistance profile of these isolates, a molecular and clinical epidemiological study was performed, including determination of patients’ clinical characteristics, genetic diversity of strains, resistance mechanisms to carbapenems, and the genetic environment of VIM-1. A total of 73 E. cloacae isolates showed resistance to extended-spectrum cephalosporins and reduced susceptibility to at least one carbapenem during 2007–2012. PCR amplification revealed the presence of bla VIM-1 gene in 37 isolates, bla VIM-2 in 1 isolate and bla KPC in 5 isolates. Molecular typing showed high clonal diversity of E. cloacae isolates carrying bla VIM-1. The genetic environment of bla VIM-1 was investigated and two integron structures were found: intI–bla VIM-1–aacA4–dfrB1–aadA1–catB2–qacEΔ1/sul1 (In624); and intI–bla VIM-1–aacA4–aadA1–qacEΔ1/sul1 (In488). Isolates belonging to three clones (A, F and G) harboured different types of integron (In624 or In488) despite belonging to the same clone. Conjugal experiments showed an association with a conjugative plasmid of ca. 300kb belonging to IncHI2 group, which is common in Spanish hospitals, suggesting that the widespread dissemination of bla VIM-1 may be due to horizontal transfer of mobile genetic determinants rather than the result of spreading of a few clones. These results have implications for infection control programmes in the hospital. [Copyright &y& Elsevier]

Abstract: Seven Enterobacter cloacae isolates and seven Klebsiella pneumoniae isolates harbouring a phenotype compatible with the production of a metallo-β-lactamase were recovered between 2009 and 2011 in three Intensive Care Units of Hospital Vall d’Hebron (Barcelona, Spain). The presence of bla VIM, bla IMP, bla NDM, bla CTX-M, aac(6′)-Ib, qnrA, qnrB and qnrS genes was screened by polymerase chain reaction (PCR) and sequencing. Clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis (PFGE) and, in the case of K. pneumoniae isolates, by multilocus sequence typing (MLST). PCR-based replicon typing, Southern hybridisation, plasmid double-locus sequence typing and MOB relaxase classification methods were used to identify and characterise the plasmids carrying the resistance genes. Transferability of the identified plasmids was tested by conjugation assays. All 14 isolates were found to carry bla VIM-1, bla CTX-M-9 (except one isolate), aac(6′)-Ib and qnrA genes. Clonality assessment demonstrated that E. cloacae isolates were distributed in three clonal clusters, whereas all of the K. pneumoniae isolates belonged to one unique clone, identified as sequence type ST252. All studied isolates harboured a large conjugative IncHI2 MOBH11 plasmid carrying all of the detected resistance genes. Plasmid DNA analysis showed that all of them belonged to the ST1 IncHI2 plasmid cluster and shared the same relaxase partial sequence. In conclusion, the present study describes the dissemination within a hospital of multiresistant E. cloacae and K. pneumoniae isolates producing VIM-1. A complex clonal epidemiology of the E. cloacae isolates was observed and plasmid DNA analysis strongly supports horizontal exchanges of the same IncHI2 plasmid between different strains and species. [Copyright &y& Elsevier]

Abstract: We report a case of long-term colonization of a carbapenemase (VIM)-producing Klebsiella pneumoniae clone in a renal transplant patient and demonstrate the in vivo transmission of a broad-host-range multidrug-resistant IncN plasmid containing bla VIM, bla SHV-12, and qnrS to Escherichia coli. [Copyright &y& Elsevier]

A study was designed to characterize a carbapenem-resistant Klebsiella pneumoniae (KPSA01) isolated from a patient in Gauteng, South Africa without recent travel outside South Africa. Molecular characterization was done using isoelectric focusing, polymerase chain reaction and sequencing for blaVIM, blaIMP, blaNDM, blaCTX-Ms, blaOXAs, blaTEMs, and blaSHV, plasmid-mediated quinolone resistance determinants, multilocus sequencing typing, plasmid replicon typing, and addiction factors. KPSA01 produced VIM-1 and belonged to the newly described sequence type ST569. The plasmid that harboured blaVIM typed within the narrow host range IncF replicon group, contained the aadA1 gene cassette, and tested positive for the vagCD and ccdAB addiction systems. This is the first report of VIM-1-producing K. pneumoniae outside Europe. It is important that surveillance studies be undertaken in Africa to determine if VIM-1-producing K. pneumoniae are present in significant numbers. [ABSTRACT FROM AUTHOR]

The in-vivo activity of imipenem against VIM-1-producing Klebsiella pneumoniae (VPKP) was assessed in a thigh infection model in neutropenic mice. Animals were infected with three VPKP isolates (imipenem MICs 2, 4 and 32 mg/L, respectively) and a susceptible clinical isolate (MIC 0.125 mg/L) that did not produce any β-lactamase with broad-spectrum activity. Bacterial density at the site of infection was determined after imipenem treatment (30 and 60 mg/kg every 2 h for 24 h). The log10 reduction in CFU/thigh was greatest for the wild-type isolate, intermediate for the two imipenem-susceptible VPKP isolates, and lowest for the imipenem-resistant VPKP isolate. Whilst in-vivo imipenem activity appeared reduced against in-vitro susceptible VIM-1 producers compared with a VIM-1-negative control, an increased drug dosage could moderate this reduction. [ABSTRACT FROM AUTHOR]

BlaVIM-1 gene was detected in four Escherichia coli clinical isolates with both reduced susceptibility to carbapenems and an ESBL phenotype. The VIM-1 determinant was located within the variable region of a Class I integron along with a 6′-N-aminoglycoside acetytransferase gene (aac(6′)-Ib) and it could be transferred by conjugation. In all four clinical isolates the VIM-1 gene cassette presented a characteristic duplication of the 3′ end coding 153 nucleotides followed by the first 14 nucleotides of the 59 base element (59be) that however did not seem to affect either the integrity of the coding sequence or the 59be of the gene cassette. These clinical isolates not only harbored the same Class I integron, but they also shared the same discrete ribotype-pattern, indicative for their clonal origin. Spread of carbapenem resistance genes among Enterobacteriaceae in hospital is a matter of great concern. [Copyright &y& Elsevier]

The objective of the study is to report a multidrug-resistant outbreak of Providencia stuartii that occurred in inpatients in the Athens area in 2012 resulting from a very successful transmissible A/C multidrug-resistant plasmid. Thirteen multidrug-resistant P . stuartii clinical isolates from 5 hospitals were studied. Molecular typing was performed by pulsed-field gel electrophoresis. Antibiotic resistance genes and their genetic surround were detected by PCR and sequencing. Plasmid analysis included conjugation experiments using liquid cultures, sizing by S1 digestion, and incompatibility replicon typing by PCR. Isolates were grouped into 2 distinct clonal types A and B, exhibiting similarity less than 70%. Isolates of type A were recovered from patients hospitalized in 4 different hospitals with no obvious epidemiological linkage, while isolates of type B were recovered from patients treated in a single hospital. Both clonal types harbored a conjugative plasmid of 130 bp and IncA/C replicon type carrying 5 β-lactamase genes bla SHV-5 , bla VEB-1 , bla VIM-1 , bla OXA-10 , and bla TEM-1 and aminoglycosides resistant determinants. All β-lactamase genes were included in stable structures as IS26, IS1999, and In-e541. The current plasmid seemed to have many common determinants with previously reported plasmids derived from P . stuartii and Proteus mirabilis clinical isolates and exhibited the ability to circulate in nosocomial bacterial populations. [ABSTRACT FROM AUTHOR]