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

Purpose Plasmids of the incompatibility group X type 3 (IncX3) were described carrying various carbapenemase genes in carbapenemase-producing Enterobacteriaceae (CPE) worldwide and in the United Arab Emirates (UAE), as well. To understand the driving force behind the emergence of such plasmids in the UAE, the relationship between IncX3 plasmids encountered locally and globally was investigated. Methods CPE strains isolated in the UAE during 2009-2014 were screened by X3 PCR-based replicon typing. The clonal relationship of CPE carrying IncX3 plasmids was determined by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Complete sequence of selected IncX3 plasmids was determined. Phylogenetic relationship between the carbapenemase carrying IncX3 plasmids from the UAE and of those reported worldwide was established by comparing the plasmid backbones. Results 10.2% of the 295 CPE tested were identified to carry IncX3 plasmids: 13 Escherichia coli, 13 Klebsiella pneumoniae, two Enterobacter cloacae, one Citrobacter freundii and one Morganella morganii isolate, respectively. Most of them were non-clonal; with small clusters of triplets and pairs of E. coli and K. pneumoniae, and a cluster of five K. pneumoniae ST11 exhibiting >90% similar PFGE patterns, respectively. The 30 isolates harbored either bla NDM-1, bla NDM-4, bla NDM-5, bla NDM-7, bla OXA-181 or bla KPC-2 carbapenemase genes on IncX3 plasmids. Phylogenetic analysis of the backbone region of IncX3 plasmids carrying various beta-lactamase genes from the UAE (n=23) and that of North-America, Europe, Asia and Australia (n=35) revealed three clusters based on the carbapenemase genes carried: plasmids harboring bla OXA-181 and bla NDM-5 formed two distinct groups, whereas backbones of plasmids with bla NDM-1, bla NDM-4 and bla NDM-7 clustered together. Each cluster contained plasmids of diverse geographical origin. Conclusion The findings suggest that different carbapenemase gene carrying IncX3 plasmids encountered in the UAE do not evolve locally, rather are subtypes of this epidemic plasmid emerging in this country due to international transfer.

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

A large outbreak of New Delhi metallo-beta-lactamase (NDM)-1-producing Klebsiella pneumoniae sequence type (ST) 147 occurred in Tuscany, Italy in 2018–2019. In 2020, ST147 NDM-9-producing K. pneumoniae were detected at the University Hospital of Pisa, Tuscany, in two critically ill patients; one developed bacteraemia. Genomic and phylogenetic analyses suggest relatedness of 2018–2019 and 2020 strains, with a change from NDM-1 to NDM-9 in the latter and evolution by colistin, tigecycline and fosfomycin resistance acquisition.

Mobile genetic elements (MGEs), especially multidrug-resistance plasmids, are major vehicles for the dissemination of antimicrobial resistance determinants. Herein, we analyse the MGEs in three extensively drug-resistant (XDR) Klebsiella pneumoniae isolates from Germany. Whole genome sequencing (WGS) is performed using Illumina and MinION platforms followed by core-genome multi-locus sequence typing (MLST). The plasmid content is analysed by conjugation, S1-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot experiments. The K. pneumoniae isolates belong to the international high-risk clone ST147 and form a cluster of closely related isolates. They harbour the blaOXA-181 carbapenemase on a ColKP3 plasmid, and 12 antibiotic resistance determinants on an multidrug-resistant (MDR) IncR plasmid with a recombinogenic nature and encoding a large number of insertion elements. The IncR plasmids within the three isolates share a high degree of homology, but present also genetic variations, such as inversion or deletion of genetic regions in close proximity to MGEs. In addition, six plasmids not harbouring any antibiotic resistance determinants are present in each isolate. Our study indicates that genetic variations can be observed within a cluster of closely related isolates, due to the dynamic nature of MGEs. The mobilome of the K. pneumoniae isolates combined with the emergence of the XDR ST147 high-risk clone have the potential to become a major challenge for global healthcare.

In this study, we investigated VIM-1-producing Escherichia coli , Klebsiella oxytoca , Klebsiella pneumoniae , Citrobacter freundii , and Enterobacter cloacae strains, isolated in 2019 during a period of active surveillance of carbapenem-resistant Enterobacterales in a large university hospital in Italy. VIM-1-producing strains colonized the gut of patients, with up to three different VIM-1-positive bacterial species isolated from a single rectal swab, but also caused bloodstream infection in one colonized patient.

Plasmids identification and classification is an essential parameter in current bacterial typing. The most widely used PCR-based methods are the PCR-based replicon typing (PBRT) and the degenerate primer MOB typing (DPMT). PBRT targets the replicons on the plasmids and DPMT targets the relaxase genes. A finer resolution of phylogenetic relatedness can be obtained by plasmid multiLocus sequence typing available for the major plasmid types occurring in Enterobacteriaceae.

Objectives We investigated the use of bacteriophages as a strategy to decolonize intestinal carriers of multidrug-resistant Escherichia coli. Methods A fermentor was used as a continuous culture system for 48 h. Two different pools of faeces (studies I and II) obtained from volunteers were spiked with a CTX-M-15-producing ST131 E. coli (strain 4901.28) susceptible to bacteriophages and challenged with three doses of INTESTI Bacteriophage cocktail administered at 2, 6 and 10 h after the inoculum. Bacterial typing was performed by implementing microdilution panels, spot test, rep-PCR and whole-genome sequencing (including cgMLST and single-nucleotide variant analysis) obtained using Nanopore and Illumina platforms. Results In study I, bacteriophages decreased the numbers of 4901.28 dramatically (≤101 CFU/mL after 6 h). In contrast, during study II, a phage-resistant mutant of 4901.28 persisted in the continuous culture (104 CFU/mL at 48 h). Whole-genome sequencing revealed the presence of two additional plasmids in the mutant as well as 11 single-nucleotide variants, including one chromosomal in a glycosyltransferase family 2 protein that is responsible for the transfer of sugars to polysaccharides and lipids. In both studies, the commensal E. coli population remained unchanged by the phage treatment maintaining itself at 108 CFU/mL. Conclusions Our data indicates that bacteriophage cocktails may be implemented to decolonize some intestinal carriers. However, the individual microbiota composition may have an impact on the development of phage resistance. Mechanisms underlying this phenomenon are likely to be various and complex. Further in vivo studies and protein expression experiments are needed to confirm our observations and hypotheses.

IncI1 has become one of the most common plasmid families in contemporary Enterobacteriaceae from both human and animal sources. In clinical epidemiology, this plasmid type ranks first as the confirmed vehicle of transmission of extended spectrum beta-lactamase and plasmid AmpC genes in isolates from food-producing animals. In this review, we describe the epidemiology and evolution of IncI1 plasmids and closely related IncIγ plasmids. We highlight the emergence of epidemic plasmids circulating among different bacterial hosts in geographically distant countries, and we address the phylogeny of the IncI1 and IncIγ family based on plasmid Multilocus Sequence Typing.

Plasmids are the main vectors of resistance and virulence genes in Enterobacteriaceae and plasmid typing is essential for the analysis of evolution, epidemiology and spread of antibacterial resistance. The PCR-Based Replicon Typing (PBRT), developed by Carattoli et al. in 2005, was an efficient method for plasmid identification and typing in Enterobacteriaceae. The 2005 PBRT scheme detected 18 replicons in 8 PCR reactions. Recently, the identification of novel replicons and plasmid types requested an update of the PBRT scheme. A commercial PBRT-KIT was devised for the identification of 28 different replicons in 8 multiplex PCRs. Here we report sensitivity and specificity of the PBRT-KIT carried out in comparison with the 2005 PBRT. The analysis of plasmid content was performed on forty-two enterobacterial strains from different sources, containing different replicon content. The 2005 PBRT identified replicons in 76.2% of the strains. The PBRT-KIT detected replicons in 100% of the analyzed strains, demonstrating increasing sensitivity and specificity of the commercial test with respect to the former 2005 PBRT scheme.

We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, bla KPC-2 was harbored on a novel variant of a Tn 4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, bla KPC-3 was on a Tn 4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion.

Objectives: Genomic characterization of the internationally spread sequence type (ST) 16 carbapenemresistant Klebsiella pneumoniae. Methods: The complete genomes of three carbapenem producing ST16 K. pneumoniae from Italian patients were analysed by single-nucleotide polymorphism-based phylogeny, core genome multilocus sequence typing, resistance, plasmid, and virulence content and compared with ten genomes of ST16 strains isolated in other countries. Plasmids carrying blaNDM-1 or blaOXA-232 carbapenemase genes were assembled and sequences were analysed. Results: The internationally spread ST16 K. pneumoniae clone showed variability in terms of distribution of NDM-1 and OXA-232 type carbapenemases. In some ST16 strains, up to six plasmids can be simultaneously present in the same cell, including ColE-like plasmids carrying blaOXA-232 and IncF plasmids carrying blaNDM-1. The differences observed in plasmid, resistance, and virulence content and core genome suggested that there is not a unique, highly conserved ST16 clone, but instead different variants of this lineage circulate worldwide. Conclusions: The ST16 K. pneumoniae clone has spread worldwide and may become a high-risk clone. (c) 2018 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

The global spread of Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been mainly associated with the dissemination of high-risk clones. In the last decade, hospital outbreaks involving KPC-producing K. pneumoniae have been predominantly attributed to isolates belonging to clonal group (CG) 258. However, results of recent epidemiological analysis indicate that KPC-producing sequence type (ST) 307, is emerging in different parts of the world and is a candidate to become a prevalent high-risk clone in the near future. Here we show that the ST307 genome encodes genetic features that may provide an advantage in adaptation to the hospital environment and the human host. Sequence analysis revealed novel plasmid-located virulence factors, including a cluster for glycogen synthesis. Glycogen production is considered to be one of the possible adaptive responses to long-term survival and growth in environments outside the host. Chromosomally-encoded virulence traits in the clone comprised fimbriae, an integrative conjugative element carrying the yersiniabactin siderophore, and two different capsular loci. Compared with the ST258 clone, capsulated ST307 isolates showed higher resistance to complement-mediated killing. The acquired genetic features identified in the genome of this new emerging clone may contribute to increased persistence of ST307 in the hospital environment and shed light on its potential epidemiological success. COL0126131

Stool samples from 38 travelers returning from India were screened for extended-spectrum cephalosporin- and carbapenem-resistant Enterobacteriaceae implementing standard selective plates. Twenty-six (76.3%) people were colonized with CTX-M or DHA producers, but none of the strains was colistin resistant and/or mcr-1 positive. Nevertheless, using overnight enrichment and CHROMagar Orientation plates supplemented with colistin, four people (10.5%) were found to be colonized with colistin-resistant Escherichia coli . One cephalosporin-susceptible sequence type 10 (ST10) strain carried a 4,211-bp IS Apl1-mcr-1 -IS Apl1 element in an IncHI2 plasmid backbone.

In this study we report the detection of the recently described mcr-4 gene in two human isolates of Salmonella enterica serovar Typhimurium. The strains were isolated from faecal samples of two Italian patients with gastroenteritis, collected in 2016. The identified mcr-4 genes (variant mcr-4.2) differed from the mcr-4 gene originally described in a Salmonella strain of swine origin from Italy. Salmonella species could represent a hidden reservoir for mcr genes.

Objectives In this study we compared the recently described mcr-4-positive Salmonella enterica monophasic variant, isolated in 2016 in two Italian patients affected by gastroenteritis, with the first mcr-4-positive Salmonella isolate identified in 2013 in a pig at slaughter in Italy. Methods WGS of the two Salmonella isolates of human origin was performed using a MiSeq instrument (Illumina). The phylogenetic analysis was performed by SNP analysis, comparing genomes of the mcr-4-positive isolates of swine and human origin with 82 Salmonella genomes downloaded from the EnteroBase Salmonella database. Complete sequences of plasmids carrying mcr-4.2 were obtained and compared. Transformation experiments were performed to transfer the mcr-4 plasmids into a colistin-susceptible Escherichia coli recipient strain. Results Comparative genomics demonstrated that the Salmonella of swine origin did not cluster with the isolates of human origin. The mcr-4.2 gene variant identified in the Salmonella of human origin was located on a ColE-like plasmid. This plasmid showed different replication and mobilization genes with respect to those previously described in the ColE plasmid carrying the mcr-4.1 variant, identified in Salmonella of swine origin. Conclusions The divergence in genomes, plasmids and gene variants demonstrated that there was not a unique mcr-4-positive, monophasic Salmonella lineage circulating in animals and causing gastroenteritis in humans in Italy. There was no horizontal transfer of the same plasmid among Salmonella strains of animal and human origin, but the mcr-4 gene and a fragment of the plasmid identified in the animal strain were mobilized by an IS1294 into a different ColE plasmid.

Campylobacter spp. is one of the main cause of bacterial gastroenteritis in the world. The increase of antibiotic resistance in this species is a threat to public health. A Campylobacter spp. surveillance study was performed in Italy in the 2013–2016 period by the Enter-Net Italia network. The most prevalent Campylobacter species identified causing gastroenteritis was Campylobacter jejuni (73.4%) and 45% of all the annual cases of campylobacteriosis were reported in the summer period. High rates of ciprofloxacin and tetracycline resistance in Campylobacter spp. have been observed. An increasing percentage of Campylobacter coli strains simultaneously resistant to ciprofloxacin, tetracycline and erythromycin has been found. Molecular mechanisms of resistance have been investigated and the role of efflux pumps evaluated. Antibiotic resistance in Campylobacter spp. is an increasing serious threat that requires coordinated action to minimize the emergence and spread of antimicrobial resistant strains from animals to humans throughout the food chain.

A Salmonella enterica serovar Corvallis strain was isolated from a wild bird in Germany. This strain carried the IncA/C 2 pRH-1238 plasmid. Complete sequencing of the plasmid was performed, identifying the bla NDM-1 , bla CMY-16 , fosA3 , sul1 , sul2 , strA , strB , aac(6 ′ )-Ib , aadA5 , aphA6 , tetA (A), mphA , floR , dfrA7 , and merA genes, which confer clinically relevant resistance to most of the antimicrobial classes, including β-lactams with carbapenems, fosfomycin, aminoglycosides, co-trimoxazole, tetracyclines, and macrolides. The strain likely originated from the Asiatic region and was transferred to Germany through the Milvus migrans migratory route.

A carbapenem-resistant sequence type 512 (ST512) Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing K. pneumoniae strain showing a novel variant plasmid content was isolated in Palermo, Italy, in 2014. ST512 is a worldwide successful clone associated with the spread of bla KPC genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain, the bla KPC-3 gene was unusually located on an IncX3 plasmid, whose complete sequence was determined. Two copies of bla KPC-3 ::Tn 4401a caused by intramolecular transposition events were detected in the plasmid.

Klebsiella pneumoniae sequence type (ST) 307, carrying bla KPC-3 , bla CTX-M-15 , bla OXA-1 , aac(6′)-Ib-cr , and qnrB 1 genes, is replacing the predominant hyperepidemic ST258 clone in Italy. Whole-genome and complete plasmid sequencing of one ST307 strain was performed and new features were identified.

The genome sequences of a number of Acinetobacter baumanii strains, including representatives of the main epidemic international lineages, have now been determined, and several others are in progress. The study of A. baumannii genomics has provided an expanded view of the adaptation and virulence capacities of this bacterial species, whilst also presenting novel insights into its intraspecies diversity and genome evolution. Genomic analyses have revealed that the current A. baumannii clinical population consists of low-grade pathogens, whose pathogenicity relies mainly on an ability to persist in the hospital setting and survive antibiotic treatment. A. baumannii has a high capacity to acquire new genetic determinants and displays an open pan genome; this feature may have played a crucial role in the evolution of this human opportunistic pathogen towards clinical success. (C) 2011 IUBMB IUBMB Life, 63(12): 1068-1074, 2011

Colonizations due to carbapenem-resistant Enterobacteriaceae (CRE) are a source of antimicrobial resistance transmission in health care settings. Eleven Citrobacter freundii strains producing KPC-3 carbapenemase were isolated from rectal swabs during a 3-year surveillance program. bla KPC-3 -carrying plasmids were found to belong to the IncX3 group in 9 of the 11 strains, and complete nucleotide sequences were obtained for 2 of them. Our results highlight the possible role of C. freundii as reservoir of resistance genes.

The nucleotide sequence of a bla KPC-2 -harboring plasmid (pKPCAPSS) from Klebsiella pneumoniae ST273 isolated in Saint Petersburg, Russia, from a patient with history of recent travel to Vietnam is presented. This 127,970-bp plasmid possessed both IncFII and IncR replicons. bla KPC-2 was localized on a hypothetical mobile element. This element was flanked by 38-bp inverted Tn 3 repeats and included a Tn 3 -specific transposase gene, macrolide resistance operon ( mphA-mrx-mphR ), and a fragment of bla TEM with unique polymorphisms.

Aim: The purpose of this study was to characterize the New Delhi metallo-beta lactamase (NDM)-7-producing Enterobacteriaceae isolated in the Arabian Peninsula. Methods: Enterobacteriaceae identified to carry blaNDM-7 in a collection of 157 NDM-producing isolates from Kuwait, Oman, Saudi Arabia, and the United Arab Emirates (UAE) were investigated for their antibiotic and disinfectant susceptibility, and resistance gene content. The virulence profile, phylogenetic and sequence types of the isolates were also determined. The plasmids carrying the blaNDM-7 were transferred, and their complete nucleotide sequence was determined. Results: Four NDM-7-producing Escherichia coli isolated in Kuwait, Oman, and the UAE, respectively, were identified. They were clonally unrelated, carried a few virulence determinants only, and belonged to clonal complexes CC10 and CC23, or ST448. They were all multi-drug resistant but remained susceptible to fosfomycin, tigecycline, and colistin. In all isolates, blaNDM-7 was located...

A novel mcr colistin resistance gene was identified in a strain of Salmonella enterica, monophasic variant of serovar Typhimurium (4,5,12:i:- ), isolated from a pig at slaughter in Italy in 2013, and in Escherichia coli strains collected during routine diagnostic of post-weaning diarrhoea in pigs from Spain and Belgium in 2015 and 2016. Immediate implementation of mcr-screening including this novel gene variant is required for Salmonella and E. coli from humans and food-producing animals in Europe.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is becoming a common cause of healthcare-associated infection in Italy, with high morbidity and mortality. Prevalent CR-KP clones and resistance mechanisms vary between regions and over time. Therapeutic approaches and their impact on mortality have to be investigated. We performed a prospective study of patients with CR-KP isolation, hospitalized in nine hospitals of Rome, Italy, from December 2010 to May 2011, to describe the molecular epidemiology, antibiotic treatment and risk factors for mortality. Overall, 97 patients (60% male, median age 69 years) were enrolled. Strains producing blaKPC-3 were identified in 89 patients, blaVIM in three patients and blaCTX-M-15 plus porin defects in the remaining five patients. Inter-hospital spread of two major clones, ST512 and ST258, was found. Overall, 36.1% and 20.4% of strains were also resistant to colistin and tigecycline, respectively. Infection was diagnosed in 91 patients who received appropriate antibiotic treatment, combination therapy and removal of the infectious source in 73.6%, 59.3% and 28.5% of cases, respectively. Overall, 23 different antibiotic regimens were prescribed. In-hospital mortality was 25.8%. Multivariate analysis adjusted for appropriate treatment, combination therapy and infectious-source removal, showed that Charlson comorbidity score, intensive-care unit onset of infection, bacteraemia and infection due to a colistin-resistant CR-KP strain were independent risk factors for mortality. The spread of clones producing K. pneumoniae carbapenemases, mainly ST258, is currently the major cause of CR-KP infection in central Italy. We observed a high rate of resistance to colistin that is independently associated with worse outcome. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Plasmids represent one of the most difficult challenge for counteracting the dissemination of antimicrobial resistance. They contribute to the spread of relevant resistance determinants, promoting horizontal gene transfer among unrelated bacteria. Undistinguishable plasmids were identified in unrelated bacterial strains isolated at huge geographically distant area, with no apparent epidemiological links. These plasmids belong to families that are largely prevalent in naturally occurring bacteria, usually carry multiple physically linked genetic determinants, conferring resistance to different classes of antibiotics simultaneously. Plasmids also harbour virulence factors and addiction systems, promoting their stability and maintenance in the bacterial host, in different environmental conditions. The characteristics of the most successful plasmids that were at the origin of the spread of carbapenemase, expanded-spectrum β-lactamase, and plasmid-mediated quinolone resistance genes are discussed in this review.

We report the case of a kidney-transplant patient, suffering an intra-abdominal abscess at the surgical site caused by a carbapenem-resistant ST258 Klebsiella pneumoniae clone, producing the KPC-3 carbapenemase. Under tigecycline treatment, the patient developed a sepsis caused by a carbapenem-susceptible ST258 K. pneumoniae strain. Complete DNA sequences of the plasmids carried by the resistant and susceptible strains from this patient were determined.The complete DNA sequences of plasmids were obtained by applying the 454 Genome Sequencer FLX-PLUS procedure on a library constructed of total plasmid DNA purified from the carbapenem-resistant and -susceptible strains.In the carbapenem-resistant strain, four plasmids encoding 24 resistance genes, including blaKPC-3, and two putative virulence clusters were detected. In the susceptible strain, large rearrangements occurred in the KPC-carrying plasmid, causing the deletion of the entire Tn4401::blaKPC-3 transposon, with the consequent reversion of the strain to carbapenem susceptibility. The patient was successfully treated with carbapenems and fully recovered.The description of the plasmid content in these two strains gives interesting insights into the plasticity of KPC-carrying plasmids in K. pneumoniae.

Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen affecting humans and a major source for hospital infections associated with high morbidity and mortality due to limited treatment options. We summarize the wide resistome of this pathogen, which encompasses plentiful chromosomal and plasmid-encoded antibiotic resistance genes (ARGs). Under antibiotic selective pressure, K. pneumoniae continuously accumulates ARGs, by de novo mutations, and via acquisition of plasmids and transferable genetic elements, leading to extremely drug resistant (XDR) strains harboring a 'super resistome'. In the last two decades, numerous high-risk (HiR) MDR and XDR K. pneumoniae sequence types have emerged showing superior ability to cause multicontinent outbreaks, and continuous global dissemination. The data highlight the complex evolution of MDR and XDR K. pneumoniae, involving transfer and spread of ARGs, and epidemic plasmids in highly disseminating successful clones. With the worldwide catastrophe of antibiotic resistance and the urgent need to identify the main pathogens that pose a threat on the future of infectious diseases, further studies are warranted to determine the epidemic traits and plasmid acquisition in K. pneumoniae. There is a need for future genomic and translational studies to decipher specific targets in HiR clones to design targeted prevention and treatment.

Increasing reports of multidrug resistance conferred by conjugative plasmids of Enterobacteriaceae necessitate a better understanding of their evolution. One such group is the narrow-host-range IncI1 plasmid type, known for their ability to carry genes encoding resistance to extended-spectrum beta lactamases. The focus of this study was to perform comparative sequencing of IncI1 plasmids from porcine enterotoxigenic Escherichia coli (ETEC), isolated irrespective of antimicrobial susceptibility phenotype. Five IncI1 plasmids of porcine ETEC origin and one IncI1 plasmid from a Salmonella enterica serovar Kentucky isolate from a healthy broiler chicken were sequenced and compared to existing IncI1 plasmid sequences in an effort to better understand the overall genetic composition of the IncI1 plasmid lineages. Overall, the sequenced porcine ETEC IncI1 plasmids were divergent from other sequenced IncI1 plasmids based upon multiple means of inferred phylogeny. High occurrences of IncI1 and IncA/C plasmid-associated genes and the blaTEM and blaCMY-2 beta lactamase genes were observed among porcine ETEC. However, the presence of blaTEM and blaCMY-2 did not strongly correlate with IncI1 plasmid possession, suggesting that these plasmids in porcine ETEC are not primarily associated with the carriage of such resistance genes. Overall, this work suggests a conservation of the IncI1 plasmid backbone among sequenced plasmids with a single locus for the acquisition of accessory genes, such as those associated with antimicrobial resistance. Furthermore, the high occurrence of IncI1 and IncA/C plasmids among clinical E. coli from commercial swine facilities is indicative of extensive horizontal gene transfer among porcine ETEC.

Ninety-six genetically diverse multidrug-resistant clinical isolates of Acinetobacter baumannii from 25 hospitals in 17 European countries were screened by PCR for specific carbapenemase-hydrolyzing class D β-lactamase (CHDL) genes and by PCR-based replicon typing for the presence of 19 different plasmid replicase ( rep ) gene homology groups (GRs). Results were confirmed by DNA sequencing where necessary. All 96 isolates contained at least 1 (with a maximum of 4) of the 19 groups of rep genes. Groups detected were GR6 ( rep Aci6; 93 isolates), GR2 (including rep Aci1 [67 isolates] and rep Aci2 [3 isolates]), GR16 ( rep ApAB49; 12 isolates), GR12 (p2ABSDF0001; 10 isolates), GR3 ( rep Aci3; 4 isolates), GR4 ( rep Aci4; 3 isolates), GR10 ( rep AciX; 1 isolate), and GR14 ( rep p4AYE; 1 isolate). Variations in rep gene content were observed even among epidemiologically related isolates. Genes encoding OXA-58-like CHDLs (22 isolates) were associated with carriage of the rep Aci1, rep Aci3, rep Aci4, and rep AciX genes, genes encoding OXA-40-like CHDLs (6 isolates) were associated with rep Aci2 and p2ABSDF0001, and genes encoding OXA-23-like CHDLs (8 isolates) were associated with rep Aci1. Most intrinsic Acinetobacter plasmids are non-self-transferable, but the almost ubiquitous rep Aci6 gene was strongly associated with a potential tra locus that could serve as a general system for plasmid mobilization and consequent horizontal transmission of plasmids and their associated antibiotic resistance genes among strains of A. baumannii .

Extensively drug-resistant (XDR) Klebsiella pneumoniae isolates usually carry a single carbapenemase (e.g. KPC, NDM, OXA-48-like). Here we describe an XDR K. pneumoniae of sequence type 101 that was detected in the screening rectal swab of a patient transferred from the intensive care unit of a hospital located in Belgrade (Serbia) to Bern University Hospital (Switzerland). The isolate was resistant to all antibiotics with the exception of colistin [minimum inhibitory concentration] (MIC ≤ 0.125 μg/mL), tigecycline (MIC = 0.5 μg/mL) and fosfomycin (MIC = 2 μg/mL). The isolate co-possessed class B (NDM-1) and class D (OXA-48) carbapenemases, class A extended-spectrum β-lactamase (CTX-M-15), class C cephalosporinase (CMY-16), ArmA 16S rRNA methyltransferase, substitutions in GyrA and ParC, loss of OmpK35 porin, as well as other genes conferring resistance to quinolones (qnrA), tetracyclines [tet(A)], sulfonamides (sul1, sul2), trimethoprim (dfrA12, dfrA14), rifampicin (arr-1), chloramphenicol (cmlA1, floR) and streptomycin (aadA1). The patient was placed under contact isolation precautions preventing the spread of this nearly untreatable pathogen.

Objectives To determine the genetic structures at the origin of the mobilization of the extended-spectrum β-lactamase (ESBL) blaGES-1 gene in an Escherichia coli clinical isolate. Methods ESBL-encoding genes and class 1 or class 3 integron-specific motifs were screened. Conjugation experiments were performed to determine whether the plasmid-carrying blaGES-1 gene was self-transferable. Plasmid sequencing was achieved by a primer-walking approach. Results The blaGES-1 gene was located in a class 3 integron. That unusual genetic structure was itself located on an ∼9 kb plasmid, pQ7, which was not self-transferable. Sequence analysis revealed that plasmid pQ7 belonged to a novel subtype of the IncQ group. Conclusions This study identified for the first time the blaGES-1 gene in E. coli and in Switzerland. It describes a novel IncQ-type plasmid subgroup that possesses original features, in particular iteron sequences that constitute a hot spot for integration of foreign DNA

The objective of this study was to understand the conjugative transmissibility of resistance plasmids present in 205 Salmonella enterica isolates from bovine sources. Polymerase chain reaction (PCR)-based replicon typing was used to type plasmid replicons. Conjugation experiments were preformed in triplicate at 30 degrees C and 37 degrees C on solid medium. PCR mapping of the A/C transfer gene operon was done on 17 Salmonella Newport isolates that were only positive for A/C. Eighty-six percent (n = 177) of the Salmonella isolates were multidrug resistant (MDR) with resistance to 3-12 antimicrobial agents. Of these, 82% (n = 146) were resistant to extended-spectrum cephalosporins and possessed a bla(CMY) gene. A/C was the predominant replicon detected, present in 90% (n = 160) of the MDR isolates. Twenty-three percent (n = 37) of the A/C-positive strains were positive for a second replicon. Replicons coresident with A/C included I1, N, B/O, HI1, and HI2. Only 31% (n = 54) of the MDR isolates produced transconjugants, and most of these donors carried multiple replicons. A/C cotransferred with B/O, N, and I1 at both 30 degrees C and 37 degrees C and with HI2 at 30 degrees C. Seven Salmonella Newport isolates that produced transconjugants possessed only the single A/C replicon and lacked bla(CMY). PCR mapping of the A/C transfer gene operon in ten Salmonella Newport isolates that carried bla(CMY) revealed a bla(CMY) inverted repeat element integrated between the traA and traC genes. These results suggest that A/C may have been a conjugative plasmid before the integration of bla(CMY) into the transfer gene operon. Additionally, transfer deficient A/C replicons may be mobilized in the presence of certain compatible conjugative plasmids.

We determined the complete nucleotide sequences of three plasmids that encode CTX-M extended-spectrum β-lactamases (ESBLs) in pulsed-field gel electrophoresis-defined United Kingdom variants (strains A, C, and D) of the internationally prevalent Escherichia coli O25:H4-ST131 clone. Plasmid pEK499 (strain A; 117,536 bp) was a fusion of type FII and FIA replicons and harbored the following 10 antibiotic resistance genes conferring resistance to eight antibiotic classes: bla CTX-M-15 , bla OXA-1 , bla TEM-1, aac6′-Ib-cr , mph (A), catB4 , tet (A), and the integron-borne dfrA7 , aadA5 , and sulI genes. pEK516 (strain D; 64,471 bp) belonged to incompatibility group IncFII and carried seven antibiotic resistance genes: bla CTX-M-15 , bla OXA-1 , bla TEM-1 , aac6′-Ib-cr , catB4 , and tet (A), all as in pEK499. It also carried aac3-IIa , conferring gentamicin resistance, and was highly related to pC15-1a, a plasmid encoding the CTX-M-15 enzyme in Canada. By contrast, pEK204 (strain C; 93,732 bp) belonged to incompatibility group IncI1 and carried only two resistance genes, bla CTX-M-3 and bla TEM-1 . It probably arose by the transposition of Tn 3 and IS Ecp1 - bla CTX-M-3 elements into a pCOLIb-P9-like plasmid. We conclude that (i) United Kingdom variants of the successful E. coli ST131 clone have acquired different plasmids encoding CTX-M ESBLs on separate occasions, (ii) the bla CTX-M-3 and bla CTX-M-15 genes on pEK204 and pEK499/pEK516 represent separate escape events, and (iii) IncFII plasmids harboring bla CTX-M-15 have played a crucial role in the global spread of CTX-M-15 ESBLs in E. coli .

Objectives: The aim of this study was to identify and characterize plasmids carrying qnrS1, qnrB2 and qnrB19 genes identified in Salmonella strains from The Netherlands. The identification of plasmids may help to follow the dissemination of these resistance genes in different countries and environments. Methods: Plasmids from 33 qnr-positive Salmonella strains were transferred to Escherichia coli and analysed by restriction, Southern blot hybridization, PCR and sequencing of resistance determinants. They were also assigned to incompatibility groups by PCR-based replicon typing, including three additional PCR assays for the IncU, IncR and ColE groups. The collection included isolates from humans and one from chicken meat. Results: Five IncN plasmids carrying qnrS1, qnrB2 and qnrB19 genes were identified in Salmonella enterica Bredeney, Typhimurium PT507, Kentucky and Saintpaul. qnrS1 genes were also located on three further plasmid types, belonging to the ColE (in Salmonella Corvallis and Anatum), IncR (in Salmonella Montevideo) and IncHI2 (in Salmonella Stanley) groups. Conclusions: Multiple events of mobilization, transposition and replicon fusion generate the complexity observed in qnr-positive isolates that are emerging worldwide. Despite the fact that the occurrence of qnr genes in bacteria from animals is scarcely reported, these genes are associated with genetic elements and located on plasmids that are recurrent in animal isolates.

Escherichia coli strains producing extended-spectrum -lactamases (ESBLs) are a major problem in many different hospitals worldwide, causing outbreaks as well as sporadic infections. The prevalence of Escherichia coli ESBL producers was analyzed in a surveillance study performed on the population attending the Policlinico Umberto I, the largest university hospital in Rome, Italy. We also investigated genotypes, pathogenicity islands, and plasmids in the ESBL-positive E. coli isolates as further markers that are useful in describing the epidemiology of the infections. In this survey, 163 nonreplicate isolates of Escherichia coli were isolated from patients from 86 different wards, and 28 were confirmed as ESBL producers. A high prevalence (26/28) of CTX-M-15 producers was observed within the bacterial population circulating in this hospital, and the dissemination of this genetic trait was associated with the spread of related strains; however, these do not have the characteristics of a single epidemic clone spreading. The dissemination was also linked to horizontal transfer among the prevalent E. coli genotypes of multireplicon plasmids showing FIA, FIB, and FII replicons in various combinations, which are well adapted to the E. coli species. The analysis of related bacteria suggests a probable interpatient transmission occurring in several wards, causing small outbreaks. A rapid dissemination of isolates producing CTX-M-type extended-spectrum -lactamases (ESBLs) has recently been reported in some European countries, including Italy, and is a matter of major concern. The blaCTX-M genes have been captured on transferable plasmids from the chromosomes of Kluyvera spp., and their products are becoming the most prevalent ESBLs encountered in Enterobacteriaceae (19). The prevalent blaCTX-M-type genes in Europe have been identified as blaCTX-M-1, blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, and blaCTX-M-15 (2, 19). Infections caused by enterobacteria producing ESBLs are associated with increased morbidity, mortality, and health care-associated costs (9, 16). In Italy, the presence of CTX-M-type ESBLs in clinical isolates of Enterobacteriaceae examined in a Italian nationwide survey (23, 27), as well as in isolates from companion animals (7), was previously reported, and the results showed that CTXM-type enzymes were common overall (around 20%) among ESBL producers (20). However, the local epidemiology of Escherichia coli producing ESBLs may differ from the national picture, with dominance of different strains. We therefore studied the prevalence and the clonality of the Escherichia coli ESBL producers in the population attending the Policlinico Umberto I, the largest university hospital in Rome, Italy, which had not been included in the nationwide survey. We also investigated virulence factors encoded on pathogenicity islands (PAIs) and plasmids carried by the E. coli ESBL producers as further markers that are useful in describing the epidemiology of nosocomially acquired infections.

Objectives To evaluate the presence of qnr genes among enterobacterial isolates carrying extended-spectrum beta-lactamases (ESBLs) in Barcelona, Spain. Methods Screening for the qnrA, qnrB and qnrS genes was carried out by PCR amplification with specific primers in 305 non-duplicate, clinically relevant ESBL-producing enterobacterial isolates obtained from February 2003 to August 2004. ESBLs from all qnr-positive isolates were characterized by isoelectric focusing, PCR amplification and DNA sequencing. Plasmid analysis was performed by S1 digestion and hybridization with specific probes for the qnr and bla genes. Plasmids containing qnr genes were transferred by conjugation or transformation. The genetic environment of qnrA1 in selected isolates was characterized by cloning experiments. Results Fifteen isolates, each from a different individual, carried qnr. Among them, 14 had qnrA1 (6 Klebsiella pneumoniae, 6 Enterobacter cloacae and 2 Escherichia coli isolates) and 1 had qnrS1 (K. pneumoniae). None of the isolates carried qnrB. Among the qnrA1-carrying isolates, 10 possessed both bla(CTX-M-9) and bla(SHV-12), 2 had both bla(CTX-M-9) and bla(SHV-92) and 2 had bla(CTX-M-9) alone. The isolate with qnrS1 possessed bla(SHV-12). The qnrA1 and ESBL genes were located together on plasmids ranging in size from 40 to 320 kb. qnrS1 and bla(SHV-12) were not located on the same plasmid. Transfer of quinolone resistance was successfully achieved from all but three isolates. The cloned region surrounding qnrA in two K. pneumoniae isolates revealed a novel genetic organization. Conclusions The prevalence of qnr among enterobacterial clinical isolates carrying ESBLs between 2003 and 2004 in Barcelona was 4.9%. qnrA1 was the most prevalent, whereas only one qnrS and no qnrB were detected.

Salmonella enterica bla CTX-M-2 and bla CTX-M-9 plasmid backbones from isolates from Belgium and France were analyzed. The bla CTX-M-2- plasmids from both human and poultry isolates were related to the IncHI2 pAPEC-O1-R plasmid, previously identified in the United States in avian Escherichia coli strains; the bla CTX-M-9 plasmids were closely related to the IncHI2 R478 plasmid.

Escherichia coli and Salmonella enterica isolates carrying the blaCTX-M-9 gene located on plasmids prevailed at the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain in the 1996–1999 period. The blaCTX-M-9-carrying plasmids showed a great variability in size, suggesting the mobilization of the gene among different plasmid scaffolds. The aim of the present work was to identify and better characterize the plasmids involved in the spread of the blaCTX-M-9 gene. Results showed that the majority of these strains carried plasmids belonging to the IncHI2 incompatibility group. The IncHI2 plasmids were further characterized, and found to be related to the reference IncHI2 plasmid R478.

Food-producing animals are the primary reservoir of zoonotic pathogens, and the rate of detection of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Salmonella strains has increased in recent years. ESBLs are widely detected in various human medical institutions, but they are not so frequently reported in bacterial populations circulating in animals. In Belgium and France the emergence of resistance to extended-spectrum cephalosporins, such as ceftriaxone and ceftiofur, has been recently reported in Salmonella enterica serovar Virchow isolates from poultry and humans (1, 15). Resistance was due to the ESBL genes blaCTX-M-2 or blaCTX-M-9 carried on large conjugative plasmids. Since 2001 a large number of strains have been isolated from poultry (more than 150 in Belgium) and a more limited number from humans (n = 15) in Belgium and France showing resistance to extended-spectrum cephalosporins by production of an ESBL not belonging to the CTX-M family and with various additional resistances to other antibiotic families. The serovars concerned were Agona, Derby, Infantis, Paratyphi B dT+, and Typhimurium. In particular, the emergence of extended-spectrum cephalosporin-resistant serovar Infantis with more than 80 strains isolated from poultry and 4 strains from humans caused some concern. The purpose of the present study was to identify the ESBL gene and its location in these strains. Strains studied are shown in Table ​Table1.1. Antibiotic susceptibility testing was done by the disk diffusion method, and the MICs of ceftriaxone and ceftiofur were determined as described previously (1, 14, 15). Resistance to extended-spectrum cephalosporins from all Salmonella strains was transferred to an E. coli recipient strain by conjugation as previously described (1, 14, 15), and all E. coli transconjugant strains showed the same antibiotic resistance profile (Table ​(Table1).1). Other resistances from multidrug-resistant strains were not transferred by conjugation. According to the MICs, the levels of resistance to ceftiofur and ceftriaxone were lower in the transconjugant strains than in the parental strains, but this was also observed in a previous study (14). PCR assays to detect ESBL genes (TEM, SHV, and CTX-M) were performed on parental and transconjugant strains using previously described primers (1, 14, 15), and nucleotide sequencing of the amplicons identified the blaTEM-52 resistance gene in all strains. Plasmids extracted from the transconjugants were further characterized by PstI restriction analysis showing that they were all identical and greater than 100 kb in size (Fig. ​(Fig.1).1). Southern blot hybridization experiment with a blaTEM-52 gene probe was performed as described previously (12). It revealed two PstI fragments of 2.9 and 2.75 kb. In fact, this PstI restriction profile corresponded exactly to that of blaTEM-52-carrying plasmids isolated in 2002 and 2003 from four isolates of S. enterica serovars Typhimurium, Enteritidis, and Panama from French patients with gastroenteritis (14). A study performed in 2001 and 2002 on Salmonella isolated from poultry, poultry products, and human patients in The Netherlands revealed that the TEM-52 variant was the most common ESBL detected in this bacterial collection (6). In particular, TEM-52-producing salmonellae of the serovars Blockley, Virchow, Typhimurium, and Paratyphi B were identified from poultry, and strains of the serovars Thompson, London, Enteritidis, and Blockley were identified from human patients (6). Several sporadic cases of E. coli TEM-52 producers were reported in animals: dogs in Portugal, rabbits in Spain, and beef meat in Denmark (2, 4, 8). These findings suggest a wide dissemination of this ESBLs in Europe in animals and humans. The presence of the blaTEM-52 gene in E. coli, as well as in different Salmonella serovars, strongly indicated that it is not due to the spread of a single clone but to the horizontal transmission of this resistance trait. To better identify the molecular mechanism of dissemination of this ESBL, the blaTEM-52-positive plasmids were typed by the PCR-based replicon typing as previously described (3), demonstrating that they all belong to the IncI1 incompatibility group. IncI1 plasmids were recently described in E. coli and Salmonella strains of different serovars isolated in the United Kingdom associated with relevant β-lactamases such as CMY-2, CMY-7, and CTX-M-15, suggesting a large prevalence of IncI1 plasmids in Europe (7). FIG. 1. Restriction analysis (PstI) (upper panel) and Southern blot hybridization with a blaTEM-52 probe (lower panel) of plasmid DNAs isolated from E. coli transconjugants. Lane 1, E. coli transconjugant 988SA01TC1; lane 2, E. coli transconjugant 2004/10101TC1; ... TABLE 1. Characteristics of the Salmonella strains and their transconjugants producing TEM-52 used in this study To identify the mobile genetic element carrying the blaTEM-52 gene, the plasmid DNA of E. coli transconjugant 04-3486TC1 extracted with a QIAfilter Midi kit (QIAGEN, Courtaboeuf, France) was digested with ClaI and ligated into the ClaI-restricted phagemid pBK-CMV (Stratagene). Recombinant plasmids were introduced into E. coli DH10B by electroporation (Bio-Rad Gene Pulser II; Bio-Rad, Marnes-La-Coquette, France) and selected on Mueller-Hinton (MH) agar (Bio-Rad) containing kanamycin (30 μg/ml) and ceftazidime (2 μg/ml). Recombinant plasmids that possessed a 4.7-kb insert were selected. Nucleotide sequencing of the insert indicated that the blaTEM-52 gene was located on a Tn3 transposon. To complete the transposon sequence, nucleotide sequencing was further performed by genome walking on the native plasmid. This Tn3 transposon structure is shown in Fig. ​Fig.2.2. Its nucleotide sequence is deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"EF141186","term_id":"119632822","term_text":"EF141186"}}EF141186. Very few sequences of complete Tn3 elements from Salmonella are currently available (10). Complete plasmid-borne Tn3 elements, however, specifying non-ESBLs have recently been described in serovar Typhimurium from a rabbit and in serovar Infantis from poultry and shown to be linked to either the tetracycline resistance gene tet(A) or the quinolone resistance gene qnrS (9, 13). FIG. 2. Genetic organization of the blaTEM-52 carrying transposon on a conjugative IncI1 plasmid from serovar Typhimurium strain 04-3486. The position and orientation of the genes are indicated by arrows. Gray arrows correspond to plasmid genes flanking the transposon. ... Among the extended-spectrum cephalosporin-resistant Salmonella strains studied, five isolates belonging to serovars Agona, Paratyphi B dT+, and Typhimurium, showed an additional multidrug resistance profile with resistances to chloramphenicol, florfenicol, streptomycin, spectinomycin, sulfonamide, tetracycline, and trimethoprim (Table ​(Table1).1). This multidrug resistance profile is characteristic of SGI1 antibiotic resistance gene clusters, which were previously identified in these serovars (11). Identification of SGI1 and mapping of its antibiotic resistance gene cluster performed as described previously (5) showed that two of the five isolates possessed SGI1 and the SGI1-A variant in serovar Typhimurium strain 04-3486 and serovar Agona strain 777SA01, respectively (Table ​(Table1).1). Serovar Agona strains with SGI1-A are frequently isolated from poultry in Belgium (5). The serovar Typhimurium isolate carrying SGI1 was further shown to be of phage type DT104, a dominant multidrug-resistant clone that has spread all over the world (11). To our knowledge, this is the first time that multidrug-resistant strains carrying SGI1 together with a plasmid-borne ESBL gene have been reported, and further surveillance of such strains is thus warranted. Since most of the strains showing extended-spectrum cephalosporin resistance were of serovar Infantis, these were further investigated for clonality by XbaI and BlnI macrorestriction pulsed-field gel electrophoresis analysis. The Infantis isolates showing the same resistance profile and whatever their origin, poultry or human, showed identical XbaI and BlnI macrorestriction profiles, indicating that these were clonal (data not shown). In conclusion, the present study showed the spread of an IncI1 plasmid carrying the blaTEM-52 gene among S. enterica serovars Agona, Derby, Infantis, Paratyphi B dT+, and Typhimurium, as well as the spread of a single Infantis clone carrying this plasmid mainly in poultry. It is thus likely that humans infected with these strains were contaminated by ingestion of undercooked poultry products. The further spread of such plasmids in multidrug-resistant strains carrying SGI1 is of concern.

A carbapenem-resistant Enterobacter cloacae strain, WCHECl-14653, causing a fatal bloodstream infection, was characterized by genome sequencing and conjugation experiments. The strain carried two carbapenemase genes, bla NDM-1 and bla KPC-2 , on separate IncF plasmids. The coexistence of bla NDM-1 and bla KPC-2 conferred slightly higher-level carbapenem resistance compared with that of bla NDM-1 or bla KPC-2 alone, and the coexistence of two IncF plasmids may generate new platforms for spreading carbapenemase genes.

This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing bla CTX-M-9 , and its association with Tn 402 , Tn 21 , and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn 402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 ( n = 26), IncP-1α ( n = 10), IncFI ( n = 4), and IncI ( n = 1) groups. Genetic platforms containing bla CTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn 402 derivatives. They were associated with Tn 21 sequences when located in IncP-1α or IncHI2 plasmids. Our study identified bla CTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn 402 derivatives, often to Tn 21 , highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of bla CTX-M-9 on broad-host-range IncP-1α plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae .

Genotyping of Salmonella strains is an important tool to discriminate among isolates and to improve epidemiological studies when an outbreak occurs. No phagetyping scheme is available for Salmonella enterica subsp. enterica serovar Abortusovis (SAO) and molecular methods previously used were not standardized and were time consuming. Among the DNA-based methods of genotyping, pulsed field gel electrophoresis (PFGE) is currently in use to subtype Salmonella isolates. In this study we evaluated the feasibility of genotyping of SAO by XbaI and BlnI restrictions. Separation of restricted fragments was performed by PFGE. To test the possibility to apply this methodology to epidemiological investigation, a collection of 38 SAO strains isolated in different regions of Italy were analyzed. Eighteen and 29 different PFGE profiles were defined for XbaI and BlnI digestions, respectively. The method demonstrated an adequate typing ability and an excellent discriminatory power. Results from this study show that PFGE may represent a powerful tool to discriminate within the SAO serovar, and provide useful information in support of traditional epidemiological investigations. In particular, this method could be used to identify the origin of infection during outbreaks within a single flock or in different herds.

Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems.

Objectives: The aim of this study was to identify chromosomally integrated genes conferring multidrug resistance to a Salmonella enterica (S.) serotype Typhimurium isolate, phage type DT193, isolated in Ireland and to compare them with resistance genes conferring plasmid-mediated multidrug resistance to a S. Enteritidis isolate from Italy. Methods: A complete DNA sequence of the regions containing the resistance genes was obtained from the chromosome of the S. Typhimurium DT193 isolate and from the IncI plasmid of the S. Enteritidis isolate. The plasmid was also characterized by conjugation and incompatibility grouping. Results: Two 10 kb multidrug resistance non-Salmonella Genomic Island 1 type clusters were independently identified in the S. Enteritidis plasmid and in the chromosome of the S. Typhimurium isolate. Detailed characterization identified an IP-type 2 integron containing a dfrA1-aadA1 gene cassette and other common resistance determinants derived from the RSF1010 plasmid. Conclusions: These multidrug resistance regions originate following chromosomal integration of key resistance markers encountered on plasmids circulating in other Salmonella serotypes. This mechanism of marker acquisition may have future implications for the evolution of similar structures in previously susceptible serotypes, leading to an increased public health risk.

A new Coronavirus (SARS‐CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in‐house ELISA based on SARS‐CoV recombinant antigens was developed. The SARS‐CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine‐tagged proteins. Six reference sera from SARS patients were used to detect virus‐specific IgG in an ELISA using each recombinant protein as coating antigen. High‐titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non‐SARS, low‐tract respiratory infections. Specific hyper‐immune sera to SARS‐CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS‐CoV infected and uninfected cells. J. Med. Virol. 76:137–142, 2005. © 2005 Wiley‐Liss, Inc.