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Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21. Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5' end of integrons was found. Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R. erythropolis SQ1.
The integron-borne bla(VEB-1) gene encodes an extended-spectrum beta-lactamase. This gene was associated mostly with IS1999 and rarely with an additional IS2000 element in Pseudomonas aeruginosa isolates from Thailand, whereas IS1999 was only very rarely associated with bla(VEB-1) in Enterobacteriaceae. Expression experiments and promoter study identified promoter sequences in IS1999 that increased the expression of VEB-1 in P. aeruginosa.
The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with P. aeruginosa clone K chromosomes at one of the two tRNA(Lys) genes, pKLC102 is incorporated into the tRNA(Lys) gene only close to the pilA locus. Targeting of the other tRNA(Lys) copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNA(Gly)-associated genome islands of P. aeruginosa clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements. Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in P. aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.
A study was designed to describe the molecular epidemiology of carbapenem-resistant (CR) Pseudomonas aeruginosa in a large well-defined geographical region with a centralized laboratory system serving one pediatric and three large adult hospitals (acute care centers I, II, and III). Molecular characterization was done using PCR with sequencing of the integron-associated gene cassettes. Pulsed-field gel electrophoresis (PFGE) using a modified combined Stenotrophomas maltophilia and Streptococcus pneumoniae protocol with SpeI was performed on CR P. aeruginosa strains isolated in the Calgary Health Region during 2002-2006. The majority (96%) of metallo-beta-lactamase (MBL)-producing isolates produced VIM-2 with gene cassettes aacC1 and aacA4, while 4% produced IMP-7 with gene cassettes aacC4 and aacC1. Eighty-six percent of VIM-2 producers belonged to a cluster (MBLV) that was responsible for nosocomial outbreaks during 2003 (intensive care unit) and 2004 (bone marrow transplant unit) at acute care center I. Environmental isolates from these units also belonged to MBLV. The majority of strains from cluster MBLVR (related to MBLV) were present in acute care center III. Isolates producing IMP-7 belonged to a different cluster (MBLI) and were related to strains described during the 1990 s. PFGE of the MBL-negative CR strains showed that 37% belonged to a closely related cluster, NMBL, whose members were predominantly isolated from acute care center II. Our findings suggest that CR and dissemination of MBL clusters among P. aeruginosa populations in large geographic healthcare regions are dynamic processes that require continuous molecular surveillance.
In Brazil, Salmonella enterica serovar Infantis resistant to various antimicrobials, including cephalosporins, has been identified as an etiological agent of severe gastroenteritis in hospitalized children since 1994. In this study, 35 serovar Infantis strains, isolated from children admitted to four different Rio de Janeiro, Brazil, hospitals between 1996 and 2001, were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing in order to determine their genetic relatedness and antimicrobial resistance profiles. Thirty-four serovar Infantis strains were resistant to at least two antibiotic classes, and all 35 strains were susceptible to fluoroquinolones, cephamycin, and carbapenem. Extended-spectrum beta-lactamase (ESBL) screening by double-disk diffusion indicated that 32 serovar Infantis strains (91.4%) produced beta-lactamases that were inhibited by clavulanic acid. Antimicrobial resistance gene profiles were determined by PCR for a subset of 11 multidrug-resistant serovar Infantis strains, and putative ESBLs were detected by isoelectric focusing. Ten serovar Infantis strains carried bla(TEM), catI, ant(3")Ia and/or ant(3")Ib, sulI and/or sulII, and tet(D) genes as well as an integron-associated aac(6')-Iq cassette. Eight strains possessed at least four different beta-lactamases with pI profiles that confirmed the presence of both ESBLs and non-ESBLs. Our PFGE profiles indicated that 33 serovar Infantis strains isolated from Rio de Janeiro hospitals came from the same genetic lineage.
In the last few years, phenotypically carbapenem resistant Acinetobacter strains have been identified throughout the world, including in many of the hospitals and intensive care units (ICUs) of Australia. Genotyping of Australian ICU outbreak-associated isolates by pulsed-field gel electrophoresis of whole genomic DNA indicated that different strains were cocirculating within one hospital. The carbapenem-resistant phenotype of these and other Australian isolates was found to be due to carbapenem-hydrolyzing activity associated with the presence of the blaOXA-23 gene. In all resistant strains examined, the blaOXA-23 gene was adjacent to the insertion sequence ISAba1 in a structure that has been found in Acinetobacter baumannii strains of a similar phenotype from around the world; blaOXA-51-like genes were also found in all A. baumannii strains but were not consistently associated with ISAba1, which is believed to provide the promoter required for expression of linked antibiotic resistance genes. Most isolates were also found to contain additional antibiotic resistance genes within the cassette arrays of class 1 integrons. The same cassette arrays, in addition to the ISAba1-blaOXA-23 structure, were found within unrelated strains, but no common plasmid carrying these accessory genetic elements could be identified. It therefore appears that antibiotic resistance genes are readily exchanged between cocirculating strains in epidemics of phenotypically indistinguishable organisms. Epidemiological investigation of major outbreaks should include whole-genome typing as well as analysis of potentially transmissible resistance genes and their vehicles.
Six distinct clones were present among Greek multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns, int1 integron structures, and presence or absence of antibiotic resistance genes ant(3'')-Ia, pse-1, and tem-1.
A total of 221 isolates of multidrug-resistant Salmonella enterica serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.
The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding alpha-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.
From 1992 to 1997, only six sporadic isolates of Salmonella enterica serotype Enteritidis from patients with cases of gastroenteritis in southern Italy exhibited resistance to broad-spectrum cephalosporins. Five isolates produced SHV-12, and one isolate encoded a class C beta-lactamase. The bla(SHV-12) gene was located in at least two different self-transferable plasmids, one of which also carried a novel class 1 integron.
Since 1996, the National Salmonella Reference Laboratory of Germany has received an increasing number of Salmonella enterica subsp. enterica serovar Paratyphi B isolates. Nearly all of these belonged to the dextrorotatory tartrate-positive variant (S. enterica subsp. enterica serovar Paratyphi B dT(+)), formerly called S. enterica subsp. enterica serovar Java. A total of 55 selected contemporary and older S. enterica subsp. enterica serovar Paratyphi B dT(+) isolates were analyzed by plasmid profiling, antimicrobial resistance testing, pulsed-field gel electrophoresis, IS200 profiling, and PCR-based detection of integrons. The results showed a high genetic heterogeneity among 10 old strains obtained from 1960 to 1993. In the following years, however, new distinct multiresistant S. enterica subsp. enterica serovar Paratyphi B dT(+) clones emerged, and one clonal lineage successfully displaced the older ones. Since 1994, 88% of the isolates investigated were multiple drug resistant. Today, a particular clone predominates in some German poultry production lines, poultry products, and various other sources. It was also detected in contemporary isolates from two neighboring countries as well.
Seventy-eight human and environmental strains of Salmonella enterica subsp. enterica serovar Typhimurium, as well as 18 isolates of other Salmonella serovars and 6 isolates of Escherichia coli, were subjected to a novel variable number of tandem repeats (VNTR)-based fingerprinting method that showed high discrimination and reproducibility for typing serovar Typhimurium isolates. The method is based on capillary separation of PCR products from fluorescence-labeled VNTR in the serovar Typhimurium genome. The serovar Typhimurium isolates displayed 54 VNTR patterns, and the VNTR assay correctly identified strains from a well-characterized outbreak. Among 37 serovar Typhimurium phage type DT104 isolates, 28 distinct VNTR patterns were found. This VNTR-based method is fast and suitable for complete automation. Our VNTR-based method was capable of high discrimination within the homogeneous serovar Typhimurium DT104 phage type and can be used to trace outbreaks and to monitor DT104 as well as other phage types. The VNTR assay was compared to XbaI pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, integron-cassette profiles and gene PCR of intI1, qacEDelta1, sulI1, and floR. The VNTR assay showed greatly improved resolution compared to all other tested methods in this study.
Twelve clonally related and multidrug-resistant Acinetobacter baumannii isolates were recovered during a 4-month period from 12 patients hospitalized at the Valenciennes Hospital in France. Antibiograms determined by the double-disk diffusion technique on cloxacillin-containing plates detected a clavulanic acid-inhibited extended-spectrum beta-lactamase (ESBL). PCR and sequencing identified the gene encoding the Ambler class A ESBL VEB-1. This gene was located on the chromosome and was part of a class 1 integron identical to that previously identified in Pseudomonas aeruginosa isolates from Thailand. Additionally, seven clonally related bla(VEB-1)-positive A. baumannii strains were identified in the immediate environment of the hospitalized patients. This is the first report of the ESBL VEB-1 in Acinetobacter spp. and the first description of VEB-1-producing strains as a source of an outbreak occurring outside Southeast Asia. This report underlines the difficulty of the identification of ESBLs in A. baumannii.
Seventeen Klebsiella pneumoniae clinical isolates carrying the bla(VIM-1) metallo-beta-lactamase gene were collected in the intensive care units of three hospitals in Athens, Greece, in 2002. They exhibited various carbapenem resistance levels (Etest MICs of imipenem ranged from 4 to 32 microg/ml). All isolates gave positive results by the imipenem-EDTA synergy Etest. The isolates were classified into four main types by pulsed-field gel electrophoresis; the majority of the isolates (5 and 10 isolates) belonged to two types. The bla(VIM-1) gene cassette was part of the variable region of a class 1 integron that also included aac6, dhfrI, and aadA. This structure was carried by transferable plasmids.
A multiresistant Klebsiella pneumoniae isolate was taken from the blood of a 75-year-old patient with nosocomial pneumonia who developed septic shock and failed therapy with imipenem. The isolate presented an MIC of imipenem of 128 microg/ml, and the production of a metallo-beta-lactamase was confirmed by phenotypic and genotypic techniques. We here report, for the first time, the detection of a metalloenzyme (IMP-1)-producing K. pneumoniae clinical strain in Latin America. The gene responsible for this phenotype was found to be bla(IMP-1), carried in a class 1 integron.
From December 2004 to March 2005, 27 Klebsiella pneumoniae clinical isolates that were positive by the imipenem-EDTA double-disk synergy test and that exhibited a single macro-restriction pattern were recovered in two distinct Greek hospitals. The isolates carried a transferable bla(VIM-1) metallo-beta-lactamase gene in a class 1 integron. Reverse transcriptase PCR showed that the gene was similarly expressed in low- and high-level carbapenem-resistant isolates, indicating the existence of additional resistance mechanisms. The clonal spread of VIM-1-producing K. pneumoniae strains in distinct regions where up to now bla(VIM-2) and bla(VIM-4) alleles were common is worrisome.
Over a 4-month period from November 2002 to February 2003, 27 ceftazidime-resistant or cefotaxime-resistant nonrepetitive Enterobacter cloacae isolates were collected from 27 patients hospitalized at HuaShan Hospital, Shanghai, People's Republic of China. The Etest did not detect extended-spectrum beta-lactamases (ESBLs) in those 27 isolates; however, screening by the NCCLS ESBL disk test and confirmatory tests detected ESBLs in 4 of 27 isolates and PCR detected ESBLs in 23 of 27 isolates. The majority of ESBL producers exhibited the same repetitive extragenic palindromic PCR pattern but harbored different ESBL genes. CTX-M-3 was the most prevalent ESBL in our study. Interestingly, 12 clonally related E. cloacae isolates possessed a novel bla(VEB)-type beta-lactamase, bla(VEB-3). Bla(VEB-3) was encoded by the chromosome and was located in an integron. Nine of the 12 isolates harbored both the bla(VEB-3) and the bla(CTX-M-3)-like ESBLs. This is the first report of a VEB-1-like ESBL in China and the first report of the simultaneous presence of VEB-1 and CTX-M-3-like ESBLs in an isolate.
Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years.