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Sequencing of the bla IMP-4 -carrying C. freundii B38 using the PacBio SMRT technique revealed that the genome contained a chromosome of 5,134,500 bp and three plasmids, pOZ172 (127,005 bp), pOZ181 (277,592 bp), and pOZ182 (18,467 bp). Plasmid pOZ172 was identified as IncFIIY, like pP10164-NDM and pNDM-EcGN174. It carries a class 1 integron with four cassettes (bla IMP-4 -qacG2-aacA4-aphA15) and a complete hybrid tni module (tniR-tniQ-tniB-tniA). The recombination of tniR from Tn402 (identical) with tniQBA from Tn5053 (99%) occurred within the res site of Tn402/5053 The Tn402/5053-like integron, named Tn6017, was inserted into Tn1722 at the res II site. The replication, partitioning, and transfer systems of pOZ181 were similar to those of IncHI2 plasmids (e.g., R478) and contained a sul1-type class 1 integron with the cassette array orf-dfrA1-orf-gcu37-aadA5 linked to an upstream Tn1696 tnpA-tnpR and to a downstream 3' conserved sequence (3'-CS) and ISCR1 A Tn2 transposon encoding a bla TEM-1 β-lactamase was identified on pOZ182. Other interesting resistance determinants encoded on the B38 chromosome included multidrug resistance (MDR) efflux pumps, an AmpC β-lactamase, and resistances to Cu, Ag, As, and Zn. This is the first report of a complete tni module linked to a bla IMP-4 -carrying class 1 integron, which, together with other recently reported non-sul1 integrons, represents the emergence of a distinct evolutionary lineage of class 1 integrons lacking a 3'-CS (qacEΔ1-sul1). The unique cassette array, complete tni module of Tn6017, and incompatibility group of pOZ172 suggest a bla IMP-4 evolutionary pathway in C. freundii B38 different from that for other bla IMP-4 genes found in Gram-negative bacteria in the Western Pacific region., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The early identification of outbreaks is crucial for the control of Clostridium difficile infection. This study aimed to determine if the number of hospital-acquired C. difficile infections could be reduced by rapidly typing C. difficile strains using multiple-locus variable-number tandem-repeat analysis (MLVA) compared to typing using PCR ribotyping. A total of 16 hospitals were recruited to the study, and all periods of increased incidence (PIIs) of C. difficile infection were identified. The hospitals were randomized into two study arms, the test and the control, with all isolates typed in the test using MLVA and in the control using PCR ribotyping. Following a PII, each hospital received a structured questionnaire regarding control measures implemented or stopped prior to or following the typing results. During the study period, there were a total of 1,682 hospital-apportioned C. difficile toxin-positive cases, with 868 in the control and 814 in the test, with modeling demonstrating no differences between the two arms. A total of 245 PIIs occurred, involving 785 patients. There was a significant difference in the mean turnaround time between the ribotyping and MLVA typing (13.6 and 5.3 days, respectively [P < 0.001]). The discriminatory ability of MLVA was greater than ribotyping, with 85 outbreaks being confirmed by ribotyping and 62 by MLVA. In the test arm, 40.6% of respondents strongly agreed that the typing result had aided their management of clusters, as opposed to 9.9% in the control. The study demonstrated the utility of rapidly typing C. difficile strains, demonstrating that it aided the management of clusters, enabling effective targeting of infection control resources.

We report on the first occurrence of high-level gentamicin resistance (MICs > or = 512 microg/ml) in seven clinical isolates of Streptococcus pasteurianus from Hong Kong. These seven isolates were confirmed to be the species S. pasteurianus on the basis of nucleotide sequencing of the superoxide dismutase (sodA) gene. Epidemiological data as well as the results of pulse-field gel electrophoresis analysis suggested that the seven S. pasteurianus isolates did not belong to the same clone. Molecular characterization showed that they carried a chromosomal, transposon-borne resistance gene [aac(6')Ie-aph(2'')Ia] which was known to encode a bifunctional aminoglycoside-modifying enzyme. The genetic arrangement of this transposon was similar to that of Tn4001, a transposon previously recovered from Staphylococcus aureus and other gram-positive isolates. Genetic linkage with other resistance elements, such as the ermB gene for erythromycin resistance, was not evident. On the basis of these findings, we suggest that routine screening for high-level gentamicin resistance should be recommended for all clinically significant blood culture isolates. This is to avoid the inadvertent use of short-course combination therapy with penicillin and gentamicin, which may lead to the failure of treatment for endocarditis, the selection of drug-resistant Streptococcus pasteurianus and other gram-positive organisms, as well as the unnecessary usage of gentamicin, a drug with potential toxicity.

A novel plasmid-mediated metallo-beta-lactamase (IMP-9) is described in seven isolates of Pseudomonas aeruginosa from Guangzhou, China, isolated in 2000. The gene was carried on a large (approximately 450-kb) IncP-2 conjugative plasmid. This is the first report of carriage of bla(IMP) genes on such large plasmids.

CTX-M-25 is a novel extended-spectrum beta-lactamase isolated from a single Canadian Escherichia coli isolate. Susceptibility testing demonstrated that this enzyme confers resistance to both cefotaxime and ceftazidime, but the level of resistance was reduced with the addition of beta-lactamase inhibitors. The bla(CTX-M-25) gene was detected on a 111-kb plasmid. It is a member of the CTX-M-8 group and has the closest amino acid identity (99%; three amino acid substitutions) with CTX-M-26. The bla(CTX-M-26) gene was detected on a 100-kb plasmid isolated from a Klebsiella pneumoniae strain from the United Kingdom, and plasmid profiling revealed that it showed some homology to the bla(CTX-M-25)-harboring plasmid. Both CTX-M genes were located downstream of ISEcp1, although the copy upstream of bla(CTX-M-25) was disrupted by IS50-A. Comparative kinetic studies of recombinant CTX-M-25 and CTX-M-26 enzymes showed that CTX-M-25 has a higher level of ceftazidime hydrolysis (kcat values, 33 and 0.005 s(-1) for CTX-M-25 and CTX-M-26, respectively).

An evaluation of the utility of IS6110-based restriction fragment length polymorphism (RFLP) typing compared to a combination of variable number tandem repeat (VNTR) typing and mycobacterial interspersed repetitive unit (MIRU) typing was undertaken. A total of 53 patient isolates of Mycobacterium tuberculosis from four presumed episodes of cross-infection were examined. Genomic DNA was extracted from the isolates by a cetyl trimethylammonium bromide method. The number of copies of tandem repeats of the five loci ETR(A) to ETR(E) and 12 MIRU loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. VNTR typing identified the major clusters of strains in the three investigations in which they occurred (each representing a different evolutionary clade: 32333, 42235, and 32433). The majority of unrelated isolates (by epidemiology and RFLP typing) were also identified by VNTR typing. The concordance between the RFLP and MIRU typing was complete, with the exception of two isolates with RFLP patterns that differed by one band each from the rest of the major epidemiologically linked groups of isolates in investigation A. All of these isolates had identical MIRU and VNTR types. A further pair of isolates differed in the number of tandem repeat copies at two MIRU alleles but had identical RFLP patterns. The speed of the combined VNTR and MIRU typing approach enabled results for some of the investigations to be supplied in "real time," influencing choices in contact tracing. The ease of comparison of results of MIRU and VNTR typing, which are recorded as single multidigit numbers, was also found to greatly facilitate investigation management and the communication of results to health care professionals.