Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as major causes of health care-associated infections worldwide. This diverse collection of organisms with various resistance mechanisms is associated with increased lengths of hospitalization, costs of care, morbidity, and mortality. The global spread of CRE has largely been attributed to dissemination of a dominant strain of Klebsiella pneumoniae producing a serine β-lactamase, termed K. pneumoniae carbapenemase (KPC). Here we report an outbreak of KPC-producing CRE infections in which the degree of horizontal transmission between strains and species of a promiscuous plasmid is unprecedented. Sixteen isolates, comprising 11 unique strains, 6 species, and 4 genera of bacteria, were obtained from 14 patients over the first 8 months of the outbreak. Of the 11 unique strains, 9 harbored the same highly promiscuous plasmid carrying the KPC gene blaKPC. The remaining strains harbored distinct blaKPC plasmids, one of which was carried in a strain of Klebsiella oxytoca coisolated from the index patient and the other generated from transposition of the blaKPC element Tn4401. All isolates could be genetically traced to the index patient. Molecular epidemiological investigation of the outbreak was aided by the adaptation of nested arbitrary PCR (ARB-PCR) for rapid plasmid identification. This detailed molecular genetic analysis, combined with traditional epidemiological investigation, provides insights into the highly fluid dynamics of drug resistance transmission during the outbreak.
Tumor necrosis factor (TNF)-alpha-dependent apoptosis of alveolar macrophages (AM) after infection with avirulent Mycobacterium tuberculosis (Mtb) results in bacillary death and the destruction of a growth niche for the pathogen. This response is minimized after infection with virulent strains of Mtb. To study the genetic control of Mtb-induced apoptosis, we used microarrays to interrogate the expression profile of infected human AM. Although we found variation in gene expression between different donors of AM, a set of genes were constant for each condition. A group of proapoptotic genes were downregulated after infection by virulent Mtb strain H37Rv, whereas infection with avirulent Mtb H37Ra led to a gene expression profile that would favor macrophage apoptosis. Neutralizing TNF in macrophage cultures infected with H37Ra changed the gene expression profile to one that resembled the profile of macrophages infected with H37Rv. These data reveal that apoptosis-related genes are regulated differently by virulent or attenuated Mtb strains, and are consistent with the hypothesis that virulent Mtb interfere with TNF death signaling. Given the importance of TNF in host defense against tuberculosis, the ability to repress the expression of genes activated by TNF may constitute a bacillary virulence mechanism.
An intervention targeting wastewater plumbing fixtures, by installation of hopper covers, demonstrated a decrease in patient KPCO acquisitions. Considering wastewater reservoirs in nosocomial transmission of multispecies carbapenemase-producing Enterobacteriaceae may be critical.
OXA-48 has emerged as a major carbapenemase associated with the Enterobacteriaceae in Europe, North Africa, and Asia. We report the first two clinical cases of OXA-48-type carbapenemase-producing Enterobacteriaceae in the United States from patients recently hospitalized in Saudi Arabia and India. Each is more carbapenem resistant than nearly all previously reported OXA-48-type-producing Enterobacteriaceae .
The Klebsiella pneumoniae carbapenemase gene (bla KPC ) is typically located within mobile transposon Tn4401. Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of bla KPC . Illumina sequences from bla KPC -positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188-bp deletion [between istB and bla KPC ]) was present in 14% (39/281) of clinical isolates. MICs showed that Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (Ն16 and Ն16, respectively), ertapenem (Ն8 and 4, respectively), and cefepime (Ն64 and 4, respectively) than E. coli strains with Tn4401b (0.5, Յ0.5, and Յ1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn4401a had a 16-fold increase and Tn4401h a 4-fold increase in bla KPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn4401a and Tn4401h promoter sequences generated higher -galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. The activity of the isolated P2 promoter was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicated that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.
The CDC screening protocol appeared to be the least expensive perirectal screening method. However, expense must be weighed against a lower sensitivity and extra labor needed for additional work-up of non-CPE isolates. The molecular test has the shortest turnaround time but the greatest expense.
The currently recommended phenotypic test for the detection of carbapenemase-producing members of the family Enterobacteriaceae is the modified Hodge test (MHT). However, the MHT lacks specificity. Here we demonstrate an alternative phenotypic test, the indirect carbapenemase test, for the detection of bla KPC -producing isolates that has specificity superior to that of the MHT for non-Klebsiella Enterobacteriaceae. The last decade has witnessed the dramatic emergence and worldwide dissemination of carbapenem resistance in Enterobacteriaceae. In the United States, the most common mechanism of carbapenem resistance is the production of the Ambler class A serine -lactamase Klebsiella pneumoniae carbapenemase (KPC) (1). The bla KPC gene product has the ability to hydrolyze all penicillins, cephalosporins, monobactams, and carbapenem antibiotics, which often leaves clinicians with few therapeutic options. As a consequence, infections due to carbapenemase-producing Enterobacteriaceae (CPE) carry a high mortality rate, reaching 40% or higher in some studies (2-5).The bla KPC gene is carried on resistance plasmids that are readily transmitted between bacterial strains and species, making the prevention of in-hospital transmission especially difficult (6-8). Accurate recognition of CPE in the clinical microbiology laboratory is central to controlling the spread of these organisms in the health care setting (9). However, identification of CPE from clinical isolates can be challenging. Molecular detection of a specific carbapenemase gene is the gold standard, but this method is impractical for many clinical laboratories.In 2009, the Clinical and Laboratory Standards Institute (CLSI) suggested phenotypic evaluation for the presence of a carbapenemase in Enterobacteriaceae with elevated MICs of one or more carbapenems by using the modified Hodge test (MHT) (9). The MHT has been found to be a useful tool for the detection of CPE but lacks specificity for serine carbapenemases (10-13). We previously reported a heterogeneous outbreak of KPC-producing Enterobacteriaceae (7,14). Starting in late 2009, there was an increase in Enterobacteriaceae isolates (mostly Enterobacter spp.) that were MHT positive but bla KPC PCR negative. This prompted us to prospectively evaluate phenotypic screening approaches for carbapenemase production. Here we compare the MHT and the indirect carbapenemase test (ICT), originally described by Moland et al. (15). Those investigators also described a direct carbapenemase test with imipenem, but we did not use it because some of our isolates expressed such high levels of resistance that no evaluable phenotypic result was possible.This study was conducted at the University of Virginia Medical Center, a 619-bed tertiary-care hospital in central Virginia, from 1 May 2010 to 31 December 2011. One hundred twenty-seven isolates of Enterobacteriaceae with ertapenem MICs of Ն1 g/ml were prospectively collected during the study period. Given the heterogeneous nature of our earlier outbreak (7), all species of En...
Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC. Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-ΔI-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated.
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