Steven H. Marshall

Gene Dosage and Linezolid Resistance in Enterococcus faecium and Enterococcus faecalis

ABSTRACT Resistance to linezolid has been associated with a G2576U mutation in domain V of the 23S rRNA. We analyzed nine clinical isolates of linezolid-resistant enterococci and showed a clear association between the number of 23S rRNA genes containing this mutation and the level of linezolid resistance expressed.

Increasing prevalence and dissemination of NDM-1 metallo-β-lactamase in India: data from the SMART study (2009)

OBJECTIVES To investigate the β-lactamase background of ertapenem non-susceptible isolates for the presence of the most commonly detected carbapenemase genes, bla(KPC), bla(OXA-48) and bla(VIM), and the newly described bla(NDM-1). METHODS Two hundred and thirty-five ertapenem-non-susceptible (MIC ≥ 0.5 mg/L) isolates of Enterobacteriaceae from the worldwide Study for Monitoring Antimicrobial Resistance Trends (SMART) 2009 programme were screened using a multiplex PCR for the presence of bla(KPC), bla(OXA-48), bla(VIM) and bla(NDM-1) genes. Extended-spectrum β-lactamase (ESBL) and AmpC genes (bla(ESBL) and bla(AmpC)) were identified using the Check-MDR CT101 microarray. DNA sequencing was performed to identify the bla(ESBL), bla(KPC) and bla(NDM-1) genes. Molecular typing was also performed to genetically characterize these isolates. RESULTS Sixty-six isolates (28%) had a carbapenemase gene, with bla(NDM-1) identified in 33 isolates including 2 isolates carrying both bla(NDM-1) and bla(OXA-48); other carbapenemase genes found included bla(KPC) (n = 23), bla(VIM) (n = 7) and bla(OXA-48) (n = 3). All bla(NDM-1)-carrying isolates were from patients in India and comprised five different species. With the exception of one isolate carrying only bla(NDM-1), all bla(NDM-1) carbapenemase-possessing isolates carried additional β-lactamases in various combinations: bla(ESBL) and bla(AmpC) (n = 18); bla(ESBL) (n = 10); bla(ESBL), bla(AmpC) and bla(OXA-48) (n = 2); and bla(AmpC) (n = 2). Except for bla(OXA-48)-carrying isolates, novel multilocus sequence types or enterobacterial repetitive intergenic consensus PCR patterns were observed along with clonal dissemination within and among sites. CONCLUSIONS A range of carbapenemase genes, associated with diverse ESBLs and/or AmpC backgrounds, were found among Enterobacteriaceae isolated during the study. Many of these ertapenem non-susceptible strains were clonally related and carried various combinations of β-lactamases.

Genomic and Transcriptomic Analyses of Colistin-Resistant Clinical Isolates of Klebsiella pneumoniae Reveal Multiple Pathways of Resistance

ABSTRACT The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Colr) is increasingly reported from clinical settings. The genetic mechanisms that lead to Colr in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Colr clinical isolates. Colr was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-l-arabinose (Ara4N) modification of lipid A was found in all Colr strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Colr mechanisms may be dependent on the genetic background.

Using Nucleic Acid Microarrays To Perform Molecular Epidemiology and Detect Novel β-Lactamases: a Snapshot of Extended-Spectrum β-Lactamases throughout the World

ABSTRACT The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008–2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla ESBL (bla SHV, bla TEM, and bla CTX-M genes of groups 1, 2, 9, and 8/25) and bla KPC genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla ESBL and bla KPC genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla ESBL gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla TEM (n = 5), bla SHV (n = 11), bla CTX-M (n = 19), and bla KPC (n = 3) were detected. A new bla SHV variant, bla SHV-129, and a new bla KPC variant, bla KPC-11, were also identified. The most common bla genes found in this study were bla CTX-M-15, bla CTX-M-14, and bla SHV-12. Using nucleic acid microarrays, we obtained a “molecular snapshot” of bla ESBL genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla SHV-129 and bla KPC-11.

Transferable Capacity for Gastrointestinal Colonization in Enterococcus faecium in a Mouse Model

A high level of gastrointestinal colonization frequently precedes invasive infection due to Enterococcus faecium. Factors other than antimicrobial resistance that promote gastrointestinal colonization by E. faecium have not been identified. We tested the ability of a colonization-proficient clinical E. faecium isolate (C68) to transfer colonizing ability to noncolonizing E. faecium recipient strains. Transconjugants derived from matings that used E. faecium D344SRF as a recipient strain colonized mouse gastrointestinal tracts in high numbers under selective pressure from clindamycin or vancomycin, compared with control strains that lacked DNA transferred from C68. We transferred DNA into a second recipient strain (E. faecium GE-1), which also colonized mice in significantly greater numbers under selective pressure from clindamycin, compared with a control strain. These results indicate that E. faecium clinical isolates express transmissible factors other than antimicrobial resistance that promote colonization of the mouse gastrointestinal tract.

Population Structure of KPC-Producing Klebsiella pneumoniae Isolates from Midwestern U.S. Hospitals

ABSTRACT Genome sequencing of carbapenem-resistant Klebsiella pneumoniae isolates from regional U.S. hospitals was used to characterize strain diversity and the blaKPC genetic context. A phylogeny based on core single-nucleotide variants (SNVs) supports a division of sequence type 258 (ST258) into two distinct groups. The primary differences between the groups are in the capsular polysaccharide locus (cps) and their plasmid contents. A strict association between clade and KPC variant was found. The blaKPC gene was found on variants of two plasmid backbones. This study indicates that highly similar K. pneumoniae subpopulations coexist within the same hospitals over time.

Sequences of MGH-1, YOU-1, and YOU-2 extended-spectrum beta-lactamase genes.

Genes for MGH-1, YOU-1, and YOU-2 extended-spectrum beta-lactamases have been cloned and sequenced. The gene for MGH-1 has the sequence of blaTEM-10, YOU-2 has that of blaTEM-12, and YOU-1 has that of blaTEM-26. All have evolved from blaTEM-1b but have the strong dual promoter sequence of blaTEM-2.

OqxAB, a Quinolone and Olaquindox Efflux Pump, Is Widely Distributed among Multidrug-Resistant Klebsiella pneumoniae Isolates of Human Origin

Use of antibiotics among livestock contributes to the selection and dissemination of multidrug resistant (MDR) bacteria ([1][1]). Olaquindox and carbadox are quinoxaline derivatives with antibacterial properties that prevent dysentery and enhance weight gain in suckling pigs ([2][2]). Resistance to

Carbapenem-resistant Enterobacter cloacae isolates producing KPC-3, North Dakota, USA.

To the Editor: Carbapenem-resistant Enterobacteriaceae (CRE) continue to emerge as a serious public health threat throughout the world (1). CRE infections in the United States are often mediated by acquisition of Klebsiella pneumoniae carbapenemase (KPC) expressed by Klebsiella spp., although KPC is also found in other genera (2). The spread of KPC-producing, gram-negative bacteria in hospitals has been linked to severity of illness, co-existing medical conditions, exposure to antimicrobial drugs, and need for chronic care (3). After reporting of CRE infections to the North Dakota Department of Health became mandatory in 2011, a total of 20 CRE cases were noted in 12 of 53 counties (2.9 cases/100,000 population [4]). Most cases involved infection with Enterobacter cloacae and occurred in Cass County, where the state’s largest city, Fargo, is located. We describe an outbreak of clonal carbapenem-resistant E. cloacae in a health care system in Fargo. Sanford Health is a 583-bed, acute-care facility, representing ≈70% of acute-care beds in Fargo. The hospital handles >27,000 admissions/year and serves as a referral center for a large area of the state, and the only long-term acute-care (LTAC) facility in the eastern half of the state operates on its campus. During December 2011–December 2012, all isolates of Enterobacteriaceae with reduced susceptibility to ertapenem (MIC ≥1 µg/mL) identified at the hospital’s clinical microbiology laboratory were screened for carbapenemase production by using the modified Hodge test (mHT), according to Clinical and Laboratory Standards Institute recommendations (5). Identification and susceptibility testing were done with the MicroScan system (Siemens Healthcare Diagnostics, Tarrytown, NY, USA); MICs of carbapenems were confirmed with Etest (bioMerieux, Durham, NC, USA). Three carbapenem-resistant E. cloacae isolates from documented cases of CRE infection at the hospital during 2010 were analyzed for comparison. To characterize carbapenem-resistant and mHT-positive isolates, we used PCR to amplify and sequence the carbapenemase genes blaIMP, blaNDM, blaVIM, and blaKPC by using established methods (6). The upstream sequence of blaKPC-positive strains was analyzed to determine the isoform of the transposon Tn4401 that harbored blaKPC (7). We investigated genetic similarity among isolates by repetitive sequence-based PCR; isolates with >95% similarity were considered clonal (6). We also sequenced the highly conserved hsp60 gene (8) and attempted conjugative transfer of the blaKPC gene by growing KPC-producing E. cloacae along with sodium azide–resistant Escherichia coli J-53. As part of the study, we examined records of patients from whom carbapenem-resistant E. cloacae was isolated. The study was approved by the Institutional Review Board at Sanford Health. During December 2011–December 2012, a total of 19 single-patient E. cloacae isolates and 1 E. aerogenes isolate had positive mHT results. blaKPC was detected in 17 of the 19 E. cloacae isolates and in the 3 carbapenem-resistant E. cloacae isolates from 2010. For all 20 of those isolates, sequencing revealed blaKPC-3 in association with isoform d of the transposon Tn4401, and all isolates were clonally related (Figure). All 20 isolates also had an identical hsp60 sequence belonging to cluster VI in the Hoffman and Roggenkamp scheme (8). Conjugation of a blaKPC-containing plasmid into E. coli J-53 was successful for 1 strain. Figure Genetic typing of carbapenem-resistant Enterobacter cloacae identified from patients at Sanford Health in Fargo, North Dakota, USA. Repetitive sequence–based PCR was used. The dendrogram at left displays the percentage similarity among band patterns ... All 20 of the patients from whom KPC-producing CRE isolates were obtained (17 from this study, 3 from 2010) had been hospitalized at Sanford Health during the 3 months before CRE isolation; 13 (65%) were admitted to intensive care. In addition, 13 (65%) patients had been admitted to the LTAC during the year before CRE isolation. Co-colonization with multidrug-resistant bacteria was documented in 16 (80%) patients, including extended-spectrum β-lactamase–producing and carbapenem-resistant organisms in 4 and 2 patients, respectively. Seven (35%) patients died; 3 (15%) deaths were attributed to CRE infection. One of the patients was a neonate 30 days of age. The finding of KPC-3–producing E. cloacae in North Dakota contrasts with the predominant epidemiology of CRE across the United States. Most CRE cases nationwide are caused by KPC-producing K. pneumoniae (2). KPC-type β-lactamases were previously identified in diverse strains of Enterobacter spp. from an urban health care system in Detroit, accounting for ≈15% of CRE (9). In contrast, our genetic analysis reveals a uniform genetic background among KPC-producing E. cloacae, which suggests horizontal dissemination of an outbreak strain. Because active surveillance programs do not exist at our facility, this study probably underestimates the extent of CRE spread. We found that patients with KPC-producing E. cloacae in this sample were exposed to an LTAC and concomitantly were colonized or infected with other multidrug-resistant organisms (9). Although the spatio-temporal origin of the outbreak (acute care vs. LTAC) remains undefined, these findings likely reflect longer exposure to the continuum of care and higher rates of co-existing conditions within the LTAC population. This outbreak of KPC-producing E. cloacae infections in a health care system in North Dakota highlights the infection control challenges of long-term care facilities and the potential role they play in CRE dissemination.

Extensively drug-resistant pseudomonas aeruginosa isolates containing blaVIM-2 and elements of Salmonella genomic island 2: a new genetic resistance determinant in Northeast Ohio

ABSTRACT Carbapenems are a mainstay of treatment for infections caused by Pseudomonas aeruginosa. Carbapenem resistance mediated by metallo-β-lactamases (MBLs) remains uncommon in the United States, despite the worldwide emergence of this group of enzymes. Between March 2012 and May 2013, we detected MBL-producing P. aeruginosa in a university-affiliated health care system in northeast Ohio. We examined the clinical characteristics and outcomes of patients, defined the resistance determinants and structure of the genetic element harboring the blaMBL gene through genome sequencing, and typed MBL-producing P. aeruginosa isolates using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST). Seven patients were affected that were hospitalized at three community hospitals, a long-term-care facility, and a tertiary care center; one of the patients died as a result of infection. Isolates belonged to sequence type 233 (ST233) and were extensively drug resistant (XDR), including resistance to all fluoroquinolones, aminoglycosides, and β-lactams; two isolates were nonsusceptible to colistin. The blaMBL gene was identified as blaVIM-2 contained within a class 1 integron (In559), similar to the cassette array previously detected in isolates from Norway, Russia, Taiwan, and Chicago, IL. Genomic sequencing and assembly revealed that In559 was part of a novel 35-kb region that also included a Tn501-like transposon and Salmonella genomic island 2 (SGI2)-homologous sequences. This analysis of XDR strains producing VIM-2 from northeast Ohio revealed a novel recombination event between Salmonella and P. aeruginosa, heralding a new antibiotic resistance threat in this regions health care system.