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ABSTRACTA 22-year-old male developed a recurrent sacral abscess associated with embedded shrapnel following a blast injury. Cultures grew extended-spectrum β-lactamase (ESBL)-producing, carbapenem-susceptible Escherichia coli. Ertapenem was administered, but the infection recurred after each course of antibiotics. Initial surgical interventions were unsuccessful, and subsequent cultures yielded E. coliand Morganella morganii, both nonsusceptible to carbapenems. The isolates were Carba NP test negative, gave ambiguous results with the modified Hodge test, and amplified the blaOXA48-like gene by real-time PCR. All E. coliisolates were sequence type 131 (ST131), carried nine resistance genes (including blaCTX-M-27) on an IncF plasmid, and were identical by genome sequencing, except for 150 kb of plasmid DNA in carbapenem-nonsusceptible isolates only. Sixty kilobases of this was shared by M. morganiiand represented an IncN plasmid harboring blaOXA-181. In M. morganii, the gene was flanked by IS3000and ISKpn19, but in all but one of the E. coliisolates containing blaOXA-181, a second copy of ISKpn19had inserted adjacent to IS3000. To the best of our knowledge, this is the first report of blaOXA-181in the virulent ST131 clonal group and carried by the promiscuous IncN family of plasmids. The tendency of M. morganiito have high MICs of imipenem, a blaOXA-181substrate profile that includes penicillins but not extended-spectrum cephalosporins, and weak carbapenemase activity almost resulted in the presence of blaOXA-181being overlooked. We highlight the importance of surveillance for carbapenem resistance in all species, even those with intrinsic resistances, and the value of advanced molecular techniques in detecting subtle genetic changes.

ABSTRACTTigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem- or colistin-resistant organisms. In Enterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated in Enterobacteriaceaeand Acinetobacterisolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Klebsiella pneumoniaeisolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 μg/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression of ramA, ramR, oqxA, acrB, marA, or rarAgenes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here named kpgABC, previously unknown to be involved in resistance. Introduction of the kpgABCgenes in a non-kpgABCbackground increased the MIC of tigecycline 4-fold and is independent of a functional AcrAB-TolC pump. This is the first report to propose a function for kpgABCand identify an insertion element whose presence correlated with the in vivodevelopment of tigecycline nonsusceptibility in K. pneumoniae.

ABSTRACTKlebsiella pneumoniaestrain MRSN2404 was isolated from the chronic wound of a soldier who had been wounded in Iraq in 2006. The strain displayed very high MICs of all aminoglycosides, including arbekacin. A gene encoding a novel 16S rRNA methyltransferase, now designated RmtH, was identified. RmtH had 64% identity with RmtB1 and RmtB2. rmtHwas bracketed by two copies of ISCR2, which may have played a role in its mobilization.

The prevalence of multidrug-resistant infections in personnel wounded in Iraq and Afghanistan has made it challenging for physicians to choose effective therapeutics in a timely fashion. To address the challenge of identifying the potential for drug resistance, we have developed the Antimicrobial Resistance Determinant Microarray (ARDM) to provide DNAbased analysis for over 250 resistance genes covering 12 classes of antibiotics. Over 70 drug-resistant bacteria from different geographic regions have been analyzed on ARDM, with significant differences in patterns of resistance identified: genes for resistance to sulfonamides, trimethoprim, chloramphenicol, rifampin, and macrolide-lincosamidesulfonamide drugs were more frequently identified in isolates from sources in Iraq/Afghanistan. Of particular concern was the presence of genes responsible for resistance to many of the last-resort antibiotics used to treat war traumaassociated infections.

ABSTRACTIn response to global concerns over the spread of the New Delhi metallo-β-lactamase gene 1, blaNDM-1, a monthly surveillance program was initiated in September 2010. All carbapenem-resistant Gram-negative strains forwarded to our facility are screened for this gene. To date, 321 carbapenem-resistant isolates, encompassing 11 bacterial species, have been tested. In February 2011, two strains of Providencia stuartii, submitted from a military hospital in Afghanistan, tested positive for blaNDM-1. Both strains were identical by pulsed-field gel electrophoresis (PFGE). blaNDM-1was carried on a large plasmid, pMR0211, which was sequenced by emulsion PCR and pyrosequencing. pMR0211 is 178,277 bp in size and belongs to incompatibility group A/C. The plasmid consists of a backbone with considerable homology to pAR060302 from Escherichia coli, and it retains many of the antibiotic resistance genes associated with it. The plasmid also shares common elements with the pNDM-HK plasmid, including blaNDM-1, armA, and sul1. However, gene orientation is reversed, and a 3-kb fragment from this region is absent from pMR0211. pMR0211 also contains additional genes, including the aminoglycoside-modifying enzyme loci aadAand aac(6′), the quinolone resistance gene qnrA, a gene with highest homology to a U32 family peptidase from Shewanella amazonensis, and the blaOXA-10gene. The finding of this gene in an intrinsically colistin-resistant species such as Providencia stuartiiis especially worrisome, as it renders the organism resistant to nearly every available antibiotic. The presence of multiple insertion sequences and transposons flanking the region containing the blaNDM-1gene further highlights the potential mobility associated with this gene.

We describe the clinical, microbiologic, and molecular features of the first series of qacA/B-containing strains of methicillin-resistant Staphylococcus aureusfrom infected US patients. All qac-carrying strains were clonally diverse, and qacAstrains exhibited increased tolerance to chlorhexidine as measured by minimum inhibitory concentrations, minimum bactericidal concentrations, and postexposure colony counts.

ABSTRACTA carbapenem-resistant Alcaligenes faecalisstrain was isolated from a surveillance swab of a service member injured in Afghanistan. The isolate was positive for blaNDMby real-time PCR. Species identification was reevaluated on three identification systems but was inconclusive. Genome sequencing indicated that the closest relative was Acinetobacter schindleriand that blaNDM-1was carried on a plasmid that shared >99% identity with one identified in an Acinetobacter lwoffiiisolate. The isolate also carried a novel chromosomally encoded class D oxacillinase.

ABSTRACTA carbapenem-resistant Acinetobacter baumanniistrain was isolated from the peritoneal fluid of a patient with complicated intra-abdominal infection and evaluated at the Multidrug-resistant Organism Repository and Surveillance Network by whole-genome sequencing and real-time PCR. The isolate was sequence type 25 and susceptible to colistin and minocycline, with low MICs of tigecycline. blaNDM-1was located on a plasmid with >99% homology to pNDM-BJ02. The isolate carried numerous other antibiotic resistance genes, including the 16S methylase gene, armA.

ABSTRACTThe increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting blaKPCand blaNDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with blaKPC- and blaNDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.

Over the past two decades, Acinetobacter baumanniihas emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumanniias a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumanniistrains for use in this endeavor.

ABSTRACTThe loss of fitness in colistin-resistant (CR) Acinetobacter baumanniiwas investigated using longitudinal isolates from the same patient. Early CR isolates were outcompeted by late CR isolates for growth in broth and survival in the lungs of mice. Fitness loss was associated with an increased susceptibility to oxidative stress since early CR strains had reduced in vitrosurvival in the presence of hydrogen peroxide and decreased catalase activity compared to that of late CR and colistin-susceptible (CS) strains.

ABSTRACTGene amplification is believed to play an important role in antibiotic resistance but has been rarely documented in clinical settings because of its unstable nature. We report a rise in MICs from 0.5 to 16 μg/ml in successive Acinetobacter baumanniiisolated over 4 days from a patient being treated with tobramycin for an infection by multidrug-resistant A. baumannii, resulting in therapeutic failure. Isolates were characterized by whole-genome sequencing, real-time and reverse transcriptase PCR, and growth assays to determine the mechanism of tobramycin resistance and its fitness cost. Tobramycin resistance was associated with two amplification events of different chromosomal fragments containing the aphA1aminoglycoside resistance gene part of transposon Tn6020. The first amplification event involved low amplification (6 to 10 copies) of a large DNA fragment that was unstable and conferred tobramycin MICs of ≤8 μg/ml. The second event involved moderate (10 to 30 copies) or high (40 to 110 copies) amplification of Tn6020. High copy numbers were associated with tobramycin MICs of 16 μg/ml, impaired fitness, and genetic instability, whereas lower copy numbers resulted in tobramycin MICs of ≤8 μg/ml and no fitness cost and were stably maintained in vitro. Exposure in vitroto tobramycin of the initial susceptible isolate and of the A. baumanniiAB0057 reference strain led to similar aphA1amplifications and elevated tobramycin MICs. To the best of our knowledge, this is the first report of in vivodevelopment of antibiotic resistance secondary to gene amplifications resulting in therapy failure.IMPORTANCEA combination of whole-genome sequencing and mapping were used to detect an antibiotic resistance mechanism, gene amplification, which has been presumed for a long time to be of major importance but has rarely been reported in clinical settings because of its unstable nature. Two gene amplification events in a patient with an Acinetobacter baumanniiinfection treated with tobramycin were identified. One gene amplification event led to high levels of resistance and was rapidly reversible, while the second event led to low and more stable resistance since it incurred low fitness cost on the host. Gene amplification, with an associated rise in tobramycin MICs, could be readily reproduced in vitrofrom initially susceptible strains exposed to increasing concentrations of tobramycin, suggesting that gene amplification in A. baumanniimay be a more common mechanism than currently believed. This report underscores the importance of rapid molecular techniques for surveillance of drug resistance.