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We explored the association of antibiotic-resistant phenotypes and genotypes in Acinetobacter spp with clinical outcomes and characteristics in 75 patients from a major military treatment facility. Amikacin resistance was associated with nosocomial acquisition of A baumannii, and carbapenem resistance and blaOXA-23 were associated with the need for mechanical ventilation. The presence of blaOXA-23 also correlated with longer hospital and ICU stay. Associations between blaOXA-23 and complexity, duration, and changes made to antibiotic regimens also existed. [ABSTRACT FROM AUTHOR]

Ledaborbactam (formerly VNRX-5236), a bicyclic boronate β-lactamase inhibitor with activity against class A, C, and D β-lactamases, is under development as an orally bioavailable etzadroxil prodrug (VNRX-7145) in combination with ceftibuten for the treatment of urinary tract infections. At ceftibuten breakpoints of ≤1 mg/L (EUCAST) and ≤8 mg/L (CLSI), 92.5% and 99.0%, respectively, of 200 carbapenem-resistant Klebsiella pneumoniae isolates, predominantly K. pneumoniae carbapenemase producing, were susceptible to ceftibuten-ledaborbactam (ledaborbactam tested at a fixed concentration of 4 mg/L) compared to 4.5% and 30.5%, respectively, to ceftibuten alone.

We characterized the molecular determinants of meropenem-vaborbactam (MV) non-susceptibility among non-metallo-β-lactamase-producing KPC- Klebsiella pneumoniae (KPC- KP ). Whole-genome sequencing was performed to identify mutations associated with MV non-susceptibility. Isolates with elevated MV MICs were found to have mutations encoding truncated or altered OmpK36 porins and increased bla KPC copy numbers. KPC- KP isolates with decreased susceptibility to MV were detected among a collection of isolates predating the availability of MV., Competing Interests: The authors declare no conflict of interest.

Taniborbactam, a bicyclic boronate β-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC), Verona integron-encoded metallo-β-lactamase (VIM), New Delhi metallo-β-lactamase (NDM), extended-spectrum beta-lactamases (ESBLs), OXA-48, and AmpC β-lactamases, is under clinical development in combination with cefepime. Susceptibility of 200 previously characterized carbapenem-resistant K. pneumoniae and 197 multidrug-resistant (MDR) Pseudomonas aeruginosa to cefepime-taniborbactam and comparators was determined by broth microdilution. For K. pneumoniae (192 KPC; 7 OXA-48-related), MIC 90 values of β-lactam components for cefepime-taniborbactam, ceftazidime-avibactam, and meropenem-vaborbactam were 2, 2, and 1 mg/L, respectively. For cefepime-taniborbactam, 100% and 99.5% of isolates of K. pneumoniae were inhibited at ≤16 mg/L and ≤8 mg/L, respectively, while 98.0% and 95.5% of isolates were susceptible to ceftazidime-avibactam and meropenem-vaborbactam, respectively. For P. aeruginosa , MIC 90 values of β-lactam components of cefepime-taniborbactam, ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam were 16, >8, >8, and >4 mg/L, respectively. Of 89 carbapenem-susceptible isolates, 100% were susceptible to ceftolozane-tazobactam, ceftazidime-avibactam, and cefepime-taniborbactam at ≤8 mg/L. Of 73 carbapenem-intermediate/resistant P. aeruginosa isolates without carbapenemases, 87.7% were susceptible to ceftolozane-tazobactam, 79.5% to ceftazidime-avibactam, and 95.9% and 83.6% to cefepime-taniborbactam at ≤16 mg/L and ≤8 mg/L, respectively. Cefepime-taniborbactam at ≤16 mg/L and ≤8 mg/L, respectively, was active against 73.3% and 46.7% of 15 VIM- and 60.0% and 35.0% of 20 KPC-producing P. aeruginosa isolates. Of all 108 carbapenem-intermediate/resistant P. aeruginosa isolates, cefepime-taniborbactam was active against 86.1% and 69.4% at ≤16 mg/L and ≤8 mg/L, respectively, compared to 59.3% for ceftolozane-tazobactam and 63.0% for ceftazidime-avibactam. Cefepime-taniborbactam had in vitro activity comparable to ceftazidime-avibactam and greater than meropenem-vaborbactam against carbapenem-resistant K. pneumoniae and carbapenem-intermediate/resistant MDR P. aeruginosa ., Competing Interests: Robert A. Bonomo reports grants from Venatorx, Entasis, Merck, Wockhardt, and Shionogi outside the submitted work. David A. Six, Greg Moeck, and Tsuyoshi Uehara are employees of Venatorx Pharmaceuticals, Inc.

Compared to other genera of Gram-negative pathogens, Pseudomonas is adept in acquiring complex non-enzymatic and enzymatic resistance mechanisms thus remaining a challenge to even novel antibiotics including recently developed β-lactam and β-lactamase inhibitor combinations. This study shows that the novel β-lactam enhancer approach enables cefepime/zidebactam to overcome both non-enzymatic and enzymatic resistance mechanisms associated with a challenging panel of P. aeruginosa . This study highlights that the β-lactam enhancer mechanism is a promising alternative to the conventional β-lactam/β-lactamase inhibitor approach in combating ever-evolving MDR P. aeruginosa ., Competing Interests: Some of the coauthors are employees of Wockhardt. R.A.B. receives grant funding from Wockhardt.

Class C Acinetobacter -derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii . Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076 , a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC β-lactamase variants with K i values <1 μM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to β -lactams. One of the most important mechanisms is the production of β -lactamase enzymes capable of hydrolyzing β -lactam antibiotics. Co-expression of multiple classes of β -lactamases is present in CRAB; therefore, the design and synthesis of "cross-class" inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical β -lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter -derived class C β -lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a K i = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other β -lactamases in A. baumannii : the cefepime-hydrolysing class C extended-spectrum β -lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1β levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1β induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.

Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

Serious infections caused by multidrug-resistant (MDR) organisms (Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii) present a critical need for innovative drug development. Herein, we describe the preclinical evaluation of YU253911, 2, a novel γ-lactam siderophore antibiotic with potent antimicrobial activity against MDR Gram-negative pathogens. Penicillin-binding protein (PBP) 3 was shown to be a target of 2 using a binding assay with purified P. aeruginosa PBP3. The specific binding interactions with P. aeruginosa were further characterized with a high-resolution (2.0 Å) X-ray structure of the compound's acylation product in P. aeruginosa PBP3. Compound 2 was shown to have a concentration >1 μg/ml at the 6 h time point when administered intravenously or subcutaneously in mice. Employing a meropenem resistant strain of P. aeruginosa, 2 was shown to have dose-dependent efficacy at 50 and 100 mg/kg q6h dosing in a mouse thigh infection model. Lastly, we showed that a novel γ-lactam and β-lactamase inhibitor (BLI) combination can effectively lower minimum inhibitory concentrations (MICs) against carbapenem resistant Acinetobacter spp. that demonstrated decreased susceptibility to 2 alone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Masson SAS.)

Successful treatment of Acinetobacter baumannii infections require early and appropriate antimicrobial therapy. One of the first steps in this process is understanding which β-lactamase (bla) alleles are present and in what combinations. Thus, we performed WGS on 98 carbapenem-resistant A. baumannii (CR Ab). In most isolates, an acquired bla OXA carbapenemase was found in addition to the intrinsic bla OXA allele. The most commonly found allele was bla OXA-23 (n = 78/98). In some isolates, bla OXA-23 was found in addition to other carbapenemase alleles: bla OXA-82 (n = 12/78), bla OXA-72 (n = 2/78) and bla OXA-24/40 (n = 1/78). Surprisingly, 20% of isolates carried carbapenemases not routinely assayed for by rapid molecular diagnostic platforms, i.e., bla OXA-82 and bla OXA-172 ; all had ISAba1 elements. In 8 CR Ab, bla OXA-82 or bla OXA-172 was the only carbapenemase. Both bla OXA-24/40 and its variant bla OXA-72 were each found in 6/98 isolates. The most prevalent ADC variants were bla ADC-30 (21%), bla ADC-162 (21%), and bla ADC-212 (26%). Complete combinations are reported., (Published by Elsevier Inc.)

Treatment of multidrug-resistant Gram-negative bacterial pathogens represents a critical clinical need. Here, we report a novel γ-lactam pyrazolidinone that targets penicillin-binding proteins (PBPs) and incorporates a siderophore moiety to facilitate uptake into the periplasm. The MIC values of γ-lactam YU253434, 1 , are reported along with the finding that 1 is resistant to hydrolysis by all four classes of β-lactamases. The druglike characteristics and mouse PK data are described along with the X-ray crystal structure of 1 binding to its target PBP3.

Background: Carbapenem-resistant Enterobacterales (CRE) are a global threat. We aimed to describe the clinical and molecular characteristics of Centers for Disease Control and Prevention (CDC)-defined CRE in the USA., Methods: CRACKLE-2 is a prospective, multicentre, cohort study. Patients hospitalised in 49 US hospitals, with clinical cultures positive for CDC-defined CRE between April 30, 2016, and Aug 31, 2017, were included. There was no age exclusion. The primary outcome was desirability of outcome ranking (DOOR) at 30 days after index culture. Clinical data and bacteria were collected, and whole genome sequencing was done. This trial is registered with ClinicalTrials.gov, number NCT03646227., Findings: 1040 patients with unique isolates were included, 449 (43%) with infection and 591 (57%) with colonisation. The CDC-defined CRE admission rate was 57 per 100 000 admissions (95% CI 45-71). Three subsets of CDC-defined CRE were identified: carbapenemase-producing Enterobacterales (618 [59%] of 1040), non-carbapenemase-producing Enterobacterales (194 [19%]), and unconfirmed CRE (228 [22%]; initially reported as CRE, but susceptible to carbapenems in two central laboratories). Klebsiella pneumoniae carbapenemase-producing clonal group 258 K pneumoniae was the most common carbapenemase-producing Enterobacterales. In 449 patients with CDC-defined CRE infections, DOOR outcomes were not significantly different in patients with carbapenemase-producing Enterobacterales, non-carbapenemase-producing Enterobacterales, and unconfirmed CRE. At 30 days 107 (24%, 95% CI 20-28) of these patients had died., Interpretation: Among patients with CDC-defined CRE, similar outcomes were observed among three subgroups, including the novel unconfirmed CRE group. CDC-defined CRE represent diverse bacteria, whose spread might not respond to interventions directed to carbapenemase-producing Enterobacterales., Funding: National Institutes of Health., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Whole genome sequencing (WGS) is replacing traditional microbiological typing methods for investigation of outbreaks in clinical settings. Here, we used a clinical microbiology laboratory core genome multilocus sequence typing (cgMLST) workflow to analyze 40 isolates of K. pneumoniae which are part of the Antimicrobial Resistance Leadership Group (ARLG) isolate collection, alongside 10 Mayo Clinic K. pneumoniae isolates, comparing results to those of pulsed-field gel electrophoresis (PFGE). Additionally, we used the WGS data to predict phenotypic antimicrobial susceptibility (AST). Thirty-one of 40 ARLG K. pneumoniae isolates belonged to the same PFGE type, all of which, alongside 3 isolates of different PFGE types, formed a large cluster by cgMLST. PFGE and cgMLST were completely concordant for the 10 Mayo Clinic K. pneumoniae isolates. For AST prediction, the overall agreement between phenotypic AST and genotypic prediction was 95.6%., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Plazomicin was tested against 697 recently acquired carbapenem-resistant Klebsiella pneumoniae isolates from the Great Lakes region of the United States. Plazomicin MIC 50 and MIC 90 values were 0.25 and 1 mg/liter, respectively; 680 isolates (97.6%) were susceptible (MICs of ≤2 mg/liter), 9 (1.3%) intermediate (MICs of 4 mg/liter), and 8 (1.1%) resistant (MICs of >32 mg/liter). Resistance was associated with rmtF -, rmtB -, or armA -encoded 16S rRNA methyltransferases in all except 1 isolate., (Copyright © 2020 American Society for Microbiology.)

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser 64 , Lys 67 , Tyr 150 , and Lys 315 ), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications., (Copyright © 2020 American Society for Microbiology.)

Background: Overcoming β-lactam resistance in pathogens such as Pseudomonas aeruginosa is a major clinical challenge. Rapid molecular diagnostics (RMDs) have the potential to inform selection of empiric therapy in patients infected by P. aeruginosa., Methods: In this study, we used a heterogeneous collection of 197 P. aeruginosa that included multidrug-resistant isolates to determine whether 2 representative RMDs (Acuitas Resistome test and VERIGENE gram-negative blood culture test) could identify susceptibility to 2 newer β-lactam/β-lactamase inhibitor (BL-BLI) combinations, ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (TOL/TAZO)., Results: We found that the studied RMD platforms were able to correctly identify BL-BLI susceptibility (susceptibility sensitivity, 100%; 95% confidence interval [CI], 97%, 100%) for both BLs-BLIs. However, their ability to detect resistance to these BLs-BLIs was lower (resistance sensitivity, 66%; 95% CI, 52%, 78% for TOL/TAZO and 33%; 95% CI, 20%, 49% for CZA)., Conclusions: The diagnostic platforms studied showed the most potential in scenarios where a resistance gene was detected or in scenarios where a resistance gene was not detected and the prevalence of resistance to TOL/TAZO or CZA is known to be low. Clinicians need to be mindful of the benefits and risks that result from empiric treatment decisions that are based on resistance gene detection in P. aeruginosa, acknowledging that such decisions are impacted by the prevalence of resistance, which varies temporally and geographically., (Published by Oxford University Press for the Infectious Diseases Society of America 2018.)

Multidrug-resistant (MDR) Acinetobacter spp. poses a significant therapeutic challenge in part due to the presence of chromosomally encoded β-lactamases, including class C Acinetobacter -derived cephalosporinases (ADC) and class D oxacillinases (OXA), as well as plasmid-mediated class A β-lactamases. Importantly, OXA-like β-lactamases represent a gap in the spectrum of inhibition by recently approved β-lactamase inhibitors such as avibactam and vaborbactam. ETX2514 is a novel, rationally designed, diazabicyclooctenone inhibitor that effectively targets class A, C, and D β-lactamases. We show that addition of ETX2514 significantly increased the susceptibility of clinical Acinetobacter baumannii isolates to sulbactam. AdeB and AdeJ were identified to be key efflux constituents for ETX2514 in A. baumannii The combination of sulbactam and ETX2514 was efficacious against A. baumannii carrying bla TEM-1 , bla ADC-82 , bla OXA-23 , and bla OXA-66 in a neutropenic murine thigh infection model. We also show that, in vitro , ETX2514 inhibited ADC-7 ( k 2 / K i 1.0 ± 0.1 × 10 6 M -1 s -1 ) and OXA-58 ( k 2 / K i 2.5 ± 0.3 × 10 5 M -1 s -1 ). Cocrystallization of ETX2514 with OXA-24/40 revealed hydrogen bonding interactions between ETX2514 and residues R261, S219, and S128 of OXA-24/40 in addition to a chloride ion occupied in the active site. Further, the C3 methyl group of ETX2514 shifts the position of M223. In conclusion, the sulbactam-ETX2514 combination possesses a broadened inhibitory range to include class D β-lactamases as well as class A and C β-lactamases and is a promising therapeutic candidate for infections caused by MDR Acinetobacter spp. IMPORTANCE The number and diversity of β-lactamases are steadily increasing. The emergence of β-lactamases that hydrolyze carbapenems poses a significant threat to our antibiotic armamentarium. The explosion of OXA enzymes that are carbapenem hydrolyzers is a major challenge (carbapenem-hydrolyzing class D [CHD]). An urgent need exists to discover β-lactamase inhibitors with class D activity. The sulbactam-ETX2514 combination demonstrates the potential to become a treatment regimen of choice for Acinetobacter spp. producing class D β-lactamases.

The activity of the siderophore cephalosporin cefiderocol is targeted against carbapenem-resistant Gram-negative bacteria. In this study, the activity of cefiderocol against characterized carbapenem-resistant Acinetobacter baumannii complex, Stenotrophomonas maltophilia , Pseudomonas aeruginosa , and Enterobacteriaceae strains was determined by microdilution in iron-depleted Mueller-Hinton broth. The MIC 90 s against A. baumannii , S. maltophilia , and P. aeruginosa were 1, 0.25, and 0.5 mg/liter, respectively. Against Enterobacteriaceae , the MIC 90 was 1 mg/liter for the group harboring OXA-48-like, 2 mg/liter for the group harboring KPC-3, and 8 mg/liter for the group harboring TEM/SHV ESBL, NDM, and KPC-2., (Copyright © 2018 American Society for Microbiology.)

In the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE), trimethoprim-sulfamethoxazole (TMP-SMX) had a limited role in the treatment of less severe carbapenem-resistant Enterobacteriaceae (CRE) infections, especially urinary tract infections. Of tested CRE, only 29% were susceptible to TMP-SMX. Development of resistance further limits the use of TMP-SMX in CRE infections.

Colistin and polymyxin B MICs were determined for 106 carbapenem-resistant Klebsiella pneumoniae (CR-Kp) isolates using Sensititre Research Use Only GNX2F plates (Thermo Fisher) and compared to CLSI broth macrodilution (BMD) as the reference method. For colistin, EUCAST breakpoints were applied and testing of isolates with very major (VM) errors was repeated in duplicate by both methods to determine a majority result. Essential agreement (MIC ± one dilution) of GNX2F with the reference method was 97.1% for polymyxin B and 92.5% for colistin (7 VM errors, 22.6%). After discrepancy testing, there were 28 colistin resistant isolates by BMD and essential agreement was 94.3% with 4 VM errors (14.3%). Colistin and polymyxin B GNX2F results showed acceptable essential agreement with BMD for MICS without interpretation. Colistin VM errors with EUCAST breakpoints were due to MIC variability in the 2 to 4 μg/mL range that could be addressed by establishing an intermediate category., (Copyright © 2018. Published by Elsevier Inc.)

Background: Carbapenem resistance is a critical healthcare challenge worldwide. Particularly concerning is the widespread dissemination of Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae harboring blaKPC (KPC-Kpn) is endemic in many areas including the United States, where the epidemic was primarily mediated by the clonal dissemination of Kpn ST258. We postulated that the spread of blaKPC in other regions occurs by different and more complex mechanisms. To test this, we investigated the evolution and dynamics of spread of KPC-Kpn in Colombia, where KPC became rapidly endemic after emerging in 2005., Methods: We sequenced the genomes of 133 clinical isolates recovered from 24 tertiary care hospitals located in 10 cities throughout Colombia, between 2002 (before the emergence of KPC-Kpn) and 2014. Phylogenetic reconstructions and evolutionary mapping were performed to determine temporal and genetic associations between the isolates., Results: Our results indicate that the start of the epidemic was driven by horizontal dissemination of mobile genetic elements carrying blaKPC-2, followed by the introduction and subsequent spread of clonal group 258 (CG258) isolates containing blaKPC-3., Conclusions: The combination of 2 evolutionary mechanisms of KPC-Kpn within a challenged health system of a developing country created the "perfect storm" for sustained endemicity of these multidrug-resistant organisms in Colombia., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Extremely drug-resistant (XDR) Acinetobacter baumannii is one of the most commonly encountered, highly resistant pathogens requiring novel therapeutic interventions., Methods: We developed C8, a monoclonal antibody (mAb), by immunizing mice with sublethal inocula of a hypervirulent XDR clinical isolate., Results: C8 targets capsular carbohydrate on the bacterial surface, enhancing opsonophagocytosis. Treating with a single dose of C8 as low as 0.5 μg/mouse (0.0167 mg/kg) markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia models of XDR A. baumannii infection. C8 was also synergistic with colistin, substantially improving survival compared to monotherapy. Treatment with C8 significantly reduced blood bacterial density, cytokine production (tumor necrosis factor α, interleukin [IL] 6, IL-1β, and IL-10), and sepsis biomarkers. Serial in vitro passaging of A. baumannii in the presence of C8 did not cause loss of mAb binding to the bacteria, but did result in emergence of less-virulent mutants that were more susceptible to macrophage uptake. Finally, we developed a highly humanized variant of C8 that retains opsonophagocytic activity in murine and human macrophages and rescued mice from lethal infection., Conclusions: We describe a promising and novel mAb as therapy for lethal, XDR A. baumannii infections, and demonstrate that it synergistically improves outcomes in combination with antibiotics., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: This study was performed to characterize the epidemiology, management, and outcomes of skin and soft tissue infection (SSTI) and colonization due to carbapenem-resistant Enterobacteriaceae (CRE)., Methods: Patients from the Consortium on Resistance Against Carbapenem in Klebsiella and Other Enterobacteriaceae (CRACKLE-1) from December 24, 2011 to October 1, 2014 with wound cultures positive for CRE were included in the study. Predictors of surgical intervention were analyzed. Molecular typing of isolates was performed using repetitive extragenic palindromic polymerase chain reaction (PCR). Carbapenemase genes were detected using PCR., Results: One hundred forty-two patients were included: 62 had SSTI (44%) and 56% were colonized. Mean age was 61 years, and 48% were male: median Charlson score was 3 (interquartile range, 1-5). Forty-eight percent of patients were admitted from long-term care facilities (LTCFs), and 31% were from the community. Two strain types (ST258A and ST258B) were identified (73% of 45 tested). Carbapenemase genes were detected in 40 of 45 isolates ( bla KPC-3 [47%], bla KPC-2 [42%]). Sixty-eight patients (48%) underwent surgical intervention, 63% of whom had SSTI. Patients admitted from LTCFs were less likely to undergo surgical intervention (odds ratio [OR], 0.36; 95% confidence interval [CI], 0.18-0.71). In multivariable analysis, among patients with SSTI, those admitted from LTCFs were less likely to undergo debridement (OR, 0.18; 95% CI, 0.04-0.93)., Conclusions: Patients admitted from LTCFs with CRE SSTI were less likely to undergo surgical intervention. Sixteen percent of the patients died, and approximately 50% of survivors required more intensive care upon discharge. These findings suggest a unique, impactful syndrome within the CRE infection spectrum. Further studies are needed to assess the role of surgical debridement in management of CRE-SSTI, particularly among LTCF residents.

Among Gram-negative bacteria, carbapenem-resistant infections pose a serious and life-threatening challenge. Here, the CRACKLE network reports a sentinel detection and characterization of a carbapenem-resistant Klebsiella pneumoniae ST147 isolate harboring bla NDM-5 and bla OXA-181 from a young man who underwent abdominal surgery in India. bla NDM-5 was located on an IncFII plasmid of ≈90 kb, whereas bla OXA-181 was chromosomally encoded. Resistome and genome analysis demonstrated multiple copies of the transposable element IS 26 and a "hot-spot region" in the IncFII plasmid., (Copyright © 2017 American Society for Microbiology.)

Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included bla IMP , bla NDM , bla OXA-48 , bla CTX-M , bla AmpC , and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log 10 -CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log 10 -CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae ., (Copyright © 2017 American Society for Microbiology.)

Background: Polymyxins including colistin are an important "last-line" treatment for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKp). Increasing use of colistin has led to resistance to this cationic antimicrobial peptide., Methods: A cohort nested within the Consortium on Resistance against Carbapenems in Klebsiella pneumoniae (CRACKLE) was constructed of patients with infection, or colonization with CRKp isolates tested for colistin susceptibility during the study period of December, 2011 to October, 2014. Reference colistin resistance determination as performed by broth macrodilution was compared to results from clinical microbiology laboratories (Etest) and to polymyxin resistance testing. Each patient was included once, at the time of their first colistin-tested CRKp positive culture. Time to 30-day in-hospital all-cause mortality was evaluated by Kaplan-Meier curves and Cox proportional hazard modeling., Results: In 246 patients with CRKp, 13% possessed ColR CRKp. ColR was underestimated by Etest (very major error rate = 35%, major error rate = 0.4%). A variety of rep-PCR strain types were encountered in both the ColS and the ColR groups. Carbapenem resistance was mediated primarily by blaKPC-2 (46%) and blaKPC-3 (50%). ColR was associated with increased hazard for in-hospital mortality (aHR 3.48; 95% confidence interval, 1.73-6.57; P < .001). The plasmid-associated ColR genes, mcr-1 and mcr-2 were not detected in any of the ColR CRKp., Conclusions: In this cohort, 13% of patients with CRKp presented with ColR CRKp. The apparent polyclonal nature of the isolates suggests de novo emergence of ColR in this cohort as the primary factor driving ColR. Importantly, mortality was increased in patients with ColR isolates., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com)

The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., bla OXA-23 , bla OXA-24/40 , and bla OXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen., (Copyright © 2016 American Society for Microbiology.)

Background.  Successful treatment of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa is thwarted by the emergence of antibiotic resistance and biofilm formation on prosthetic devices. Our aims were to decipher the molecular basis of resistance in a unique case of prosthetic valve endocarditis (PVE) caused by MDR P. aeruginosa . Methods.  Five sequential MDR P. aeruginosa blood isolates collected during a 7-month period were recovered from a patient suffering from PVE previously exposed to β-lactam antibiotics. Minimum inhibitory concentrations (MICs) of several classes of antibiotics were used to indicate clinical resistance characteristics; relatedness of the isolates was determined using multilocus sequence typing and repetitive sequence-based polymerase chain reaction. Amplification and sequencing of regulatory and resistance genes was performed. Results.  All isolates belonged to ST 298, possessed bla PDC-16 , and were resistant to fluoroquinolones and carbapenems. In the course of therapy, we observed a >2-fold increase in cephalosporin resistance (4 µg/mL to >16 µg/mL). Sequencing of the AmpC regulator, amp R, revealed a D135N point mutation in cephalosporin-resistant isolates. Common carbapenemase genes were not identified. All isolates demonstrated a premature stop codon at amino acid 79 of the outer membrane protein OprD and mutations in the quinolone resistance-determining regions of gyr A and par C. Point mutations in nal C, an efflux pump regulator, were also observed. Conclusions.  In this analysis, we chart the molecular evolution of β-lactam resistance in a case of PVE. We show that mutations in regulatory genes controlling efflux and cephalosporinase production contributed to the MDR phenotype.

The medical community needs systematic and pragmatic approaches for evaluating the benefit-risk trade-offs of diagnostics that assist in medical decision making. Benefit-Risk Evaluation of Diagnostics: A Framework (BED-FRAME) is a strategy for pragmatic evaluation of diagnostics designed to supplement traditional approaches. BED-FRAME evaluates diagnostic yield and addresses 2 key issues: (1) that diagnostic yield depends on prevalence, and (2) that different diagnostic errors carry different clinical consequences. As such, evaluating and comparing diagnostics depends on prevalence and the relative importance of potential errors. BED-FRAME provides a tool for communicating the expected clinical impact of diagnostic application and the expected trade-offs of diagnostic alternatives. BED-FRAME is a useful fundamental supplement to the standard analysis of diagnostic studies that will aid in clinical decision making., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

We report complete genome sequences of four blaNDM-1-harboring Gram-negative multidrug-resistant (MDR) isolates from Colombia. The blaNDM-1 genes were located on 193-kb Inc FIA, 178-kb Inc A/C2, and 47-kb (unknown Inc type) plasmids. Multilocus sequence typing (MLST) revealed that these isolates belong to sequence type 10 (ST10) (Escherichia coli), ST392 (Klebsiella pneumoniae), and ST322 and ST464 (Acinetobacter baumannii and Acinetobacter nosocomialis, respectively). Our analysis identified that the Inc A/C2 plasmid in E. coli contained a novel complex transposon (Tn125 and Tn5393 with three copies of blaNDM-1) and a recombination "hot spot" for the acquisition of new resistance determinants., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians., Methods: We compared the performance characteristics of 4 RMD platforms for detecting resistance against β-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against β-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making., Results: In PRIMERS I, the 4 RMD platforms detected β-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem., Conclusions: RMD platforms can help inform empiric β-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Staphylococcus intermedius and Staphylococcus pseudintermedius are difficult to distinguish using conventional microbiological methods. Molecular diagnostic tools change our understanding of the epidemiology of these 2 organisms. In this study, we present (1) a detailed review of the current literature on molecular diagnostics and (2) a case series in which misidentification was proven in 1 case. We conclude that S pseudintermedius is a more common human pathogen than previously recognized.

Background: Acinetobacter baumannii is one of the most antibiotic-resistant pathogens. Defining mechanisms driving pathogenesis is critical to enable new therapeutic approaches., Methods: We studied virulence differences across a diverse panel of A. baumannii clinical isolates during murine bacteremia to elucidate host-microbe interactions that drive outcome., Results: We identified hypervirulent strains that were lethal at low intravenous inocula and achieved very high early, and persistent, blood bacterial densities. Virulent strains were nonlethal at low inocula but lethal at 2.5-fold higher inocula. Finally, relatively avirulent (hypovirulent) strains were nonlethal at 20-fold higher inocula and were efficiently cleared by early time points. In vivo virulence correlated with in vitro resistance to complement and macrophage uptake. Depletion of complement, macrophages, and neutrophils each independently increased bacterial density of the hypovirulent strain but insufficiently to change lethality. However, disruption of all 3 effector mechanisms enabled early bacterial densities similar to hypervirulent strains, rendering infection 100% fatal., Conclusions: The lethality of A. baumannii strains depends on distinct stages. Strains resistant to early innate effectors are able to establish very high early bacterial blood density, and subsequent sustained bacteremia leads to Toll-like receptor 4-mediated hyperinflammation and lethality. These results have important implications for translational efforts to develop therapies that modulate host-microbe interactions., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of β-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC β-lactamase enzymes and that blaPDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Genome sequencing of carbapenem-resistant Klebsiella pneumoniae isolates from regional U.S. hospitals was used to characterize strain diversity and the bla(KPC) 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 bla(KPC) 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

An increase in the number of infections with fluoroquinolone (FQ)-resistant Escherichia coli following transrectal ultrasound-guided biopsy of the prostate (TRUBP) was observed in Louis Stokes Cleveland Department of Veterans Affairs Medical Center. This study investigated whether these infections were caused by a single strain of E. coli possessing distinct resistance and virulence determinants. Of 15 patients with urinary tract infection, 5 were complicated with bacteraemia and 1 with prostate abscess. Thirteen FQ-resistant isolates demonstrated mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC but did not contain plasmid-mediated quinolone resistance determinants; bla CTX-M and bla CMY as well as genes coding for extended-spectrum β-lactamases were also absent. Genes encoding aminoglycoside-modifying enzymes were discovered in an isolate that was gentamicin-resistant. The most prevalent sequence type (ST) was ST43 ( n = 7), corresponding to ST131 in Achtman's multilocus sequence typing (MLST) scheme. These isolates (i) were distinguished as >95% similar by repetitive sequence-based PCR (rep-PCR), (ii) belonged to the virulent phylogenetic group B2 and (iii) contained plasmid types FIB, FIA and Frep. Several other strain types were present (ST2, ST27, ST30, ST44, ST472, ST494, ST511 and ST627). Non-ST43 isolates infected patients with more co-morbidities but contained similar virulence factors ( kpsMTII , iutA , papAH / papC and sfa / focDE ). In our hospital, E. coli isolates causing TRUBP-related infection are quite heterogeneous (ST131 and other ST types) and are part of phylogenetic groups containing multiple virulence factors.

Unlabelled: Acinetobacter baumannii is a globally important nosocomial pathogen characterized by an increasing incidence of multidrug resistance. Routes of dissemination and gene flow among health care facilities are poorly resolved and are important for understanding the epidemiology of A. baumannii, minimizing disease transmission, and improving patient outcomes. We used whole-genome sequencing to assess diversity and genome dynamics in 49 isolates from one United States hospital system during one year from 2007 to 2008. Core single-nucleotide-variant-based phylogenetic analysis revealed multiple founder strains and multiple independent strains recovered from the same patient yet was insufficient to fully resolve strain relationships, where gene content and insertion sequence patterns added additional discriminatory power. Gene content comparisons illustrated extensive and redundant antibiotic resistance gene carriage and direct evidence of gene transfer, recombination, gene loss, and mutation. Evidence of barriers to gene flow among hospital components was not found, suggesting complex mixing of strains and a large reservoir of A. baumannii strains capable of colonizing patients., Importance: Genome sequencing was used to characterize multidrug-resistant Acinetobacter baumannii strains from one United States hospital system during a 1-year period to better understand how A. baumannii strains that cause infection are related to one another. Extensive variation in gene content was found, even among strains that were very closely related phylogenetically and epidemiologically. Several mechanisms contributed to this diversity, including transfer of mobile genetic elements, mobilization of insertion sequences, insertion sequence-mediated deletions, and genome-wide homologous recombination. Variation in gene content, however, lacked clear spatial or temporal patterns, suggesting a diverse pool of circulating strains with considerable interaction between strains and hospital locations. Widespread genetic variation among strains from the same hospital and even the same patient, particularly involving antibiotic resistance genes, reinforces the need for molecular diagnostic testing and genomic analysis to determine resistance profiles, rather than a reliance primarily on strain typing and antimicrobial resistance phenotypes for epidemiological studies.

We report the 3.78-Mbp high-quality draft assembly of the genome from a clinical isolate of Acinetobacter nosocomialis called strain M2 (previously known as Acinetobacter baumannii strain M2).

We report the sequences of two Klebsiella pneumoniae clinical isolates, strains JHCK1 and VA360, from a newborn with meningitis in Buenos Aires, Argentina, and from a tertiary care medical center in Cleveland, OH, respectively. Both isolates contain one chromosome and at least five plasmids; isolate VA360 contains the Klebsiella pneumoniae carbapenemase (KPC) gene.

Comparison of genome-wide, high-resolution restriction maps of Klebsiella pneumoniae clinical isolates, including an NDM-1 producer, and in silico-generated restriction maps of sequenced genomes revealed a highly heterogeneous region we designated the 'high heterogeneity zone' (HHZ). The HHZ consists of several regions, including a 'hot spot' prone to insertions and other rearrangements. The HHZ is a characteristic genomic area that can be used in the identification and tracking of outbreak-causing strains., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (bla(KPC)) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection., Methods: Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed., Results: The DNA microarray detected bla(KPC) in all 12 isolates, and bla(KPC-3) was identified by PCR amplification and sequencing of the amplicon. In addition, a bla(SHV-11) gene was detected in all isolates. Immunoblotting revealed "low-level" production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all bla(KPC-3)-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with bla(KPC) carriage. Plasmids from 3 representative isolates readily transferred the bla(KPC-3) to Escherichia coli J-53 recipients., Conclusions: Our findings reveal the "silent" dissemination of bla(KPC-3) as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing bla(KPC-3) in an LTCF caring for neurologically impaired children and young adults.

Various combinations of antibiotics are reported to show synergy in treating nosocomial infections with multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii). Here, we studied hospital-acquired outbreak strains of MDR A. baumannii to evaluate optimal combinations of antibiotics. One hundred and twenty-one strains were grouped into one major and one minor clonal group based on repetitive PCR amplification. Twenty representative strains were tested for antibiotic synergy using Etest(®). Five strains were further analyzed by analytical isoelectric focusing and PCR to identify β-lactamase genes or other antibiotic resistance determinants. Our investigation showed that the outbreak strains of MDR A. baumannii belonged to two dominant clones. A combination of colistin and doxycycline showed the best result, being additive or synergistic against 70% of tested strains. Antibiotic additivity was observed more frequently than synergy. Strains possessing the same clonality did not necessarily demonstrate the same response to antibiotic combinations in vitro. We conclude that the effect of antibiotic combinations on our outbreak strains of MDR A. baumannii seemed strain-specific. The bacterial response to antibiotic combinations is probably a result of complex interactions between multiple concomitant antibiotic resistance determinants in each strain., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Genotyping methods are essential to understand the transmission dynamics of Acinetobacter baumannii. We examined the representative genotypes of A. baumannii at different time periods in select locations in Ohio, using two rapid automated typing methods: PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), a form of multi-locus sequence typing (MLST), and repetitive-sequence-based-PCR (rep-PCR). Our analysis included 122 isolates from 4 referral hospital systems, in 2 urban areas of Ohio. These isolates were associated with outbreaks at 3 different time periods (1996, 2000 and 2005-2007). Type assignments of PCR/ESI-MS and rep-PCR were compared to each other and to worldwide (WW) clone types. The discriminatory power of each method was determined using the Simpson's index of diversity (DI). We observed that PCR/ESI-MS sequence type (ST) 14, corresponding to WW clone 3, predominated in 1996, whereas ST 12 and 14 co-existed in the intermediate period (2000) and ST 10 and 12, belonging to WW clone 2, predominated more recently in 2007. The shift from WW clone 3 to WW clone 2 was accompanied by an increase in carbapenem resistance. The DI was approximately 0.74 for PCR/ESI-MS, 0.88 for rep-PCR and 0.90 for the combination of both typing methods. We conclude that combining rapid automated typing methods such as PCR/ESI-MS and rep-PCR serves to optimally characterize the regional molecular epidemiology of A. baumannii. Our data also sheds light on the changing sequence types in an 11 year period in Northeast Ohio.

We have investigated the reproducibility of DiversiLab rep-PCR fingerprints between two laboratories with the aim of determining if the fingerprints and clustering are laboratory-specific or portable. One-hundred non-duplicate Acinetobacter baumannii isolates were used in this study. DNA isolation and rep-PCR were each performed separately in two laboratories and rep-PCR patterns generated in laboratory A were compared with those from laboratory B. Twelve A. baumannii isolates processed in laboratory A showed ≥98 % pattern similarity with the corresponding 12 isolates tested in laboratory B and were considered identical. Sixty-four isolates showed 95-97.9 % similarity with their corresponding isolates. Twenty-three isolates showed 90-94 % similarity with the corresponding isolates, while one isolate showed only 87.4 % similarity. However, intra-laboratory clustering was conserved: isolates that clustered in laboratory A also clustered in laboratory B. While clustering was conserved and reproducible at two different laboratories, demonstrating the robustness of rep-PCR, interlaboratory comparison of individual isolate fingerprints showed more variability. This comparison allows conclusions regarding clonality to be reached independent of the laboratory where the analysis is performed.

Objectives: We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans., Methods: Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various β-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed., Results: Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried bla(OXA-66), bla(ADC-25), bla(TEM-1), aacC2 and IS1133. Strains of ST15 possessed bla(OXA-69), bla(ADC-11), bla(TEM-1) and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of bla(ADC) (one ST10 and one ST12) and/or bla(OXA-66) (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection., Conclusions: STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumannii.