Fosfomycin maintains activity against most clinical isolates, but the growth of colonies within the zone of inhibition around the fosfomycin disk is occasionally observed upon susceptibility testing. We aimed to estimate the frequency of such nonsusceptible inner colony mutants and identify the underlying resistance mechanisms. Disk diffusion testing of fosfomycin was performed on 649 multidrug-resistant clinical isolates collected between 2011 and 2015. For those producing inner colonies inside the susceptible range, the parental strains and their representative inner colony mutants were subjected to MIC testing, whole-genome sequencing, reverse transcription-quantitative PCR (qRT-PCR), and carbohydrate utilization studies. Of the 649 clinical isolates, 5 (0.8%) consistently produced nonsusceptible inner colonies. Whole-genome sequencing revealed the deletion of encoding hexose-6-phosphate antiporter in 4 of the inner colony mutants, while the remaining mutant contained a nonsense mutation in The expression of was absent in the mutant strains with deletion and was not inducible in the strain with the mutation, unlike in its parental strain. All 5 inner colony mutants had reduced growth on minimal medium supplemented with glucose-6-phosphate. In conclusion, fosfomycin-nonsusceptible inner colony mutants can occur due to the loss of function or induction of UhpT but are rare among multidrug-resistant clinical strains. Considering that these mutants carry high biological costs, we suggest that fosfomycin susceptibility of strains that generate inner colony mutants can be interpreted on the basis of the zone of inhibition without accounting for the inner colonies.

Acinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected Acinetobacter baumannii complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate A. baumannii complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in A. baumannii complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics.

Carbapenem resistant Enterobacteriaceae are a significant public health concern, and genes encoding the Klebsiella pneumoniae carbapenemase (KPC) have contributed to the global spread of carbapenem resistance. In the current study, we used whole-genome sequencing to investigate the diversity of blaKPC-containing plasmids and antimicrobial resistance mechanisms among 26 blaKPC-containing Escherichia coli, and 13 blaKPC-containing Enterobacter asburiae, Enterobacter hormaechei, K. pneumoniae, Klebsiella variicola, Klebsiella michiganensis, and Serratia marcescens strains, which were isolated from the same patients as the blaKPC-containing E. coli. A blaKPC-containing IncN and/or IncFIIK plasmid was identified in 77% (30/39) of the E. coli and other bacterial species analyzed. Complete genome sequencing and comparative analysis of a blaKPC-containing IncN plasmid from one of the E. coli strains demonstrated that this plasmid is present in the K. pneumoniae and S. marcescens strains from this patient, and is conserved among 13 of the E. coli and other bacterial species analyzed. Interestingly, while both IncFIIK and IncN plasmids were prevalent among the strains analyzed, the IncN plasmids were more often identified in multiple bacterial species from the same patients, demonstrating a contribution of this IncN plasmid to the inter-genera dissemination of the blaKPC genes between the E. coli and other bacterial species analyzed.

cTreatment options for infections due to carbapenem-resistant Acinetobacter baumannii are extremely limited. Minocycline is a semisynthetic tetracycline derivative with activity against this pathogen. This study compared susceptibility testing methods that are used in clinical microbiology laboratories (Etest, disk diffusion, and Sensititre broth microdilution methods) for testing of minocycline, tigecycline, and doxycycline against 107 carbapenem-resistant A. baumannii clinical isolates. Susceptibility rates determined with the standard broth microdilution method using cation-adjusted Mueller-Hinton (MH) broth were 77.6% for minocycline and 29% for doxycycline, and 92.5% of isolates had tigecycline MICs of <2 g/ml. Using MH agar from BD and Oxoid, susceptibility rates determined with the Etest method were 67.3% and 52.3% for minocycline, 21.5% and 18.7% for doxycycline, and 71% and 29.9% for tigecycline, respectively. With the disk diffusion method using MH agar from BD and Oxoid, susceptibility rates were 82.2% and 72.9% for minocycline and 34.6% and 34.6% for doxycycline, respectively, and rates of MICs of <2 g/ml were 46.7% and 23.4% for tigecycline. In comparison with the standard broth microdilution results, very major rates were low (ϳ2.8%) for all three drugs across the methods, but major error rates were higher (ϳ5.6%), especially with the Etest method. For minocycline, minor error rates ranged from 14% to 37.4%. For tigecycline, minor error rates ranged from 6.5% to 69.2%. The majority of minor errors were due to susceptible results being reported as intermediate. A cinetobacter baumannii has become a major health care-associated pathogen over the past 2 decades, due to its intrinsic resistance to several classes of antimicrobial agents, its propensity to acquire resistance to other drug classes, and its ability to resist desiccation in environments typically found in hospitals (1). A. baumannii causes a variety of infections, with respiratory tract infections being the most common (1). Significant clinical challenges are posed by carbapenem-resistant A. baumannii, which accounted for over 75% of A. baumannii clinical isolates tested in a recent global survey (2). Mortality rates for carbapenem-resistant A. baumannii infections may be as high as 76% (3). Treatment options for carbapenem-resistant A. baumannii infections have not been well defined but generally include polymyxins (colistin and polymyxin B), tigecycline, or sulbactam, alone or in combination with a second agent, such as rifampin or a carbapenem, with the expectation of synergistic activities (4). However, toxicity (polymyxins), suboptimal pharmacokinetics (tigecycline), and the propensity for development of resistance (sulbactam) limit these options (1).Minocycline is a semisynthetic tetracycline derivative that was introduced into clinical practice in the 1960s (5). With the recent reintroduction of an intravenous formulation of minocycline, there is increasing interest in this agent as an additional treatment option for carbapenem-resi...

Objective:Describe the epidemiological and molecular characteristics of an outbreak of Klebsiella pneumoniae carbapenemase (KPC)–producing organisms and the novel use of a cohorting unit for its control.Design:Observational study.Setting:A 566-room academic teaching facility in Milwaukee, Wisconsin.Patients:Solid-organ transplant recipients.Methods:Infection control bundles were used throughout the time of observation. All KPC cases were intermittently housed in a cohorting unit with dedicated nurses and nursing aids. The rooms used in the cohorting unit had anterooms where clean supplies and linens were placed. Spread of KPC-producing organisms was determined using rectal surveillance cultures on admission and weekly thereafter among all consecutive patients admitted to the involved units. KPC-positive strains underwent pulsed-field gel electrophoresis and whole-genome sequencing.Results:A total of 8 KPC cases (5 identified by surveillance) were identified from April 2016 to April 2017. After the index patient, 3 patients acquired KPC-producing organisms despite implementation of an infection control bundle. This prompted the use of a cohorting unit, which immediately halted transmission, and the single remaining KPC case was transferred out of the cohorting unit. However, additional KPC cases were identified within 2 months. Once the cohorting unit was reopened, no additional KPC cases occurred. The KPC-positive species identified during this outbreak included Klebsiella pneumoniae, Enterobacter cloacae complex, and Escherichia coli. blaKPC was identified on at least 2 plasmid backbones.Conclusions:A complex KPC outbreak involving both clonal and plasmid-mediated dissemination was controlled using weekly surveillances and a cohorting unit.