Characterization of Ceftazidime Resistance Mechanisms in Clinical Isolates of Burkholderia pseudomallei from Australia

Burkholderia pseudomallei is a Gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ≥256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen

Catalytic and Non-Catalytic Roles for the Mono-ADP-Ribosyltransferase Arr in the Mycobacterial DNA Damage Response

Recent evidence indicates that the mycobacterial response to DNA double strand breaks (DSBs) differs substantially from previously characterized bacteria. These differences include the use of three DSB repair pathways (HR, NHEJ, SSA), and the CarD pathway, which integrates DNA damage with transcription. Here we identify a role for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. Arr is transcriptionally induced following DNA damage and cellular stress. Although Arr is not required for induction of a core set of DNA repair genes, Arr is necessary for suppression of a set of ribosomal protein genes and rRNA during DNA damage, placing Arr in a similar pathway as CarD. Surprisingly, the catalytic activity of Arr is not required for this function, as catalytically inactive Arr was still able to suppress ribosomal protein and rRNA expression during DNA damage. In contrast, Arr substrate binding and catalytic activities were required for regulation of a small subset of other DNA damage responsive genes, indicating that Arr has both catalytic and noncatalytic roles in the DNA damage response. Our findings establish an endogenous cellular function for a mono-ADP-ribosyltransferase apart from its role in mediating Rifampin resistance

Evolution of Burkholderia pseudomallei in Recurrent Melioidosis

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis

Validation of Open-Heart Intraoperative Risk score to predict a prolonged intensive care unit stay for adult patients undergoing cardiac surgery with cardiopulmonary bypass

Sirirat Tribuddharat,1 Thepakorn Sathitkarnmanee,1 Kriangsak Ngamsaengsirisup,1 Chawalit Wongbuddha2 1Department of Anesthesiology, 2Division of Cardiothoracic Surgery, Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand Background: A prolonged stay in an intensive care unit (ICU) after cardiac surgery with cardiopulmonary bypass (CPB) increases the cost of care as well as morbidity and mortality. Several predictive models aim at identifying patients at risk of prolonged ICU stay after cardiac surgery with CPB, but almost all of them involve a preoperative assessment for proper resource management, while one – the Open-Heart Intraoperative Risk (OHIR) score – focuses on intraoperative manipulatable risk factors for improving anesthetic care and patient outcome.Objective: We aimed to revalidate the OHIR score in a different context.Materials and methods: The ability of the OHIR score to predict a prolonged ICU stay was assessed in 123 adults undergoing cardiac surgery (both coronary bypass graft and valvular surgery) with CPB at two tertiary university hospitals between January 2013 and December 2014. The criteria for a prolonged ICU stay matched a previous study (ie, a stay longer than the median).Results: The area under the receiver operating characteristic curve of the OHIR score to predict a prolonged ICU stay was 0.95 (95% confidence interval 0.90–1.00). The respective sensitivity, specificity, positive predictive value, and accuracy of an OHIR score of ≥3 to discriminate a prolonged ICU stay was 93.10%, 98.46%, 98.18%, and 95.9%.Conclusion: The OHIR score is highly predictive of a prolonged ICU stay among intraoperative patients undergoing cardiac surgery with CPB. The OHIR comprises of six risk factors, five of which are manipulatable intraoperatively. The OHIR can be used to identify patients at risk as well as to improve the outcome of those patients. Keywords: predictive models, validation studies, cardiac surgical procedures, cardiopulmonary bypass, intensive care units, OHIR scor