In vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli ST131: Characterization of an Acquired Extended-Spectrum AmpC (ESAC) Conferring Resistance to Cefepime.

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance are reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical E. coli isolates obtained from a patient. The CMY-2-producing E. coli (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli (CMY-33-Ec) was detected in bronchoalveolar lavage. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime.CMY-33-Ec and CMY-2-Ec were identical by rep-PCR, being of hyperepidemic ST131, but showed different β-lactam MICs (e.g., cefepime 16 vs. ≤0.5 μg/ml). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but cefepime MICs were higher for CMY-33 than CMY-2 (8 vs. 0.25 μg/ml). The kcat/Km or kinact/KI (μM(-1) s(-1)) indicated that CMY-33 possesses an ESBL-like spectrum compared to CMY-2 (cefoxitin: 0.2 vs. 0.4; ceftazidime: 0.2 vs. not measurable; cefepime: 0.2 vs. not measurable; tazobactam 0.0018 vs. 0.0009). Using molecular modeling, we show that a widened active site (∼4 Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide, therefore awareness that cefepime treatment may select for resistant isolates is critical.

Exposure assessment of extended-spectrum beta-lactamases/AmpC beta-lactamases-producing Escherichia coli in meat in Denmark

INTRODUCTION Extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases (AmpC) are of concern for veterinary and public health because of their ability to cause treatment failure due to antimicrobial resistance in Enterobacteriaceae. The main objective was to assess the relative contribution (RC) of different types of meat to the exposure of consumers to ESBL/AmpC and their potential importance for human infections in Denmark. MATERIAL AND METHODS The prevalence of each genotype of ESBL/AmpC-producing E. coli in imported and nationally produced broiler meat, pork and beef was weighted by the meat consumption patterns. Data originated from the Danish surveillance program for antibiotic use and antibiotic resistance (DANMAP) from 2009 to 2011. DANMAP also provided data about human ESBL/AmpC cases in 2011, which were used to assess a possible genotype overlap. Uncertainty about the occurrence of ESBL/AmpC-producing E. coli in meat was assessed by inspecting beta distributions given the available data of the genotypes in each type of meat. RESULTS AND DISCUSSION Broiler meat represented the largest part (83.8%) of the estimated ESBL/AmpC-contaminated pool of meat compared to pork (12.5%) and beef (3.7%). CMY-2 was the genotype with the highest RC to human exposure (58.3%). However, this genotype is rarely found in human infections in Denmark. CONCLUSION The overlap between ESBL/AmpC genotypes in meat and human E. coli infections was limited. This suggests that meat might constitute a less important source of ESBL/AmpC exposure to humans in Denmark than previously thought - maybe because the use of cephalosporins is restricted in cattle and banned in poultry and pigs. Nonetheless, more detailed surveillance data are required to determine the contribution of meat compared to other sources, such as travelling, pets, water resources, community and hospitals in the pursuit of a full source attribution model.

Persistência de plasmídeos que codificam carbapenemases do tipo New-Delhi-Metalo-β-Lactamase

As metalo-β-lactamases (MBL) são capazes de hidrolisar os carbapenêmicos, a classe de antimicrobianos com maior potência para o tratamento de infecções graves e de maior uso clinico. Dentre as MBL, o grupo mais recentemente descrito e que apresentou rápida disseminação em todo o mundo é o da New-Delhi-Metalo- β-lactamases (NDM). Nas enterobactérias, os genes que codificam essas enzimas estão mais frequentemente localizados em plasmídeos. O estudo da estabilidade de plasmídeos que albergam o gene blaNDM-1 é importante para entender a predominância de espécies que carregam esses plasmídeos, desvendar mecanismos moleculares envolvidos na sua persistência e para desenvolver novas drogas que possam diminuir a sua persistência. Estudos recentes sobre estabilidade plasmidial evidenciaram que a maprotilina é capaz de induzir perda plasmidial de até 90% em E. coli K12. Neste trabalho, foi estudado o efeito da maprotilina na indução de cura de plasmídeos, que albergam o gene blaNDM-1, em diferentes espécies da família Enterobacteriaceae. Nove isolados pertencentes a diferentes espécies foram incluídas no estudo. Os plasmídeos foram caracterizados quanto ao seu tamanho por eletroforese e por sequenciamento de DNA no sistema Illumina. A persistência plasmidial foi determinada pelo método de contagem em placa em LB ágar com e sem tratamento com maprotilina em concentrações sub-inibitórias (50mg/L). O experimento foi conduzido por 10 dias, representando aproximadamente 100 gerações. Neste estudo evidenciou-se que o grupo das enterobactérias estão envolvidas na disseminação de plasmídeos com blaNDM-1, sendo que plasmídeos do grupo IncF estão mais relacionados a essa dispersão. A maprotilina teve efeito de cura plasmidial em todos os isolados exceto em E. hormaechei \"subsp. oharae\" e C. freundii. O isolado P. rettgeri apresentou maior taxa de perda plasmidial e a análise comparativa da sequência nucleotídica do plasmídeo indicou que a presença da IS5 pode estar relacionada com a diminuição da persistência plasmidial. Diferenças na persistência plasmidial, quando tratados com maprotilina, entre E. hormaechei \"subsp. steigerwaltii\" e E. hormaechei \"subsp. oharae\" sugerem que E. hormaechei \"subsp. oharae\" pode ser um possível disseminador de plasmídeos albergando blaNDM-1, devido a processos de adaptação co-evolutivos.

Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

Background In Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link. Results Pseudomonas aeruginosa PAO1 has two ampG paralogs, PA4218 (ampP) and PA4393 (ampG). Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates ampP andampG encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both ampP and ampG are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either ampP or ampG. Both ampG and ampP are the second members of two independent two-gene operons. Analysis of the ampG and ampPoperon expression using β-galactosidase transcriptional fusions showed that in PAO1, ampGoperon expression is β-lactam and ampR-independent, while ampP operon expression is β-lactam and ampR-dependent. β-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP. Conclusions In P. aeruginosa, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an ampPand ampG dependent pathway, and at high concentrations where although both ampP and ampGplay a role, ampG may be of greater importance. Both ampP and ampG are required for maximum induction. Similar to ampC, ampP expression is inducible in an ampR-dependent manner. Importantly, ampP expression is autoregulated and ampP also regulates expression of ampG. Both AmpG and AmpP have topologies consistent with functions in transport. Together, these data suggest that the mechanism of β-lactam resistance of P. aeruginosa is distinct from well characterized systems in Enterobacteriaceae and involves a highly complicated interaction between these putative permeases and known Amp proteins.

Infecções por enterobacteriaceae resistente aos carbapenêmicos em hospital de ensino: epidemiologia e caracterização molecular

Introduction: The production of KPC (Klebsiella pneumoniae carbapenemase) has become an important mechanism of carbapenem-resistance among Enterobacteriaceae strains. In Brazil, KPC is already widespread and its incidence has increased significantly, reducing treatment options. The “perfect storm” combination of the absence of new drug developmentand the emergence of multidrug-resistant strains resulted in the need for the use of older drugs, with greater toxicity, such as polymyxins. Aims: To determine the occurrence of carbapenemase-producing strains in carbapenem-resistant Enterobacteriaceae isolated from patients with nosocomial infection/colonization during September/2014 to August/2015, to determine the risk factors associated with 30-day- mortality and the impact of inappropriate therapy. Materials and Methods: We performed a case control study to assess the risk factors (comorbidities, invasive procedures and inappropriate antimicrobial therapy) associated with 30-day-mortality, considering the first episode of infection in 111 patients. The resistance genes blaKPC, blaIMP, blaVIM and blaNDM-1 were detected by polymerase chain reaction technique. Molecular typing of the strains involved in the outbreak was performed by pulsed field gel electrophoresis technique. The polymyxin resistance was confirmed by the microdilution broth method. Results: 188 episodes of carbapenem-resistant Enterobacteriaceae infections/colonizations were detected; of these, 122 strains were recovered from the hospital laboratory. The presence of blaKPC gene were confirmed in the majority (74.59%) of these isolates. It was not found the presence of blaIMP , blaVIM and blaNDM-1 genes. K. pneumoniae was the most frequent microorganism (77,13%), primarily responsible for urinary tract infections (21,38%) and infections from patients of the Intensive Care Unit (ICU) (61,38%). Multivariate statistical analysis showed as predictors independently associated with mortality: dialysis and bloodstream infection. The Kaplan-Meier curve showed a lower probability of survival in the group of patients receiving antibiotic therapy inappropriately. Antimicrobial use in adult ICU varied during the study period, but positive correlation between increased incidence of strains and the consumption was not observed. In May and July 2015, the occurrence rates of carbapenem-resistant Enterobacteriaceae KPC-producing per 1000 patient-days were higher than the control limit established, confirming two outbreaks, the first caused by colistin-susceptible KPC-producing K. pneumoniae isolates, with a polyclonal profile and the second by a dominant clone of colistin-resistant (≥ 32 μg/mL) KPC-producing K. pneumoniae. The cross transmission between patients became clear by the temporal and spatial relationships observed in the second outbreak, since some patients occupied the same bed, showing problems in hand hygiene adherence among healthcare workers and inadequate terminal disinfection of environment. The outbreak was contained when the ICU was closed to new admissions. Conclusions: The study showed an endemicity of K. pneumoniae KPC-producing in adult ICU, progressing to an epidemic monoclonal expansion, resulted by a very high antibiotic consumption of carbapenems and polymyxins and facilitated by failures in control measures the unit.

Producing new Asian masculinities

An exploration of issues of Asian masculinity in white cultures, integrating film and literary analysis, autobiography and postcolonial theory.

A review of rationale for allocating costs and payments in producing and supplying public sector information

Presentation given by Dr John S Cook at the Queensland Spatial Conference 2008, Global Warming: What’s Happening in Paradise?, held at Holiday Inn, Surfers Paradise,Queensland from 17-19 July, 2008 This presentation provides some semblance of an information infrastructure that is aligned generally to problems of governance in complex organisations.

The key lessons learnt from producing the ABC programme Talking Heads a talk show/documentary hybrid in a fast turnaround environment

The following exegesis will detail the key advantages and disadvantages of combining a traditional talk show genre with a linear documentary format using a small production team and a limited budget in a fast turnaround weekly environment. It will deal with the Australian Broadcasting Corporation series Talking Heads, broadcast weekly in the early evening schedule for the network at 18.30 with the presenter Peter Thompson. As Executive Producer for the programme at its inception I was responsible for setting it up for the ABC in Brisbane, a role that included selecting most of the team to work on the series and commissioning the music, titles and all other aspects required to bring the show to the screen. What emerged when producing this generic hybrid will be examined at length, including: „h The talk show/documentary hybrid format needs longer than 26¡¦30¡¨ to be entirely successful. „h The type of presenter ideally suited to the talk show/documentary format requires someone who is genuinely interested in their guests and flexible enough to maintain the format against tangential odds. „h The use of illustrative footage shot in a documentary style narrative improves the talk show format. iv „h The fast turnaround of the talk show/documentary hybrid puts tremendous pressure on the time frames for archive research and copyright clearance and therefore needs to be well-resourced. „h In a fast turnaround talk show/documentary format the field components are advantageous but require very low shooting ratios to be sustainable. „h An intimate set works best for a talk show hybrid like this. Also submitted are two DVDs of recordings of programmes I produced and directed from the first and third series. These are for consideration in the practical component of this project and reflect the changes that I made to the series.

Experience mapping : producing spatially continuous environment representations using RatSLAM

RatSLAM is a system for vision-based Simultaneous Localisation and Mapping (SLAM) inspired by models of the rodent hippocampus. The system can produce stable representations of large complex environments during robot experiments in both indoor and outdoor environments. These representations are both topological and metric in nature, and can involve multiple representations of the same place as well as discontinuities. In this paper we describe a new technique known as experience mapping that can be used online with the RatSLAM system to produce world representations known as experience maps. These maps group together multiple place representations and are spatially continuous. A number of experiments have been conducted in simulation and a real world office environment. These experiments demonstrate the high degree to which experience maps are representative of the spatial arrangement of the environment.

Performing for (and against) the microphone : personal takes : producing a credible vocal

This contribution proposes the effect of the studio practice compiling vocals from many takes on the performance of vocalists.

A review of rationale for allocating costs and payments in producing and supplying public sector information PART A of 2 Parts – Chapters 1, 2 and 3) 13 March 2011 version

This work reviews the rationale and processes for raising revenue and allocating funds to perform information intensive activities that are pertinent to the work of democratic government. ‘Government of the people, by the people, for the people’ expresses an idea that democratic government has no higher authority than the people who agree to be bound by its rules. Democracy depends on continually learning how to develop understandings and agreements that can sustain voting majorities on which democratic law making and collective action depends. The objective expressed in constitutional terms is to deliver ‘peace, order and good government’. Meeting this objective requires a collective intellectual authority that can understand what is possible; and a collective moral authority to understand what ought to happen in practice. Facts of life determine that a society needs to retain its collective competence despite a continual turnover of its membership as people die but life goes on. Retaining this ‘collective competence’ in matters of self-government depends on each new generation: • acquiring a collective knowledge of how to produce goods and services needed to sustain a society and its capacity for self-government; • Learning how to defend society diplomatically and militarily in relation to external forces to prevent overthrow of its self-governing capacity; and • Learning how to defend society against divisive internal forces to preserve the authority of representative legislatures, allow peaceful dispute resolution and maintain social cohesion.