Identification of a novel metallo-ß-lactamase, CAM-1, in clinical Pseudomonas aeruginosa isolates from Canada.

OBJECTIVES: To identify the ß-lactamase responsible for the positive detection of carbapenemase production in four clinical isolates of Pseudomonas aeruginosa that were negative by PCR for KPC, OXA-48, NDM, VIM, IMP, GES and NMC/IMI carbapenemase genes. METHODS: WGS using short-read and long-read methods was used to characterize the isolates. Bioinformatic analysis was used to identify the potential gene encoding a carbapenemase. Cloning, antimicrobial susceptibility testing and biochemical and phenotypic characterization were used to determine metallo-enzyme activity. Single-nucleotide variant (SNV) typing was used to determine strain relatedness. Conjugation experiments were used to determine transmissibility of the novel carbapenemase-encoding gene. RESULTS: WGS analysis revealed a novel class B ß-lactamase gene, blaCAM-1 (Central Alberta Metallo-ß-lactamase), located in a 73 kb integrative element, named IMEPaCAM-1, in the chromosome of four clinical isolates of P. aeruginosa. The cloned blaCAM-1 gene conferred carbapenem resistance to Escherichia coli TOP10. The four isolates, which were all closely related, were from three patients, all of whom spent time in the same hospital in 2008 and/or 2009. IMEPaCAM-1 could not be transferred by conjugation. CONCLUSIONS: A novel metallo-enzyme, CAM-1, is encoded on an integrative element, IMEPaCAM-1, located in the chromosome of clinical isolates of P. aeruginosa. No additional isolates harbouring CAM-1 have been identified in Alberta since 2007.

Headache Characteristics in Children With Pseudotumor Cerebri Syndrome, Elevated Opening Pressure Without Papilledema, and Normal Opening Pressure: A Retrospective Cohort Study.

BACKGROUND: Certain headache characteristics and associated symptoms are commonly attributed to increased intracranial pressure, but they have not been systematically studied among children in the context of revised diagnostic criteria for pseudotumor cerebri syndrome (PTCS). METHODS: We performed a retrospective cohort study of patients treated for suspected or confirmed PTCS. Charts were reviewed for PTCS and headache diagnostic criteria and associated characteristics. Chi-squared or Fisher's exact tests were used to compare the frequency of headache characteristics between groups. RESULTS: One hundred and twenty-seven individuals were identified: 61 had definite PTCS, 10 had probable PTCS, 31 had elevated opening pressure (OP) without papilledema, and 25 had normal OP without papilledema. Eleven children had no headache (6 with definite PTCS, 5 with probable PTCS). Headache pattern was episodic in 49% (95% CI: 34-64%) of those with definite PTCS, 18% (95% CI 6-37%) of those with elevated OP without papilledema, and 16% (5-36%) of those with normal OP without papilledema. Headache location was more likely to involve the head along with neck or shoulders in those with definite PTCS compared with elevated OP without papilledema (OR = 7.2, 95% CI: 1.9-27.6) and normal OP (OR = 4.5, 95% CI: 1.3-15.6) groups. DISCUSSION: While missing data and small cohort size are limitations, this study suggests that headache in PTCS is more likely to involve the head along with neck/shoulders, and that headache in PTCS may be episodic or constant. Headache is occasionally absent in PTCS.

Infections Caused by Actinomyces neuii: A Case Series and Review of an Unusual Bacterium.

Background. Actinomyces neuii is a Gram-positive bacillus rarely implicated in human infections. However, its occurrence is being increasingly recognized with the use of improved identification systems. Objective. To analyse A. neuii infections in Alberta, Canada, and review the literature regarding this unusual pathogen. Methods. Cases of A. neuii were identified in 2013-2014 in Alberta. Samples were cultured aerobically and anaerobically. A predominant catalase positive Gram-positive coryneform bacillus with no branching was isolated in each case. Testing was initially done with API-CORYNE® (bioMérieux) and isolates were sent to the Provincial Laboratory for Public Health for further testing. Isolates' identities were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry microbial identification system (MALDI-TOF MS MIS; bioMérieux) and/or DNA sequencing. Results. Six cases of A. neuii infection were identified. All patients had soft tissue infections; typically, incision and drainage were done followed by a course of antibiotics. Agents used included cephalexin, ertapenem, ciprofloxacin, and clindamycin. All had favourable outcomes. Conclusions. While A. neuii is infrequently recognized, it can cause a diverse array of infections. Increased use of MALDI-TOF MS MIS is leading to increased detection; thus, understanding the pathogenicity of this bacterium and its typical susceptibility profile will aid clinical decision-making.

Comparison of Uriswab to alternative methods for urine culture collection and transport: confirmation of standard culture methodology for investigation of urinary tract infections.

The ability to isolate and identify causative agents of urinary tract infections relies primarily on the quality of the urine sample that is submitted to the microbiology. The most important factors are the method of collection, the maintenance of viability of the potential pathogens during transport, and standardization of the culturing of the urine sample. This report is a composite of several investigations comparing collection and transport on urine culture paddles, with a preservative urine sponge (Uriswab), and a comparison of Uriswab with the BD preservative transport tube as methods of preservation of urinary pathogens. Primary studies showed that Uriswab maintained significantly more urinary pathogens than the urine culture paddle with fewer mixed or contaminated cultures. The two preservative transport systems were comparable for maintenance of viability of the pathogens, but there were fewer mixed cultures when samples were collected with Uriswab. This study confirms the importance of a standard volume of 1 µL of urine for culture.

Variability of ß-lactam susceptibility testing for Streptococcus pneumoniae using 4 commercial test methods and broth microdilution.

Limited data are available that verify the performance of commercial susceptibility methods for Streptococcus pneumoniae following the 2008 Clinical and Laboratory Standards Institute revision of the ß-lactam breakpoints. We compared the performance of Etest, M.I.C. Evaluator (M.I.C.E), Vitek, and Sensititre systems to broth microdilution for S. pneumoniae susceptibility testing of penicillin, ceftriaxone, meropenem, and amoxicillin. Essential agreement was ≥90% for the majority of the ß-lactams and methods tested, particularly for penicillin and ceftriaxone. Categorical agreements (CAs) for penicillin using meningeal and nonmeningeal breakpoints were ≥90%; CAs using penicillin oral breakpoints were 84-89%. Ceftriaxone CAs using nonmeningeal and meningeal breakpoints were 68-88% for Etest, M.I.C.E., and Vitek2 with 6-12% very major errors (VMEs) using meningeal breakpoints. Sensititre CAs for ceftriaxone, amoxicillin, and meropenem were ≥90% with no VMEs. In the context of the current guidelines, there exists considerable method-dependent variability in the susceptibility of S. pneumoniae to ß-lactams.

Effects of breakpoint changes on carbapenem susceptibility rates of Enterobacteriaceae: Results from the SENTRY Antimicrobial Surveillance Program, United States, 2008 to 2012.

In the absence of clinical resistance, breakpoints for many antimicrobial agents are often set high. Clinical failures following use of the agents over time requires re-evaluation of breakpoints. This is based on patient response, pharmacokinetic/pharmacodynamic information and in vitro minimal inhibitory concentration data. Data from the SENTRY Antimicrobial Surveillance Program has shown that Clinical and Laboratory Standards Institute breakpoint changes for carbapenems that occurred between 2008 and 2012 in North America have resulted in decreased levels of susceptibility for some species. In particular, reduced susceptibility to imipenem was observed for Proteus mirabilis (35%) and Morganella morganii (80%). Minor decreases in susceptibility were also noted for Enterobacter species with ertapenem (5%) and imipenem (4.3%), and Serratia species with imipenem (6.4%). No significant decreases in susceptibility were observed for meropenem following the breakpoint changes. There were no earlier breakpoints established for doripenem. Very few of these Enterobacteriaceae produce carbapenamase enzymes; therefore, the clinical significance of these changes has not yet been clearly determined. In conclusion, ongoing surveillance studies with in vitro minimum inhibitory concentration data are essential in predicting the need for breakpoint changes and in identifying the impact of such changes on the percent susceptibility of different species.

En l'absence de résistance clinique, la résistance de nombreux antimicrobiens est souvent fixée à un seuil élevé. En raison de l'échec clinique de certains de ces médicaments, il faut en réévaluer les seuils de résistance, d'après la réponse du patient, l'information pharmacocinétique et pharmacodynamique et les données relatives à la concentration minimale inhibitrice in vitro. Les données du programme de surveillance antimicrobienne SENTRY ont révélé que les changements au seuil de résistance des carbapénèmes établis par le Clinical and Laboratory Standards Institute entre 2008 et 2012 en Amérique du Nord ont entraîné une diminution de la susceptibilité de certaines espèces. Notamment, les chercheurs ont observé une susceptibilité réduite du Proteus mirabilis (35 %) et du Morganella morganii (80 %) à l'imipénem. Ils ont également remarqué de légères diminutions de la susceptibilité des espèces d'Enterobacter à l'ertapénem (5 %) et à l'imipénem (4,3 %), ainsi que des espèces de Serratia à l'imipénem (6,4 %). La susceptibilité du méropénem n'a pas diminué de manière significative, tandis qu'aucun seuil de résistance n'avait été établi auparavant pour le doripénem. Puisque très peu de ces entérobactériacés produisent des enzymes de carbapénémase, la signification clinique de ces changements n'est pas encore claire. Bref, il est essentiel de poursuivre les études de surveillance pour colliger des données sur les concentrations minimales inhibitrices in vitro afin de prédire la nécessité de changer le seuil de résistance et de déterminer les conséquences de ces changements sur le pourcentage de susceptibilité des diverses espèces.

In vitro efficacy of N-acetylcysteine on bacteria associated with chronic suppurative otitis media.

BACKGROUND: The safety and efficacy of Ciprodex® has been demonstrated for treatment of chronic suppurative otitis media (CSOM). However, symptoms fail to resolve in 9-15% of patients. The objective of this study is to evaluate the efficacy of N-acetylcysteine (NAC) on S. aureus, and planktonic and sessile (biofilm forming) P. aeruginosa in vitro using clinical isolates from patients with CSOM. METHODS: 1) Stability was assessed using liquid chromatography-mass spectrometry for each component in a prepared mixture of Ciprodex® and NAC over 15 days. Sterility was assessed by measuring bacterial growth on a blood agar plate. Efficacy was assessed using a disc diffusion method by inoculating plates with S. aureus ATCC 29513 and P. aeruginosa ATCC 27853, and measuring the clearance zone.2) Fifteen P. aeruginosa strains were isolated from patients with CSOM and tested in vitro using the bioFILM PA™ antimicrobial susceptibility assay. Treatment solutions included Ciprodex® & ciprofloxacin +/- NAC, and NAC alone (0.25%, 0.5% & 1.25%). RESULTS: 1) NAC combined with Ciprodex® demonstrated stability, sterility, and efficacy over a two-week period2) P. aeruginosa strains in the sessile (33%-40%) and planktonic (13%) state demonstrated resistance to Ciprodex® and ciprofloxacin. When NAC ≥0.5% was used in isolation or as an adjunct to either of these medications, no resistance was found in the sessile or planktonic state among all 15 strains. CONCLUSION: 1) Ciprodex® combined with NAC has a shelf life of at least two weeks given the documented preservation of stability, sterility, and clinical efficacy of the mixed compounds.2) P. aeruginosa strains demonstrated resistance to both Ciprodex® and ciprofloxacin. NAC ≥0.5% overcomes issues with resistance and shows promise in the treatment of CSOM.

Current and future challenges in the development of antimicrobial agents.

Micro-organisms exist to survive. Even in the absence of antimicrobial agents, many have determinants of resistance that may be expressed phenotypically, should the need arise. With the advent of the antibiotic age, as more and more drugs were developed to treat serious infections, micro-organisms (particularly bacteria) rapidly developed resistance determinants to prevent their own demise.The most important determinants of resistance have been in the Gram-positive and Gram-negative bacteria. Among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) have taxed researchers and pharmaceutical companies to develop new agents that are effective against these resistant strains. Among the Gram-negative bacteria, extended-spectrum beta-lactamase (ESBL) enzymes, carbapenemases (CREs) and the so-called amp-C enzymes that may be readily transferred between species of enterobacteriaceae and other facultative species have created multi-drug resistant organisms that are difficult to treat. Other resistance determinants have been seen in other clinically important bacterial species such as Neisseria gonorrhoeae, Clostridium difficile, Haemophilus influenzae and Mycobacterium tuberculosis. These issues have now spread to fungal agents of infection.A variety of modalities have been used to stem the tide of resistance. These include the development of niche compounds that target specific resistance determinants. Other approaches have been to find new targets for antimicrobial activity, use of combination agents that are effective against more than one target in the cell, or new delivery mechanism to maximize the concentration of antimicrobial agents at the site of infection without causing toxicity to the host. It is important that such new modalities have been proved effective for clinical therapy. Animal models and non-mammalian systems have been developed to determine if new agents will reach sufficient concentrations at infection sites to predict clinical efficacy without toxicity. It will also be key to consider antimicrobial stewardship as an important component of the continuing battle to prevent the development of antimicrobial resistance.

A ß-mannan trisaccharide conjugate vaccine aids clearance of Candida albicans in immunocompromised rabbits.

A ß-(1 → 2)-linked mannose trisaccharide epitope that is the optimal inhibitor of two protective monoclonal antibodies specific for the Candida albicans cell wall phosphomannan was used to create a synthetic conjugate vaccine. Two injections of the trisaccharide-tetanus toxoid conjugate administered with alum induced a robust secondary antibody response in rabbits with trisaccharide specific IgG ELISA titers in excess of 1:100,000. Fluorescent labeling studies demonstrated these antibodies (i) recognized the cell wall ß-mannan of C. albicans on hyphae and budding cells and (ii) C. albicans incubated with immune sera bound complement factor C3. The synthetic conjugate vaccine but not the carrier protein, tetanus toxoid reduced Candida load in vaccinated rabbits subsequently rendered leukocytopenic by injection of cyclophosphamide and then challenged with live C. albicans. These data support the contention that antibody mediated immunity plays a role in combating C. albicans infections and suggests that a surprisingly simple, readily accessible synthetic conjugate vaccine may have therapeutic potential.

Design of a Candida albicans disaccharide conjugate vaccine by reverse engineering a protective monoclonal antibody.

A disaccharide-chicken serum albumin conjugate vaccine against Candida albicans infections has been developed by reverse engineering a protective monoclonal antibody, C3.1. The binding site of C3.1 binds short oligosaccharides of ß1,2-linked mannopyranose residues present in the fungal cell wall phosphomannan. By delineating the fine detail of the molecular recognition of the cell wall ß-mannan antigen, a disaccharide epitope was deduced to be the minimum size epitope that should induce the formation of protective antibody. Sequential functional group replacement of disaccharide hydroxyl groups to yield a series of monodeoxy and mono-O-methyl ß1,2-linked mannobioside congeners established that three hydroxyl groups are essential for binding. Two of these, O-3 and O-4, are located on the internal mannose residue of the disaccharide, and a third, O-3', is located on the terminal mannose. Synthesis of a series of trisaccharides that mandate binding of either the reducing or nonreducing disaccharide epitopes provided the final indication that a disaccharide protein conjugate should have the potential to induce protective antibody. When disaccharide was conjugated to chicken serum albumin this vaccine produced antibodies in rabbits that recognized the native cell wall phosphomannan. In proof of concept protection experiments, three immunized rabbits showed a reduction in fungal burden when challenged with live C. albicans.

Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata.

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.

Prevalence of antimicrobial resistant pathogens from blood cultures from Canadian hospitals: results of the CANWARD 2007-2009 study.

This study assessed the epidemiology and antimicrobial resistance of pathogens associated with bloodstream infections in Canadian hospitals between 2007 and 2009. Tertiary-care medical centers representing 8 of 10 Canadian provinces submitted bloodstream infection pathogens from patients attending hospital clinics, emergency rooms, medical/surgical wards, and intensive care units. Over 8,000 blood culture pathogens were collected. The 10 most common pathogens (representing 80.9% of all isolates) were Escherichia coli (1856 [22.6%]), Staphylococcus aureus (1457 [17.7%] including 1101 methicillin-susceptible Staphylococcus aureus and 356 methicillin-resistant Staphylococcus aureus), coagulase-negative staphylococci (907 [11.0%]), Klebsiella pneumoniae (600 [7.3%]), Streptococcus pneumoniae (470 [5.7%]), Enterococcus faecalis (360 [4.4%]), Pseudomonas aeruginosa (333 [4.0%]), viridans group streptococci (321 [3.9%]), Enterobacter cloacae (193 [2.3%]), and Streptococcus pyogenes (159 [1.9%]). The most active agents against Gram-negative bacilli were carbapenems (e.g., meropenem and ertapenem) and piperacillin-tazobactam, while for Gram-positive cocci, they were vancomycin, linezolid, and daptomycin.

Three swipes and you're out: how many swipes are needed to decontaminate plastic with disposable wipes?

At our hospital, health care workers use commercially available wipes to reduce bacterial counts on plastic surfaces. The workers use the wipes in a cursory fashion--swiping objects once for one to two seconds. We sought to measure the ability of wipes to reduce bacterial counts when swiped across plastic surfaces using various routines.

Canadian clinical practice guidelines for invasive candidiasis in adults.

Candidemia and invasive candidiasis (C/IC) are life-threatening opportunistic infections that add excess morbidity, mortality and cost to the management of patients with a range of potentially curable underlying conditions. The Association of Medical Microbiology and Infectious Disease Canada developed evidence-based guidelines for the approach to the diagnosis and management of these infections in the ever-increasing population of at-risk adult patients in the health care system. Over the past few years, a new and broader understanding of the epidemiology and pathogenesis of C/IC has emerged and has been coupled with the availability of new antifungal agents and defined strategies for targeting groups at risk including, but not limited to, acute leukemia patients, hematopoietic stem cell transplants and solid organ transplants, and critical care unit patients. Accordingly, these guidelines have focused on patients at risk for C/IC, and on approaches of prevention, early therapy for suspected but unproven infection, and targeted therapy for probable and proven infection.

Caspofungin Etest endpoint for Aspergillus isolates shows poor agreement with the reference minimum effective concentration.

The Clinical and Laboratory Standards Institute (CLSI) M38-A2 reference broth microdilution (BMD) method for the antifungal susceptibility testing of filamentous fungi now includes guidelines for testing echinocandin activity using the minimum effective concentration (MEC) as the endpoint measurement. In this study, we compared the caspofungin Etest MIC on RPMI agar and Mueller-Hinton agar (supplemented with glucose and methylene blue [MGM]) to the BMD MEC for 345 clinical Aspergillus isolates, including A. flavus, A. fumigatus, A. nidulans, A. niger, and A. terreus. The essential agreement (+/-1 log(2) dilution) of the Etest on MGM and RPMI agar with the reference BMD MEC was 18 and 26%, respectively. The geometric mean values for BMD MEC and MGM Etest were 0.137 and 0.024 microg/ml, respectively, and the geometric mean values for BMD and RPMI agar were 0.128 and 0.031 microg/ml, respectively. Comparatively, 91% of paired MGM and RPMI Etest results were within 2 log(2) dilutions of each other and consistently produced clearly defined endpoints. In conclusion, the caspofungin Etest MIC, like the BMD MEC, is a reproducible endpoint but is markedly lower than the reference BMD. In anticipation of susceptibility breakpoint assignments, optimization studies will be required to improve the concordance of these two assays so that the potential for underreporting echinocandin resistance in Aspergillus is mitigated.