Assessment of a newly developed immunochromatographic assay for NDM-type metallo-ß-lactamase producing Gram-negative pathogens in Myanmar.

BACKGROUND: To detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-ß-lactamases (NDMs) was developed. METHODS: The immunochromatographic assay for New Delhi metallo-ß-lactamases producers was developed using rat monoclonal antibodies against NDMs. The assessment was performed using 350 isolates of Gram-negative bacteria, including Acinetobacter baumannii (51 isolates), Enterobacteriaceae (163 isolates), and Pseudomonas aeruginosa (136 isolates) obtained from 2015 to 2017 in medical settings in Myanmar. Of them, 302 isolates were resistant to carbapenems, including imipenem and/or meropenem. The blaNDM genes were identified by PCR and sequencing. RESULTS: Of the 350 clinical isolates tested, 164 (46.9%) (60 isolates of Escherichia coli, 51 isolates of Klebsiella pneumoniae, 25 isolates of Enterobacter cloacae, 23 isolates of P. aeruginosa, and 5 isolates of A. baumannii) were positive on this assay, and all the positive isolates harbored genes encoding NDM-1, - 4, - 5 and - 7. The remaining 186 (53.1%) isolates negative on the assay did not harbor genes encoding NDMs. The assay had a specificity of 100% and a sensitivity of 100%. The assessment revealed that more than 90% of carbapenem-resistant Enterobacteriaceae produced NDMs. CONCLUSIONS: The immunochromatographic assay is an easy-to-use and reliable kit for detection of NDMs-producing Gram-negative bacteria. The assay revealed that NDM-producing Enterobacteriaceae isolates are wide-spread in medical settings in Myanmar.

Validation of a clinical decision tree to predict if a patient has a bacteraemia due to a ß-lactamase producing organism.

BACKGROUND: In recent years, several scores and algorithms have been developed in order to guide empirical antibiotic treatment in patients with gram-negative bacilli (GNB) bacteraemia according to the risk of extended-spectrum ß-lactamase (BL) producing. Some of these algorithms do not have easy applicability or present some limitations in their validation. The aim of our study was to validate a recently designed decision tree in our prospective cohort of bacteraemia due to gram-negative bacilli. METHODS: We prospectively identified and analyzed all bacteraemia due to gram-negative bacilli in adult patients in our centre between January 2015 and December 2016. Previously developed clinical decision tree was used to classify patients in each of the terminal nodes. Patients were classified as BL group according to whether they were producers of any type of BL. The statistical power of the tree was analyzed by receiver operating characteristics (ROC) curve and by calculation of C-statistics. RESULTS: A total of 448 episodes of bacteraemia were included; 132 (29.5%) were BL group; 68 (15.1%) ESBL producing, 43 (9.6%) due to AmpC and 21 (4.7%) isolates of Pseudomonas aeruginosa. The original clinical decision tree was modified according to the results of our multivariate analysis. The modified tree has a sensitivity of 71%, specificity of 92%, predictive positive value (PPV) of 79% and predictive negative value (NPV) of 88% generating an ROC curve with a C-statistic of 0.76. CONCLUSIONS: An easy-to-apply clinical decision tree could be used at the exact moment of diagnosis and adjust the empirical antibiotic treatment in patients with gram-negative bacilli bacteraemia.

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

BACKGROUND: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). METHODS: 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. RESULTS: Among the carbapenem-resistant isolates the genes bla KPC, bla VIM, bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC, bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. CONCLUSION: The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

Development of a Luminex xTAG® assay for cost-effective multiplex detection of ß-lactamases in Gram-negative bacteria.

OBJECTIVES: The objective of this study was to design and validate a genotyping method for multiplex identification of ESBLs and carbapenemases in Gram-negative bacilli. This assay had to be (i) superior to traditional (multiplex) PCR/sequencing-based tests in turn-around time, gene coverage and the ability to detect multiple variants of the same allele, and (ii) significantly more cost-effective than commercial microarrays and WGS. The targeted ß-lactamases include ESBLs (CTX-M families and subtypes, ESBL and non-ESBL SHV- and TEM-likes, OXA-1/2/7-likes, PER, VEB, GES), plasmid-mediated cephalosporinases (CMY, MOX, FOX, ACC, DHA, MIR/ACT) and carbapenemases (OXA-48, NDM, KPC, VIM, IMP). METHODS: A modular multiplex oligonucleotide ligation-PCR procedure was used, with read-out on a Luminex MAGPIX(®) platform. We designed 46 xTAG(®)-compatible probes targeting ß-lactamase alleles and allele variants, and one probe targeting a conserved 16S rRNA region serving as a DNA extraction control. The assay was optimized using a collection of 48 reference strains and further validated using 105 foodborne ESBL-producing Escherichia coli isolates. RESULTS: The specificity and selectivity of the test are 100% and 99.4%, respectively. Multiple variants of the same allele were successfully discriminated, as exemplified by five E. coli strains encoding both blaTEM-1 and blaTEM-52 genes. The turn-around time from single colony to result is 5 h and total consumable costs remained <€5 per sample. CONCLUSIONS: We designed and validated the first Luminex-compatible genotyping assay that reliably and rapidly identifies a broad range of ESBL, pAmpC and carbapenemase producers in culture.

Epidemiology and Management of Emerging Drug-Resistant Gram-Negative Bacteria: Extended-Spectrum ß-Lactamases and Beyond.

Worldwide prevalence of antimicrobial resistance is rapidly increasing, primarily a result of antibiotic misuse in the medical community. Resistant infections involving the urinary tract are typically caused by gram-negative bacteria. When treating these infections, clinicians have few effective antimicrobials to choose from and many are associated with significant adverse effects. There are now situations when clinicians are tasked with managing infections from pan-resistant organisms; thus, it is of paramount importance that spread of resistance be controlled. This review discusses common gram-negative resistance classes, highlighting the mechanisms of resistance, risk factors, type of infections, treatment, and outcomes associated with each class.

Evaluation of the Rapidec Carba NP Test Kit for Detection of Carbapenemase-Producing Gram-Negative Bacteria.

Recently, bioMérieux, France, introduced the Rapidec Carba NP test kit for rapid detection of carbapenemase-producing Gram-negative bacteria. This kit was evaluated in this study, and we report sensitivity, specificity, and positive and negative predictive values of 92.6%, 96.2%, 95.83%, and 92.6%, respectively. The test was easy to perform and interpret and relatively inexpensive ($5/Rs 300 per test) and provides a practical solution for early detection of carbapenemase-producing, multidrug-resistant Gram-negative bacteria.

The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods.

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

Prevalence and predictive factors of harboring fluoroquinolone-resistant and extended-spectrum ß-lactamase-producing rectal flora in Hong Kong Chinese men undergoing transrectal ultrasound-guided prostate biopsy.

OBJECTIVE: To study the prevalence of fluoroquinolone-resistant (FQ-resistant) and extended-spectrum ß-lactamase-producing (ESBL-producing) bacteria in the rectums of patients undergoing transrectal ultrasound-guided prostate biopsy (TRUS-Bx), identifying predictive factors for such carriage and to correlate with the microbiology of those who developed postbiopsy infection (PBI). METHODS: A total of 371 men undergoing TRUS-Bx were prospectively enrolled from August 2011 to March 2012. Rectal swab was obtained before antimicrobial prophylaxis on the day of biopsy and grown in selective media for resistant bacteria. Standard FQ prophylaxis was used without guidance from rectal swab results. Univariate and multivariate analyses were performed to identify predictive factors of either FQ-resistant or ESBL-producing bacteria carriage. RESULTS: A total of 199 of 371 patients (53.6%) carried antimicrobial-resistant rectal flora, with 150 (40.4%) and 152 (41.0%) patients having FQ-resistant and ESBL-producing bacteria, respectively. Diabetes mellitus (odds ratio, 2.075; P = .028) and the use of antimicrobials within the prior 5 years (odds ratio, 1.550; P = .047) were independent predictors of rectal carriage of such flora. PBI occurred in 9 patients, of which 7 harbored prebiopsy antimicrobial-resistant bacteria, which completely matched the microbiological data collected during the patients' PBI episodes. CONCLUSION: A high prevalence of FQ-resistant and ESBL-producing rectal flora in Chinese men undergoing TRUS-Bx was found. Diabetes mellitus and prior antimicrobial use within 5 years were significant predictors for resistant bacterial carriage. Despite the high-resistant bacteria prevalence, PBI rate remained low. A targeted approach of antimicrobial prophylaxis using prebiopsy culture swab in areas with high prevalence of resistant bacteria should be further investigated.

Evaluation of Ertapenem use with impact assessment on extended-spectrum beta-lactamases (ESBL) production and gram-negative resistance in Singapore General Hospital (SGH).

BACKGROUND: Ertapenem (preferred choice for ESBL-producing organisms) use exhibited an increasing trend from 2006 to 2008. As extensive use of ertapenem might induce the mutation of resistant bacteria strains to ertapenem, we aimed to assess the appropriateness and impact of ertapenem-use, on ESBL production, the trends of gram-negative bacterial resistance and on the utilization of other antibiotics in our institution. METHODS: Inpatients who received a dose of ertapenem during 1 January 2006 to 31 December 2008, were reviewed. Pertinent patient clinical data was extracted from the pharmacy databases and assessed for appropriateness based on dose and indication. Relevant data from Network for Antimicrobial Resistance Surveillance (Singapore) (NARSS) was extracted, to cross-correlate with ertapenem via time series to assess its impact on hospital epidemiology, trends of gram-negative resistance and consumption of other antibiotics from 2006 to mid-2010. RESULTS: 906 cases were reviewed. Ertapenem therapy was appropriate in 72.4% (93.7% success rate). CNS adverse events were noted in 3.2%. Readmission rate (30-day) due to re-infection (same pathogen) was 5.5%. Fifty cases had cultures growing Pseudomonas aeruginosa within 30 days of ertapenem initiation, with 25 cases growing carbapenem-resistant Pseudomonas aeruginosa.Ertapenem use increased from 0.45 DDD/100 patient days in 2006 to 1.2 DDD/100 patient days in mid-2010. Overall, the increasing trend of ertapenem consumption correlated with 1) increasing incidence-densities of ciprofloxacin-resistant/cephalosporin-resistant E. coli at zero time lag; 2) increasing incidence-densities of ertapenem-resistant Escherichia. coli and Klebsiella spp. at zero time lag; 3) increasing incidence-density of carbapenem-resistant Pseudomonas aeruginosa, at zero time lag.Increasing ertapenem consumption was significantly correlated with decreasing consumption of cefepime (R2 = 0.37344) 3 months later. It was significantly correlated with a decrease in imipenem consumption (R2 = 0.31081), with no time lag but was correlated with subsequent increasing consumption of meropenem (R2 = 0.4092) 6 months later. CONCLUSION: Ertapenem use was appropriate. Increasing Ertapenem consumption did not result in a decreasing trend of ESBL producing enterobacteriaceae and could result in the selection for multi-drug resistant bacteria.

Development of rapid phenotypic system for the identification of Gram-negative oxidase-positive bacilli in resource-limited settings.

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology. The diagnosis and surveillance of diseases is dependent, to a great extent, on laboratory services, which cannot function without effective reliable reagents and diagnostics. Despite the advancement in microbiology diagnosis globally, resourcelimited countries still struggle to provide an acceptable diagnosis quality which helps in clinical disease management and improve their mortality and morbidity data. During this study an indigenous product, Quick Test Strip (QTS) NE, was developed for the rapid identification of biochemically slower group of Gram-negative oxidase-positive bacilli that covers 19 different bacterial genera. Some of the members belonging to these groups are well-established human pathogens, e.g. various species of Vibrio, Pseudomonas, Burkholderia, Aeromonas, Achromobacter and Stenotrophomonas. This study also evaluates the performance of QTS-NE by comparing with genotypic characterization methods. A total of 232 clinical and reference bacterial isolates were tested by three different methods. QTSNE provides 100 percent concordant results with other rapid identification and molecular characterization methods and confirms the potential to be used in clinical diagnosis.

Effect of air drying on bacterial viability: A multiparameter viability assessment.

The effect of desiccation on the viability of microorganisms is a question of great interest for a variety of public health questions and industrial applications. Although viability is traditionally assessed by plate counts, cultivation-independent methods are increasingly applied with the aim to gain more insight into why cells might not form colonies and to optimize production processes. To evaluate their usefulness, we applied in this study a multiparameter viability assay to selected bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterococcus hirae, and Staphylococcus aureus) subjected to air-drying in the absence or presence of supplements. Tests included growth on solid culture medium and the measurement of membrane integrity, membrane potential, esterase and respiratory activities using fluorescent dyes. All measured parameters were responsive to desiccation stress. Results suggested that extending plate count analysis with cultivation-independent methods can greatly enhance resolution especially for moderate stress conditions, which do not get reflected in plate counts due to cellular recovery. Whereas plate counts reflect the final effect on viability, immediate measurement of cellular functions provides a snapshot picture of the fitness status at a specific point in time. Special emphasis was given to MgCl(2) which in concentrations≥50mM dramatically increased the bacterial susceptibility to desiccation in the case of the gram-negative bacteria and to a lesser extent also for the gram-positive bacteria. The study in addition confirmed a good agreement of results obtained with the recently developed real-time viability (RTV) assay and the BacLight LIVE/DEAD method in combination with a fluorescence plate reader.

Fourteen years in resistance.

Resistance trends have changed greatly over the 14 years (1997-2011) whilst I was Director of the UK Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL). Meticillin-resistant Staphylococcus aureus (MRSA) first rose, then fell with improved infection control, although with the decline of one major clone beginning before these improvements. Resistant pneumococci too have declined following conjugate vaccine deployment. If the situation against Gram-positive pathogens has improved, that against Gram-negatives has worsened, with the spread of (i) quinolone- and cephalosporin-resistant Enterobacteriaceae, (ii) Acinetobacter with OXA carbapenemases, (iii) Enterobacteriaceae with biochemically diverse carbapenemases and (iv) gonococci resistant to fluoroquinolones and, latterly, cefixime. Laboratory, clinical and commercial aspects have also changed. Susceptibility testing is more standardised, with pharmacodynamic breakpoints. Treatments regimens are more driven by guidelines. The industry has fewer big profitable companies and more small companies without sales income. There is good and bad here. The quality of routine susceptibility testing has improved, but its speed has not. Pharmacodynamics adds science, but over-optimism has led to poor dose selection in several trials. Guidelines discourage poor therapy but concentrate selection onto a diminishing range of antibiotics, threatening their utility. Small companies are more nimble, but less resilient. Last, more than anything, the world has changed, with the rise of India and China, which account for 33% of the world's population and increasingly provide sophisticated health care, but also have huge resistance problems. These shifts present huge challenges for the future of chemotherapy and for the edifice of modern medicine that depends upon it.

On the number of recovered individuals in the SIS and SIR stochastic epidemic models.

The basic models of infectious disease dynamics (the SIS and SIR models) are considered. Particular attention is paid to the number of infected individuals that recovered and its relationship with the final epidemic size. We investigate this descriptor both until the extinction of the epidemic and in transient regime. Simple and efficient methods to obtain the distribution of the number of recovered individuals and its moments are proposed and discussed with respect to the previous work. The methodology could also be extended to other stochastic epidemic models. The theory is illustrated by numerical experiments, which demonstrate that the proposed computational methods can be applied efficiently. In particular, we use the distribution of the number of individuals removed in the SIR model in conjunction with data of outbreaks of ESBL observed in the intensive care unit of a Spanish hospital.

Ertapenem administered as outpatient parenteral antibiotic therapy for urinary tract infections caused by extended-spectrum-beta-lactamase-producing Gram-negative organisms.

OBJECTIVES: Infections with extended-spectrum-beta-lactamase-producing organisms are an increasing public health concern. We reviewed the use of an outpatient parenteral antibiotic therapy (OPAT) programme to facilitate the early discharge from hospital of patients with ESBL-associated urinary tract infections. METHODS: A retrospective review of patients treated for urinary tract infections caused by ESBL-producing organisms through the OPAT programme at the Royal Hallamshire Hospital, Sheffield, UK over a 4 year period to January 2010 was conducted. Data on patient demographics, clinical presentation and laboratory results were collected. RESULTS: Twenty-four OPAT episodes involving 11 patients were identified. Six patients (54.5%) had an underlying urological abnormality on presentation to OPAT. All patients were treated with parenteral ertapenem. Two patients had multiple infections treated by OPAT. The mean duration of the OPAT episodes was 9.9 days (range 3-42). A total of 238 inpatient bed days were avoided, with resultant cost savings. CONCLUSIONS: Ertapenem administration through OPAT may help to decrease the costs associated with ESBL infections by reducing the number of inpatient bed days required for their successful treatment.

Rapid detection of ESBL-producing gram-negative bacteria isolated from blood: a reasonable and reliable tool for middle and low resource countries.

INTRODUCTION: Delay in appropriate treatment in patients with bacteraemia can increase morbidity, mortality, and health expenditures. We compared the Rapid Direct Test (RDT) designed to detect ESBL-producing gram-negative bacteria (GNB) directly from positive blood cultures bottles, with two conventional ESBL detection tests: Screening and Confirmatory Disk Diffusion Assay (SC-DDA) and an MIC Screening and ESBL E-test (MIC/ET). MATERIAL AND METHODS: We screened all blood cultures in a tertiary care facility from August to December 2005. We only included one positive bottle per patient in which GNB were observed. RDT: Blood from each bottle was inoculated on Mueller-Hinton agar. Ceftazidime and cefotaxime disks with and without clavulanic acid were added and incubated at 35 degrees C +/- 2 degrees C for 24 h. Results were interpreted according to CLSI recommendations for the SC-DDA and MIC/ET. All methods were performed simultaneously. Time for reporting as an ESBL-producer and cost of the tests were recorded. RESULTS: We isolated 124 GNB in 114 episodes of bacteraemia, 10 of them (8.8%) polymicrobial; 79 (63.7%) of the GNB were enteric bacteria, 44 (35.5%) glucose non-fermenter GNB and one Haemophilus influenzae. The most common microorganism was Escherichia coli in 56 episodes (45.2%), followed by Pseudomonas aeruginosa in 24 (19.3%), and Klebsiella pneumoniae in 13 (10.5%). Of the 114 episodes, 41 (36%) had at least one GNB resistant to 3rd generation cephalosporins, and 25 (21.9%) were caused by an ESBL-producing GNB. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the RDT were 96%, 98.9%, 96% and 98.9%, respectively. Agreement by kappa index between RDT and SC-DDA was 0.95 and between the RDT and MIC/ET was 0.92. The RDT detected 24/25 ESBL-producing bacteria. The mean time to detect an isolate as an ESBL producer after a positive blood culture bottle signal was 1.02 +/- 0.19 days when using the RDT, and 3.40 +/- 0.59 days when using any other method. The difference in reporting time was 2.38 +/- 0.63 days (p < 0.0001). Our estimated cost per test was $1.54 for RDT, $2.32 for screening/ confirmatory SC-DDA, and $49.65 for MIC screening and MIC/ET. Conclusions. The RDT is a rapid, reliable and easy analysis to perform, as well as cost-effective.

Rapid, specific and sensitive electrochemical detection of foodborne bacteria.

Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius, is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2-ODN reporter conjugate. The detection limit corresponds to approximately 500 colony forming units (cfu) Escherichia coli. Beside high sensitivity, the application of electrochemical biochips allows discrimination of two gram-negative and two gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli. This detection system has the capability to be applied for monitoring of bacterial food contamination.

Impact and cost of infection control measures to reduce nosocomial transmission of extended-spectrum beta-lactamase-producing organisms in a non-outbreak setting.

We evaluated the impact of infection control interventions to reduce nosocomial extended-spectrum beta-lactamase (ESBL) transmission in a non-outbreak setting. This study was conducted at a tertiary 1200-bed hospital in Canada. The incidence of ESBLs was based on recovery of clinical isolates and assessed prospectively from 1999 to 2005. The incidence increased significantly from 0.28 to 0.67 per 1000 admissions during this period (P<0.001), reflecting an increase in the regional ESBL incidence from 1.32 to 9.28 per 100 000 population (P<0.001). Despite this increase, nosocomial ESBL rates increased only marginally, suggesting that infection control measures had an impact on nosocomial transmission. Infection control measures consisted of isolating all ESBL patients, as well as implementing the use of contact precautions for those with a high risk for transmission. The cost of these measures was CN$138 046.00 per year and CN$3191.83 per case admitted. A combination of control measures including active surveillance cultures, contact precautions for all colonized or infected patients and antimicrobial stewardship is required to significantly reduce the incidence of ESBLs.

Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems.

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.

Implementation of sequential therapy programs--a microbiologist's view.

Sequential antimicrobial therapy is not new, but confusion about the timing and nature of the switch often negates perceived advantages. A common problem is the choice of oral antibiotic to follow empirical administration of an intravenous second or third generation cephalosporin. Where guidelines do not exist, particularly when data are lacking as the the best option, the Delphi technique of obtaining a consensus agreement by review of a series of case histories is recommended. Majority verdicts are used to determine what is acceptable practice, and as such the approach is also suitable for audit. Savings through reduced drug acquisition costs and shorter lengths of stay have been highlighted. However, other less obvious potential benefits of sequential antimicrobial therapy include reduced incidence of intravascular catheter infection because of shorter line dwell times and less endoluminal contamination. Sequential antimicrobial therapy may also be used as part of a policy to reduce the selective pressure, particularly due to cephalosporin use, for endemic hospital pathogens such as Clostridium difficile and extended spectrum producing gram-negative baccilli.

Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant alpha-amylase.

Alpha-Amylase from the antarctic psychrophile Altermonas haloplanktis is synthesized at 0 +/- 2 degrees C by the wild strain. This heat-labile alpha-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18 degrees C.