OXY-2-15, a novel variant showing increased ceftazidime hydrolytic activity.

OBJECTIVES: Klebsiella oxytoca is a member of the family of Enterobacteriaceae and often contains the ß-lactamase blaOXY gene. Although this ß-lactamase does not naturally hydrolyse ceftazidime, this study describes possible in vivo selection of a clinical K. oxytoca isolate showing increased MICs of ceftazidime. METHODS: To reveal the molecular mechanism underlying this unusual resistance phenotype, WGS, cloning, overexpression, MIC and steady-state kinetic studies were performed. RESULTS: A patient was treated for a septic episode with ceftazidime (4 g/day). This therapy was based on earlier culture results in which, amongst others, a K. oxytoca (Velp-1) isolate was identified. After 11 days of treatment, K. oxytoca Velp-2 was isolated from a pus sample drained from the wound. The isolate showed increased resistance to ceftazidime (MIC ≥64 mg/L) compared with the original K. oxytoca isolate (Velp-1). WGS revealed the presence of a novel blaOXY-2 allele, designated blaOXY-2-15, with a two amino acid deletion at Ambler positions 168 and 169 compared with OXY-2-2. Cloning blaOXY-2-15 into Escherichia coli TOP10 resulted in increased MICs of ceftazidime, but reduced MICs of most other ß-lactams compared with OXY-2-2. Steady-state kinetics confirmed the results of the MIC data, showing clearly significant ceftazidime hydrolysis. CONCLUSIONS: This report shows the risk of in vivo selection of ceftazidime-resistant K. oxytoca isolates after prolonged ceftazidime treatment. Furthermore, it is the first known report of a K. oxytoca isolate conferring resistance to ceftazidime by a two amino acid deletion in the omega loop of OXY-2-2.

Distribution of extended-spectrum beta-lactamase genes using a commercial DNA micro-array system.

Extended-spectrum beta-lactamase (ESBL) genes are distributed worldwide and their epidemiology is complex. Using the Check-ESBL assay, the distribution of class A ESBL genes in clinical isolates of aerobic Gram-negative bacilli from three laboratories in the East of The Netherlands was determined. Four patient categories were distinguished: (i) patients admitted to an intensive care unit (ICU); (ii) non-ICU inpatients; (iii) outpatients admitted less than a year before collection of the isolate, (<1); (iv) outpatients admitted more than one-year prior to isolate collection or who had never been hospitalized (>1). From February 2009 until March 2010, out of 491 putative ESBL-positive isolates detected by the Vitek2 or Phoenix automated sensitivity testing systems, ESBL genes were detected in 247 (50.3%) by the Check-ESBL assay. Of these, 116 were from hospitalized patients (35 ICU, 81 non-ICU) and 131 were from outpatients (43 <1, 88 >1). In all, 274 ESBL genes were identified in these 247 isolates: 153 CTX-M-1 group (predominantly in E. coli and K. pneumoniae, 70.4% and 51.6% respectively), 67 CTX-M-9 group (predominantly in E. cloacae, 57.9%), 32 SHV, 14 TEM and 8 CTX-M-2 group. ESBL-producing E. cloacae were significantly more common in hospitalized patients than in outpatients, 20.7% and 3.8% respectively (P=0.001). CTX-M-9 group ESBLs were significantly more prevalent in ICU patients (P=0.003), whereas SHV ESBLs were more common in hospitalized patients than in outpatients (P<0.001). There was no significant difference in distribution of ESBL genes between the two outpatient groups.

Rapid diagnostic testing of methicillin-resistant Staphylococcus aureus carriage at different anatomical sites: costs and benefits of less extensive screening regimens.

Multiple body site screening and pre-emptive isolation of patients at risk for methicillin-resistant Staphylococcus aureus (MRSA) carriage are considered essential for control of nosocomial spread. The relative importance of extranasal screening when using rapid diagnostic testing (RDT) is unknown. Using data from a multicentre study evaluating BD GeneOhm™ MRSA PCR (IDI), Xpert MRSA (GeneXpert) and chromogenic agar, added to conventional cultures, we determined cost-effectiveness assuming isolation measures would have been based on RDT results of different hypothetical screening regimes. Costs per isolation day avoided were calculated for regimes with single or less extensive multiple site RDT, regimes without conventional back-up cultures and when PCR would have been performed with pooling of swabs. Among 1764 patients at risk, MRSA prevalence was 3.3% (n = 59). In all scenarios the negative predictive value is above 98.4%. With back-up cultures of all sites as a reference, the costs per isolation day avoided were €15.19, €30.83 and €45.37 with 'nares only' screening using chromogenic agar, IDI and GeneXpert, respectively, as compared with €19.95, €95.77 and €125.43 per isolation day avoided when all body sites had been screened. Without back-up cultures costs per isolation day avoided using chromogenic agar would range from €9.24 to €76.18 when costs per false-negative RDT range from €5000 up to €50 000; costs for molecular screening methods would be higher in all scenarios evaluated. In conclusion, in a low endemic setting chromogenic agar screening added to multiple site conventional cultures is the most cost-effective MRSA screening strategy.

Rapid screening of methicillin-resistant Staphylococcus aureus using PCR and chromogenic agar: a prospective study to evaluate costs and effects.

Pre-emptive isolation of suspected methicillin-resistant Staphylococcus aureus (MRSA) carriers is considered essential for controlling the spread of MRSA, but noncolonized patients will be isolated unnecessarily as a result of a delay in diagnosis of 3-5 days with conventional cultures. We determined costs per isolation day avoided, and incremental costs of rapid MRSA screening tests when added to conventional screening, but with decisions on isolation measures based on PCR results. A prospective multicentre study evaluating BD GeneOhm MRSA PCR (`IDI') (BD Diagnostics, San Diego, CA, USA), Xpert MRSA (`GeneXpert') (Cepheid, Sunnyvale, CA, USA) and chromogenic agar (MRSA-ID) (bioMérieux, Marcy-l'Etoile, France) was performed in 14 Dutch hospitals. Among 1764 patients at risk, MRSA prevalence was 3.3% (n=59). Duration of isolation was 19.7 and 16.1 h with IDI and GeneXpert, respectively, and would have been 30.0 and 76.2 h when based on chromogenic agar and conventional cultures, respectively. Negative predictive values (at a patient level) were 99.5%, 99.1% and 99.5% for IDI, GeneXpert and chromogenic agar, respectively. Numbers of isolation days were reduced by 60% and 47% with PCR-based and chromogenic agar-based screening, respectively. The cost per test was €56.22 for IDI, €69.62 for GeneXpert and €2.08 for chromogenic agar, and additional costs per extra isolation day were €26.34. Costs per isolation day avoided were €95.77 (IDI) and €125.43 (GeneXpert). PCR-based decision-making added €153.64 (IDI) and €193.84 (GeneXpert) per patient to overall costs and chromogenic testing would have saved €30.79 per patient. Rapid diagnostic testing safely reduces the number of unnecessary isolation days, but only chromogenic screening, and not PCR-based screening, can be considered as cost saving.

[Septic arthritis in two young children caused by unusual gram-negative pathogens]. / Septische artritis bij twee jonge kinderen veroorzaakt door ongebruikelijke gram-negatieve verwekkers.

Two children, a girl aged 2 years and a boy aged 10 months, were moderately ill with signs of inflammation of the left and the right knee, respectively. Both had had pharyngitis, and the boy also had paronychia of the right foot. The Gram preparation of synovial fluid showed Gram-positive cocci in the girl, while Kingella kingae was cultured. In the boy, a Moraxella was cultured from the synovial fluid using an aerobic blood culture system. Both recovered without sequelae after adequate antibiotic treatment. The micro-organisms cultured were Gram-negative bacteria, which are rarely seen in septic arthritis and are difficult to demonstrate. In young children, septic arthritis often presents with mild symptoms and inconclusive laboratory findings. Even if the Gram preparation of the synovial fluid shows no micro-organisms, unusual pathogens may be isolated by means of an aerobic blood culture system.

Cytotoxic effects of topical antimicrobial and antiseptic agents on human keratinocytes in vitro.

The cytotoxicity of commonly used antimicrobial and antiseptic agents to cultured human keratinocytes was investigated, combining a morphologic assay with a quantitative neutral red (NR) spectrophotometric assay. Main outcome criteria of response were the initial cytotoxicity (NR 90) and midpoint cytotoxicity (NR 50) and the highest tolerated dose (HTD), a concentration causing the first observed morphologic alterations. These values were compared with commonly administered clinical doses or calculated doses. Thirty-five agents were evaluated; five agents were also subjected to an assay for cell morphologic alterations, and HTD and percentage of cells taking up trypan blue and annihilation dose were assessed. These studies show that in clinical concentrations many, but not all, agents exert profound cytotoxic effects. The results suggest that under certain conditions cultured epithelial grafts may be exposed to clinical concentrations of neomycin, clindamycin, framycetin, erythromycin, gentamicin, and 0.1% solutions of povidone-iodine. Tetrachlorodecaoxygen anion complex (TCDO) should be further evaluated as a likely effective cleansing agent before culture grafting.