Lennart E. Nilsson

Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae.

Extended‐spectrum β‐lactamases (ESBLs) are often mediated by bla‐SHV, blaTEM and blaCTX‐M genes in Enterobacteriaceae and other Gram‐negative bacteria. Numerous molecular typing methods, including PCR‐based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between bla‐SHV, blaTEM and blaCTX‐M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of bla‐SHV, blaTEM and blaCTX‐M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of blaSHV, blaTEM and blaCTX‐M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well‐established CTX‐M primer pair used here to reveal plasmid‐encoded blaCTX‐M genes would also amplify the chromosomally located K‐1 enzyme gene in all Klebsiella oxytoca strains included in the study.

Pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs.

The pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were investigated by studying the postantibiotic effect (PAE) and initial killing. The influence of Ca2+ and albumin on these drugs was also evaluated. The PAE was studied by use of bioluminescence assay of bacterial ATP. Daptomycin at clinically achievable concentrations produced a dose-dependent PAE on E. faecalis (0.6 to 6.7 h) and S. aureus (1.0 to 6.3 h). The long PAE of daptomycin was seen simultaneously with a potent dose-dependent initial killing assayed by viable count determination. The initial change in bacterial ATP was not as extensive as the decrease in viability. Vancomycin at corresponding concentrations produced shorter PAEs on E. faecalis (0.5 to 1.0 h) and S. aureus (1.3 to 1.8 h). This coincides with a weak non-dose-dependent initial change in viability and intracellular ATP. The MICs of vancomycin were not influenced by different Ca2+ concentrations or by the addition of albumin to the broth. The MICs of daptomycin for both strains were lowered, and the PAEs were prolonged with increasing concentrations of Ca2+ in the broth. The PAE of daptomycin was Ca2+ dependent to the same extent as the MIC was. In the presence of physiological concentrations of albumin and free Ca2+, the PAEs of daptomycin on both strains were reduced and the MICs were increased in comparison with the results obtained in pure Mueller-Hinton broth with approximately the same free Ca2+ concentration. This decrease in daptomycin activity was considered to be due to the albumin binding of daptomycin. Despite the albumin binding of daptomycin, the PAE produced on E. faecalis and S. aureus in the presence of a physiological free Ca2+ concentration was still over 6 h at clinically achievable concentrations.

Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae: incidence and risk factors

OBJECTIVES To study the acquisition of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) among the faecal flora during travel, with a focus on risk factors, antibiotic susceptibility and ESBL-encoding genes. METHODS An observational prospective multicentre cohort study of individuals attending vaccination clinics in south-east Sweden was performed, in which the submission of faecal samples and questionnaires before and after travelling outside Scandinavia was requested. Faecal samples were screened for ESBL-PE by culturing on ChromID ESBL and an in-house method. ESBL-PE was confirmed by phenotypic and genotypic methods. Susceptibility testing was performed with the Etest. Individuals who acquired ESBL-PE during travel (travel-associated carriers) were compared with non-carriers regarding risk factors, and unadjusted and adjusted ORs after manual stepwise elimination were calculated using logistic regression. RESULTS Of 262 enrolled individuals, 2.4% were colonized before travel. Among 226 evaluable participants, ESBL-PE was detected in the post-travel samples from 68 (30%) travellers. The most important risk factor in the final model was the geographic area visited: Indian subcontinent (OR 24.8, P < 0.001), Asia (OR 8.63, P < 0.001) and Africa north of the equator (OR 4.94, P = 0.002). Age and gastrointestinal symptoms also affected the risk significantly. Multiresistance was seen in 77 (66%) of the ESBL-PE isolates, predominantly a combination of reduced susceptibility to third-generation cephalosporins, trimethoprim/sulfamethoxazole and aminoglycosides. The most common species and ESBL-encoding gene were Escherichia coli (90%) and CTX-M (73%), respectively. CONCLUSION Acquisition of multiresistant ESBL-PE among the faecal flora during international travel is common. The geographical area visited has the highest impact on ESBL-PE acquisition.

Morphologic conversion of Helicobacter pylori from bacillary to coccoid form

The morphologic conversion ofHelicobacter pylori from bacillary to coccoid form was studied by microscopy, viable count on agar plates, and bioluminescence assay of bacterial ATP. When morphologic conversion from bacillary to coccoid form was detected by microscopy, the viable counts and the bacterial ATP decreased. No viable count was found after nine days of incubation, but bacterial ATP was still present. In these cultures in which only the coccoid form ofHelicobacter pylori was present, there was no accumulation of extracellular ATP, indicating no leaky cells. During the transition phase from the bacillary to the coccoid form ofHelicobacter pylori, the addition of fresh medium increased the intracellular ATP 26-fold. The coccoid form ofHelicobacter pylori had a 1000-fold lower ATP level per cell compared to the bacillary form, which indicates a decreased metabolic activity in the coccoid form. Addition of fresh medium to the coccoid cultures from days 9 and 10 increased the ATP level twofold. However, no conversion from coccoid to bacillary form was found in these cultures during prolonged incubation in fresh broth for four weeks. Such conversion needs to be demonstrated before it is proven that the coccoid form ofHelicobacter pylori is responsible for transmission and relapse of infection.

Bacteria classification based on feature extraction from sensor data

Data evaluation and classification have been made on measurements by an electronic nose on the headspace of samples of different types of bacteria growing on petri dishes. The chosen groups were: Escherichia coli, Enterococcus sp., Proteus mirabilis, Pseudomonas aeruginosa, and Staphylococcus saprophytica. An approximation of the response curve by time was made and the parameters in the curve fit were taken as important features of the data set. A classification tree was used to extract the most important features. These features were then used in an artificial neural network for classification. Using the ‘leave-one-out’ method for validating the model, a classification rate of 76% was obtained.

Postantibiotic effect of beta-lactam antibiotics on Escherichia coli evaluated by bioluminescence assay of bacterial ATP.

The in vitro postantibiotic effects (PAE) of aztreonam, ceftazidime, cefuroxime, imipenem, and piperacillin on Escherichia coli ATCC 25922 were studied by a bioluminescence assay of bacterial ATP. In parallel with the PAE investigation, viability and morphology studies were performed. The strain was exposed for 2 h to different concentrations of beta-lactam antibiotics. The antibiotic activity was eliminated by 10(-4) dilutions, and regrowth of bacteria was monitored hourly by the bioluminescence assay of bacterial ATP. The length of PAE was dose dependent for ceftazidime (0.5 to 2.6 h), cefuroxime (0.4 to 2.6 h), and imipenem (0.3 to 4.5 h). The long PAE for these antibiotics at higher concentrations was associated with a potent initial killing and the presence of spheroplasts. Aztreonam and piperacillin produced a short, non-dose-dependent PAE (0.4 to 0.95 h). Short PAEs (below 1 h) were seen concomitantly with production of filaments, except in the case of imipenem, which only produced spheroplasts. The bioluminescence method was not jeopardized by filament formation, in contrast to the viable count assay which is normally used for PAE investigations. This makes it possible to study PAE for beta-lactam antibiotics on gram-negative bacteria with bioluminescence.

Molecular detection of aggregation substance, enterococcal surface protein, and cytolysin genes and in vitro adhesion to urinary catheters of Enterococcus faecalis and E. faecium of clinical origin

It has been hypothesized that nosocomial enterococci might have virulence factors that enhance their ability to colonise hospitalised patients. The objectives of this study were to investigate the prevalence of genes encoding 3 virulence factors: aggregation substance (asa1), enterococcal surface protein (esp), and 5 genes within the cytolysin operon (cylA, cylB, cylM, cylL(L), cylL(S)) and cytolysin production in 115 enterococcal clinical isolates (21 Enterococcus faecium and 94 E. faecalis). Adhesion to siliconized latex urinary catheters in relation to presence of esp was analysed in a subset of isolates. The isolates were previously characterised by pulsed-field gel electrophoresis (PFGE). esp was the only virulence gene found in E. faecium. It was found in 71% of the 21 E. faecium isolates. asa1, esp, and the cyl operon were found in 79%, 73% and 13% respectively, of the 94 E. faecalis isolates. There was a complete agreement between presence of the cyl operon and phenotypic cytolysin production. Isolates belonging to a cluster of genetically related isolates carried esp and asa1 more often when compared to unique isolates. No difference was found with respect to cyl genes. E. faecalis isolates adhered with higher bacterial densities than E. faecium. E. faecalis isolates within the same PFGE cluster adhered with similar bacterial densities, but there was no association between adhesion and the presence of esp when isolates within the same cluster were compared. In conclusion, E. faecalis isolates with high-level gentamicin resistance (HLGR) belonging to clusters of genetically related isolates widely distributed in Swedish hospitals, were likely to carry both esp and asa1. Adhesion was not affected by esp.

High antibiotic susceptibility among bacterial pathogens in Swedish ICUs Report from a nation-wide surveillance program using TA90 as a novel index of susceptibility

Local infection control measures, antibiotic consumption and patient demographics from 1999–2000 together with bacteriological analyses were investigated in 29 ICUs participating in the ICU-STRAMA programme. The median antibiotic consumption per ICU was 1147 (range 605–2143) daily doses per 1000 occupied bed d (DDD1000). Antibiotics to which >90% of isolates of an organism were susceptible were defined as treatment alternatives (TA90). The mean number of TA90 was low (1–2 per organism) for Enterococcus faecium (vancomycin:VAN), coagulase negative staphylococci (VAN), Pseudomonas aeruginosa (ceftazidime:CTZ, netilmicin: NET) and Stenotrophomonas maltophilia (CTZ, trimethoprim-sulfamethoxazole: TSU), but higher (3–7) for Acinetobacter spp. (imipenem:IMI, NET, TSU), Enterococcus faecalis (ampicillin:AMP, IMI, VAN), Serratia spp. (ciprofloxacin:CIP, IMI, NET), Enterobacter spp. (CIP, IMI, NET, TSU), E. coli (cefuroxime:CXM, cefotaxime/ceftazidime:CTX/CTZ, CIP, IMI, NET, piperacillin-tazobactam:PTZ, TSU), Klebsiella spp. (CTX/CTZ CIP, IMI, NET, PTZ, TSU) and Staphylococcus aureus (clindamycin, fusidic acid, NET, oxacillin, rifampicin, VAN). Of S. aureus isolates 2% were MRSA. Facilities for alcohol hand disinfection at each bed were available in 96% of the ICUs. The numbers of TA90 available were apparently higher than in ICUs in southern Europe and the US, despite a relatively high antibiotic consumption. This may be due to a moderate ecological impact of the used agents and the infection control routines in Swedish ICUs.

Antibiotic prescription practices, consumption and bacterial resistance in a cross section of Swedish intensive care units

Background: The purpose of this work was to study usage of antibiotics, its possible determinants, and patterns of bacterial resistance in Swedish intensive care units (ICUs).

Post-antibiotic effect of beta-lactam antibiotics on gram-negative bacteria in relation to morphology, initial killing and MIC

The in vitro post-antibiotic effect (PAE) of cefepime, cefotaxime, ceftazidime and imipenem on reference strains ofEscherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa andSerratia marcescens were evaluated by bioluminescence assay of bacterial ATP. In parallel with the PAE determination, initial killing and morphology studies were performed. Imipenem produced>1 h PAE on all strains tested, cefepime and cefotaxime on four strains and ceftazidime only on one of the strains tested. The length of the PAE on different strains did not correlate in the same way to MIC. Imipenem induced>1 h PAE at 1/4-2 MIC while the cephalosporins caused>1 h PAE at 4−256 × MIC. A PAE exceeding 1.2 h was seen concomitantly with spheroplasts but there was not necessarily strong (≥99 %) initial killing at the same time. The PAE duration at≥99 % initial killing varied between 2.0 h and 5.0 h. When the cephalosporins produced<1 h PAEs, this was seen concomitantly with production of filaments and weak initial killing. The bioluminescence method was not jeopardized by filament formation and no negative PAE was found in contrast to the viable count method. The study showed that neither a certain multiple of MIC, the presence of spheroplasts nor strong initial killing can predict the length of PAE for β-lactam antibiotics on gram-negative bacteria.