The effectiveness of clinical pathway software in inpatient settings: A systematic review.

BACKGROUND: Various studies have assessed the effectiveness of clinical pathways (CPs) in inpatient settings and provided systematic evidence that they positively affect patient outcomes and efficiency of care, thus lowering costs. In recent years, CP implementation is often combined or extended with clinical pathway software (CPS). Until now, no systematic literature review appears to exist which synthesizes the evidence on the effectiveness of CPS in inpatient settings, in relation to the CPs they support. OBJECTIVES: The purpose of this study was to systematically review evidence on (perceived) effectiveness of clinical pathway software (CPS) and investigate mechanisms explaining the effects of CPS implementation on outcomes. METHODS: We searched MEDLINE via PubMed and Scopus, for English-language original articles. Articles were included if they examined the effectiveness and/or the perceived effectiveness of CPS in the inpatient setting. They were analyzed for evidence on structure, process and outcome effects, as well as for mechanisms explaining such effects in relation to contextual factors. RESULTS: From 2904 articles, 12 studies met our inclusion criteria. The seven studies reporting on adherence provide conclusive evidence that CPSs can improve adherence. We also found conclusive evidence of improvement of process related measures regarding appropriate diagnostics, timeliness of care, and length of stay (LOS). Evidence on costs and outcomes is weak and/or less conclusive. This holds true both for patient outcomes (e.g. mortality/patient satisfaction) and caregiver outcomes (e.g. user satisfaction). The studies presented no direct evidence on mechanisms explaining how CPS relate to process and outcome improvements. CONCLUSIONS: The primary effects of CPS to increase adherence may in turn positively impact other process indicators such as LOS, timeliness of care, and diagnostic effectiveness. Subsequent effects on costs, outcomes for patients, physicians and nurses remain inconclusive and call for further research. Further research should explicitly take context into account. The scarce and weak evidence-base relating CPS implementation to process and outcome effects needs development along the same lines.

Clinical pharmacokinetics of cefamandole and ceftazidime administered by continuous intravenous infusion.

In view of the results of animal studies as well as theoretical considerations, continuous administration of beta-lactam antibiotics should be superior to intermittent administration because of the close relationship between efficacy and the duration of time in which the concentration of unbound antibiotics in plasma remains above the MIC. The aim of the present study was to establish the pharmacokinetic parameters of cefamandole and ceftazidime for patients receiving these cephalosporins by continuous infusion. The interindividual differences in the concentrations in plasma at the steady state were mainly attributable to variations in renal function, as estimated by the rate of creatinine clearance. Using these results, we derived formulas for both cephalosporins that can be used to determine on an individual basis the total daily dose needed to obtain a therapeutic concentration in plasma. These formulas were tested with a group of subsequent patients and proved to be practical and fairly reliable. For some patients, a correction for a possible underestimation of the renal clearance at presentation might be required.

Glycosylation of the Enterobacter cloacae outer membrane protein OmpX in eukaryotic cells.

The topological model of the Enterobacter cloacae outer membrane protein OmpX showed three putative glycosylation sites. When OmpX was expressed in bacteria that were cultured under aerated conditions, no glycosylation was observed. The coupling of carbohydrate chains to the ompX gene product was also investigated in the eukaryotic baculovirus expression system. For this purpose, a recombinant ompX gene-containing baculovirus was made. Infection of insect cells with this recombinant virus resulted in the production of sufficient amounts of OmpX to study glycosylation. In this system, all potential N-glycosylation sites of OmpX were utilized. Furthermore, it became clear that glycosylated OmpX was retained in the insect cells and was not secreted in the medium. Given the fact that OmpX plays a role in the invasion of E. cloacae in rabbit enterocytes, glycosylation of this protein occurring only under specific conditions may be involved in this process.

Expression of the Pseudomonas aeruginosa gentamicin resistance gene aacC3 in Escherichia coli.

The Pseudomonas aeruginosa aacC3 gene was expressed in Escherichia coli after cloning of the single gene behind the strong tac promoter. In the original Pseudomonas strain, aacC3 is preceded by cysC; together they form a single transcription unit. The ribosome-binding site and start codon of aacC3 are involved in a putative intercistronic hairpin, the stability of which interfered with the aminoglycoside resistance level. In Northern blots, full-length transcripts comprising both cysC and aacC3 could not be detected either in the original Pseudomonas strain or in E. coli harboring a plasmid with the cloned operon. In contrast, cysC transcripts were abundant. Cloning of the operon between the tac promoter and a transcription termination signal resulted in higher mRNA levels and phenotypic expression in E. coli. The absence of a transcription termination signal in the wild-type cysC-aacC3 sequence is associated with transcripts of heterogeneous size that were undetected in Northern blots. Our results shed more light on the expression of this gentamicin resistance determinant, although the discrepancies between its expression in E. coli and Pseudomonas are not fully solved.

Immuno-detection of the virulence determinant OmpX at the cell surface of Enterobacter cloacae.

A model for the topology of the Enterobacter cloacae outer membrane protein OmpX has been proposed, based on the primary sequence and on analogy to homologous proteins. According to this model the membrane embedded part of the protein consists of eight antiparallel beta-strands. Four random coil loops are located at the bacterial surface and three beta-turns at the periplasmic side of the membrane. Antibodies were raised against synthetic peptides representing five OmpX domains, four of which are putative peripheral and one located in the membrane. The accessibilities of OmpX to these antibodies were tested in intact cells by immuno-gold electron microscopy. This study showed that OmpX is indeed an outer membrane protein, the N-proximal loop of which forms an IgG-accessible epitope at the cell surface.

Changing susceptibilities of coagulase-negative staphylococci to teicoplanin in a teaching hospital.

The susceptibility of two collections of coagulase-negative staphylococci (CNS) isolated from clinical specimens for teicoplanin and vancomycin were compared. They comprised 91 and 101 isolates, collected in 1985 and 1994 respectively, from different departments of a teaching hospital. MICs of vancomycin and teicoplanin were determined by a modified Etest method. Additionally, a disc diffusion test was performed for teicoplanin. All isolates were susceptible to vancomycin (MIC < or = 4 mg/L). Two of the 91 isolates collected in 1985 were intermediate to teicoplanin (MIC between 8 and 32 mg/L), whereas in 1994 the number of intermediate isolates was 20 out of 101 (P < 0.01). The correlation between MICs, as determined by the modified Etest assay, and disc diffusion zones was poor (r = -0.35). Results show that resistance to teicoplanin in CNS has increased in the study hospital over a period of 9 years. This increase is likely to be correlated with the introduction of teicoplanin. Furthermore, a disc diffusion method does not appear to be the first method of choice for detection of strains of CNS with diminished susceptibility to teicoplanin.

Mechanisms of resistance to piperacillin of Enterobacter cloacae strains differ by antibiotic prescription policy.

During a study of piperacillin resistance among aerobic Gram-negative bacteria, 18 resistant strains of Enterobacter cloacae were obtained from a General Hospital in Rotterdam and 13 from a University Hospital in Amsterdam. The patterns of antibiotic susceptibilities were different: the Amsterdam strains were generally resistant to penicillins, the third generation cephalosporins and temocillin, whereas the Rotterdam strains were more often sensitive to the third generation cephalosporins and temocillin but more resistant to penicillins. Isoelectric focusing and substrate profiles showed the presence of chromosomal Class 1 beta-lactamase in ten of the Amsterdam strains: in three strains a plasmid mediated TEM-1 enzyme was detected. In contrast 15 of the 18 Rotterdam strains possessed a plasmid mediated beta-lactamase, ten of which were TEM-2. Eight of the ten strains with the TEM-2 enzyme harboured a transferable plasmid coding for resistance to piperacillin. Endonuclease analysis of plasmid DNA from these eight strains revealed an identical pattern in seven strains. Different selective pressures were operative in each hospital. In Amsterdam the general use of cefotaxim and piperacillin favoured emergence of strains with derepressed chromosomal Class 1 beta-lactamase, whereas in Rotterdam the use of cefuroxime favoured the spread of a plasmid, encoding TEM-2 beta-lactamase.

Inhibition of the hemolytic activity of the first component of complement C1 by an Escherichia coli C1q binding protein.

Molecular mimicry is a well established mechanism via which bacteria protect themselves from complement-mediated killing. We have previously demonstrated that a number of human cells express receptors for C1q (C1qR) and that the soluble form of this receptor inhibits activation of the classical pathway of complement. We now investigated whether Escherichia coli possesses a C1qR-like protein that protects these bacteria from complement-mediated injury. By FACS analysis it was shown that approximately 60% of the bacteria bound C1q directly in the absence of Abs. With ELISA we confirmed that the bacterial cell envelope was able to bind C1q in a dose-dependent fashion. We isolated a cell envelope associated C1q binding protein (C1qBP) by C1q affinity chromatography, then by anion exchange chromatography and gel filtration chromatography. On SDS-PAGE, the m.w. of C1qBP appeared to be 57 kDa and 51 kDa under reducing and nonreducing conditions, respectively. It was demonstrated that C1qBP specifically binds C1q and inhibits the hemolytic activity of C1q in both a dose- and time-dependent fashion. The binding of C1qBP to C1q is inhibited by C1q itself and also by the collagen-like stalks and the globular heads of C1q. In this respect, bacterial C1qBP is different from human C1qR because the binding of C1q to C1qR is only inhibited by the collagen-like stalks of C1q and not by the globular heads of C1q. C1qBP, when bound to C1q, prevents the assembly with C1r and C1s to form a functional C1 complex. The occurrence of C1qBP is not limited to certain E. coli strains, but is also found on Staphylococcus aureus, Citrobacter freundii, and Pseudomonas aeruginosa. Also, the binding of 125(I)-labeled C1q to these bacteria is specific because the binding of C1q to these bacteria is inhibitable with isolated soluble C1qBP. These findings provide evidence for the existence of a C1qR-like protein on bacteria that might protect them from complement-mediated damage.

Antibiotic resistance mechanisms in Salmonella species causing bacteraemia in Malawi and Kenya.

In two studies on the causative agents of bacteraemia in Malawi and Kenya, 33 Salmonella strains were isolated. Fourteen strains of Salmonella typhimurium and Salmonella enteritidis were found to exhibit resistance to amoxicillin, amoxicillin/clavulanic acid and cotrimoxazole as well as decreased susceptibility to a range of aminoglycosides. The resistant strains were studied to establish their resistance mechanisms. Beta-lactamase co-focusing with TEM-1 was present in 12 strains. In two strains, both S. typhimurium from Kenya, an OXA-1 beta-lactamase was detected. The aminoglycoside-modifying enzyme ANT(2") was found in 10 strains. The presence of the encoding genes was confirmed by PCR. For comparison, susceptibility records of 73 Salmonella strains isolated during the past 14 years in our hospital were studied retrospectively. Only one of these strains was resistant to amoxicillin. This resistance was acquired during therapy.

Susceptibility of Streptococcus pyogenes to azithromycin, clarithromycin, erythromycin and roxithromycin in vitro.

The susceptibility of 180 clinical isolates of Streptococcus pyrogenes from six regions of The Netherlands to the macrolide antibiotics azithromycin, clarithromycin, erythromycin and roxithromycin was analysed. The results of a microbroth MIC method, the E-test method and a disk diffusion assay were compared, and the MBC determined. In addition, the susceptibility to erythromycin of 436 clinical isolates of S. pyogenes from the Leiden region was determined. The microbroth MIC90s of azithromycin, clarithromycin, erythromycin and roxithromycin for group A streptococci were < or = 0.5 mg/L. Erythromycin had the lowest MIC90 (0.09 mg/L). The MIC data obtained with the E-test method suggested that clarithromycin and erythromycin had slightly higher anti-streptococcal activity than azithromycin and roxithromycin in vitro. MICs obtained with the E-test were lower than those found with the microbroth method. Only minor discrepancies were observed among the three methods. The MBC50 for both clarithromycin and erythromycin was 0.75 mg/L and 5.0 mg/L for azithromycin and roxithromycin. None of the 180 strains and two of the collection of 436 strains (0.5%) were resistant to erythromycin and the other macrolides tested; MICs ranged from 1 to 16 mg/L. The erythromycin-resistant strains showed an inducible type of macrolide-lincosamide-streptogramin B (MLS) resistance.

Effects of mutations and deletions on expression of the Enterobacter cloacae ompX gene.

The Enterobacter cloacae outer membrane protein OmpX is involved in resistance to beta-lactams, and possesses virulence characteristics. To gain more insight into the genetic elements that are important for OmpX expression, several mutations were introduced at, and immediately upstream of, the N-terminus of the OmpX coding sequence. These mutations enabled us to delete the 5' untranslated region and the signal peptide coding sequence. The former led to decreased ompX expression, indicating an unexpected and hitherto unexplained role for this region. Deletion of the signal peptide coding sequence blocked transport across the cytoplasmic membrane, indicating that translocation of OmpX across the cytoplasmic membrane is mediated by the general secretory pathway.

Differences in penicillin-binding protein patterns of penicillin tolerant and non-tolerant group A streptococci.

The penicillin-binding proteins (PBPs) of five penicillin tolerant group A streptococci and their isogenic non-tolerant strains, and seven unrelated non-tolerant group A streptococci were compared. PBPs from late logarithmic cultures were labelled in vitro with 3H-benzylpenicillin and analysed by SDS-PAGE and fluorography. The PBP patterns for all non-tolerant strains were identical. This pattern differed markedly from that for penicillin tolerant strains, both qualitatively and quantitatively. The most striking change in penicillin tolerant strains was decreased binding of 3H-penicillin to PBP 3 and increased binding to PBP 5, while PBP 2a was replaced by a new PBP (PBP 2a') of lower electrophoretic mobility. Tolerance was lost during storage but could be restored by consecutive transfers on to penicillin gradient agar plates. At the same time the PBP profiles of these strains became identical to those found for stable tolerant strains. These results suggest the possibility that PBP 2a' and PBP 5 in combination with other PBP alterations play a role in penicillin tolerance found in group A streptococci.

Invasion of rabbit ileal tissue by Enterobacter cloacae varies with the concentration of OmpX in the outer membrane.

The outer membrane protein OmpX of Enterobacter cloacae shows high amino acid homology with virulence proteins PagC and Rck from Salmonella typhimurium and with Ail from Yersinia enterocolitica. Here we demonstrate a role for OmpX in the invasion of rabbit ileal tissue by E. cloacae. An organ culture system was used for maintenance of rabbit gut tissue during the experiments. The invasiveness of three E. cloacae strains, which differed in OmpX content, were compared with each other and with that of Salmonella typhimurium TML (a highly invasive strain) and S. typhimurium LT7 (a noninvasive strain). There was no significant difference between the invasiveness of the wild type and that of an ompX deletion mutant strain of E. cloacae; they were equally as invasive or less invasive than S. typhimurium LT7. The invasiveness of an OmpX overproducer strain of E. cloacae was 10-fold higher than that of its immediate parent carrying only the multicopy plasmid, higher than that of S. typhimurium LT7, but lower than that of S. typhimurium TML. The invasiveness of E. cloacae thus varied directly with the level of OmpX in the outer membrane in rabbit ileal enterocytes challenged in situ.

High-level aminoglycoside resistance among enterococci and group A streptococci.

The occurrence of high-level aminoglycoside resistance (HLAmR) was determined for 73 enterococci and 54 group A streptococci by the high-load disc method, tube macrodilution and the polymerase chain reaction (PCR). The PCR method revealed the presence of genes coding for aminoglycoside-3'-O-phosphoryltransferase-III (APH(3')-III), aminoglycoside-6'-N-acetyltransferase/2''-O-phosphoryltransferase (AAC(6')/APH(2'')), or both, in 20.6%, 9.6% and 4.1% of the enterococci, respectively. The prevalence of HLAmR to at least one aminoglycoside among local enterococci was 37% (27/73). Only one of 54 Streptococcus pyogenes isolates produced APH(3')-III and exhibited high-level resistance to kanamycin and streptomycin. In general, the three methods yielded comparable results, with only three discrepancies among the 127 isolates examined. High-load disc screening and tube macrodilution proved to be practical, reliable and reproducible, and thus suitable for routine screening. Of 20 Enterococcus faecalis strains tested, all were penicillin-tolerant. Only one of seven penicillin-tolerant S. pyogenes strains was HLAmR. No association between the two forms of resistance was found.

Localisation of the aminoglycoside-(3)-N-acetyltransferase isoenzyme II in Escherichia coli.

For the location of the aminoglycoside-(3)-N-acetyltransferase isoenzyme II (AAC(3)-II) in the bacterial cell, two strains were studied: Escherichia coli HB101(pJV03), producing the 31-kDa AAC (3)-II enzyme, and E. coli HB101, which served as a control. From each strain five protein fractions were prepared: culture supernatant, and proteins occurring in the periplasm, cytoplasm, inner membrane and outer membrane. All fractions were tested for enzymatic activity of AAC(3)-II. Most of the acetylating activity was found in the cytoplasmic fraction. The distribution of marker enzymes showed a good separation between the periplasmic and the cytoplasmic fraction.

Nucleotide sequence of the aacC3 gene, a gentamicin resistance determinant encoding aminoglycoside-(3)-N-acetyltransferase III expressed in Pseudomonas aeruginosa but not in Escherichia coli.

A chromosomal gentamicin resistance determinant from Pseudomonas aeruginosa was cloned on a 2.4-kb fragment in the broad-host-range vector pLAFR3. Substrate profiles for eight aminoglycosides at three concentrations showed that resistance was due to aminoglycoside-(3)-N-acetyltransferase III. This enzyme was produced in Pseudomonas strains but not in an Escherichia coli strain bearing the aacC3 gene. Nucleotide sequencing revealed two contiguous open reading frames (ORFs) preceded by a potential promoter and a ribosome-binding site. ORF-1 was 642 bp in length and encoded a protein of unknown function with a molecular mass of 23.9 kDa. ORF-2 was 813 bp in length and encoded a protein of 29.6 kDa. From deletion mutagenesis, in vitro transcription-translation data, and protein analysis of bacterial lysates, it was inferred that this 29.6-kDa protein represents the aminoglycoside-(3)-N-acetyltransferase III enzyme. A polymerase chain reaction with two specific intragenic 20-mer primers was developed to detect the aacC3 gene. A BstEII restriction site in the amplified DNA region was used to demonstrate the specificity of the reaction. Tests of 23 reference strains, which produced 12 different aminoglycoside-modifying enzymes, confirmed the specificities of the primers. The gene proved to be absent from a collection of 50 gentamicin-resistant P. aeruginosa strains selected at random in The Netherlands.

Variation in induction of chromosomal beta-lactamase expression in strains of Enterobacter cloacae.

Eight strains of Enterobacter cloacae with varying patterns of susceptibility to beta-lactam antibiotics were studied to compare the inducibility and expression of their beta-lactamase genes. These strains included two isolates from one patient, which differed in their susceptibility to cefamandole. A resistant strain constitutively produced large amounts of beta-lactamase; a sensitive strain produced an inducible beta-lactamase. Study of induction and growth characteristics of all strains revealed that the induction and the expression of inducible beta-lactamase genes may vary considerably among strains of E. cloacae.

Molecular characterization of an Enterobacter cloacae outer membrane protein (OmpX).

A chromosomal gene of Enterobacter cloacae encoding an outer membrane protein (OmpX) has been cloned. Overproduction of the OmpX protein decreased the quantity of porins in the outer membrane of the parental strain and of Escherichia coli HB101. The ompX gene was located by insertions of the gamma delta sequence into the recombinant plasmid. The polarity of the gene was determined by in vitro transcription and translation of the gamma delta-containing plasmids. The nucleotide sequence of the ompX gene was elucidated by using both inverted terminal repeats of the gamma delta sequence as starting points for M13 dideoxy sequencing. The gene was found to encode a precursor of the OmpX protein consisting of 172 amino acid residues with a molecular mass of 18.6 kDa. The protein contains an N-terminal signal sequence of 23 amino acid residues. The exact cleavage point was established by sequencing the N-terminal part of the mature protein. The OmpX protein has several characteristics in common with outer membrane proteins of gram-negative bacteria. The protein is rather hydrophilic and is devoid of long hydrophobic stretches. On the basis of these results, we present a model for the OmpX protein folding in an outer membrane.