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Objectives: Little is known about the validity and reliability of expert assessments of the quality of antimicrobial prescribing, despite their importance in antimicrobial stewardship. We investigated how infectious disease doctors' assessments compared with a reference standard (modal expert opinion) and with the assessments of their colleagues., Methods: Twenty-four doctors specialized in infectious diseases or clinical microbiology (16 specialists and 8 residents) from five hospitals were asked to assess the appropriateness of antimicrobial agents prescribed for a broad spectrum of indications in 56 paper cases. They were instructed how to handle guideline applicability and deviations. We created a reference standard of antimicrobial appropriateness using the modal assessment of 16 specialists. We calculated criterion validity and interrater and intrarater overall and specific agreement with an index expert (senior infectious disease physician) and analysed the influence of doctor characteristics on validity., Results: Specialists agreed with the reference standard in 80% of cases (range 75%-86%), with a sensitivity and specificity of 75% and 84%, respectively. This did not differ by clinical specialty, hospital or years of experience, and residents had similar results. Specialists agreed with the index expert in 76% of cases and the index expert agreed with his previous assessments in 71% of cases., Conclusions: Doctors specialized in infectious diseases and clinical microbiology assess the appropriateness of antimicrobials prescribed for a broad spectrum of indications with acceptable agreement and validity, regardless of their experience or hospital of employment. However, there is room for improvement, which merits attention in multidisciplinary discussions and education., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The objectives of this study were to determine the prevalence of carriage of ESBL-producing Enterobacteriaceae (ESBL-E) in a representative sample of the general adult Dutch community, to identify risk factors and to gain understanding of the epidemiology of these resistant strains., Methods: Adults enrolled in five general practices in Amsterdam were approached by postal mail and asked to fill in a questionnaire and to collect a faecal sample. Samples were analysed for the presence of ESBL-E. ESBL genes were characterized by PCR and sequencing. Strains were typed using MLST and amplified fragment length polymorphism (AFLP) and plasmids were identified by PCR-based replicon typing. Risk factors for carriage were investigated by multivariate analysis., Results: ESBL-E were found in 145/1695 (8.6%) samples; 91% were Escherichia coli. Most ESBL genes were of the CTX-M group (blaCTX-M-1 and blaCTX-M-15). MLST ST131 was predominant and mainly associated with CTX-M-15-producing E. coli. One isolate with reduced susceptibility to ertapenem produced OXA-48. In multivariate analyses, use of antimicrobial agents, use of antacids and travel to Africa, Asia and Northern America were associated with carriage of ESBL-E, in particular strains with blaCTX-M-14/15., Conclusions: This study showed a high prevalence of ESBL-E carriage in the general Dutch community. Also, outside hospitals, the use of antibiotics was a risk factor; interestingly, use of antacids increased the risk of carriage. A major risk factor in the general population was travel to countries outside Europe, in particular to Asia, Africa and Northern America., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Background: The worldwide prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing rapidly both in hospitals and in the community. A connection between ESBL-producing bacteria in food animals, retail meat, and humans has been suggested. We previously reported on the genetic composition of a collection of ESBL-producing Escherichia coli (ESBL-EC) from chicken meat and humans from a restricted geographic area. Now, we have extended the analysis with plasmid replicons, virulence factors, and highly discriminatory genomic profiling methods., Methods: One hundred forty-five ESBL-EC isolates from retail chicken meat, human rectal carriers, and blood cultures were analyzed using multilocus sequence typing, phylotyping, ESBL genes, plasmid replicons, virulence genes, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE)., Results: Three source groups overlapped substantially when their genetic composition was compared. A combined analysis using all variables yielded the highest resolution (Wilks lambda [Λ]: 0.08). Still, a prediction model based on the combined data classified 40% of the human isolates as chicken meat isolates. AFLP and PFGE showed that the isolates from humans and chicken meat could not be segregated and identified 1 perfect match between humans and chicken meat., Conclusions: We found significant genetic similarities among ESBL-EC isolates from chicken meat and humans according to mobile resistance elements, virulence genes, and genomic backbone. Therefore, chicken meat is a likely contributor to the recent emergence of ESBL-EC in human infections in the study region. This raises serious food safety questions regarding the abundant presence of ESBL-EC in chicken meat.

The two major primary antibody deficiency disorders are X-linked hypogammaglobulinaemia (XLA) and common variable immunodeficiency (CVID). CVID patients have an elevated risk for gastric cancer and extra-nodal marginal zone lymphoma. Both diseases are associated with Helicobacter pylori infection. We investigated whether antibody deficiency leads to defective serum bactericidal activity against H. pylori. We also investigated the correlation with immunoglobulin (Ig)M levels and observed the terminal complement complex (TCC) activity. Sera of 13 CVID patients (four H. pylori positive), one patient with hyper-IgM syndrome, one patient with Good syndrome (both H. pylori positive), five XLA patients, four H. pylori seropositive controls, four H. pylori seronegative controls and a sample of pooled human serum (PHS) were incubated in vitro with bacterial suspensions of H. pylori for 30 min. After 72 h of culture, colony-forming units were counted. TCC formation was measured by enzyme-linked immunosorbent assay. We found that normal human serum is bactericidal for H. pylori, whereas heat-inactivated serum shows hardly any killing of H. pylori. Serum (1%) of hypogammaglobulinaemia patients has a decreased bactericidal activity against H. pylori. Helicobacter pylori-positive (HP(+)) normal individuals show more than 90% killing of H. pylori, whereas CVID patients show 35% killing (P = 0.007) and XLA patients only 19% (P = 0.003). Serum (1%) of HP(+) volunteers showed significantly better killing compared with serum of H. pylori-negative (HP(-)) volunteers (P = 0.034). No correlation between (substituted) IgG levels and serum bactericidal activity was found, but a weak correlation between total serum IgM and serum bactericidal activity was found. In conclusion, serum bactericidal activity against H. pylori is decreased in patients with hypogammaglobulinaemia. Heat treatment of the serum abolished the bactericidal capacity, indicating that complement activity is essential for the bactericidal effect.

Background: Antibiotic resistance among microbes urgently necessitates the development of novel antimicrobial agents. Since ancient times, honey has been used successfully for treatment of infected wounds, because of its antibacterial activity. However, large variations in the in vitro antibacterial activity of various honeys have been reported and hamper its acceptance in modern medicine., Methods: We assessed the in vitro bactericidal activity of Revamil (Bfactory), a medical-grade honey produced under controlled conditions, and assessed its efficacy for reduction of forearm skin colonization in healthy volunteers in a within-subject-controlled trial., Results: With Bacillus subtilis as a test strain, we demonstrated that the variation in bactericidal activity of 11 batches of medical-grade honey was <2-fold. Antibiotic-susceptible and -resistant isolates of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella oxytoca were killed within 24 h by 10%-40% (vol/vol) honey. After 2 days of application of honey, the extent of forearm skin colonization in healthy volunteers was reduced 100-fold (P < .001), and the numbers of positive skin cultures were reduced by 76% (P < .001)., Conclusions: Revamil is a promising topical antimicrobial agent for prevention or treatment of infections, including those caused by multidrug-resistant bacteria.

In several bacterial species that show natural transformation, dprA has been described as a competence gene. The DprA protein has been suggested to be involved in the protection of incoming DNA. However, members of the dprA gene family (also called smf) can be detected in virtually all bacterial species, which suggests that their gene products have a more general function. We examined the function of the DprA/Smf homologue of Escherichia coli. Escherichia coli dprA/smf is able to partially restore transformation in a Haemophilus influenzae dprA mutant, which shows that dprA/smf genes from competent and noncompetent species are interchangeable with respect to their involvement in natural transformation. From this, we conclude that natural transformation is probably an additional function of these genes. Subsequently, the dprA/smf gene was deleted in various recombination mutants of E. coli, and the resultant phenotype was tested. All the resultant E. coli dprA/smf mutants did not differ from their parent strains with respect to transformation, Hfr-conjugation, recombination and DNA repair. Therefore, a role of DprA/Smf in DNA recombination could not be established and the basic function of dprA/smf remains unclear.

Objectives: During treatment with selective decontamination of the digestive tract (SDD), four multidrug-resistant (MDR) strains, three different Escherichia coli and one Klebsiella pneumoniae, were isolated from four patients not known as carriers of such MDR strains before their admission to the intensive care unit (ICU) in the Academic Medical Center (AMC) in Amsterdam. These isolates were extended-spectrum beta-lactamase (ESBL)-positive. We investigated whether this was due to interspecies transfer of resistance genes., Methods: The MDR strains were typed by amplified fragment length polymorphism (AFLP) analysis. The plasmids from these strains were characterized by restriction fragment length polymorphism and the resistance genes were characterized by PCR and sequence analysis., Results: The strains were genetically unrelated and contained identical plasmids with ESBL genes., Conclusions: We identified an outbreak of plasmid-mediated ESBL genes during SDD treatment in the ICU. The use of third-generation cephalosporins in SDD is associated with the emergence of ESBLs. We conclude that identification of emerging MDR Gram-negative bacteria and recognition of resistance plasmid transfer during SDD treatment are crucial for optimal application of this regimen in ICUs.

Objective: To estimate the effect of multicentre surveillance for nosocomial infections on patients' risk of surgical site infection (SSI)., Design: Prospective multi-centre cohort study, from January 1996 to December 2000., Setting: Acute care hospitals in The Netherlands., Study Participants: All 50 hospitals performing surveillance for one of seven selected procedures in the Dutch surveillance network for nosocomial infections PREZIES were invited. Thirty-seven hospitals participated (74%) and provided information on 21 920 operations, after which 885 (4%) SSI occurred., Interventions: The surveillance comprised the following: Development of surveillance methodology by multidisciplinary team; use of a standardized registration protocol and software; regular training of data collectors; anonymous inter-hospital comparison of infection rates and feedback of results; appointment of one contact person per hospital, responsible for data collection; and dissemination of results to other health care professionals. Regular discussion of both successful and failing prevention strategies that had been instituted based on the surveillance results., Outcome Measure: Risk of SSI., Results: The risk of infection was reduced for patients who had an operation during the fourth surveillance year (RR = 0.69; 95% confidence interval (CI) = 0.52-0.89) and decreased further for patients operated on during the fifth surveillance year (RR = 0.43; CI = 0.24-0.76) as compared with patients who underwent surgery within one year of the start of surveillance in their hospital. No significant risk reduction was observed for patients operated on during the second and third surveillance years., Conclusion: Surveillance, supported by participation in a surveillance network, reduced the risk of SSI in surgical patients registered in the Dutch surveillance network PREZIES. Our results suggest that infection control teams need to be perseverant and that surveillance programmes should be given time before evaluation.

We screened 14,008 adult nonsurgical patients for Staphylococcus aureus nasal carriage at hospital admission and assessed them for invasive S. aureus disease and in-hospital mortality. Multilocus sequence typing was performed on endogenous invasive strains and nasal strains of matched asymptomatic carriers to investigate whether virulent clones could be identified in nasal carriers. Clonal complex (CC) 45 was significantly underrepresented (odds ratio [OR], 0.16 [95% confidence interval {CI}, 0.04-0.59]) and CC30 was overrepresented (not statistically significant) among invasive strains (OR, 1.91 [95% CI, 0.91-4.0]). The distribution of CCs of invasive S. aureus strains in noncarriers did not differ from that in carriers. Those infected with S. aureus strains belonging to a CC had higher mortality than those infected with strains not belonging to a CC (P<.05), which indicates the coevolution of S. aureus virulence and spread in humans.

Background: Recently, it has been suggested that Chlamydia pneumoniae possibly plays a possible role in the pathogenesis of atherosclerosis. We investigated whether treatment with clarithromycin prior to coronary artery bypass graft (CABG) surgery would prevent subsequent cardiovascular events and mortality., Methods: Patients who were scheduled for CABG surgery were randomly assigned to receive either clarithromycin or placebo until the day of surgery in a double-blind trial. During the 2 years of follow-up, mortality and cardiovascular events were assessed., Results: Follow-up at 2 years was achieved for 473 patients. The mean duration of treatment was 16 days. Patient characteristics at baseline were well balanced between the 2 treatment groups. Mortality was equal in the 2 groups: 10 (4.2%) of 238 patients in the clarithromycin group and 9 (3.8%) of 235 patients in the placebo group (relative risk, 1.10; 95% CI, 0.42-2.89; P=1.0). Also, there were no significant differences in the proportion of patients who experienced cardiovascular events during the follow-up period: 20 (8.4%) of 238 patients in the clarithromycin group and 19 (8.1%) of 235 patients in the placebo group (relative risk, 1.04; 95% CI, 0.55-1.98; P=1.0). The overall rate of such events was 58 (12.3%) of 473 patients., Conclusions: Treatment with clarithromycin in patients scheduled for CABG surgery did not reduce the subsequent occurrence of cardiovascular events or mortality during a 2-year follow-up period.

In Ecuador, Helicobacter pylori infections are highly prevalent. A total of 42 H. pylori clinical isolates from 86 patients attending the outpatient clinic of the gastroenterology department of the university hospital of Guayaquil in Ecuador were characterized. Their susceptibility, and cagA and vacA status were determined. Resistance to metronidazole and clarithromycin was found in 80.9% and 9.5% of strains, respectively. Neither amoxicillin- nor tetracycline-resistant strains were found. The most prevalent genotype was the cagA(+), vacA s1b,m1 type. This genotype was associated with gastric cancer and peptic ulcer. Typing by random amplified polymorphic DNA showed no genetic relationship among the strains.

Helicobacter pylori infection results in chronic gastritis, which is initiated by the release of cytokines like interleukin (IL)-12 and IL-8 from mononuclear cells, and IL-8 from gastric epithelial cells. The severity of gastritis is influenced both by host factors and by bacterial factors such as the Cag proteins and the vacuolating cytotoxin VacA. Amounts of IL-12 and IL-8 produced by monocytic THP-1 cells differed considerably between the eight H. pylori isolates tested, but in contrast to H. pylori-induced IL-8 production by gastric epithelial cells, did not correlate to the Cag and VacA types of the strains. Apparently, in addition to Cag and VacA, other bacterial factors determine the extent in which H. pylori induced IL production in monocytes.

To persist in the hostile acidic environment of the stomach, Helicobacter pylori must survive acid shock and grow at acidic pH. Of a library of 1250 random mutants screened for isolates unable to grow at low pH, 10 mutants were detected that were unable to grow at pH 4.8. However, all 10 mutants were resistant to acid shock. Four mutants had an insertion in genes of unknown function. One mutant was affected in lepA, an orthologue of a membrane GTPase. Three mutants were disrupted in loci involved in the transport of H(+) ions or other cations (FRaseI, czcA, and aldo-keto reductase). Two mutants were affected in loci that contribute to acid resistance in other microorganisms (uvrA and atpF'). Thus, at least 10 loci not related to urease are essential for the growth of H. pylori under acidic conditions and should be critical for lifelong infection by this pathogen.

Successful treatment of Helicobacter pylori infection is becoming compromised by emerging resistance. We report the prevalence rates of H. pylori resistance to metronidazole, clarithromycin, amoxycillin, tetracycline and trovafloxacin in The Netherlands. A total of 231 H. pylori clinical isolates were collected throughout the country over a period of 6 months during 1997-1998. The MICs of the above-mentioned antibiotics were determined in a single laboratory. The overall percentage of resistance for clarithromycin and metronidazole was 1.7% and 21.2%, respectively. None of the strains was resistant to amoxycillin or tetracycline. The primary resistance rate of trovafloxacin was as high as 4.7%. Since trovafloxacin has not yet been introduced on to the Dutch market, the resistance is probably induced by the use of other quinolones. Our data indicate that treatment outcome would benefit from susceptibility testing before starting therapy, especially when prescribing metronidazole.

To investigate the early events of Helicobacter pylori infection in a mouse model, CD1 mice were infected with a type I (CagA+/VacA+) H. pylori strain. Up to 4 weeks after infection the majority of gastric tissue biopsies were positive in culture. Immunohistochemical analysis showed that inflammatory changes started to occur after 3 weeks. Four weeks after infection a significant increase in T cells was observed in the cardia/corpus region of the stomachs of infected mice. These T cells were CD4+ and CD8+, and they were located in an area with increased expression of MHC class II antigens. In 50% of the infected mice also an increased number of mast cells was seen. Furthermore, aggregates of B and T cells were present in the submucosa. Characterization of cytokines by immunohistochemistry showed an increase in IL-5-secreting cells in the inflamed area of the infected stomach. No difference was observed between interferon-gamma (IFN-gamma)-, IL-4- and IL-10-secreting cells in control and infected mice. These results suggest that no polarized T-helper cell response was present at this early phase of infection. Infection with H. pylori also induced a serum response and especially IgG was increased after 4 weeks of infection. However, no particular increase in IgG1, IgG2a or IgG3 isotype was observed. Part of the serum antibodies was directed against lipopolysaccharide (LPS), but no evidence for anti-Lewis antibodies or antibodies against epitopes on the gastric mucosa was found.

Acid resistance is considered an important virulence factor of the human pathogen Helicobacter pylori. The enzyme urease plays an important role in this acid resistance, but there are indications that other systems are present. We set out to establish the relevance of these urease-independent acid-resistance systems for growth at low pH. Four mutants out of a total of 1000 UV-mutants were urease positive, grew identical to wild-type on pH 7 plates, but did not grow on pH 5 plates. Whereas transformation of a mutant with its own chromosomal DNA did not restore growth at pH 5, transformation with wild-type DNA or DNA of one of the other mutants did restore the growth. From these complementation studies, we conclude that in H. pylori a urease-independent acid-resistance system, probably depending on the expression of more than one gene, is involved in the growth at low pH.

The presence of sequences related to the agr of Staphylococcus aureus was demonstrated in Staphylococcus epidermidis by agr-specific PCR, and Southern blot. The agr-like locus of S. epidermidis A086 was cloned and sequenced. An overall homology of 68% was found between the agr locus from S. epidermidis and S. aureus. The agr locus from S. epidermidis was organized similar to those from S. aureus and S. lugdunensis. The putative RNAII molecule contains four open reading frames, agr A, B, C and D. AgrA was a response regulator. AgrB showed homology with transducer and translocase molecules. AgrC is expected to act as a histidine protein kinase in which a leucine zipper is present. AgrD is presumably processed into an autoinducer peptide. The putative RNAIII molecule contained an open reading frame encoding a putative 26 amino acid (aa) polypeptide, which differed in 3 aa from the RNAIII encoded delta-toxin of S. aureus. Kinetic studies showed that the production of this RNAIII was elevated during the post-exponential phase. delta-Toxin activity was demonstrated for 21 of 23 tested S. epidermidis strains. Kinetic studies of the production of delta-toxin showed that the toxin was produced during the post-exponential phase. Sequencing of S. epidermidis A097, which showed a delayed agr-response, revealed a truncated AgrC lacking the histidine kinase domain. These data indicate that an agr-like locus is active in S. epidermidis during the post-exponential phase.

Seventy-three Helicobacter pylori-positive patients were treated with a combination of clarithromycin and ranitidine in order to eradicate the bacterium. Eradication was successful in 79.5%. In 15 patients eradication failed, and in 11 cases this was due to clarithromycin resistance. In one patient the infecting strain was resistant at the onset of treatment, while in the remaining 10 patients resistance developed during therapy. These isolates had also become resistant to various other antibiotics. Random amplified polymorphic DNA and restriction fragment end-labeling analysis of the isolates showed close genetic relatedness between pre- and post-treatment isolates, indicating that resistance was the result of selection of variants of the infecting strain rather then infection with an exogenous resistant strain. Nucleotide sequence comparisons revealed that all resistant isolates had a single base pair mutation in the 23S rRNA. Since this single point mutation results in co-resistance to various antibiotics at high frequencies, caution should be taken when using clarithromycin as a single antibiotic.

To evaluate the importance of nasal carriage of Staphylococcus aureus as a risk factor for the development of wound infection at the sternotomy site after cardiac surgery, a case-control study was done. The study population consisted of 1980 consecutive patients. Cases were all patients who developed a sternal wound infection from which S. aureus was cultured. Forty cases were identified, and 120 controls were selected. Preoperative nasal carriage of S. aureus, insulin-dependent diabetes mellitus, and younger age were identified as significant risk factors. The crude odds ratio of nasal carriage was 9.6 (95% confidence interval, 3.9-23.7). The median postoperative length of hospital stay for cases was 30 days longer than for controls. Mortality was also significantly higher for cases than for controls (10.0% and 0.8%, respectively).