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Vibrio vulnificus is a potentially zoonotic bacterial pathogen of fish, which can infect humans (causing necrotic fasciitis). We analysed 24 V. vulnificus isolates (from 23 severe eel disease outbreaks in 8 Dutch eel farms during 1996 to 2009, and 1 clinical strain from an eel farmer) for genetic correlation and zoonotic potential. Strains were typed using biotyping and molecular typing by high-throughput multilocus sequence typing (hiMLST) and REP-PCR (Diversilab®). We identified 19 strains of biotype 1 and 5 of biotype 2 (4 from eels, 1 from the eel farmer), that were subdivided into 8 MLST types (ST) according to the international standard method. This is the first report of V. vulnificus biotype 1 outbreaks in Dutch eel farms. Seven of the 8 STs, of unknown zoonotic potential, were newly identified and were deposited in the MLST database. The REP-PCR and the MLST were highly concordant, indicating that the REP-PCR is a useful alternative for MLST. The strains isolated from the farmer and his eels were ST 112, a known potential zoonotic strain. Antimicrobial resistance to cefoxitin was found in most of the V. vulnificus strains, and an increasing resistance to quinolones, trimethoprim + sulphonamide and tetracycline was found over time in strain ST 140. Virulence testing of isolates from diseased eels is recommended, and medical practitioners should be informed about the potential risk of zoonotic infections by V. vulnificus from eels for the prevention of infection especially among high-risk individuals. Additional use of molecular typing methods such as hiMLST and Diversilab® is recommended for epidemiological purposes during V. vulnificus outbreaks.

The purpose of this investigation was to determine the prevalence of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) and Panton-Valentine leucocidin (PVL)-positive S. aureus in general practice (GP) patients with skin and soft tissue infections (SSTI) in the northern (Groningen and Drenthe) and southern (Limburg) regions of The Netherlands. Secondary objectives were to assess the possible risk factors for patients with SSTI caused by S. aureus and PVL-positive S. aureus using a questionnaire-based survey. From 2007 to 2008, wound and nose cultures were obtained from patients with SSTI in general practice. These swabs were analysed for the presence of S. aureus and the antibiotic susceptibility was determined. The presence of the PVL toxin gene was determined by polymerase chain reaction (PCR) and the genetic background with the use of spa typing. A survey was performed to detect risk factors for S. aureus infection and for the presence of PVL toxin.S. aureus was isolated from 219 out of 314 (70%) patients with SSTI, of which two (0.9%) patients were MRSA-positive. In 25 (11%) patients, the PVL toxin gene was found. A higher prevalence of PVL-positive S. aureus of patients with SSTI was found in the northern region compared to the south (p < 0.05). Regional differences were found in the spa types of PVL-positive S. aureus isolates, and for PVL-negative S. aureus isolates, the genetic background was similar in both regions. The prevalence of CA-MRSA in GP patients with SSTI in The Netherlands is low. Regional differences were found in the prevalence of PVL-positive S. aureus isolates from GP patients with SSTI. Household contacts having similar symptoms were found to be a risk factor for SSTI with S. aureus.

Gemella haemolysans, Streptococcus equinus and Tropheryma whipplei are rare Gram-positive bacteria which may cause endocarditis and are associated with gastrointestinal disorders. We report on three patients with infective endocarditis caused by these microorganisms. G. haemolysans and S. equinus (Streptococcus bovis group) were isolated from blood cultures, and T. whipplei was diagnosed by molecular typing of an excised heart valve. The association between endocarditis caused by these microorganisms and gastrointestinal disorders warranted further examination. Endoscopic examination revealed a colonic carcinoma in the patient with G. haemolysans endocarditis and diverticulosis in the patient with S. equinus endocarditis. No gastrointestinal tract disorders were found in the patient with T. whipplei endocarditis, but this does not exclude Whipple's disease. Examination of the gastrointestinal tract for a focus of infection should be considered in patients with endocarditis caused by G. haemolysans, S. equinus and T. whipplei if no other source of bacteraemia is apparent.

Objective: To determine how many family members of methicillin resistant Staphylococcus aureus (MRSA) patients were colonised with MRSA and how this colonisation developed over time., Design: Descriptive, prospective., Method: Two laboratories notified the Public Health Services of newly-diagnosed MRSA patients in three provinces of the Netherlands. These persons and their family members were screened for MRSA colonisation at baseline, after 3 to 4 months and after 6 to 12 months. No advice on medical intervention was given. Relevant medical interventions by general practitioners were registered., Results: Nineteen index patients and their families were included. A total of 41% of the family members (n = 44) proved MRSA positive on at least one of the three tests. At second follow-up the proportion of colonised family members had decreased slightly from 32% to 27%; by the end of the study only a third of the index patients were still MRSA positive. Colonisation of index patients was more persistent if family members were colonised as well., Conclusion: A large and changing number of family contacts of MRSA-positive patients were shown to be colonised over time, and sometimes in the longer term.

Vibrio vulnificus is a zoonotic bacterium that can be found in raw fish (mainly eel and oysters) and seawater. Human infections may extend from wound infections to fasciitis necroticans or primary sepsis with a mortality rate of more than 50%. Although V. vulnificus is mainly found in the USA, its worldwide spread is also likely to involve the Netherlands, as demonstrated by an increasing number of infected fish farms. Since 2007, V. vulnificus infections have become a notifiable infectious disease in the USA. Due to the high mortality rate and an increase in the number of elderly people with known risk factors for infection, we argue that human V. vulnificus infections should become a notifiable infectious disease in the Netherlands as well. This would provide reliable information on the epidemiology and facilitate correct risk assessment for public health.

Conventional diagnostic methods for the detection of Salmonella enterica and Campylobacter jejuni are laborious and time-consuming procedures, resulting in final results, for the majority of specimens, only after 3 to 4 days. Molecular detection can improve the time to reporting of the final results from several days to the next day. However, molecular assays for the detection of gastrointestinal pathogens directly from stool specimens have not made it into the routine clinical microbiology laboratory. In this study we have assessed the feasibility of a real-time PCR-based molecular screening method (MSM), aimed at S. enterica and C. jejuni, in the daily practice of a routine clinical microbiology laboratory. We have prospectively analyzed 2,067 stool specimens submitted for routine detection of gastrointestinal bacterial pathogens over a 7-month period. The MSM showed 98 to 100% sensitivity but routine culture showed only 77.8 to 86.8% sensitivity when an extended "gold standard" that included all culture-positive and all MSM-positive specimens, as confirmed by an independent secondary PCR of a different target gene, was used. An overall improvement in the rate of detection of both pathogens of 15 to 18% was observed. Both approaches performed nearly identically with regard to the specificity, positive predictive value, and negative predictive value, with the values for MSM being 99.7%, 93.1 to 96.6%, and 99.8 to 100%, respectively, and those for routine culture being 100%, 100%, and 97.6 to 99.5%, respectively. Finally, the final results were reported between 3 and 4 days earlier for negative specimens compared to the time of reporting of the results of routine culture. Positive specimens, on the other hand, required an additional 2 days to obtain a final result compared to the time required for routine culture, although preliminary MSM PCR-positive results were reported, on average, 2.9 to 3.8 days before the final routine culture results were reported. In conclusion, MSM can be incorporated into the daily practice of a routine clinical microbiology laboratory with ease. Furthermore, it provides an improvement in the screening for S. enterica and C. jejuni and substantially improves the time to the reporting of negative results.

Infections caused by community-acquired methicillin-resistant Staphylococcus aureus (MRSA) are emerging as a major public health problem. In this study, we describe the distribution of 54 Panton-Valentine leucocidin (PVL)-carrying MRSA isolates in the northern Netherlands between 1998 and 2005, of which 43 (80%) consisted of the European PVL-positive strain multi locus sequence type 80 with staphylococcal cassette chromosome mec type IVc (ST80). Individual cases and small clusters of ST80 predominated in the community (74%), but ST80 was also found in nursing homes (16%) and hospitals (9%). Long-term carriership (months to years) and reinfection of patients with ST80 has probably led to the strain spreading in the community and subsequently to further migration to health care environments.

Two 41-year-old women, who had previously been splenectomized, were admitted to the intensive-care unit due to fulminant sepsis. On admission, petechiae and ecchymoses characterised the clinical presentation of both patients. Laboratory tests revealed the presence of renal insufficiency and thrombocytopenia with disseminated intravascular coagulation. Streptococcus pneumoniae with serotypes (24 and 38) not included in the current polyvalent pneumococcal vaccine were found in blood cultures from both patients. One patient died as a result of a refractory septic shock. The other patient, who had never been vaccinated with the polyvalent pneumococcal vaccine, survived the sepsis. The clinical course of a fulminant Streptococcus pneumoniae sepsis in both asplenic patients underlines the importance of vaccination. It is also important to educate the patient about seeking immediate medical help if an infection is suspected on the basis of these symptoms. Vaccination does not provide complete protection.

The first documented Dutch case of an ovine Chlamydia psittaci infection concerned a 20-year-old woman in the 26th week of pregnancy, following contact with lambing sheep. She had a severe sepsis and had to be artificially ventilated. Finally, the patient made a full recovery. The preterm born child died directly after birth. The placenta showed an acute intervillitis in which chlamydial antigen was demonstrated immunohistologically. Infection with an ovine C. psittaci was confirmed by sequence analysis of amplified chlamydial DNA from the placenta. Infections with C. psittaci are typically associated with contact with (sick) birds. However, mammals also may act as a source of human infection, especially sheep in which C. psittaci is an important cause of abortion. Infections with ovine C. psittaci are a particular hazard for pregnant women, in whom there is severe placentitis and frequently foetal loss. Such infections are mainly associated with contact with lambing sheep.

Objective: To study the cefpirome pharmacokinetics of patients with sepsis and multiple organ failure treated with CVVH., Design: Measurements of serum and ultrafiltrate (UF) concentrations and in vitro sensitivity testing of isolated micro-organisms., Setting: University hospital-based, single ICU., Patients: Six critically ill CVVH-dependent patients with sepsis and multiple organ dysfunction syndrome in need of antimicrobial therapy. Age range: 60-75 years; APACHE II score for severity of illness on admission: 19-30. One patient survived., Interventions: Cefpirome i.v. was started at 2 g in 30 min, then continued 1 g i.v.b.i.d., Measurements: The UF rate was 27 +/- 7 ml/min on day 1 and 34 +/- 2 ml/min on day 2. Serum and ultrafiltrate samples were measured by a validated high performance liquid chromatography assay. Volume of distribution: 23 x 5(SD +/- 4 x 6) l. Total cefpirome clearance was 32 +/- 6 x 3 ml/min; cefpirome CVVH clearance (ClCVVH): 17 +/- 4.2 ml/min; mean serum half-life (t1/2): 8.8 +/- 2.3 h; mass transfer on day 1: 660 +/- 123 mg/12 h (33 +/- 6% of administered dose) and day 2: 642 +/- 66 mg/12 h (64 +/- 7%). Estimated sieving coefficient (ClCVVH/UF rate): 64 +/- 11%. In vitro sensitivity of isolated microbes was excellent except for two non-sensitive enterococci and Candida spp., Conclusions: The sieving coefficient (64%) indicates that a substantial fraction of the drug is not filtered; clearance by pathways other than CVVH mounted to 50% of the total clearance and increased on day 2, indicating that the dosing schedule used is appropriate for this setting. Cefpirome appeared to be safe in these patients and effective for most of the nosocomial microbial isolates. During more than 90% of the time, serum levels were maintained above killing concentrations for susceptible micro-organisms.

Two males aged 41 and 32 years developed pneumonia which responded inadequately to antibiotic treatment and necessitated mechanical ventilation. It was only after surgical and digital opening, drainage of pus pockets and daily pleural lavage that the clinical picture improved. The microorganisms cultured from both patients included Streptococcus milleri, probably acquired by aspiration. Thoracic empyema as a complication of pneumonia is clinically recognised by lack of response to antimicrobial agents. For the diagnosis, ultrasonographic and CT imaging, followed by pleural puncture are used. Simple parapneumonic effusions are managed by drainage with or without rinsing with normal saline, while in advanced empyema, instillations with fibrinolytic agents have proved safe and effective. Sometimes, video-assisted thoracoscopic or conventional surgery is necessary to clear the pleural space, while in complicated cases, extensive surgical procedures are warranted.