Human carriage of ESBL/pAmpC-producing Escherichia coli and Klebsiella pneumoniae in relation to the consumption of raw or undercooked vegetables, fruits, and fresh herbs.

We investigated to what extent the consumption of raw or undercooked vegetables, fruits, and fresh herbs influences carriage rates of ESBL/pAmpC-producing Escherichia coli and Klebsiella pneumoniae (ESBL-E/K) in the general population. We assessed long-term carriage and changes in ESBL-E/K prevalence over time, by comparing the results to findings in the same population 5 years earlier. Between July and December 2021, participants sent in two fecal samples and questionnaires, 3 months apart. Food frequency questionnaires were sent on a monthly basis. Fecal samples were cultured and screened for ESBL-E/K, and phenotypically positive isolates were sequenced. Multivariable logistic regression models were established to assess the association between the consumption of fresh produce and ESBL-E/K carriage. The ESBL-E/K prevalence was 7.6% [41/537; 95% confidence interval (CI): 5.7-10.2] in the first sampling round and 7.0% (34/489; 95% CI: 5.0-9.6) in the second. Multivariable models did not result in statistical significance for any of the selected fruit and vegetable types. Trends for increased carriage rates were observed for the consumption of raspberry and blueberry in the summer period. ESBL-E/K prevalence was comparable with the prevalence in the same cohort 5 years earlier (7.5%; 95% CI: 5.6-10.1%). In six persons (1.2%) a genetically highly homologous ESBL-E/K was found. In conclusion, the contribution of the consumption of raw fruits, vegetables, and herbs to ESBL-E/K carriage in humans in the Netherlands is probably low. Despite COVID-19 containment measures (e.g., travel restrictions, social distancing, and hygiene) the ESBL-E/K prevalence was similar to 5 years earlier. Furthermore, indications for long-term carriage were found.IMPORTANCEESBL-producing bacteria are resistant against important classes of antibiotics, including penicillins and cephalosporines, which complicates treatment of infections. Food is one of the main routes of transmission for carriage of these bacteria in the general population. Although fruits, vegetables, and herbs are generally less frequently contaminated with ESBL-producing bacteria compared to meat, exposure might be higher since these products are often eaten raw or undercooked. This research showed that the contribution of the consumption of raw or undercooked fresh produce to ESBL-E/K carriage in the general Dutch population was low. No specific types of fruit or vegetables could be identified that gave a higher risk of carriage. In addition, we demonstrated the presence of genetically highly homologous ESBL-E/K in six persons after a period of 5 years, indicative for long-term carriage.

Colistin-resistant Enterobacterales among veterinary healthcare workers and in the Dutch population.

Objectives: Plasmid-mediated colistin resistance can be transferred from animals to humans. We investigated the prevalence of carriage of mcr-mediated colistin-resistant Escherichia coli and Klebsiella pneumoniae (ColR-E/K) in veterinary healthcare workers and in the general population in the Netherlands. Methods: Two cross-sectional population studies were performed: one among veterinary healthcare workers and one in the general population. Participants sent in a faecal sample and filled in a questionnaire. Samples were analysed using selective enrichment and culture. Mobile colistin resistance genes (mcr) were detected by PCR and ColR-E/K were sequenced using Illumina and Nanopore technologies. Results: The prevalence of mcr-mediated ColR-E/K was 0.2% (1/482, 95% CI 0.04%-1.17%) among veterinary personnel and 0.8% (5/660, 95% CI 0.3%-1.8%) in the population sample. mcr-1 was found in E. coli from four persons, mcr-8 in K. pneumoniae from one person and another person carried both mcr-1 and mcr-8 in a K. pneumoniae isolate. mcr-1 was found on different plasmid types (IncX4, IncI1 and IncI2), while mcr-8 was found on IncF plasmids only. Conclusions: mcr-mediated ColR-E/K resistance was uncommon in both populations. Professional contact with animals does not increase the chance of carriage of these bacteria in the Netherlands at present. mcr-8 was found for the first time in the Netherlands. Surveillance of colistin resistance and its underlying mechanisms in humans, livestock and food is important in order to identify emerging trends in time.

Prevalence and risk factors for colonization of Clostridium difficile among adults living near livestock farms in the Netherlands.

A cross-sectional study was performed among 2494 adults not living or working on a farm to assess prevalence of Clostridium difficile (CD) colonization and risk factors in a livestock dense area. CD prevalence was 1·2%. Twenty-one persons were colonized with a toxigenic strain and nine with a non-toxigenic strain. CD-positive persons did not live closer to livestock farms than individuals negative for CD. Antibiotic exposure in the preceding 3 months was a risk factor for CD colonization (odds ratio 3·70; 95% confidence interval 1·25-10·95).

Extended-spectrum ß-lactamase- and pAmpC-producing Enterobacteriaceae among the general population in a livestock-dense area.

OBJECTIVES: In the Netherlands there is an ongoing debate regarding environmental health risks of livestock farming for neighbouring residents. This explorative study aims to determine the prevalence of carriage of extended-spectrum ß-lactamase and/or plasmid-mediated AmpC-producing Enterobacteriaceae (ESBL/pAmpC-E) in the general population living in a livestock-dense area, and to study associations between determinants, including exposure through contact with animals and the environment, and human carriage of ESBL/pAmpC-E. METHODS: A cross-sectional study was performed among 2432 adults (aged 20-72 years) in 12 temporary research centres in the south of the Netherlands, consisting of a questionnaire and analysis of a faecal sample to assess carriage of ESBL/pAmpC-E. Risk factors were analysed using logistic regression. RESULTS: The prevalence for carriage of ESBL/pAmpC-E was 4.5% (109/2432; 95% CI 3.7-5.4) ranging from 1.4% to 10.9% among the research centres. ESBL/pAmpC resistance genes were detected in Escherichia coli and Klebsiella pneumoniae isolates obtained from these 109 persons and the most common ESBL-resistance genes were blaCTX-M-15, blaCTX-M-14/17 and blaCTX-M-1, originating from 76 participants. Travel in the previous 12 months to Africa, Asia or Latin America (OR 2.82; 95% CI 1.71-4.63), having kept cows for a hobby in the previous 5 years (OR 3.77; 95% CI 1.22-11.64), usage of proton-pump inhibitors (OR 1.84; 95% CI 1.05-3.23), and living within 1000 m of a mink farm (OR 2.26; 95% CI 1.28-3.98) were identified as risk factors. Exposure to poultry was not identified as a risk factor. CONCLUSIONS: Overall, living in close proximity to livestock animals and farms does not seem to be a risk factor for carriage of ESBL/pAmpC-E.

Transmission of MRSA between humans and animals on duck and turkey farms.

OBJECTIVES: The objectives of this study were to estimate the prevalence of MRSA on duck and turkey farms, identify risk factors for human carriage and study transmission between animals and humans. METHODS: On 10 duck and 10 turkey farms, samples were taken from animals, poultry houses, home residences and humans and cultured using pre-enrichment and selective enrichment. MRSA isolates were typed by spa typing and multiple-locus variable number tandem repeat analysis (MLVA) typing. A subset of isolates from animals and humans was investigated using whole-genome mapping. RESULTS: MRSA was found on one duck farm and three turkey farms. On duck farms, all humans were MRSA negative. On turkey farms, 5 of 11 farmers, 2 of 32 family members and 15 of 49 samples from the home residences were MRSA positive. Individuals with daily contact with turkeys were significantly more often MRSA positive than individuals without daily contact. All MRSA isolates belonged to livestock-associated MLVA complex 398, belonged to spa type t011, were negative for Panton-Valentine leucocidin, were mecC negative and were mecA positive. Whole-genome mapping proved a valuable tool to study the transmission of livestock-associated MRSA and showed that on two turkey farms the isolates from the animals and humans were indistinguishable or closely related, indicating transmission. CONCLUSIONS: MRSA carriage in individuals in daily contact with turkeys was significantly higher than that in individuals only living on the farms or than in the general Dutch population. Therefore, persons with a high degree of contact with turkeys have an increased risk of MRSA carriage, and we propose that they should be screened prior to hospitalization in order to decrease the risk of nosocomial transmission.

B-cell activating factor in the pathophysiology of multiple myeloma: a target for therapy?

Multiple myeloma (MM) is a currently incurable malignancy of plasma cells. Malignant myeloma cells (MMCs) are heavily dependent upon the bone marrow (BM) microenvironment for their survival. One component of this tumor microenvironment, B-Cell Activating Factor (BAFF), has been implicated as a key player in this interaction. This review discusses the role of BAFF in the pathophysiology of MM, and the potential of BAFF-inhibitory therapy for the treatment of MM. Multiple studies have shown that BAFF functions as a survival factor for MMCs. Furthermore, MMCs express several BAFF-binding receptors. Of these, only Transmembrane Activator and CAML Interactor (TACI) correlates with the MMC's capability to ligate BAFF. Additionally, the level of expression of TACI correlates with the level of the MMC's BM dependency. Ligation of BAFF receptors on MMCs causes activation of the Nuclear Factor of κ-B (NF-κB) pathway, a crucial pathway for the pathogenesis of many B-cell malignancies. Serum BAFF levels are significantly elevated in MM patients when compared to healthy controls, and correlate inversely with overall survival. BAFF signaling is thus an interesting target for the treatment of MM. Several BAFF-inhibitory drugs are currently under evaluation for the treatment of MM. These include BAFF-monoclonal antibodies (tabalumab) and antibody-drug conjugates (GSK2857916).

The Presence of Borrelia miyamotoi, A Relapsing Fever Spirochaete, in Questing Ixodes ricinus in Belgium and in The Netherlands.

Borrelia miyamotoi is a tick-borne bacterium that may cause relapsing fever in humans. As this pathogen has been discovered in Europe only recently, only little is known about its local impact on human health and its spatial distribution. In this study, we show the results of PCR screenings for B. miyamotoi in flagged Ixodes ricinus from Belgium and the Netherlands. B. miyamotoi was detected in nine of thirteen, and three of five locations from the Netherlands and Belgium, respectively. These outcomes indicate that B. miyamotoi is more spread than previously thought. The mean infection rate B. miyamotoi was 1.14% for Belgium and 3.84% for the Netherlands.

Prevalence of methicillin-resistant Staphylococcus aureus carrying mecA or mecC in dairy cattle.

In the Netherlands, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been found in pigs, veal calves, horses and poultry. However, little is known about its prevalence in healthy dairy cattle. Recently, a new mec gene, called mecC, has been found in methicillin-resistant S. aureus (MRSA) isolates from humans and animals in several countries. The objective of our pilot study was to investigate the prevalence of MRSA (mecA and mecC) in dairy cows at a large slaughterhouse. Samples from the skin between the udder and hind leg were taken from 411 cows. The samples were incubated in Mueller-Hinton enrichment broth with 6.5% NaCl, followed by selective enrichment and plated onto Columbia agar with 5% sheep blood, Brilliance MRSA 2 agar and Baird-Parker agar. Suspected colonies were tested by PCR for a S. aureus specific DNA fragment, the mecA and mecC genes and the Panton-Valentine leucotoxin (PVL) genes. All MRSA isolates and methicillin-susceptible S. aureus (MSSA) isolates were typed by spa typing and MLVA typing. Sixteen of 411 (3.9%) cows, all originating from different farms, were found to be MRSA positive and this prevalence is lower than in Dutch pigs, veal calves and broilers. All MRSA isolates belonging to livestock-associated MLVA complex 398, were PVL-negative and spa type t011 predominated. MSSA isolates (n=39) were of many different MLVA types and spa type t543 was found most often. Four MSSA isolates belonging to MLVA clonal complex 398 and spa types t011 (n=2), t108 and t034 were isolated from different MRSA-negative animals. In conclusion, the prevalence of MRSA in dairy cows was low and isolates carrying the mecC gene were not found, indicating that it is absent or has a low prevalence (<0.73%) in Dutch dairy cows.

Prevalence of livestock-associated MRSA on Dutch broiler farms and in people living and/or working on these farms.

This study aimed to determine the prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) on 50 Dutch broiler farms. Of 145 persons living and/or working on these farms, eight tested positive for MRSA (5.5%). Investigation of 250 pooled throat samples of broilers and 755 dust samples resulted in four farms where MRSA-positive samples were present (8.0%). All isolates belonged to the CC398 complex. Living and/or working on a MRSA-positive farm was a risk for MRSA carriage; 66.7% of people on positive farms were MRSA positive vs. 1.5% on negative farms (P<0.0001). Due to the low number of positive farms and persons, and high similarity in farm management, it was impossible to draw statistically valid conclusions on other risk factors. For broiler farming, both farm and human MRSA prevalence seem much lower than for pig or veal farming. However, MRSA carriage in people living and/or working on broiler farms is higher compared to the general human population in The Netherlands (5.5% vs. <0.1%). As broiler husbandry systems are not unique to The Netherlands, this might imply that people in contact with live broilers are at risk for MRSA carriage worldwide.

Prevalence of livestock-associated MRSA in broiler flocks and risk factors for slaughterhouse personnel in The Netherlands.

To determine methicillin-resistant Staphylococcus aureus (MRSA) carriage in poultry and slaughterhouse personnel, 40 Dutch broiler flocks, in six slaughterhouses and 466 personnel were sampled. Of the employees, 26 were positive (5.6%), indicating a higher risk of exposure when compared to the general Dutch population (0.1%). This risk was significantly higher for personnel having contact with live animals (5.2%) - especially hanging broilers on the slaughterline (20.0%) - than for all other personnel (1.9%). Conventional electric stunning conferred a significantly higher risk of MRSA carriage for employees than CO2 stunning (9.7% vs. 2.0%). A total of 405 broilers were sampled upon their arrival at the slaughterhouse, of which 6.9% were positive. These broilers originated from 40 Dutch slaughter flocks of which 35.0% were positive. MRSA contamination in the different compartments of slaughterhouses increased during the production day, from 8% to 35%. Of the 119 MRSA isolates, predominantly livestock-associated MRSA ST398 was found, although 27.7% belonged to ST9 (spa type t1430). There is an increased risk of MRSA carriage in personnel working at broiler slaughterhouses, particularly those having contact with live animals.

Occurrence of methicillin-resistant Staphylococcus aureus in rats living on pig farms.

In The Netherlands, MRSA ST398 has emerged in hospitals and human carriers have been associated with exposure to pigs and cattle. High prevalences of MRSA ST398 in pigs and pig farmers have been determined and the transmission routes of MRSA on pig farms need to be elucidated. In the south of the Netherlands, in recent years, the black rat (Rattus rattus) has emerged as a prominent rodent on livestock farms. From March till May 2008, a survey on MRSA in rats living on livestock farms in the south of The Netherlands and the north of Belgium was conducted. In total, 40 black rats (R. rattus) and 3 brown rats (Rattus norvegicus) were collected on 12 farms including five pig farms, five poultry farms, one mixed pig and veal farm and one goat farm. MRSA ST398 was detected in black rats captured at two of the five pig farms as well as in a black rat living on the mixed pig and veal farm. From one black rat captured at another pig farm MRSA ST 97 was isolated. Considering the behaviour of rats on livestock farms, it is concluded that rats might play a role in the spread and persistence of MRSA on pig farms.

Iron mobilisation with chelating agents during acute viral hepatitis.

During acute viral hepatitis, disturbances in iron metabolism occur. To obtain more insight into this, DFO and DTPA induced urinary iron excretion was studied during acute viral hepatitis. It was found that liver cell damage enhances iron excretion, in proportion to the extent of liver cell disintegration: a highly significant correlation was shown between the DFO as well as DTPA induced urinary iron excretion, and the SGPT. Also, a correlation existed between sideruria and the serum iron level, as well as with the serum bilirubin concentration, provided the test was performed within 10 days after the onset of the jaundice. It is suggested that during liver cell damage, iron, which is liberated during ferritin catabolism, forming a transit pool within the hepatocyte and/or at the cell membrane, is the immediate source of iron donation to DFO and/or DTPA. The practical importance for interpretation of iron mobilisation tests is pointed out.

Some aspects of iron metabolism during acute viral hepatitis.

In the acute phase of acute viral hepatitis high serum iron and serum ferritin levels were found in all patients. The normalisation of the serum ferritin concentration parallelled that of the serum glutamic pyruvic transaminase activity. However serum iron levels remained elevated for a long period of time. Chemical analysis of liver tissue showed a low total liver depot iron concentration during the first two weeks of the disease, indicating that the high serum iron levels are caused by iron liberation from disintegrated hepatocytes. Patients studied after two weeks showed higher liver iron concentrations, in particular the non-ferritin iron fraction, reflecting iron accumulation in the reticulo-endothelial system. Indeed, histological examination in the patients studied after two weeks showed sinusoidal lining cell siderosis in addition to "diffuse iron" in clusters lining cells which in electron microscopical studies proved to be macrophages. These cells showed a positive immunohistological reaction for ferritin protein. It is suggested that during acute viral hepatitis two mechanisms act together, i.e. changes in iron metabolism caused by damage of the main iron depot organ (specific liver pathology) and changes in iron metabolism common to all infectious processes.

Follow-up of blood donors positive for hepatitis B surface antigen.

From 1973 to 1977 in Amsterdam the incidence of hepatitis B surface antigen (HBsAg) in blood donations from new donors was 0.224 and from known donors 0.034%. 65 donors, previously found positive for HBsAg, were re-examined. Persistence of HBsAg in new donors (28 of 31) occurred significantly (p less than 0.0005) more often than in known donors (15 of 34). All carriers were classified into HBeAg (21%) or anti-HBe (79%) by a sensitive Elisa technique. Abnormal liver function tests (LFTs) were observed in 30% of the carriers and were significantly (p less than 0.005) more often found in HBeAg than in anti-HBe-positive carriers. When the LFTs remained abnormal, in almost all (8 of 9) carriers moderate to severe histological liver disease was diagnosed.

On the iron content of human serum ferritin, especially in acute viral hepaptitis and iron overload.

A new method has been developed to measure ferritin iron in human serum. The ferritin is bound to antibodies to ferritin, which are coupled to Sepharose 4-B and separated from the serum by centrifugation. The iron is liberated from the bound ferritin and measured colorimetrically. The detection limit of this method proved to be approx. 15 ng Fe/ml serum. The serum ferritin iron concentration has been compared with the serum ferritin protein concentration. During liver disease with liver cell leakage the mean iron content of serum ferritin proved to be less than the mean iron content in the liver ferritin. The mean iron content in the liver ferritin was 16%, range 7%--32% (n = 8). The mean iron content in serum ferritin from the same patients was 5%, range 0%--14%. Also in two cases of haemosiderosis the serum ferritin iron content was low. It is suggested that ferritin loses part of its iron on passage from the tissue cells to the blood. In some cases of severe liver cell (or other cell) leakage the mean iron content in serum ferritin might be high, because then more or less complete ferritin molecules enter the blood.

An enzyme-linked immunoassay for ferritin in human serum and rat plasma and the influence of the iron in serum ferritin on serum iron measurement, during acute hepatitis.

To measure human serum ferritin and rat plasma ferritin a non-competitive enzyme-linked immunoassay has been developed using horseradish peroxidase as the enzyme. In this assay it proved necessary to use heated rat plasma to obtain reproducible ferritin values. The heating procedure caused a loss of 38% of the plasma ferritin. Rat plasma ferritin values have been corrected for this loss. The standard deviation, from duplicate normal human and rat samples is 10 ng ferritin/ml serum and 69 ng/ml plasma, respectively. (The mean ferritin concentrations are: in human sera, 82 ng/ml and in rat plasma 762 ng/ml.) Mean recovery of added liver ferritin in the human serum is 104% +/- 4% (+/-S.E.M') and in the rat plasma 101% +/- 3% (+/- S.E.M.). Normal ferritin concentrations varied in the human material between 30 ng/ml and 300 ng/ml serum, and in the rat plasma between 500 ng/ml and 1300 ng/ml. During increased body iron and acute hepatitis the ferritin concentrations, in patients as well as in rats, exceeded the upper limit of the normal values in most cases. During human hepatitis high serum ferritin levels combined with high serum iron levels were measured. The high serum iron concentrations could not be explained by the high serum ferritin concentrations, even if the iron content of the ferritin is supposed to be high.

A method for measurement of liver iron fractions in needle biopsy specimens and some results in acute liver disease.

Methods are described for measurement of total tissue iron, ferritin iron, haem iron and ferritin protein in approx. 15 mg of tissue obtained by liver biopsy. The validity of these methods is examined by comparison with the values observed in larger samples of the same post-mortem derived liver tissue. Correlation coefficients vary between 0.80 and 0.99 (n = 11--16). It appears that in post-mortem liver tissue the haem iron concentration is higher than in biopsy specimens from patients. Analysis of liver biopsy specimens from patients with hepatitis showed a large variation in the mean iron content of the liver ferritin molecules. Also, the non-ferritin depot iron concentration and ferritin protein concentration is quite variable. It is suggested that in cases of advanced ferritin catabolism during hepatitis the mean percentage of iron in ferritin molecules often increases while at the same time the non-ferritin depot iron fraction decreases, probably because of iron release from the liver.