Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014-2018 national laboratory surveillance.

OBJECTIVES: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. METHODS: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). RESULTS: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with blaOXA-48 being predominant (38%, 336/892), followed by blaNDM-1 (16%, 145/892). For the first time in the Netherlands, blaOXA-181, blaOXA-232 and blaVIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. CONCLUSIONS: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are blaOXA-48 and blaNDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem.

Extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: prevalence, risk factors and molecular characteristics.

OBJECTIVES: The objectives of this study were to: estimate the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli carriage among broiler farmers, their family members and employees; identify and quantify risk factors for carriage, with an emphasis on contact with live broilers; and compare isolates from humans and broilers within farms with respect to molecular characteristics to gain insight into transmission routes. METHODS: A cross-sectional prevalence study was conducted on 50 randomly selected Dutch broiler farms. Cloacal swabs were taken from 20 randomly chosen broilers. Faecal swabs were returned by 141 individuals living and/or working on 47 farms. ESBL/AmpC-producing E. coli were isolated and, for selected isolates, phylogenetic groups, plasmids and sequence types were determined. Questionnaires were used for risk factor analysis. RESULTS: All sampled farms were positive, with 96.4% positive pooled broiler samples. The human prevalence was 19.1%, with 14.3% and 27.1% among individuals having a low and a high degree of contact with live broilers, respectively. Five pairs of human-broiler isolates had identical genes, plasmid families and E. coli sequence types, showing clonal transmission. Furthermore, similar ESBL/AmpC genes on the same plasmid families in different E. coli sequence types in humans and broilers hinted at horizontal gene transfer. CONCLUSIONS: The prevalence among people on broiler farms was higher than in previous studies involving patients and the general population. Furthermore, an increased risk of carriage was shown among individuals having a high degree of contact with live broilers. The (relative) contribution of transmission routes that might play a role in the dissemination of ESBL/AmpC-encoding resistance genes to humans on broiler farms should be pursued in future studies.

Extended-spectrum ß-lactamase producing Klebsiella spp. in chicken meat and humans: a comparison of typing methods.

Recently, chicken meat was identified as a plausible source of extended-spectrum ß-lactamase (ESBL) -producing Escherichia coli in humans. We investigated the relatedness of ESBL-producing Klebsiella spp. in chicken meat and humans. Furthermore, we tested the performance of SpectraCell RA(®) (River Diagnostics), a new typing method based on Raman spectroscopy, in comparison with multilocus sequence typing (MLST) for Klebsiella pneumoniae. Twenty-seven phenotypically and genotypically confirmed ESBL-producing Klebsiella spp. isolates were typed with MLST and SpectraCell RA. The isolates derived from chicken meat, human rectal swabs and clinical blood cultures. In the 22 ESBL-producing K. pneumoniae isolates, CTX-M15 was the predominant genotype, found in five isolates of human origin and in one chicken meat isolate. With MLST, 16 different STs were found, including five new STs. Comparing the results of SpectraCell RA with MLST, we found a sensitivity of 70.0% and a specificity of 81.8% for the new SpectraCell RA typing method. Therefore, we conclude that SpectraCell RA is not a suitable typing method when evaluating relationships of ESBL-producing Klebsiella spp. at the population level. Although no clustering was found with isolates of chicken meat and human origin containing the same ESBL genes, MLST showed no clustering into distinctive clones of isolates from chicken meat and human origin. More studies are needed to elucidate the role of chicken meat in the rise of ESBL-producing Klebsiella spp. in humans.

Prevalence of extended-spectrum ß-lactamase-producing Enterobacteriaceae in humans living in municipalities with high and low broiler density.

Prevalence of, and risk factors for, carriage of extended-spectrum ß-lactamase (ESBL) -producing Enterobacteriaceae were determined for 1025 Dutch adults in municipalities with either high or low broiler densities. Overall prevalence of ESBL carriage was 5.1%. The hypothesis that individuals in areas with high broiler densities are at greater risk for ESBL carriage was rejected, as the risk was lower (OR = 0.45; p 0.009) for these individuals. Owning a horse increased the risk (OR = 4.69; p ≤0.0001), but horse owners often owned multiple species of companion animals. Routes of transmission from animals to humans in the community, and the role of poultry in this process, remain to be elucidated.

Effect of aspirin on the Wnt/beta-catenin pathway is mediated via protein phosphatase 2A.

Nonsteroidal anti-inflammatory drugs show chemopreventive efficacy in colon cancer, but the mechanism behind this remains unclear. Elucidating this mechanism is seen as vital to the development of new chemopreventive agents. We studied the effects of aspirin on the oncogenic Wnt/beta-catenin pathway activity in colorectal cancer cell lines and observed that aspirin dose-dependently decreased the activity of this pathway, as judged by TCF-driven luciferase activity, reduced Wnt target gene expression and increased phosphorylation of beta-catenin by immunoblotting. Furthermore, the ubiquitination and cytoplasmic levels of beta-catenin were assessed by immunoblotting, and also the localization of beta-catenin was shown by green fluorescent protein-tagged beta-catenin and time-lapse fluorescent imaging. Importantly, aspirin treatment caused increased phosphorylation of protein phosphatase 2A (PP2A), an event associated with inhibition of PP2A enzymatic activity, which was confirmed by a reduction in enzymatic PP2A activity. Moreover, this inhibition of PP2A enzymatic activity was essential for the effects of aspirin on the Wnt/beta-catenin pathway as shown by transient transfection with PP2A constructs. The findings in this article provide a molecular explanation for the efficacy of aspirin in chemoprevention of colorectal cancer and shows biochemical evidence that PP2A is an important regulator of Wnt/beta-catenin pathway activity in these cells.

Panton-Valentine leukocidin positive MRSA in 2003: the Dutch situation.

Analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolates in the Netherlands in 2003 revealed that 8% of the hospital isolates carried the loci for Panton-Valentine leukocidin (PVL). Molecular subtyping showed that most Dutch PVL-MRSA genotypes corresponded to well-documented global epidemic types. The most common PVL-MRSA genotypes were sequence type ST8, ST22, ST30, ST59 and ST80. MRSA with ST8 increased in the Netherlands from 1% in 2002 to 17% in 2003. It is emphasised that PVL-MRSA might not only emerge in the community, but also in the hospital environment.

Panton-Valentine leukocidin positive MRSA in 2003: the Dutch situation.

Analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolates in the Netherlands in 2003 revealed that 8% of the hospital isolates carried the loci for Panton-Valentine leukocidin (PVL). Molecular subtyping showed that most Dutch PVL-MRSA genotypes corresponded to well-documented global epidemic types. The most common PVL-MRSA genotypes were sequence type ST8, ST22, ST30, ST59 and ST80. MRSA with ST8 increased in the Netherlands from 1% in 2002 to 17% in 2003. It is emphasised that PVL-MRSA might not only emerge in the community, but also in the hospital environment.

Surveillance of antibiotic resistance in Neisseria gonorrhoeae in The Netherlands, 1977-95.

OBJECTIVE: To evaluate the prevalence and epidemiology of penicillinase producing Neisseria gonorrhoeae (PPNG) and tetracycline resistant N gonorrhoeae (TRNG) in the period 1977-95 in the Netherlands. To compare auxotypes, serovars, and antibiograms of PPNG, non-PPNG, and TRNG. To identify determinants in patient characteristics for the epidemic spread of TRNG/PPNG. METHODS: With respect to the national gonococcal surveillance all PPNG isolates from 30 laboratories over the country in 1977-90 and all gonococcal isolates from five sentinel laboratories (during 1 month per quarter) in 1991-5 were collected. Isolates were auxotyped and serotyped, the susceptibility for various antibiotics was tested and plasmid contents were evaluated. Additional data on PPNG infected individuals were collected retrospectively during a microepidemic of TRNG/PPNG. Univariate and multivariate analyses were performed to identify risk factors for TRNG/PPNG infections. RESULTS: In 1995 an overall high prevalence of PPNG infection (27%) and TRNG among PPNG infection (24%) was found in the Netherlands. Importantly, PPNG were found to have higher MICs for ceftriaxone and ciprofloxacin than non-PPNG; clinically relevant resistance to these antibiotics (or related agents) may emerge first among these strains. The observed diversity of strains (123 auxo/serovar classes since 1988) indicates a continuous introduction of new strains into the community. The epidemic increase of TRNG/PPNG was mainly caused by A/S classes NR/1B-6, PRO/1A-3, and PRO/1A-6, suggesting a clonal spread of a few strains; the rapid spread was associated with transmission in high risk individuals (that is, prostitutes and their clients). CONCLUSION: The prevalence of PPNG in the Netherlands remains high and reduced sensitivity to other antimicrobials was detected among the PPNG strains. This underlines the necessity for a continuous national surveillance of resistance in gonococci including limited epidemiological information.

Comparison between self-report measures and clinical observations of functional disability in ankylosing spondylitis, rheumatoid arthritis and fibromyalgia.

OBJECTIVE: To study concordance between self-report measures and clinical observations of functional disability in ankylosing spondylitis (AS), rheumatoid arthritis (RA), and fibromyalgia (FM). METHODS: 35 patients with AS completed 9 selected items of the Functional Index questionnaire, whereas 12 patients with RA and 13 with FM completed 7 selected items of the Arthritis Impact Measurement Scales. Five days later, all 60 patients and 4 controls actually performed the selected activities, which were recorded on video. The tapes were assessed in random order by 12 observers (6 occupational therapists and 6 physicians). Both patients and observers indicated functional disability on a 10 cm visual analog scale (VAS). RESULTS: Interobserver agreement was high (Cronbach's alpha 0.98). All observers scored the 4 healthy controls as having no disability at all. Mean discordance scores (VAS patients minus VAS observers) for the selected items were negligible in AS [-0.17 cm (p = 0.30)], moderate in RA [+1.10 cm (p = 0.06)] and high in FM [+2.44 cm (p < 0.01)]. CONCLUSION: Discordance between self-report questionnaires and observed functional disability is a feature most striking in FM. In validation of self-report questionnaires of functional disability the appropriate spectrum of rheumatological diagnoses should be considered.

Interfering with implantation by postcoital estrogen administration. II. Endometrium epithelial cell ultrastructure.

Secretory human endometrium was studied by electron microscopy after high-dose ethinylestradiol administration, a method used for interception (morning-after pill). Medicated (experimental) cycles were compared to non-medicated (control) cycles in the same volunteers. The nucleolar channel system typically present in the nucleus of the epithelial cell in secretory phase of the human endometrium disappears completely as a result of this treatment, resulting in a nucleolar basket structure. Glycogen deposits were prominent at the basal membrane and in the apex of the cell during the experimental cycle. Giant mitochondria were observed to occur to similar extent during control and treatment cycles. This study confirms the morphological delay found in light microscopic studies, published earlier with diethylstilbestrol and with ethinylestradiol.

PIP: High-dose ethinylestradiol is used as a postcoital contraceptive. Its mechanism of action is to delay the secretory development of the endometrial epithelial cells. This study, using 4 normal volunteers, was undertaken to see the effects of high-dose ethinylestradiol on the ultrastructure of the endometrial cells and on glycogen metabolism during the luteal phase of the menstrual cycle. Ovulation was determined from plasma luteinizing hormone levels, and the subjects were given 5 mg ethinylestradiol for 5 consecutive days thereafter. The nucleolar channel system, which typically appears in the cell during the secretory phase, completely disappeared after treatment with ethinylestradiol. Moreover, the Golgi complexes were enlarged, and large amounts of glycogen accumulated at the basal membrane and in apical vacuoles of the cells. These changes show that large doses of ethinylestradiol given after ovulation interfere with the synthesis and secretion of glycogen in the endometrial epithelial cells.

Postcoital interception with steroids.

At present there is a high demand for PCI. The psychological distress experienced by women after unprotected intercourse while awaiting the onset of her next menses could be avoided with effective postcoital treatment. As demonstrated in countries with good health services, the availability of PCI leads to low abortion rates. PCI is designed for use as an emergency method after unprotected intercourse as an isolated incident. Women who have frequent intercourse however, do better to use a regular, very effective, method of contraception such as a combined oral contraceptive. Postcoital progestagens can well be used in infrequent intercourse since they are to be taken after each coitus, if applicable several times a month. Such use however leads to menstrual cycle disturbances and irregular bleedings. The estrogen-only and the estrogen-progestin combination are recommended, but need to be given within 2 or 3 days after the event of unprotected intercourse, preferably periovulatory. The latest development of the anti-progestins as a morning-after pill is promising. Taken from day 27 through 30 of the menstrual cycle it induces menstruation at the expected day. Side effects are minimal and the efficacy is good. Because of the imperfectness of PCI to prevent all pregnancies, sofar this regimen cannot be recommended for monthly use and does not replace regular oral contraceptives. PCI has a definite place in family-planning and fertility regulation. Since different methods are available today careful assessment of individual needs can help to decide for the best suitable method for the individual person.

Interception. III: Postcoital luteal contragestion by an antiprogestin (mifepristone, RU 486) in 62 women.

The new antiprogestin mifepristone (RU 486) was studied as an emergency postcoital contragestive. An actual pregnancy rate of 1.6% was observed and was related to the actual conception rate. The follow-up rate was 100%. The patterns of onset and duration of the induced menstruation after mifepristone treatment are described. This method provides a good new interceptive technique when the time for use of postcoital steroids or for a postcoital IUD insertion has lapsed.

Interception. IV: Failure of mifepristone (RU 486) as a monthly contragestive, "Lunarette".

The new antiprogestin mifepristone (RU 486) was studied as a contragestive for continuous fertility control in 24 menstrual cycles. Two pregnancies out of three conceptions continued in spite of antiprogestin treatment. To date, mifepristone at the doses used appears to be inadequate for monthly use.

Post coital contraception.

PIP: The experience of the Utrecht State University with postcoital estrogens in high and low combined doses and with postcoital placement of IUDs since 1964 with over 4000 patients is summarized. The high dose postcoital estrogen treatment consists of 5 mg ethinyl estradiol for 5 days, either orally, or in case of vomiting not controlled by an antiemetic, estradiol benzoate 30 mg by injection. Side effects recorded in 3016 women were nausea in 54%, vomiting in 24%, tender breasts in 23%, menorrhagia in 11%, altered cycle length in 24%. Complications were 1 case of non-fatal pulmonary edema and 1 case of an 8 kg weight gain during treatment. There were 3 pregnancies. The overall failure rate in the whole series was 0.15%, with 10% ectopic pregnancies. There were no thromboembolisms or teratogenic effects. The combined estrogen treatment consisted of 50 mc ethinyl estradiol with 250 mc levonorgestrel (Neogynon oral contraceptive), 2 pills followed by 2 pills 12 hours later. A double-blind randomized trial resulted in no significant differences in pregnancy rates or side effects between the high and low dose regimens. The alternate treatment, if the woman presents more than 72 hours after intercourse, or if estrogens are contraindicated, is postcoital insertion of an IUD. The Dept. of Obstetrics and Gynecology does not place an IUD in a woman with infection nor in case of rape unless there is time for a complete work-up. Nulliparas are informed of the increased risk of pelvic inflammatory disease. Recently, the Multiload-copper 250 and later ML 375 were used exclusively, to achieve better blastocidal effect and lower expulsion rates. The ethical debate over use of postcoital methods centers around the morality of "procuring a miscarriage," but this argument is not relevant since these methods will not terminate a pregnancy once implantation has occurred. In the Netherlands, 25% of all abortion clients become pregnant during their 1st intercourse. In 1982, 35,000 postcoital contraceptives were administered, (roughly 16% of all pregnancies), compared to 15,000 abortions (7% of pregnancies; a total of 23% of pregnancies terminated). Compare these figures with 29% unwanted pregnancies all terminated by abortion in Sweden in that year. The postcoital methods are cheap, effective, and invaluable in emergency cases of rape, incest, intoxication, failure of barrier contraceptives, or unwanted pregnancy in women fearful or opposed to abortion.^ieng

Interception II: postcoital low-dose estrogens and norgestrel combination in 633 women.

The new low-dose hormonal postcoital method, a combination of 200 mcg ethinylestradiol and 2 mg norgestrel was used in 633 women, and a statistically significantly lower observed pregnancy rate was found compared to the expected number of pregnancies if no contraception was used. Patterns of menstruation, its onset and duration, after use of this morning-after pill, are documented in respect to follicular, midcycle and luteal administration. Individual side effects such as nausea, vomiting and mastalgia are noted, but antiemetics did not reduce the incidence. Though efficacy of this technique is not found to be favorable in comparison to the 5 mg ethinylestradiol treatment, the low-dose of steroid, the one-day treatment and its lesser side effects show this alternative morning-after pill suitable for use as a first choice in case of an unprotected sexual encounter.

A comparison of high-dose estrogens versus low-dose ethinylestradiol and norgestrel combination in postcoital interception: a study in 493 women.

Ethinylestradiol (EE), at a dosage of 5 mg/day for 5 consecutive days (5 mg EE), has generally been used for interception. A combination of 200 micrograms EE and 2 mg dl-norgestrel (EE + NG) was proposed as an effective alternative. Efficacy and tolerance of these methods were compared in a randomized, double-blind study. A group of 465 women was studied with a follow-up rate of 94.3%. In the 5 mg EE group a pregnancy rate of 0.9% was observed, and in the EE + NG group a rate of 0.4% was found. These rates differ significantly from the expected rates (P less than 0.0005, in both series). Nausea was noted in 59.1% of the 5 mg EE group and in 54.0% of the EE + NG series. Nausea and vomiting occurred in 20.8% and 15.8%, respectively. The efficacy of both methods as alternative morning-after medication was confirmed. The new method is preferable because treatment is limited to only 1 day.

PIP: Ethinyl estradiol (EE), at a dosage of 5 mg/day for 5 consecutive days (5 mg EE) has generally been used for interception. A combination of 200 mcg EE and 2 mg dl--norgestrel (EE+NG) was porposed as an effective alternative. Efficacy and tolerance of these methods were compared in a randomized, double-blind study. A group of 465 women was studied with a follow-up rate of 94.3%. In the 5 mg EE group, a pregnancy rate of 0.9% was observed, and in the EE+NG group, a rate of 0.4% was found. These rates differ significantly from the expected rates (P0.0005, in both series). Nausea was noted in 59.1% of the 5 mg EE group in and 54.0% of the EE+NG group. Nausea and vomiting occurred in 20.8% and 15.8% respectively. The efficacy of both methods as alternative to morning-after medication was confirmed. The new method is preferable since treatment is limited to only 1 day.

[Postcoital IUD insertion, a review]. / Postkoitale IUP-Einlage, ein Uberblick.

Following the development of hormonal interception after coitus the post-coital insertion of an intrauterine contraceptive device was proposed by Tatum . The advantage of this treatment is the avoidance of the ingestion of large doses of estrogen which causes much nausea and vomiting although it is a very effective post-coital method of contraception. The recently proposed alternative administration of 200 micrograms Ethynol Estradiol combined with 2 mg of DL norgesterol in 2 equal doses at 12 hour intervals has the same disadvantage of a high percentage of side effects. The post-coital insertion of an intrauterine contraceptive device is the first method which is effective up to five days following unprotected intercourse which is three days longer than treatment by estrogen. In addition the method can be offered to women who would want to continue to wear the intrauterine contraceptive device for long term contraception. The disadvantage of the post-coital insertion of an intrauterine contraceptive device is the ability of serious complications if the patient has a vaginal or venereal infection or an asymptomatic cervicitis or salpingitis. Following appropriate physical examination women who present themselves for post-coital treatment are selected. Cases of rape are usually not suitable for treatment with intrauterine contraceptives devices. However, when cases of rape are seen early enough the appropriate investigations may be done and the treatment with the intrauterine device started within five days. The potential risk of future infertility must be considered since salpingitis is 7 times more common in nulliparous wearers of intrauterine devices than in nulliparous non-wearers. Young sexually active nulliparous women especially of lower socio economic background are patients with a high risk. Over 70% of the women who present themselves for interception treatment are nulliparous.(ABSTRACT TRUNCATED AT 250 WORDS)