Prenatal exposure to mixtures of per- and polyfluoroalkyl substances and organochlorines affects cognition in adolescence independent of postnatal exposure.

BACKGROUND: Studies on cognitive and neurodevelopmental outcomes have shown inconsistent results regarding the association with prenatal exposure to perfluoroalkyl substance (PFAS) and organochlorines. Assessment of mixture effects of correlated chemical exposures that persist in later life may contribute to the unbiased evaluation and understanding of dose-response associations in real-life exposures. METHODS: For a subset of the 4th Flemish Environment and Health Study (FLEHS), concentrations of four PFAS and six organochlorines were measured in respectively 99 and 153-160 cord plasma samples and 15 years later in adolescents' peripheral serum by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Sustained and selective attention were measured at 14-15 years with the Continuous Performance Test (CPT) and Stroop Test as indicators of potential neurodevelopmental deficits. Quantile g-computation was applied to assess the joint associations between prenatal exposure to separate and combined groups of PFAS and organochlorines and performance in the CPT and Stroop Test at adolescence. Subsequently, individual effects of each chemical compound were analyzed in mixed effects models with two sets of covariates. Analytical data at birth and at the time of cognitive assessment allowed for off-setting postnatal exposure. RESULTS: In mixtures analysis, a simultaneous one-quantile increase in the natural log-transformed values of PFAS and organochlorines combined was associated with a decrease in the mean reaction time (RT) and the reaction time variability (RTV) in the CPT (ß = -15.54, 95% CI:-29.64, -1.45, and ß = -7.82, 95% CI: -14.97, -0.67 respectively) and for the mixture of PFAS alone with RT (ß = -11.94, 95% CI: -23.29, -0.60). In the single pollutant models, these results were confirmed for the association between perfluorohexanesulfonate (PFHxS) with RT (ß = -17.95, 95% CI = -33.35, -2.69) and hexachlorobenzene with RTV in the CPT (ß = -5.78, 95% CI: -10.39, -0.76). Furthermore, the participants with prenatal exposure above the limit of quantification for perfluorononanoic acid (PFNA) had a significantly shorter RT and RTV in the CPT (ß = -23.38, 95% CI: -41.55, -5.94, and ß = -9.54, 95% CI: -19.75, -0.43, respectively). CONCLUSION: Higher prenatal exposure to a PFAS mixture and a mixture of PFAS and organochlorines combined was associated with better sustained and selective attention during adolescence. The associations seemed to be driven by PFHxS and were not linked to exposure levels at the time of assessment.

Harmonization of Human Biomonitoring Studies in Europe: Characteristics of the HBM4EU-Aligned Studies Participants.

Human biomonitoring has become a pivotal tool for supporting chemicals' policies. It provides information on real-life human exposures and is increasingly used to prioritize chemicals of health concern and to evaluate the success of chemical policies. Europe has launched the ambitious REACH program in 2007 to improve the protection of human health and the environment. In October 2020 the EU commission published its new chemicals strategy for sustainability towards a toxic-free environment. The European Parliament called upon the commission to collect human biomonitoring data to support chemical's risk assessment and risk management. This manuscript describes the organization of the first HBM4EU-aligned studies that obtain comparable human biomonitoring (HBM) data of European citizens to monitor their internal exposure to environmental chemicals. The HBM4EU-aligned studies build on existing HBM capacity in Europe by aligning national or regional HBM studies. The HBM4EU-aligned studies focus on three age groups: children, teenagers, and adults. The participants are recruited between 2014 and 2021 in 11 to 12 primary sampling units that are geographically distributed across Europe. Urine samples are collected in all age groups, and blood samples are collected in children and teenagers. Auxiliary information on socio-demographics, lifestyle, health status, environment, and diet is collected using questionnaires. In total, biological samples from 3137 children aged 6-12 years are collected for the analysis of biomarkers for phthalates, HEXAMOLL® DINCH, and flame retardants. Samples from 2950 teenagers aged 12-18 years are collected for the analysis of biomarkers for phthalates, Hexamoll® DINCH, and per- and polyfluoroalkyl substances (PFASs), and samples from 3522 adults aged 20-39 years are collected for the analysis of cadmium, bisphenols, and metabolites of polyaromatic hydrocarbons (PAHs). The children's group consists of 50.4% boys and 49.5% girls, of which 44.1% live in cities, 29.0% live in towns/suburbs, and 26.8% live in rural areas. The teenagers' group includes 50.6% girls and 49.4% boys, with 37.7% of residents in cities, 31.2% in towns/suburbs, and 30.2% in rural areas. The adult group consists of 52.6% women and 47.4% men, 71.9% live in cities, 14.2% in towns/suburbs, and only 13.4% live in rural areas. The study population approaches the characteristics of the general European population based on age-matched EUROSTAT EU-28, 2017 data; however, individuals who obtained no to lower educational level (ISCED 0-2) are underrepresented. The data on internal human exposure to priority chemicals from this unique cohort will provide a baseline for Europe's strategy towards a non-toxic environment and challenges and recommendations to improve the sampling frame for future EU-wide HBM surveys are discussed.

Determinants of Chronic Biological Stress, Measured as Hair Cortisol Concentration, in a General Population of Adolescents: From Individual and Household Characteristics to Neighborhood Urbanicity.

Chronic biological stress may adversely affect adolescents' physical and mental health, but insight in the personal and environmental factors that determine chronic stress is limited. We measured 3-month cumulative hair cortisol concentration (HCC) in 419 adolescents, participating in the Flemish Environment and Health Study. Adolescents' health and lifestyle characteristics, household and neighborhood socio-economic status as well as neighborhood urbanicity were assessed as potential determinants of HCC, using multiple linear regression models. We additionally explored heterogeneity of our results by sex. HCC were significantly higher in boys from densely populated neighborhoods, the association was not significant in girls. Accordingly, boys living outside cities had significantly lower HCC than boys, living in cities. HCC was significantly lower in adolescents with an optimal vitality, a measure of a positive mental health status. In adolescent girls, menarcheal status (pre-/postmenarche) was a significant determinant of HCC. Our findings are the first to suggest that residential urbanicity may have an impact on chronic biological stress in a general population of adolescent boys.

Urinary Polycyclic Aromatic Hydrocarbon Metabolites Are Associated with Biomarkers of Chronic Endocrine Stress, Oxidative Stress, and Inflammation in Adolescents: FLEHS-4 (2016-2020).

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants of public health concern. Multiple biological mechanisms have been hypothesized to contribute to PAHs-associated adverse health effects. Little is known about the impact of PAHs on endocrine stress and inflammation in adolescence. We examined 393 Flemish adolescents (14-15 years) cross-sectionally, measured urinary concentrations of hydroxylated naphthalene, fluorene, phenanthrene and pyrene metabolites, and calculated the sum of all measured metabolites. We determined hair cortisol concentration (HCC) as endocrine stress biomarker, leucocyte counts and neutrophil-lymphocyte ratio (NLR) in peripheral blood as inflammatory biomarkers, and urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) concentration as oxidative stress biomarker. Exposure-response associations were analyzed by multiple regression, adjusted for a priori selected covariates. A doubling of 1-hydroxypyrene concentration was associated with a factor of 1.13 (95% CI: 1.03, 1.24) increase in HCC and a factor of 1.07 (95% CI: 1.02, 1.13) increase in 8-oxodG. Doublings of 2- and 3-hydroxyphenanthrene concentrations were associated with a factor of 1.08 (95% CI: 1.02, 1.14) and 1.06 (95% CI: 1.00, 1.12) increase in 8-oxodG, respectively. Doubling of 2-hydroxyphenanthrene and of the sum of 2- and 3-hydroxyfluorene was associated with, respectively, a factor of 1.08 (95% CI: 1.02, 1.14) and 1.06 (95% CI: 1.01, 1.13) increase in NLR. Our results indicate the glucocorticoid pathway as a potential target for PAH exposure in adolescents and suggest oxidative stress, endocrine stress, and inflammation in adolescence as underlying mechanisms and early markers for PAH-related adverse health effects.

Long-term residential exposure to air pollution is associated with hair cortisol concentration and differential leucocyte count in Flemish adolescent boys.

BACKGROUND: Exposure to air pollution and traffic noise are associated with adverse health outcomes in adolescents. Chronic endocrine stress and systemic inflammation have been hypothesized to underlie the adverse health effects. Simultaneous assessment of inflammation and chronic endocrine stress in epidemiological studies is lacking. The aim of the study was to investigate biomarkers of chronic endocrine stress and inflammation in relation to long-term residential exposure to air pollution and traffic noise in adolescents. METHODS: In Flemish adolescents (14-15 years), we determined hair cortisol concentration (HCC) as a chronic stress biomarker in 3-cm scalp-near hair sections (n = 395), and leucocyte and leucocyte subtype counts (neutrophils, monocytes, lymphocytes) as inflammatory biomarkers in peripheral blood (n = 385). Daily particulate matter (PM2.5, PM10), nitrogen dioxide (NO2) and black carbon (BC) concentrations were modelled at the residential address and averaged over 3-month and 1-year periods prior to sampling. Residential traffic noise level was estimated and classified in 5 dB intervals. Sex-specific associations between residential exposures and effect biomarkers were studied using linear regression models, adjusted for a priori selected covariates. RESULTS: In boys, HCC increased with a factor 1.30 (95% CI: 1.10, 1.54) for an increase in 1-year mean NO2 from the 25th to 75th percentile (p75/p25), after adjustment for age, BMI, personal and neighborhood socioeconomic status. The corresponding estimate for PM10 was 1.24 (95% CI: 1.02, 1.51). Total leucocyte count in boys, adjusted for the aforementioned covariates and recent health complaints, was positively associated with PM2.5, PM10, NO2 and BC. In particular, the neutrophil count increased with a factor 1.11 (95% CI: 1.03, 1.19) for a (p75/p25)-factor increase in 1-year mean BC, corresponding estimates for PM2.5, PM10 and NO2 were 1.10 (95% CI: 1.01, 1.19), 1.10 (95% CI: 1.01, 1.20) and 1.08 (95% CI: 1.00, 1.16). Lymphocyte count increased with a factor 1.05 (95% CI: 1.01, 1.10) for a (p75/p25)-factor increase in 1-year mean NO2. Similar results were observed for 3-month mean exposures. Results were robust to adjustment for recent air pollution exposure. In girls, air pollutants were not associated with HCC or differential leucocyte count. Residential traffic noise level was not associated with HCC or leucocyte counts in boys nor girls. CONCLUSIONS: Long-term residential exposure to air pollutants was positively associated with chronic endocrine stress and inflammation in adolescent boys, not in girls. This study may contribute to a better understanding of the early pathophysiological changes that may underlie adverse health effects of air pollution exposure in adolescents.

Biomarkers of phthalates and alternative plasticizers in the Flemish Environment and Health Study (FLEHS IV): Time trends and exposure assessment.

Restrictions on the use of legacy phthalate esters (PEs) as plasticizer chemicals in several consumer products has led to the increased use of alternative plasticizers (APs), such as di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH) and di-(2-ethylhexyl) terephthalate (DEHTP). In the fourth cycle of the Flemish Environment and Health Study (FLEHS IV, 2016-2020), we monitored exposure to seven PEs (diethyl phthalate (DEP), di-(2-ethylhexyl) phthalate (DEHP), di-isobutyl phthalate (DiBP), di-n-butyl phthalate (DnBP), butylbenzyl phthalate (BBzP, di-isononyl phthalate (DINP), and di-isodecyl phthalate (DIDP))and three APs (DINCH, DEHTP, and di-(2-ethylhexyl) adipate (DEHA)) by measuring multiple biomarkers in urine of 416 adolescents from Flanders, Belgium (14-15 years old). The reference values show that exposure to PEs is still widespread, although levels of several PE metabolites (e.g., sum of DEHP metabolites, mono-normal-butyl phthalate (MnBP) and mono-benzyl phthalate (MBzP)) have decreased significantly compared to previous human biomonitoring cycles (2003-2018). On the other hand, metabolites of DINCH and DEHTP were detected in practically every participant. Concentrations of AP exposure biomarkers in urine were generally lower than PE metabolites, but calculations of estimated daily intakes (EDIs) showed that exposure to DINCH and DEHTP can be considerable. However, preliminary risk assessment showed that none of the EDI or urinary exposure levels of APs exceeded the available health-based guidance values, while a very low number of participants had levels of MiBP and MnBP exceeding the HBM value. Several significant determinants of exposure could be identified from multiple regression models: the presence of building materials containing PVC, ventilation habits, socio-economic status and season were all associated with PE and AP biomarker levels. Cumulatively, the results of FLEHS IV show that adolescents in Flanders, Belgium, are exposed to a wide range of plasticizer chemicals. Close monitoring over the last decade showed that the exposure levels of restricted PEs have decreased, while newer APs are now frequently detected in humans.

Planarians Customize Their Stem Cell Responses Following Genotoxic Stress as a Function of Exposure Time and Regenerative State.

Aiming to in vivo characterize the responses of pluripotent stem cells and regenerative tissues to carcinogenic stress, we employed the highly regenerative organism Schmidtea mediterranea. Its broad regenerative capacities are attributable to a large pool of pluripotent stem cells, which are considered key players in the lower vulnerability toward chemically induced carcinogenesis observed in regenerative organisms. Schmidtea mediterranea is, therefore, an ideal model to study pluripotent stem cell responses with stem cells residing in their natural environment. Including microenvironmental alterations is important, as the surrounding niche influences the onset of oncogenic events. Both short- (3 days) and long-term (17 days) exposures to the genotoxic carcinogen methyl methanesulfonate (50 µM) were evaluated during homeostasis and animal regeneration, two situations that render altered cellular niches. In both cases, MMS-induced DNA damage was observed, which provoked a decrease in proliferation on the short term. The outcome of DNA damage responses following long-term exposure differed between homeostatic and regenerating animals. During regeneration, DNA repair systems were more easily activated than in animals in homeostasis, where apoptosis was an important outcome. Knockdown experiments confirmed the importance of DNA repair systems during carcinogenic exposure in regenerating animals as knockdown of rad51 induced a stem cell-depleted phenotype, after regeneration was completed.

Phthalate-induced oxidative stress and association with asthma-related airway inflammation in adolescents.

BACKGROUND: In Belgium, around 8.5% of the children have asthmatic symptoms. Increased asthma risk in children has been reported in relation to exposure to phthalate plasticizers but the underlying mechanisms are largely unknown. AIM: The aim of this study was to identify if oxidative stress, assessed by excision of 8-hydroxydeoxyguanosine (8-OHdG) from damaged DNA, is an intermediate marker for the association between phthalate exposure and doctor-diagnosed asthma. MATERIAL AND METHODS: In 418 14-15-year-old youngsters, recruited as a representative sample of residents of Flanders (Belgium), personal exposure to phthalates was assessed by measuring phthalate metabolites in urine: mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-n-butyl phthalate (MnBP), mono-benzyl phthalate (MBzP), mono-isobutyl phthalate (MiBP) and mono-ethyl phthalate (MEP). Analysis of 8-OHdG in urine was used as a sensitive biomarker of oxidative stress at the level of DNA. The presence of doctor-diagnosed asthma was elicited by a self-administered questionnaire. Associations were assessed using multiple linear and logistic regression models. Mediation was tested using Baron and Kenny's regression approach. RESULTS: A significant increased risk of a youngster being diagnosed with asthma was found for both urinary MnBP (metabolite of dibutyl phthalate (DBP)) and the sum of the three di(2-ethylhexyl) phthalate metabolites (ΣDEHP=MEHP+MEHHP+MEOHP), with respective odds ratio of 1.84 [95% CI: 1.02, 3.32] for MnBP and 1.94 [95% CI: 1.07, 3.51] for ΣDEHP. In addition, we observed significant associations between all urinary phthalate metabolites and increased urinary levels of 8-OHdG. The associations were stronger in girls than in boys. We did not found evidence that 8-OHdG was associated with doctor-diagnosed asthma. CONCLUSION: The results of our study are in line with other findings from epidemiological surveys and raise further concern about DEHP and DBP as risk factors for asthma, however, the underlying mechanisms are not yet well understood.

Environmental exposure to human carcinogens in teenagers and the association with DNA damage.

BACKGROUND: We investigated whether human environmental exposure to chemicals that are labeled as (potential) carcinogens leads to increased (oxidative) damage to DNA in adolescents. MATERIAL AND METHODS: Six hundred 14-15-year-old youngsters were recruited all over Flanders (Belgium) and in two areas with important industrial activities. DNA damage was assessed by alkaline and formamidopyrimidine DNA glycosylase (Fpg) modified comet assays in peripheral blood cells and analysis of urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Personal exposure to potentially carcinogenic compounds was measured in urine, namely: chromium, cadmium, nickel, 1-hydroxypyrene as a proxy for exposure to other carcinogenic polycyclic aromatic hydrocarbons (PAHs), t,t-muconic acid as a metabolite of benzene, 2,5-dichlorophenol (2,5-DCP), organophosphate pesticide metabolites, and di(2-ethylhexyl) phthalate (DEHP) metabolites. In blood, arsenic, polychlorinated biphenyl (PCB) congeners 118 and 156, hexachlorobenzene (HCB), dichlorodiphenyltrichloroethane (DDT) and perfluorooctanoic acid (PFOA) were analyzed. Levels of methylmercury (MeHg) were measured in hair. Multiple linear regression models were used to establish exposure-response relationships. RESULTS: Biomarkers of exposure to PAHs and urinary chromium were associated with higher levels of both 8-OHdG in urine and DNA damage detected by the alkaline comet assay. Concentrations of 8-OHdG in urine increased in relation with increasing concentrations of urinary t,t-muconic acid, cadmium, nickel, 2,5-DCP, and DEHP metabolites. Increased concentrations of PFOA in blood were associated with higher levels of DNA damage measured by the alkaline comet assay, whereas DDT was associated in the same direction with the Fpg-modified comet assay. Inverse associations were observed between blood arsenic, hair MeHg, PCB 156 and HCB, and urinary 8-OHdG. The latter exposure biomarkers were also associated with higher fish intake. Urinary nickel and t,t-muconic acid were inversely associated with the alkaline comet assay. CONCLUSION: This cross-sectional study found associations between current environmental exposure to (potential) human carcinogens in 14-15-year-old Flemish adolescents and short-term (oxidative) damage to DNA. Prospective follow-up will be required to investigate whether long-term effects may occur due to complex environmental exposures.

Peripheral blood collection: the first step towards gene expression profiling.

A crucial challenge for gene expression analysis in human biomonitoring studies on whole blood samples is rapid sample handling and mRNA stabilization. This study was designed to evaluate the impact of short bench times (less than 30 min) on yield, quality and gene expression of mRNA in the presence of different stabilization buffers (Tempus(TM) Blood RNA tube and RNAlater(®) Stabilization Reagent). Microarray analyzes showed significant changes over short periods of time in expression of a considerate part of the transcriptome (2356 genes) with a prominent role for NFкB-, cancer- and glucocorticoid-mediated networks, and specifically interleukin-8 (IL-8). These findings suggest that even short bench times affect gene expression, requiring to carry out blood collection in a strictly standardized way.

Acquired immune responses to three malaria vaccine candidates and their relationship to invasion inhibition in two populations naturally exposed to malaria.

BACKGROUND: Malaria still represents a major cause of morbidity and mortality predominantly in several developing countries, and remains a priority in many public health programmes. Despite the enormous gains made in control and prevention the development of an effective vaccine represents a persisting challenge. Although several parasite antigens including pre-erythrocytic antigens and blood stage antigens have been thoroughly investigated, the identification of solid immune correlates of protection against infection by Plasmodium falciparum or clinical malaria remains a major hurdle. In this study, an immuno-epidemiological survey was carried out between two populations naturally exposed to P. falciparum malaria to determine the immune correlates of protection. METHODS: Plasma samples of immune adults from two countries (Ghana and Madagascar) were tested for their reactivity against the merozoite surface proteins MSP1-19, MSP3 and AMA1 by ELISA. The antigens had been selected on the basis of cumulative evidence of their role in anti-malarial immunity. Additionally, reactivity against crude P. falciparum lysate was investigated. Purified IgG from these samples were furthermore tested in an invasion inhibition assay for their antiparasitic activity. RESULTS: Significant intra- and inter- population variation of the reactivity of the samples to the tested antigens were found, as well as a significant positive correlation between MSP1-19 reactivity and invasion inhibition (p < 0.05). Interestingly, male donors showed a significantly higher antibody response to all tested antigens than their female counterparts. In vitro invasion inhibition assays comparing the purified antibodies from the donors from Ghana and Madagascar did not show any statistically significant difference. Although in vitro invasion inhibition increased with breadth of antibody response, the increase was not statistically significant. CONCLUSIONS: The findings support the fact that the development of semi-immunity to malaria is probably contingent on the development of antibodies to not only one, but a range of antigens and that invasion inhibition in immune adults may be a function of antibodies to various antigens. This supports strategies of vaccination including multicomponent vaccines as well as passive vaccination strategies with antibody cocktails.

Genetic diversity of pneumococcal surface protein A of Streptococcus pneumoniae meningitis in German children.

Pneumococcal surface protein A (PspA) is a possible candidate for the development of a pneumococcal vaccine that has the potential to offer a broad range of protection. PspA genes of pneumococcal meningitis isolates (n=40) isolated as part of an ongoing population-based nation-wide surveillance program on invasive pneumococcal disease in children in Germany were analyzed to expand our knowledge on the distribution of PspA families of this important vaccine candidate in Germany. The serotype distribution of the strains was as follows: serotype 4 (n=3), 6B (5), 9V (2), 14 (8), 18C (6), 19F (5), 23F (6), and 7F (5). The pspA genes of these strains could be assigned to 2 families containing 20 pneumococcal strains each. Family I could be subdivided into 2 clades with 17 strains in clade 1 and 3 strains in clade 2, and family II could be subdivided into 3 subgroups (clades 3-5) containing 16, 3, and 1 strain, respectively. Pneumococcal serotypes were distributed evenly over all clades and families. Interestingly, the distribution of the PspA gene families in Germany was seen to differ slightly that found in other countries.

ISSag1 in streptococcal strains of human and animal origin.

The chromosomal region of Streptococcus agalactiae harboring the C5a peptidase and the lmb genes displays the structure of a composite transposon. Its presence in a streptococcal strain is associated with the origin of this strain from a human host. In S. agalactiae it is flanked by two copies of the insertion element ISSag2, and the nucleotide sequence for a third IS element (ISSag1) can be found in this region. Based on amino acid sequence similarity of the deduced transposase ISSag1 belongs to the IS3 family. It is 1251 bp long and flanked by 37 bp imperfect inverted repeats. Horizontal gene transfer among different bacterial species is facilitated by mobile genetic elements. To investigate if ISSag1 homologues are also present in other streptococcal species, various species of pyogenic streptococci from animal and human origin were analyzed by Southern blot hybridization and PCR. Among the different streptococcal species, multiple copies of an ISSag1 homologue could only be detected in S. dysgalactiae subsp. dysgalactiae strains of animal origin. All of the S. agalactiae strains harbored only a single copy, that was always found in strains with the scpB-lmb composite transposon. A single copy of an ISSag1 homologue could also be detected in some of the S. pyogenes and S. dysgalactiae subsp. equisimilis strains. Nucleotide sequencing of the IS element in S. dysgalactiae subsp. dysgalactiae strains revealed several different variants. One of the variants showed the features of a regular IS3 element. The other two variants that were observed displayed a 500-bp deletion and a mosaic structure composed of ISSag1 and ISSag2 homologues. This mosaic structure suggests that recombination and horizontal gene transfer events in S. dysgalactiae strains of bovine origin could have played a role in the assembly of the scpB-lmb composite transposon structure.

Molecular characterisation of macrolide resistance mechanisms of Streptococcus pneumoniae and Streptococcus pyogenes isolated in Germany, 2002-2003.

In the present study, a real-time PCR protocol was developed for the detection of macrolide resistance determinants and was validated in a nationwide study in Germany covering a total of 236 Streptococcus pyogenes and 241 Streptococcus pneumoniae strains collected from children < or = 16 years of age with community-acquired infections. Macrolide resistance was observed in 19.9% of pneumococcal strains and 14% of S. pyogenes isolates. Of the erythromycin A-resistant S. pyogenes strains, 93.9% showed the efflux type mef(A); 62.5% of the S. pneumoniae strains were mef(A)- and 37.5% erm(B)-positive. The correlation of the results of real-time PCR assay genotyping in the present study compared with those of conventional PCR genotyping and resistance phenotyping was 100%. Macrolide resistance is of growing concern in Germany. This highly sensitive and specific PCR assay to detect macrolide resistance has the potential to provide sufficiently rapid results to improve antibiotic treatment of streptococcal infections.