How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches.

Next-generation sequencing (NGS) has instigated the research on the role of the microbiome in health and disease. The compositional nature of such microbiome datasets makes it however challenging to identify those microbial taxa that are truly associated with an intervention or health outcome. Quantitative microbiome profiling overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. Both cell-based methods (e.g., flow cytometry) and molecular methods (qPCR) have been used to determine the absolute microbial abundances, but to what extent different quantification methods generate similar quantitative microbiome profiles has so far not been explored. Here we compared relative microbiome profiling (without incorporation of microbial quantification) to three variations of quantitative microbiome profiling: (1) microbial cell counting using flow cytometry (QMP), (2) counting of microbial cells using flow cytometry combined with Propidium Monoazide pre-treatment of fecal samples before metagenomics DNA isolation in order to only profile the microbial composition of intact cells (QMP-PMA), and (3) molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene. Although qPCR and flow cytometry both resulted in accurate and strongly correlated results when quantifying the bacterial abundance of a mock community of bacterial cells, the two methods resulted in highly divergent quantitative microbial profiles when analyzing the microbial composition of fecal samples from 16 healthy volunteers. These differences could not be attributed to the presence of free extracellular prokaryotic DNA in the fecal samples as sample pre-treatment with Propidium Monoazide did not improve the concordance between qPCR-based and flow cytometry-based QMP. Also lack of precision of qPCR was ruled out as a major cause of the disconcordant findings, since quantification of the fecal microbial load by the highly sensitive digital droplet PCR correlated strongly with qPCR. In conclusion, quantitative microbiome profiling is an elegant approach to bypass the compositional nature of microbiome NGS data, however it is important to realize that technical sources of variability may introduce substantial additional bias depending on the quantification method being used.

Cross-border dissemination of methicillin-resistant Staphylococcus aureus, Euregio Meuse-Rhin region.

Because the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) differs among the 3 countries forming the Euregio Meuse-Rhin (EMR) region (Belgium, Germany, and the Netherlands), cross-border healthcare requires information about the spread of MRSA in the EMR. We investigated the emergence, dissemination, and diversity of MRSA clones in the EMR by using several typing methods. MRSA associated with clonal complexes 5, 8, 30, and 45 was disseminated throughout the EMR. Dutch isolates, mainly associated with sequence types (ST) ST5-MRSA-II, ST5-MRSA-IV, ST8-MRSA-IV, and ST45-MSRA-IV had a more diverse genetic background than the isolates from Belgium and Germany, associated with ST45-MRSA-IV and ST5-MRSA-II, respectively. MRSA associated with pigs (ST398-MRSA-IV/V) was found in the Dutch area of the EMR. Five percent of the MRSA isolates harbored Panton-Valentine leukocidin and were classified as community-associated MRSA associated with ST1, 8, 30, 80, and 89.

An exploration of the gut and environmental resistome in a community in northern Vietnam in relation to antibiotic use.

Background: Antibiotic resistance is a major global public health threat. Antibiotic use can directly impact the antibiotic resistant genes (ARGs) profile of the human intestinal microbiome and consequently the environment through shedding. Methods: We determined the resistome of human feces, animal stools, human food and environmental (rain, well, and irrigative water) samples (n = 304) in 40 households within a community cohort and related the data to antibiotic consumption. Metagenomic DNA was isolated and qPCR was used to determine presence of mobile colistin resistance (mcr) genes, genes encoding extended-spectrum ß-lactamases (ESBL), carbapenemases and quinolone resistance genes. Results: Nearly 40 % (39.5%, 120/304) of samples contained ESBL genes (most frequent were CTX-M-9 (23.7% [72/304]), CTX-M-1 (18.8% [57/304]). Quinolone resistance genes (qnrS) were detected in all human and 91% (41/45) of animal stool samples. Mcr-1 and mcr-3 were predominantly detected in human feces at 88% (82/93) and 55% (51/93) and animal feces at 93% (42/45) and 51% (23/45), respectively. Mcr-2, mrc-4 and mcr-5 were not detected in human feces, and only sporadically (< 6%) in other samples. Carbapenemase-encoding genes were most common in water (15% [14/91]) and cooked food (13% [10/75]) samples, while their prevalence in human and animal stools was lower at 4% in both human (4/93) and animal (2/45) samples. We did not find an association between recent antibiotic consumption and ARGs in human stools. Principal component analysis showed that the resistome differs between ecosystems with a strong separation of ARGs profiles of human and animal stools on the one hand versus cooked food and water samples on the other. Conclusions: Our study indicated that ARGs were abundant in human and animal stools in a rural Vietnamese community, including ARGs targeting last resort antibiotics. The resistomes of animal and human stools were similar as opposed to the resistomes from water and food sources. No association between antibiotic use and ARG profiles was found in a setting of high background rates of AMR.

Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR.

To determine the influence of either exclusive breast-feeding or formula feeding on both composition and quantity of the gut microbiota in infants, we have developed real-time, quantitative PCR assays for the detection of Bifidobacterium spp. and Clostridium difficile. Furthermore, we have monitored the prevalence and counts of Escherichia coli by applying a previously described real-time PCR assay. We found all 100 infants tested to be colonized by Bifidobacterium spp. The bifidobacterial counts were comparable between the 50 breast-fed and 50 formula-fed infants with median values of 10.56 log10 and 10.24 log10 CFU g(-1) wet weight faeces, respectively. C. difficile was detected in 14% of the breast-fed and 30% of the formula-fed infants. In addition, the C. difficile counts were significantly lower in breast-fed infants than in the formula-fed group (median values of 3.28 log10 and 7.43 log10 CFU g(-1), respectively; p=0.03). The prevalence of E. coli in the breast-fed and formula-fed group was 80% and 94%, respectively. Also, the E. coli counts in colonized infants was significantly lower in the breast-fed infants than in the formula-fed group (median values of 9.11 log10 and 9.57 log10 CFU g(-1), respectively; p=0.004). We conclude that the prevalence and counts of C. difficile as well as E. coli are significantly lower in the gut microbiota of breast-fed infants than in that of formula-fed infants, whereas the prevalence and counts of Bifidobacterium spp. is similar among both groups.

The prevalence of the Staphylococcus aureus tst gene among community- and hospital-acquired strains and isolates from Wegener's Granulomatosis patients.

To allow rapid identification of toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus strains, a real-time PCR assay for the detection of the tst gene, which encodes TSST-1, was developed. The assay was applied to S. aureus isolates from patients with Wegener's Granulomatosis (WG), as well as isolates that were classified as either community- (CA) or hospital-acquired (HA). No significant difference in the percentage of tst-positive strains was observed between isolates from WG patients and CA isolates (24% and 25%, respectively). In contrast, only 14% of the HA isolates were tst-positive (p<0.05). Investigation of the clonal relationship between tst-positive CA and HA strains could indicated the recent emergence of a virulent S. aureus clone in the community.

Rapid detection of Panton-Valentine leukocidin from clinical isolates of Staphylococcus aureus strains by real-time PCR.

To allow rapid identification of Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus strains, a real-time PCR assay for detection of PVL was developed. This assay is convenient, since it can be applied directly on bacterial suspensions and does not require previous DNA purification. Furthermore, the assay was found to be highly reproducible, robust and specific, since positive results were generated exclusively with PVL-positive S. aureus strains, and neither with PVL-negative strains nor staphylococci other than S. aureus.

Prevalence and spread of multidrug-resistant Escherichia coli including ST131 in different patient populations in the Euroregion Meuse-Rhine.

AIM: To investigate the prevalence and genetic background of Escherichia coli collected from different patient populations in the Euroregion Meuse-Rhine. MATERIALS & METHODS: Susceptibility testing was performed on 1651 E. coli isolates with broth microdilution. Their genetic background was determined using pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: The prevalence of resistance varied significantly between the populations. Approximately 10% of the E. coli isolates were multidrug-resistant and/or a ß-lactamase producer. The most prevalent extended-spectrum ß-lactamase type was CTX-M-15 and ST131 was the most prevalent multilocus sequence typing type. RESULTS from pulsed-field gel electrophoresis of the ST131 isolates indicate the spread of these isolates in the Euroregion. CONCLUSION: E. coli ST131 was the most prevalent sequence type in our Euroregional study. It is essential to control the spread of these resistant strains (e.g., with infection-control policies, antibiotic stewardship programs and antibiotic resistance surveillance). In this way we could observe shifts in the prevalence of resistance of the E. coli population and act accordingly.

Staphylococcus aureus carriage among GPs in The Netherlands.

BACKGROUND: The extent to which GPs serve as a reservoir for antibiotic-resistant Staphylococcus aureus is unknown and not well studied. AIM: To determine the prevalence of nasal S. aureus carriage among GPs in the Netherlands, as well as the antimicrobial resistance and the genotypes of isolated S. aureus. DESIGN OF STUDY: Observational, point-prevalence, and cross-sectional study. SETTING: GPs attending the annual conference of the Dutch College of General Practitioners in 2006. METHOD: Nasal swabs were randomly taken from 395 GPs and analysed for the presence of S. aureus. Antimicrobial susceptibility was determined by a microbroth dilution method and the genotypes by spa typing, which was associated with multilocus sequence typing. RESULTS: Of the GPs, 129/395 (33%; 95% confidence interval [CI] = 28 to 37%) were carriers of S. aureus. No meticillin-resistant S. aureus (MRSA) was found. Resistance was observed to penicillin (71%; 95% CI = 63 to 79%), fusidic acid (7%; 95% CI = 3 to 13%), and clarithromycin (6%; 95% CI = 3 to 12%). In 72% of the isolates, an MRSA-related genotype of S. aureus was found. CONCLUSION: The low antibiotic resistance found among S. aureus of GPs suggests that GPs are not a reservoir of antibiotic-resistant S. aureus strains. The relatively high resistance to fusidic acid, which has not previously been described in the Netherlands and is mostly because of antibiotic use, suggests that patients infect GPs and not the other way round. GPs may be at risk for nasal carriage of S. aureus with an MRSA-related genotype.

Different clonal complexes of methicillin-resistant Staphylococcus aureus are disseminated in the Euregio Meuse-Rhine region.

The Euregio Meuse-Rhine (EMR) is formed by the border regions of Belgium, Germany, and The Netherlands. Cross-border health care requires infection control measures, in particular since the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) differs among the three countries. To investigate the dissemination of MRSA in the EMR, 152 MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), SCCmec typing, and multilocus sequence typing. PFGE revealed major clonal groups A, G, L, and Q, suggesting dissemination of MRSA in the EMR. Group A harbored mainly SCCmec type III and sequence types (STs) 239 and 241. The majority of the strains from group G harbored SCCmec type I and ST8 and ST247, whereas most strains from group L carried either SCCmec type IV or type I. Within group L, ST8 and ST228 were found, belonging to clonal complexes 8 and 5, respectively. Most strains from group Q included SCCmec type II and were sequence typed as ST225. Both ST225-MRSA-II and ST241-MRSA-III were novel findings in Germany. In addition, the SCCmec type of two isolates has not been described previously. One strain was classified as SCCmec type III but harbored the pls gene and the dcs region. Another strain was characterized as SCCmec type IV but lacked the dcs region. In addition, one isolate harbored both SCCmec type V and Panton-Valentine leukocidin. Finally, the SCCmec type of the strains was found to be correlated with the antibiotic susceptibility pattern.

Different levels of genetic homogeneity in vancomycin-resistant and -susceptible Enterococcus faecium isolates from different human and animal sources analyzed by amplified-fragment length polymorphism.

The genetic relationship among fecal vancomycin-resistant Enterococcus faecium (VREF) and vancomycin-susceptible E. faecium (VSEF) isolates (n = 178) from the same populations of pigs, human healthy volunteers, and hospitalized patients (from The Netherlands) and chickens (from The Netherlands and Greece) was studied by amplified-fragment length polymorphism (AFLP). The majority of VREF isolates from pigs, healthy volunteers, and hospitalized patients grouped together (genetic similarity, >or=65%). In a previous AFLP study by our group the VREF isolates from hospitalized patients grouped separately, most likely because these were clinical and not fecal isolates as in the present study. Furthermore, VSEF isolates from humans and pigs were found much more genetically diverse than VREF isolates, whereas VREF and VSEF isolates from chickens clustered together in a separate genogroup (genetic similarity, >or=65%), a pattern clearly distinct from the patterns for human and pig isolates. The present study suggests that pigs are a more important source of VREF for humans than chickens and that human- and pig-derived VSEF isolates seem much more heterogeneous than VREF isolates.