Clinical relevance of colonization with antimicrobial-resistant bacteria (AMRB) and methicillin susceptible Staphylococcus aureus (MSSA) for mothers during pregnancy.

PURPOSE: The impact of colonization with antimicrobial-resistant bacteria (AMRB) and methicillin-sensitive Staphylococcus aureus (MSSA) of healthy pregnant women is not described in detail in Germany. In this study, we screened for MSSA and AMRB, especially for methicillin-resistant S. aureus (MRSA) as well as extended-spectrum beta-lactamase (ESBL)-producing E. coli. Potential risk factors for colonization with AMRB/MSSA and the potential effects of colonization with these on the obstetric population were investigated. METHODS: From October 2013 until December 2015 pregnant women were screened before birth for colonization with AMRB/MSSA from the mammillae, nose, perianal and vaginal area. Before birth, the expectant mother was administered a standardized interview questionnaire by a trained interviewer. Data from the hospital admission records were also included. RESULTS: Samples from 651 pregnant women were analyzed. Colonization with MSSA was detected in 14.3% (n = 93), AMRB in 3.5% [(n = 23); MRSA: n = 3/ESBL: n = 20]. Significantly more colonization of AMRB/MSSA could be detected in women who had previously given birth compared to women who were nulliparous (p < 0.05). MSSA colonization was significantly associated with self-reported respiratory diseases during pregnancy (p < 0.05), but AMRB/MSSA colonization was not statistically associated with other types of infection. CONCLUSION: Our results demonstrate a low overall rate of colonization with AMRB/MSSA, as well as a low percentage of colonized pregnant women who developed infections. Multiparous women are at higher risk for colonization with MSSA/MRSA or ESBL. Because the prevalence of AMRB/MSSA is low, this study suggests that general screening of pregnant women without risk factors is not recommended.

Evaluation of sampling locations in pregnant women and newborns for the detection of colonisation with antibiotic-resistant bacteria.

Up to now, little has been known about the prevalence and clinical relevance of colonisation of asymptomatic pregnant women with methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) or extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. In this two-centre cross-sectional study, we evaluated the performance and importance of screening at different times and different locations for colonisation in pregnant women and newborns. Between October 2013 and December 2015, four samples were collected from pregnant women, two from newborns at birth and three from 3-day-old newborns. Samples were screened on culturing media and were confirmed with molecular methods. MSSA was used as a surrogate for MRSA, as the two share most microbiologic characteristics and colonisation patterns. Of 763 pregnant women, 14.5% (111) were colonised with MSSA, 0.4% (3) with MRSA and 2.6% (20) with ESBL-producing E. coli. Of 658 newborns, 0.9% (10) were colonised with MSSA at birth and 13.1% (70) at 3 days old, 0.5% (3) were colonised with MRSA and 2.6% (17) with ESBL-producing E. coli. Nasal sampling identified 91.0% of MSSA-colonised pregnant women and 60.0% of newborns. In newborns, nasal and umbilical sampling at 3 days after birth discovered 84.0% of colonised cases. For ESBL-producing E. coli, the perianal region was positive in all colonised pregnant women and in 88.2% of colonised newborns. Combining nasal and perianal swabs is optimal when screening for antibiotic-resistant bacteria in pregnant women. Nasal, perianal and umbilical sample collection from 3-day-old newborns significantly increased the sensitivity compared to screening immediately after birth.

Single-nucleotide polymorphism in the SCCmec-orfX junction distinguishes between livestock-associated MRSA CC398 and human epidemic MRSA strains.

A number of real-time PCR assays for direct detection of methicillinresistant (MRSA) in clinical specimens are targeting staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. When testing 184 MRSA strains of human and animal origin from geographically distinct locations, we identified several characteristic single-nucleotide polymorphisms (SNPs) within the SCCmec-orfX junction of livestock-associated (LA) MRSA CC398 which serve as suitable strain markers for screening purposes. Within an assay time of 60 minutes and an additional 10 minutes for the melting curve analysis, all MRSA CC398 isolates were correctly identified by their characteristic T(m) value in the commercial LightCycler MRSA Advanced test. Studies to confirm the diagnostic accuracy of the SNP-based strain identification assay with a larger collection of clinical and LA-MRSA strains are ongoing.

Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.

Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.

Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments.

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and beta-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, beta-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species.

Specific detection of green sulfur bacteria by in situ hybridization with a fluorescently labeled oligonucleotide probe.

An oligodeoxynucleotide probe (GSB-532) specific for green sulfur bacteria was developed. Highly stringent hybridization conditions were established using whole cells of Chlorobium limicola DSM249 immobilized on glass slides. At a formamide concentration of 10%, the optimum specificity was reached at 47 degrees C. When a conventional fixation procedure was used, a conspicuous autofluorescence developed within the cells. This autofluorescence was due to the liberation of bacteriochlorophyll by the detergent Triton X-100 and a subsequent conversion to bacteriophenophytin and related compounds. The signal-to-noise ratio could be increased by a final dehydration of the samples with methanol. Finally, the method was adapted to the hybridization of natural samples collected on polycarbonate membrane filters. In situ hybridization of pure cultures, various enrichments, and natural samples from the chemocline of a freshwater lake confirmed that probe GSB-532 hybridized exclusively to cells of green sulfur bacteria. Our protocol allows the highly specific detection of green sulfur bacteria in water samples and a rapid screening of natural bacterial communities. Employing probe GSB-532, the phylogenetic affiliation of the epibionts in "Chlorochromatium aggregatum" and "Pelochromatium roseum" could be demonstrated for the first time.

The ecological niche of the consortium "Pelochromatium roseum"

A dense accumulation of the phototrophic consortium "Pelochromatium roseum" in a small, eutrophic, freshwater lake (Dagowsee, Brandenburg, Germany) was investigated. Within the chemocline, the number of epibionts of the consortia represented up to 19% of the total number of bacteria. Per "P. roseum" a mean value of 20 epibionts was determined. Similar to other aquatic habitats, consortia in the Dagowsee were found only at low light intensities (< 7 &mgr;mol quanta m-2 s-1) and low sulfide concentrations (0-100 &mgr;M). In dialysis cultures of "P. roseum", bacterial cells remained in a stable association only when incubated at light intensities between 5 and 10 &mgr;mol quanta m-2 s-1. Intact consortia from natural samples had a buoyant density of 1046.8 kg m-3, which was much higher than that of ambient chemocline water (995.8 kg m-3). Under environmental conditions and without motility, this density difference would result in rapid sedimentation of consortia toward the lake bottom. Our results indicate that (1) consortia are adapted to a very narrow regime of light intensities and sulfide concentrations, (2) motility and tactic responses must be of ecological significance for the colonization of the free water column of lakes, and (3) phototrophic growth of consortia can be explained only by a cycling of sulfur species in the chemocline, possibly within the consortia themselves.

Phylogeny and molecular fingerprinting of green sulfur bacteria.

The 16S rDNA sequences of nine strains of green sulfur bacteria (Chlorobiaceae) were determined and compared to the four known sequences of Chlorobiaceae and to sequences representative for all eubacterial phyla. The sequences of the Chlorobiaceae strains were consistent with the secondary structure model proposed earlier for Chlorobium vibrioforme strain 6030. Similarity values > 90.1% and Knuc values < 0.11 indicate a close phylogenetic relatedness among the green sulfur bacteria. As a group, these bacteria represent an isolated branch within the eubacterial radiation. In Chlorobiaceae, a similar morphology does not always reflect a close phylogenetic relatedness. While ternary fission is a morphological trait of phylogenetic significance, gas vesicle formation occurs also in distantly related species. Pigment composition is not an indicator of phylogenetic relatedness since very closely related species contain different bacteriochlorophylls and carotenoids. Two different molecular fingerprinting techniques for the rapid differentiation of Chlorobiaceae species were investigated. The 16S rDNA fragments of several species could not be separated by denaturing gradient gel electrophoresis. In contrast, all strains investigated during the present work gave distinct banding patterns when dispersed repetitive DNA sequences were used as targets in PCR. The latter technique is, therefore, well suited for the rapid screening of isolated pure cultures of green sulfur bacteria.