Impact of expression mode and timing of sample collection, relative to milk ejection, on human milk bacterial DNA profiles.

AIM: To investigate the impact of expression mode: electric breast pump or hand expression, and timing of sample collection: pre- and post-milk ejection on human milk (HM) bacterial DNA profiles. METHODS AND RESULTS: Three HM samples from the same breast were collected from 30 breastfeeding mothers: a pre-milk ejection pump-expressed sample (pre-pump), a post-milk ejection pump-expressed sample (post-pump) and a post-milk ejection hand-expressed sample (post-hand). Full-length 16S rRNA gene sequencing was used to assess milk bacterial DNA profiles. Bacterial profiles did not differ significantly based on mode of expression nor timing of sample collection. No significant differences were detected in the relative abundance of any OTUs based on expression condition (pre-pump/ post-pump and post-pump/post-hand) with univariate linear mixed-effects regression analyses (all P-values > 0·01; α = 0·01). Similarly, no difference in richness was observed between sample types (number of observed OTUs: post-pump/post-hand P = 0·13; pre-pump/post-pump P = 0. 45). CONCLUSION: Bacterial DNA profiles of HM did not differ according to either expression method or timing of sample collection. SIGNIFICANCE AND IMPACT OF THE STUDY: Hand or pump expression can be utilized to collect samples for microbiome studies. This has implications for the design of future HM microbiome studies.

DNA extraction method influences human milk bacterial profiles.

AIMS: To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM). METHODS AND RESULTS: Human milk DNA was extracted using the following methods: (i) Qiagen MagAttract Microbial DNA Isolation Kit (kit QM), (ii) Norgen Milk Bacterial DNA Isolation Kit (kit NM), (iii) Qiagen MagAttract Microbiome DNA/RNA Isolation Kit (kit MM) and (iv) TRIzol LS Reagent (method LS). The full-length 16S rRNA gene was sequenced. Kits MM and method LS were unable to extract detectable levels of DNA in 9/11 samples. Detectable levels of DNA were recovered from all samples using kits NM (mean = 0·68 ng µl-1 ) and QM (mean = 0·55 ng µl-1 ). For kits NM and QM, the greatest number of reads were associated with Staphylococcus epidermidis, Streptococcus vestibularis, Propionibacterium acnes, Veillonella dispar and Rothia mucilaginosa. Contamination profiles varied substantially between kits, with one bacterial species detected in negative extraction controls generated with kit QM and six with kit NM. CONCLUSIONS: Kit QM is the most suitable of the kits tested for the extraction of bacterial DNA from human milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of extraction method impacts the efficiency of bacterial DNA extraction from human milk and the resultant bacterial community profiles generated from these samples.

Detection of group B Streptococcus during antenatal screening in Western Australia: a comparison of culture and molecular methods.

AIM: Global screening strategies for Group B Streptococcus (GBS) include risk- or culture-based methods to guide intrapartum prophylaxis. In Western Australia (WA), antenatal culture-based screening is routine; however, numerous culture methods exist, in addition to molecular methods. We aimed to assess the comparability of research and diagnostic screening approaches. METHODS AND RESULTS: Vaginal and rectal swabs were self-collected by pregnant women (n = 531) from King Edward Memorial Hospital, WA, in parallel to routine screening (35-37 weeks of gestation). Research methods involved culture (Strep B Carrot Broth™ and StrepB CHROMagar™) and molecular methods (real-time PCR) and were compared to routine diagnostic screening (Lim Broth and Granada agar). Overall, GBS detection was comparable between research and diagnostic approaches (3-5% discrepancy, kappa = 0·76). Specificity/sensitivity of Carrot Broth™ was 100%/89%, while that of CHROMagar™ was 73%/100%, respectively. Direct PCR was unable to detect GBS in ~18% of specimens which were culture positive; however, it exhibited 100% specificity. CONCLUSIONS: This clinical evaluation of GBS screening methods provides support for current practice. SIGNIFICANCE AND IMPACT OF THE STUDY: Although CHROM was highly sensitive, further testing is recommended due to a high false-positive rate. Molecular assays are useful for rapid detection; however, low-titre samples may require additional enrichment prior to molecular analysis to improve sensitivity.

Re: "Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community" (2018) 6:87, https://doi.org/10.1186/s40168-018-0475-7.

A recent publication by Lim et al. 2018 (Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community) strongly concluded that the microbiome of amniotic fluid primarily originates from reagent contamination. However, upon closer inspection of the methods used and data presented in this study, in particular the supplementary data, such conclusions do not appear to be supported by the results. We outline such methodological/data interpretation concerns and invite the authors to discuss these.

Characterization of the bacterial microbiome in first-pass meconium using propidium monoazide (PMA) to exclude nonviable bacterial DNA.

Numerous studies have reported bacterial DNA in first-pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell-free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first-pass meconium (n = 5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20%. PMA-treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P = 0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell-free/nonviable DNA. The mean Sørensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus. SIGNIFICANCE AND IMPACT OF THE STUDY: DNA-based microbiome studies performed using 16S rRNA gene sequencing are limited by their inability to discriminate between live bacterial cells, dead bacterial cells and cell-free DNA. Here we use propidium monoazide (PMA) to exclude nonviable bacteria from microbiome analysis of first-pass meconium samples and thereby reveal that the majority of the purported fetal gut microbiome is from intact bacterial cells. This work demonstrates the importance of excluding nonviable bacteria when analysing the microbial community in low-biomass samples such as meconium.

Identification and removal of contaminating microbial DNA from PCR reagents: impact on low-biomass microbiome analyses.

Reagent-derived contamination can compromise the integrity of microbiome data, particularly in low microbial biomass samples. This contamination has recently been attributed to the 'kitome' (contamination introduced by the DNA extraction kit), prior to which attention was mostly paid to potential contamination introduced by PCR reagents. In this study, we assessed the proportion to which our DNA extraction kit and PCR master mix introduce contaminating microbial DNA to bacterial microbial profiles generated by 16S rRNA gene sequencing. Utilizing a commercial dsDNase treatment protocol to decontaminate the PCR master mix, we demonstrated that the vast majority of contaminating DNA was derived from the PCR master mix. Importantly, this contamination was almost completely eliminated using the simple dsDNase treatment, resulting in a 99% reduction in contaminating bacterial reads. We suggest that dsDNase treatment of PCR reagents should be explored as a simple and effective way of reducing contamination in low-biomass microbiome studies and producing more robust and reliable data. SIGNIFICANCE AND IMPACT OF THE STUDY: Reagent contamination with microbial DNA is a major problem in microbiome studies of low microbial biomass samples. Levels of such contaminating DNA often outweigh what is present in the sample and heavily confound subsequent data analysis. Previous studies have suggested this contamination is primarily derived from DNA extraction kits. Here, we identified the PCR master mix as the primary source of contamination, and showed that enzymatic removal of the contamination drastically reduced the blank signal and improved precision. Decontamination of PCR master mixes may have the potential to improve the sensitivity and accuracy of low-biomass microbiome studies.

One-step simultaneous detection of Ureaplasma parvum and genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology.

Ureaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high-resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species-level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one-step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 × 10-5  ng DNA (SV3) and 0.4 × 10-6  ng DNA (U. parvum, SV1 and SV6). Compared to our HRM PCR, the PlexPCR defined genotype distribution in all seven cases previously reported as 'mixed', and detected another eight cases where multiple genotypes (two) were present in samples previously reported as single genotypes using HRM PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Ureaplasma spp. have been associated with prematurity for decades, however, only a minority of studies have examined this beyond the genus level. In those that have, Ureaplasma parvum has been strongly associated with preterm birth. We recently demonstrated this in Australian women and further showed that U. parvum genotype SV6 was of particular significance. Our PlexPCR assay allows rapid detection and concurrent genotyping of U. parvum in clinical samples and may be of particular interest to obstetricians, particularly those caring for women at a high risk of preterm birth, and any other disease phenotypes where U. parvum is of interest.

Modelling the bacterial communities associated with cystic fibrosis lung infections.

In many human diseases that cystic fibrosis (CF) patients suffer from, for example, lung infections, bacteria have been considered to grow as biofilms. The ability of key CF pathogens such as Pseudomonas aeruginosa to resist antibiotic therapies may be due to the poor drug penetration of these biofilms. The overall aim of this study was to develop biofilm models in vitro that resembled the bacterial species composition of CF sputa. Here, this was a step towards a longer term goal of forming multiple bacterial biofilm models in vitro that would serve, in turn, as better assays of antibiotic susceptibilities than conventionally grown cells. Biofilm models were constructed from 31 CF sputum samples, using a modified microtitre plate assay. Three forms of assessment of these biofilms were made, namely, the mass, microscopic analysis and species composition. Species composition in sputa and biofilms, characterised by terminal restriction fragment length polymorphism (T-RFLP) analysis of ribosomal gene polymerase chain reaction (PCR) products amplified from directly extracted nucleic acids, indicated that the bacterial community in sputa was well reproduced in the biofilm models. Typically, fresh sputa contained 4.6 +/- 2.3 bacterial species, with the species number decreasing to 4.0 +/- 1.6 over 5 days-this was not statistically significant (p = 0.29). This study outlines a novel methodology by which to generate and study bacterial biofilms communities. It is also hoped that the versatility of this in vitro approach, combined with its simplicity and high reproducibility, will make it an effective system to study CF sputum biofilm development and, in the longer term, serve as a means of assessing antibiotic susceptibilities.

Microbial diversity of mid-stage Palinurid phyllosoma from Great Barrier Reef waters.

AIMS: This study aimed to determine the bacterial community associated with wild-caught, mid-stage larvae of spiny lobsters (Palinuridae) in their native oligotrophic marine environment, and to compare their diversity and composition with communities associated with aquaculture-reared larvae of the tropical rock lobster Panulirus ornatus. METHODS AND RESULTS: Bacterial clone libraries constructed from wild P. ornatus (two libraries) and Panulirus penicillatus (one library) larvae (phyllosoma) revealed a dominance of alpha-proteobacterial sequences, with Sulfitobacter spp.-affiliated sequences dominating both P. ornatus libraries and constituting a major portion of the P. penicillatus library. Vibrio-related sequences were rarely detected from wild phyllosoma clone libraries in contrast to similar studies of aquaculture-reared animals. Scanning electron microscopy analysis revealed low levels of bacterial colonization on the external carapace of wild phyllosoma, again in contrast to aquaculture-reared animals, which are often colonized with filamentous bacteria (mainly Thiothrix sp.) that compromise their health. Fluorescence in situ hybridization of sectioned wild phyllosoma tissue displayed low overall abundance of bacteria within the tissue and on external surfaces, with alpha-, beta-, and gamma-Proteobacteria being confirmed as members of this bacterial community. CONCLUSIONS: The consistency in predominant clone sequences retrieved from the three libraries indicated a conserved microbiota associated with wild phyllosoma. In addition, the observed differences in the microbial composition and load of reared and wild phyllosoma are indicative of the different environments in which the animals live. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial disease during early larval stages is a major constraint currently hindering the development of an aquaculture industry for the ornate rock lobster P. ornatus. Knowledge of the microbial community associated with wild animals will be advantageous for the identification of bacteria that may promote animal health.

Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W.

A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.

Parathyroid hyperplasia: an unusual cause of neonatal hypercalcemia.

Primary hyperparathyroidism (PHPT) in infants is caused by parathyroid chief cell hyperplasia. Patients present with symptoms of chronic hypercalcemia, such as failure to thrive, irritability, abdominal pain, and anorexia. Medical therapy is inadequate, often resulting in chronic hypercalcemia or death. Partial or total surgical removal of the parathyroid gland is the preferred treatment. We describe a case of a 7-month-old infant with PHPT secondary to hyperplasia successfully treated with a subtotal parathyroidectomy.

Cloning and nucleotide sequence of amidase gene from Pseudomonas putida.

Amidases are a class of enzymes which convert amides to acids and have potential value in the development of commercial bioprocesses for the production of useful chemicals. A gene encoding an amidase in Pseudomonas putida 5B has been cloned, sequenced, and overexpressed in Escherichia coli. An additional open reading frame (P38K) encoding a putative protein of 38 kDa was found immediately upstream of the amidase gene. This work continues our characterization of a P. putida operon, which now appears to include P38K, amidase, and a stereo-specific nitrile hydratase. This characterization underlies continuing efforts in biocatalyst development.

Engineering Pichia pastoris for biocatalysis: co-production of two active enzymes.

High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp). In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6. MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase. This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.

A stereoselective cobalt-containing nitrile hydratase.

Nitrile hydratase from Pseudomonas putida NRRL-18668 has been purified and characterized. The purified enzyme catalyzes the hydration of 2(S)-(4'-chlorophenyl)-3-methylbutyronitrile at least fifty times faster than that of 2(R)-(4'-chlorophenyl)-3-methylbutyronitrile. This enzyme is a member of the class of nitrile hydratase that contains cobalt. Visible absorption and CD spectra suggest the cobalt exists as a non-corrin low-spin Co3+ ion in a tetragonally-distorted octahedral ligand field. Chemical reduction of the native enzyme results in a species with the EPR signature of a low-spin Co2+ complex. Like the other cobalt-containing nitrile hydratases, this enzyme is relatively stable, maintaining its activity below 35 degrees C, and it shows a broad activity optimum between pH 7.2 and 7.8. The structural genes for this enzyme have been cloned and sequenced. The deduced amino acid sequences for the alpha and beta subunits show 48-63% and 35-41% homology, respectively, to other sequenced nitrile hydratases. In particular, the cysteine residues in the alpha subunit that have been suggested to coordinate the metal ion in the iron-containing nitrile hydratases [Brennan, B. A., Cummings, J. G., Chase, D. B., Turner, I. M., Jr., & Nelson, M. J. (1996) Biochemistry 35, 10068-10077] are conserved in this enzyme, suggesting that this nitrile hydratase, like the enzyme from Rhodococcus rhodochrous J1, is a member of a newly described class of metalloenzymes with Co3+-thiolate ligation [Brennan, B. A., Alms, G., Nelson, M. J., Durney, L. T., & Scarrow, R. C. (1996) J. Am. Chem. Soc. 118, 9194-9195].

Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein.

The stereoselective nitrile hydratase (NHase) from Pseudomonas putida 5B has been over-produced in Escherichia coli. Maximal enzyme activity requires the co-expression of a novel downstream gene encoding a protein (P14K) of 127 amino acids, which shows no significant homology to any sequences in the protein database. Nitrile hydratase produced in transformed E. coli showed activity as high as 472 units/mg dry cell (sixfold higher than 5B), and retained the stereoselectivity observed in the native organism. Separated from the end of the beta subunit by only 51 bp, P14K appears to be part of an operon that includes the alpha and beta structural genes of nitrile hydratase, and other potential coding sequences.

High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: engineering a biocatalyst.

Glycolate oxidase (GO) is a flavo-enzyme that catalyzes the oxidation of glycolate, and is useful for the biocatalytic production of glyoxylate. We have produced high levels of spinach GO in the methylotrophic yeast Pichia pastoris (Pp), by chromosomal integration of multiple copies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter. Under fermentation conditions, greater than 250 units of GO per gram of cells (wet weight) was obtained, corresponding to roughly 20-30% of soluble cell protein. This recombinant Pp strain was used as a whole-cell biocatalyst for conversion of glycolic acid to glyoxylic acid.

Sphingosine regulates Ca(2+)-ATPase and reloading of intracellular Ca2+ stores in the pancreatic acinar cell.

The purpose of present study was to examine the effects of sphingosine on cellular Ca2+ transports using dispersed rat pancreatic acini. The results demonstrated that sphingosine had a specific effect to inhibit Ca2+ uptake into the cell's agonist-sensitive pool as well as inhibiting microsomal Ca(2+)-ATPase. The ability of sphingosine to inhibit Ca2+ uptake resulted in both augmentation of Ca2+ release from the pool by inositol 1,4,5-trisphosphate (IP3) and conversion of the Ca2+ release by inositol 1,4,5-trisphosphate from a transient response to a sustained response. Furthermore, by preventing Ca2+ pool refilling sphingosine mimicked the effect of the agonist, carbachol, to maintain an increased [Ca2+]i during sustained stimulation. These results suggest that regulation of Ca(2+)-ATPase by sphingosine or a sphingosine-like agent mediates some of the effects of agonist on cell Ca2+ transports.

Use of alkaline phosphatase fusions to study protein secretion in Bacillus subtilis.

We have constructed a vector designed to facilitate the study of protein secretion in Bacillus subtilis. This vector is based on a translational fusion between the expression elements and signal sequence of Bacillus amyloliquefaciens alkaline protease and the mature coding sequence for Escherichia coli alkaline phosphatase (phoA). We show that export of alkaline phosphatase from B. subtilis depends on a functional signal sequence and that alkaline phosphatase activity depends upon secretion. The vector design facilitates the insertion of heterologous coding sequences between the signal and phoA to generate three-part translational fusions. Such phoA fusions are easily analyzed by monitoring alkaline phosphatase activity on agar plates or in culture supernatants or by immunological detection. Exploitation of this methodology, which has proven to be extremely useful in the study of protein secretion in E. coli, has a variety of applications for studying protein secretion in B. subtilis.

Dual pathways for agonist-stimulated arachidonic acid release in pancreatic acini: roles in secretion.

The present experiments were performed to determine pathways responsible for arachidonic acid release stimulated by cholecystokinin (CCK) and phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), and the roles of pathways in the secretory response in dispersed acini from guinea pig pancreas. Both CCK-octapeptide (CCK-OP) and PMA increased intracellular arachidonic acid. To determine the source of released arachidonic acid, we measured the effects of PMA and CCK-OP on cellular 1,2-diacylglycerol and lysophosphatidylcholine (LPC) and of diglyceride lipase inhibitor RHC 80267 on [3H]arachidonic acid release. Both PMA and CCK-OP increased 1,2-diacylglycerol and LPC. RHC 80267 had no effect on LPC but inhibited the increase in [3H]arachidonic acid release with a concentration of CCK-OP that was maximal for enzyme secretion. The increase in [3H]arachidonic acid release with PMA or a supramaximal concentration of CCK-OP was not inhibited by RHC 80267. In parallel fashion, RHC 80267 inhibited amylase release caused by maximally effective concentrations of CCK-OP but not that caused by PMA or by supramaximally effective concentrations of CCK-OP. Arachidonic acid stimulated amylase release. Exogenous addition of phospholipase A2 caused increases in [3H]arachidonic acid release, LPC formation, and amylase release. The results indicate that there are at least two pathways responsible for the increase in free cellular arachidonic acid stimulated by pancreatic agonists. One is sequential action of phospholipase C and diglyceride lipase on phosphatidylinositol. The other is a phospholipase A action on phosphatidylcholine. The results also suggest a stimulatory role for both pathways in the secretory response.

Cyclic GMP regulates free cytosolic calcium in the pancreatic acinar cell.

The present studies were performed in order to measure the effects of cyclic GMP (cGMP) on the regulation of free cytosolic calcium [( Ca2+]i) in the pancreatic acinar cell. In guinea pig dispersed pancreatic acini the findings demonstrated that the Ca2+ ionophore, Br A23187, caused a sustained increase in [Ca2+]i in the presence of 3 mM CaCl2 in the media and a transient 20 fold rise in cellular cGMP followed by a sustained 3-4 fold rise in cellular cGMP. Increasing cellular cGMP with nitroprusside, hydroxylamine or dibutyryl cGMP had no effect on resting [Ca2+]i. However, these agents attenuated the increase in [Ca2+]i resulting from Br A23187-induced Ca2+ influx. Nitroprusside also attenuated the carbachol-induced sustained rise in [Ca2+]i that resulted from Ca2+ influx. The nitroprusside effect on carbachol-stimulated acini occurred without decreasing Ca2+ influx across the plasma membrane or alteration in the mobilization of Ca2+ from the intracellular agonist-sensitive pool. Inhibition of the increase in cellular cGMP caused by Br A23187 by the guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), resulted in augmentation of the increase in [Ca2+]i. This augmentation was reversed with dibutyryl cGMP. These results indicated that cGMP regulated [Ca2+]i in the pancreatic acinar cell. The mechanism involves the removal of Ca2+ from the cytoplasm.