Peera Hemarajata

Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling.

Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation:

Recent explorations of the human gut microbiota suggest that perturbations of microbial communities may increase predisposition to different disease phenotypes. Dietary nutrients may be converted into metabolites by intestinal microbes that serve as biologically active molecules affecting regulatory functions in the host. Probiotics may restore the composition of the gut microbiome and introduce beneficial functions to gut microbial communities, resulting in amelioration or prevention of gut inflammation and other intestinal or systemic disease phenotypes. This review describes how diet and intestinal luminal conversion by gut microbes play a role in shaping the structure and function of intestinal microbial communities. Proposed mechanisms of probiosis include alterations of composition and function of the human gut microbiome, and corresponding effects on immunity and neurobiology.

The Human Gut Microbiome and Body Metabolism: Implications for Obesity and Diabetes

BACKGROUND Obesity, metabolic syndrome, and type 2 diabetes are major public health challenges. Recently, interest has surged regarding the possible role of the intestinal microbiota as potential novel contributors to the increased prevalence of these 3 disorders. CONTENT Recent advances in microbial DNA sequencing technologies have resulted in the widespread application of whole-genome sequencing technologies for metagenomic DNA analysis of complex ecosystems such as the human gut. Current evidence suggests that the gut microbiota affect nutrient acquisition, energy harvest, and a myriad of host metabolic pathways. CONCLUSION Advances in the Human Microbiome Project and human metagenomics research will lead the way toward a greater understanding of the importance and role of the gut microbiome in metabolic disorders such as obesity, metabolic syndrome, and diabetes.

First Report of Ceftazidime-Avibactam Resistance in a KPC-3-Expressing Klebsiella pneumoniae Isolate

ABSTRACT Ceftazidime-avibactam is the first antimicrobial approved by the U.S. FDA for the treatment of carbapenem-resistant Enterobacteriaceae. Avibactam, a non-β-lactam β-lactamase inhibitor, inactivates class A serine carbapenemases, including Klebsiella pneumoniae carbapenemase (KPC). We report a KPC-producing K. pneumoniae isolate resistant to ceftazidime-avibactam (MIC, 32/4 μg/ml) from a patient with no prior treatment with ceftazidime-avibactam.

Lactobacillus reuteri-Specific Immunoregulatory Gene rsiR Modulates Histamine Production and Immunomodulation by Lactobacillus reuteri

Human microbiome-derived strains of Lactobacillus reuteri potently suppress proinflammatory cytokines like human tumor necrosis factor (TNF) by converting the amino acid l-histidine to the biogenic amine histamine. Histamine suppresses mitogen-activated protein (MAP) kinase activation and cytokine production by signaling via histamine receptor type 2 (H2) on myeloid cells. Investigations of the gene expression profiles of immunomodulatory L. reuteri ATCC PTA 6475 highlighted numerous genes that were highly expressed during the stationary phase of growth, when TNF suppression is most potent. One such gene was found to be a regulator of genes involved in histidine-histamine metabolism by this probiotic species. During the course of these studies, this gene was renamed the Lactobacillus reuteri-specific immunoregulatory (rsiR) gene. The rsiR gene is essential for human TNF suppression by L. reuteri and expression of the histidine decarboxylase (hdc) gene cluster on the L. reuteri chromosome. Inactivation of rsiR resulted in diminished TNF suppression in vitro and reduced anti-inflammatory effects in vivo in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of acute colitis. A L. reuteri strain lacking an intact rsiR gene was unable to suppress colitis and resulted in greater concentrations of serum amyloid A (SAA) in the bloodstream of affected animals. The PhdcAB promoter region targeted by rsiR was defined by reporter gene experiments. These studies support the presence of a regulatory gene, rsiR, which modulates the expression of a gene cluster known to mediate immunoregulation by probiotics at the transcriptional level. These findings may point the way toward new strategies for controlling gene expression in probiotics by dietary interventions or microbiome manipulation.

Development of a novel real-time PCR assay with high-resolution melt analysis to detect and differentiate OXA-48-like β-lactamases in carbapenem-resistant Enterobacteriaceae

ABSTRACT The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using blaOXA-48-like-specific primers coupled with an unlabeled 3′-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including blaKPC, blaSME, blaIMP, blaNDM-1, blaVIM, blaOXA-48, blaOXA-162, blaOXA-181, blaOXA-204, blaOXA-244, blaOXA-245, and blaOXA-232. Our assay identified the presence of blaOXA-48-like β-lactamase genes and clearly distinguished between blaOXA-48 and its variants in control strains, including between blaOXA-181 and blaOXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.

Resistance to Ceftazidime-Avibactam in Klebsiella pneumoniae Due to Porin Mutations and the Increased Expression of KPC-3.

We reported the first clinical case of a ceftazidime-avibactam resistant KPC-3producing Klebsiella pneumoniae (1), from a patient with no history of ceftazidimeavibactam therapy. We now present data documenting mechanisms of ceftazidimeavibactam resistance in this isolate. Whole-genome sequencing (WGS) was performed on two isolates: KP1245 (ceftazidime-avibactam MIC, 4 g/ml; from blood on hospital day 1; referred to as isolate 1 in our previous report [1]) and KP1244 (ceftazidimeavibactam MIC, 32 g/ml; from blood on hospital day 2; referred to as isolate 2 in our previous report [2]), using MiSeq (Illumina, San Diego, CA) and PacBio RSII (Menlo Park, CA) systems (2). The in silico multilocus sequence type (ST) was ST258. Single nucleotide polymorphism (SNP) analysis revealed 17 SNPs between KP1245 and KP1244, indicating that the isolates were related but that significant diversity existed in this patient (2). Nonsynonymous mutations are shown in Table 1; the most striking of these is in the OmpK36 porin gene. KP1244 contained a missense mutation predicted to encode a T333N mutation. Both isolates also harbored a mutation predicted to encode R191L in OmpK36 and had a nonfunctional OmpK35, due to a frameshift mutation that truncated the protein at amino acid 42, common to K. pneumoniae ST258 (3). Association between mutations in ompK36 and elevated ceftazidime-avibactam MICs has been shown previously (4). However, T333N, found in one of the -sheet domains of the OmpK36 subunit, has not been described in K. pneumoniae; as such, further validation is required to confirm the role of the OmpK36 mutation in this isolate’s ceftazidimeavibactam resistance phenotype. Both isolates harbored blaSHV-11 on the chromosome and blaKPC-3 and blaSHV-12 on the sole plasmid. The plasmid was a 205-kb plasmid, pUCLAKPC with an IncX3 origin of replication. Two copies of the blaKPC-3 Tn4401d transposon were in both isolates (5). Plasmid copy number was estimated, from WGS read coverage data and real-time PCR comparing efficiency-corrected cycle thresholds (CT) between blaKPC-3 and the 16S rRNA genes (6), to be 4 for both isolates. Real-time quantitative PCR from 3 experiments demonstrated that the blaKPC-3 expression of KP1244 was 3.80.2-fold that of KP1245. This correlated with meropenem MICs of 512 g/ml versus 16 g/ml. Of note, no mutations in the -loop were found in blaKPC-3 genes. Surveillance studies continue to document very low rates of resistance to ceftazidimeavibactam among the Enterobacteriaceae (7). However, testing of ceftazidime-avibactam resistance is not common in microbiology laboratories, and resistance may be underappreciated. While our patient had no exposure to ceftazidime-avibactam, she was treated with meropenem and cefepime (1), providing the selective pressure required for mutation to OmpK36. Emergence of mutations to OmpK36 during treatment with carbapenems is relatively common (8, 9). While OmpK35 and OmpK36 are not thought Accepted manuscript posted online 10 April 2017 Citation Humphries RM, Hemarajata P. 2017. Resistance to ceftazidime-avibactam in Klebsiella pneumoniae due to porin mutations and the increased expression of KPC-3. Antimicrob Agents Chemother 61:e00537-17. https://doi.org/10.1128/AAC.00537-17. Copyright

Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae

ABSTRACT In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

Resistance to ceftazidime-avibactam is due to transposition of KPC in a porin-deficient strain of Klebsiella pneumoniae with increased efflux activity.

ABSTRACT Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 μg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.

Implementation of a Rapid Genotypic Assay to Promote Targeted Ciprofloxacin Therapy of Neisseria gonorrhoeae in a Large Health System

Multidrug-resistant Neisseria gonorrhoeae is a top threat to public health. In November 2015, UCLA Health introduced a rapid gyrase A (gyrA) genotypic assay for prediction of Neisseria gonorrhoeae susceptibility to ciprofloxacin. We found a significant reduction in ceftriaxone use with a concomitant increase in targeted therapy.