Exploring secretory proteome and cytokine kinetic of human peripheral blood mononuclear cells exposed to methicillin-resistant Staphylococcus aureus biofilms and planktonic bacteria.

Staphylococcus aureus is a highly successful pathogen infecting various body parts and forming biofilms on natural and artificial surfaces resulting in difficult-to-treat and chronic infections. We investigated the secreted cytokines and proteomes of isolated peripheral blood mononuclear cells (PBMCs) from healthy volunteers exposed to methicillin-resistant S. aureus (MRSA) biofilms or planktonic bacteria. Additionally, the cytokine profiles in sera from patients with community-acquired pneumonia (CAP) caused by S. aureus were investigated. The aim was to gain insights into the immune response involved and differentiate between the planktonic and sessile MRSA forms. We identified 321 and 298 targets that were significantly differently expressed in PBMCs when exposed to planktonic or biofilm-embedded bacteria, respectively. PBMCs exposed to planktonic MRSA cells secreted increased levels of TNF-α, while IL-18 was elevated when exposed to the biofilm. The machine-learning analyses of the cytokine profiles obtained for the in vitro PBMCs and CAP sera distinguished between the two types of bacteria forms based on cytokines IL-18, IL12, and IL-17, and with a lower importance IL-6. Particularly, IL-18 which has not been correlated with S. aureus biofilms so far might represent a suitable marker for monitoring chronification during MRSA infection to individualize the therapy, but this hypothesis must be proved in clinical trials.

Bacteriophage-mediated decolonization of Klebsiella pneumoniae in a novel Galleria mellonella gut colonization model with Enterobacteriaceae.

Galleria mellonella larvae have emerged as an invertebrate model for investigating bacterial pathogenesis and potential therapies, addressing ethical concerns related to mammalian models. This model has the advantage of having a simple gut microbiome, which is suitable for gut colonization studies. Intestinal colonization by Enterobacteriaceae significantly contributes to the spread of antibiotic resistance. This study aimed to establish a novel Enterobacteriaceae gut colonization larval model and assess its suitability for evaluating distinct antimicrobial efficacies. Larvae were force-fed sequentially with bacterial doses of K. pneumoniae and E. coli at 0, 24, and 48 h, with survival monitoring at 24 h intervals. Bacterial counts were assessed after 48 h and 120 h of force-feeding. Successfully colonized larvae were subjected to one-time force feeding of a bacteriophage cocktail (107 PFU/larvae) or MIC-based meropenem and ciprofloxacin. The colonized bacterial load was quantified by CFU count. Three doses of 106 CFU/larvae resulted in stable gut colonization, independent of the K. pneumoniae or E. coli strain. Compared with the control, force-feeding of the bacteriophage reduced the colonization of the strain Kp 419614 by 5 log10 CFU/larvae, while antibiotic treatment led to a 3 log10 CFU/larval reduction. This novel G. mellonella model provides a valuable alternative for gut colonization studies, facilitating proof-of-concept investigations and potentially reducing or replacing follow-up experiments in vertebrate models.

The Impact of Pseudomonas aeruginosa Infection in Adult Cystic Fibrosis Patients-A Single Polish Centre Study.

BACKGROUND: Pseudomonas aeruginosa (PA) is one of the most predominant pathogens of lung infections, often causing exacerbations in adult patients with cystic fibrosis (CF). MATERIALS AND METHODS: Microbiological characterization of 74 PA isolates and to evaluate the correlations between the bacterial features and 44 adult Polish CF cohort clinical parameters. RESULTS: The most common variant in the CF transmembrane conductance regulator (CFTR) gene was F508del (76.3%), followed by 3849+10kbC>T (26.3%). A total of 39.4% of the PA isolates showed multiple resistances. In patients with parameters pointing to a decline in lung function, there was a statistically significant moderate correlation with ß-lactam resistance and a weak correlation between hospital frequency and colistin resistance. The mucoidity did not correlate with the biofilm formation ability, which showed 41.9% of the isolates. Proteolytic activity, observed in 60.8% of the clinical isolates, was weakly associated with motility detected in 78.4% of the strains. The genetic profiles of the PA were highly heterogeneous, and a weak positive correlation was established between cluster group and biofilm formation. CONCLUSION: The findings suggest that there is a high variety in P. aeruginosa populations in adult CF patients. There is a need to monitor PA strains in groups of patients with cystic fibrosis, in particular, in terms of the occurrence of antibiotic resistance related to a decline in lung function.

Biocide Susceptibility and Antimicrobial Resistance of Escherichia coli Isolated from Swine Feces, Pork Meat and Humans in Germany.

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum ß-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials.

LSPR-Based Biosensing Enables the Detection of Antimicrobial Resistance Genes.

The development of rapid, simple, and accurate bioassays for the detection of nucleic acids has received increasing demand in recent years. Here, localized surface plasmon resonance (LSPR) spectroscopy for the detection of an antimicrobial resistance gene, sulfhydryl variable ß-lactamase (blaSHV), which confers resistance against a broad spectrum of ß-lactam antibiotics is used. By performing limit of detection experiments, a 23 nucleotide (nt) long deoxyribonucleic acid (DNA) sequence down to 25 nm was detected, whereby the signal intensity is inversely correlated with sequence length (23, 43, 63, and 100 nt). In addition to endpoint measurements of hybridization events, the setup also allowed to monitor the hybridization events in real-time, and consequently enabled to extract kinetic parameters of the studied binding reaction. Performing LSPR measurements using single nucleotide polymorphism (SNP) variants of blaSHV revealed that these sequences can be distinguished from the fully complementary sequence. The possibility to distinguish such sequences is of utmost importance in clinical environments, as it allows to identify mutations essential for enzyme function and thus, is crucial for the correct treatment with antibiotics. Taken together, this system provides a robust, label-free, and cost-efficient analytical tool for the detection of nucleic acids and will enable the surveillance of antimicrobial resistance determinants.

Label-free multimodal imaging of infected Galleria mellonella larvae.

Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner.

What the Phage: a scalable workflow for the identification and analysis of phage sequences.

Phages are among the most abundant and diverse biological entities on earth. Phage prediction from sequence data is a crucial first step to understanding their impact on the environment. A variety of bacteriophage prediction tools have been developed over the years. They differ in algorithmic approach, results, and ease of use. We, therefore, developed "What the Phage" (WtP), an easy-to-use and parallel multitool approach for phage prediction combined with an annotation and classification downstream strategy, thus supporting the user's decision-making process by summarizing the results of the different prediction tools in charts and tables. WtP is reproducible and scales to thousands of datasets through a workflow manager (Nextflow). WtP is freely available under a GPL-3.0 license (https://github.com/replikation/What_the_Phage).

Raman Spectroscopic Differentiation of Streptococcus pneumoniae From Other Streptococci Using Laboratory Strains and Clinical Isolates.

Streptococcus pneumoniae, commonly referred to as pneumococci, can cause severe and invasive infections, which are major causes of communicable disease morbidity and mortality in Europe and globally. The differentiation of S. pneumoniae from other Streptococcus species, especially from other oral streptococci, has proved to be particularly difficult and tedious. In this work, we evaluate if Raman spectroscopy holds potential for a reliable differentiation of S. pneumoniae from other streptococci. Raman spectra of eight different S. pneumoniae strains and four other Streptococcus species (S. sanguinis, S. thermophilus, S. dysgalactiae, S. pyogenes) were recorded and their spectral features analyzed. Together with Raman spectra of 59 Streptococcus patient isolates, they were used to train and optimize binary classification models (PLS-DA). The effect of normalization on the model accuracy was compared, as one example for optimization potential for future modelling. Optimized models were used to identify S. pneumoniae from other streptococci in an independent, previously unknown data set of 28 patient isolates. For this small data set balanced accuracy of around 70% could be achieved. Improvement of the classification rate is expected with optimized model parameters and algorithms as well as with a larger spectral data base for training.

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore's adaptive sequencing.

Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective 'human host depletion' resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

Uncovering antimicrobial resistance in three agricultural biogas plants using plant-based substrates.

Antimicrobial resistance (AMR) is becoming an increasing global concern and the anaerobic digestion (AD) process represents a potential transmission route when digestates are used as fertilizing agents. AMR contaminants, e.g. antibiotic-resistant bacteria (ARB) and plasmid-mediated antibiotic resistance genes (ARGs) have been found in different substrates and AD systems, but not yet been investigated in plant-based substrates. AMR transfer from soils to vegetable microbiomes has been observed, and thus crop material potentially represents a so far neglected AMR load in agricultural AD processes, contributing to AMR spread. In order to test this hypothesis, this study examined the AMR situation throughout the process of three biogas plants using plant-based substrates only, or a mixture of plant-based and manure substrates. The evaluation included a combination of culture-independent and -dependent methods, i.e., identification of ARGs, plasmids, and pathogenic bacteria by DNA arrays, and phylogenetic classification of bacterial isolates and their phenotypic resistance pattern. To our knowledge, this is the first study on AMR in plant-based substrates and the corresponding biogas plant. The results showed that the bacterial community isolated from the investigated substrates and the AD processing facilities were mainly Gram-positive Bacillus spp. Apart from Pantoea agglomerans, no other Gram-negative species were found, either by bacteria culturing or by DNA typing array. In contrast, the presence of ARGs and plasmids clearly indicated the existence of Gram-negative pathogenic bacteria, in both substrate and AD process. Compared with substrates, digestates had lower levels of ARGs, plasmids, and culturable ARB. Thus, digestate could pose a lower risk of spreading AMR than substrates per se. In conclusion, plant-based substrates are associated with AMR, including culturable Gram-positive ARB and Gram-negative pathogenic bacteria-associated ARGs and plasmids. Thus, the AMR load from plant-based substrates should be taken into consideration in agricultural biogas processing.

In vitro additive effects of dalbavancin and rifampicin against biofilm of Staphylococcus aureus.

Dalbavancin is a novel glycopeptide antibiotic approved for the treatment of acute bacterial skin and skin structure infections (ABSSSIs). It is characterized by a potent activity against numerous Gram-positive pathogens, a long elimination half-life and a favorable safety profile. Most recently, its application for the treatment of periprosthetic joint infections (PJIs) was introduced. The aim of this study was to proof our hypothesis, that dalbavancin shows superior efficacy against staphylococcal biofilms on polyethylene (PE) disk devices compared with vancomycin and additive behavior in combination with rifampicin. Staphylococcus aureus biofilms were formed on PE disk devices for 96 h and subsequently treated with dalbavancin, vancomycin, rifampicin and dalbavancin-rifampicin combination at different concentrations. Quantification of antibacterial activity was determined by counting colony forming units (CFU/ml) after sonification of the PE, serial dilution of the bacterial suspension and plating on agar-plates. Biofilms were additionally life/dead-stained and visualized using fluorescence microscopy. Dalbavancin presented superior anti-biofilm activity compared to vancomycin. Additive effects of the combination dalbavancin and rifampicin were registered. Dalbavancin combined with rifampicin presents promising anti-biofilm activity characteristics in vitro. Further in vivo studies are necessary to establish recommendations for the general use of dalbavancin in the treatment of PJIs.

Biochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy.

Biochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte's specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.

In Vitro Synergism of Penicillin and Ceftriaxone against Enterococcus faecalis.

Enterococcus faecalis infective endocarditis is commonly treated with intravenous ampicillin/ceftriaxone combination therapy. Ampicillin, however, is unsuitable for outpatient parenteral antibiotic therapy (OPAT) regimens due to its instability in 24 h continuous infusors, and has been successfully replaced by benzylpenicillin used together with ceftriaxone in a few small case series. Since in vitro synergy data of penicillin/ceftriaxone against E. faecalis are still lacking, checkerboard assays were performed for 28 clinical E. faecalis isolates and one laboratory standard strain. Synergistic effects (both lowest and median FICI) were observed for penicillin/ceftriaxone in 15/29 isolates, while ampicillin/ceftriaxone exhibited synergism in 22/29 isolates. For isolates with ceftriaxone MICs ≤ 256 mg/L, the addition of free ceftriaxone trough concentrations to penicillin or ampicillin resulted in comparable synergistic effects for both combinations. In contrast, for isolates with ceftriaxone MICs ≥ 512 mg/L free ceftriaxone trough concentrations were only sufficient to exhibit synergistic effects in combination with ampicillin, but not penicillin. This study suggests that benzylpenicillin/ceftriaxone would be expected to be suitable for the OPAT treatment of enterococcal endocarditis for E. faecalis isolates with ceftriaxone MICs ≤ 256 mg/L. However, combination therapy would be expected to provide no advantage over benzylpenicillin monotherapy for isolates with ceftriaxone MICs ≥ 512 mg/L. Further investigation is required to analyse the relationship between ceftriaxone susceptibility and penicillin/ceftriaxone synergy, especially for isolates with ceftriaxone MICs of 64 to 512 mg/L.

Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification.

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.

Assessment of Phenotype Relevant Amino Acid Residues in TEM-ß-Lactamases by Mathematical Modelling and Experimental Approval.

Single substitutions or combinations of them alter the hydrolytic activity towards specific ß-lactam-antibiotics and ß-lactamase inhibitors of TEM-ß-lactamases. The sequences and phenotypic classification of allelic TEM variants, as provided by the NCBI National Database of Antibiotic Resistant Organisms, does not attribute phenotypes to all variants. Some entries are doubtful as the data assessment differs strongly between the studies or no data on the methodology are provided at all. This complicates mathematical and bioinformatic predictions of phenotypes that rely on the database. The present work aimed to prove the role of specific substitutions on the resistance phenotype of TEM variants in, to our knowledge, the most extensive mutagenesis study. In parallel, the predictive power of extrapolation algorithms was assessed. Most well-known substitutions with direct impact on the phenotype could be reproduced, both mathematically and experimentally. Most discrepancies were found for supportive substitutions, where some resulted in antagonistic effects in contrast to previously described synergism. The mathematical modelling proved to predict the strongest phenotype-relevant substitutions accurately but showed difficulties in identifying less prevalent but still phenotype transforming ones. In general, mutations increasing cephalosporin resistance resulted in increased sensitivity to ß-lactamase inhibitors and vice versa. Combining substitutions related to cephalosporin and ß-lactamase inhibitor resistance in almost all cases increased BLI susceptibility, indicating the rarity of the combined phenotype.

The Emergence of Klebsiella pneumoniae with Reduced Susceptibility Against Third Generation Cephalosporins and Carbapenems in Lagos Hospitals, Nigeria.

This study investigated the prevalence of Klebsiella (K.) pneumoniae isolates among clinical samples of patients in four medical centers in Lagos, Nigeria and the burden of extended-spectrum beta-lactamases (ESBL) and carbapenem-resistant K. pneumoniae (CRKP) strains. Different samples (stool, blood, urine, wound swabs and nasal swabs) from 127 patients with suspected Gram-negative infections based on on-site performed Gram-stain from four public hospitals between March and September 2015 were analyzed. K. pneumoniae was identified in 43 (34%) patients. Resistance rates of these 43 strains according to the CLSI breakpoints were as followed: cotrimoxazole (90.7%), cefuroxime (74.4%), ofloxacin (55.8%), ceftazidime (46.5%), and cefixime (35%). Three isolates (7%) were resistant to imipenem. All isolates were susceptible to amoxicillin/clavulanic acid and nitrofurantoin. The prevalence of ESBL-producing, MDR and CRKP strains was 69.8%, 62.8%, and 7.0%, respectively. Of the ESBL-producing isolates, two K. pneumoniae isolates obtained from urine harbored both blaSHV and blaCTX-M-1, and a third isolate from urine harbored only the blaCTX-M-1. This study revealed the emergence of CRKP isolates and blaCTX-M-1 and blaSHV co-harboring K. pneumoniae strains in Lagos hospitals. The emergence of CRKP strains is an early warning signal for carbapenem antibiotics' prudent use with concern for their efficacies.

Lethal Neonatal Respiratory Failure by Perinatal Transmission of Ureaplasma Parvum after Maternal PPROM.

A primiparous pregnant woman was admitted due to preterm premature rupture of membranes (PPROM) at 27+0 week of gestational age (WGA). Conventional vaginal microbiological analysis had no pathological finding. Management decisions based on national guidelines included antenatal corticoids, tocolytics and antibiotics. Unstoppable efforts of preterm labor in 28+0 WGA and supposed amniotic infection syndrome necessitated emergency cesarean section. The preterm infant underwent NICU therapy, developed an early-onset neonatal sepsis and therapy-refractory pulmonary insufficiency with consecutive right heart failure, resulting in death on the 36th day of life. Microbiota analyses by 16Sr DNA sequencing was performed from maternal vaginal swabs and from neonatal pharyngeal swabs. Maternal antibiotic treatment resulted in depletion of physiological vaginal colonization with Lactobacillus crispatus. Ureaplasma parvum became the dominant vaginal microorganism at delivery and was detected in high relative abundance in the neonatal specimen. Progressive radiological air-space changes and interstitial pathologies associated with Ureaplasma infection (bronchopulmonary dysplasia type III) were seen early at the 3rd and distinctly from 14th day of life. This clearly demonstrates the need of vaginal colonization diagnostics in PPROM patients and awareness of the consecutive risks in the preterm. Vaginal microbiome analysis may allow individualized and targeted maternal and fetal diagnostic, prophylactic and therapeutic strategies to identify, protect and treat the high-risk neonates after PPROM.

Human Milk Oligosaccharides Exhibit Biofilm Eradication Activity Against Matured Biofilms Formed by Different Pathogen Species.

Human milk oligosaccharides (HMOs) have been shown to exhibit plenty of benefits for infants, such as prebiotic activity shaping the gut microbiota and immunomodulatory and anti-inflammatory activity. For some pathogenic bacteria, antimicrobial activity has been proved, but most studies focus on group B streptococci. In the present study, we investigated the antimicrobial and antibiofilm activities of the total and fractionated HMOs from pooled human milk against four common human pathogenic Gram-negative species (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia cenocepacia) and three Gram-positive species (Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis). The activity of HMOs against enterococci and B. cenocepacia are addressed here for the first time. We showed that HMOs exhibit a predominant activity against the Gram-positive species, with E. faecalis being the most sensitive to the HMOs, both in planktonic bacteria and in biofilms. In further tests, we could exclude fucosyllactose as the antibacterial component. The biological significance of these findings may lie in the prevention of skin infections of the mother's breast as a consequence of breastfeeding-induced skin laceration and/or protection of the infants' nasopharynx and lung from respiratory pathogens such as staphylococci.

Evaluation of Th2 and Th17 Immunity-Related Factors as Indicators of Brucellosis.

Objective: Brucellosis is a common bacterial zoonotic infection, and greater than half a million new cases are diagnosed annually. This study investigates the expression of Th2 and Th17 immunity-related factors (Th2-LCR lncRNA, IL-25, TRAF3IP2, and IL-17RB) in different stages of Brucella infections. Material and Methods: In total, 99 brucellosis patients were divided into three groups (acute = first infection before treatment, relapse = before treatment, and treated = after treatment for 6-8 weeks with doxycycline and rifampin). Thirty-three healthy volunteers represented the control group. Gene expression levels were assessed by quantitative amplification in reference to the 18S rRNA gene and statistically evaluated. Results: No significant differences in the expression of these genes were observed between the control group and patients after completion of antibiotic treatment. Compared to these two groups, only Th2-LCR lncRNA and TRAF3IP2 were significantly more highly expressed in the acute group. Th2-LCR lncRNA was also significantly elevated in the relapse group. TRAF3IP2 expression was additionally significantly increased in the acute group compared to the relapse group. Conclusion: IL-25 and IL-17RB failed to differentiate between the infected and noninfected groups. TRAF3IP2 and Th2-LCR lncRNA might be good indicators of brucellosis during the acute phase, but the expression levels varied strongly among patients. To verify the suitability of these factors as an indicator for brucellosis, acute infection or relapse should be investigated in further studies on larger cohorts with well-defined inclusion criteria.