Lower Bacterial Load Research Articles

Analysis of Xpert MTB/RIF results in retested patients with very low initial bacterial loads: A retrospective study in China

BackgroundThe Xpert MTB/RIF (Xpert) assay has been widely used to diagnose suspected active tuberculosis (TB) and rifampicin-resistant TB cases. Despite its excellent performance record, false-positive Xpert rifampicin (RIF) resistance results are obtained for specimens with extremely low bacterial loads. ObjectiveWe aimed to study the feasibility of repeat Xpert testing as a strategy for reducing the odds of obtaining false-positive results when testing paucibacillary TB patients. MethodsWe enrolled previously tested TB patients with very low initial bacterial loads from May 2016 to February 2022 for Xpert retesting. A total of 251 TB patients were retested using the Xpert assay. ResultsRIF resistance was noted in 65 (25.9 %) patients when tested by Xpert at initial diagnosis. Only 107 (42.6 %) of 251 patients tested positive for MTB when retested via Xpert. The majority (98.6 %) of RIF-susceptible cases were still susceptible to RIF when retested. Initial Xpert testing yielded 35 positive results for MTB in the RIF-resistant group, of whom 25 (71.4 %) still exhibited RIF resistance when retested. All culture-positive MTB isolates in the RIF-susceptible group were also RIF-susceptible by phenotypic DST. In the RIF-resistant group, 10 of 14 culture-positive MTB isolates exhibited RIF resistance, of which 4 isolates were deemed RIF-susceptible by phenotypic DST. The proportion of double mutations within the MTB rpoB RRDR sequence, as detected by hybridization of Xpert D and E probes, was significantly higher in the RIF-susceptible group than in the RIF-susceptible group. ConclusionsOur results demonstrated that initial RIF-susceptible results were more accurate than RIF-resistant results. Additionally, patients with double mutations that delayed probe D/E hybridization were more likely to have false-positive Xpert results. Our findings emphasize that repeat Xpert MTB/RIF testing is necessary for TB patients with extremely low bacterial loads who are at high risk for RIF-resistant TB.

Anthropogenic impact and antibiotic resistance among the indicator and pathogenic bacteria from several industrial and sewage discharge points along the coast from Pydibhimavaram to Tuni, East Coast of India

Increasing urbanisation and industrialisation of the Visakhapatnam region have brought domestic sewage and industrial wastewater discharge into the coastal ocean. This study examines the indicator and pathogenic bacteria's quantitative abundance and antibiotic susceptibility. This study collected surface and subsurface water samples from ten different regions (147 stations; 294 samples), including 12 industrial discharge points, surrounding stations and two harbours from the coast of Pydibheemavaram to Tuni. Physicochemical parameters like salinity, temperature, fluorescence, pH, total suspended matter, nutrients, chlorophyll-a and dissolved oxygen showed a difference between regions. We noticed the presence of indicator (Escherichia coli and Enterococcus faecalis) and pathogenic (Aeromonas hydrophila, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella and Shigella, Vibrio cholera and Vibrio parahaemolyticus) bacteria among the samples. Waters from the near harbour and Visakhapatnam steel plant showed lower bacterial load with no direct input from industries to the coastal water. Samples collected during the industrial discharge period had a higher bacterial load, including E. coli. Enteric bacteria were found in higher numbers at most stations. Some isolates were resistant to multiple antibiotics with higher antibiotic resistance and multiple antibiotic resistance indexes compared with the other coastal water habitats in the Bay of Bengal. The occurrence of these bacteria above the standard limits and with multiple antibiotic resistance in the study region may pose a potential threat to the local inhabitants. It can create an alarming situation in the coastal waters in the study region.

Assessment and Relationship between Chemical Composition and Microbial Load in Corn Tortillas Sampled from Different Vending Points in Two Regions of Mexico

During tortilla production, different heating stages help to ensure low bacterial loads that contribute to their safety. However, there is an increasing concern about these products’ stability. This work is aimed at analyzing corn tortillas collected at different vending points from two regions of the country, Nuevo Leon and Guanajuato. A total of 80 samples were analyzed, and the bacterial presence was related to the origin of the raw materials. Guanajuato region resulted with the highest concentration ( 6.04 ± 0.07 Log UFC/g). Tortillas made with nixtamalized corn masa flour (NMF) had significantly lower microbial loads ( p < 0.05 ) than those made with 100% nixtamal masa or mixtures of NMF/nixtamal masa. In all samples, there were no pathogenic bacteria. On 28 selected samples, through PCA analysis, a strong influence of the microbial loads (which include foodborne bacteria) and their changes in chemical composition being reflected in the free sugars, free amino nitrogen (FAN), and peroxides contents, respectively, could be related with the vending point good manufacturing practices.

Control of Mycobacterium avium subsp. paratuberculosis load within infected bovine monocyte-derived macrophages is associated with host genetics.

The genetic loci influencing individual resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are still largely unknown. In the current study, we searched for genetic loci associated with resistance to MAP infection by evaluating the performance of monocyte-derived macrophages (MDMs) isolated from the peripheral blood of 75 healthy Holsteins cows and infected ex vivo with MAP. Bacterial load (log colony-forming units, log CFUs) within MDMs was quantified at 2h and 7 days p. i. using a BACTEC MGIT 960 instrument. In addition, the expression levels of some genes with important roles in the innate immune response including epiregulin (EREG), complement component C3 (C3), galectin-9 (Gal9), and nitric oxide (NO-) were measured in the supernatant of the infected cells. DNA from peripheral blood samples of the animals included in the study was isolated and genotyped with the EuroG MD bead Chip (44,779 single nucleotide-polymorphisms, SNPs). Linear mixed models were used to calculate the heritability (h2 ) estimates for each indicator of MDM performance, MAP load within MDMs and EREG, C3, Gal9, and NO-expression. After performing a genome-wide association study, the only phenotypes that showed SNPs with a significant association were the bacterial load within MDMs at 2h (h2 = 0. 87) and 7 days (h2 = 0.83) p.i. A total of 6 SNPs, 5 candidate genes, and one microRNA on the Bos taurus chromosomes BTA2, BTA17, BTA18, and BTA21 were associated with MAP load at 2h p.i. Overlap was seen in two SNPs associated with the log CFUs at 2h and 7d p.i. The identified SNPs had negative regression coefficients, and were, therefore, associated with a low bacterial load within MDMs. Some of the identified SNPs were located within QTLs previously associated with longevity, reproductive, and udder health traits. Some of the identified candidate genes; Oxysterol Binding Protein Like 6, Cysteine and Serine Rich Nuclear Protein 3, and the Coiled-Coil Domain Containing 92 regulate cellular cholesterol trafficking and efflux, apoptosis, and interferon production, respectively. Taken together, our results define a heritable and distinct immunogenetic profile in MAP-infected macrophages designed to limit bacterial load early after infection.

Diagnostic accuracy of an immunomagnetic separation-PCR assay to detect pathogenic Leptospira spp. in urine from dairy cattle, using a Bayesian latent class model

Leptospirosis is a zoonotic disease that has spread worldwide and causes significant economic losses in the dairy industry. The causal agents of this infectious disease are members of the genus Leptospira, known as pathogenic Leptospira spp. Specific clinical signs of the infection are difficult to detect. Therefore, the disease is normally under-diagnosed, mostly due to the lack of a cost-effective technique for diagnosing animals with a low bacterial load in their urine. The aim of this study was to assess the diagnostic accuracy of a qPCR coupled with a previous Immunomagnetic separation (IMS) step (IMS-qPCR) against a qPCR without using IMS, using a Bayesian latent class model (2 tests, 3 populations) to determine the leptospirosis infectious status in naturally infected dairy cattle. The results revealed that IMS qPCR had a sensitivity (Se) of 95.7% (95% Probability Interval (PI) = 85.0; 99.4%) and a specificity (Sp) of 98% (95% PI = 96.1; 99.4%), indicating that it is more sensitive than conventional qPCR (Se = 69.7% (95% PI = 59.2; 79.0%); median difference = 25.2% (Monte Carlo Error = 10.2%); and the Sp = 98.8% (95% PI = 97.6; 99.5%), median difference = 0.8% (Monte Carlo Error = 2.1%). Therefore, results shows that IMS-qPCR is a more useful diagnostic tool in terms of accuracy for detecting infectious animals with pathogenic Leptospira in their urine.

Time to detection of Mycobacterium tuberculosis using culture filtrate H37rv supplementation on MGIT 960 System

Background: The fundamental priority program for controlling Tuberculosis (TB) is accurate and rapid diagnosis. Variable Mycobacterium tuberculosis (MTB) phenotypes were discovered in sputum samples from pulmonary TB (PTB) patients. Active replicating bacteria and differentially culturable tubercle bacteria (DCTB) are present in the sputum. Differentially culturable tubercle bacilli did not grow on the standard medium. Culture filtrate (CF) H37Rv supplementation containing resuscitation-promoting factors (Rpfs) has the potential to increase sensitivity and reduce time to detection (TTD) on culture-based diagnostics. Methods: This study was a true experimental laboratory. A clinical sample was taken from the sputum of 15 PTB patients, which was "MTB detected" by Xpert MTB/RIF assay. Sputum samples were divided into two groups and inoculated on MGIT 960 in one group with CF-H37Rv supplementation and without in the other as standard MGIT medium. Results: On both standard MGIT medium and MGIT+CF H37Rv, the recovery rate was 100%. Seven samples (47%) show a growth promotion effect with CF H37Rv supplementation (ΔTTD > 10 hours), while eight samples (53%) show no change in TTD (ΔTTD < 10 hours). There is no evidence that CF H37Rv supplementation inhibits growth. Six of the seven samples with ΔTTD > 10 jam (85,7%) were dominated by samples with acid-fast bacilli (AFB) grading 1+, which could be an assumption related to re-treatment PTB cases, chronic cases, treatment stages, TB-HIV, or follow up of treatments. Conclusion: There was no difference in recovery rate or TTD between standard MGIT medium and MGIT+CF H37Rv. The supplementation of culture filtrate H37Rv has a greater advantage on samples with low bacterial load (AFB 1+).

LDH as an adjuvant makes Brucella outer-membrane vesicles and outer-membrane vesicle-associated proteins highly protective in mice.

Existing Brucella vaccines are attenuated and can cause vaccine-associated brucellosis; and these safety concerns have affected their application. Although subunit vaccines have the advantages of safety, efficacy, low cost, and rapid production, they are usually poorly immunogenic and insufficient to trigger persistent immunity. Therefore, we added layered double hydroxide (LDH) as an adjuvant to Brucella subunit vaccine formulations to enhance the immune response to the antigen. LDH and Freund's adjuvant were combined with Brucella outer-membrane vesicles (OMVs) and OMV-associated proteins to form a subunit vaccine, respectively. The immunogenicity of LDH as an adjuvant was assessed in BALB/c mice. We examined levels of immunoglobulin G, G1, and G2a (IgG, IgG1, and IgG2a) antibodies (aBs); percentages of Cluster of Differentiation 4-positive (CD4+) and CD8+ T cells in peripheral-blood lymphocytes; and secretion of cytokines in mouse spleen lymphocytes. Finally, splenic index and splenic bacterial load were assessed via Brucella challenge experiments on mice. The LDH subunit vaccine also produced high levels of specific aBs in mice compared with Freund's adjuvant subunit vaccine and induced mainly T-helper 1 cell (Th1)-type immune responses. In addition, mice in the LDH subunit vaccine group had significantly lower bacterial loads in their spleens than those in the Freund's adjuvant subunit vaccine group, and the LDH-OMV vaccine offered a higher level of protection against Brucella attack. LDH as an adjuvant-paired vaccine provided a high level of protection against Brucella infection.

Hyperbaric oxygen therapy augments ciprofloxacin effect against Pseudomonas aeruginosa biofilm infected chronic wounds in a mouse model

IntroductionChronic wounds have a compromised microcirculation which leads to restricted gas exchange. The majority of these hypoxic wounds is infested with microorganisms congregating in biofilms which further hinders the antibiotic function. We speculate whether this process can be counteracted by hyperbaric oxygen therapy (HBOT). MethodologyTwenty-eight BALB/c mice with third-degree burns were included in the analyses. Pseudomonas aeruginosa embedded in seaweed alginate beads was injected under the eschar to mimic a biofilm infected wound. Challenged mice were randomized to receive either 4 days with 1 x ciprofloxacin combined with 2 × 90 min HBOT at 2.8 standard atmosphere daily, 1 x ciprofloxacin as monotherapy or saline as placebo. The mice were clinically scored, and wound sizes were estimated by planimetry daily. Euthanasia was performed on day 8. Wounds were surgically removed in toto, homogenized and plated for quantitative bacteriology. Homogenate supernatants were used for cytokine analysis. ResultsP. aeruginosa was present in all wounds at euthanasia. A significant lower bacterial load was seen in the HBOT group compared to either the monotherapy ciprofloxacin group (p = 0.0008), or the placebo group (p < 0.0001). IL-1β level was significantly lower in the HBOT group compared to the placebo group (p = 0.0007). Both treatment groups had higher osteopontin levels than the placebo group (p = 0.002 and p = 0.004). The same pattern was seen in the S100A9 analysis (p = 0.01 and p = 0.008), whereas no differences were detected between the S100A8, the VEGF or the MMP8 levels in the three groups. ConclusionThese findings show that HBOT improves the bactericidal activity of ciprofloxacin against P. aeruginosa wound biofilm in vivo. HBOT in addition to ciprofloxacin also modulates the host response to a less inflammatory phenotype.

Pseudomonas aeruginosa PAO1 outer membrane vesicles-diphtheria toxoid conjugate as a vaccine candidate in a murine burn model

Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.

Microbiological characteristics of bread dough and nutritional quality of “Tabnen-naow,” ethnic artisan bread in Burkina Faso

AbstractThis study aimed to evaluate the microbiological characteristics of bread dough and the nutritional quality of “Tabnen-naow,” an ethnic artisan bread consumed mainly in Burkina Faso. Originating in Boussé, this bread is spreading to the outskirts of Ouagadougou for several reasons, such as the increase in the price of industrial bread and especially the supposed low glycemic index of artisan bread. However, there is insufficient information in the literature about the nutritional quality and technology of artisan bread in Burkina Faso. For this purpose, surveys were conducted among artisan bakers in five artisan bread production units in Ouagadougou, using survey forms to draw the production diagram. The microbiological characteristics of bread dough were evaluated using standard microbiology methods. Lastly, the nutritional quality of ethnic artisan bread was determined using standard techniques. All the bakers were male, between 22 and 34 years old, with an average age of 27.8 ± 5.1 years. They all used wheat flour, mainly (80%) from the Grand Moulin du Faso (GMF). The Baker’s dough fermentation was done at room temperature using dry or lyophilized yeast. The interesting microorganisms (yeasts, lactic acid bacteria) were present in all the bread dough samples. The yeast (2.5 108 CFU/g) and lactic acid bacteria (2.27 107 CFU/g) were abundant apart from the one bread dough, which had a low lactic acid bacterial load. The ethnic artisan bread contains carbohydrates (88.20–89.05%), proteins (9.60–10.60%), and minerals such as iron (11.56 to 89.27 mg/kg), calcium (00.00–290.03 mg/kg), and zinc (69.18 ± 36.74 to 389.10 ± 32.89 mg/kg). The nutritional composition revealed that ethnic artisan bread could contribute significantly to a healthy diet and food security.

Accuracy of the InnowaveDX MTB/RIF test for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre study

ABSTRACT Early and accurate diagnosis of tuberculosis (TB) is necessary to initiate proper therapy for the benefit of the patients and to prevent disease transmission in the community. In this study, we developed the InnowaveDX MTB/RIF (InnowaveDX) to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance simultaneously. A prospective multicentre study was conducted to evaluate the diagnostic performance of InnowaveDX for the detection MTB in sputum samples as compared with Xpert and culture. The calculated limit of detection (LOD) for InnowaveDX was 9.6 CFU/ml for TB detection and 374.9 CFU/ml for RIF susceptibility. None of the other bacteria tested produced signals that fulfilled the positive TB criteria, demonstrating a species-specificity of InnowaveDX. Then 951 individuals were enrolled at 7 hospitals, of which 607 were definite TB cases with positive culture and/or Xpert results, including 354 smear-positive and 253 smear-negative cases. InnowaveDX sensitivity was 92.7% versus bacteriologically TB standard. Further follow-up revealed that 61 (91.0%) out of 67 false-positive patients with no bacteriological evidence met the criteria of clinically diagnosed TB. Among 125 RIF-resistant TB patients diagnosed by Xpert, 108 cases were correctly identified by InnowaveDX, yielding a sensitivity of 86.4%. Additionally, the proportion of very low bacterial load in the discordant susceptibility group was significantly higher than in the concordant susceptibility group (P = 0.029). To conclude, we have developed a novel molecular diagnostic with promising detection capabilities of TB and RIF susceptibility. In addition, the discordant RIF susceptibility results between InnowaveDX and Xpert are more frequently observed in samples with very low bacterial load.

Dynamic evolution of membrane biofouling in feed channels affected by spacer–membrane clearance and the induced hydrodynamic conditions

Membrane biofouling is regarded as a Gordian knot in applying reverse osmosis and nanofiltration for water treatment. It occurs in the feed spacer channels of spiral wound membrane elements. The influence of feed spacer structure on hydrodynamic condition and biofouling development remains yet to be fully understood. This study aims to quantify the dynamic evolution and spatial distribution of biomass in feed spacer channels, and reveal the great importance of spacer–membrane clearance in the biofouling development. Four kinds of feed spacers with the same thickness (28 mil) but different clearance width, by varying filament diameter (0.4, 0.45, 0.55 and 0.6 mm), were used. A relatively long-term (20 d) biofouling experiment was conducted by using a feed solution of lower bacterial load and nutrient dosage. Results showed that the “shear stress effect” dominated the early stage of biofouling as biofilm was first attached to the low-shear regions, regardless of the spacer–membrane clearance width. In the middle and late stage, the “blocking effect” emerged with the continuous accumulation of biomass, as was especially the case in the feed channel with narrow spacer–membrane clearance. In this period, biofilm was prone to deposit and block the narrow flow passage between the spacer filaments and membrane surface, which decreased the uniformity of flow field, expanded the flow “dead zone” area, boosted the feed channel pressure (FCP) drop, and resultantly accelerated the rate of membrane biofouling. Enhancement of the “shear stress effect” under an appropriate range of spacer–membrane clearance width and thus channel porosity, as well as customed design of feed spacer geometries according to specific fouling locations are suggested for the future optimization of feed spacer.

Current innovations in medical laboratory diagnosis of Tuberculosis

Tuberculosis (TB) is a major global health problem, ranking as the second highest cause of death from an infectious disease globally, after the human immunodeficiency virus (HIV). Nigeria ranked first in Africa with burden of TB. Tuberculosis is a major public health problem in Nigeria with about 407,000 people infected. The World Health Organization estimates that 120,000 people developed TB in 2019, with 154,000 death of whom, 13% were HIV positive individuals. Among the incident cases, 56% were from the South-East Asia and Western Pacific Regions and one quarter were from Africa. The African continent accounts for the highest rates of cases and deaths relative to population. Traditionally, tuberculosis is mostly being diagnosed by a combination of chest X-rays, the staining of sputum with special dyes followed by microscopy, the growth of Mycobacterium tuberculosis in culture and the Mantoux test. The sputum smear microscopy is easy to do and is very cheap and combined with chest X-rays has been used for a long time by TB control agencies worldwide. However, the sputum smear microscopy (sputum AFB) test has some problems in HIV-positive patients and children also in patients with low bacterial load and in latent TB infections. TB continues to be a complex disease to diagnose and manage due to the chronicity of the disease, the nature of the host-pathogen relationship, and the resulting diversity in its clinical manifestations. Therefore, this review considers recent innovations in TB laboratory diagnosis in conventional (traditional), non- conventional (molecular), serological (immunological) methods and the latest methods of TB diagnosis.

Extracellular DNAses Facilitate Antagonism and Coexistence in Bacterial Competitor-Sensing Interference Competition.

Over the last 4 decades, the rate of discovery of novel antibiotics has decreased drastically, ending the era of fortuitous antibiotic discovery. A better understanding of the biology of bacteriogenic toxins potentially helps to prospect for new antibiotics. To initiate this line of research, we quantified antagonists from two different sites at two different depths of soil and found the relative number of antagonists to correlate with the bacterial load and carbon-to-nitrogen (C/N) ratio of the soil. Consecutive studies show the importance of antagonist interactions between soil isolates and the lack of a predicted role for nutrient availability and, therefore, support an in situ role in offense for the production of toxins in environments of high bacterial loads. In addition, the production of extracellular DNAses (exDNases) and the ability to antagonize correlate strongly. Using an in domum-developed probabilistic cellular automaton model, we studied the consequences of exDNase production for both coexistence and diversity within a dynamic equilibrium. Our model demonstrates that exDNase-producing isolates involved in amensal interactions act to stabilize a community, leading to increased coexistence within a competitor-sensing interference competition environment. Our results signify that the environmental and biological cues that control natural-product formation are important for understanding antagonism and community dynamics, structure, and function, permitting the development of directed searches and the use of these insights for drug discovery. IMPORTANCE Ever since the first observation of antagonism by microorganisms by Ernest Duchesne (E. Duchesne, Contribution à l'étude de la concurrence vitale chez les microorganisms. Antagonism entre les moisissures et les microbes, These pour obtenir le grade de docteur en medicine, Lyon, France, 1897), many scientists successfully identified and applied bacteriogenic bioactive compounds from soils to cure infection. Unfortunately, overuse of antibiotics and the emergence of clinical antibiotic resistance, combined with a lack of discovery, have hampered our ability to combat infections. A deeper understanding of the biology of toxins and the cues leading to their production may elevate the success rate of the much-needed discovery of novel antibiotics. We initiated this line of research and discovered that bacterial reciprocal antagonism is associated with exDNase production in isolates from environments with high bacterial loads, while diversity may increase in environments of lower bacterial loads.

Efficacy of phage therapy in preclinical models of bacterial infection: a systematic review and meta-analysis

Antimicrobial resistance of bacterial pathogens is an increasing clinical problem and alternative approaches to antibiotic chemotherapy are needed. One of these approaches is the use of lytic bacterial viruses known as phage therapy. We aimed to assess the efficacy of phage therapy in preclinical animal models of bacterial infection. In this systematic review and meta-analysis, MEDLINE/Ovid, Embase/Ovid, CINAHL/EbscoHOST, Web of Science/Wiley, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and Google Scholar were searched from inception to Sept 30, 2021. Studies assessing phage efficacy in animal models were included. Only studies that assessed the efficacy of phage therapy in treating established bacterial infections in terms of survival and bacterial abundance or density were included. Studies reporting only in-vitro or ex-vivo results and those with incomplete information were excluded. Risk-of-bias assessment was performed using the Systematic Review Centre for Laboratory Animal Experimentation tool. The main endpoints were animal survival and tissue bacterial burden, which were reported using pooled odds ratios (ORs) and mean differences with random-effects models. The I2 measure and its 95% CI were also calculated. This study is registered with PROSPERO, CRD42022311309. Of the 5084 references screened, 124 studies fulfilled the selection criteria. Risk of bias was high for 70 (56%) of the 124 included studies; therefore, only studies classified as having a low-to-moderate risk of bias were considered for quantitative data synthesis (n=32). Phage therapy was associated with significantly improved survival at 24 h in systemic infection models (OR 0·08 [95% CI 0·03 to 0·20]; I2=55% [95% CI 8 to 77]), skin infection (OR 0·08 [0·04 to 0·19]; I2 = 0% [0 to 79]), and pneumonia models (OR 0·13 [0·06 to 0·31]; I2=0% [0 to 68]) when compared with placebo. Animals with skin infections (mean difference -2·66 [95% CI -3·17 to -2·16]; I2 = 95% [90 to 96]) and those with pneumonia (mean difference -3·35 [-6·00 to -0·69]; I2 = 99% [98 to 99]) treated with phage therapy had significantly lower tissue bacterial loads at 5 ± 2 days of follow-up compared with placebo. Phage therapy significantly improved animal survival and reduced organ bacterial loads compared with placebo in preclinical animal models. However, high heterogeneity was observed in some comparisons. More evidence is needed to identify the factors influencing phage therapy performance to improve future clinical application. Swiss National Foundation and Swiss Heart Foundation.

Efficacy and Immune Correlates of OMP-1B and VirB2-4 Vaccines for Protection of Dogs from Tick Transmission of Ehrlichia chaffeensis.

Ehrlichia chaffeensis, an obligatory intracellular bacterium, causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. Here, we investigated the vaccine potential of OMP-1B and VirB2-4. Among the highly expressed and immunodominant E. chaffeensis porin P28s/OMP-1s, OMP-1B is predominantly expressed by E. chaffeensis in A. americanum ticks, whereas VirB2-4 is a pilus protein of the type IV secretion system essential for E. chaffeensis infection of host cells. Immunization with recombinant OMP-1B (rOMP-1B) or recombinant VirB2-4 (rVirB2-4) protected mice from E. chaffeensis infection as effectively as Entry-triggering protein of Ehrlichia immunization. Dogs vaccinated with a nanoparticle vaccine composed of rOMP-1B or rVirB2-4 and an immunostimulating complex developed high antibody titers against the respective antigen. Upon challenge with E. chaffeensis-infected A. americanum ticks, E. chaffeensis was undetectable in the blood of rOMP-1B or rVirB2-4 immunized dogs on day 3 or 6 post-tick attachment and for the duration of the experiment, whereas dogs sham-vaccinated with the complex alone were persistently infected for the duration of the experiment. E. chaffeensis exponentially replicates in blood-feeding ticks to facilitate transmission. Previously infected ticks removed from OMP-1B-immunized dogs showed significantly lower bacterial load relative to ticks removed from sham-immunized dogs, suggesting in-tick neutralization. Peripheral blood leukocytes from rVirB2-4-vaccinated dogs secreted significantly elevated amounts of interferon-γ soon after tick attachment by ELISpot assay and reverse transcription-quantitative PCR, suggesting interferon-γ-mediated Ehrlichia inhibition. Thus, Ehrlichia surface-exposed proteins OMP-1B and VirB2-4 represent new potential vaccine candidates for blocking tick-borne ehrlichial transmission. IMPORTANCE Ehrlichia are tick-borne pathogens that cause a potentially fatal illness-ehrlichiosis-in animals and humans worldwide. Currently, no vaccine is available for ehrlichiosis, and treatment options are limited. Ticks are biological vectors of Ehrlichia, i.e., Ehrlichia exponentially replicates in blood-sucking ticks before infecting animals. Ticks also inoculate immunomodulatory substances into animals. Thus, it is important to study effects of candidate vaccines on Ehrlichia infection in both animals and ticks and the immune responses of animals shortly after infected tick challenge. Here, we investigated the efficacy of vaccination with functionality-defined two surface-exposed outer membrane proteins of Ehrlichia chaffeensis, OMP-1B and VirB2-4, in a mouse infection model and then in a dog-tick transmission model. Our results begin to fill gaps in our understanding of Ehrlichia-derived protective antigens against tick-transmission and immune correlates and mechanisms that could help future development of vaccines for immunization of humans and animals to counter tick-transmitted ehrlichiosis.

The comparative effects of erythromycin and amikacin on acute respiratory Pseudomonas aeruginosa infection.

One of the most common causes of pneumonia is Pseudomonas aeruginosa (P. aeruginosa). As with other microbial pathogens, this bacterium tends to develop resistance to various antibiotics. Amikacin and erythromycin, which are from the aminoglycoside and macrolide antibiotic families, are used to treat respiratory infections caused by P. aeruginosa. This study explored whether amikacin, erythromycin or a combination of both works better against P. aeruginosa acute lung infection. For this study, 32 rats were used. The trachea of rats was exposed aseptically and their lung was infected with P. aeruginosa through trachea. Then, according to the group, they received amikacin, erythromycin or a combination of both for 1 week. Finally, they were euthanised on the 3rd and 7th days post-infection. The macroscopic and microscopic evaluations of the lungs, kidney and liver were performed. The right lung was collected for in vivo bacteriological analysis. The amikacin group (A group) had a statistically significantly lower macroscopic and microscopic scores than the other groups (p < 0.05). In vivo bacteriological test revealed that the A group had significantly lower lung bacterial load (p < 0.05). In summary, it was concluded that amikacin could help alleviate the respiratory infection caused by P. aeruginosa solely, and it was more effective than erythromycin.

Pooling Rectal, Pharyngeal, and Urine Samples to Detect Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium Using Multiplex Polymerase Chain Reaction Is as Effective as Single-Site Testing for Men Who Have Sex With Men.

Screening for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) at pharyngeal, urogenital, and anorectal sites is recommended for men who have sex with men (MSM). Pooling samples is a promising technique, but no data are available when pooled screening also includes Mycoplasma genitalium (MG). The main objective of this study was to examine the sensitivity of pooled samples for detecting CT, NG, and MG in MSM using nucleic acid amplification versus single-site testing. In this multicenter study, MSM with a positive result for CT, NG, or MG were recalled to the clinic for treatment and were asked to participate in this study. Separate samples were sent to a central virological department that proceeded to form the pooled samples. Testing was performed using the multiplex real-time polymerase chain reaction Allplex STI Essential Assay (Seegene, Seoul, Korea), which can simultaneously detect 7 pathogens. A total of 130 MSM with at least 1 positive test for CT, NG, or MG were included. A total of 25.4% had a coinfection. The sensitivities of pooled-sample testing were 94.8% for CT, 97.0% for NG, and 92.3% for MG. Pooling failed to detect 8 infections, but pooled-sample analysis missed detecting only samples with a low bacterial load (cycle threshold >35). Pooling samples from MSM to detect CT, NG, and MG is as sensitive as individual-site testing for these 3 pathogens using the Allplex assay. Missed infections with a very low bacterial load could have a low impact on further transmission. Clinical Trials Registration. NCT03568695.

A High Daptomycin Dose Is Associated with Better Bacterial Clearance in Infections Caused by Vancomycin-Resistant Enterococcus faecium Regardless of Daptomycin Minimum Inhibitory Concentration in a Rat Infective Endocarditis Model.

ABSTRACTA high daptomycin dose has been suggested for treating vancomycin-resistant Enterococcus faecium (VREf) infections. However, even a 12 mg/kg daptomycin dose might be insufficient for treating VREf with high daptomycin minimum inhibitory concentrations (MICs). Additionally, animal pharmacodynamic and infection models to confirm the efficacy of 12 mg/kg daptomycin are lacking. Male Wistar rats were used for pharmacokinetic profiling and for the development of an infective endocarditis (IE) model. Daptomycin-susceptible dose-dependent VREf (DSE) (MIC of 0.5 mg/L) and daptomycin nonsusceptible VREf (DNSE) (MIC of 8 mg/L) were used for the IE models. The bacterial load of vegetation was the primary outcome and was evaluated after 3 days of daptomycin treatment. Daptomycin administered subcutaneously (s.c.) at 45 and 90 mg/kg, which corresponded to maximum serum concentrations (Cmax) of 122.6 mg/L and 178.5 mg/L, respectively, was equivalent to doses of 8 mg/kg and 12 mg/kg, respectively, in humans. The Cmax/MIC value was correlated with the bacterial load of vegetation after treatment (r = −0.88, P < 0.001). The 90 mg/kg s.c. group showed a significantly lower bacterial load of vegetation (log10 CFU/g) than the 45 mg/kg s.c. group against DSE (0 versus 4.75, P < 0.001) and DNSE (5.12 versus 6.98, P = 0.002). The 90 mg/kg s.c. group did not sterilize the vegetation against DNSE. Although the human equivalent dose of 12 mg/kg daptomycin was more effective than the smaller dose in reducing the bacterial load in DSE and DNSE IE, the dose could not sterilize the vegetation during a DNSE treatment. Further treatment strategies by which to manage severe VREf infections, especially at high daptomycin MICs, are urgently needed.IMPORTANCE Using a rat IE model with pharmacokinetic analysis, the treatment response of VREf IE was found to be daptomycin dose-dependent, presented as Cmax/MIC or as the 24 h area under the concentration-time curve (AUC0–24)/MIC. Daptomycin 90 mg/kg s.c. significantly reduced the bacterial load against DSE and DNSE. It also showed significant activity against DSE and DNSE, compared to 45 mg/kg s.c. Although daptomycin 90 mg/kg can eradicate the bacterial load after 3 days of treatment against DSE, eradication cannot be achieved with 90 mg/kg daptomycin against DNSE.

Evaluation of a droplet digital PCR assay for quantification of Mycobacterium avium subsp. paratuberculosis DNA in whole-blood and fecal samples from MAP-infected Holstein cattle.

Bovine paratuberculosis (PTB) is an infectious disease that affects ruminants worldwide and is a burden on the dairy industry. PTB control measures include culling of Mycobacterium avium subsp. paratuberculosis (MAP)-infected animals from the herd and the enhancement of farm-biosecurity measures. Diagnostics tools for the direct detection of MAP are fecal real-time qPCR and bacteriological culture, the last one being considered the gold standard. However, both show limitations for detecting subclinical MAP-infected cattle with low bacterial load in feces and gut tissues. Droplet digital polymerase chain reaction (ddPCR) is a third-generation PCR method that shows high reproducibility for the quantification of low DNA copy numbers. The objective of this study was to design a ddPCR assay to detect and quantify a fragment of the F57 MAP-specific sequence in samples of naturally MAP-infected Holstein cattle. DNA was isolated from whole-blood and fecal samples from control cows with a negative ELISA and qPCR result (N = 75) and from cows with PTB-associated focal (N = 32), multifocal (N = 21), and diffuse lesions (N = 17) in gut tissues. After ddPCR, the DNA extracted from fecal samples of cows with diffuse lesions showed higher mean copies per microliter (13,791.2 copies/μl) than samples from cows with multifocal lesions (78.8 copies/μl), focal lesions (177.1 copies/μl) or control cows (4.8 copies/μl) (P ≤ 0.05). Significant differences in mean DNA copies/μl were also observed in the blood samples from cows with focal lesions (47.7 copies/μl) when compared with cows with multifocal and diffuse lesions; 18.1 and 12.4 copies/μl, respectively. Using a principal component analysis, the results of the fecal ddPCR clustered together with the results of a commercial ELISA for the specific detection of MAP antibodies, fecal and tissue qPCR, and bacteriological culture results. In contrast, blood ddPCR results clustered together with the results of an ELISA for the detection of a biomarker of subclinical PTB, the ABCA13 transporter. Blood ddPCR was the most sensitive tool (sensitivity 71%, specificity 100%) of all the quantitative methods used in the study for the detection of subclinical cows with focal lesions.