Outer Membrane Protein Gene Research Articles

Transcriptional profiling of extraintestinal Escherichia coli exposed to cranberry pomace extracts alone or in combination with ceftriaxone

Extraintestinal pathogenic Escherichia coli (ExPEC) includes several serotypes that have been associated with colibacillosis in poultry, as well as urinary tract infections and newborn meningitis in humans. This study investigated the antimicrobial activities of ceftriaxone (AXO) and cranberry pomace extracts (CRAN) alone or in combination (CC) against multidrug-resistant (MDR) ExPEC from broiler. The growth-inhibitory activity of CRAN and synergy tests by a checkerboard method were determined in cation-adjusted Mueller–Hinton broth (CAMHB). The transcriptomic profile of the MDR E. coli O7:H18 (ST38) grown in CAMHB supplemented with sub-inhibitory concertation of CRAN and AXO alone or in combination was obtained by RNA-seq. The MIC of CRAN for all isolates was 16 mg/mL. An additive activity was observed between 4 mg/mL of CRAN and 4 μg/mL of AXO. Compared to the control, the transcriptomic analysis revealed that 4 mg/ml of (1/4MIC) CRAN and its combination with 4 μg/mL of (1/8MIC) AXO (CC) exposures resulted in 727 and 712 differentially expressed genes, respectively (false discovery rate < 0.001 and log2-fold change > 2), in the studied E. coli. Major virulence genes including adhesins (fim, flg, csg, and yad), protectins (omp, tra, waa, and hly), secretion systems (hof, pho, and vir), and quorum sensing (lsr), which are energetically expensive for bacteria, were downregulated. Most importantly, 1/4MIC of CRAN or CC downregulated the β-lactamase blaCMY-2 and efflux pump including tolC, mdtEIJ, gadEW, and their regulator gene evgS, while upregulating the cysteine biosynthesis and oxidative stress-related regulatory genes including cys, dmlA, sbp, nrdGHI, soxSR, and rpoH. Downregulation of multiple enzymes involved in TCA cycles and upregulation of Fe–S cluster coordinated by Cys and Isc proteins reflect the regulation of energy metabolism of the studied E. coli upon CRAN or CC exposure. The downregulation of outer membrane protein genes that control permeability barriers, along with different antimicrobial resistance genes, demonstrates that CRAN may have the unique potential to enhance the antimicrobial activities of third-generation cephalosporins such as AXO against MDR E. coli.

Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

High Frequency of blaOXA-48like Producing Klebsiella pneumoniae Isolated from Nosocomial Infection in Azerbaijan, Iran.

Klebsiella pneumoniae is one of the significant agents of hospital-acquired infections. In recent years, carbapenem-resistant K. pneumoniae (CRKP) isolates have been found in numerous epidemics of nosocomial infections. This study aimed to determine carbapenem resistance mechanisms and molecular epidemiological of CRKP infections in Azerbaijan, Iran. A total of 50 non-duplicated CRKP from January 2020 to December 2020 were isolated form Sina and Imam Reza Hospitals in Tabriz, Iran. Antimicrobial susceptibility testing was performed by the disk-diffusion method. The carbapenem resistance mechanisms were determined by the phenotypic and PCR procedures. CRKP isolates were typed by the Random Amplified Polymorphic DNA PCR (RAPD-PCR) technique. Amikacin was the most effective antibiotics against CRKP isolates. AmpC overproduction was observed in five CRKP isolates. Efflux pump activity was found in one isolate by the phenotypic method. Carba NP test could find carbapenemases genes in 96% of isolates. The most common carbapenemases gene in CRKP isolates were blaOXA-48-like (76%) followed by blaNDM (50%), blaIMP (22%), blaVIM (10%), and blaKPC (10%). The outer membrane protein genes (OmpK36 and OmpK35) were identified in 76% and 82% of CRKP isolates, respectively. RAPD-PCR analysis yielded 37 distinct RAPD-types. Most blaOXA-48-like positive CRKP isolates were obtained from patients hospitalized in intensive care unit (ICU) wards with urinary tract infections. The blaOXA-48-like is the main carbapenemase among CRKP isolates in this area. Most blaOXA-48-like producer CRKP strains were collected from the ICU ward and urine samples. To control infections due to CRKP, a strict control program in hospital settings is required.

Effects of Toxin-Antitoxin System HicAB on Biofilm Formation by Extraintestinal Pathogenic E. coli

The type II toxin-antitoxin (T-A) HicAB system is abundant in several bacteria and archaea, such as Escherichia coli, Burkholderia Pseudomallei, Yersinia pestis, Pseudomonas aeruginosa, and Streptococcus pneumoniae. This system engages in stress response, virulence, and bacterial persistence. This study showed that the biofilm-forming ability of the hicAB deletion mutant was significantly decreased to moderate ability compared to the extra-intestinal pathogenic Escherichia coli (ExPEC) parent strain and the complemented strain, which are strong biofilm producers. Congo red assay showed that the hicAB mutant maintained the ability to form curli fimbriae. Using RNA-seq and comparative real-time quantitative RT-PCR, we observed the difference in gene expression between the hicAB mutant and the parent strain, which was associated with biofilm formation. Our data indicate that the HicAB type II T-A system has a key role in biofilm formation by ExPEC, which may be associated with outer membrane protein (OMP) gene expression. Collectively, our results indicate that the hicAB type II T-A system is involved in ExPEC biofilm formation.

Helicobacter pylori-Associated Iron Deficiency Anemia in Childhood and Adolescence-Pathogenesis and Clinical Management Strategy.

Many epidemiological studies and meta-analyses show that persistent Helicobacter pylori infection in the gastric mucosa can lead to iron deficiency or iron deficiency anemia (IDA), particularly in certain populations of children and adolescents. Moreover, it has been demonstrated that H. pylori infection can lead to and be closely associated with recurrent and/or refractory iron deficiency and IDA. However, the pathogenesis and specific risk factors leading to this clinical outcome in H. pylori-infected children remain poorly understood. In general, most of pediatric patients with H. pylori-associated IDA do not show evidence of overt blood loss due to gastrointestinal hemorrhagic lesions. In adult populations, H. pylori atrophic gastritis is reported to cause impaired iron absorption due to impaired gastric acid secretion, which, subsequently, results in IDA. However, significant gastric atrophy, and the resultant substantial reduction in gastric acid secretion, has not been shown in H. pylori-infected children. Recently, it has been hypothesized that competition between H. pylori and humans for iron availability in the upper gastrointestinal tract could lead to IDA. Many genes, including those encoding major outer membrane proteins (OMPs), are known to be involved in iron-uptake mechanisms in H. pylori. Recent studies have been published that describe H. pylori virulence factors, including specific OMP genes that may be associated with the pathogenesis of IDA. Daily iron demand substantively increases in children as they begin pubertal development starting with the associated growth spurt, and this important physiological mechanism may play a synergistic role for the microorganisms as a host pathogenetic factor of IDA. Like in the most recent pediatric guidelines, a test-and-treat strategy in H. pylori infection should be considered, especially for children and adolescents in whom IDA is recurrent or refractory to iron supplementation and other definitive causes have not been identified. This review will focus on providing the evidence that supports a clear biological plausibility for H. pylori infection and iron deficiency, as well as IDA.

Antibacterial Effect of Various Fermentation Products and Identification of Differentially Expressed Genes of E. coli

Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) are typical opportunistic pathogens. Moreover, these bacteria are known to possess multidrug-resistant (MDR) properties. This study investigates the antimicrobial activity of six fermented products, which have varying efficacies against P. aeruginosa, E. coli, and S. aureus. To identify novel candidate genes, differential expression analysis was performed using an annealing control primer. In the disk diffusion method, Fig vinegar (FV) and Diospyros kaki Thunb. vinegar (DTV) showed the greatest increase in inhibition compared to other fermented products, whereas fermented Korean traditional nature herb (FKTNH) had no antibacterial effect. This study identified down-regulation of Escherichia coli O157:H7 OmpW gene for outer membrane protein W, whereas gene for synthetic construct Lao1 gene for L-amino acid oxidase were up-regulated in E. coli treated with 5% FV. Consuming fermented vinegar helps to prevent bacterial infections. In particular, FV and DTV are potentially useful alternative natural products for multidrug resistance. Furthermore, both are expected to be used as effective natural antimicrobial agents, such as disinfectants.

Removal of antibiotic resistant bacteria, genes and inhibition of plasmid-mediated horizontal transfer by peroxymonosulfate: Efficiency and mechanisms

As the emerging contaminants, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have caught considerable research attention, horizontal gene transfer is one of the most primary contributors to antibiotic resistance dissemination. The application of the oxidant peroxymonosulfate (PMS) as a disinfection alternative has been systemically explored. Also, the effects of various process parameters on ARB inactivation and plasmid-mediated horizontal transfer inhibition, initial bacterial concentration, pH value and temperature, were systemically investigated. The changes in cell substances (cell membrane integrity and intracellular reactive oxygen species) were determined by flow cytometry. The results suggested that the inactivation efficiency of Escherichia coli DH5α (donor) and Pseudomonas sp. HLS-6 (recipient) by 0.8 mM PMS treatment reached 4.45 log and 4.15 log in 30 min, respectively. The conjugative transfer didn’t arise after treatment for only 1 min when the dosage of PMS exceeded 0.4 mM. PMS could work in a wide pH and ambient temperature range. It also showed significant removal efficiency on intracellular ARGs and extracellular ARGs. To identify the underlying mechanisms of restraining conjugative transfer, the mRNA expression of conjugative genes, oxidative stress genes, save our souls (SOS) response-related gene, cell outer membrane proteins genes and global regulatory genes were evaluated. The results demonstrated that the expressions of these genes were altered by 0.8 mM PMS treatment. PMS has the advantages of low cost, easy operation and technical applicability. This study illustrated that PMS treatment showed great potential for controlling the antibiotic resistance for wastewater treatment.

Sublethal concentrations of heavy metals Cu2+ and Zn2+ can induce the emergence of bacterial multidrug resistance

The emergence of multidrug resistance has become an environmental safety issue of worldwide concern. Numerous studies have confirmed that heavy metals can promote the spread of bacterial resistance. However, in addition to co-resistance plasmids, the role and mechanism of metals at sublethal levels (< minimum inhibitory concentration [MIC]) on antibiotic resistance remain poorly defined. Here we investigated the alteration of resistance phenotype under Cu 2 + and Zn 2 + exposure and explored the molecular mechanism by which heavy metals affect antibiotic resistance. The results showed that the MIC of Escherichia coli and Staphylococcus aureus on antibiotics, especially norfloxacin and tetracyclines, was significantly increased after seven consecutive days of exposure to Cu 2 + and Zn 2 + and exhibited a higher cross-resistance rate, which meant that heavy metals can induce bacteria to develop antibiotic resistances that were not previously available. It was observed the downregulation of outer membrane protein gene omp C after heavy metal exposure, while the expression of other efflux pump and resistance genes in E. coli was upregulated, especially tet B, tol C and arc AB genes, suggesting that heavy metals might enhance antibiotic resistance by altering the expression of efflux pump genes. Moreover, many point mutations were found in the efflux system of resistant mutant strains. In particular, quinolone-resistant mutant strains showed a point mutation in the conversion of serine (Ser) to phenylalanine (Phe) at residue 464 of the gyr B gene, which may also be an important cause of increased quinolone resistance. This study provides an explanation for the effect of heavy metals on bacterial resistance. • The MIC of E. coli and S. aureus on antibiotics increased after Cu 2+ and Zn 2+ exposure. • After exposure to heavy metals, the cross resistance rates also showed an increase. • Heavy metals may enhance antibiotic resistance by altering efflux pump expression. • Many point mutations were found in the efflux system of resistant mutant strains.

Effects of Cinnamon Essential Oil on Oxidative Damage and Outer Membrane Protein Genes of Salmonella enteritidis Cells.

Salmonella is an important pathogen causing food poisoning. Food safety and health are the themes of today′s society. As a class of food-borne pathogens, Salmonella enteritidis had become one of the common zoonotic pathogens. Cinnamon essential oil (CEO) had been reported as an antibacterial agent, but there are few studies on its antibacterial mechanism. This study investigated the effects of CEO on oxidative damage and outer membrane protein genes of Salmonella enteritidis cells. First, the reactive oxygen species content in bacteria treated with different concentrations of cinnamon essential oil was determined by fluorescence spectrophotometry, and the effects of superoxide dismutase (SOD), catalase (CAT) and superoxide dismutase (SOD), and catalase (CAT) and peroxidase (POD) were determined by the kit method. The activity of POD and the content of malondialdehyde (MDA) were investigated to investigate the oxidative damage of CEO to Salmonella enteritidis cells. By analyzing the effect of CEO on the Salmonella enteritidis cell membrane’s outer membrane protein gene expression, the mechanism of CEO′s action on the Salmonella enteritidis cell membrane was preliminarily discussed. The results showed that CEO treatment had an obvious oxidative damaging effect on Salmonella enteritidis. Compared with the control group, the increase in CEO concentration caused a significant increase in the bacteria ROS content. The observation technique experiment found that with the increase in CEO concentration, the number of stained cells increased, which indicated that CEO treatment would increase the ROS level in the cells, and it would also increase with the increase in CEO concentration, thus causing the oxidation of cells and damage. In addition, CEO treatment also caused the disruption of the balance of the cellular antioxidant enzymes (SOD, CAT, POD) system, resulting in an increase in the content of MDA, a membrane lipid metabolite, and increased protein carbonylation, which ultimately inhibited the growth of Salmonella enteritidis. The measurement results of cell membrane protein gene expression levels showed that the Omp genes to be detected in Salmonella enteritidis were all positive, which indicated that Salmonella enteritidis carried these four genes. Compared with the control group, the relative expressions of OmpF, OmpA and OmpX in the CEO treatment group were significantly increased (p < 0.05), which proved that the cell function was disturbed. Therefore, the toxicity of CEO to Salmonella enteritidis could be attributed to the damage of the cell membrane and the induction of oxidative stress at the same time. It was speculated that the antibacterial mechanism of CEO was the result of multiple effects. This work was expected to provide a theoretical basis for the development of new natural food preservatives and the prevention and control of Salmonella enteritidis.

Efficacy of recombinant subunit OMP and hly vaccines against Aeromonas hydrophila in Rohu (Labeo rohita).

The main aim of the current study was to clone and express a new outer membrane protein (OMP) and haemolysin (hly) from a pathogenic Aeromonas hydrophila and to investigate their potential as a vaccine candidate against A. hydrophila infection in Rohu (Labeo rohita). The OMP and hly genes were cloned in pET-30b vector and recombinant plasmids pET-30b-OMP and pET-30b-hly were constructed, which were then transferred into Escherichia coli BL21 (DE3). The recombinant E.coli BL21 (DE3) was induced by IPTG, and the OMP and hly proteins were expressed highly. The proteins OMP and hly were estimated in 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their molecular weights were found to be 40 kD and 68 kD. The expressed proteins OMP and hly were purified by Ni-NTA His-Bind Resin column, and the immunogenicity was confirmed by Western blotting. The fishes (L.rohita) were divided into IV groups, and the group I fishes were treated with phosphate saline, the II and III group were immunized with the purified OMP and hly recombinant proteins, and the fishes were treated IV group with combined OMP and hly for 10 days. After 10 days of treatment, the fishes of all the four groups were challenged with virulent A.hydrophila. The results revealed that vaccinated fish showed significantly improved haematological profile, phagocytic activity, myeloperoxidase activity and total immunoglobulin levels on the 5th and 10th days. The non-vaccinated group (Group I) showed 100% mortality, whereas the mixture of recombinant OMP (r-OMP) and hly (r-hly) protein-treated groups (Group IV) exhibited higher survival rate (80%). Relatively, expression of pro- and anti-inflammatory cytokines (IL-1β, IL-10 and TGF-β), c-type and g-type lysozymes were significantly up-regulated in heart and kidney of vaccinated groups compared with the non-vaccinated group. Our results revealed that OMP and hly genes were effective vaccine candidates in the aquaculture system and could be used as recombinant subunit vaccine for diseases caused by pathogenic A.hydrophila.

Presence of bromide and iodide promotes the horizontal transfer of antibiotic resistance genes during chlorination: A preliminary study

Chlorination was reported to have a great potential to increase horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs), which poses a great threat to global human health. Bromide (Br−) and iodide (I−) ions are widely spread ions in water and wastewater. In chlorination, Br− and I− can be oxidized to active bromine and iodine species. The influence of the co-existing different halogen oxidants (chlorine + bromine or iodine species) on HGT of ARGs were rarely investigated. In this study, the conjugative transfer of ARGs between a donor strain E. coli K12 and a recipient strain E. coli HB101 was investigated in chlorination without/with the presence of Br− or I−. Immediately after the addition of sodium hypochlorite, 53–88 % of the dosed chlorine was rapidly consumed, 10 %–42 % fast transformed into organic combined chloramines, and only low levels of free chlorine (0.02–0.8 mg/L as Cl2) left in the diluted cultural medium. Conjugative transfer mediated by the RP4 plasmid was not significantly enhanced in chlorination without the presence of Br− or I−. With the presence of Br− (0.5–5.0 mg/L) or I− (0.05–0.5 mg/L) in chlorination, the co-existing free halogen oxidants and their organic combined ones up-regulated the mRNA expression of the oxidative stress-regulatory gene (rpoS), outer membrane protein gene (ompC), and conjugation-relevant genes (trbBp and trfAp), and caused more damage to cell entirety. As a result, the co-existing reactive halogen oxidants enhanced the HGT of ARGs probably via conjugative transfer and transformation. This study showed that the presence of Br− and I− of common levels in aquatic environment promoted HGT of ARGs in chlorination, thus accelerating the transmission and prevalence of ARGs.

Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation

Cefoperazone/sulbactam (CSL) and piperacillin/tazobactam (TZP) are commonly used in clinical practice in China because of their excellent antimicrobial activity. CSL and TZP-nonsusceptible Enterobacteriaceae are typically resistant to extended-spectrum cephalosporins such as ceftriaxone (CRO). However, 11 nonrepetitive Enterobacteriaceae strains, which were resistant to CSL and TZP yet susceptible to CRO, were collected from January to December 2020. Antibiotic susceptibility tests and whole-genome sequencing were conducted to elucidate the mechanism for this rare phenotype. Antibiotic susceptibility tests showed that all isolates were amoxicillin/clavulanic-acid resistant and sensitive to ceftazidime, cefepime, cefepime/tazobactam, cefepime/zidebactam, ceftazidime/avibactam, and ceftolozane/tazobactam. Whole-genome sequencing revealed three of seven Klebsiella pneumoniae strains harbored blaSHV-1 only, and four harbored blaSHV-1 and blaTEM-1B. Two Escherichia coli strains carried blaTEM-1B only, while two Klebsiella oxytoca isolates harbored blaOXY-1-3 and blaOXY-1-1, respectively. No mutation in the β-lactamase gene and promoter sequence was found. Outer membrane protein (Omp) gene detection revealed that numerous missense mutations of OmpK36 and OmpK37 were found in all strains of K. pneumoniae. Numerous missense mutations of OmpK36 and OmpK35 and OmpK37 deficiency were found in one K. oxytoca strain, and no OmpK gene was found in the other. No Omp mutations were found in E. coli isolates. These results indicated that narrow spectrum β-lactamases, TEM-1, SHV-1, and OXY-1, alone or in combination with Omp mutation, contributed to the resistance to CSL and TZP in CRO-susceptible Enterobacteriaceae.Antibiotic susceptibility testsAntibioticsBreakpoint, (μg/ml)Klebsiella pneumoniaeEscherichia couKlebriehd axyocaE1E3E4E7E9E10E11E6E8E2E5CRO≤1≥4≤0.5≤0.5≤0.5≤0.5 1≤0.51≤0.5≤0.511CAZ4 ≥161214444241 1FEP≤2 216 110.2512220.521 1AMC≤8 ≥32≥128≥128≥128≥128≥128≥128≥128≥128≥128≥128≥128CSL≤16 ≥6464646464≥128128≥12864128128≥128TZP≤16 ≥128≥256≥256≥256≥25622562256≥256≥256≥256≥256≥256FPT≤2 ≥1610.50.060.1252120.2510.1250.25FPZ≤2 2160.250.250.060.1250.250.25 10.1250.250.1250.125CZA≤8 216 10.50.250.2510.2510.50.50.50.25CZT≤2 28210.5 1222 1122CROceftriaxone, CAZceftazidime, FEPcefepime, AMC:amoxicillin clavulanic-acid, CSLcefoperazone/sulbactam, TZP:piperadllin/tazobactam, FPT:cefepime tazobactam, FPZ:cefepime/zidebactam, CZA:ceftazidime/avibactam, CZTceftolozane/tazobactam Gene sequencing resultsNumberStrainSTp-Lactamase genePromoter sequence mutationOmp mutationElKpn45blaSHV-1, blaTEM-lBnoneOmpK36, OmpK3 7E3Kpn45blaSHV-1, blaTEM-lBnoneOmpK36. OmpK3 7E4Kpn2854blaSHV-1noneOmpK36, OmpK3 7E7Kpn2358blaSHV-1 - blaTEM-lBnoneOmpK36, OmpK3 7E9Kpn2358blaSHV-1. blaTEM-lBnoneOmpK36. OmpK3 7E10Kpn 189blaSHV-1noneOmpK36. OmpK3 7EllKpn45blaSHV-1noneOmpK36, OmpK3 7E6Eco88blaTEM-lBnonenoneESEco409blaTEM-1BnonenoneE2Kox194blaOXY-1-3noneOmpK36 mutations. OmpK35 and OmpK 37 deficiencyE5Kox 11blaOXY-1-1noneno OmpK (OmpK3 5, OmpK36 and OmpK37) gene found

Effect of sulfamethazine on the horizontal transfer of plasmid-mediated antibiotic resistance genes and its mechanism of action

As a new type of environmental pollutant, antibiotic resistance genes (ARGs) pose a huge challenge to global health. Horizontal gene transfer (HGT) represents an important route for the spread of ARGs. The widespread use of sulfamethazine (SM2) as a broad-spectrum bacteriostatic agent leads to high residual levels in the environment, thereby increasing the spread of ARGs. Therefore, we chose to study the effect of SM2 on the HGT of ARGs mediated by plasmid RP4 from Escherichia coli (E. coli) HB101 to E. coli NK5449 as well as its mechanism of action. The results showed that compared with the control group, SM2 at concentrations of 10 mg/L and 200 mg/L promoted the HGT of ARGs, but transfer frequency decreased at concentrations of 100 mg/L and 500 mg/L. The transfer frequency at 200 mg/L was 3.04 × 10−5, which was 1.34-fold of the control group. The mechanism of SM2 improving conjugation transfer is via enhancement of the mRNA expression of conjugation genes (trbBP, trfAP) and oxidative stress genes, inhibition of the mRNA expression of vertical transfer genes, up regulation of the outer membrane protein genes (ompC, ompA), promotion of the formation of cell pores, and improvement of the permeability of cell membrane to promote the conjugation transfer of plasmid RP4. The results of this study provide theoretical support for studying the spread of ARGs in the environment.

How heavy metal stress promotes dissemination of antibiotic resistance genes in the activated sludge process

Heavy metals have been recently revealed as promoters to antibiotic resistance gene (ARG) dissemination in water environment, but their influence on ARG transfer in the activated sludge process has not been clear. In this study, a set of sequencing batch reactors (SBRs) and micro-scale microfluidic chips were established to quantify the impacts of heavy metals (0.5 mM of Pb, 0.1 mM of As, and 0.005 mM of Hg) on the ARG spreading in the activated sludge consortium. Under heavy metal stress, transfer frequencies were 1.7–3.6 folds increase compared to the control. Gram-negative bacteria increased significantly after heavy metal added, which were more prone to receiving resistant plasmid from donors. Meanwhile, the relative expression of genes related to conjugation changed in activated sludge, especially the expression of outer membrane protein and oxidative stress regulatory genes increased by 2.9–7.4 folds and 7.8–13.1 folds, respectively. Furthermore, using microfluidic chips, the dynamics of ARG transfer was observed at single cell level under heavy metal pressure. Heavy metals firstly promoted conjugation and then vertical gene transfer played an important part for ARG spreading. The results provided in-depth understanding of the influence of heavy metals on ARG behavior in the environment.

Distribution characteristics of the sabA, hofC, homA, homB and frpB-4 genes of Helicobacter pylori in different regions of China.

BackgroundHelicobacter pylori (H. pylori) encodes numerous outer membrane proteins (OMPs), with considerable geographic heterogeneity and related to different clinical outcomes. This study aimed to investigate the distribution characteristics of five important OMP genes (sabA, hofC, homA, homB and frpB-4) in different regions of China.Materials and methodA total of 266 strains were isolated from 348 stomach biopsy specimens in Shandong, Guangxi, Heilongjiang, Hunan, and Qinghai provinces. The presence of sabA, hofC, homA, homB and frpB-4 gene was detected by polymerase chain reaction (PCR) from H. pylori genomic DNA.ResultsAmong the strains in five regions, the prevalence of frpB-4 was 100% and that of hofC was 97.7%. The prevalence of homB in the isolates from Qinghai (45.5%) was significantly lower than that in Shandong (75.3%), Guangxi (76.9%) and Hunan (69.6%) (P<0.05). The frequency of homA in Shandong (30.1%) was significantly lower than in Guangxi (57.7%) and Qinghai (63.6%) (P<0.05). The prevalence of the sabA gene in Shandong, Guangxi, Heilongjiang, Hunan and Qinghai provinces was 21.9%, 59.7%, 45.9%, 52.2%, and 18.2%, respectively (P<0.05). The sabA “on” status was significantly more frequent in isolates from Guangxi (46.8%), Heilongjiang (37.8%), and Hunan (47.8%) than Qinghai (3.0%) (P<0.05). The presence of homA and sabA genes may be negatively correlated with the development of gastritis. There was no significant association between the frpB-4, hofC, homB gene and clinical outcomes.ConclusionThe prevalence of homA, homB, and sabA genes and the sabA “on” or “off” status have significant geographical differences among five provinces in China. The presence of homA and sabA genes may be protective factors of gastritis.

Avian Pathogenic Escherichia coli through Pfs Affects the Tran-Scription of Membrane Proteins to Resist β-Lactam Antibiotics.

Avian pathogenic Escherichia coli (APEC) is a causative agent of colibacillosis, one of the principal causes of morbidity and mortality in poultry worldwide. Nowadays, antibiotics are mainly used to prevent and control poultry colibacillosis, but the situation of drug resistance is serious. 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing (QS) pathways. In this study, compared with the APEC wild-type strain DE17, the pfs deletion strain DE17Δpfs was more susceptible to β-lactam antibiotics (amoxicillin, ceftazidime, cefuroxime) by drug sensitivity test and minimum inhibitory concentration (MIC), and the MIC of the DE17Δpfs was half that of the DE17. Quorum sensing signal molecule AI-2 is involved in antibiotic resistance. In the case of pfs inactivation, the DE17Δpfs cannot synthesize AI-2, so it is necessary to add AI-2 to study whether it affects APEC resistance. When the exogenous AI-2 was added, the MIC of all APEC did not change. Transcriptome sequencing indicated that the transcription levels of a lot of outer membrane protein genes and metabolic genes had changed due to the deletion of pfs. Moreover, the transcription levels of the efflux pump gene tolC and penicillin binding protein (fstI and mrcA) were significantly reduced (p < 0.05), while the transcription levels of the porin protein genes (ompF, ompC, and ompD) were significantly increased (p < 0.05). In addition, it was also found that the outer membrane permeability of the DE17Δpfs was significantly increased (p < 0.05). The results indicated that pfs does not affect APEC strain DE17 resistance to β-lactam antibiotics through AI-2, but pfs affects the sensitivity of APEC to β-lactam antibiotics by affecting antibiotic-related genes. This study can provide a reference for screening new drug targets.

Vector and rodent surveillance for Orientia tsutsugamushi in north India.

Scrub typhus or chigger borne typhus, caused by Orientia tsutsugamushi is an emerging vector-borne disease as large numbers of cases have been reported in various tropical countries. It is transmitted to humans through bites of infected chiggers (larval mites). The knowledge about the vector, its distribution, density and habitat are important so as to understand the epidemiology of scrub typhus in a given area. To control rickettsial infections, regular rodent-vector surveillance should be planned in areas where the disease transmission is occurring and it will also help to strengthen the existing entomological data related to the vector of scrub typhus in northern India. In the present study, rodent-vector surveillance was planned for one whole year, covering both mite active and non-active seasons (October 2019-December 2020) in selected areas of Chandigarh and Punjab in north India. Rodent tissues and mites were also examined for the presence of O. tsutsugamushi by nested PCR for 56 kDa gene and real-time PCR for 47 kDa outer membrane protein gene. 18S gene PCR was performed for molecular identification of mites. In the surveillance, three types of ectoparasite, viz. mites, fleas and ticks were obtained in rodents. All mites found were of Laelapidae family. None of the pooled rodent tissue samples as well as mite samples were found positive for O. tsutsugamushi by nested PCR for rickettsial DNA. In the present study, we did not get any evidence of carriage of O. tsutsugamushi in either mites or rodents collected and sampled in selected regions in Chandigarh and Punjab. We need to strengthen the entomological surveillance over a broader region and increase the frequency of trapping rodents to increase clarity on vector-reservoir dynamics in this geographical region.

Quinolone Resistance of Actinobacillus pleuropneumoniae Revealed through Genome and Transcriptome Analyses.

Actinobacillus pleuropneumoniae is a pathogen that infects pigs and poses a serious threat to the pig industry. The emergence of quinolone-resistant strains of A. pleuropneumoniae further limits the choice of treatment. However, the mechanisms behind quinolone resistance in A. pleuropneumoniae remain unclear. The genomes of a ciprofloxacin-resistant strain, A. pleuropneumoniae SC1810 and its isogenic drug-sensitive counterpart were sequenced and analyzed using various bioinformatics tools, revealing 559 differentially expressed genes. The biological membrane, plasmid-mediated quinolone resistance genes and quinolone resistance-determining region were detected. Upregulated expression of efflux pump genes led to ciprofloxacin resistance. The expression of two porins, OmpP2B and LamB, was significantly downregulated in the mutant. Three nonsynonymous mutations in the mutant strain disrupted the water–metal ion bridge, subsequently reducing the affinity of the quinolone–enzyme complex for metal ions and leading to cross-resistance to multiple quinolones. The mechanism of quinolone resistance in A. pleuropneumoniae may involve inhibition of expression of the outer membrane protein genes ompP2B and lamB to decrease drug influx, overexpression of AcrB in the efflux pump to enhance its drug-pumping ability, and mutation in the quinolone resistance-determining region to weaken the binding of the remaining drugs. These findings will provide new potential targets for treatment.

Protective effect of Lactobacillus reuteri Lb11 from chicken intestinal tract against Salmonella Enteritidis SE05 in vitro.

Salmonella infections in eggs with increasing morbidity and mortality exhibit worldwide prevalence. The present study intends to evaluate the efficacy of Lactobacillus reuteri Lb11 (L. reuteri Lb11, isolated from chicken intestinal tract) in inhibiting the growth of multi-drug resistant (MDR) Salmonella Enteritidis SE05 (obtained from egg content). The cell-free cell lysates (CFCL) of L. reuteri Lb11 obtained by the agar spot test performed well on inhibition of the MDR (Multi-DrugResistant) Salmonella Enteritidis SE05, The heat-inactivated (HI) fraction of L. reuteri Lb11 showed no inhibition activity. By co-culturing with L. reuteri Lb11 in vitro, the growth of S. Enteritidis SE05 decreased along with time, while, the pH value decreased significantly. Furthermore, In order to evaluate the mechanism of action of CFCL of L.reuteri Lb11, the genes related to the transcription level of AcrAB-TolC efflux pump, outer membrane protein OMPs genes and drug resistance genes have been quantified by real-time PCR, when the S. Enteritidis was SE05 exposed to the CFCL of L. reuteri Lb11 (1 × 1012CFU/mL). Almost all of the AcrAB-TolC efflux pump genes, outer membrane protein genes and antibiotic resistance genes were down-regulated. Especially, the level of ramA, tetA and tetB genes were down-regulated -20.77, -15.85 and -12.42 folds, respectively. L. reuteri Lb11 can effectively prevent the formation of efflux pump to inhibit the production of multidrug-resistant Salmonella Enteritidis in eggs.

Molecular Detection of Bacterial Agents of Atypical Pneumonia among patients from six hospitals in Suez Canal region

Background: Reliable diagnosis of atypical pneumonia microbial agents is crucial since they do not respond to beta-lactams but to other groups of antibiotics. Therefore, empiric treatment with beta lactam groups will be ineffective for their eradication if no additional antibiotics as macrolides are administered to the management plan. This study aimed to determine prevalence of Mycoplasma pneumoniae (M. pneumoniae), Chlamydophila pneumoniae (C. pneumoniae) and Legionella pneumophila (L. pneumophila) among patients with atypical pneumonia, and drawing physicians' attention to the role of these pathogens as etiologic agents of atypical pneumonia in the Suez Canal region. Methods: Eighty-four atypical pneumonia cases were enrolled in this study. Respiratory samples were collected. Part of each specimen was inoculated onto blood, MacConkey, and chocolate agar plates, another part of specimens was processed for DNA extraction and multiplex PCR assay for detection of M. pneumoniae p1adhesion gene, C. pneumoniae outer membrane protein (ompA) gene, and L. pneumophila macrophage infectivity potentiator (mip) gene. Results: Out of the 84 atypical pneumonia cases, atypical bacteria were detected by multiplex conventional PCR in 12 (14%) cases and they all were L. pneumophila, 3 cases (4%) were mixed with Staphylococcus aureus, and 2 (2%) cases were mixed with Streptococcus pyogens. Mycoplasma pneumoniae and C. pneumoniae were not detected by PCR in our samples. Conclusions: Legionella pneumophila incidence is not low in our geographical region in patients with atypical pneumonia; so it is of pivotal importance to recruit sensitive and reliable molecular based techniques to detect and control this infection in healthcare environments.