Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Historical and Molecular Perspectives on the Presence of Helicobacter pylori in Latin America: A Niche to Improve Gastric Cancer Risk Assessment.

Helicobacter pylori (H. pylori) is responsible for causing chronic gastritis, which can cause peptic ulcer and premalignant lesions such as atrophic gastritis, intestinal metaplasia, and dysplasia, with the risk of developing gastric cancer. Recent data describe that H. pylori colonizes the gastric mucosa of more than 50% of the world's population; however, this bacterium has been described as infecting the human population since its prehistory. This review focuses on the populations and subpopulations of H. pylori, differentiated by the polymorphisms present in their constitutive and virulence genes. These genes have spread and associated with different human populations, showing variability depending on their geographical distribution, and have evolved together with the human being. The predominant genotypes worldwide, Latin America and Chile, are described to understand the genetic diversity and pathogenicity of H. pylori in different populations and geographic regions. The high similarity in the sequence of virulence genes between H. pylori strains present in Peruvian and Spanish natives in Latin America suggests a European influence. The presence of cagA-positive strains and vacA s1 m1 allelic variants is observed with greater prevalence in Chilean patients with more severe gastrointestinal diseases and is associated with its geographical distribution. These findings highlight the importance of understanding the genetic diversity of H. pylori in different regions of the world for a more accurate assessment of the risk of associated diseases and their potential impact on health.

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution.

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Inheritance and fitness cost of laboratory-selected resistance to Vip3Aa in Helicoverpa zea (Lepidoptera: Noctuidae).

The polyphagous pest Helicoverpa zea (Lepidoptera: Noctuidae) has evolved practical resistance to transgenic corn and cotton producing Cry1 and Cry2 crystal proteins from Bacillus thuringiensis (Bt) in several regions of the United States. However, the Bt vegetative insecticidal protein Vip3Aa produced by Bt corn and cotton remains effective against this pest. To advance knowledge of resistance to Vip3Aa, we selected a strain of H. zea for resistance to Vip3Aa in the laboratory. After 28 generations of continuous selection, the resistance ratio was 267 for the selected strain (GA-R3) relative to a strain not selected with Vip3Aa (GA). Resistance was autosomal and almost completely recessive at a concentration killing all individuals from GA. Declines in resistance in heterogeneous strains containing a mixture of susceptible and resistant individuals reared in the absence of Vip3Aa indicate a fitness cost was associated with resistance. Previously reported cases of laboratory-selected resistance to Vip3Aa in lepidopteran pests often show partially or completely recessive resistance at high concentrations and fitness costs. Abundant refuges of non-Bt host plants can maximize the benefits of such costs for sustaining the efficacy of Vip3Aa against target pests.

Assessment of the mixed origin of the gastric epithelial extracellular vesicles in acellular transfer of Helicobacter pylori toxins and a systematic review.

BACKGROUND: H. pylori are generally considered as extracellular organisms, with exclusive colonization of the gastric milieu. Yet, several extra gastric manifestations are associated with this infection. The aim of the present study was to investigate the feasibility of toxin transfer by extracellular vesicles, from bacterial and epithelial origins. METHODS: Tox-positive H. pylori and its two cagA and vacA mutant strains were used to produce bacterial vesicles (BVs) and to infect AGS cells. The produced BVs and the infected cell vesicles (ICVs) were collected by ultracentrifugation and evaluated by western blotting, DLS and electron microscopy. These two sets of vesicles were applied to a second set of recipient AGS cells, in which the acellular transfer of toxins, IL-8 production and downstream morphologic changes were assessed, by western blotting, ELISA and light microscopy, respectively. RESULTS: The BVs were positive for H. pylori membrane markers (BabA and UreB), VacA and CagA toxins, except for from the corresponding mutant strains. The ICVs were larger in size and positive for bacterial markers, as well as epithelial markers of CD9, LGR5, but negative for nuclear (Ki76) or cytoplasmic (ß-actin) markers. Bacteria-independent transfer of CagA and VacA into the recipient cells occurred upon treatment of cells with BVs and ICVs, followed by cellular vacuolation and elongation. IL-8 production was induced in recipient AGS cells, treated with BVs (1279.4 ± 19.79 pg/106 cells), early (8 h, 1171.4 ± 11.31 pg/106 cells) and late (48 h, 965.4 ± 36.77 pg/106 cells) ICVs (P < 0.0001). CONCLUSION: Our data indicates that ICVs, with mixed bacterial and epithelial constituents, similar to BVs, are capable of transferring bacterial toxins into the recipient cells, inducing IL-8 production and subsequent morphologic changes, in an acellular manner.

Inheritance and Fitness Costs of Laboratory-Selected Resistance to Gpp34/Tpp35Ab1 Corn in Western Corn Rootworm (Coleoptera: Chrysomelidae).

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn and is currently managed with corn hybrids that produce insecticidal proteins derived from the bacterium Bacillus thuringiensis (Bt). Bt corn kills rootworm larvae and reduces larval feeding injury to corn roots. The Bt protein Gpp34/Tpp35Ab1, previously named Cry34/35Ab1, has been widely used in transgenic Bt corn for management of western corn rootworm, and field-evolved resistance has been found in some populations. In the United States, the refuge strategy is used to manage Bt resistance, with refuges of non-Bt host plants serving as a source of Bt-susceptible individuals, which in turn reduce the frequency of homozygous resistant individuals within a population. As such, the dominance of resistance strongly influences resistance evolution, with faster evolution of resistance when resistance is not recessive. Additionally, selection for resistance by a Bt crop leads to the accumulation of resistance alleles within refuge populations, thereby reducing the capacity of refuges to delay resistance. However, fitness costs can remove resistance alleles from refuge populations and preserve the dynamic of refuges producing Bt-susceptible genotypes. Bt-susceptible and Gpp34/Tpp35Ab1-resistant western corn rootworm were used to quantify the inheritance and fitness costs of resistance. We found that Gpp34/Tpp35Ab1 resistance was not recessive and had the accompanying fitness costs of slower developmental rate to adulthood and lower egg viability. This research will help improve insect resistance management by providing a better understanding of the risk of western corn rootworm evolving resistance to transgenic corn that produces Gpp34/Tpp35Ab1.

Directed carbapenemase testing is no longer just for Enterobacterales: cost, labor, and workflow assessment of expanding carbapenemase testing to carbapenem-resistant P. aeruginosa.

Molecular carbapenem-resistance testing, such as for the presence of carbapenemases genes, is commonly implemented for the detection of carbapenemase-producing Enterobacterales. Carbapenemase-producing P. aeruginosa is also associated with significant morbidity and mortality, although; prevalence may be underappreciated in the United States due to a lack of carbapenemase testing. The present study sought to compare hands-on time, cost and workflow implementation of carbapenemase gene testing in Enterobacterales and P. aeruginosa isolates versus sending out isolates to a public health laboratory (PHL) for testing to assess if in-house can provide actionable results. The time to carbapenemase gene results were compared. Differences in cost for infection prevention measures were extrapolated from the time of positive carbapenemase gene detection in-house versus PHL. The median time to perform carbapenemase gene testing was 7.5 min (range 5-14) versus 10 min (range 8-22) for preparation to send isolates to the PHL. In-house testing produced same day results compared with a median of 6 days (range 3-14) to receive results from PHL. Cost of in-house testing and send outs were similar ($46.92 versus $40.53, respectively). If contact precautions for patients are implemented until carbapenemase genes are ruled out, in-house testing can save an estimated $76,836.60 annually. Extension of in-house carbapenemase testing to include P. aeruginosa provides actionable results 3-14 days earlier than PHL Standard Pathway testing, facilitating guided therapeutic decisions and infection prevention measures. Supplemental phenotypic algorithms can be implemented to curb the cost of P. aeruginosa carbapenemases testing by identifying isolates most likely to harbour carbapenemases.

Critical Assessment of the Prospects of Quorum-Quenching Therapy for Staphylococcus aureus Infection.

Staphylococcus aureus is an important pathogen that causes a high number of infections and is one of the leading causes of death in hospitalized patients. Widespread antibiotic resistance such as in methicillin-resistant S. aureus (MRSA) has prompted research into potential anti-virulence-targeted approaches. Targeting the S. aureus accessory gene regulator (Agr) quorum-sensing system, a master regulator of virulence, is the most frequently proposed anti-virulence strategy for S. aureus. While much effort has been put into the discovery and screening for Agr inhibitory compounds, in vivo analysis of their efficacy in animal infection models is still rare and reveals various shortcomings and problems. These include (i) an almost exclusive focus on topical skin infection models, (ii) technical problems that leave doubt as to whether observed in vivo effects are due to quorum-quenching, and (iii) the discovery of counterproductive biofilm-increasing effects. Furthermore, potentially because of the latter, invasive S. aureus infection is associated with Agr dysfunctionality. Altogether, the potential of Agr inhibitory drugs is nowadays seen with low enthusiasm given the failure to provide sufficient in vivo evidence for their potential after more than two decades since the initiation of such efforts. However, current Agr inhibition-based probiotic approaches may lead to a new application of Agr inhibition strategies in preventing S. aureus infections by targeting colonization or for otherwise difficult-to-treat skin infections such as atopic dermatitis.

Assessment of the influence of selected stress factors on the growth and survival of Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes are Gram-positive rods, which are the etiological factor of listeriosis. L. monocytogenes quickly adapts to changing environmental conditions. Since the main source of rods is food, its elimination from the production line is a priority. The study aimed to evaluate the influence of selected stress factors on the growth and survival of L. monocytogenes strains isolated from food products and clinical material. RESULTS: We distinguished fifty genetically different strains of L. monocytogenes (PFGE method). Sixty-two percent of the tested strains represented 1/2a-3a serogroup. Sixty percent of the rods possessed ten examined virulence genes (fbpA, plcA, hlyA, plcB, inlB, actA, iap, inlA, mpl, prfA). Listeria Pathogenicity Island 1 (LIPI-1) was demonstrated among 38 (76.0%) strains. Majority (92.0%) of strains (46) were sensitive to all examined antibiotics. The most effective concentration of bacteriophage (inhibiting the growth of 22 strains; 44.0%) was 5 × 108 PFU. In turn, the concentration of 8% of NaCl was enough to inhibit the growth of 31 strains (62.0%). The clinical strain tolerated the broadest pH range (3 to 10). Five strains survived the 60-min exposure to 70˚C, whereas all were alive at each time stage of the cold stress experiment. During the stress of cyclic freezing-defrosting, an increase in the number of bacteria was shown after the first cycle, and a decrease was only observed after cycle 3. The least sensitive to low nutrients content were strains isolated from frozen food. The high BHI concentration promoted the growth of all groups. CONCLUSIONS: Data on survival in stress conditions can form the basis for one of the hypotheses explaining the formation of persistent strains. Such studies are also helpful for planning appropriate hygiene strategies within the food industry.

Assessment of horizontal gene transfer-mediated destabilization of Synechococcus elongatus PCC 7942 biocontainment system.

Biological containment is a biosafety strategy that prevents the dispersal of genetically modified organisms in natural ecosystems. We previously established a biocontainment system that makes bacterial growth dependent on the availability of phosphite (Pt), an ecologically rare form of phosphorus (P), by introducing Pt metabolic pathway genes and disrupting endogenous phosphate and organic phosphate transporter genes. Although this system proved highly effective, horizontal gene transfer (HGT) mediated recovery of a P transporter gene is considered as a potential pathway to abolish the Pt-dependent growth, resulting in escape from the containment. Here, we assessed the risk of HGT driven escape using the Pt-dependent cyanobacterium Synechococcus elongatus PCC 7942. Transformation experiments revealed that the Pt-dependent strain could regain phosphate transporter genes from the S. elongatus PCC 7942 wild-type genome and from the genome of the closely related strain, S. elongatus UTEX 2973. Transformed S. elongatus PCC 7942 became viable in a phosphate-containing medium. Meanwhile, transformation of the Synechocystis sp. PCC 6803 genome or environmental DNA did not yield escape strains, suggesting that only genetic material derived from phylogenetically-close species confer high risk to generate escape. Eliminating a single gene necessary for natural competence from the Pt-dependent strain reduced the escape occurrence rate. These results demonstrate that natural competence could be a potential risk to destabilize Pt-dependence, and therefore inhibiting exogenous DNA uptake would be effective for enhancing the robustness of the gene disruption-dependent biocontainment.

Comparative evaluation of phenotypic and genotypic methods for the rapid and cost-effective detection of carbapenemases in extensively drug resistant Klebsiella pneumoniae.

PURPOSE: Carbapenemases are the enzymes that can hydrolyze carbapenems and other ß-lactam antibiotics. These enzymes confer resistance to multiple antibiotics and act as a stumbling block in the treatment of infections caused by gram-negative bacteria. Therefore, rapid and specific detection of these enzymes is crucial for deciding the course of treatment and better clinical outcomes. MATERIAL AND METHODS: This study was conducted to compare various phenotypic and PCR based methods for the detection of carbapenemases in carbapenem- and colistin-resistant Klebsiella pneumoniae. One hundred clinical isolates of extensively resistant Klebsiella pneumoniae were included in the study. Phenotypic detection for carbapenemases was performed by Rapidec® Carba NP (Biomerieux), modified carbapenem inactivation method (mCIM), imipenem-ethylenediaminetetraacetic acid disk synergy (EDS), double disk synergy test using mercaptopropionic acid (DDST-MPA), and combined disk method (CD) and for colistin by microbroth dilution method. Genotypic detection for carbapenemases and colistin resistance was performed by targeted PCR. RESULTS: The sensitivity of Carba NP test and mCIM were positive in 95% and 96% respectively and specificity was 100% for both methods. The sensitivity of EDS, DDST-MPA, and CD were 55.6%, 88.9% and 54.5% respectively. Among the carbapenem resistance genes, blaOXA-48 (82%) genes were the most prevalent. Among metallo-beta lactamases, blaVIM (56%) was most common followed by blaNDM (54%) and blaIMP (20%). The mcr-1 gene for colistin resistance was not detected in any isolate. CONCLUSION: Among the five phenotypic assays analyzed, the mCIM is the most simple, inexpensive, accurate and reproducible method for carbapenemase detection in Klebsiella pneumoniae. The DDST-MPA test provides the best sensitivity for the detection of carbapenemases, although specificity is low. These tests, when applied in a clinical laboratory and assessed by the microbiologist, can help in guiding the course of treatment.

Klebsiella Species and Enterobacter cloacae Isolates Harboring blaOXA-181 and blaOXA-48: Resistome, Fitness Cost, and Plasmid Stability.

IncX3 and IncL plasmids have been named as catalysts advancing dissemination of blaOXA-181 and blaOXA-48 genes. However, their impact on the performance of host cells is vastly understudied. Genetic characteristics of blaOXA-48- and blaOXA-181-containing Klebsiella pneumoniae (EFN299), Klebsiella quasipneumoniae (EFN262), and Enterobacter cloacae (EFN743) isolated from clinical samples in a Ghanaian hospital were investigated by whole-genome sequencing. Transfer of plasmids by conjugation and electroporation, plasmid stability, fitness cost, and genetic context of blaOXA-48, blaOXA-181, and blaDHA-1 were assessed. blaOXA-181 was carried on two IncX3 plasmids, an intact 51.5-kb IncX3 plasmid (p262-OXA-181) and a 45.3-kb IncX3 plasmid (p743-OXA-181) without replication protein sequence. The fluoroquinolone-resistant gene qnrS1 region was also excised, and unlike in p262-OXA-181, the blaOXA-181 drug-resistant region was not found on a composite transposon. blaOXA-48 was carried on a 74.6-kb conjugative IncL plasmid with unknown ~10.9-kb sequence insertion. This IncL plasmid proved to be highly transferable, with a conjugation efficiency of 1.8 × 10-2. blaDHA-1 was present on an untypeable 22.2 kb genetic structure. Plasmid stability test revealed plasmid loss rate between 4.3% and 12.4%. The results also demonstrated that carriage of IncX3-blaOXA-181 or IncL-blaOXA-48 plasmids was not associated with any fitness defect, but rather an enhanced competitive ability of host cells. This study underscores the significant contribution of IncX3 and IncL plasmids in the dissemination of resistance genes and their efficient transfer calls for regular monitoring to control the expansion of resistant strains. IMPORTANCE The growing rate of antibiotic resistance is an important global health threat. This threat is exacerbated by the lack of safe and potent alternatives to carbapenems in addition to the slow developmental process of newer and effective antibiotics. Infections by carbapenem-resistant Gram-negative bacteria are becoming almost untreatable, leading to poor clinical outcomes and high mortality rates. OXA-48-like carbapenemases are one of the most widespread carbapenemases accounting for resistance among Enterobacteriaecae. We characterized OXA-48- and OXA-181-producing Enterobacteriaecae to gain insights into the genetic basis and mechanism of resistance to carbapenems. Findings from the study showed that the genes encoding these enzymes were carried on highly transmissible plasmids, one of which had sequences absent in other similar plasmids. This implies that mobile genetic elements are important players in the dissemination of resistance genes. Further characterization of this plasmid is warranted to determine the role of this sequence in the spread of resistance genes.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

Safety of Bacillus thuringiensis Cry1Ah and Vip3Aa toxins for the predatory stink bug Arma custos (Hemiptera: Pentatomidae).

The widespread adoption of Bt crops expressing insecticidal proteins derived from Bacillus thuringiensis has created a need to assess the potential effects of these toxins on non-target organisms, especially species such as Arma custos, a generalist predator that provides important biological control services in many field crops in Asia. Direct dietary exposure of A. custos to Cry1Ah and Vip3Aa proteins produced no adverse effects on life history traits, despite continuous exposure throughout development and early adult life to concentrations significantly higher than the Bt protein concentration likely encountered by A.custos in the field, even when feeding directly on Bt plants. Enzyme-linked immunosorbent assay confirmed the presence of Bt proteins in A. custos midguts, but quantitative real-time PCR analysis of 12 genes associated with detoxification, antioxidative responses, immune responses, and metabolism revealed no significant changes in expression in adult bugs. Indirect exposure to these toxins via consumption of intoxicated prey, larvae of Helicoverpa armigera (Hübner), likewise produced no negative impacts on survival, development, adult weight, or female fecundity in either the F0 (exposed) or F1 (unexposed) generation, but female fresh weight was reduced in the F0 generation by the Cry1Ah (50 µg/g) treatment. Finally, a competitive binding assay with labelled protein and a ligand blotting assay both demonstrated that the Cry1Ah protein could not bind to receptors on the midgut brush border membrane vesicles (BBMVs) of A. custos adults. Therefore, we conclude that Cry1Ah and Vip3Aa proteins are unlikely to have significant negative effects on A. custos populations if employed as plant-incorporated protectants in field crops.

Risk assessment of gastric cancer in the presence of Helicobacter pylori cagA and hopQII genes.

Helicobacter pylori bacterium is one of the most common bacterial infections globally and is the leading cause of indigestion, gastric and duodenal ulcers, and gastric cancer. This bacterium can escape the antibacterial effects of stomach acid by adapting to the inner layers of the stomach. It combines with the natural sugars in the gastric mucosa. The compound is so effective that it makes bacterium resistant. For genes related to the pathogenesis of H. pylori, using the existence of genes such as cagA, hopQI, and hopQII, PCR is performed on some of these genes to amplify fragments of different lengths. One of the less-studied cases is that two or more pathogenic genes are simultaneously associated with H. pylori. This study examined the frequency of diseases and healthy individuals infected with H. pylori and cagA and hopQII genotypes. To diagnose H. pylori infection in healthy and stomach cancer patients, the PCR products are electrophoresed on the agarose gel after glmM gene amplification by PCR. To this aim, stomach tissue biopsies were used for patients, and saliva was used for healthy individuals. For this purpose, 150 gastric biopsy samples from stomach cancer patients and 150 saliva samples from healthy people were collected. Data showed a significant relationship between the coexistence of two genes, cagA and hopQII, and stomach cancer. 34.2% of patients and 10.1% of healthy individuals showed two genotypes, while other healthy people (89.9%) infected with H. pylori did not have this genotype. Therefore, the simultaneous presence of these two bacterial genes in human societies can be an essential biomarker for the diagnosis and prognosis of gastric cancer.

Direct Colorimetry of Imipenem Decomposition as a Novel Cost-Effective Method for Detecting Carbapenemase-Producing Enterobacteria.

In the absence of a molecule that would collectively inhibit both metallo-ß-lactamases and serine-reactive carbapenemases, containment of their genes is the main weapon currently available for confronting carbapenem resistance in hospitals. Cost-effective methodologies rapidly detecting carbapenemase-producing enterobacteria (CPE) would facilitate such measures. Herein, a low-cost CPE detection method was developed that was based on the direct colorimetry of the yellow shift caused by the accumulation of diketopiperazines-products of the acid-catalyzed imipenem oligomerization-induced by carbapenemase action on dense solutions of imipenem/cilastatin. The reactions were studied by spectrophotometry in the visible spectrum using preparations of ß-lactamases from the four molecular classes. The effects of various buffers on reaction mixtures containing the potent carbapenemases NDM-1 and NMC-A were monitored at 405 nm. Optimal conditions were used for the analysis of cell suspensions, and the assay was evaluated using 66 selected enterobacteria, including 50 CPE as well as 16 carbapenemase-negative strains overexpressing other ß-lactamases. The development of the yellow color was specific for carbapenemase-containing enzyme preparations, and the maximum intensity was achieved in acidic or unbuffered conditions in the presence of zinc. When applied on bacterial cell suspensions, the assay could detect CPE with 98% sensitivity and 100% specificity, with results being comparable to those obtained with the Carba NP technique. Direct colorimetry of carbapenemase-induced imipenem decomposition required minimum reagents while exhibiting high accuracy in detecting CPE. Therefore, it should be considered for screening purposes after further clinical evaluation. IMPORTANCE Currently, the spread of multidrug-resistant (MDR) carbapenemase-producing enterobacteria (CPE), mostly in the clinical setting, is among the most pressing public health problems worldwide. In order to effectively control CPE, use of reliable and affordable methods detecting carbapenemase genes or the respective ß-lactamases is of vital importance. Herein, we developed a novel method, based on a previously undescribed phenomenon, that can detect CPE with few reagents by direct colorimetry of bacterial suspensions and imipenem/cilastatin mixtures.

Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis.

The diagnosis of abdominal tuberculosis (aTB) is challenging and there is an urgent need for an accurate diagnostic test. We have developed a high affinity DNA aptamer against GlcB antigen of Mycobacterium tuberculosis (Mtb). We further compared the diagnostic utility of in-house-generated high affinity DNA aptamers and polyclonal antibodies against two Mtb antigens, namely GlcB and HspX, in ascitic fluid samples. These diagnostic reagents were assessed in patients (n = 94) who were categorized as 'Definite TB', 'Probable TB', 'Possible TB' (taken together as aTB) and 'Non-TB' disease. Receiver operating characteristic curves were used to derive cut-off values to provide ≥93% specificity. Aptamer Linked Immobilized Sorbent Assay (ALISA) for HspX and GlcB exhibited a sensitivity of ∼84% and 50%, respectively (p-value <0.01). In contrast, antibody-based ELISA exhibited a lower sensitivity of ∼18% and ∼28% for HspX and GlcB, respectively (p-value <0.0001 and p = 0.05 for HspX and GlcB ELISA vs. ALISA, respectively). HspX ALISA detected 32/38 aTB cases, while Xpert detected only 9 samples. In conclusion, HspX aptamer-based test was found to be superior to the other tests for diagnosing aTB and it nearly fulfils the sensitivity criteria of WHO's 'Target Product Profile' for extrapulmonary tuberculosis (sensitivity ≥80%, specificity 98%).

Extensively Drug-Resistant Klebsiella pneumoniae Counteracts Fitness and Virulence Costs That Accompanied Ceftazidime-Avibactam Resistance Acquisition.

The ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages. IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

Phage resistance mutation triggered by OmpC deficiency in Klebsiella pneumoniae induced limited fitness costs.

Outer membrane proteins (OMPs) play an important role in bacterial fitness costs. Derived from the interaction between Klebsiella pneumoniae K7 and phage GH-K3, K7RB is an outer membrane porin-deficient phage-resistant mutant strain triggered by ompC712 deletion, exhibits expression inhibition of OmpC, OmpN, KPN_02430 and OmpF, but its fitness costs and regulatory mechanism remains unknown. In this study, compared with K7, K7RB showed almost unaffected growth rate, slightly decreased virulence, and increased resistance to some antibiotics. Transcriptome analysis showed that the pathways of glycerolipid metabolism and nitrogen metabolism in K7RB were significantly inhibited, while the transcription of permeases belonging to ABC transporters tended to be active, nutrient uptakes such as citrate and phenylalanine were also enhanced. However, transcriptional up-regulation in K7RB was inhibited by overexpression of OmpC, OmpN, KPN_02430 and OmpF in general. Overexpression of OmpN, KPN_02430 and OmpF, respectively, restoring the sensitivity of strains to antibiotics to varying degrees, while OmpC overexpression aggravated the bacterial drug-resistance especially to ß-lactam antibiotics. Besides, unlike OmpC and OmpF, overexpression of OmpN and KPN_02430 reduced bacterial virulence. In brief, by revealing the limited fitness costs of phage-resistant mutant K. pneumoniae with porin-deficiency, our study providing a reference for the design and development of drugs to inhibit the ways of bacterial metabolic rewiring and to increase fitness costs.

Prediction of Monomeric and Dimeric Structures of CYP102A1 Using AlphaFold2 and AlphaFold Multimer and Assessment of Point Mutation Effect on the Efficiency of Intra- and Interprotein Electron Transfer.

The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer (AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (ket), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor ß, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains. The structures of monomers and homodimers of A83F and A83I mutants were also calculated, and their structures were compared with the wild-type protein. Significant differences in the structure of all isolated monomers with respect to the structures of monomers in homodimers were also found for them, and at the same time, insignificant differences were revealed for all homodimers. Comparative analysis for CYP102A1/WT between the calculated intra- and interprotein distances FAD→FMN→HEME and the rate constants of hydroxylation in these proteins showed that the distance between prosthetic groups both in the monomer and in the dimer allows the implementation of electron transfer between PGs, which is consistent with experimental literature data about kcat. For the mutant form of monomer A83I, an increase in the distance between PGs was obtained, which can restrict electron transportation compared to WT; however, for the dimer of this protein, a decrease in the distance between PGs was observed compared to the WT form, which can lead to an increase in the electron transfer rate constant and, accordingly, kcat. For the monomer and homodimer of the A83F mutant, the calculations showed an increase in the distance between the PGs compared to the WT form, which should have led to a decrease in the electron transfer rate, but at the same time, for the homodimer, the approach of the aromatic group F262 with heme can speed up transportation for this form and, accordingly, the rate of hydroxylation.