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The rectangular microstrip patch antenna (RMPA) had designed and manufactured to operate in two working areas of the worldwide interoperability for microwave access (WiMAX) communication system. Flame retardant (FR-4) material had used for implementation, and the total antenna size was 57.22 × 1.6 mm 3 . The chemical method was used to implement the RMPA. The proposed antenna is capable of working at frequencies 2.51 GHz and 3.87 GHz experimentally. The results were -21.62 dB of return loss, and 50 MHz of bandwidth for the first frequency. Also, for second frequency was -20.01 dB of return loss, and 80 MHz of bandwidth.

Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen. The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA . The Δ rmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains. IMPORTANCE Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

Capsule is a critical virulence factor in Klebsiella pneumoniae , in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene ( rmpD ) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits. Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: classical strains that are a notable problem in health care settings due to multidrug resistance, and hypervirulent (hv) strains that are historically drug sensitive but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene, rmpA , encodes a transcriptional regulator required for maximal capsule ( cps ) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and Δ rmpC mutants have reduced cps expression but retain HMV, suggesting that capsule production and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both Δ rmpD and Δ rmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC , within an operon regulated by RmpA. These data, combined with our previous work, suggest a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression. IMPORTANCE Capsule is a critical virulence factor in Klebsiella pneumoniae , in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene ( rmpD ) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.

Gastrointestinal carriage of Klebsiella pneumoniae is a predisposing factor for liver abscess in several Asian countries. To determine whether hypervirulent K. pneumoniae in the gut may be transmitted through food, we screened a range of raw and ready-to-eat retail food by culture and recovered K. pneumoniae in 21% (147 of 698) of samples tested. Based on PCR, no K. pneumoniae isolates carried the rmpA gene linked to community-acquired pyogenic liver abscess, providing no evidence of a link between food and liver disease. However, phenotypic resistance to multiple antibiotic classes was seen through disk diffusion tests, and carriage of genetic elements (wcaG and capsule types K1, K2, and K54) associated with increased virulence (8%, 11 of 147) was observed by PCR. Multidrug-resistant isolates were from raw vegetables, chicken or pork liver, and a ready-to-eat poultry dish; one multidrug-resistant K. pneumoniae isolate from raw bean sprouts was resistant to a third-generation cephalosporin (ceftriaxone). Although K. pneumoniae may be present in food without causing harm, we found isolates belonging to the K1 capsular serotype coexisting with the wcaG gene, one also conferring multidrug resistance. K. pneumoniae that carry antibiotic resistance genes, regardless of pathogenicity, may increase the available genetic pool of resistance along the food chain. Hygienic food handling practices are necessary to lower risks of acquiring K. pneumoniae and other opportunistic pathogens. .

Virulence plasmids are associated with hypervirulent types of Klebsiella pneumoniae, which generally do not carry antibiotic resistance genes. In contrast, nosocomial isolates are often associated with resistance, but rarely with virulence plasmids. Here, we describe virulence plasmids in nosocomial isolates of “high-risk” clones of sequence types (STs) 15, 48, 101, 147 and 383 carrying carbapenemase genes. The whole genome sequences were determined by long-read nanopore sequencing. The 12 isolates all contained hybrid plasmids containing both resistance and virulence genes. All carried rmpA/rmpA2 and the aerobactin cluster, with the virulence plasmids of two of three representatives of ST383 carrying blaNDM-5 and seventeen other resistance genes. Representatives of ST48 and ST15 had virulence plasmid-associated genes distributed between two plasmids, both containing antibiotic resistance genes. Representatives of ST101 were remarkable in all sharing virulence plasmids in which iucC and terAWXYZ were missing and iucB and iucD truncated. The combination of resistance and virulence in plasmids of high-risk clones is extremely worrying. Virulence plasmids were often notably consistent within a lineage, even in the absence of epidemiological links, suggesting they are not moving between types. However, there was a common segment containing multiple resistance genes in virulence plasmids of representatives of both STs 48 and 383.

Hypervirulent produce an increased amount of capsular substance and are associated with a hypermucoviscous phenotype. Capsule production is regulated by capsular regulatory genes and capsular gene cluster variations. In the present study, we focus on the effect of and on capsule biosynthesis. Phylogenetic trees were constructed to analyze wcaJ and rmpA sequence diversity in different serotypes hypervirulent strains. Then mutant strains (K2044 , K2044 , K2044 and K2044 ) were used to verify the effects of wcaJ and its diversity on capsule synthesis and strain virulence. Furthmore, the role of rmpA in capsular synthesis and its mechanisms were detected in K2044 strain. RmpA sequences are conversed in different serotypes. And rmpA promoted the production of hypercapsules by simultaneously acting on three promoters in cps cluster. Whereas w , its sequences are different in different serotypes, and its loss result in the termination of capsular synthesis. Moreover, the results verified that K2 could form hypercapsule in K2044 strains (K1 serotype), but K64 w could not. The interaction of multiple factors is involved in capsule synthesis, including w and r . RmpA, an known conserved capsular regulator gene, acts on cps cluster promoters to promote the production of the hypercapsule. WcaJ as initiating enzyme of CPS biosynthesis, its presence determines the synthesis of capsule. Besides, different from rmpA, w sequence consistency is limited to the same serotype, which cause wcaJ functioning in different serotype strains with sequence recognition specificity.

This study aimed to analyze the influence of the main aerobactin-encoding gene iucB and the regulator of mucoid phenotype rmpA on the virulence of Klebsiella pneumoniae causing liver abscess. In addition, the possible regulatory effects of the main encoding gene iucB on the regulator of mucoid phenotype rmpA were explored, thus providing novel strategies for the prevention and control of hypervirulent K. pneumoniae (hvKp) causing liver abscess. The virulence-related genes iucB and rmpA of K. pneumoniae were detected by PCR. iucB and rmpA were cloned into K. pneumoniae strain by using plasmid pET28b as vector. Quantitative real-time PCR (RT-qPCR) was employed to detect the relative expression of rmpA gene in K. pneumoniae . We investigated the potential effects of aerobactin coding gene iucB and regulator of mucoid phenotype rmpA on the virulence of K. pneumoniae by establishing the Galleria mellonella infection model. Capsule quantitative experiment was conducted to investigate the impact of aerobactin-encoding gene iucB on the modulation of regulator of mucoid phenotype rmpA . The results of the G. mellonella infection model indicated that iucB gene could significantly enhance the virulence of K. pneumoniae , but the presence of rmpA gene did not markedly affect the virulence of K. pneumoniae . RT-qPCR showed that iucB inhibited the expression of rmpA gene. Quantitative capsulation experiments showed that the presence of rmpA gene could not increase the capsulation production of K. pneumoniae . The main encoding gene of aerobactin, namely iucB , could substantially enhance the virulence of K. pneumoniae . The gene iucB might be involved in the biosynthesis of the capsular polysaccharide through an unknown mechanism instead of the gene rmpA . Overall, these findings provide important theoretical support for the treatment of infections caused by hvKp.

ABSTRACT Respiratory diseases are emerging concerns of animals including camels which result in considerable loss in production, elevated mortalities and increased cost of treatment. K. pneumoniae is a substantial opportunistic pathogen that induces a wide spectrum of respiratory infections in human and animals. In the present study, a total of 116 nasal swabs and 89 lung tissue samples were collected from 33 apparently healthy and 83 respiratory ill camels. Samples were screened for the isolation of bacteria, and positive samples were subjected to classical and API 20 E biochemical-based characterization of Klebsiella pneumoniae. Additionally, obtained isolates were assessed for antibiotic susceptibility, the existence of antibiotic resistance genes (blaTEM and aadB) and virulence factors (magA, rmpA). Out of 205 examined samples, 15 isolates (7.31%) were culturally and biochemically confirmed as K. pneumonia. All isolates appeared to be resistant to amoxicillin, ampicillin and gentamycin, however, showed variable susceptible to levofloxacin, imipenem (100%), norfloxacin (93.3%) and ceftriaxon (73%). The application of uniplex PCR on the selected K. pneumoniae isolates revealed the detection of antibiotic resistance genes (blaTAM and aadB) in all isolates. The virulence genes including magA and rmpA were found in 40% and 0% of samples, respectively. In conclusion, the data highligh the existence multidrug resistant K. pneumoniae among Egyptian camels and may represent a theat to public health.

To investigate the phenotypic and genomic characteristics of the multi-drug resistant and hypervirulent strain recovered from bacteremia. Antimicrobial susceptibility testing (AST) was performed by the microdilution method. Antimicrobial resistance genes, virulence-associated genes, multilocus sequence typing (MLST), and plasmid replicon were characterized by next-generation sequencing (NGS) and nanopore sequencing. S1 nuclease-pulsed field gel electrophoresis (S1-PFGE) and Southern blotting were performed to characterize the plasmid profile. The hypervirulent colistin- and carbapenem-resistant strain DY2009 was identified as ST5571, co-carrying , and . In silico analysis found that it was K2 serotype. AST results revealed that DY2009 was resistant to carbapenems, cephalosporins, ciprofloxacin, chloramphenicol, and colistin but remained susceptible to aztreonam, gentamicin, amikacin, and tigecycline. Through the whole-genome analysis, a variety of virulence determinants were identified, including . Plasmid analysis confirmed that the and gene harbored a ~33 kb IncX4 plasmid and a ~44 kb IncX3 plasmid. In contrast, was encoded by chromosome. To the best of our knowledge, we first report the clinical hypervirulent isolate co-producing MCR-1, NDM-1, and OXA-10 causing bacteremia. We found that and genes were located on two self-conjugative epidemic plasmids, contributing to the widespread MCR-1 and NDM-1 in China. The results of this work improve our understanding of the genetic background of colistin- and carbapenem-resistant isolate from bacteremia and the resistance mechanisms. Our findings highlight the urgent need for infection control of such strain to prevent it from becoming an extensive-drug resistant clone.

( ) is a Gram-negative bacterium that is predominantly associated with liver abscesses in global diabetic patients. High levels of glucose in the surrounding of increase its pathogenicity including capsular polysaccharide (CPS) and fimbriae. Other important virulent factors include outer membrane protein A (ompA) and regulator mucoid phenotype A (rmpA). The objective of this investigation was to elucidate the effects of high glucose on and gene expression and serum resistance of causing liver abscess. The clinical history of 57 patients suffering from -caused liver abscesses (KLA) was acquired and their clinical and laboratory manifestations in the presence or absence of diabetes were analyzed. The antimicrobial susceptibility, serotypes, and virulence genes were tested. Clinical isolates of 3 serotype-K1 hypervirulent (hvKP) were used to detect the effect of exogenous high glucose on , and genes expression, and bacterial serum resistance. KLA patients with diabetes showed higher C-reactive protein (CRP) compared to non-diabetic KLA patients. Furthermore, the diabetic group showed increased incidences of sepsis and invasive infections, and their length of hospital stay was also prolonged. Pre-incubation of in high glucose (0.5%) concentration up-regulated , and genes expression. However, cAMP supplementation, which was inhibited by environmental glucose, reversed the increase of and in a cAMP-dependent manner. Moreover, hvKP strains incubated in high glucose also exhibited enhanced protection from serum killing. High glucose levels reflected by poor glycemic control has increased gene expression of and in hvKP by the cAMP signaling pathway and enhanced its resistance to serum killing, thus providing a new and reasonable explanation for the high incidences of sepsis and invasive infections in KLA patients with diabetes.