Resistance Methylase Research Articles

EFFECT OF PENICILLIUM SPECIES ON THE ANTIBIOTIC RESISTANCE PROFILE OF ALCALIGENES FAECALIS.

Infectious diseases due to antibiotic resistant pathogens are a global public health problem. This study aimed at determining the potential effect of bacterial-fungal interaction on the antibiotic susceptibility profile of Alcaligenes faecalis. Alcaligenes faecalis was isolated from water samples. The isolate was identified using the conventional biochemical tests and the 16S rRNA molecular sequencing technique. Additionally, Penicillium species was isolated and identified based on colony morphological characteristics and microscopic features. Standardized isolates were co-cultured in broth medium. Antibiotic susceptibility evaluation of the Alcaligenes faecalis from the co-culture and the original Alcaligenes faecalis was carried out using the Kirby bauer disk diffusion method. The antibiotic susceptibility profile of Alcaligenes faecalis before and after co-culture remained largely unchanged except in the case of chloramphenicol, where the isolate showed reduced susceptibility. Molecular analysis of resistance gene revealed the absence of tested gene encoding antibiotic resistance, including the streptomycin resistance (str) genes (stra and strb) and the erythromycin resistance methylase (erm) gene. The result of this study showed that there is a minimal influence of Penicillium cultures on the susceptibility of A. faecalis. Further research involving a wide spectrum of microorganisms and their interactions should be conducted to acquire a thorough understanding of the influence of microbial interactions on antibiotic susceptibility profiles in order to pave way for novel strategies to combat antimicrobial resistance.

An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance.

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

First report of coexistence of blaKPC-2 and blaNDM-1 in carbapenem-resistant clinical isolates of Klebsiella aerogenes in Brazil.

Klebsiella aerogenes is an important opportunistic pathogen with the potential to develop resistance against last-line antibiotics, such as carbapenems, limiting the treatment options. Here, we investigated the antibiotic resistance profiles of 10 K. aerogenes strains isolated from patient samples in the intensive-care unit of a Brazilian tertiary hospital using conventional PCR and a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. All isolates were completely resistant to β-lactam antibiotics, including ertapenem, imipenem, and meropenem with differencing levels of resistance to aminoglycosides, quinolones, and tigecycline also observed. Half of the strains studied were classified as multidrug-resistant. The carbapenemase-producing isolates carried many genes of interest including: β-lactams (blaNDM-1, blaKPC-2, blaTEM-1, blaCTX-M-1 group, blaOXA-1 group and blaSHVvariants in 20-80% of the strains), aminoglycoside resistance genes [aac(6')-Ib and aph(3')-VI, 70 and 80%], a fluoroquinolone resistance gene (qnrS, 80%), a sulfonamide resistance gene (sul-2, 80%) and a multidrug efflux system transporter (mdtK, 70%) while all strains carried the efflux pumps Acr (subunit A) and tolC. Moreover, we performed a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. The draft genome assembly of the CRK317 had a total length of 5,462,831 bp and a GC content of 54.8%. The chromosome was found to contain many essential genes. In silico analysis identified many genes associated with resistance phenotypes, including β-lactamases (blaOXA-9, blaTEM-1, blaNDM-1, blaCTX-M-15, blaAmpC-1, blaAmpC-2), the bleomycin resistance gene (bleMBL), an erythromycin resistance methylase (ermC), aminoglycoside-modifying enzymes [aac(6')-Ib, aadA/ant(3")-Ia, aph(3')-VI], a sulfonamide resistance enzyme (sul-2), a chloramphenicol acetyltransferase (catA-like), a plasmid-mediated quinolone resistance protein (qnrS1), a glutathione transferase (fosA), PEtN transferases (eptA, eptB) and a glycosyltransferase (arnT). We also detected 22 genomic islands, eight families of insertion sequences, two putative integrative and conjugative elements with a type IV secretion system, and eight prophage regions. This suggests the significant involvement of these genetic structures in the dissemination of antibiotic resistance. The results of our study show that the emergence of carbapenemase-producing K. aerogenes, co-harboring blaKPC-2 and blaNDM-1, is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms.

Characteristics of the ErmK Protein of Bacillus halodurans C-125.

Bacillus halodurans C-125 is an alkaliphilic microorganism that grows best at pH 10 to 10.5. B. halodurans C-125 harbors the erm (erythromycin resistance methylase) gene as well as the mphB (macrolide phosphotransferase) and putative mef (macrolide efflux) genes, which confer resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. The Erm protein expressed in B. halodurans C-125 could be classified as ErmK because it shares 66.2% and 61.2% amino acid sequence identity with the closest ErmD and Erm(34), respectively. ErmK can be regarded as a dimethylase, as evidenced by reverse transcriptase analysis and the antibiotic resistance profile exhibited by E. coli expressing ermK. Although ErmK showed one-third or less in vitro methylating activity compared to ErmC', E. coli cells expressing ErmK exhibited comparable resistance to erythromycin and tylosin, and a similar dimethylation proportion of 23S rRNA due to the higher expression rate in a T7 promoter-mediated expression system. The less efficient methylation activity of ErmK might reflect an adaption to mitigate the fitness cost caused by dimethylation through the Erm protein presumably because B. halodurans C-125 has less probability to encounter the antibiotics in its favorable growth conditions and grows retardedly in neutral environments. IMPORTANCE Erm proteins confer MLSB antibiotic resistance (minimal inhibitory concentration [MIC] value up to 4,096 μg/mL) on microorganisms ranging from antibiotic producers to pathogens, imposing one of the most pressing threats to clinics. Therefore, Erm proteins have long been speculated to be plausible targets for developing inhibitor(s). In our laboratory, it has been noticed that there are variations in enzymatic activity among the Erm proteins, Erm in antibiotic producers being better than that in pathogens. In this study, it has been observed that Erm protein in B. halodurans C-125 extremophile is a novel member of Erm protein and acts more laggardly, compared to that in pathogen. While this sluggishness of Erm protein in extremophile might be evolved to reduce the fitness cost incurred by Erm activity adapting to its environments, this feature could be exploited to develop the more potent and/or efficacious drug to combat formidably problematic antibiotic-resistant pathogens.

Alterations of gut microbial pathways and virulence factors in hemodialysis patients.

Alterations in gut microbiota might contribute to uremic toxicity and immune dysregulation in patients with end-stage renal disease. Hemodialysis patients are prone to infection and higher mortality following sepsis. The virulence factors in the gut metagenome have not been well studied in hemodialysis patients, which could be employed by microorganisms to successfully thrive and flourish in their hosts. In this study, we performed shotgun metagenomics sequencing on fecal DNA collected from 16 control subjects and 24 hemodialysis patients. Our analysis shows that a number of microbial species, metabolic pathways, antibiotic resistance, and virulence factors were significantly altered in hemodialysis patients compared with controls. In particular, erythromycin resistance methylase, pyridoxamine 5-phosphate oxidase, and streptothricin-acetyl-transferase were significantly increased in hemodialysis patients. The findings in our study laid a valuable foundation to further elucidate the causative role of virulence factors in predisposing HD patients to infection and to develop treatment strategies to reduce the genetic capacities of antibiotic resistance and virulence factors in HD patients.

Co-expression of 16S rRNA Methyltransferase and Carbapenemase in Multidrug Resistant Gram Negative Bacteria

Introduction: The drug resistance caused by bacteria has complicated the fight against the infectious diseases and recovery of the patients from the diseases. Multiple mode of drug resistant mechanisms including epigenetic and enzymatic mode have been involved in the genesis of drug resistance leaving only fewer antibiotics for the treatment. Studies has shown the presence of 16S methyltransferase which is associated with carbapenem drug resistance. Early detection of different multimode of bacterial drug resistance helps in reducing the dissemination rate and treatment failures. Aim: To detect the genes related to the methylation mode of bacterial drug resistant mechanism including armA (aminoglycoside resistance methylase), rmtA, rmtB, rmtC and rmtD (rmt-RNA methyltrasferase) as well as genes OXA-48 (Oxacillinase), VIM (Verona intergron metallo-β lactamase) and KPC (Klebsiella pneumoniae carbapenemase) in the production of carbapenemases and also to identify the co-expression of these genes in the gram negative bacterial isolates. Materials and Methods: Prospective study was conducted through application of multiplex Polymerase Chain Reaction (PCR)for detection of 16S methyltransferase genes including armA, rmtA, rmtB, rmtC and rmtD and carbpenemase VIM, KPC and OXA-48 genes, respectively. A total of 200 multidrug resistant gram negative bacilli were evaluated for presence of these genes. The antibiotic sensitivity was evaluated using Kirby-Bauer disc diffusion method. Results: The gram negative bacteria which were isolated includes Acinetobacter spp. 20, E.coli-85, Klebsiella spp.-55 and Pseudomonas aeruginosa-40 from several specimens such as urine, endotracheal tube-secretion, wound swab, sputum, pus etc. A total of 121 (61%) of the bacterial isolates were resistant to either gentamicin or amikacin and netilimycin and 50 (25%) bacterial isolates were resistant to either imipenem or carbapenem. A total of 48 (24%) isolates were resistant to both aminoglycosides and carbapenems. The 16S methyltransferase genes including armA gene was detected in 16 isolates, rmtB in 15 isolates, rmtC in 10 isolates and rmtD detected in 13 isolates The carbapenem genes detected including VIM in 20 isolates, OXA-48 in 25 isolates and KPC was detected in 13 bacterial isolates. Co-expression of both methyltransferase and carbapenemase were detected in nine isolates. Conclusion: The presence of multiple mode of bacterial drug resistance mechanisms including epigenetic and enzymatic modes have been found to be associated with resistance to different classes of antibiotics. The application of multiplex PCR helps in detection of multiple genes involved in bacterial drug resistance and helps in prevention of rapid spread of bacterial drug resistance and prevention of overuse of antibiotics.

High-Level Resistance of Toxigenic Clostridioides difficile Genotype to Macrolide-Lincosamide- Streptogramin B in Community Acquired Patients in Eastern China.

BackgroundClostridioides difficile resistant to macrolide-lincosamide-streptogramin B (MLSB) has not been reported in China.MethodsIn a cross-sectional study in two tertiary hospitals, C. difficile isolates from stool specimens from community-onset, hospital-associated diarrheal patients were analyzed for toxin genes, genotype, and antibiotic resistance, and the patients’ clinical charts were reviewed.ResultsA total of 190 (15.2%) isolates (102 A+B+ and 88 A−B+) from 1250 community acquired (CA) patients were recovered and all were susceptible to vancomycin and metronidazole. High-level resistance (minimum inhibitory concentration > 128 mg/L) to erythromycin and clindamycin was recorded in 77.9% and 88.4% of the tested isolates, respectively. Furthermore, 89.3% (159/178) of the isolates resistant to MLSB carried the erythromycin resistance methylase gene (ermB). The statistically significant factors associated with C. difficile infection (CDI) induced by A−B+ isolates with MLSB resistance included a severity score of >2 (odds ratio [95% confidence interval], 7.43 [2.31–23.87]) and platelet count (cells × 109 cells/L) < 100 [5.19 (1.58–17.04)]. The proportion of A−B+ increased with enhanced CDI severity (x2 = 21.62, P < 0.001), which was significantly higher than that of ermB-positive A+B+ in severity score of 4 (x2 = 8.61, P = 0.003). The average severity score of ermB-positive isolates was significantly higher than that of ermB-negative isolates in A−B+ (Z = −2.41, P = 0.016).ConclusionThe ermB-positive A−B+ C. difficile with MLSB resistance is described for the first time as a potential epidemic clone inducing severe CDI in CA diarrheal patients in Eastern China.

Short-term and long-term impacts of Helicobacter pylori eradication with reverse hybrid therapy on the gut microbiota.

Anti-Helicobacter pylori therapy may lead to the growth of pathogenic or antibiotic-resistant bacteria in the gut. The study aimed to investigate the short-term and long-term impacts of H.pylori eradication with reverse hybrid therapy on the components and macrolide resistance of the gut microbiota. Helicobacter pylori-related gastritis patients were administered a 14-day reverse hybrid therapy. Fecal samples were collected before treatment and at the end of week 2, week 8, and week 48. The V3-V4 region of the bacterial 16S rRNA gene in fecal specimens was amplified by polymerase chain reaction and sequenced on Illumina MiSeq platform. Additionally, amplification of erm(B) gene (encoding erythromycin resistance methylase) was performed. Reverse hybrid therapy resulted in decreased relative abundances of Firmicutes (from 62.0% to 30.7%; P<0.001) and Actinobacteria (from 3.4% to 0.6%; 0.032) at the end of therapy. In contrast, the relative abundance of Proteobacteria increased from 10.2% to 49.1% (0.002). These microbiota alterations did not persist but returned to the initial levels at week 8 and week 48. The amount of erm(B) gene in fecal specimens was comparable with the pretreatment level at week 2 but increased at week 8 (0.025) and then returned to the pretreatment level by week 48. Helicobacter pylori eradication with reverse hybrid therapy can lead to short-term gut dysbiosis. The amount of erm(B) gene in the stool increased transiently after treatment and returned to the pretreatment level at 1-year post-treatment.

Studies of antimicrobial resistance in rare mycobacteria from a nosocomial environment

BackgroundNontuberculous mycobacteria (NTM) are ubiquitous in nature and recognized agents of opportunistic infection, which is often aggravated by their intrinsic resistance to antimicrobials, poorly defined therapeutic strategies and by the lack of new drugs. However, evaluation of their prevalence in anthropogenic environments and the associated antimicrobial resistance profiles have been neglected. In this work, we sought to determine minimal inhibitory concentrations of 25 antimicrobials against 5 NTM isolates recovered from a tertiary-care hospital surfaces. Antimicrobial susceptibilities of 5 other Corynebacterineae isolated from the same hospital were also determined for their potential clinical relevance.ResultsOur phylogenetic study with each of the NTM isolates confirm they belong to Mycobacterium obuense, Mycobacterium mucogenicum and Mycobacterium paragordonae species, the latter initially misidentified as strains of M. gordonae, a species frequently isolated from patients with NTM disease in Portugal. In contrast to other strains, the M. obuense and M. mucogenicum examined here were resistant to several of the CLSI-recommended drugs, suggestive of multidrug-resistant profiles. Surprisingly, M. obuense was susceptible to vancomycin. Their genomes were sequenced allowing detection of gene erm (erythromycin resistance methylase) in M. obuense, explaining its resistance to clarithromycin. Remarkably, and unlike other strains of the genus, the Corynebacterium isolates were highly resistant to penicillin, ciprofloxacin and linezolid.ConclusionsThis study highlights the importance of implementing effective measures to screen, accurately identify and control viable NTM and closely related bacteria in hospital settings. Our report on the occurrence of rare NTM species with antibiotic susceptibility profiles that are distinct from those of the corresponding Type strains, along with unexpected resistance mechanisms detected seem to suggest that resistance may be more common than previously thought and also a potential threat to frail and otherwise vulnerable inpatients.

The potential impact of naturally produced antibiotics, environmental factors, and anthropogenic pressure on the occurrence of erm genes in urban soils

The occurrence of environmental antibiotic resistance genes (ARGs) are often attributed to selective pressure from antibiotics from point source pollution. However, the potential effects of natural production of antibiotics, environmental factors, and anthropogenic pressure on the development and spread of ARGs have not been fully investigated. This study evaluated the occurrence and distribution of erythromycin resistance methylase (erm) genes in urban soils. The ermA, ermB, ermC, ermD, ermF, ermG, ermT, and ermY genes were detected with detection frequencies ranging from 20% to 80% and abundances ranging between 5.95 × 101 and 6.94 × 106 copies g−1 dw soil. Both polyketide synthase (PKS) type I and type II biosynthesis genes—which are responsible for biosynthesis of polyketides, such as erythromycin—were detected in all soil samples with a range between 5.77 × 102 and 9.39 × 106 copies g−1 dw soil. The abundances of PKS genes were significantly correlated with 16S rRNA genes (r = 0.487 to 0.741, p < 0.001) and absolute abundances of ermB, ermC, ermD, ermG, and ermY (r = 0.302–0.490, p < 0.05), suggesting that the wide occurrence of ARGs in soils could be potentially driven by naturally produced antibiotics. Erythromycin was strongly correlated with ermB, ermC, ermF and ermY genes (r = 0.462 to 0.667, p < 0.05), but no significant correlation was observed between macrolides and PKS genes, suggesting other environmental factors may have contributed to detected macrolides. The fact that erm gene presented higher extent of variability than PKS genes in different land use types suggests that anthropogenic activity might also influence the occurrence of erm genes in urban soils.

Update on Tetracycline Susceptibility of Pediococcus acidilactici Based on Strains Isolated from Swiss Cheese and Whey

Bacterial strains used as starter cultures in the production of fermented foods may act as reservoirs for antibiotic resistance (AbR) genes. To avoid the introduction of such genes into the food chain, the presence of acquired AbR in bacterial strains added to food must be tested. Standard protocols and microbiological cut-off values have been defined to provide practitioners with a basis for evaluating whether their bacterial isolates harbor an acquired resistance to a given antibiotic. Here, we tested the AbR of 24 strains of Pediococcus acidilactici by using the standard protocol and microbiological cut-off values recommended by the European Food Safety Authority. Phenotypic data were complemented by searching for known AbR genes using an in silico analysis of whole genomes. The majority (54.2%) of the strains were able to grow at a tetracycline concentration above the defined cut-off, even though only one strain carried a known tetracycline resistance gene, tetM. The same strain also carried the AbR gene of an erythromycin resistance methylase, ermA, and displayed resistance toward clindamycin and erythromycin. Our results bolster the scarce data on the sensitivity of P. acidilactici to tetracycline and suggest that the microbiological cut-off recommended by the European Food Safety Authority for this antibiotic should be amended.

A Low Prevalence of Inducible Macrolide, Lincosamide, and Streptogramin B Resistance Phenotype among Methicillin-Susceptible Staphylococcus aureus Isolated from Malaysian Patients and Healthy Individuals.

BackgroundAntibiotic resistance among Staphylococcus aureus is of great concern worldwide. This resistance is further complicated by the ability of S. aureus to confer cross-resistance to other antibiotics due to the presence of resistance genes, such as erythromycin resistance methylase (erm) genes, which render the bacterium resistant to macrolide-lincosamide-streptogramin B (MLSB) antibiotics. Resistance to these antibiotics can lead to therapeutic failure, resulting in significant morbidity and mortality in patients with S. aureus infections.ObjectivesThis study was performed to examine the distribution of MLSB-resistant strains of methicillin-susceptible S. aureus (MSSA), which were obtained from hospitalized patients and normal healthy individuals (carriers) using phenotypic methods, such as the double-disk diffusion (D-test) and the genotypic method by polymerase chain reaction (PCR).MethodsA total of 183 nonduplicative MSSA isolates obtained from hospitalized patients (133) and carriers (50) in our previous studies were randomly selected for the D-test. The guidelines of the Clinical and Laboratory Standards Institute (CLSI) were used for the interpretation of the results of this test. The detection of ermA, ermB, ermC and msrA genes by PCR was performed for isolates that had positive D-test results and that were resistant to erythromycin.ResultsOf the 183 MSSA isolates, 97.2% and 98.4% were highly susceptible to erythromycin and clindamycin, respectively. MSLB resistance was detected in four isolates (2.2%). Of the 133 MSSA isolated from hospitalized patients, only 3.0% (4/133) and 2.3% (3/133) exhibited resistance to erythromycin and clindamycin, respectively. With regard to the MLSB resistance phenotypes, only 1.6% and 0.6% exhibited inducible MLSB (iMLSB) and MS phenotypes, respectively. The ermC gene was detected in all three iMLSB phenotypes, and the msrA gene was detected in the MS phenotype. Surprisingly, all MSSA isolates (100%) from carriers exhibited extremely high susceptibility to both antibiotics.ConclusionsThe prevalence rates of iMLSB MSSA isolates vary according to geographical locations and the local antibiotic policy. The low prevalence rate of iMLSB MSSA isolates could probably be related to the judicious use of antibiotics for treating S. aureus infections in our studied population. Nonetheless, continuous antibiotic surveillance is still necessary to control any emergence of resistance isolates so that targeted therapy and effective control can be implemented accordingly.

Isolation of Rapidly Growing Nontuberculous Mycobacteria in Wounds Following Combat-Related Injury.

Rapidly growing nontuberculous mycobacteria (RGNTM) have yet to be described in combat-related injuries. This study investigates the epidemiology, clinical findings, treatment, and outcomes of RGNTM infections among combat casualties wounded in Afghanistan from 2010 to 2012. Patients with RGNTM were identified from the Department of Defense Trauma Registry through the Trauma Infectious Disease Outcomes Study. Trauma history, surgical management, and clinical data were collected. Six isolates from patients requiring antimycobacterial therapy were sequenced. Seventeen cases were identified. Six cases, predominantly associated with Mycobacterium abscessus, required aggressive debridement and a median of 180 days of multidrug antimycobacterial therapy that included clofazimine. M. abscessus isolates expressed the erythromycin resistance methylase (erm(41)) gene for inducible macrolide resistance, yet there were no clinical treatment failures when macrolides were utilized in combination therapy. No clonal similarity between M. abscessus isolates was found. Eleven cases had positive wound cultures, but did not require antimycobacterial therapy. The median duration of time of injury to first detection of a RGNTM was 57 days. This represents the first report of RGNTM infections in war-wounded patients. RGNTM should be recognized as potential pathogens in grossly infected combat wounds. Surgical debridement and multidrug antimycobacterial therapy, when clinically indicated, was associated with satisfactory clinical outcomes.

Discovery of Novel MLSB Resistance Methylase Genes and Their Associated Genetic Elements in Staphylococci

Erythromycin resistance methylases (Erm) confer resistance to three classes of clinically important antibiotics, the macrolides, the lincosamides, and the streptogramins B (MLSB). Erm methylases are located on acquired genetic elements and are widespread in Staphylococcus and other bacterial species. The latest erm determinants have been identified predominantly in coagulase-negative staphylococci using whole-genome sequencing (WGS) and the subsequent search for sequence similarity to erm methylases and specific amino acid motifs. This review presents workflows facilitating the identification and characterization of novel methylase genes, as well as the current status of dissemination of erm genes and their associated genetic elements in staphylococci. Discovery of novel antibiotic resistance genes is necessary to give new insight into molecular epidemiology of antibiotic resistance, for the establishment of better identification and surveillance systems, as well as to continuously improve molecular diagnostic of antibiotic resistance.

Revealing the role of VEGFA in clear cell sarcoma of the kidney by protein-protein interaction network and significant pathway analysis.

Despite clear cell sarcoma of the kidney (CCSK) being the second most common renal tumor in children, its mechanism has not yet been fully investigated. The aim of the present study was to investigate the potential role of vascular endothelial growth factor A (VEGFA) in CCSK development. Following preprocessing of the original GSE2712 data, the differentially-expressed genes (DEGs) between 14 CCSK and 3 fetal kidney samples were identified through Significance Analysis of Microarrays, using the R package. Pathway enrichment analysis was then performed on the DEGs. A protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins database and the DEGs that were enriched in the most significant pathways. Following this, gene ontology analysis was performed on the VEGFA-associated genes, whilst transcription factor binding site analysis was conducted on the hot genes. A total of 2,681 DEGs, including 543 upregulated and 2,138 downregulated genes, were identified, and these were significantly enriched in pathways associated with cancer and focal adhesion. Furthermore, VEGFA, integrin β1, integrin αV, v-akt murine thymoma viral oncogene homolog 1 and endothelial growth factor receptor were identified as hot genes in the PPI network. In addition, the upregulated VEGFA-associated genes, cyclin D1 and cyclin-dependent kinase inhibitor 1B, affected kinase regulation, and the downregulated VEGFA-associated genes, receptor tyrosine-protein kinase erbB-2, mesenchymal-epithelial transition tyrosine kinase receptor and kinase insert domain receptor, were enriched in the protein tyrosine kinase process. It was identified that VEGFA was regulated by restorer of fertility, erythromycin resistance methylase, GA binding protein subunit α, norepinephrine transporter, nuclear factor κB and Sp2 transcription factor genes. Overall, VEGFA and its associated genes serve important roles during CCSK development, and alongside transcription factors, they may function as novel therapeutic targets for disease treatment.

Molecular basis of resistance to macrolides, lincosamides and streptogramins in Staphylococcus hominis strains isolated from clinical specimens.

Coagulase-negative staphylococci (CoNS) are the most frequently isolated bacteria from the blood and the predominant cause of nosocomial infections. Macrolides, lincosamides and streptogramin B (MLSB) antibiotics, especially erythromycin and clindamycin, are important therapeutic agents in the treatment of methicillin-resistant staphylococci infections. Among CoNS, Staphylococcus hominis represents the third most common organism. In spite of its clinical significance, very little is known about its mechanisms of resistance to antibiotics, especially MLSB. Fifty-five S. hominis isolates from the blood and the surgical wounds of hospitalized patients were studied. The erm(C) gene was predominant in erythromycin-resistant S. hominis isolates. The methylase genes, erm(A) and erm(B), were present in 15 and 25 % of clinical isolates, respectively. A combination of various erythromycin resistance methylase (erm) genes was detected in 15 % S. hominis isolates. The efflux gene msr(A) was detected in 18 % of isolates, alone in four isolates, and in different combinations in a further six. The lnu(A) gene, responsible for enzymatic inactivation of lincosamides was carried by 31 % of the isolates. No erythromycin resistance that could not be attributed to the genes erm(A), erm(B), erm(C) and msr(A) was detected. In S. hominis, 75 and 84 %, respectively, were erythromycin resistant and clindamycin susceptible. Among erythromycin-resistant S. hominis isolates, 68 % of these strains showed the inducible MLSB phenotype. Four isolates harbouring the msr(A) genes alone displayed the MSB phenotype. These studies indicated that resistance to MLSB in S. hominis is mostly based on the ribosomal target modification mechanism mediated by erm genes, mainly the erm(C), and enzymatic drug inactivation mediated by lnu(A).

The new macrolide-lincosamide-streptogramin B resistance gene erm(45) is located within a genomic island in Staphylococcus fleurettii.

Genome alignment of a macrolide, lincosamide, and streptogramin B (MLSB)-resistant Staphylococcus fleurettii strain with an MLSB-susceptible S. fleurettii strain revealed a novel 11,513-bp genomic island carrying the new erythromycin resistance methylase gene erm(45). This gene was shown to confer inducible MLSB resistance when cloned into Staphylococcus aureus. The erm(45)-containing island was integrated into the housekeeping gene guaA in S. fleurettii and was able to form a circular intermediate but was not transmissible to S. aureus.

First report in China of Enterobacteriaceae clinical isolates coharboring blaNDM-1 and blaIMP-4 drug resistance genes.

To describe the identification of two carbapenem-resistant, NDM-1 and IMP-4, carbapenemases coproducing Enterobacteriaceae isolates recovered from hospitalized patients in China. Both Klebsiella pneumoniae clinical isolates (Kpn922 and Kpn9599) were resistant to meropenem and imipenem and were subjected to additional antibiotic susceptibility testing. Polymerase chain reaction (PCR) and sequence analyses were used to characterize bacterial carbapenemase resistance genes, extended-spectrum β-lactamases, plasmid-mediated AmpC enzymes, quinolone resistance, and 16s RNA methylase. Genetic relatedness was determined using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were analyzed by S1-PFGE and Southern blot. PCR analyses revealed that the Kpn922 isolate carried blaNDM-1, blaIMP-4, blaTEM-1, and blaSHV-1 genes, while Kpn9599 carried blaNDM-1, blaIMP-4, blaTEM-1, and blaSHV-12 genes. MLST determined that the two isolates were ST1043 and ST571 sequence types. Southern blot analyses revealed that metallo-β-lactamase genes were plasmid borne in both isolates. Plasmids ∼300 kb simultaneously carried blaNDM-1 and blaIMP-4. Coexistence of blaNDM-1 and blaIMP-4 in these clinical isolates may herald the emergence of a new pattern of drug resistance. Surveillance of carbapenemases, particularly metallo-β-lactamases, in Enterobacteriaceae is urgently needed to control and prevent the spread of these resistance determinants in China.

Molecular investigation of antibiotic resistance genes in methicillin resistant Staphylococcus aureus isolated from nasal cavity in pediatric service

Methicillin-resistant staphylococci may also be resistant to some other antibiotics as well as β-lactams. Co-existence of resistance to methicillin and macrolides was genetically investigated in Staphylococcus aureus. A total of 59 S. aureus strains isolated from nasal cavity of patients hospitalized in the pediatric service were investigated for their susceptibility to oxacillin and erythromycin. In addition, the β-lactams resistance gene (mecA and blaZ), the erythromycin resistance methylase genes (ermA, ermB, and ermC) and macrolide efflux genes msrA were analysed by multiplex PCR. The minimal inhibitory concentration (MIC) values revealed that 98 and 97% of isolates were resistant to oxacillin and erythromycin, respectively. In addition, the frequency of resistant strains for tested genes was 66% (mecA), 100% (blaZ), 15% (ermA), 30% (ermB), 22% (ermC) and 36% (msrA). A high rate of macrolide resistance was determined in methicillin-resistant staphylococci. The erm gene was the most frequently detected. Results of susceptibility testing and the carriage of resistance genes did not match. Thus, polymerase chain reaction (PCR) multiplex identification of antibiotic resistance genes, as a rapid and accurate technique, could be a supplementary method, which can be easily incorporated into the diagnostic molecular microbiology laboratory work flow. Key words: Staphylococcus aureus, multidrug resistance, multiplex polymerase chain reaction (PCR).

Treatment of Mycobacterium abscessus

Treatment of <i>Mycobacterium abscessus</i>