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International audience; A Gram-positive, anaerobic coccus isolated from a human surgical site infection was previously shown to belong to an unknown species of the genus Peptoniphilus initially proposed as ‘Peptoniphilus nemausus’ sp. nov., based on both 16S rRNA gene sequence identity of 97.9% with the most closely related species Peptoniphilus coxii and an individualized phylogenetic branching within the genus Peptoniphilus. A polyphasic characterization of the novel species is proposed herein. Whole genome sequence analysis showed an average nucleotide identity value of 84.75% and digital DNA-DNA hybridization value of 28.9% against P. coxii type strain. The strain displayed unique features among members of the genus Peptoniphilus, as it was able to hydrolyze aesculin, and produced acetate as the major metabolic end-product without associated production of butyrate. Growth was observed under microaerophilic conditions. From all these data, the isolate is confirmed as belonging to a new Peptoniphilus species, for which the name Peptoniphilus nemausensis sp. nov. is proposed. The type strain is 1804121828T (= LMG 31466T = CECT 9935T). A database survey using a highly polymorphic partial sequence of the 16S rRNA gene of P. nemausensis revealed P. nemausensis to be a particularly rare skin-associated species in humans. An emendated description of the Peptoniphilus genus is proposed based on a review of the characteristics of the 12 new species with validly published names since the genus description in 2001 and of P. nemausensis. Finally, the relationships between members of the genus Peptoniphilus were explored based on whole genome sequence analysis in order to clarify the taxonomic status of not yet validly published species showing that three pairs of species should be considered as synonyms: Peptoniphilus timonensis and ‘Peptoniphilus phoceensis’, Peptoniphilus lacydonensis and ‘Peptoniphilus rhinitidis’, Peptoniphilus tyrrelliae and Peptoniphilus senegalensis.

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Therapeutic options for coronaviruses remain limited. To address this unmet medical need, we screened 5406 compounds, including United States Food and Drug Administration (FDA)-approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6, representing effective drugs. The time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs which acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI &gt, 100), atovaquone, an anti-malarial (TI &gt, 34), and ciclesonide, an inhalable corticosteroid (TI &gt, 6). Furthermore, utilizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we tested combinations of remdesivir with selected drugs in Vero-E6 and Calu-3 cells, in lung organoids, and identified ciclesonide, nelfinavir, and camostat to be at least additive in vitro. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.

Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment., The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic Escherichia coli (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments.

Background The only licensed live Bacille Calmette-Guérin (BCG) vaccine used to prevent severe childhood tuberculosis comprises genetically divergent strains with variable protective efficacy and rates of BCG-induced adverse events. The whole-genome sequencing (WGS) allowed evaluating the genome stability of BCG strains and the impact of spontaneous heterogeneity in seed and commercial lots on the efficacy of BCG-vaccines in different countries. Our study aimed to assess sequence variations and their putative effects on genes and protein functions in the BCG-1 (Russia) seed lots compared to their progeny isolates available from immunocompetent children with BCG-induced disease (mainly, osteitis). Results Based on the WGS data, we analyzed the links between seed lots 361, 367, and 368 used for vaccine manufacture in Russia in different periods, and their nine progeny isolates recovered from immunocompetent children with BCG-induced disease. The complete catalog of variants in genes relative to the reference genome (GenBank: CP013741) included 4 synonymous and 8 nonsynonymous single nucleotide polymorphisms, and 3 frameshift deletions. Seed lot 361 shared variants with 2 of 6 descendant isolates that had higher proportions of such polymorphisms in several genes, including ppsC, eccD5, and eccA5 involved in metabolism and cell wall processes and reportedly associated with virulence in mycobacteria. One isolate preserved variants of its parent seed lot 361 without gain of further changes in the sequence profile within 14 years. Conclusions The background genomic information allowed us for the first time to follow the BCG diversity starting from the freeze-dried seed lots to descendant clinical isolates. Sequence variations in several genes of seed lot 361 did not alter the genomic stability and viability of the vaccine and appeared accumulated in isolates during the survival in the human organism. The impact of the observed variations in the context of association with the development of BCG-induced disease should be evaluated in parallel with the immune status and host genetics. Comparative genomic studies of BCG seed lots and their descendant clinical isolates represent a beneficial approach to better understand the molecular bases of efficacy and adverse events during the long-term survival of BCG in the host organism.

Background Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. Result By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase β gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. Conclusion The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal β-lactam antibiotics., With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal β-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in β-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa.

the tuberculosis (tB) disease is still widely found even though BCg vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. objective of the research was to determine the immunogenicity of katg protein of M. tuberculosis clinical iso-late L19 in mice. the katg protein as antigen was prepared by expression of the katg gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the katg was vaccinated to mice to detect its immunogenicity. the expression of katg in e.coli BL21 could result in katg protein with molecular weight 80 kda in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of Igg1, Igg2a, Igg2b, Igg2c, Igg3, and IgM. the highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. the katg protein exhibited a high immunogenici ty in mice, so it is possible to develop as a vaccine candidate for tB. a clinical test should be performed in a future to ensure its safety as a therapeutic protein.

Over the last two decades worldwide levels of antibiotic resistance have risen leading to the appearance of multidrug resistant microorganisms. Acinetobacter baumannii is a known skin pathogen which has emerged as a major cause of nosocomial outbreaks due to its capacity to colonize indwelling medical devices and natural antibiotic resistance. With chitosan being an effective antimicrobial agent against antibiotic resistant microorganisms, the aim of this work was to access its potential as an alternative to traditional antimicrobials in the management of A. baumannii growth. What the results showed was that both chitosan MW’s tested were active upon A. baumannii ’s planktonic and sessile growth. For planktonic growth MICs and MBCs were obtained at relatively low concentrations (0.5–2 mg/mL) while for sessile growth chitosan proved to be an effective inhibitor of A. baumannii ’s adhesion and biofilm formation. Considering these results chitosan shows a high potential for control of A. baumannii infections.

Lassa virus, the causative agent of Lassa fever, is a zoonotic pathogen causing annual outbreaks in West African countries. Human patients can develop lethal hemorrhagic fever in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few available countermeasures, such as antiviral drugs or vaccines. Moreover, the fact that animal models are not readily accessible and the fact that mostly laboratory viruses, which have been passaged many times after isolation, are used for studies further limits the successful development of countermeasures. In this study, we demonstrate that a human isolate of Lassa virus causes lethal infection uniformly in Hartley guinea pigs. This novel animal model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics., Lassa virus (LASV), a member of the family Arenaviridae, is the causative agent of Lassa fever. Lassa virus is endemic in West African countries, such as Nigeria, Guinea, Liberia, and Sierra Leone, and causes outbreaks annually. Lassa fever onset begins with “flu-like” symptoms and may develop into lethal hemorrhagic disease in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few licensed vaccines or therapeutics against Lassa fever. The fact that animal models are limited and the fact that mostly laboratory-derived viruses are used for studies limit the successful development of countermeasures. In this study, we demonstrated that the LASV isolate LF2384-NS-DIA-1 (LF2384), which was directly isolated from a serum sample from a fatal human Lassa fever case in the 2012 Sierra Leone outbreak, causes uniformly lethal infection in outbred Hartley guinea pigs without virus-host adaptation. This is the first report of a clinically isolated strain of LASV causing lethal infection in outbred guinea pigs. This novel guinea pig model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics. IMPORTANCE Lassa virus, the causative agent of Lassa fever, is a zoonotic pathogen causing annual outbreaks in West African countries. Human patients can develop lethal hemorrhagic fever in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few available countermeasures, such as antiviral drugs or vaccines. Moreover, the fact that animal models are not readily accessible and the fact that mostly laboratory viruses, which have been passaged many times after isolation, are used for studies further limits the successful development of countermeasures. In this study, we demonstrate that a human isolate of Lassa virus causes lethal infection uniformly in Hartley guinea pigs. This novel animal model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics.

Whole-genome sequencing (WGS) was used to investigate the genetic features of the recently identified lsa(E) gene in porcine S. aureus ST9 isolates. Three quinupristin/dalfopristin-resistant isolates harboring the lsa(E) gene (two MRSA and one MSSA) were sequenced. Phylogenetic analysis of 184 S. aureus genomes showed that ST9 porcine isolates belong to a distinct sequence cluster. Further analysis showed that all isolates were deficient in the recently described type IV restriction-modification system and SCCmec type XII was identified in the two MRSA isolates, which included a rare class C2 mec gene complex. A 24 kb Psi SCC fragment was found in the MRSA and MSSA isolates sharing 99% nucleotide sequence homology with the Psi SCCJCSC6690 (O-2) element of a ST9 MRSA isolate from Thailand (accession number AB705453). Comparison of these ST9 isolates with 181 publically available S. aureus genomes identified 24 genes present in all (100%) ST9 isolates, that were absent from the most closely related human isolate. Our analysis suggests that the sequenced quinupristin/dalfopristin-resistant ST9 lineage represent a reservoir of mobile genetic elements associated with resistance and virulence features. (C) 2016 Elsevier GmbH. All rights reserved.

The first human parechovirus 3 (HPeV3 VGHKS-2007) in Taiwan was identified from a clinical specimen from a male infant. The entire genome of the HPeV3 isolate was sequenced and compared to known HPeV3 sequences. Genome alignment data showed that HPeV3 VGHKS-2007 shares the highest nucleotide identity, 99%, with the Japanese strain of HPeV3 1361K-162589-Yamagata-2008. All HPeV3 isolates possess at least 97% amino acid identity. The analysis of the genome sequence of HPeV3 VGHKS-2007 will facilitate future investigations of the epidemiology and pathogenicity of HPeV3 infection.

Listeria monocytogenes is a foodborne pathogen that infects the placenta and can cause pregnancy complications. Listeriosis usually occurs as a sporadic infection, but large outbreaks are also reported., Listeria monocytogenes is a foodborne pathogen that infects the placenta and can cause pregnancy complications. Listeriosis usually occurs as a sporadic infection, but large outbreaks are also reported. Virulence from clinical isolates is rarely analyzed due to the large number of animals required, but this knowledge could help guide the response to an outbreak. We implemented a DNA barcode system using signature tags that allowed us to efficiently assay variations in virulence across a large number of isolates. We tested 77 signature-tagged clones of clinical L. monocytogenes strains from 72 infected human placentas and 5 immunocompromised patients, all of which were isolated since 2000. These strains were tested for virulence in a modified competition assay in comparison to that of the laboratory strain 10403S. We used two in vivo models of listeriosis: the nonpregnant mouse and the pregnant guinea pig. Strains that were frequently found at a high abundance within infected organs were considered hypervirulent, while strains frequently found at a low abundance were considered hypovirulent. Virulence split relatively evenly among hypovirulent strains, hypervirulent strains, and strains as virulent as 10403S. The laboratory strain was found to have an intermediate virulence phenotype, supporting its suitability for use in pathogenesis studies. Further, we found that splenic virulence and placental virulence are closely linked in both the guinea pig and mouse models. This suggests that outbreak and sporadic pregnancy-associated L. monocytogenes strains are not generally more virulent than lab reference strains. However, some strains did show consistent and reproducible virulence differences, suggesting that their further study may reveal deeper insights into the biological underpinnings of listeriosis.

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC 50 ], 50 , 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro . In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC 50 , 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections.

Antimicrobial peptides (AMPs) are promising candidates for new antibiotic classes but often display an unacceptably high toxicity towards human cells. A naturally produced C-terminal fragment of PGLa, named PGLa-H, has been reported to have a very low haemolytic activity while maintaining a moderate antibacterial activity. A sequential tandem repeat of this fragment, diPGLa-H, was designed, as well as an analogue with a Val to Gly substitution at a key position. These peptides showed markedly improved in vitro bacteriostatic and bactericidal activity against both reference strains and multidrug resistant clinical isolates of Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells as assessed by haemolysis, cell viability, and DNA damage assays. The glycine substitution analogue, kiadin, had a slightly better antibacterial activity and reduced haemolytic activity, which may correlate with an increased flexibility of its helical structure, as deduced using molecular dynamics simulations. These peptides may serve as useful lead compounds for developing anti-infective agents against resistant Gram-negative and Gram-positive species. Copyright A 2016 Elsevier B.V. All rights reserved.

A Gram-positive bacterial strain, 99221/2016T, was isolated from blood of a patient with bacteraemia at the Institute of Medical Microbiology, Göttingen, Germany. The strain was rod-shaped with a palisade arrangement of cells, non-spore-forming, non-lipophilic, catalase-positive and oxidase-negative. It grew well at 37 °C on Columbia blood agar and showed good growth under aerobic, microaerophilic and anaerobic conditions. The colonies were white-cream, circular and convex with a shiny, smooth surface. The predominant respiratory quinones were MK-8(H2) and MK-9(H2). The polar lipids profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. Two unidentified phospholipids and several unidentified lipids were also detected. The prevalent cellular fatty acids comprised cis-9-octadecenoic acid (C18 : 1 ω9c), hexadecanoic acid (C16 : 0) and pentadecanoic acid (C15 : 0). Corynemycolates with 28-36 carbons in length were present. The whole-cell hydrolysate contained meso-diaminopimelic acid and arabinose, glucose, galactose and ribose as major sugars. Analysis of the 16S rRNA gene sequence identities revealed that the strain is most closely related to Corynebacterium imitans DSM 44264T (98.0 %), Corynebacterium lipophiloflavum DSM 44291T (96.9 %), Corynebacterium afermentans subsp. afermentans DSM 44280T (96.9 %) and Corynebacterium afermentans subsp. lipophilum DSM 44282T (96.8 %). The identity with Corynebacterium diphtheriae DSM 44123T, the type species of the genus, was 94 %. The DNA G+C content was 69.2 mol%. DNA-DNA hybridization with Corynebacterium imitans DSM 44264T revealed a value of 34 %, confirming that the strain represents a novel species. The type strain 99221/2016T (DSM 103494T=JCM 31931T) is proposed to represent a novel species of the genus Corynebacterium with the name Corynebacterium gottingense. peerReviewed

Whole genome sequencing (WGS) can provide a comprehensive analysis of Mycobacterium tuberculosis mutations that cause resistance to anti-tuberculosis drugs. With the deployment of bench-top sequencers and rapid analytical software, WGS is poised to become a useful tool to guide treatment. However, direct sequencing from clinical specimens to provide a full drug resistance profile remains a serious challenge. This article reviews current practices for extracting M. tuberculosis DNA and possible solutions for sampling sputum. Techniques under consideration include enzymatic digestion, physical disruption, chemical degradation, detergent solubilization, solvent extraction, ligand-coated magnetic beads, silica columns, and oligonucleotide pull-down baits. Selective amplification of genomic bacterial DNA in sputum prior to WGS may provide a solution, and differential lysis to reduce the levels of contaminating human DNA is also being explored. To remove this bottleneck and accelerate access to WGS for patients with suspected drug-resistant tuberculosis, it is suggested that a coordinated and collaborative approach be taken to more rapidly optimize, compare, and validate methodologies for sequencing from patient samples.

TEM-154, identified in Portugal in 2004, associated the substitutions observed in the extended-spectrum β-lactamase (ESBL) TEM-12 and in the inhibitor-resistant penicillinase (IRT) TEM-33. This enzyme exhibited hydrolytic activity against ceftazidime and a low level of resistance to clavulanic acid. Surprisingly, the substitution Met69Leu enhanced the catalytic efficiency of oxyimino β-lactams conferred by the substitution Arg164Ser. Its discovery confirms the dissemination of the complex mutant group of TEM enzymes in European countries.

Introduction: Pseudomonads are very important as an opportunistic bacterium among gram-negative bacteria. Class B beta-lactamases such as Verona imipenemase (VIM) and Imipenemase (IMP) are the most important contributors to the resistance of gram-negative bacteria like Pseudomonas aeruginosa. These enzymes were isolated from burn wounds in Zahedan due to extended spectrum beta-lactamase (ESBL) activity and high resistance to β-lactamase antibodies (IMP and VIM) in P. aeruginosa isolates. Objectives: This study was aimed to determine the frequency of VIM, IMP beta-lactamase genes in clinical isolates of P. aeruginosa isolated from therapeutic centers of Zahedan. Materials and Methods: In this descriptive cross-sectional study, 125 samples of P. aeruginosa from burn wounds were collected from patients who were hospitalized in Zahedan as well as were cultured. Suspected colonies of P. aeruginosa were identified using common biochemical methods. The Kirby Bayer method was used to measure antibiotic susceptibility, and then the minimum inhibitory concentration (MIC) of these strains was examined. The genotypes of the β-lactamase genes of IMP and VIM were investigated in polymerase chain reaction (PCR) strains. Results: In this study, 125 isolates of P. aeruginosa were found in 32 isolates (26%) producing ESBL. In general, the resistance of P. aeruginosa to the antibiotics of imipenem, piperacillin, ceftazidime, cefotaxime, ceftriaxone, levofloxacin, ciprofloxacin, gentamicin, amikacin, streptomycin, tetracycline, and carbenicillin were 88%, 93%, 89%, 98%, 97%, 89%, 84%, 82%, 97%, 91%, 90% and 95%, respectively. According to the PCR test, 32 isolates with ESBL enzymes were 43.75 and 18.75, respectively. Conclusion: The prevalence of ESBLs and antibiotic resistance in the burn injuries of hospital is high and requires such as antibiotic susceptibility test, rational administration of antibiotics and the control of beneficial factors. The results showed that most of the samples were resistant to the drug and the frequency of blaVIM genes was higher than blaIMP among the strains producing ESBLs.

Antimicrobial resistance against colistin has emerged worldwide threatening the efficacy of one of the last-resort antimicrobials used for the treatment of Enterobacteriaceae. To investigate the presence of the recently identified colistin resistance genes (mcr-4, mcr-5) in China, we established PCRs to detect mcr-4 and mcr-5 on 213 anal and 1,339 nasal swabs from apparently healthy pigs (n = 1,454) in nine provinces, and 1,696 cloacal and 1,647 oropharyngeal samples from poultry (n = 1,836) at live-bird markets in 24 provinces of China. The prevalence of the mcr-4 in swine swabs (41.4%; 642/1,552) was significantly higher than in swabs from poultry (11.5%; 384/3,343). The mcr-4 gene was found in geese (49.5%, 54/109), chickens (17.2%, 257/1,498), pigeons (17.2%, 17/99) and ducks (15.4%, 20/130). In a similar trend, the prevalence of the mcr-5 in swine swabs (33.1%; 514/1552) was significantly higher than in swabs from poultry (5.6%; 187/3,343). The mcr-5 was identified in geese (17.4%, 19/109), chickens (9.9%, 148/1,498), ducks (7.7%, 10/130) and pigeons (3%, 3/99). The mcr-4 prevalence in the nasal swabs from pigs (59.2%, 58/98) was significantly higher than that in anal swabs (29.6%, 29/98) (P

Background Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. Results We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. Conclusion We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.