Shigella are ranked among the most prevalent aetiologies of diarrhoeal disease worldwide, disproportionately affecting young children in developing countries and high-risk communities in developed settings. Antimicrobial treatment, most commonly with fluoroquinolones, is currently recommended for Shigella infections to alleviate symptoms and control disease transmission. Resistance to fluoroquinolones has emerged in differing Shigella species  since the turn of the 21st century, originating in endemic areas, and latterly spreading into non-endemic regions. Despite occurring independently, the emergence of fluoroquinolone resistance in these different Shigella species shares striking similarities regarding their epidemiology and resistance mechanisms. Here, we review and discuss the current epidemiology of fluoroquinolone-resistant Shigella species, particularly in the light of recent genomic insights. Shigella is a genus of human-adapted bacterial pathogens that cause dysenteric diarrhoea (shigellosis) in developing and developed countries. A specific class of antimicrobials, known as the fluoroquinolones, is recommended for the treatment of shigellosis, but resistance to this group of antimicrobials is rising rapidly within this genus. Here, we have combined available epidemiological and high-resolution genomic data to outline common themes that define the emergence and circulation of fluoroquinolone-resistant Shigella species. The information gathered in this review will be useful for determining optimal shigellosis treatment regimens and to tailor public health measures for alerting, containing and preventing the future spread of these antimicrobial-resistant enteric pathogens.Shigella, a pathogenic genus within the extensive Gram-negative family Enterobacteriaceae, is a major cause of diarrhoeal disease worldwide [Shigella does not comprise a monophyletic group of organisms but is formed of multiple discrete Escherichia coli lineages, all of which harbour a signature virulence plasmid responsible for the distinctive invasive pathogenesis [S. dysenteriae, boydii, flexneri and sonnei), which differ significantly in their epidemiology. Toxigenic S. dysenteriae serotype 1 (Sd1) is the causative agent of the now rare, often fatal, epidemic bacillary dysentery. S. boydii is only sporadically isolated from diarrhoeal cases in the Indian subcontinent [S. flexneri and S. sonnei, which predominantly circulate in developing and developed regions, respectively [orldwide . The gloorldwide . The disogenesis . Currentontinent . The oveectively .Shigella. Although the disease is self-limiting, antimicrobial treatment is recommended to prevent further complications, assist recovery and restrict faecal shedding [gyrA and gyrB) and topoisomerase IV (encoded by parC and parE) to inhibit functional replication and induce bacterial cell death [Shigella, estimating that resistance increased from 0.6 % in 1998–2000 to 29 % in 2007–2009 of the endemic shigellosis in Asia and Africa [Shigella on the list of global priority pathogens that urgently need focused development of novel antimicrobials [Shigella species, highlighting insights provided through genome sequencing and phylogenetic reconstruction. Due to its low prevalence and research focus, S. boydii will be excluded from this discussion.Shigellosis usually results in profuse diarrhoea, often accompanied by mucous or bloody discharge. This clinical presentation is associated with disruption of the intestinal epithelium, which is mediated by intracellular proliferation of the infecting shedding . One of ll death . Routined Africa  Fig. 1)Shigella.d Africa . Resistacrobials . This regyrA-S83L/D87G and S83L/D87N, which were associated with different geographical distributions [gyrA-S83L mutation as early as 1994, later diverging into two FQR sub-populations, which were then characterized by differing secondary mutations. Indeed, a genomic investigation of the global phylogeny of Sd1 concluded that resistance to fluoroquinolones was acquired only once during the species’ evolutionary history, conferred by the co-occurrence of parC-S80I, gyrA-S83L and a secondary gyrA-D87 mutation between 1995 and 2002 [S. dysenteriae serotypes is limited, probably due to their low prevalence, even in regions where the disease was once highly endemic.Resistance against fluoroquinolones had not been previously observed in Sd1 until FQR organisms were isolated during a dysentery outbreak arising in India and Bangladesh in 2002–2003  Fig. 2)Fig. 2). ibutions . These dand 2002 . This FQand 2002 . InformaS. flexneri has been conducted in South Asia and China, where the pathogen’s burden remains significant. The first incidences of FQR in S. flexneri were documented in eastern and northern China in 2001–2002 [gyrA-S83L, gyrA-D87G and parC-S80I QRDR mutations [S. flexneri underlined a worrying rising trend of fluoroquinolone resistance, which was almost exclusively found in serotype 2a [S. flexneri by 2010. These Bangladeshi isolates differed from their Chinese counterparts by a secondary QRDR mutation, harbouring gyrA-D87N instead of G. Furthermore, FQR S. flexneri with identical mutation profiles were recovered during a decade-long surveillance in Switzerland, highlighting the occurrence of fluoroquinolone resistance in non-endemic regions [The majority of epidemiological research on 001–2002 , and a dutations . Subsequotype 2a . Althoug regions .S. flexneri of various serotypes, including 1a, 1c, 2a, 2b, 2av, 4a, 4c and X [S. flexneri [S. flexneri serotypes. Despite being present in a wide range of serotypes and locations, identical QRDR mutations have been frequently encountered in S. flexneri in China, encompassing gyrA-S83L, gyrA-D87G/N and parC-S80I. Furthermore, these mutations are commonly accompanied by an unusual mutation (gyrA-H211Y), which was also present in the aforementioned Bangladeshi FQR isolates [S. flexneri from these two countries were also found to exhibit a close genetic relationship via PFGE [S. flexneri probably belong to one dominant widespread clone, where the gyrA-H211Y, gyrA-S83L and parC-S80I mutations arose prior to geographical divergence. Later, a secondary mutation in gyrA delineated the Bangladeshi (gyrA-D87N) and the Chinese (gyrA-D87G) FQR isolates. However, the increasing isolation frequency of the gyrA-D87N variant in parts of China may be the result of a higher degree of trans-border dissemination from South Asia and/or the separate, indigenous emergence of a competent FQR subclone [S. flexneri, the true nature of such events cannot be easily measured using low-resolution molecular typing methods.Routine dysentery surveillance in China has reported a steady increase of FQR 4c and X . This obflexneri . Availabisolates . FQR S. via PFGE . This cosubclone . Due to S. flexneri to S. sonnei) has been observed concurrently in multiple Asian countries as they undergo rapid economic transition; this has been recorded in Bangladesh, China, Thailand and Vietnam [S. sonnei worldwide, making antimicrobial resistance in this species a focal target for monitoring. Surveillance studies in developed countries have identified strong epidemiological links between FQR S. sonnei and a travel history to India [S. sonnei recovered in South Asia [S. sonnei are clonal and have evolved and spread in the region before disseminating intercontinentally. Indeed, phylogenetic analysis on representative extant FQR S. sonnei confirmed this hypothesis, concluding that South Asia was the most likely origin of these organisms [S. sonnei circulating in Cambodia, Vietnam and California [S. sonnei is generally determined by the sequential accumulation of three mutations: gyrA-S83L, parC-S80I and gyrA-D87G [gyrA-D87N instead of D87G) and the synergy between the plasmid-mediated qnrB gene and gyrA mutations, have also been identified [Shigella should be closely monitored in MSM networks, especially in the wake of increased shigellosis incidence, HIV infection and resistance to other antimicrobials, such as azithromycin, within this high-risk group [A shift in species dominance (from  Vietnam . This into India . Moreoveuth Asia . These rrganisms . Furtherlifornia . FluoroqyrA-D87G . Howeverentified . It is oentified . Therefosk group .Shigella species. (1) FQR is almost exclusively determined by sequential QRDR mutations in the following order: gyrA-S83L, parC-S80I and gyrA-D87G/N. (2) To date, the majority of FQR isolates identified within an individual species are clonal despite their wide geographical distribution. (3) South Asia, and potentially China, serve as likely reservoirs for the rise and spread of resistant clones. These interpretations are currently deduced from genomic insights into Sd1 and S. sonnei, and are subjected to various confounders, including geographical bias in sample collection. However, the extensive genetic diversity within S. flexneri may present an alternative scenario, which will benefit from large-scale molecular epidemiology data generated through whole-genome sequencing.The presented evidence reveals striking similarities between the emergence of fluoroquinolone resistance among the discrete Shigella only began to emerge in the early 2000s. The intervening period witnessed the emergence of Shigella exhibiting resistance to multiple antimicrobials including co-trimoxazole, ampicillin and nalidixic acid [Shigella species. The ordered QRDR mutations are selected in favour of those in efflux regulatory machinery due to their co-optimization for non-susceptibility (MIC levels) and fitness cost [in vitro generated and clinical isolates, resistance to fluoroquinolones almost exclusively commences with an initial mutation, gyrA-S83L. This mutation has arisen independently on multiple occasions for different Shigella lineages, possibly as an adaptive strategy for resistance against the first-generation quinolone, nalidixic acid [parC-S80I, which occurs much less frequently during evolution but gives the bacterium a significant increase in fluoroquinolone MIC and potentially a non-inferior fitness. Such a mutation is suggested to be favourably selected in abundance of mutation supply, fulfilled either by a large population size or a high mutation rate, when antimicrobial pressure is high [Shigella is relatively stable, the first scenario appears to be more plausible [Shigella transmission, resulting in a large bacterial population. Suboptimal public health measures in the region, exemplified by the fact that only 40 % of the Indian population has access to improved sanitation [Shigella. This expansion has arisen on a backdrop of rapidly increasing fluoroquinolone use for treatment of multiple enteric and febrile diseases since the turn of this century [Shigella and Salmonella Typhi [The first widely used fluoroquinolone, ciprofloxacin, was introduced to clinical practice in 1987. However, resistance in xic acid . Therefoxic acid . Recent ess cost . For botxic acid . However is high . Given tlausible . High ponitation , further century . Indeed, century . These cla Typhi .Shigella has been quickly followed by the expansion and, for S. sonnei, rapid international spread. Furthermore, co-resistance to other first-line antimicrobials, such as the macrolides and third-generation cephalosporins, is frequently identified among these bacteria [Enterobacteriaceae, even in the absence of fluoroquinolone pressure [Staphylococcus aureus, Klebsiella pneumoniae, E. coli, Clostridium difficile and Neisseria gonorrhoeae [Shigella in the transmission chain. However, future research is warranted to challenge this hypothesis, as well as to develop the best practices for controlling and treating new emerging antimicrobial-resistant clones.The emergence of FQR bacteria . These apressure . Alternaorrhoeae . All the


Acinetobacter pittii has emerged as an important hospital pathogenthat is associated with outbreaks and drug resistance. In cystic fibrosis (CF)patients, the detection of Acinetobacter spp. is rare; however, weisolated the A. pittii sequence type ST643 in several Brazilian CFpatients treated in the same centre. The current study describes the draft genome ofA. pittii ST643. Acinetobacter spp. are important nosocomial pathogens with A.baumannii being the most frequently observed species among clinical isolates, from which they are rarely isolated (Acinetobacter spp. strains from Brazilian CF patients. Theidentification of isolates to the species level by polymerase chain reaction (PCR)amplification and sequencing of a segment of the rpoB gene , in 2009. Agenomic library was constructed by transposon tagmentation with the Nextera XT DNA SamplePrep kit . Two sequence runs were performed with the 500- and 300-cycleMiSeq Reagent v. 2 kits on a MiSeq benchtop sequencer (Illumina Inc. USA). A total of1,445,561 and 1,232,439 paired-end reads for the 500- and 300-cycle kits, respectively,were obtained. Reads were corrected and assembled ades 3.5 . TheRappgaA, pgaB,pgaC and pgaD), as well as genes involved in capsularpolysaccharide biosynthesis and assembly , toxin production (dedA andcolicinV) and protein secretion systems .Genes associated with resistance to betalactams (blaOXA417 and blaADC-18) and to quinolones  were also identified. Althoughmutations in DNA gyrase and topoisomerase IV genes have also been associated with adecreased susceptibility to ciprofloxacin , chloramphenicol (ceoB) and macrolides wereobserved.Annotation results indicate the presence of genes associated with virulence, such as thoseinvolved in adhesin biosynthesis (floxacin , A. pittA. pittii in CFpatients. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank underthe accession LBCP00000000. The version described in this paper is versionLBCP01000000.This genome sequence provides the basis for a future detailed genomic analysis that willcertainly increase our understanding of the role of


Staphylococcus sciuri (MRSS) epidemiology in European healthcare settings. The aim of the study was to determine the prevalence of nasal and pharyngeal carriage and diversity of MRSS among inpatients and healthcare workers (HCWs) in the largest healthcare centre in Serbia, and to assess performance of different methods for MRSS screening. Nasal and pharyngeal swabs were obtained from 195 patients and 105 HCWs in different departments. Each swab was inoculated directly onto MRSA-ID, oxacillin-resistance screening agar and mannitol salt agar (MSA) with 2 mg/L of oxacillin. After inoculation, each swab was dipped in Mueller-Hinton broth with 6.5% NaCl and after overnight incubation, subcultured onto oxacillin-MSA. Characterisation of isolated MRSS strains was determined by antimicrobial susceptibility testing, PFGE, SCCmec typing and antimicrobial resistance genes detection. MRSS nasal and pharyngeal carriage rate was high (5%) in our hospital and department-variable. PFGE revealed a possible cross-transmission of MRSS between a patient and an HCW, and dissemination across hospital wards. All analysed isolates were multidrug resistant. Fusidic acid resistance was discovered in 93.7% of isolates, but fusA mutations in EF-G and fusB/C genes were not detected. SCCmec regions of MRSS contained elements of classic methicillin-resistant S. aureus type III. Broth enrichment prior to isolation on oxacillin-MSA was superior to direct cultivation on different media with a sensitivity/specificity of 100% and 88.5%, respectively. MRSS is a significant coloniser of patients and HCWs in the hospital. Further research is needed to investigate the clinical significance of the bacterium in our settings.There has been a paucity of data on methicillin-resistant  Staphylococcus sciuri belongs to the group of oxidase-positive, novobiocin-resistant coagulase-negative staphylococci (CoNS) [S. sciuri is only occasionally isolated from humans, it has been associated with a number of serious infections such as septicemia, endocarditis, peritonitis, pelvic inflammatory disease, urinary tract infections and wound infections [S. sciuri [mecA homologue  from which the mecA gene of methicillin-resistant staphylococci may have developed [mecA1 does not confer methicillin resistance in S. sciuri, but some strains may become resistant by acquisition of the mecA or mecC genes, identical to ones found in other staphylococci [i (CoNS) . This bai (CoNS) –5. It hai (CoNS)  and althfections ,2,7,8. A. sciuri . It has eveloped ,9,10. meylococci ,7,11.Staphylococcus aureus (MRSA) has emerged as a major pathogen in the healthcare and community settings worldwide and where MRSA infections are often preceded by a period of carriage [S. sciuri (MRSS), is scarce.Methicillin-resistant carriage . A numbecarriage –14. In acarriage . Still, S. sciuri has long been the focus of our investigation [tigation –3,6,8, aA cross-sectional study was conducted between November 2010 and January 2011 at the Clinical Centre of Serbia, which has over 3000 beds and represents the main referral healthcare centre in our country. Sample collections were performed in three departments: Emergency Department (ED), Surgical Department (SD) and Medical Department (MD).Participation was voluntary and all participants signed informed consent form prior to their inclusion in the study. Descriptive information regarding participants' age, gender, diagnosis, department and period of hospitalisation was collected . The stuSpecimens were taken from 195 hospitalised patients  and 105 HCWs . Samples for each person included two swabs, one taken from both anterior nares and one from the throat.After the collection, all samples were processed within 2 h. Each swab was inoculated directly onto MRSA-ID , oxacillin resistance screening agar  and mannitol salt agar  supplemented with 2 mg/L of oxacillin. All inoculated media were incubated aerobically at 35°C and observed after 24 h and 48 h of incubation. The order of plating first alternated between the three media for every 100 samples. After inoculation, each swab was dipped in 3 mL of Mueller-Hinton broth  supplemented with 6.5% NaCl and after incubation for 24 h at 35°C, subcultured onto MSA supplemented with 2 mg/L of oxacillin, which was thereafter incubated for up to 48 h at 35°C aerobically.S. sciuri is a mannitol-positive organism [mecA gene [S. sciuri mecA homologue was detected with mecA primers included in the PCR designed by Kondo et al. [S. sciuri subsp. sciuri CCM 3473, S. sciuri subsp. rodentium CCM 4657, S. sciuri subsp. carnaticus CCM 4835, S. vitulinus CCM 4511, S. lentus CCM 3472 and S. aureus ATCC 25923 were used as control.organism ,7, whichorganism  and confecA gene . S. sciuo et al. . Strainshttp://www.eucast.org). In order to identify MLSb phenotypes, a double-disk diffusion test (D test) was performed with erythromycin (15 μg) and clindamycin (2 μg), following the procedure recommended by EUCAST. Determination of minimum inhibitory concentration (MIC) of fusidic acid was performed by broth microdilution method in accordance with EUCAST recommendation. The molecular mechanism(s) of fusidic acid resistance in fusidic acid resistant isolates was investigated by testing isolates for the presence of the fusB and fusC genes using a multiplex PCR and by investigating isolates for the presence of fusA gene mutations by nucleotide sequencing [Susceptibility to cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, fusidic acid, erythromycin, gentamicin, kanamycin, linezolid, mupirocin, quinupristin-dalfopristin, penicillin, rifampin, tetracycline, tobramycin and trimethoprim/sulfamethoxazole  was tested by disk diffusion method and to vancomycin and teicoplanic by Etest  in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendation , macrolide-lincosamide-streptogramin (MLS) group of antibiotics (erm(A), erm(B), erm(C), lnu(A), mph(A), and mph(C)), tetracycline (tet(K), tet(M), tet(L) and tet(O)) and chloramphenicol  resistance genes [All isolates were screened for aminoglycoside (ce genes –21.PFGE was performed as described previously . Accordimec types was carried out by previously described PCR protocol for mec class and ccr type [S. aureus strains HT20020290 (SCCmec type I), HT20020285 (SCCmec type II), HT20030826 (SCCmec type III), HT20040068 (SCCmec type IV), HT20060580 (SCCmec type V) and HT20020274 (SCCmec type VI), kindly provided by the Centre National de Référence des Staphylocoques, Lyon, France, were used as control. SCCmec typing was also performed on 37 MRSS strains previously isolated from humans, dogs, hospital environment and public transportation [Determination of SCCccr type . S. aureortation ,2,6,8.Demographic characteristics (age and gender) of patients and HCWs and descriptive information regarding department, underlying diagnosis and duration of hospitalisation  were desMRSS carrier prevalence in the two groups was 12/195 (6.1%) in patients and 3/105 (2.9%) in HCWs. Distribution of MRSS carriers and non-carriers stratified by population characteristics is shown in The presence of MRSS in nasal and throat swab specimens obtained from patients and HCWs is presented in In total, 16 MRSS isolates were further analysed and the results of phenotypic and genotypic characterisation are presented in Based on PFGE typing, genetically unrelated MRSS could be divided into nine different pulsotypes (A-I). The largest group consisted of strains belonging to pulsotype B (25%) only found in patients from different departments (ED and SD).mec typing revealed that most of the MRSS strains (75%) from the study contained elements of classic SCCmec type III (mec class A and ccr type 3). Among 37 additional MRSS strains investigated from our collection, the combination of mec class A and ccr type 3 was detected in 17/37 (45.9%) strains, while only the presence of mec class A gene complex was identified in the remaining 20/37 (54.1%) strains.SCCAll tested isolates were resistant to two or more antibiotics classes besides beta-lactam antibiotics, i.e. they were multidrug resistant. High levels of resistance were observed for aminoglycosides (93.8%) and fusidic acid (93.8%). Two (12.5%) and four (25%) isolates were resistant to ciprofloxacin and chloramphenicol, respectively. Only one isolate (6.3%) was resistant to rifampicin, tetracycline, mupirocin, erythromycin and clindamycin, while the remaining 15 (93.8%) strains exhibited intermediate resistance to clindamycin. All MRSS isolates were susceptible to trimethoprim/sulfamethoxazole, vancomycin, teicoplanin, linezolid and quinupristin-dalfopristin.aacA-aphD, aphA3 and aadC genes were detected in 100%, 46.7%, and 20% isolates, respectively. The erm(C) gene was responsible for the constitutive MLSb resistance in 6.3% of isolates, whereas the lnu(A) gene was revealed in 31.3% of MRSS isolates. The tet(M) gene was identified in 6.3% of MRSS isolates that were also phenotypically resistant to tetracycline. The cat221 gene was discovered in a chloramphenicol-resistant isolate which was grouped in pulsotype D. However, in the remaining three isolates resistant to chloramphenicol (pulsotypes F and H), the PCR product with primers specific for cat221 were obtained, but the products had a higher molecular weight than the positive control.Of the 15 MRSS isolates that exhibited resistance to aminoglycosides, the fusB like resistance mechanisms. However, no amplification was obtained with the applied fusB/fusC PCR. Neither did sequencing of the EFG-G reveal mutations that correlated with the obtained MIC values. However, alterations in EFG-G defined two different clusters (A and B) and two single isolates with deviating amino acid substitutions (MICs for fusidic acid showed low- to intermediate- resistance (2–16 mg/L), indicating presence of a itutions , whereasmecA gene, also discover the presence of S. sciuri mecA homologue. The expected product of 286 bp was observed not only in isolated MRSS but also in methicillin-susceptible S. sciuri strains, including reference strains S. sciuri subsp. sciuri CCM 3473, S. sciuri subsp. rodentium CCM 4657 and S. sciuri subsp. carnaticus CCM 4835. Additionally, this 286 bp product was not obtained with S. vitulinus CCM 4511, S. lentus CCM 3472, or S. aureus ATCC 25923, which do not possess mecA homologue.It was noticed that a pair of primers designed by Kondo et al.  to detecS. sciuri isolates found in one swab taken from the anterior nares of a hospitalised patient were classified into pulsotypes B and G.There was a good concordance between the observed antibiotic resistance profiles and the pulsotypes: pulsotype B and F . Two S. S. sciuri, including methicillin-resistant strains, from nose of healthy and hospitalised individuals has previously been reported in low rates among healthy children (0.3%) [Isolation of n (0.3%) , at hospn (0.3%) , or amonn (0.3%) . To the S. sciuri in very low numbers, while the remaining isolates produced only one to seven colonies during primary isolation.To determine the exact pattern of carriage in investigated individuals, non-carriers, intermittent carriers, or persistent carriers, longitudinal studies are required. However, persistent carriers are usually colonised by a higher load of a single strain over a long period, while intermittent carriers may carry different strains over time . SemiquaS. sciuri in humans is concerned, its transmission may occur via contact with animals. Another possible source for colonisation of humans with S. sciuri is food. In a previous study, a high rate of colonisation (10.5%) of hospital environment with S. sciuri was shown [S. sciuri strains isolated from that hospital environment, 64.3% were resistant to methicillin. These results confirm the previous hypothesis of Couto et al. [S. sciuri strains isolated from nasal passages and pharynx are acquired from the environment rather than from other sources. Yet, human to human transmission should also be considered.As far as the origin of as shown . Among So et al.  that S. S. sciuri strains isolated from hospital environment has also been reported in a previous study [PFGE analysis revealed nine pulsotypes within the population of MRSS strains. A high genetic diversity of us study . In the S. sciuri may remain controversial , the capacity of this bacterium to carry resistance determinants is well established [S. sciuri shows low level of natural resistance to lincosamides, i.e. licomycin and clindamycin [S. scuiri isolates has also been confirmed in recent studies in animal species, pointing to a natural resistance of S. scuiri [fusA gene or by presence of the fusB, fusC and fusD genes which encode EF-G proteins protected from fusidic acid binding [fusA mutations in EF-G, but could not find any. MICs of analysed isolates were in the low to intermediate range, pointing to a fusB-like mechanism, however fusB/fusC genes were not detected. Additionally, Schoenfelder et al. tested S. sciuri for fusD gene, which was also negative, indicating the presence of different mechanism mediating fusidic acid resistance that is still to be determined [Despite the fact that the clinical significance of ablished ,27. Thisndamycin , and the. scuiri ,28. Fusi binding ,27. Thertermined .mec elements for S. aureus. Therefore, we analysed SCCmec regions of isolated MRSS strains. SCCmec typing revealed mec class A in all tested MRSS strains, but ccr type 3 was discovered in 12 (75%) strains. This mec class/ccr type combination corresponds to SCCmec type III [ccr type could not be determined. Presence of SCCmec type III or only mec class A in MRSS was also observed in other studies [mec class complex A. Finally, we tested 37 previously described MRSS strains isolated from humans, dogs, hospital environment and public transportation [mec type III, i.e. combination of mec class A and ccr type 3, was detected in 45.9% of strains, while in the remaining strains only the presence of mec class A gene complex could be demonstrated. The high prevalence of non-typeable SCCmec cassettes in S. sciuri confirms the potential divergence of ccr and mec complexes as previously suggested [mec elements [CoNS are believed to constitute a reservoir of SCCtype III . In rema studies –32. The ortation ,8. SCCmeuggested  and indielements .S. sciuri is CoNS that can be easily misidentified as S. aureus because both species are mannitol-fermenting organisms that may grow as yellow colonies on blood agar and may give positive Slidex Staph Plus agglutination test and Staphaurex test [rex test ,7. Furthrex test .S. scuiri may serve as a reservoir and an important hub for exchange of the mecA gene and other resistance genes among staphylococci. In order to improve the detection of MRSS, broth enrichment prior to isolation on MSA with oxacillin represented the optimal available choice for isolating this bacterium.MRSS is a significant nasal and pharyngeal coloniser of patients and HCWs in this hospital. Molecular typing revealed the possibility of cross-transmission of MRSS between a patient and an HCW and dissemination through hospital. S1 TableDistribution of MRSS carriers and non-carriers stratified by population characteristics and characteristics of MRSS strains isolated from carriers.(DOCX)Click here for additional data file.


Synoeca spp.) are found throughout the tropical regions of South America, are much feared due to their aggressive nest defence and painful sting. There are only five species of Synoeca, all construct distinctive nests that consist of a single sessile comb built onto the surface of a tree or rock face, which is covered by a ribbed envelope. Although locally common, research into this group is just starting. We studied eight colonies of Synoeca septentrionalis, a species recently been described from Brazil. A new colony is established by a swarm of 52 to 140 adults that constructs a colony containing around 200 brood cells. The largest colony collected containing 865 adults and over 1400 cells. The number of queen’s present among the eight colonies varied between 3 and 58 and no clear association between colony development and queen number was detected. Workers and queens were morphologically indistinguishably, but differences in their cuticular hydrocarbons were detected, particularly in their (Z)-9-alkenes. The simple cuticular profile, multiple queens, large size and small number of species makes the ‘Warrior wasps’ an excellent model group for further chemical ecology studies of swarm-founding wasps.Swarm-founding ‘Warrior wasps’ ( Vespa] and yellow-jackets [Vespula]) are replaced by the swarm-founding Polistinae. In the Neo-tropics, these swarm-founding wasps have undergone a spectacular radiation. So, although only 25% of Polistinae species are swarm-founding, they are the most taxonomical diverse, exhibit the greatest diversity of nest structures and range of colony sizes  con conS. seharacter . So, theharacter  are vitaS. septentrionalis colonies, comparisons with previous studies on S. surinama [S. cyanea [Synoeca undergoes cyclical oligogyny, as suggested for other Epiponini wasps [S. cyanea, suggest that an entire colony re-located, possibly due to their previous colony been destroyed or disturbed.Although, this is the first study on surinama , 21–24 a. cyanea  coloniesni wasps , 4, althni wasps . New nesSynoeca were constructed at heights between 1m to over 6m. The nest construction is unusual among social wasps, since the base of the sessile comb is built first then the walls of the envelope are constructed. The ribbed structure results from the extension of the edge effect of hexagonal shaped comb cells [All nests of mb cells . This alS. septentrionalis, just like in S. chalibea [S. surinama [S. cyanea [S. septentrionalis is a similar size than S. cyanea (correcting for the x10 error in the measurements in) [S. surinama using the measurements in [S. surinama collected from the study area had workers that were clearly larger than S. septentrionalis suggesting a large variation in body size among each species, although within a colony, sizes are very consistent.This study found no significant morphological differences between queens and workers in chalibea , S. surisurinama , 24 and . cyanea . Howeverents in)  and bothSynocea group, with only one previous study [S. surinama. Both species of Synocea had a CHC based on a series of C21-C33 n-alkanes and (Z)-9-alkenes, and dienes although the double bond positions have only been determined in S. surinama as the Z9 [S. septentrionalis (this study). The presence of olefins  in social wasps is unusual since wasps have predominantly diversified their methyl-branched alkanes production [Mischocyttarus cassununga whose CHC is dominated by mono and di-methyl-alkanes [Very little is known about the chemical ecology of the us study  on S. sus the Z9 , i.e. thoduction , such as-alkanes , typical-alkanes .S. septentrionalis CHC data both caste and colony differences occur, these are more distinct in the alkenes, but difficult to understand within a limited number of colonies sampled. Although, in both S. septentrionalis  and workers, has also been reported in other social wasp species, including S. surinama [Polistes dominulus [P. fuscatus [Polybia micans [Apis mellifera [Within, the rionalis  and S. ssurinama  (Z)-9-C2surinama , Polisteominulus , P. fuscfuscatus  and Polya micans  as well ellifera , 30, indS1 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


Cladosporium cladosporioides had a less extensive colonization in isolation (control) than in dual interactions (coexistence) with other species. Phoma eupyrena exhibited the highest colonization success rate and competitive dominance among all tested species. X-ray diffraction (XRD) and scanning electron microscope (SEM) analyses revealed that Pestalotiopsis maculans and Paraconiothyrium sp. produced calcium oxalate crystals during their growth on coupon surfaces, both in isolation and in dual interactions. Our results demonstrate that interactions between abundant fungal species influence the fungal colonization on substrates, the biomineralization and the fungal community assemblage growing in limestone biofilms.The assembly of fungal communities on stone materials is mainly influenced by the differential bioreceptivity of such materials and environmental conditions. However, little is known about the role of fungal interactions in the colonization and establishment of fungal species. We analyzed the effects of intra- and interspecific interactions between 11 species of fungi in oligotrophic and copiotrophic media and on limestone coupons. In a previous study, these species were the most frequently isolated in the epilithic biofilms of limestone walls exposed to a subtropical climate. In the culture media, we found a greater frequency of intra- and interspecific inhibitory effects in the oligotrophic medium than in the copiotrophic medium. On the limestone coupons, all fungi were able to establish; however, the colonization success rate varied significantly.  Studies on the fungal communities that colonize lithic substrates have revealed a diverse assemblage of species –3, whoseThe types of interactions among fungal species, as well as the responses of particular fungal species to such interactions, depend on the availability of nutrients in the fungal substrate and on environmental conditions , 16. ForInterspecific fungal interactions can have various effects on substrates. In some cases, interspecific interactions enable the optimal degradation of wood substrates . On stonBased on a culture-dependent approach, we previously identified the fungal species that were frequently isolated from biofilms adhered to fragments of limestone used to construct buildings, considering the distinct exposure times of these limestone blocks to a subtropical environment in Campeche, México . We obseThe fungal community assemblage considered in this study was previously analyzed in Gómez-Cornelio et al. , in whicThese species were identified by their micro- and macroscopic characteristics in Gómez-Cornelio et al.  and in t3), which is the main component of limestone ) for each crystalline phase registered in the diffractogram, whose sum was required to reach 100%. Therefore, the following equation was applied: % Intensity of F1 = 100. In addition, fragments of coupons were deposited on standard carbon tape for observation, and images of these were taken under low-vacuum conditions (secondary electrons imaging). Finally, the elemental analysis of the crystals formed on the coupon surfaces was carried out via field emission scanning electron microscopy .The interaction responses of fungi on culture media and limestone coupons were subjected to analyses of variance (ANOVA) given the established classification criterion. The means of treatments were compared with Tukey's multiple comparison tests . The LevThe fungal species involved in this study were previously identified in a fungal community assemblage on limestone in a subtropical environment of Campeche, Mexico . In partThe growth of the evaluated fungi in oligotrophic and copiotrophic culture media showed a wide range of interaction types  that werCurvularia clavata, Fusarium oxysporum, Hyphomycete sp. and Pestalotiopsis maculans, whereas the rest of the species displayed competitive interactions , while 32% of species grew up to and around the challenge species (B2 and E2). Mutual inhibition at a distance (category D) was observed in 34% of the interactions in the CACO medium yet in only 2.5% in the MEAC medium , and then growth stopped. This was the most frequent interaction in both MEAC (41%) and CACO (34%) media . Hyphal C medium .C. cladosporioides in the MEAC medium, which obtained a low antagonism index score (19). This species exhibited mutual intermingling (category A) with one species and presented interactions characteristic of type B1 with four species and of types B2 and C with three species each. At the other extreme, F. redolens in the MEAC medium presented interactions of types E1 and E2 with four and five species, respectively, and also presented type-C interactions with two species; this behavior corresponds with its high score (152) on the AI  , which eI Tables . The AI M. roridum and F. redolens showed the highest inhibition percentages in relation to their challenging species in both culture media  in both culture media  in the presence of  control . In mostre media .C. cladosporioides, the colonization of coupons was greater than 70%. Significant differences were observed between C. lunata, P. maculans and P. eupyrena with respect to F. redolens and C. cladosporioides  or two species Tables  and 5. Iorioides .P. eupyrena displayed greater dominance during colonization than C. cladosporioides, F. redolens, Paraconiothyrium sp. and P. maculans. In the interaction between P. eupyrena and M. roridum, we observed competition for space during colonization: Each fungus colonized 39.3% of the coupon surface, yet the fungi also coexisted across a small portion (12.7%) of the coupon surface. In turn, C. lunata dominated the limestone surface when interacting with F. redolens, Paraconiothyrium sp. and P. maculans (P<0.05) and was able to coexist with C. cladosporioides, M. roridum and P. eupyrena in the same space on the stone surface (with a recovery rate of higher than 54%). Furthermore, F. redolens displayed greater dominance during colonization than C. cladosporioides, M. roridum and P. maculans. However, P. maculans dominated the stone surface when interacting with Paraconiothyrium sp. and C. cladosporioides, and M. roridum dominated Paraconiothyrium sp. and P. maculans. Finally, C. cladosporioides coexisted with M. roridum across 54% of the coupon surface  and dihydrated (weddellite) calcium oxalate , whereas soil may be its realized niche [P. maculans produced both types of calcium oxalate (whewellite and weddellite). However, in some interactions, this fungus produced only one type of calcium oxalate, while no crystals were observed in other interactions Click here for additional data file.S2 TableC. cladosporioides, Cucl: C. clavata, Culu: C. lunata, Fuox: F. oxysporum, Fure: F. redolens, Hyph: Hyphomycete sp., Myro: M. roridum, Para: Paraconiothyrium sp., Pema: P. maculans, Pheu: P. eupyrena, Scco: S. constrictum.M = MEAC medium; C = CACO medium. Means  followed by the same letter do not differ significantly between the two media according to Tukey’s post hoc test at P ≤ 0.05. Clcl: (DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(ZIP)Click here for additional data file.


Meloidogyne enterolobii is one of the obligate biotrophic root-knot nematodes that has the ability to reproduce on many economically-important crops. We carried out de novo sequencing of the transcriptome of M. enterolobii using Roche GS FLX and obtained 408,663 good quality reads that were assembled into 8193 contigs and 31,860 singletons. We compared the transcripts in different nematodes that were potential targets for biological control. These included the transcripts that putatively coded for CAZymes, kinases, neuropeptide genes and secretory proteins and those that were involved in the RNAi pathway and immune signaling. Typically, 75 non-membrane secretory proteins with signal peptides secreted from esophageal gland cells were identified as putative effectors, three of which were preliminarily examined using a PVX (pGR107)-based high-throughput transient plant expression system in Nicotiana benthamiana (N. benthamiana). Results showed that these candidate proteins suppressed the programmed cell death (PCD) triggered by the pro-apoptosis protein BAX, and one protein also caused necrosis, suggesting that they might suppress plant immune responses to promote pathogenicity. In conclusion, the current study provides comprehensive insight into the transcriptome of M. enterolobii for the first time and lays a foundation for further investigation and biological control strategies. Meloidogyneenterolobii (M. enterolobii) is an important obligate biotrophic plant parasite, infestation of which can lead to yellowing and stunting as a result of the nutritional and water stress caused by the physical root damage of nematode feeding [M. enterolobii was first discovered in Danzhou, China, in 1983 [M. enterolobii can infect a variety of host plants, including economic crops, such as cotton, pepper, tobacco and watermelon [Root-knot nematodes include over eighty species. These nematodes can negatively affect host plants in three main ways: damaging the plant roots, competing for nutrients and causing infection from other microbes induced by root damage . Meloido feeding . M. entetermelon ,4, and hM. enterolobii has received great attention because of its ability to reproduce on a variety of host cultivars bred for nematode resistance. A PCR-based detection method can be used to identify M. enterolobii accurately [M. enterolobii. Many efficient chemical-based nematicides have been banned or restricted for environmental and health concerns [M. enterolobii for such control strategies is still absent [curately . Currentconcerns ,7. Biolol absent .Pratylenchus coffeae [Meloidogyne graminicola [Pratylenchus thornei [Heterodera avenae [Aphelenchoides besseyi [M. enterolobii is lacking. Therefore, the objective of the current research was to systematically study the transcriptome of M. enterolobii and to determine the possible mechanism of pathology of M. enterolobii in plants, as well as any important genes that might be used as RNAi targets for biological control. To achieve this objective, we collected the M. enterolobii, sequenced its transcriptome and carried out a series of bioinformatics analyses by comparing its transcriptome with those with known transcriptomic information.The interaction between plants and parasites is complicated and delicate. Plant parasitic nematodes have evolved to possess a variety of strategies that allow them to successfully infect and survive their hosts. For example, plant-parasitic nematodes can secrete numerous proteins, termed effectors, into their hosts that have various functions, including suppression of plant defenses and interference with plant signaling or hormone pathways to promote the formation of nematode feeding sites . RNA seq coffeae , Meloidominicola , Pratyle thornei , Heteroda avenae  and Aphe besseyi , whereasM. enterolobii generated 408,663 reads, for a total of 165,040,879 base pairs, with an average length of 403 bp, of which 355,760 reads were assembled into 8193 contigs with an average length of 1202 bp. Additionally, 31,860 singletons were obtained, with an average length of 380 bp. We obtained 8143 putative operons from the contigs and 29,403 from the singletons , cellular metabolic process (52%), membrane-bounded organelle (44%), ion binding (44%), primary metabolic process (41%), organic substance metabolic process (41%), single-organism cellular process (41%), nitrogen compound metabolic process (39%) and biosynthetic process 31%; .M. enterolobii with several nematodes that have available genetic information, including M. incognita, M. hapla, B. xylophilus, G. pallida and C. elegans. Of the genes pooled from these nematodes, we identified 17,966 orthologous families and 42,156 non-orthologous families. Approximately 51% of the operons  from M. enterolobii belonged to 11,926 orthologous families, of which 2826 gene families, including 6999 operons, were unique to M. enterolobii. Phylogenetic analysis of 1022 conserved single-copy orthologous families showed that M. enterolobii had the closest relationship with M. incognita, followed by M. hapla , β-lactam resistance (KO_00312), bisphenol degradation (KO_00363), peptidoglycan biosynthesis (KO_00550), atrazine degradation (KO_00791), steroid degradation (KO_00984), biosynthesis of the siderophore group nonribosomal peptides (KO_01053) and malaria (KO_05144)  . The numematodes .M. enterolobii had only 249 CAZyme modules, which was the lowest among those of the six nematodes analyzed , we found that analyzed . Typicalectively .M. enterolobii had protein kinases in all twelve types of kinases. Compared with other nematodes, M. enterolobii had fewer kinases in most groups of kinases; for example, M. enterolobii had only one “RGC” kinase and two “atypical” kinases, which indicated the low abundance of these enzymes in M. enterolobii  encoding putative chemotaxis participators for host-seeking and location, amphid protein map-1, SXP/RAL-2; (II) encoding plant reprogramming regulators for feeding cells formation and maintenance, expansin (EXP), cellulose-binding proteins (CBP); (III) encoding immunomodulatory components for protecting nematodes against plant defense response, chorismate mutase (CM), fatty acid and retinol binding protein (FAR), venom allergen-like protein (VAP), annexins, etc. Moreover, we also identified some esophageal gland cell secretory proteins that were conserved in plant parasitic nematodes (PPNs), but without functional characterization yet  and secreted through the stylet into plant cells play essential roles in the parasitism initiation and maintenance especially. From our analysis results,  species . Blastintion yet .M. enterolobii with the effectors reported, we found 241 transcripts that could be translated into effectors. These putative effectors were involved in cell wall degradation, proteinase, defense or suppression of the host plant immune system, participation in the signaling of the host plants and other similar functions. Of the 241 putative effectors, 75 were biosynthesized from the esophageal gland cells (Agrobacterium tumefaciens GV3101 expressing vector pGR107-candidate gene or buffer as a control was injected into the leaves of N. benthamiana 24 h before the injection of genes expressing pGR107-BAX. The results showed that the No. 5 and No. 10 proteins could effectively suppress BAX-induced programmed cell death (PCD). Interestingly, the No. 8 protein not only caused necrosis, but also suppressed BAX-induced PCD  may endow M. incognita with more competitive ability in host cells. What is worth special mention is malaria (KO_05144), which suggested that M. enterolobii may have evolved to have homologous elements involved in the KO_05144 pathway of human malaria parasites, providing clues to reveal the genetic and pathologic mechanism for its strong parasitism ability. Furthermore, M. incognita  had fewer genes expressed overall than did M. enterolobii , but more than M. hapla . This may be due to differences in their living conditions and the evolutionary stress during environmental adaptation.We compared the transcripts obtained from M. enterolobii to parasitize a host plant is to destroy the plant cell walls. Glycoside hydrolases are enzymes that can degrade cellulose, which is the main component of plant cell walls. We found 19 putative glycoside hydrolases expressed in M. enterolobii, of which 10 putative glycoside hydrolases  were expressed in all six nematodes. It should be noted that these genes were expressed in both free-living nematodes (C. elegans) and parasitic nematodes, which is possible because GH genes can be acquired via horizontal gene transfer [CAZymes are enzymes that are involved in the synthesis, metabolism and transport of carbohydrates. The first important step for transfer .M. enterolobii had fewer kinases than C. elegans did, especially in the RGC and atypical categories. RGC is an abbreviation for receptor guanylate cyclase, which plays an important role in neuronal signaling systems [M. enterolobii was isotig04408, which was annotated as having multiple functions, such as guanylate cyclase activity, protein tyrosine kinase activity and ATP binding.Protein kinases are another group of influential enzymes that participate in nearly every biochemical reaction. The current research showed that  systems . The onlC. elegans [M. enterolobii and other parasitic root nematodes had many fewer flp and nlp genes expressed than did C. elegans. However, these neuropeptides may still play an important role in their life cycle. For example, the inactivation of flp-1 can led to many behavioral dysfunctions [Neuropeptides play an essential role in chemoreception in nematodes. To date, 113 genes coding for 250 neuropeptides have been identified in  elegans . Our datunctions ,19.M. enterolobii will be helpful to perform RNAi experiments effectively for this nematode. Different from C. elegans, information is limited on the RNAi pathways of these parasitic nematodes. However, we found that most of the genes involved in the RNAi pathway were conserved in these parasitic nematodes, indicating their essential roles in the living cycle of these nematodes. For example, the genes of RNaseIII , RNA helicase , export protein  and amplification factors  were shown to be expressed in all of the nematodes analyzed.In the recent ten years, RNAi has been widely employed in functional genomic research on PPNs and has also been perceived as an effective strategy to engineer PPN resistance for host plants. Therefore, identification of the components known to be involved in the RNAi pathway in M. enterolobii has a unique immune system in that it avoids other pathogens. For example, M. enterolobii can avoid attack from Pasteuria penetrans, which is effective for infecting other nematodes [C. elegans, a well-studied nematode model [dbl-1 gene was expressed in all six nematodes that were analyzed. The DBL-1 pathway must work with several factors, including the SMA-2/SMA-3/SMA-4 complex and SMA-9 [M. enterolobii, M. incognito and B. xylophilus had sma-2, sma-3 and sma-4 expressed, whereas M. hapla and G. pallida seemed not to be capable of forming the SMA-2/SMA-3/SMA-4 complex, which might lead to the disruption of this pathway.ematodes ,21,22. Ide model ,24. In tnd SMA-9 . Our datM. enterolobii can overcome the anti-parasitic genes that are effective in prevention in other nematodes, including the tomato Mi resistance gene and pepper N resistance gene. In addition, M. enterolobii could not be infected by Pasteuria penetrans, which has potential as a biocontrol agent for Meloidogyne species. Therefore, M. enterolobii is considered to be a great threat to these crops. Our transcriptome data displayed the unique gene expression pattern of M. enterolobii compared with other nematodes. This information may work as a guideline to determine key points  for further biological control of these parasites. Despite different lifestyles, the nematodes shared common effectors that enable them to invade root tissues and suppress host immune responses for survival. Esophageal gland cells, including one dorsal gland cell and two subventral gland cells [M. enterolobii. These were many more than those from the other two Meloidogyne species, which partly explained why M. enterolobii can infect a wide spectrum of host plants.Different from other nematodes, nd cells , are impnd cells ,27. Thernd cells ,33,34,35nd cells ,11. The nd cells ,13. JacoMeloidogyne genus, and their homologues had been suggested to be exclusively expressed in esophageal gland cells of M. incognita by in situ mRNA hybridization [M. enterolobii had multiple strategies to neutralize host immune reactions. To understand their regulation patterns, further research is still needed to confirm the molecular functions of these effectors.The pro-apoptotic mouse protein BAX is a death-promoting member of the Bcl-2 family that is involved in programmed cell death (PCD). Because of the common mechanism between the animal and plant cell death program, BAX can also trigger plant PCD (BT-PCD), physiologically resembling that associated with the hypersensitive response , so the dization . TherefoM. enterolobii, we noticed that these parasitic nematodes showed very similar gene expression patterns. Interestingly, these parasitic nematodes missed a variety of genes that were readily expressed in C. elegans, which is a free-living nematode. This might be because C. elegans was better armed to adapt to the tough free-living environment, whereas the parasitic nematodes were more focused on the defense avoidance of the host plants. It should be noted that the present bioinformation analysis was performed based on M. enterolobii expressed sequence tag sequences, which may have led to misidentification because of the potential limitations in transcriptomic coverage and assembly facticity. In the future, the comparative bioinformatics performed on deep/full-length transcriptome or genome sequences, combined with genes’ functional analysis, will facilitate the comprehensive and reliable identification for M. enterolobii-specific and Meloidogyne conserved genes. Either way, our extensive analyses exposed the specific and conserved genes that are essential to M. enterolobii parasitism or survival, and these genes represent the promising targets for novel efficient biocontrol strategies that are friendlier to the environment.By analyzing the transcriptome data of M. enterolobii stain “MeLD1” was collected from infested tomato roots growing in Ledong, China, whose genetic homogeneity had been identified previously by microscopical observation of perineal patterns [Lycopersicum esculentum cv. “Baiguo”) in axenic cultures starting from a single-egg mass in a greenhouse of the nematode laboratory in China Agricultural University. Egg masses on root galls were handpicked and hatched over sterile water at 25 °C on improved Baermann pans for 72 h to yield preparasitic second-stage juveniles (pre-J2s). Mixed parasitic stages of M. enterolobii were collected from infected tomato roots by root blending and sieving as previous described [patterns ,45 and bpatterns . The genescribed , then thM. enterolobii at mixed stages was treated with DNase I at 37 °C for 25 min to remove any residual DNA. The cDNA was then synthesized using SuperScriptTM III Reverse Transcriptase (Invitrogen) at 42 °C for 1 h. The synthesized cDNA was fragmented into 300–800 bp using an ultrasound sonicator  and purified using Ampure beads . The purified cDNA was prepared with a GS DNA Library Preparation kit , amplified using the GS emPCR kit (Roche Applied Science) and then sequenced in a Roche 454 Genome Sequencer FLX machine.RNA was extracted using TRIzol . A Micropoly (A) Purist TM mRNA purification kit  was used to purify the mRNA under the manufacturer’s instructions. The mRNA from E value < 1 × 10−5. The functional annotation of all predicted proteins of M. enterolobii was performed with Blast2GO [The reads were assembled using Newbler 2.7  at the default setting. Genes and associated ORFs were predicted using GetORF from the EMBOSS tool kit and then BLASTed against the Swiss-Prot and TrEMBL databases at an Blast2GO . The GO Blast2GO  with theMeloidogyne enterolobii, Meloidogyne hapla, Meloidogyne incognita, Bursaphelenchus xylophilus, Globodera pallida and Caenorhabditis elegans by orthAgogue [http://www.genome.jp/tools/kaas/) with the BBH  method, we mapped the pathways of the three evolutionarily-closest nematodes, i.e., M. enterolobii, M. hapla and M. incognita, and compared the differences in KEGG Ontology (KO) numbers.Based on sequence similarity, orthologous families were constructed for predicted proteins of thAgogue  at the dthAgogue  for eachthAgogue . The phythAgogue  using thhttp://csbl.bmb.uga.edu/dbCAN/download/). Kinomes were identified using Kinannote [Meloidogyne enterolobii, Meloidogyne hapla, Meloidogyne incognita, Bursaphelenchus xylophilus, Globodera pallida and Caenorhabditis elegans [C. elegans. Putative effectors were predicted from the secretory proteins based on two conditions , as previously reported using SignaIP 4.1 [We compared the transcripts in different nematodes that putatively coded for CAZymes, kinases, neuropeptide genes and secretory proteins and those that were involved in the RNAi pathway and immune signaling. Genes encoding putative carbohydrate-active enzymes were identified using the hmmscan program in the HMMER 3 package  to searcinannote . The neu elegans ,57,58,59naIP 4.1  and TMHMnaIP 4.1 .A. tumefaciens GV3101. The verification method was modified from Wang et al. [Agrobacterium strain GV3101 harboring different constructs was grown in liquid LB medium with suitable antibiotics at 28 °C in a shaking incubator at 200 rpm. After 16 h incubation, the bacterial cells were collected by centrifugation at 5000× g for 5 min, washed with 10 mM MgCl2 three times and suspended in infiltration medium  to an OD600 of 0.4 and kept at room temperature for 1–3 h prior to infiltration into the four-week-old N. benthamiana leaves. The cultured A. tumefaciens cells carrying candidate genes and GFP were initially infiltrated into leaves of N. benthamiana. After 24 h, the same infiltration site was then challenged with A. tumefaciens cells carrying the BAX. The leaves were boiled six days later in 95% ethanol to clear the background for photographing. All experiments were repeated.Of the predicted 75 esophageal gland secretory proteins, 3 proteins with signal peptides were cloned. These putative proteins were amplified using the primers listed in g et al. . Briefly


Helicobacter pylori (H. pylori) which colonizes the stomach can cause a wide array of gastric disorders, including chronic gastritis, peptic ulcer, and gastric cancer. Recently, accumulating evidence has implicated H. pylori infection in extragastrointestinal diseases such as cardiovascular diseases, neurological disorders, and metabolic diseases. At the same time, many scholars have noted the relationship between H. pylori infection and non-alcoholic fatty liver disease (NAFLD). Despite the positive association between H. pylori and NAFLD reported in some researches, there are opposite perspectives denying their relationship. Due to high prevalence, unclear etiology and difficult treatment of NAFLD, confirming the pathogenicity of H. pylori infection in NAFLD will undoubtedly provide insights for novel treatment strategies for NAFLD. This paper will review the relationship between H. pylori infection and NAFLD and the possible pathogenic mechanisms. Helicobacter pylori (H. pylori) is a gram-negative microaerophilic bacterium that colonizes the stomach of humans. In 1983, Australian researchers Marshall and Warren  lymphoma, and gastric cancer. Recently, accumulating evidence implicates H. pylori infection existing in the patients with extragastrointestinal diseases, including idiopathic thrombocytopenic purpura, ischemic heart diseases, obesity, type 2 diabetes mellitus (T2DM)   has been detected in patients with various etiologies of chronic liver diseases (CLD), including hepatitis, liver fibrosis, and HCC  and H. pylori infection could independently predict NAFLD in logistic regression analysis. It is indicated that H. pylori infection may represent one more hit contributing to the pathogenesis of NAFLD . Besides, the total NAFLD activity score (NAS) and the grade of hepatocyte ballooning were higher in patients with H. pylori IgG seropositivity than in those without (p = 0.03). This study also found that H. pylori infection could independently predict NASH in logistic regression analysis (p = 0.003). And it further confirmed that H. pylori infection may represent a contributing factor to NAFLD  than in patients in the non-IR group . Multiple linear regression analysis showed that H. pylori infection was significantly correlated with the HOMA-IR . The authors therefore proposed that H. pylori infection may be an important independent risk factor for the development of IR  and log (HOMA-IR) (p = 0.007) could independently predict NAFLD. Moreover, the study also examined the levels of glucose, insulin, HOMA-IR, ALT, AST, and TNF-α, and found that all of these items were higher in H. pylori-IgG positive group compared to negative group. Therefore, the author put forward that H. pylori infection may contribute to the pathogenesis of NAFLD, mainly by adding to the first hit and this process may be achieved indirectly, or directly, though increasing IR  and HOMA-IR (p < 0.01) were significantly lower than before treatment, indicating that H. pylori eradication improved IR and may prevent the occurrence of MS, and NAFLD , tumor necrosis factor (TNF)-α, and interleukin (IL)-6 -1β . While, 6 weeks after eradication therapy, the two indicators were significantly decreased from the pretreatment level  that sensitized the liver to a second metabolic insult triggering a cascade of tissue damage (inflammation) resulting in fibrosis. Although, the etiology of NAFLD is maltifactorial and remains largely enigmatic, it is well-accepted that inflammation is a central component of NAFLD pathogenesis . H. pylori infection may influence leptin production  and hepatic insulin sensitivity , indicating that chronic H. pylori infection may influence the serum lipid profile  and higher in TG  or liver biopsy measuring liver-fat, demonstrated that a serum-lipid signature comprising a lipid triplet  could estimate the percentage of liver fat. The significant associations of specific lipid and polar metabolite concentrations with liver-fat content suggested that circulating molecular lipids may be predictive of liver fat , while HOMA-IR, TC, TG, LDL-C, and CRP levels in patients with successful eradication were significantly decreased compared to the pretreatment levels (P < 0.05). This study showed beneficial effects of H. pylori eradication on insulin resistance, atherogenic lipid abnormalities, and low-grade inflammation. These results suggest that H. pylori eradication may prevent metabolic syndrome including NAFLD  concentrations were significantly higher in the males with positive IgG antibody titers for F ≥ 2 patients, whereas Prevotella abundance was decreased, and Ruminococcus abundance was significantly higher in F ≥ 2 patients. By multivariate analysis, Bacteroides abundance was independently associated with NASH and Ruminococcus with F ≥ 2 fibrosis  and specific pathogen free (SPF) mice in the presence and absence of H. pylori. The results also indicated that ongoing crosstalk occurs between H. pylori and the normal gut microbiota, which is associated with metabolism and gut inflammation. Thus, some scholars speculate the mechanisms of the development of NAFLD caused by H. pylori related-gut microbiota dysbiosis are follows; H. pylori invasion into intestinal mucosa might increase gut permeability and gut microbiota disorder and then facilitate the passage of bacterial endotoxin (mainly LPS) via the portal vein to the liver, and promote inflammation response .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fusarium and Aspergillus) were isolated from the samples. Numerous Fusarium isolates were recovered and identified to the species level by comparison of translation elongation factor 1-α gene sequences to verified strains. Fusariumfujikuroi was the most abundant species recovered (239 isolates), followed by F. proliferatum (52), F. incarnatum-equiseti (14), F. oxysporum (7), F. concentricum (1), and F. solani (1). In vitro assays quantified fumonisin production for representative isolates via liquid chromatography-tandem mass spectrometry. Surprisingly, nearly all F. fujikuroi isolates produced fumonisins B1, B2, and B3 at levels comparable to both the F. proliferatum isolates and the positive control, Fusariumverticillioides. Such capacity for fumonisin production refutes the generally accepted notion that F.fujikuroi produces undetectable or low levels of fumonisins and provides evidence to reconsider this species as a mycotoxigenic threat to economically significant crops.Mycotoxins pose a challenge to a safe food supply worldwide, and their threat is expected to worsen with our changing climate. The need for diligence is exemplified by the discovery of fumonisin B2 in wine, which joins ochratoxin A as a mycotoxin of concern in the grape-wine chain. To elucidate the mycotoxin risk in southeastern American wine, grape samples were collected from vineyards during harvest in 2013 and potentially mycotoxigenic fungi ( Aspergillus species such as Aspergillus carbonarius and Aspergillus niger [A. niger [Fusarium verticillioides [The possibility exists that mycotoxins, toxins produced by fungi having adverse effects on humans and animals, can survive the winemaking process to contaminate bottled wine even after the fungi themselves are eliminated from the must. The mycotoxin ochratoxin A (OTA) was discovered in 1965 by van der Merwe et al. . In 1996us niger ,8, and pus niger . The souA. niger ,12. Thisllioides . FB1 is llioides .Fusarium species on winegrapes. One Slovakian survey revealed the presence of Fusarium proliferatum on grapes [F. proliferatum from both surface-sterilized and non-sterilized grape berries, indicating that the fungus is capable of being present not only on the exterior of the grapes, but also within the berries [F. proliferatum [Winegrapes are well established as a host for potentially mycotoxigenic black-spored aspergilli , but onln grapes . A secon berries . A surve berries  and lateiferatum .Fusarium species can produce the B series of fumonisins in significant concentrations, with F. verticillioides and F. proliferatum being the most notable. Despite having a fumonisin biosynthetic gene cluster (FUM) nearly identical to that of F. verticillioides and F. proliferatum [Fusarium fujikuroi is widely noted in the literature to produce low amounts, if any, of B-type fumonisins except in rare cases [F. fujikuroi that correlated with minor fumonisin production in all Japanese isolates studied. This species is best known as a pathogen of rice contributing to Bakanae disease, or the “foolish seedling disease,” caused by the gibberellin secondary metabolites produced by the fungus [Only a few iferatum , Fusariure cases ,23,24,25re cases  found a e fungus .Vitis vinifera grapes are Alabama, Georgia, North Carolina, Tennessee, Kentucky, and Virginia. Popular grape varieties are Cabernet franc, Cabernet Sauvignon, and Merlot, although other varieties such as Sangiovese, Malbec, and Tannat are also grown. The Southeast represents a challenging environment for winegrapes, due to both disease and weather. Pierce’s disease, vectored by the glassy-winged sharpshooter insect, is widespread, and a strict fungicide regimen is generally necessary to mitigate the effects of mildews and rots [Aspergillus and Fusarium spp. [A. carbonarius produces OTA more efficiently on damaged berries than on intact ones [Aspergillus and Fusarium species, the presence of mycotoxigenic fungi in the vineyards was evaluated in the interest of public health safety and to fill a knowledge gap since vineyards within this region have not been extensively surveyed for these fungi. Thus, the objectives of this work were to isolate potentially mycotoxigenic fungi from southeastern U.S. red V. vinifera winegrapes, to identify these fungi to the species level, and to determine if they produce toxins in vitro. To the best of our knowledge, this study is the first report of F. fujikuroi isolated from winegrapes and the first report of a population of F. fujikuroi producing significant levels of fumonisins comparable to F. verticillioides and F. proliferatum.The southeastern American states currently producing commercially available red and rots . Weatherium spp. . In factact ones . Since wAspergillus isolates were recovered from the winegrape samples, each one from a different vineyard. None of these isolates had the morphological characteristics of the main OTA producer, A. carbonarius, and thus were not further investigated for mycotoxin production. In contrast, a total of 314 Fusarium isolates were recovered from the winegrape samples using dichloran rose bengal chloramphenicol (DRBC) and 2-benzoxazolinone (BOA) selective media. Based on sequencing of the translation elongation factor 1-α (TEF) region and BLASTN searches of three nucleotide databases for species identification, F. fujikuroi was the most abundant species recovered , followed by F. proliferatum , F. incarnatum-equiseti , F. oxysporum , F. concentricum , and F. solani . F. fujikuroi isolates were recovered from every vineyard (n = 8) and every grape variety (n = 10) sampled (F. proliferatum was recovered from fewer vineyards (n = 3) and grape varieties (n = 4), and only from the states of North Carolina and Alabama. The importance of collecting more than one isolate from a single sample replicate was demonstrated, as multiple species were found in distinct replicates.A total of three black-spored  sampled , whereasF. fujikuroi or F. proliferatum, percent identity matrices were generated from ClustalW nucleotide alignments of winegrape isolates compared to multiple reference strains of each species. Identity of ≥97% was used to make species determinations, and F. fujikuroi was clearly distinguishable from F. proliferatum  produced fumonisins in the range of 1125–4653 ppm (µg/g) total fumonisins . Average total fumonisins for F. proliferatum isolates was 1922 ppm. All F. fujikuroi isolates (50/50) produced fumonisins with an average total fumonisins of 1997 ppm (range of 1.09–3921 ppm). The average total fumonisins produced by the positive control, F. verticillioides, was 1675 ppm. No F. oxysporum isolates tested produced fumonisins in this study. There were no correlations with fumonisin production and state, vineyard, grape variety, or media isolation type (DRBC or BOA). Furthermore, isolates with the TEF SNP corresponding with non-production according to Suga et al. [A representative group of ed assay . Nearly a et al.  did prodF. proliferatum is a well-known fumonisin producer, the general consensus in the Fusarium research community has been that F. fujikuroi produces little or no fumonisins. Surprisingly, all but two F. fujikuroi winegrape isolates tested with the rice assay produced significant levels of total fumonisins . The levels of fumonisins produced by the F. fujikuroi winegrape isolates are comparable to the levels produced by the F. proliferatum winegrape isolates and to the F. verticillioides positive controls. This is the first report of a large population of F. fujikuroi isolates with the ability to produce high amounts of fumonisins.Whereas F. fujikuroi population, further testing with verified F. fujikuroi and F. proliferatum isolates from the NRRL Culture Collection was conducted to determine if the rice assay method of in vitro fumonisin production, less common than the alternative cracked corn assay, was perhaps the cause of such production. One of these verified F. fujikuroi isolates, NRRL 31883, was isolated from a grape host in NY, USA. This one isolate, but none of the other NRRL F. fujikuroi reference strains we tested, produced high amounts of fumonisins similar to the southeastern winegrape isolates (F. fujikuroi NRRL reference strains produced undetectable to low amounts of fumonisins (<5 ppm). The species identity of all NRRL isolates tested was verified through sequencing of the TEF region as described earlier. Therefore, the rice assay testing method was not the cause of the high fumonisin production by F. fujikuroi winegrape isolates, which represent a truly unique population for this species. Differential regulation of the fumonisin biosynthetic gene cluster within this population may be the basis for the enhanced production of the mycotoxin. The grape host or the associated vineyard pest management practices may create an environment conducive to activation of the biosynthetic genes, perhaps related to nitrogen availability, which was shown to affect expression of the gene cluster [Due to the rarity of a high fumonisin-producing isolates . All othFusarium isolates is difficult due to the great diversity amongst in vitro fumonisin testing assays. Methods include a variety of growth media , incubation periods, toxin extraction methods, and means of reporting the toxin levels . Therefore, a more accurate and comprehensive approach is to obtain standard strains from a well-curated repository, verify their identity through sequencing, and test these strains in-house alongside collected experimental isolates. To this end, 35 of the winegrape Fusarium isolates were deposited into the permanent ARS Culture Collection. The NRRL numbers of these winegrape isolates are listed in F. fujikuroi secondary metabolite profile and its regulation. Given the wide host range of Fusarium species, this newly discovered high fumonisin-producing population of F. fujikuroi in vineyards in the Southeast raises concern for fumonisin contamination in other crops as well. Closer attention should be paid to the mycotoxigenic potential of F. fujikuroi worldwide.Comparing published results of fumonisin production by V. vinifera winegrapes were obtained from eight commercial vineyards in the southeastern U.S.  during harvest in 2013. This harvest represented a vintage with both late season rains and high disease pressure. Grape varieties sampled included Cabernet Sauvignon, Cabernet franc, Malbec, Merlot, Petit Verdot, Pinot noir, Sangiovese, Tannat, and Touriga Nacional. One additional vineyard, planted with the non-vinifera variety of Norton, was also sampled. Up to three different grape varieties were sampled per vineyard. The majority of grape varieties sampled in each vineyard consisted of six clusters in total, each cluster randomly selected from a different non-adjacent grapevine on an individual vineyard row, forming up to three sample replicates of two clusters each. In total, 56 sample replicates were collected and processed. All clusters were brought to the laboratory on ice in re-sealable plastic bags and prepared for further analysis within 12 h from being cut from the vine. Stems were removed from clusters and grapes were homogenized  into a must slurry. The grapes were not surface sterilized in order to recover greater mycoflora diversity and to allow recovery of ochratoxigenic A. carbonarius or other black-spored Aspergillus species that typically sporulate on the grape berry exterior [Samples of red, exterior . A steri® 20 . Undiluted and serially diluted must samples were plated in duplicate onto two types of selective media: DRBC agar [Aspergillus and cottony-white Fusarium colonies were transferred onto potato dextrose agar (PDA) and incubated at 27 °C or 30 °C for purification and further identification. As Fusarium spp. were abundant, a maximum of 10 Fusarium colonies were isolated from each sample replicate and media type combination. Pure cultures of either Aspergillus or Fusarium were single-spore isolated via the dilution plating method on PDA [® 20 at −80 °C for further analysis.Thawed grape must sample replicates (1–3 per variety per vineyard) were serially diluted in sterile reverse osmosis (RO) water with 0.10% TweenRBC agar  and BOA RBC agar . Negativd on PDA . IsolateFusarium isolates were grown on PDA for 2–4 days at 27 °C and genomic DNA was extracted using the previously described thermolysis procedure [® 2X Master Mix with Standard Buffer  were used to PCR-amplify a portion of the TEF gene for species identity [® version 5.4.1 . The TEF sequences were compared with verified strains in the Fusarium MLST  [Fusarium-ID [F. fujikuroi and F. proliferatum reference sequences from the databases using ClustalW implemented in Geneious analysis software  [F. fujikuroi or F. proliferatum. Detailed phylogenetic characterization of these isolates will be published separately.Single-spored rocedure . The prirocedure , along widentity . PCR conerlands) , Fusariuarium-ID , and NCBarium-ID  databaseZealand) . Percentn = 73) of the total recovered potentially mycotoxigenic Fusarium winegrape isolates (n = 291) was selected for in vitro fumonisin analysis. This representative subset included both F. proliferatum (n = 22) and F. fujikuroi (n = 51) isolates from different states, vineyards, grape varieties, sample replicates, and media isolation types. Additionally, to test the Japanese isolate hypothesis that a SNP indicates fumonisin production or lack thereof, representative isolates with both producing and non-producing SNP types were included [F. oxysporum winegrape isolates were assayed as well, though fumonisin B production is extremely rare for this species. Isolates of F. incarnatum-equiseti and F. concentricum, also from winegrapes, were included as negative controls. Additionally, strains of F. verticillioides, F. proliferatum, and F. fujikuroi were obtained from the USDA-ARS Culture Collection (NRRL) for inclusion in the fumonisin analysis  [F. verticillioides strains  were used as positive controls, and one winegrape F. fujikuroi isolate (SLB 6326) that produced fumonisins in the first assay was used as an internal assay control for all subsequent assays. Therefore, each experimental run included testing potential fumonisin-producing Fusarium isolates from winegrapes and/or verified strains from the NRRL Culture Collection, a winegrape isolate negative control, a sterile water negative control, a F. verticillioides positive control, and an internal assay control. All isolates and strains analyzed, including controls, were tested in triplicate in each experiment. Assays were repeated on different days, and every winegrape isolate and verified strain was tested for fumonisin production in at least two independent experiments.Following species identification, a subset  . Fumonis® 20) was used as inoculum for 50 mL of sterile potato dextrose broth (PDB) in a 125 mL Erlenmeyer shaking flask. Culture flasks were incubated for 3 days (±1 h) at 27 °C with shaking at 200 rpm in darkness. To a 20 mL glass scintillation vial, 3 g (±0.004 g) of rice  and 3 mL of reverse osmosis water were added. Vials were topped with foam plugs, autoclaved , and cooled to room temperature just prior to use. In triplicate, 250 µL of high inoculum spore suspension (~107 spores per mL) from incubated PDB flasks (or sterile water control) were added to each rice vial replicate. Inoculated rice vials were incubated for 7 days (±1 h) at 27 °C in darkness.For fumonisin analysis, fungal isolates were grown on sterile rice using an assay modified from . For eac®, speed 6.5, Stovall Life Science, Inc., Greensboro, NC, USA) to extract any fumonisins. The resulting extraction solution was centrifuge-filtered , diluted , and analyzed via LC-MS/MS (Thermo LTQ XL). All analyses were multiple reaction monitoring (MRM) performed in positive ion mode, and the instrument was tuned using FB1 . An Imtakt Cadenza CW-C18 column , maintained at 30 °C, was used for separations. Solvent A was distilled water with 3% acetonitrile plus 0.1% formic acid, and solvent B was acetonitrile with 3% distilled water plus 0.1% formic acid. The solvent program began at 30:70 A:B, and increased linearly to 100% B over 9 min, held at 100% B for 2 min, and then returned to starting conditions. Samples were run along with a blank control and standard solutions of fumonisins B1, B2, and B3 . Analytes were identified by retention time, as compared to standards, and fragmentation patterns : distilled water extraction solution containing 5% formic acid  was added to each vial. Samples were vigorously shaken and frozen to −20 °C for at least 10 h. Prior to LC-MS/MS analysis for fumonisins, rice cultures were broken apart and vials were rocked for 3 h (The Belly Dancerpatterns . Levels