Aap Gene Research Articles

Sequencing a CC239-MRSA-III with a novel composite SCC mec element from Kuwait.

Staphylococcus aureus CC239-MRSA-III is an ancient pandemic strain of hospital-associated, methicillin-resistant S. aureus that spread globally for decades and that still can be found in some parts of the world. In Kuwait, microarray-based surveillance identified from 2019 to 2022 a series of isolates of a hitherto unknown variant of this strain that carried a second set of recombinase genes, ccrA/B-2. To elucidate the structure of its SCCmec element, two isolates were subjected to nanopore sequencing. This revealed, in addition to ccrA/B-2, several SCC-associated genes including speG (spermidine N acetyltransferase) and a gene encoding a large "E-domain containing protein" (dubbed as edcP-SCC). This gene contained three regions consisting of multiple repeating units. In terms of sequence and structure it was similar but not identical to the biofilm-related aap gene from S. epidermidis. A review of published sequences identified edcP-SCC in eighteen genome sequences of S. aureus, S. epidermidis and S. capitis, and frequently it appears in a similar cluster of genes as in the strains sequenced herein. Isolates also carried a prophage with the adhesion factor sasX/sesI and aminoglycoside resistance genes. This is consistent with an affiliation to the "South-East Asian" Clade of CC239. The emergence of edcP-SCC and sasX-positive CC239 strain shows that, against a global trend towards community-associated MRSA, the ancient pandemic CC239 hospital strain still continues to evolve and to cause outbreaks.

Biofilm formation of two genetically diverse Staphylococcus aureus isolates under beta-lactam antibiotics.

Our aim was to evaluate the biofilm formation of 2 genetically diverse Staphylococcus aureus isolates, 10379 and 121940, under different concentrations of beta-lactam antibiotics on biomass content and biofilm viability. Biofilm formation and methicillin resistance genes were tested using PCR and multiplex PCR. PCR was combined with bioinformatics analysis to detect multilocal sequence typing (MLST) and SCCmec types, to study the genetical correlation between the tested strains. Then, the crystal violet (CV) test and XTT were used to detect biomass content and biofilm activity. Antibiotic susceptibility was tested using a broth dilution method. According to their specific MIC, different concentrations of beta-lactam antibiotics were used to study its effect on biomass content and biofilm viability. Strain 10379 carried the icaD, icaBC, and MRSA genes, not the icaA, atl, app, and agr genes, and MLST and SCCmec typing was ST45 and IV, respectively. Strain 121940 carried the icaA, icaD, icaBC, atl, and agr genes, not the aap gene, and MLST and SCCmec typed as ST546 and IV, respectively. This suggested that strains 10379 and 121940 were genotypically very different. Two S. aureus isolates, 10379 and 121940, showed resistance to beta-lactam antibiotics, penicillin, ampicillin, meropenem, streptomycin and kanamycin, some of which promoted the formation of biofilm and biofilm viability at low concentrations. Despite the large differences in the genetic background of S. aureus 10379 and 121940, some sub-inhibitory concentrations of beta-lactam antibiotics are able to promote biomass and biofilm viability of both two isolates.

Molecular Detection of Virulence Associated Genes in Coagulase Negative Staphylococci Isolated from Blood Culture.

Coagulase-negative staphylococci (CoNS) are one of the most important causes of infections. Unlike Staphylococcus aureus, less is known about their pathogenic mechanisms. In the present study, we aimed to evaluate the presence of virulence genes among 98 CoNS isolated from blood cultures of inpatients. The isolates were identified by MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). PCR was performed to detect 29 virulence factors using specific primers for icaA, icaB, icaC, icaD, icaADB, aap, fbe, aae, sesI, atIE, hla, hlb, hld, gehC, gehD, sea, seb, sec, sed, see, seg, seh, sei, tst, eta, etb, etd, etx, and pvl genes. The VITEK2 system (bio-Merieux, France) and the BD Phoenix™ System (Becton Dickinson, USA) were used for antimicrobial susceptibility testing. Staphylococcus epidermidis was found to be the most virulent CoNS species. All isolates were negative for eta, etb, etd, sea, seb, sed, see, seg, sei, and pvl virulence genes. We detected up to 15 virulence genes in a single isolate. The most common gene was icaC (73.5%), followed by icaA (57.1%), icaD (56.1%), aap (55.1%), aae (52.0%), sesl (51.0%), gehC (50.0%), hld (50.0%), hlb (49.0%), fbe (44.9%), atIE (37.8%), icaADB (37.8%), gehD (34.7%), icaB (31.6%), hla (30.6%), etx (2.0%), sec (1.0%), seh (1.0%), and tst (1.0%). We determined high rates of genes encoding biofilm formation. Only four isolates did not possess either the ica operon or aap gene. Although we found low rates of toxin-related genes, our data indicates that apart from biofilm formation, the CoNS isolates could express various virulence genes similar to those of Staphylococcus aureus.

Detection of the agr System and Resistance to Antimicrobials in Biofilm-Producing S. epidermidis.

The ability of Staphylococcus epidermidis to produce virulence factors, such as biofilm, added to its increased resistance to antimicrobials can cause infections that are difficult to treat. Many staphylococcal virulence factors are under the control of the accessory gene regulator (agr). The objective of this study was to establish the agr locus and susceptibility of biofilm-producing S. epidermidis specimens to antimicrobial agents, through PCR reactions, reverse transcription polymerase chain reaction (RT-PCR), and the determination of minimum inhibitory concentration (MIC), and to analyze the clonal profile of 300 strains isolated from blood culture specimens from inpatients at a University Hospital in Brazil, over a 20-year period by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques. The ica operon expression was shown in 83.6% strains, bhp gene in 11.5%, and aap gene in 32.8%. Oxacillin resistance was detected in 90.1%, while 4.9% showed tigecycline resistance, and intermediate resistance to quinupristin/dalfopristin was identified in 0.4%. Clonal profile determination showed 11 clusters, with the ST2 type determined as the major cluster. The S. epidermidis biofilm producer demonstrated a predominance of agr I locus, oxacillin resistance, and SCCmec III as well as the potential dissemination of pathogenic clones in hospital settings over long periods.

Expression study of an Amino Acid Permease-like gene in Phaseolus vulgaris L.

Amino acid permease-like (AAP-like) gene plays a critical role in absorbing amino acids through roots in plants. A number of studies have been done on amino acids uptake in plants but till date there is no report about the expression of AAP gene in Phaseolus under field allied condition. The aim of this study is to measure the expression of AAP-like gene on alanine, glycine and proline amino acid uptake capacity in Phaseolus vulgaris at field relevant concentrations. Amongst three amino acids, a drastic significant increase of 63.15 fold in expression of AAP-like gene is observed in 50 µM alanine at 2 hr. At 50 µM of proline and 25 µM of alanine, AAP-like gene expression also shows high expression of 43.71 fold at 2 hr and 42.50 fold at 1 hr respectively. This study elucidated the dose dependent relationship of glycine, alanine and proline with the expression of AAP-like gene in amino acid transport in natural conditions in roots of P. vulgaris. Additionally, this research is also useful in identification of plants needing less surplus nitrogen additions and helpful in optimizing fertilizers by tailoring AAP gene expression to match plant uptake capacities in agriculture.

Evaluation of Antibacterial Activity of Zataria multiflora Against the Expression of icaADB and aap Gene and Biofilm Formation in Staphylococcus epidermidis

Background: This study aimed at evaluating the effect of the extract and essential oil of Zataria multiflora on reducing phenotypes and genotypes of biofilm formation in clinical isolates of Staphylococcus epidermidis. Methods: The ability to produce biofilm was evaluated by microtiter plate (MtP) for phenotype and through the presence of icaADB and aap by PCR for genotype among the 153 clinical isolates of Staphylococcus epidermidis. The impact of ethanol extract and essential oil of Zataria multiflora were measured on biofilm formation in phenotypic evaluation based on MtP and on the expression of ica operon and aap by real time-PCR. Results: The results showed that 71.2% of isolates were able to produce biofilms. The PCR results showed that 52.2% and 88.9% of the isolates had icaABD and aap, respectively. In addition, the relative mRNA expression of icaA, icaD, and aap genes were significantly reduced compared to the negative control after treating the Staphylococcus epidermidis RP62A with subMIC concentration of essential oil and extract (P < 0.001). Conclusions: Given the significant inhibitory effect of the extract and essential oil of Zataria multiflora on biofilm formation, it seems that these substances are good options for studies related to controlling biofilm formation in Staphylococcus epidermidis

Biofilm-associated accumulation-associated protein (Aap): A contributing factor to the predominant growth of Staphylococcus epidermidis in platelet concentrates.

Staphylococcus epidermidis is a predominant contaminant of platelet concentrates (PCs), outcompeting other skin flora bacteria such as Staphylococcus capitis. The accumulation-associated protein (Aap), encoded by the aap gene, is involved in formation of bacterial aggregates (biofilms) in S.epidermidis and is absent in S.capitis. In this study, the role of S.epidermidis aap in enhancing biofilm formation and conferring an advantageous growth in PCs was investigated. Biofilm formation assays of S.epidermidis 1457, S.epidermidis 1457∆aap, S.capitis 517 and S.capitis 517 carrying S.epidermidis aap (S.capitis 517/pRBaap) were performed in glucose-supplemented trypticase soy broth (TSBg) and PCs. Additionally, competition assays with paired cultures (1:1 ratio) of S.epidermidis and S.capitis strains were seeded in PCs, followed by determination of viable counts of each organism at the end of PC storage. Staphylococcus epidermidis aap had no effect on biofilm formation in TSBg. By contrast in PCs, S.epidermidis 1457 showed higher biofilm formation than S.epidermidis 1457∆aap (P<0·05). Biofilm formation was also enhanced in S.capitis 517/pRBaap compared to S.capitis 517 (P=0·054). Competition assays showed that S.epidermidis 1457 outcompeted S.capitis 517, and importantly, S.capitis 517/pRBaap outcompeted S.capitis 517 and S.epidermidis 1457∆aap. This study demonstrated that S.epidermidis aap plays a role in biofilm formation in PCs conferring an advantageous proliferation to skin flora bacteria in this milieu. The molecular mechanisms of action of Aap merit further investigation.

Genetic and Phenotypic Traits of Staphylococcus Epidermidis Strains Causing Postcataract Endophthalmitis Compared to Commensal Conjunctival Flora

The aim of this study was to compare the virulence and antibiotic resistance traits of Staphylococcus epidermidis strains causing acute postcataract endophthalmitis to those isolated from the conjunctiva of uninfected control patients. Case-control study. We isolated an S epidermidis strain from each of the 22 endophthalmitis patients, and from 43 of the 72 controls. Species identification was confirmed using both Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and tuf gene amplification and sequencing. Antibiotic susceptibilities were evaluated using the AST-P631 card and the Vitek II automated system. The S epidermidis strains were tested for the presence of 7 virulence genes (icaA, icaB, icaC, icaD, atlE, aap, and capA), the insertion sequence IS256, and the mecA gene. The S epidermidis strains from the endophthalmitis patients displayed higher prevalence rates for aap, atlE, and mecA gene carriage compared to those of the control group (77% vs 42%, P= .007; 100% vs 79%, P= .02; and 54% vs 11%, P < .001, respectively). They also harbored the combination of the mecA and icaA genes more frequently compared to the control group (13% vs 2%, P= .01). They were significantly more resistant than control strains to methicillin, fluoroquinolones, and the aminoglycosides. A higher capacity of adhesion to the intraocular lens and formation of biofilms as well as greater resistance to antibiotics were found in S epidermidis strains causing postcataract endophthalmitis. The usefulness of such virulence and antibiotic resistance markers warrants further evaluation for prevention, treatment, and prognostic evaluation of S epidermidis endophthalmitis.

High prevalence of diarrheagenic Escherichia coli carrying toxin-encoding genes isolated from children and adults in southeastern Brazil

BackgroundDiarrheagenic Escherichia coli (DEC) are important bacterial causes of childhood diarrhea in Brazil, but its impact in adults is unknown. This study aimed at investigating DEC among children and adults living in endemic areas.MethodsA total of 327 stools specimens were collected from children (n = 141) and adults (n = 186) with diarrhea attending health centers. Diarrheagenic E. coli (DEC) were identified by their virulence genes (multiplex polymerase chain reaction) and HEp-2 cell adherence patterns.ResultsDEC were detected in 56 (40%) children and 74 (39%) adults; enteroaggregative E. coli (EAEC) (23%) was the most prevalent pathotype, followed by diffusely adherent E. coli (DAEC) (13%), and occurred at similar frequencies in both diarrheal groups. Atypical enteropathogenic E. coli (aEPEC) strains were recovered more frequently from children (6%) than from adults (1%). Twenty-six percent of the EAEC were classified as typical EAEC possessing aggR gene, and carried the aap gene. EAEC strains carrying aggR-aap-aatA genes were significantly more frequent among children than adults (p < 0.05). DAEC strains possessing Afa/Dr. genes were detected from children (10%) and adults (6%). EAEC and DAEC strains harboring genes for the EAST1 (astA), Pet, Pic, and Sat toxins were common in both diarrheal groups. The astA and the porcine AE/associated adhesin (paa) genes were found in most of aEPEC strains. High levels of resistance to antimicrobial drugs were found among DAEC and aEPEC isolates.ConclusionThe results show a high proportion of EAEC and DAEC carrying toxin-encoding genes among adults with diarrhea.

Methicillin resistance and virulence genes in invasive and nasal Staphylococcus epidermidis isolates from neonates

BackgroundStaphylococcus epidermidis is an opportunistic pathogen involved in hospital-acquired infections, particularly in those related to medical devices. This study characterized 50 genetically unrelated S. epidermidis isolates from bloodstream infections (BSIs, n = 31) and nares (n = 19) of neonates in relation to staphylococcal chromosomal cassette mec (SCCmec) type, biofilm production and associated genes, and the arginine catabolic mobile elements (ACME), in order to detect virulence factors that could discriminate a potential invasiveness isolate or predict an increasing pathogenicity.ResultsIsolates from both groups showed no difference for biofilm production and ACME genes detection. However, BSI isolates harbored more frequently the sdrF and sesI genes (p < 0.05), whereas biofilm producer isolates were associated with presence of the aap gene. The sdrF gene was also significantly more in the biofilm producer isolates from BSI. The SCCmec type IV and the ccr2 complex were related to BSI isolates (p < 0.05), while 83% of the nasal isolates were non-typeable for the SCCmec elements, with the mec complex and ccr undetectable as the most frequent profile.ConclusionsDespite the great clonal diversity displayed by S. epidermidis isolates from neonates, BSI isolates harbored more frequently the sdrF and sesI adhesin genes, while nasal isolates were very variable in SCCmec composition. These aspects could be advantageous to improve colonization in the host increasing its pathogenicity.

Evaluation of Prevalence, Homology and Immunogenicity of Dispersin among Enteroaggregative Escherichia coli Isolates from Iran.

Background:Diarrhea, caused by enteroaggregative Escherichia coli (EAEC), is an important infection leading toillness and death. Numerous virulent factors have been described in EAEC. However, their prevalence was highly variable among EAECs of distinct geographic locations. Studies have shown that dispersin (antiaggregation protein, aap) is one of the important and abundant virulent factors in EAEC. In this study, we aimed to determine the presence, conservation, and immunogenicity of aap gene in EAEC isolated from Iranian patients.Methods:PCR amplification of aap gene in the EAEC isolates was performed, and the aap gene was cloned in pBAD-gIIIA vector. The sequence of aap gene was analyzed using the ExPASy and BLAST tools. The expression of aap gene was performed in E. coli Top10, and expression confirmation was carried out by SDS-PAGE and Western-blot techniques. Rabbits were immunized with purified dispersin protein emulsified with Freund’s adjuvant. Sera were collected and examined for antibody response. Finally, in vitro efficacy of dispersin and anti-dispersin was evaluated.Results:The results of PCR showed the presence of aap gene in all of the EAEC isolates with significant homology. Finally, the significant difference between the levels of IgG response in dispersin-injected rabbits and control group was observed.Conclusion:Our results were in accordance with other studies that reported the presence of dispersin in the EAEC isolates with high conservation and immunogenicity. Hence, dispersin could be a promising candidate for any probable prevention against EAEC infections.

302. Molecular AAV Capsid Evolution Is Not Primarily Restricted by Inadvertent Shuffling of the Assembly-Activating Protein AAP

A 2010 report that Adeno-associated virus 2 (AAV2) encodes an additional protein in the second open reading frame of the cap(sid) gene significantly changed our perception of the biology of this virus. This so-called assembly-activating protein or AAP appears to interact with the major viral capsid protein 3 (VP3) and to chaperone its assembly into virus-like particles, via mechanisms that remain unknown. Moreover, it was shown that deliberate mutation of the AAP gene diminishes AAV particle production.As the AAP open reading frame fully overlaps with that of the AAV capsid proteins VP1/VP2 (and partially VP3), it is likely that the AAP gene is inadvertently disrupted or mutated during molecular AAV evolution protocols that involve capsid gene shuffling (homology-directed recombination of cap gene fragments). To study this possibility comprehensively and to provide solutions, if needed, we first investigated the role of AAP for 9 further AAV serotypes other than AAV2. Therefore, we knocked out AAP expression in serotypes 1, 3-9 and rh10 while leaving the rep and cap genes intact. Indeed, all mutants were markedly impaired in particle production, confirming that functional AAP is critically required for at least 10 different AAV serotypes.Notably, all vectors were fully restored upon co-delivery of the corresponding AAP via an extra plasmid during particle production. Furthermore, when testing the cross-reactivity of all AAPs with the AAV2-AAP mutant (AAV2mut), we observed a broad interchangeability except for AAP4 and 5 which predominantly rescued their cognate serotype.These results were confirmed independently in a baculovirus AAV2 vector production system where we also observed a dramatic reduction in particle yields upon AAP mutation. Interestingly, in both, mammalian and insect cells, we consistently noted that absence of AAP reduced the steady-state levels of VP proteins, suggesting a similar biological function of AAP in the two heterologous cellular systems. As we could rule out an effect on the transcriptional level, we postulate that AAP-mediated chaperoning of AAV virion assembly indirectly stabilizes VP proteins in cells of different species.To explicitly dissect the role of AAP in the context of molecular AAV evolution, we produced five different AAV capsid libraries with increasing complexities, subcloned 46 randomly selected shuffled AAPs from these and tested them with the AAV-AAP mutants. Out of these 46 shuffled AAPs, 37 could rescue AAV2mut, and two more efficiently complemented an AAV5-AAP mutant. In total, 84.8 % of all tested AAPs were functional.Of note, the highest proportion of defect AAPs was found in libraries that contained the diverse capsids of AAV4 and 5. We thus shuffled serotypes 2, 4, 5, 8 and 9 and produced viral libraries with or without a cocktail of excess AAP from all five serotypes. Strikingly, albeit up to 40% of all AAP genes were predicted to be non-functional as a result of inadvertent shuffling, AAP trans-complementation had no significant effect on particle yields.Collectively, our data suggest that albeit AAP is disrupted to some extent during AAV capsid gene shuffling, this is likely not a major restriction for the efficiency and success of this technology.

Detección de genes de virulencia del patotipo enteroagregativo en cepas de Escherichia coli aisladas de fuentes de agua subterránea de la provincia del Chaco, Argentina

Groundwater is an important source of drinking water for many communities in Northern Argentina; particularly, in the province of Chaco, where about 14% of households use this natural resource. Enteroaggregative Escherichia coli is an emerging pathogen whose global importance in public health has increased in recent years. Despite the significant risk of disease linked to contaminated water exposure, the prevalence of E. coli pathotypes in aquatic environments is still not so well defined. The aim of the present study was to detect the presence of typical enteroaggregative E. coli through the recognition of its virulence factors aap, AA probe and aggR by molecular techniques. A total of 93 water samples from different small communities of Chaco were analyzed. E. coli was identified in 36 (38.7%) of the tested samples. Six strains isolated from different samples harbored the studied genes. Of these 6 isolates, 3 carried the aap gene, 2 the AA probe and the last one the combination of aap/aggR genes. The prevalence of E. coli isolates harboring enteroaggregative virulence genes in groundwater sources was 6.4%. This work represents the first contribution to the study of the presence and distribution of virulence genes of EAEC in groundwater sources in this region of Argentina.

Role of the two-component regulatory system arlRS in ica operon and aap positive but non-biofilm-forming Staphylococcus epidermidis isolates from hospitalized patients

The ica operon and aap gene are important factors for Staphylococcus epidermidis biofilm formation. However, we found 15 out of 101 S. epidermidis strains isolated from patients had both the ica operon and the aap gene in the genome but could not form biofilms (ica+aap+/BF− isolates). Compared with standard strain RP62A, the 15 ica+aap+/BF− isolates had similar growth curves and initial attachment abilities, but had much lower apparent transcription levels of the icaA gene and significantly less production of polysaccharide intercellular adhesion (PIA). Furthermore, the expression of accumulation-associated protein in ica+aap+/BF− isolates was much weaker than in RP62A. The mRNA levels of icaADBC transcription-related regulatory genes, including icaR, sarA, rsbU, srrA, arlRS and luxS, were measured in the 15 ica+aap+/BF− clinical isolates. The mRNA levels of arlR and rsbU in all of the ica+aap+/BF− isolates were lower than in RP62A at 4 h. At 10 h, 14/15 of the isolates showed lower mRNA levels of arlR and rsbU than shown by RP62A. However, expression of sarA, luxS, srrA and icaR varied in different ica+aap+/BF− isolates.To further investigate the role of arlRS in biofilm formation, we analyzed icaA, sarA and rsbU transcription, PIA synthesis, Aap expression and biofilm formation in an arlRS deletion mutant of S. epidermidis strain 1457 and all were much less than in the wild type strain. This is consistent with the hypothesis that ArlRS may play an important role in regulating biofilm formation by the ica+aap+/BF−S. epidermidis clinical isolates and operate via both ica-dependent and Aap-dependent pathways.

Coagulase-negative staphylococcal bloodstream and prosthetic-device-associated infections: the role of biofilm formation and distribution of adhesin and toxin genes.

Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and Staphylococcus haemolyticus, have emerged as opportunistic pathogens in immunocompromised patients and those with indwelling medical devices. In this study, CNS recovered from patients with bloodstream infections (BSIs) or prosthetic-device-associated infections (PDAIs) were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, and carriage of adhesin and toxin genes. A total of 226 CNS isolates (168 S. epidermidis and 58 S. haemolyticus) recovered from hospital inpatients with BSIs (100 isolates) or PDAIs (126 isolates) were tested for biofilm formation, antimicrobial susceptibility, and mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, sec) genes. The selected CNS were classified into pulsotypes by PFGE and assigned to sequence types by multilocus sequence typing. In total, 106/226 isolates (46.9%) produced biofilm, whereas 150 (66.4%) carried the ica operon. Most isolates carried mecA and were multidrug resistant (90.7%). CNS recovered from BSIs were significantly more likely to produce biofilm (P=0.003), be resistant to antimicrobials and carry mecA (P<0.001), as compared with isolates derived from PDAIs. CNS from PDAIs were more likely to carry the aap and bap genes (P=0.006 and P=0.045, respectively). No significant differences in the carriage of toxin genes were identified (P>0.05). Although PFGE revealed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by multilocus sequence typing revealed three major clones (ST2, ST5 and ST16). A clonal relationship was found with respect to antimicrobial susceptibility and ica and aap gene carriage, reinforcing the premise of clonal expansion in hospital settings. The results of this study suggest that the pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAIs are related to the adhesion capabilities of S. epidermidis and S. haemolyticus strains.

Accumulation-associated protein gene and TGF-beta 1 affects the formation of lung cancer-related biological material Staphylococcus epidermidis biofilm

背景与目的研究聚集相关蛋白（accumulation-associated protein, Aap）基因及转化生长因子β1（TGF-β1）对肺癌相关生物材料表皮葡萄球菌（SE）生物膜形成的影响。方法种属鉴定分离临床肺癌患者植入材料感染表皮葡萄球菌株，PCR法检测生物膜形成相关基因Aap，检测表皮葡萄球菌Aap基因株生物形成能力。密度梯度法提取非小细胞肺癌患者外周血单个核细胞，与人肺腺癌细胞A549在不同浓度（10 ng/mL, 20 ng/mL, 40 ng/mL）TGF-β1共培养30 h后，取上清液分别加入SE Aap+株、SE Aap-株下与医用硅橡胶培养30 h，半定量粘附试验测各组细菌生物膜形成的情况，扫描电镜观察材料表面生物膜微观情况。结果Aap基因的与表皮葡萄球菌生物膜的形成密切相关（P < 0.01）。在（10 ng/mL, 20 ng/mL, 40 ng/mL）TGF-β1浓度组医用硅橡胶表面SE Aap+生物膜的厚度大于空白组（P < 0.01）。SE Aap+株在TGF-β1浓度组间生物膜的厚度无明显差异（P > 0.05）。在不同浓度TGF-β1因子刺激下SE Aap-株均不能形成明显细菌生物膜。结论在肺癌患者植入材料引起感染中表皮葡萄球菌Aap基因表达阳性株较易形成细菌生物膜，TGF-β1对SE Aap阳性形成细菌生物膜有一定促进作用。

Staphylococcus epidermidis with the icaA− /icaD− /IS256 − genotype and protein or protein/extracellular-DNA biofilm is frequent in ocular infections

In ocular infections (OIs) caused by Staphylococcus epidermidis, biofilms composed mainly of poly-N-acetylglucosamine (PNAG) have been widely studied, but PNAG-independent biofilms have not. Therefore, we searched for a relationship between the ica operon (involved in PNAG-biofilm) and the biochemical composition of biofilms in isolates from OI. Isolates from OI (n = 62), from healthy conjunctiva (HC; n = 45) and from healthy skin (HS; n = 53), were used to detect icaA and icaD genes, and the insertion sequence 256 (IS256) using PCR. The compositions of the biofilms were determined by treatment with NaIO₄, proteinase K and DNase I. Multilocus sequence typing (MLST) was performed to characterize the isolates, and the expression of aap and embp genes was determined by real-time qPCR. A strong relationship between the icaA(-)/icaD(-)/IS256(-) genotype and protein- or protein/extracellular DNA (eDNA)-biofilm composition was found in the isolates from OI (53.6%), whereas the icaA(+)/icaD(+)/IS256(-) genotype and carbohydrate-biofilm was most prevalent in isolates from HC (25%) and HS (25%). Isolates with an icaA(-)/icaD(-)/IS256(-) genotype and protein-biofilm phenotype were predominantly of the ST2 lineage, while carbohydrate-biofilm-producing strains were mainly of the ST9 lineage. The protein-biofilm-producing strains had higher expression levels of aap gene than carbohydrate-biofilm-producing strains; while embp gene did not have the same pattern of expression. These results suggest that S. epidermidis strains with icaA(-)/icaD(-)/IS256(-) genotype and protein- or protein/eDNA-biofilms have a stronger ability to establish in the eye than S. epidermidis strains with icaA(+)/icaD(+)/IS256(-) genotype and PNAG-biofilms.

The AAP gene family for amino acid permeases contributes to development of the cyst nematode Heterodera schachtii in roots of Arabidopsis

The beet cyst nematode Heterodera schachtii is able to infect Arabidopsis plants and induce feeding sites in the root. These syncytia are the only source of nutrients for the nematodes throughout their life and are a nutrient sink for the host plant. We have studied here the role of amino acid transporters for nematode development. Arabidopsis contains a large number of different amino acid transporters in several gene families but those of the AAP family were found to be especially expressed in syncytia. Arabidopsis contains 8 AAP genes and they were all strongly expressed in syncytia with the exception of AAP5 and AAP7, which were slightly downregulated. We used promoter::GUS lines and in situ RT-PCR to confirm the expression of several AAP genes and LHT1, a lysine- and histidine-specific amino acid transporter, in syncytia. The strong expression of AAP genes in syncytia indicated that these transporters are important for the transport of amino acids into syncytia and we used T-DNA mutants for several AAP genes to test for their influence on nematode development. We found that mutants of AAP1, AAP2, and AAP8 significantly reduced the number of female nematodes developing on these plants. Our study showed that amino acid transport into syncytia is important for the development of the nematodes.

Prevalence of biofilm-producing Staphylococcus epidermidis in the healthy skin of individuals in Tamil Nadu, India

Purpose:Staphylococcus epidermidis is a major commensal bacteria. Various strains of S. epidermidis are capable of forming biofilms by attaching to several surfaces. Biofilm-forming ability of this organism is found to be associated with many hospital-acquired infections and can even impair wound healing. S. epidermidis strains producing polysaccharide-biofilms possess the intercellular adhesion (ica) operon while strains forming the protein adhesion-mediated biofilms possess the accumulation associated protein (aap) gene. We screened for biofilm-forming S. epidermidis in the skin of healthy individuals in Tamil Nadu in order to determine the risk of acquiring S. epidermidis infections in hospital settings. Materials and Methods: Skin swabs were taken from seventy two subjects residing in Chennai with healthy skin who showed no visible signs of skin lesions or allergies. S. epidermidis was isolated from 58 samples out of the 72 collected. The presence of ica operon in S. epidermidis isolates was determined by PCR and biofilm production was examined using quantitative tissue culture plate assay. Results: Majority of the samples (47/72; 65.3%) showed pure S. epidermidis growth, (14/72; 19.4%) showed pure Staphylococcus aureus growth and the remainder (11/72; 15.3%) showed mixed growth. Biofilm-forming S. epidermidis were found in the majority of samples (53/58; 91.4%) and ica operon was detected in 19 samples out of 58 (32.8%) which is a significantly higher percentage when compared to other studies conducted at different parts of the globe (P = 0.0003). Conclusion: We inferred that ica operon and biofilm-forming S. epidermidis are common in the healthy skin of individuals in Tamil Nadu. Measures have to be taken to reduce the risk of hospital-acquired S. epidermidis infections.

Comparison of Staphylococcus epidermidis isolated from prosthetic joint infections and commensal isolates in regard to antibiotic susceptibility, agr type, biofilm production, and epidemiology

Staphylococcus epidermidis is the predominant bacterial species in the normal flora of the human skin and superficial mucosal membranes. However, it has also emerged as the most important pathogen in infections related to foreign-body materials, such as prosthetic joints and heart valves. The aims of this study were to characterise S. epidermidis isolated from prosthetic joint infections (PJI; n=61) and commensal isolates from healthy individuals (n=24) in regard to antimicrobial sensitivity, agr type, hld gene presence, biofilm production including presence of ica and aap genes involved in the biofilm formation process and epidemiology using both phenotypic (the PhenePlate-system) and genotypic [multilocus sequence typing (MLST)] methods. Among the PJI isolates, the majority (67%) were multidrug-resistant. Two major clusters of PJI isolates could be identified; 44% belonged to MLST sequence type (ST) 2, all but one were of agr type 1, and 31% were assigned ST215 and were of agr type 3. Of the commensal isolates, only one isolate was multidrug-resistant, and they were more molecular epidemiologically diverse with mainly MLST singletons and a maximum of 3 isolates assigned to the identical ST. Biofilm production was detected in 41% of the PJI isolates and 58% of the commensal isolates, with the aap gene (95%) more frequently detected than the ica genes (62%) in the biofilm-positive isolates. In conclusion, S. epidermidis isolated from PJIs and commensal isolates differed regarding antimicrobial sensitivity and molecular epidemiological typing using MLST, but not substantially in the distribution of agr types, biofilm production, or the presence of ica and aap genes.