Aap Genes Research Articles

Kanamycin promotes biofilm viability of MRSA strains showing extremely high resistance to kanamycin

Staphylococcus aureus is widely distributed in environment and can cause various human infection and food poisoning cases. Also, this pathogen is a typical biofilm former, which further complicates its pathogenicity. Antibiotics have been widely used to eliminate pathogenic bacteria, but their indiscriminate use has also led to the widespread emergence of drug-resistant bacteria, such as Methicillin-Resistant Staphylococcus aureus (MRSA). In this study, the effect of antibiotics on biofilm formation of MRSA strains 875 and 184 was explored. Firstly, MRSA 875 belongs to SCCmec type IV, ST239, carrying the atl, icaA, icaD, icaBC, and aap genes, and MRSA 184 belongs to SCCmec type II, ST5, carrying the atl, icaD, icaBC, aap, and agr genes. Then, a total of 8 antibiotics have been selected, including kanamycin, gentamycin, cipprofloxacin, erythromycin, meropenem, penicillin G, tetracycline, vancomycin. Minimum inhibitory concentrations (MICs) of each antibiotic were determined, and MIC of MRSA 875 and 184 to kanamycin/gentamicin are 2048/64 μg/mL and 2048/4 μg/mL, respectively. A total of 10 concentrations, ranging from 1/128 to 4 MIC with 2-fold, were used to study biofilm formation. Biofilm biomass and viability were determined during different phases, including initial adhesion (8 h), proliferation (16 h), accumulation (24 h) and maturation (48 h). Importantly, kanamycin at specific concentrations showed significant promotion of biofilm biomass and biofilm viability, with none of such observation acquired from other antibiotics. This study provides scientific basis and new research ideas for the quality control technology of microorganisms and safety prevention of MRSA.

Evaluation of anti-biofilm activity of Lactobacillus rhamnosus GG and Nisin on the expression of aap, ica-A and ica-D as biofilm-associated genes of Staphylococcus epidermidis.

In the present study, the anti-biofilm activity of Lactobacillus rhamnosus GG and Nisin was investigated on biofilm-forming abilities of Staphylococcus epidermidis strains and the expression of the biofilm-associated genes. In this study, the standard strain of L. rhamnosus GG (ATCC 53103) and Nisin were used to assess their anti-microbial and anti-biofilm effects on S. epidermidis (RP62A). The MIC and MBC analysis showed that Nisin at 256 μg/mL and 512 μg/mL, and L. rhamnosus GG at 1×107 CFU/mL and 1×108 CFU/mL have anti-microbial activity compared to the negative control respectively. L. rhamnosus GG bacteria and Nisin inhibited the biofilm formation of S. epidermidis based on optical density of at 570 nm (P <0.001). The relative mRNA expression of aap, icaA, and icaD genes was significantly reduced compared to the negative control after treating S. epidermidis with sub-MIC of Nisin (0.44, 0.25 and 0.6 fold, respectively) (P>0.05). In addition, the relative expression of aap and icaA genes, but not icaD (P>0.05), was significantly lower than the negative control (0.62 and 0.7 fold, respectively) (P>0.05), after exposure to the sub MIC of L. rhamnosus GG. Nisin and L. rhamnosus GG exhibit potent activity against biofilm-forming abilities of S. epidermidis and these agents could be utilized as an anti-biofilm agents against S. epidermidis infections.

Molecular Analysis of Pathogenicity, Adhesive Matrix Molecules (MSCRAMMs) and Biofilm Genes of Coagulase-Negative Staphylococci Isolated from Ready-to-Eat Food.

This paper provides a snapshot on the pathogenic traits within CoNS isolated from ready-to-eat (RTE) food. Eighty-five strains were subjected to biofilm and slime production, as well as biofilm-associated genes (icaA, icaD, icaB, icaC, eno, bap, bhp, aap, fbe, embP and atlE), the insertion sequence elements IS256 and IS257 and hemolytic genes. The results showed that the most prevalent determinants responsible for the primary adherence were eno (57.6%) and aap (56.5%) genes. The icaADBC operon was detected in 45.9% of the tested strains and was correlated to slime production. Moreover, most strains carrying the icaADBC operon simultaneously carried the IS257 insertion sequence element. Among the genes encoding for surface proteins involved in the adhesion to abiotic surfaces process, atlE was the most commonly (31.8%) followed by bap (4.7%) and bhp (1.2%). The MSCRAMMs, including fbe and embp were detected in the 11.8% and 28.2% of strains, respectively. A high occurrence of genes involved in the hemolytic toxin production were detected, such as hla_yiD (50.6%), hlb (48.2%), hld (41.2%) and hla_haem (34.1%). The results of the present study revealed an unexpected occurrence of the genes involved in biofilm production and the high hemolytic activity among the CoNS strains, isolated from RTE food, highlighting that this group seems to be acquiring pathogenic traits similar to those of S. aureus, suggesting the need to be included in the routine microbiological analyses of food.

The nitric oxide synthase gene negatively regulates biofilm formation in Staphylococcus epidermidis.

Staphylococcus epidermidis (S. epidermidis) is a clinically important conditioned pathogen that can cause a troublesome chronic implant-related infection once a biofilm is formed. The nitric oxide synthase (NOS) gene, which is responsible for endogenous nitric oxide synthesis, has already been found in the genome of S. epidermidis; however, the specific mechanisms associated with the effects of NOS on S. epidermidis pathogenicity are still unknown. The purpose of the current study was to investigate whether the NOS gene has an impact on biofilm formation in S. epidermidis. Bioinformatics analysis of the NOS gene was performed, and homologous recombination was subsequently employed to delete this gene. The effects of the NOS gene on biofilm formation of S. epidermidis and its underlying mechanisms were analyzed by bacterial growth assays, biofilm semiquantitative determination, Triton X-100-induced autolysis assays, and bacterial biofilm dispersal assays. Additionally, the transcription levels of fbe, aap, icaA, icaR and sigB, which are related to biofilm formation, were further investigated by qRT-PCR following NOS deletion. Phylogenetic analysis revealed that the NOS gene was conserved between bacterial species originating from different genera. The NOS deletion strain of S. epidermidis 1457 and its counterpart were successfully constructed. Disruption of the NOS gene resulted in significantly enhanced biofilm formation, slightly retarded bacterial growth, a markedly decreased autolysis rate, and drastically weakened bacterial biofilm dispersal. Our data showed that the fbe, aap and icaA genes were significantly upregulated, while the icaR and sigB genes were significantly downregulated, compared with the wild strain. Therefore, these data strongly suggested that the NOS gene can negatively regulate biofilm formation in S. epidermidis by affecting biofilm aggregation and dispersal.

Flavaspidic acid BB combined with mupirocin improves its anti-bacterial and anti-biofilm activities against Staphylococcus epidermidis

BackgroundThe increase in drug-resistant opportunistic pathogenic bacteria, especially of antibiotic-resistant Staphylococcus epidermidis (S. epidermidis), has led to difficulties in the treatment of skin and soft tissue infections (SSTI). The major reason for bacterial resistance is the formation of bacterial biofilm. Here, we report a promising combination therapy of flavaspidic acid BB (BB) and mupirocin, which can effectively eradicate the biofilm of S. epidermidis and eliminate its drug resistance.ResultThe susceptibility test showed that the combination of BB and mupirocin has good antibacterial and antibiofilm activities, and the fractional inhibitory concentration index (FICI) of BB combined with mupirocin was 0.51 ± 0.00 ~ 0.75 ± 0.05, showing synergistic effect. Moreover, the time-kill curve assay results indicated that the combination of drugs can effectively inhibit the planktonic S. epidermidis. After drugs treatment, the drug-combination showed significantly inhibitory effects on the metabolic activity and total biomass in each stage of biofilm formation. The synergistic effect is likely related to the adhesion between bacteria, which is confirmed by field emission scanning electron microscope. And the expression level of aap, sarA and agrA genes were detected by real-time quantitative PCR (qRT-PCR).ConclusionOur study provides the experimental data for the use of BB for the clinical treatment of skin infections and further demonstrate the potential of BB as a novel biofilm inhibitor.

Persistence of Staphylococcus spp. in milk from cows undergoing homeopathy to control subclinical mastitis

BackgroundMastitis is one of the major diseases in dairy cattle, as it causes great economic losses to producers due to the reduction of milk production and changes in the quality of the product. The disease is mainly caused by bacteria of the genus Staphylococcus spp., these microorganisms can express various virulence factors, such as biofilms for example. In herds with organic management, producers and technicians use unconventional ways to treat and control the disease, such as homeopathy. However, it is not known if this type of treatment is able to control pathogenic bacteria such as those of the genus Staphylococcus, of relevance to animal and human health. Thus, the objective of this study was to investigate the production of biofilm in vitro and its genes by Staphylococcus spp. isolated in the milk of cows treated with homeopathy, as well as the persistence of microorganisms in animals.MethodsNinety-nine isolates of Staphylococcus spp. from cows treated and not treated with homeopathy were identified by internal transcribed space-polymerase chain reaction and investigated for the presence of the icaABCD, bap, aap, atlE, and bhp genes and in vitro biofilm production using the adhesion method on polystyrene plates. The enzyme restriction profile was determined by Pulsed-Field Gel Electrophoresis. Clusters of S. aureus and S. epidermidis with three or more isolates had an isolate selected for Multilocus Sequence Typing.ResultsThe frequency of S. aureus isolations was similar in treated and untreated cows, while 71.4% of the coagulase-negative identified were isolated in cows treated with homeopathy. The distribution of the operon ica genes was similar in animals with and without treatment, except for the icaD gene, more frequent in treated cows. Production of biofilm was associated with presence of one or more genes from the icaADBC operon. S. aureus revealed a greater diversity and greater dissemination in cows treated and not treated with homeopathy. Sequence Types ST1, ST5, and ST126 were identified in S. aureus.ConclusionsThe presence of biofilm-associated genes and the in vitro production of biofilms, combined with the persistence of clonal profiles of Staphylococcus spp. demonstrate other forms of control for bovine mastitis should be researched for organic production herds.

Comparative profiling of agr locus, virulence, and biofilm-production genes of human and ovine non-aureus staphylococci

BackgroundIn a collaboration between animal and human health care professionals, we assessed the genetic characteristics shared by non-aureus staphylococci (NAS) infecting humans and dairy ewes to investigate their relatedness in a region concentrating half of the total National sheep stock. We examined by PCR 125 ovine and 70 human NAS for biofilm production, pyrogenic toxins, adhesins, autolysins genes, and accessory gene regulator (agr) locus. The microtiter plate assay (MPA) was used for the phenotypic screening of biofilm production. Ovine NAS included S. epidermidis, S. chromogenes, S. haemolyticus, S. simulans, S. caprae, S. warneri, S. saprophyticus, S. intermedius, and S. muscae. Human NAS included S. haemolyticus, S. epidermidis, S. hominis, S. lugdunensis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis.ResultsPhenotypically, 41 (32.8%) ovine and 24 (34.3%) human isolates were characterized as biofilm producers. Of the ovine isolates, 12 were classified as biofilm-producing while the remaining 29 as weak biofilm-producing. All 24 human isolates were considered weak biofilm-producing. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA), while the bhp, aap, and embp genes coding biofilm accumulation proteins were present in both non-producing and biofilm-producing isolates. Fifty-nine sheep NAS (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the agr locus: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans.Concerning virulence genes, 40, 39.2, 47.2%, 52.8, 80 and 43.2% of the sheep isolates carried atlE, aae, sdrF, sdrG, eno and epbS respectively, against 37.1, 42.8, 32.8, 60, 100 and 100% of human isolates. Enterotoxins and tsst were not detected.ConclusionsConsiderable variation in biofilm formation ability was observed among NAS isolates from ovine and human samples. S. epidermidis was the best biofilm producer with the highest prevalence of adhesin-encoding genes.

Prevention, inhibition, and degradation effects of melittin alone and in combination with vancomycin and rifampin against strong biofilm producer strains of methicillin-resistant Staphylococcus epidermidis

Methicillin-resistant Staphylococcus epidermidis (MRSE) bacteria are being recognized as true pathogens as they are able to resist methicillin and commonly form biofilms. Recent studies have shown that antimicrobial peptides (AMPs) are promising agents against biofilm-associated bacterial infections. In this study, we aimed to explore the antibiofilm activity of melittin, either alone or in combination with vancomycin and rifampin, against biofilm-producing MRSE strains. Minimum biofilm preventive concentration (MBPC), minimum biofilm inhibition concentration (MBIC), and minimum biofilm eradication concentration (MBEC), as well as fractional biofilm preventive-, inhibitory-, and eradication concentrations (FBPCi, FBICi, and FBECi), were determined for the antimicrobial agents tested. Cytotoxicity and hemolytic activity of melittin at its synergistic concentration were examined on human embryonic kidney cells (HEK-293) and Red Blood Cells (RBCs), respectively. The effect of melittin on the downregulation of biofilm-associated genes was explored using Real-Time PCR. MBPC, MBIC, and MBEC values for melittin were in the range of 0.625–20, 0.625–20, and 10–40 μg/μL, respectively. Melittin showed high synergy (FBPCi, FBICi and FBECi < 0.5). The synergism resulted in a 64–512-fold, 2–16 and 2–8-fold reduction in melittin, rifampicin and vancomycin concentrations, respectively. The synergistic melittin concentration found to be effective did not manifest either cytotoxicity on HEK-293 or hemolytic activity on RBCs. Results showed that melittin downregulated the expression of biofilm-associated icaA, aap, and psm genes in all isolates tested, ranging from 0.04-folds to 2.11-folds for icaA and from 0.05 to 3.76-folds for aap and psm. The preventive and therapeutic indexes of melittin were improved 8-fold when combined with vancomycin and rifampin. Based on these findings, the combination of melittin with conventional antibiotics could be proposed for treating or preventing biofilm-associated MRSE infections.

The frequency of hybrid Enteroaggregative/Uropathogenic Escherichia coli isolated from clinical samples of Isfahan hospitals, Iran

BackgroundIt has not been long since hybrid Escherichia coli pathotypes have been reported in scientific literatures. These isolates harbor a higher number of virulence factors and have greater pathogenicity. Here, for the first time, we reported the hybrid Enteroaggregative/Uropathogenic E. coli (EAEC/UPEC) isolates from clinical samples of hospitalized patients of Isfahan, Iran. MethodsIn this cross-sectional study, 196 E. coli isolates were collected from hospitals of Isfahan. PCR was performed for the detection of fimH, chuA, fyuA, and papc genes in UPEC isolates as well as aap, aggR, and aatA genes in EAEC isolates. DNA sequencing was conducted to confirm the presence of virulence genes in EAEC and UPEC pathotypes. Data were statistically analyzed by chi-square (X2) test. ResultsBased on our results, EAEC and UPEC pathotypes were isolated from 17 (8.6%) and 49 (25%) clinical samples, respectively. Also, according to the results, from a total of 196 E. coli isolates, 135 (68.8%), 113 (57.6%), 89 (45.4%), 73 (37.2%), 40 (20.4%), 26 (13.2%) and 15 (7.6%) carried the fimH, chuA, fyuA, papc, aap, aggR, and aatA genes, respectively. Interestingly, according to our data, 26 (13.2%) isolates were identified as hybrid UPEC/EAEC and this was confirmed by DNA sequencing and the BLAST results. Statistical analysis showed a significant correlation between EAEC or UPEC pathotypes (p = 0.00) and their isolation source. ConclusionThere is an urgency to identify heteropathogen EAEC/UPEC isolates that can carry different virulence factors attributed to both diarrheagenicity and uropathogenicity.

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.

Comparative analysis of virulence determinants, phylogroups, and antibiotic susceptibility patterns of typical versus atypical Enteroaggregative E. coli in India.

Enteroaggregative Escherichia coli (EAEC) is an evolving enteric pathogen that causes acute and chronic diarrhea in developed and industrialized nations in children. EAEC epidemiology and the importance of atypical EAEC (aEAEC) isolation in childhood diarrhea are not well documented in the Indian setting. A comparative analysis was undertaken to evaluate virulence, phylogeny, and antibiotic sensitivity among typical tEAEC versus aEAEC. A total of 171 EAEC isolates were extracted from a broad surveillance sample of diarrheal (N = 1210) and healthy children (N = 550) across North India. Polymerase chain reaction (PCR) for the aggR gene (master regulator gene) was conducted to differentiate tEAEC and aEAEC. For 21 virulence genes, we used multiplex PCR to classify possible virulence factors among these strains. Phylogenetic classes were identified by a multiplex PCR for chuA, yjaA, and a cryptic DNA fragment, TspE4C2. Antibiotic susceptibility was conducted by the disc diffusion method as per CLSI guidelines. EAEC was associated with moderate to severe diarrhea in children. The prevalence of EAEC infection (11.4%) was higher than any other DEC group (p = 0.002). tEAEC occurrence in the diarrheal group was higher than in the control group (p = 0.0001). tEAEC strain harbored more virulence genes than aEAEC. astA, aap, and aggR genes were most frequently found in the EAEC from the diarrheal population. Within tEAEC, this gene combination was present in more than 50% of strains. Also, 75.8% of EAEC strains were multidrug-resistant (MDR). Phylogroup D (43.9%) and B1 (39.4%) were most prevalent in the diarrheal and control group, respectively. Genetic analysis revealed EAEC variability; the comparison of tEAEC and aEAEC allowed us to better understand the EAEC virulence repertoire. Further microbiological and epidemiological research is required to examine the pathogenicity of not only typical but also atypical EAEC.

Characteristics of oral methicillin-resistant Staphylococcus epidermidis isolated from dental plaque

The oral microbial community is widely regarded as a latent reservoir of antibiotic resistance genes. This study assessed the molecular epidemiology, susceptibility profile, and resistance mechanisms of 35 methicillin-resistant Staphylococcus epidermidis (MRSE) strains isolated from the dental plaque of a healthy human population. Broth microdilution minimum inhibitory concentrations (MICs) revealed that all the isolates were nonsusceptible to oxacillin and penicillin G. Most of them were also resistant to trimethoprim (65.7%) and erythromycin (54.3%). The resistance to multiple antibiotics was found to be largely due to the acquisition of plasmid-borne genes. The mecA and dfrA genes were found in all the isolates, mostly dfrG (80%), aacA-aphD (20%), aadD (28.6%), aphA3 (22.9%), msrA (5.7%), and the ermC gene (14.3%). Classical mutational mechanisms found in these isolates were mainly efflux pumps such as qacA (31.4%), qacC (25.7%), tetK (17.1%), and norA (8.6%). Multilocus sequence type analysis revealed that sequence type 59 (ST59) strains comprised 71.43% of the typed isolates, and the eBURST algorithm clustered STs into the clonal complex 2-II(CC2-II). The staphyloccoccal cassette chromosome mec (SCCmec) type results showed that 25 (71.43%) were assigned to type IV. Moreover, 88.66% of the isolates were found to harbor six or more biofilm-associated genes. The aap, atlE, embp, sdrF, and IS256 genes were detected in all 35 isolates. This research demonstrates that biofilm-positive multiple-antibiotic-resistant ST59-SCCmec IV S. epidermidis strains exist in the dental plaque of healthy people and may be a potential risk for the transmission of antibiotic resistance.

Antibiotic resistance and virulence patterns of pathogenic Escherichia coli strains associated with acute gastroenteritis among children in Qatar

BackgroundThe treatment of Enterobacteriaceae family including diarrheagenic E. coli (DEC) has been increasingly complicated due to the emergence of resistant strains. Here we report on the phenotypic resistance profiles and ESBL genotype and virulence profiles of Enteroaggregative E. coli (EAEC) and Enteropathogenic E. coli (EPEC) isolated from children hospitalized with acute gastroenteritis in Qatar (AGE).ResultsE. coli were isolated and characterized from 76 diarrheagenic stool positive samples, collected from hospitalized children less than 10 years old. Isolates were tested for antibiotic susceptibility against eighteen clinically relevant antibiotics using E-test method. Conventional PCR was performed to detect genes encoding ESBL and virulence factors. Chi-square test was performed to compare the individual antibiotic resistance between EPEC and EAEC.A significant percentage (73.7%) of isolates were resistant to at least one antibiotic. Overall, high resistance (70%) was reported to the first-line antibiotics such as ampicillin, tetracycline (46.4%), and sulfamethoxazole-trimethoprim (42.9%). Further, 39.5% of the isolates were multidrug resistant (MDR), with 22.4% being ESBL producers. On the other hand, all isolates were susceptible to carbapenem, fosfomycin, amikacin and colistin. The incidences of resistance to the 18 antibiotics between EPEC and EAEC were not significantly different by Pearson chi -square test (P > 0.05). Genetic analysis revealed that 88.23% of ESBL production was blaCTX-M-G1 (blaCTX-M-15, blaCTX-M-3) - encoded. Several different combinations of virulence markers were observed, however, there was no specific trend among the isolates apart from absence of the bundle-forming pilus (bfpA) gene, which encodes the type IV fimbriae in EPEC adherence factor (EAF) plasmid (pEAF), among all EPEC (atypical). 15% of the EAEC strains were positive for a combination of astA, aap & capU, while 10% were positive for three different combinations. The aap, aatA, capU and aggR virulence genes showed the highest frequency of 65, 60, 55 and 55% respectively. Others genes, east, astA, and aai, showed frequencies of 35, 30 and 20% respectively.ConclusionsAtypical EPEC and EAEC were the primary etiological agents of diarrhea in children among DEC pathotypes. Our results indicated high rate of antimicrobial resistance pattern of DEC strains, which necessities the development of regulatory programs and reporting systems of antimicrobial resistance in DEC and other AGE-associated bacteria to insure effective control of diarrheal diseases. Results from this study demand a further research on identifying the phenotypic and genotypic profiles of more DEC pathotypes in various clinical samples.

Wide genetic heterogeneity and low antimicrobial resistance of enteroaggregative Escherichia coli isolates from several rural communities.

Enteroaggregative Escherichia coli (EAEC), a highly heterogeneous pathotype of E. coli classified as typical and atypical, are an emerging cause of acute and persistent diarrhea. We aimed to investigate whether population living in rural geographic areas, impacts in the heterogeneity, dissemination and antimicrobial susceptibility of EAEC strains. EAEC isolates (n=73) were analysed for the presence of 23 putative virulence factors, plasmid and antimicrobial resistance profiles, biofilm formation, pulsedfield gel electrophoresis (PFGE) and by multilocus sequence typing (MLST). The agg3A, agg4A, agn43, aap, shf, astA, pet, pic/set1A and sat genes, biofilm forming and antimicrobial resistance were statistically associated with typical EAEC. A low frequency of all isolates was resistant or showed a multidrug-resistance profile. No isolate showed the same plasmid profile. In total, 58 different pulsotypes were observed. Sixteen isolates analysed by MLST belonged to 15 different sequence types (ST) and showed a different PFGE pattern and virulence-gene profile. The fact that the communities are semi-isolated did not impact on the peculiar heterogeneity of EAEC, being characterized as epidemiologically independent strains.

Biofilm formation capacity in common SCCmec types of coagulase-negative staphylococci isolated from hospitalized patients and health-care workers in northwest of Iran

The present study characterized the distribution; biofilm formation ability of common SCCmec (staphylococcal cassette chromosome mec) types of CoNS isolates from hospitalized patients (HP) and health-care workers (HCW) in northwest of Iran. Thirteen species of CoNS were identified by using of the conventional microbiological tests included S. epidermidis, S. haemolyticus, S. hominis S. warneri S. auricularis S. saprophyticus, S. lugdunensis, S. caprae, S. scheliferi, S. xylosus, S. capitis, S. simulans and S. cohnii. High prevalence of methicillin-resistant (MR-CoNS) isolates (76.2%) was observed. The prevalence of biofilm-producing isolates was higher in HP (93%) than HCW (82.5%). There was not a significant statistically difference between the presence of icaA, icaD and aap genes in these groups (p-value ˃ 0.05). However, a significant statistically difference was observed in the presence of IS256 between two groups (p-value ˂ 0.05). No significant association was observed between any SCCmec type and biofilm formation or the presence of biofilm related genes. The high prevalence of MR-CoNS and consequently, MDR isolates, as well as, their high ability to form slime or biofilm, is a common concern in our hospitals. Biofilm formation ability or the presence of biofilm related genes was not a SCCmec type trait.

Non-aureus staphylococci in fecal samples of dairy cows: First report and phenotypic and genotypic characterization

The aims of this study were to determine whether non-aureus staphylococci (NAS) are present in rectal feces of healthy dairy cows, and if so, to delineate species to which they belong and to study several phenotypic and genotypic traits as a first step toward determining the potential impact of fecal shedding of NAS on bovine udder health. Fecal samples were aseptically collected from the rectum of 25 randomly selected clinically healthy dairy cows in a commercial dairy herd using an automated milking system. Fecal NAS were isolated and then identified at the species level using transfer RNA-intergenic spacer PCR and sequencing of the 16S rRNA housekeeping gene. Strain typing was performed using random amplification of polymorphic DNA (RAPD)-PCR. The antimicrobial resistance profiles, biofilm formation, and growth and inhibitory characteristics of all NAS isolates were evaluated. Half of the cows were shedding NAS, resulting in 31 NAS isolates belonging to 11 different species. The most prevalent species were Staphylococcus rostri (23%, n = 7), Staphylococcus cohnii (16%, n = 5), and Staphylococcus haemolyticus (13%, n = 4) with all Staphylococcus agnetis, Staphylococcus chromogenes, and Staph. rostri isolates belonging to the same strain according to RAPD banding patterns. Acquired antimicrobial resistance was observed in 28 of the 31 NAS isolates, mainly due to β-lactamase production. Most of the isolates (84%, n = 27) had a weak biofilm-forming potential, but only 2 contained the bap gene. The ica and aap genes were not detected in any of the isolates. In vitro growth of Staphylococcus aureus and Streptococcus dysgalactiae was inhibited by Staph. agnetis isolates, and Staph. chromogenes isolates were able to inhibit the growth of Strep. dysgalactiae and Streptococcus uberis. All fecal isolates were able to grow when oxygen and iron were limitedly available, mimicking the growth conditions in the mammary gland.

Prevalence and virulence factors of coagulase negative Staphylococcus causative of prosthetic joint infections in an orthopedic hospital of Mexico.

To determine the prevalence and virulence factors of coagulase-negative Staphylococci (CNS) in prosthetic joint infections (PJI). CNS were isolated of 66 hip and knee PJI from Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra, México City. Antimicrobial susceptibility and biofilm formation in CNS were determined; icaADBC, aap, bap and embp genes were determined by PCR. Staphylococcus and Staphylococcus hominis were the most prevalent with 82 y 80% respectively. Staphylococcus lugdunensis, Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus sciuri and Staphylococcus lentus were less frequent. The majority of isolates were resistant to β-lactam antibiotics, fluoroquinolone, and erythromycin. 41% of CNS were biofilm former and 59% were non-biofilm former (p = 0.0551). Biofilm former Staphylococcus epidermidis showed a high presence of icaADBC, aap and embp operons compared to the non-biofilm former isolates (p < 0.05). In contrast, non-S. epidermidis CNS had only the aap gen. S. haemolyticus, S. sciuri and S. lentus are new isolates of PJI not previously reported with virulence factors similar to CNS isolates.

Association between biofilm formation, structure and antibiotic resistance in Staphylococcus epidermidis isolated from neonatal septicemia in southwest Iran.

Background: Staphylococcus epidermidis has emerged as the pathogen from neonatal septicemia. Antibiotic resistance and the capability of biofilm formation make these infections much harder to treat. Hence, the aim of this study was to investigate the association between biofilm formation, structure and antibiotic resistance in S. epidermidis isolated from neonatal septicemia.Methods: Overall, 65 S. epidermidis isolates were recovered from blood cultures of neonatal septicemia. Antibiotic resistance pattern and the biofilm production were determined using phenotypic methods. The presence of ica operon, the bhp, the aap genes and SCCmec types were screened using PCR.Results: Most S.epidermidis isolates were resistant to erythromycin, while all isolates were sensitive to linezolid and vancomycin. Fifty-three percent of S.epidermidis isolates were resistant to methicillin. SCCmec types II was found commonly among methicillin-resistant S. epidermidis (MRSE) strains. The biofilm formation was observed in 65% of S.epidermidis isolates and the majority have polysaccharide matrix. icaA and icaD genes were found in 40% and 19% of isolates. Twenty-three isolates (62%) produced dissolvable polysaccharide intercellular adhesion (PIA)-dependent biofilms in SM after growth in TSB with NaCl and 14 (37%) isolates produced dissolvable protein-dependent biofilms in PK after growth in TSB with glucose. Three isolates (62%) produced dissolvable polysaccharide intercellular adhesion.Conclusion: Our data indicate the high rates of antibiotic resistance and the capability of biofilm formation among S. epidermidis isolates. Hence, the transmission of these strains can cause an increased risk of serious nosocomial infections.

GATA-type transcription factors play a vital role in radiation sensitivity of Cryptococcus neoformans by regulating the gene expression of specific amino acid permeases

Cryptococcus neoformans is a basidiomycete fungus that is highly resistant to ionizing radiation and has been identified in highly radioactive environments. Transcription factors (TFs) are master regulators of gene expression by binding to specific DNA sequences within promoters of target genes. A library of 322 signature-tagged gene deletion strains for 155 C. neoformans TF genes has been established. Previous phenome-based functional analysis of the C. neoformans TF mutant library identified key TFs important for various phenotypes, such as growth, differentiation, virulence-factor production, and stress responses. Here, utilizing the established TF mutant library, we identified 5 TFs that are important for radiation sensitivity, including SRE1, BZP2, GAT5, GAT6, and HCM1. Interestingly, BZP2, GAT5 and GAT6 all belong to the GATA-type transcription factors. These factors regulate transcription of nitrogen catabolite repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. In addition to radiation, we found that specific GATA factors are important for other stressors such as rapamycin, fluconazole, and hydroxyurea treatment. Using real-time PCR method, we studied the expression of GATA down-stream genes after radiation exposure and identified that AAP4, AAP5 and URO1 were differentially expressed in the GAT5 and GAT6 mutants compared to the wild type cells. In summary, our data suggest that GATA TFs are important for radiation sensitivity in C. neoformans by regulating specific downstream AAP genes.

Biofilm production, quorum sensing system and analysis of virulence factors of Staphylococcus epidermidis collected from sheep milk samples

Intramammary infections are a serious problem in dairy sheep farming, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 199 S. epidermidis isolates collected in Sardinia (Italy) from sheep milk samples and used for the preparation of inactivated autogenous vaccines were characterized with phenotypic and genotypic methods. The potential for biofilm production was evaluated phenotypically by assessing colony characteristics on Congo Red Agar (CRA), and genotypically by PCR for i) the icaA/D genes coding the polysaccharide intercellular adhesin (PIA) and ii) for the bhp, aap and embp genes coding biofilm accumulation proteins. Isolates were also investigated by PCR for the presence of autolysins (atlE and aae genes), microbial surface components recognizing adhesive matrix molecules (MSCRAMMs, sdrG and sdrF genes), enterotoxins (sea, seb, sec, sed and see genes), and the toxic shock syndrome toxin (tsst genes). In addition, the staphylococcal accessory gene regulator (agr), the most important locus responsible for the regulation of virulence factors, was characterized for the first time. As a result, only two isolates showed biofilm production on CRA and harbored the icaA/D genes. Four isolates, phenotypically classified as non-biofilm producers, possessed the icaA/D genes (n = 3) or only the icaA gene (n = 1). All isolates harbored the atlE, aae, embp and sdrG genes, but none harbored the genes encoding enterotoxins and tsst. Most isolates belonged to the agr-3se group (56.3%), followed by agr-1se (41.2%) and agr-2se (2.5%). All six isolates that harbored ica genes belonged to agr-1se.In conclusion, this study provides a detailed characterization of the putative virulence genes present in S. epidermidis strains circulating in Sardinian sheep. Because of their role in ovine mastitis pathogenesis, these might represent valuable candidates for vaccine development.