IIIa Genes Research Articles

Frequency of Vancomycin and Aminoglycoside Resistance Genes in Enterococcus spp. Isolated from Chicken Raw Meat Samples

Background: Enterococci are main normal microbial flora of both humans and animals and can survive in a diverse range of environments. These bacteria carry out aminoglycoside and vancomycin resistance genes and spread them in environment by many routs such as chicken meat products. The present study was aimed to determine the frequency of aminoglycoside and vancomycin resistant genes in Enterococcus species isolated from chicken meat specimens. Methods: A total of 250 chicken raw meat specimens was prepared from slaughterhouses at Zanjan province, cultured at BHI broth and incubated at 37°C for 24h. The positive cultures were sub-cultured in blood agar plates and grown colonies identified using phenotypical and biochemical tests. Antibiotic susceptibility was determined according to the Clinical and Laboratory Standards Institute (CLSI) standards and PCR assays were performed to detect vanA, vanB, aph (2")1c, aph (2")1b, aph (2")1d, ant(3'), aph(3')IIIa, ant(4')1a, ant(6') and aac(6') genes. Results: In total, 100 Enterococcus species isolated from 250 specimens and 35% of them belonged to E. faecalis and the others were E. faecium (65%). The prevalence of the vanA, vanB, aph (2")1c, aph (2")1b, aph (2")1d, ant(3'), aph(3')IIIa, ant(4')1a, ant(6') and aac(6') genes among the 100 Enterococcus species was 14%, 12%, 10%, 1%, 2%, 50%, 26%, 9%, 18% and 22%, respectively. Conclusion: The current study revealed that the rate of antimicrobial resistance genes to aminoglycosides and vancomycin was worrying and health measurements in meat products industries must be performed to prevent spread of antimicrobial resistance elements among bacteria.

Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6′)Ie-aph(2″)Ia and aph(3′)-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6′)-Ie-aph(2″)-Ia or aph(3’)-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

Identification of major sequence types among aminoglycoside resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical samples.

Aminoglycosides have been widely used for treating severe staphylococcal infections. Production aminoglycoside modifying enzymes (AMEs) is the main mechanism of resistance to this antibiotic. The aim of this study was to determine the prevalence of AME genes and molecular characterization of aminoglycoside-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical specimens in Iran. A total of 42 clinical isolates of Gram-positive cocci (20 S. aureus and 22 S. epidermidis) with resistance to gentamicin were tested for antimicrobial resistance and differentiated by multilocus sequence typing (MLST). All 42 isolates were resistant to methicillin, kanamycin, and most of them were also resistant to amikacin (98%), tobramycin (98%) and netilmycin (78.5%). Overall, aac(6')-Ie-aph(2")-Ia was the dominant AME gene found in 100% of isolates, followed by aph(3')IIIa found in 90% of isolates. MLST classified S. aureus and S. epidermidis into 5 and 9 distinct sequence types (ST), respectively. The majority of the strains belonged to ST239 (50%) for S. aureus and ST2 (36%) for S. epidermidis. The resistance to aminoglycosides was mainly due to the presence of the aac(6')-Ie-aph(2")-Ia and aph(3') IIIa genes as well as the ST239 for S. aureus and ST2 for S. epidermidis have become the predominant clones in the selected university hospital of Tehran, Iran. Thus, it is critical that clinicians and healthcare workers are aware of the population of S. aureus and S. epidermidis present in order to make decisions for appropriate treatment and infection control practices.

A One Health Approach Molecular Analysis of Staphylococcus aureus Reveals Distinct Lineages in Isolates from Miranda Donkeys (Equus asinus) and Their Handlers

Donkeys (Equus asinus) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate S. aureus and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of S. aureus strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in S. aureus isolates. From the 49 donkey samples, 4 S. aureus (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of S. aureus and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the mecA, aph (3′)-IIIa, ant (4′)-Ia, tetM, tetK, lnuA, ermB, ermC, dfrA and dfrG genes. S. aureus isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the blaZ, aph (3′)-IIIa, tetL, tetM and tetK genes. All S. aureus isolates from donkeys belonged to ST49 and spa-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 spa-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant S. sciuri. S. aureus transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.

Molecular Characterization of High-Level Aminoglycoside Resistance among Enterococcus Species.

Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminoglycoside by producing aminoglycoside modifying enzymes (AMEs). In enterococci, high level resistance to aminoglycosides is mediated by acquisition of plasmid mediated genes encoding for aminoglycoside modifying enzymes (AMEs). High level gentamicin resistance (MIC ≥ 500μg /mL) is predominantly mediated by aac(6′)-Ie-aph(2″)-Ia, encoding the bifunctional aminoglycoside modifying enzyme AAC(6′)-APH(2″). This enzyme eliminates the synergistic activity of gentamicin when combined with a cell wall active agent. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id and ant(4′)-1a have also been detected in enterococci. Objective This study was carried out to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of Enterococci . Materials and Methods A total number of 150 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion. Minimum Inhibitory Concentration (MIC) was ascertained by agar dilution method. Polymerase chain reaction was done to screen the following AMEs (aac(6′)-Ie-aph(2″)-Ia; aph(2″)-Ib; aph(2″)-Ic; aph(2″)-Id and aph(3′)- IIIa genes) . Results 51.3% of the study isolates exhibited high level gentamicin resistance. Polymerase chain reaction revealed that aph(3′)-111a is the most prevalent AME, followed by aac(6′)-1e-aph(2″)-1a . The combination of both the genes were detected in 44.1% of the study isolates. The rest of the AMEs and their combinations were not encountered in this study. 8.6% of the study isolates did not harbour any AME genes screened for, but was phenotypically resistant to gentamicin. In contrast 31.3% anchored the AME genes but phenotypically appeared susceptible to gentamicin. Conclusion This study indicates the high- level aminoglycoside resistance disseminated among Enterococci in our geographical region. It also emphasizes the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbour one or more AMEs genes leading to therapeutic failure.

High Frequency of the EMRSA-15 Clone (ST22-MRSA-IV) in Hospital Wastewater.

Hospital wastewaters often carry multidrug-resistant bacteria and priority pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Pathogens and antibiotic resistance genes present in wastewaters may reach the natural environment facilitating their spread. Thus, we aimed to isolate MRSA from wastewater of 3 hospitals located in the north of Portugal and to characterize the isolates regarding the antimicrobial resistance and genetic lineages. A total of 96 wastewater samples were collected over six months. The water was filtered, and the filtration membrane was immersed in BHI broth supplemented with 6.5% of NaCl and incubated. The inoculum was streaked in ORSAB agar plates for MRSA isolation. The isolates susceptibility testing was performed against 14 antimicrobial agents. The presence of resistance and virulence genes was accessed by PCR. Molecular typing was performed in all isolates. From the 96 samples, 28 (29.2%) were MRSA-positive. Most isolates had a multidrug-resistant profile and carried the mecA, blaZ, aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa, ermA, ermB, ermC, tetL, tetM, dfrA dfrG and catpC221 genes. Most of the isolates were ascribed to the immune evasion cluster (IEC) type B. The isolates belonged to ST22-IV, ST8-IV and ST105-II and spa-types t747, t1302, t19963, t6966, t020, t008 and tOur study shows that MRSA can be found over time in hospital wastewater. The wastewater treatment processes can reduce the MRSA load. The great majority of the isolates belonged to ST22 and spa-type t747 which suggests the fitness of these genetic lineages in hospital effluents.

Identification of SNPs in crucial starch biosynthesis genes in rice

Rice is the foremost crop catering to the major calorific requirement of the human population but has the disadvantage of having high glycaemic index (GI). The fine quality rice varieties, BPT and RNR have been recently identified as having low GI in nature and are grown mostly in southern parts of India. Starch (80%) is the major component of rice endosperm attributing to GI. The study aimed to unravel the molecular basis of low GI through targeted pooled amplicon sequencing of major starch biosynthetic genes. A total of 13 candidate genes involved in starch synthesis were amplified and pooled in equimolar proportion for sequencing. Single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) were detected in both coding and noncoding regions. Among the genes that are under study, the highest number of variations were identified in starch synthase I (SSI) followed by starch synthase IIIA (SSIIIA) genes. Nonsynonymous SNPs with high probability of effecting gene function were validated by Sanger sequencing and molecular docking. Identified causative SNPs were mapped on 3000 rice genome database and their allele frequencies were obtained. The outcome of this study has a potential to be applied in breeding programmes to obtain low GI rice varieties with added beneficial traits.

Ready-to-eat dairy products as a source of multidrug-resistant Enterococcus strains: Phenotypic and genotypic characteristics

The enterococci are ubiquitous bacteria able to colonize the human and animal gastrointestinal tracts and fresh and fermented food products. Their highly plastic genome allows Enterococcus spp. to gain resistance to multiple antibiotics, making infections with these organisms difficult to treat. Food-borne enterococci could be carriers of antibiotic resistance determinants. The goal of this work was to study the characteristics of Enterococcus spp. in fermented milk products from Poland and their antibiotic resistance gene profiles. A total of 189 strains were isolated from 182 dairy products out of 320 samples tested. The predominant species were Enterococcus faecium (53.4%) and Enterococcus faecalis (34.4%). Isolates were resistant to streptomycin (29.1%), erythromycin (14.3%), tetracycline (11.6%), rifampicin (8.7%), and tigecycline (8.1%). We also detected 2 vancomycin-resistant and 3 linezolid-resistant strains; however, no vanA or vanB genes were identified. A total of 57 high-level aminoglycoside resistance strains (30.2%) were identified, most of which have the ant(6')-Ia gene, followed by the aac(6')-Ie-aph(2″)-Ia and aph(3″)-IIIa genes. Resistance to tetracycline was most often conferred by tetM and tetL genes. Macrolide resistance was most frequently encoded by ermB and ermA genes. Conjugative mobile genetic element (transposon Tn916-Tn1545) was identified in 15.3% of the strains, including 96.3% of strains harboring the tetM gene. This study found that enterococci are widely present in retail ready-to-eat dairy products in Poland. Many isolated strains are antibiotic resistant and carry transferable resistance genes, which represent a potential source of transmission of multidrug-resistant bacteria to humans.

Clonal Structure and Antibiotic Resistance of Enterococcus spp. from Wild Birds in Poland.

The purpose of this study was to analyze the antibiotic resistance and genetic diversity of 27 enterococci (Enterococcus faecium, Enterococcus hirae, Enterococcus durans, and Enterococcus casseliflavus) isolated from wild bird species. Resistance to lincomycin was most common, followed by erythromycin, ciprofloxacin, tetracyclines, high level of aminoglycoside, and β-lactam antibiotics. No vancomycin- and chloramphenicol-resistant isolates were identified. The antibiotic resistance was linked to the tet(M), tet(L), erm(A), erm(B), msr(A/B), pbp5, ant(6)-Ia, and aph(3')-IIIa genes. Tn916/Tn1545-like transposons were detected. The high-level resistance to gentamicin was associated with the presence of gene aph(2″)-Id. All 18 E. faecium isolates were divided into 16 pulsotypes and 17 sequence types (STs), among which 7 STs were newly assigned (ST1266-ST1272). A majority of E. faecium isolates possess multilocus sequence typing profiles belonging to clonal complex 17 (CC17), the major epidemic lineage responsible for nosocomial infections. Two ST17 and newly described ST1267 and ST1271 (an SLV and DLV of ST17, respectively) of E. faecium isolates carried the type 1 allele of the housekeeping gene purK detected in hospital-related strains. Our results indicated that wild birds could be a source of resistant E. faecium isolates, belonging to CC17 and may represent a hazard to human health by transmission of these isolates.

Association of Fc gamma-receptor genes polymorphisms with chronic periodontitis and Peri-Implantitis.

This study was conducted on 87 patients with chronic periodontitis (CP), 50 patients with peri-implantitis and 90 periodontally healthy individuals referring to the Department of Periodontics for evaluating the association between Fc gamma-receptor genes polymorphisms with CP and peri-implantitis. After obtaining consent, venous blood samples (5cc) were obtained from patients and DNA was extracted using Miller's salting-out method. Polymerase chain reaction (PCR)-restriction fragment length polymorphism and tetra-primer amplification refractory mutation system-PCR methods were used to assess the polymorphisms of FcγRs IIa, IIIa, and IIIb genes. Analyzing showed a significant association between specific genotypes with increasing CP and peri-implantitis risks in codominant and dominant models. For FcγR IIIa, analyzing revealed a significant association between specific genotypes with increasing CP and peri-implantitis risks in codominant, dominant, and recessive models. For FcγR IIIb, we also detected a significant association between specific genotypes with increasing CP and peri-implantitis risks in codominant, dominant, and recessive models ( P < 0.05). According to the results of this study, the FCGRIIa (rs1801274), FCGRIIIa (rs396991), and FCGRIIIb (rs1050501) polymorphisms were significantly associated with CP and peri-implantitis and may have a role in the pathogenesis of these diseases.

High prevalence of vancomycin and high-level gentamicin resistance in Enterococcus faecalis isolates.

Multiple drug-resistant enterococci are major cause of healthcare-associated infections due to their antibiotic resistance traits. Among them, Enterococcus faecalis is an important opportunistic pathogen causing various hospital-acquired infections. A total of 53 E. faecalis isolates were obtained from various infections. They were identified by phenotypic and genotypic methods. Determination of antimicrobial resistance patterns was done according to CLSI guidelines. The isolates that were non-susceptible to at least one agent in ≥3 antimicrobial categories were defined as multidrug-resistant (MDR). Detection of antimicrobial resistance genes was performed using standard procedures. According to MDR definition, all of the isolates were MDR (100%). High-level gentamicin resistance was observed among 50.9% of them (MIC ≥ 500 μg/ml). The distributions of aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa genes were 47.2% and 69.8%, respectively. The aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id, and ant(4')-Ia genes were not detected. Vancomycin resistance was found in 45.3% of strains. The vanA gene was detected in 37.7% of isolates, whereas vanB and vanC1 genes were not observed in any strain. Erythromycin resistance rate was 79.2% and the frequencies of ermB and ermC genes were 88.6% and 69.8%, respectively. The ermA and msrA genes were not present in any of the isolates. Our data indicate a high rate of MDR E. faecalis strains. All of high-level gentamicin-resistant isolates carried at least one of aac(6')-Ie-aph(2'')-Ia or aph(3')-IIIa genes. Distribution of vanA was notable among the isolates. In addition, ermB and ermC were accountable for resistance to erythromycin.

Occurrence of enterococci harbouring clinically important antibiotic resistance genes in the aquatic environment in Gauteng, South Africa

The development of antibiotic resistance and dissemination of its determinants is an emerging public health problem as it compromises treatment options of infections that were, until recently, treatable. Investigation of outbreaks of vancomycin resistant enterococci (VRE) suggests that the environment serves as a significant reservoir for antibiotic resistance genes (ARGs). However, there is a paucity of data regarding the presence of ARGs in the water sources in South Africa. In this study, water samples collected from wastewater treatment plants (WWTPs), surface water and hospital sewage were screened for enterococci harbouring genes conferring resistance to four classes of antibiotics. Enterococci isolates harbouring ARGs were detected in raw influent and treated wastewater discharge from WWTPs and hospital sewage water. Plasmid and transposon encoded ermB (macrolide), tetM and tetL (tetracycline) as well as aph(3’)-IIIa (aminoglycosides) genes were frequently detected among the isolates, especially in E. faecalis. The presence of enterococci harbouring ARGs in the treated wastewater suggest that ARGs are discharged into the environment where their proliferation could be perpetuated. Among the enterococci clonal complexes (CCs) recovered from wastewater were E. faecium CC17 (ST18), which is frequently associated with hospital outbreaks and a novel E. faecalis sequence type (ST), ST780.

Molecular investigation of methicillin-resistant staphylococcus aureus isolates from blood: USA600 emerges as the major type.

The widespread emergence of methicillin-resistant Staphylococcus aureus is turning into a real worry in public health. The goals of the present study were to identify resistance and virulence encoding genes and molecular characteristics of methicillin-resistant S. aureus bloodstream isolates. A cross-sectional study was conducted on 84 S. aureus bloodstream isolates during a 10-month period. To evaluate antibiotic susceptibility of the isolates, we used Kirby-Bauer disk diffusion method. In addition, the prevalence of antimicrobial resistance and toxins genes was assessed using polymerase chain reaction. Isolates were typed according to polymorphisms seven housekeeping genes by MLST. All the isolates were resistant to methicillin. The most prevalent resistance gene was mecA gene (100%) followed by tetM (57.1%), aac (6΄)-Ie/aph (2˝) (53.6%), ant (4΄)-Ia (46.4%), ermA (45.2%), msrA (35.7%), msrB (33.3%), aph (3΄)-IIIa (33.3%), ermB (31%), ermC (16.7%), and mupA (14.3%) genes. The presence of toxin encoding genes tst, pvl, eta, and etb were detected in 25%, 14.3%, 3.6% and 3.6%, respectively. The isolates were classified into five different sequence types: ST45 (29.8%), ST239 (27.4%), ST858 (21.4%), ST22 (17.8%), and ST59 (3.6%). All the high-level mupirocin-resistant (HLMUPR) strains belonged to ST239, while the low-level mupirocin resistant (LLMUPR) strains belonged to ST22 (13%) and ST239 (6%). To the best of our knowledge, the present study is the first report of ST59 in MRSA bloodstream isolates in Iran. Our data demonstrated the need for thorough epidemiological monitoring to detect emergence and dissemination of MDR- MRSA types in our hospitals.

Prevalence of antibiotic resistance genes in bacteriophage DNA fraction from Funan River water in Sichuan, China

To better understand the role that bacteriophages play in antibiotic resistance genes (ARGs) dissemination in the aquatic environment, 36 water samples were collected from the Funan River in Sichuan, China. The occurrence of 15 clinically relevant ARGs and one class 1 integron gene int1 in phage-particle DNA were evaluated by PCR. The abundance of ARGs (blaCTX-M, sul1, and aac-(6′)-1b-cr) was determined by quantitative PCR (qPCR). High prevalence of the int1 gene (66.7%) was found in the phage-particle DNA of tested samples, followed by sul1 (41.7%), sul2 (33.3%), blaCTX-M (33.3%), aac-(6′)-lb-cr (25%), aph(3′)-IIIa (16.7%), and ermF (8.3%). The qPCR data showed higher gene copy (GC) numbers in samples collected near a hospital (site 7) and a wastewater treatment plant (WWTP) (site 10) (P < .05). Particularly the absolute abundance of aac-(6′)-lb-cr gene was significantly higher than the blaCTX-M and sul1 genes with the gene copy (GC) numbers of 5.73 log10 copy/mL for site 7 and 4.99 log10 copy/mL for site 10. To our best knowledge, this is the first study to report the presence of sul2, aac-(6′)-lb-cr, ermF and aph(3′)-IIIa genes in bacteriophage DNA derived from aquatic environments. Our findings highlight the potential of ARGs to be transmitted via bacteriophages in the aquatic environment.

WILD BIRDS AS A POTENTIAL SOURCE OF KNOWN AND NOVEL MULTILOCUS SEQUENCE TYPES OF ANTIBIOTIC-RESISTANT ENTEROCOCCUS FAECALIS.

We assessed the antibiotic resistance and genetic diversity of 27 Enterococcus faecalis isolates from 25 wild bird species in Poland. Resistance to lincomycin (100%) was most common followed by tetracycline (48%), erythromycin (44%), and ciprofloxacin (22%). High-level resistance to streptomycin and kanamycin was observed in 19% and 15% of isolates, respectively. One isolate (4%) exhibited low-level resistance to penicillin and vancomycin, and all isolates were susceptible to gentamicin and chloramphenicol. Antibiotic resistance was linked to the tet(M), tet(L), erm(A), erm(B), msr(A/B), ant(6)-Ia, and aph(3')-IIIa genes. None of the tested van ( vanA, vanB, vanC1, vanC2/C3, vanD, vanE, vanG) genes were found in the vancomycin-resistant isolate. Based on pulsed-field gel electrophoresis and multilocus sequence typing analysis, the E. faecalis population from wild birds revealed high genetic diversity. All isolates were divided into 22 pulsotypes and 18 sequence types (STs), among which seven STs were newly assigned (ST748-ST753 and ST764). The most-prevalent STs were ST290 and ST374 followed by ST287 and ST34. The coexistence of strains assigned to the same STs in wild birds and in nonwildlife populations strongly indicated that many wild bird species could constitute a source of E. faecalis for infections in humans, pets, and farm animals.

Interaction among CYP2C8, GPIIIa and P2Y12 variants increase susceptibility to ischemic stroke in Chinese population.

PurposeGenetic variants in cytochrome P450 (CYP), platelet membrane receptor (P2Y12, P2Y1), and glycoprotein IIIa (GPIIIa) genes are associated with the efficacy of clopidogrel and adverse clinical events on ischemic stroke (IS) patients. However, few studies have assessed whether gene-gene interactions among these genes influence the risk of IS. The aim of the present study was to investigate the association of fifteen variants with IS and to determine whether these gene-gene interactions increase the risk of IS.MethodsFifteen variants in CYP3A4, CYP3A5, CYP2C8, CYP2C9, CYP2C19, P2Y12, P2Y1 and GPIIIa genes were examined using mass spectrometry methods in 396 patients with IS and 378 controls. Gene-gene interactions were analyzed using generalized multifactor dimensionality reduction (GMDR) methods.ResultsSingle-gene variant analysis showed no significant differences in the genotype distributions of the fifteen variants between IS patients and controls using the single-locus analytical approach. However, GMDR analysis showed a significant gene-gene interaction among rs17110453A>C, rs2317676A>G, and rs16863323C>T, which scored 10 for cross-validation consistency and 9 for the sign test (P = 0.016). Logistic regression analysis showed that high-risk interactions among rs17110453A>C, rs2317676A>G, and rs16863323C>T were independent risk factor for IS after adjusting for age, hypertension, diabetes mellitus, and hemoglobin A1C (OR=2.24, 95% CI: 1.17–5.62, P=0.005).ConclusionsThe rs17110453A>C, rs2317676A>G, and rs16863323C>T three-loci interaction may confer a higher risk for IS. The combinatorial analysis used in this study may be helpful to elucidate complex genetic risk factors for IS.

Detection of tetracycline resistance genes, aminoglycoside modifying enzymes, and coagulase gene typing of clinical isolates of Staphylococcus aureus in the Southwest of Iran.

Objective(s):The aim of the present study was to determine the aminoglycoside modifying enzymes (AMEs) encoded genes, tetracycline resistance genes, and the coa based typing of Staphylococcus aureus isolates in the Southwest of Iran.Materials and Methods:Antimicrobial susceptibility of isolates was carried out by agar disk diffusion methods. Two sets of multiplex PCR mixture were used for detection of AME genes and tet genes. All of the isolates were typed with the coagulase gene typing method. Of the 121 isolates, 29.75% and 47.93% were resistant to at least one aminoglycosides and tetracyclines, respectively.Results:The aac(6’)-Ie-aph(2”) was the most frequent gene (97.22%), and aph (3’)-IIIa and ant (4’)-Ia genes were detected in 61.11% and 11.11% of aminoglycoside resistant isolates, respectively. The tetK and tetM genes were detected in 82.75% and 56.9% of tetracycline resistant isolates, respectively. Overall 31.4% of isolates were MRSA. Totally 17 distinct coa gene RFLP patterns, numbered C1 to C17, were observed. The C5 was the most frequent coa type with 31 isolates.Conclusion:The aac(6’)-Ie-aph(2”) and aph (3’)-IIIa genes were the most important genes contributing to aminoglycosides resistance, while resistance to tetracyclines was mediated by tetK and tetM genes. Interestingly all S. aureus with C5 as the most prevalent coa-type were resistant to at least one of the aminoglycoside antibiotics and tetracycline simultaneously. Moreover, 30 out of 31 isolates with this coa type were MRSA, indicating the importance of the C5 coa-type in MRSA strains and also in isolates that were resistant to aminoglycosides and tetracycline.

Conjugal transfer of aac(6')Ie-aph(2″)Ia gene from native species and mechanism of regulation and cross resistance in Enterococcus faecalis MCC3063 by real time-PCR.

High level aminoglycoside resistance (HLAR) in the lactic acid bacteria (LAB) derived from food animals is detrimental. The aim of this study was to investigate the localization and conjugal transfer of aminoglycoside resistance genes, aac(6')Ie-aph(2″)Ia and aph(3')IIIa in different Enterococcus species. The cross resistance patterns in Enterococcus faecalis MCC3063 to clinically important aminoglycosides by real time PCR were also studied. Southern hybridization experiments revealed the presence of aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes conferring HLAR in high molecular weight plasmids except in Lactobacillus plantarum. The plasmid encoded bifunctional aac(6')Ie-aph(2″)Ia gene was transferable from Enterococcus avium (n=2), E.cecorum (n=1), E. faecalis (n=1) and Pediococcus lolii (n=1) species into the recipient strain; E.faecalis JH2-2 by filter mating experiments thus indicating the possible risks of gene transfer into pathogenic strains. Molecular analysis of cross resistance patterns in native isolate of E.faecalis MCC3063 carrying aac(6')Ie-aph(2″)Ia and aph(3')IIIa gene was displayed by quantification of the mRNA levels in this study. For this, the culture was induced with increasing concentrations of gentamicin, kanamycin and streptomycin (2048, 4096, 8192, 16384μg/mL) individually. The increasing concentrations of gentamicin and kanamycin induced the expression of the aac(6')Ie-aph(2″)Ia and aph(3')IIIa resistance genes, respectively. Interestingly, it was observed that induction with streptomycin triggered a significant fold increase in the expression of the aph(3')IIIa gene which otherwise was not known to modify the aminoglycoside. This is noteworthy as streptomycin was found to confer cross resistance to structurally unrelated kanamycin. Also, expression of the aph(3')IIIa gene when induced with streptomycin, revealed that bacteria harbouring this gene will be able to overcome streptomycin bactericidal action at specific concentrations. HLAR in E.faecalis MCC3063 may be due to the combined expression of both the aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes which could be therapeutically challenging. A combined expression of both the genes in E.faecalis MCC3063 may yield HLAR which could be therapeutically challenging. The study highlights the significant alterations in the mRNA expression levels of aac(6')Ie-aph(2″)Ia and aph(3')IIIa in resistant pathogens, upon exposure to clinically vital aminoglycosides.

Multidrug-resistant enterococci in the hospital environment: detection of novel vancomycin-resistant E. faecium clone ST910.

The role of the hospital environment as a reservoir of resistant bacteria in Tunisia has been poorly investigated; however, it could be responsible for the transmission of multidrug-resistant bacteria. The objective was to study the prevalence of Enterococcus in the environment of a Tunisian hospital and the antibiotic resistance phenotype/genotype in recovered isolates, with special reference to vancomycin resistance. A total of 300 samples were taken (March-June, 2013) and inoculated in Slanetz-Bartley agar plates supplemented or not supplemented with 8 µg/mL of vancomycin. Antibiotic resistance genes were tested by polymerase chain reaction (PCR). The clonal relatedness of the vanA isolates was assessed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence testing (MLST). Enterococci were recovered in 33.3% of tested samples inoculated in SB medium. E faecium was the most prevalent species, followed by E. faecalis and E. casseliflavus. Antimicrobial resistance genes detected were as follows (number of isolates): erm(B) (71), tet(M) (18), aph(3')-IIIa (27), ant(6)-Ia (15), cat(A) (4), and van(C2) (6). Vancomycin-resistant-enterococci (VRE) were recovered from 14 samples (4.7%), when tested in SB-VAN. The 14 VRE (one per positive sample) were identified as E. faecium and contained the van(A),erm(B), tet(M), ant(6)-Ia, and aph(3')-IIIa genes. Thirteen of the VRE strains were ascribed by PFGE and MLST to a novel clone (new ST910), and only one VRE strain was typed as ST80 included in CC17. The emergence and spread of new clones of VRE, especially in the hospital environment in this country, could become particularly problematic.

Significant correlation of angiotensin converting enzyme and glycoprotein IIIa genes polymorphisms with unexplained recurrent pregnancy loss in north of Iran

Spontaneous abortion is considered as the most complex problem during pregnancy. Thrombophilia is resumed as a cause of recurrent pregnancy loss (RPL). Glycoprotein IIIa (GPIIIa) gene is involved in thrombosis and abortion. Angiotensin converting enzyme (ACE) converts angiotensin I to angiotensin II and is involved in thrombosis. The most common polymorphism in this gene is the insertion/deletion (I/D). In this study, we analyzed the association between ACE I/D and GPIIIa c.98C >T polymorphisms in women with unexplained RPL from the north of Iran. Sample population consisted of 100 women with unexplained RPL and 100 controls. The ACE I/D and GPIIIa c.98C>T polymorphisms were genotyped by TETRA-ARMS PCR. The association between genotypes frequency and RPL were analyzed using χ(2) and exact fisher tests. Associated risk with double genotype combinations was also investigated by binary logistic regression. There was significant association between ACE DD genotype and RPL (OR=2.04; 95% CI=0.94-4.44; p=0.036). ACE D Allele was also significantly associated with the RPL (OR=1.59; 95% CI=1.05-2.41; p=0.013). No significant association was observed between GPIIIa c.98C>T polymorphism and RPL. ACE I/D polymorphism may probably be a prognostic factor in female family members of women with the history of recurrent abortion.