The Effect of Pimpinella Anisum and Origanum Vulgare Extracts Against Streptococcus Sanguinis, Streptococcus Mutans, and Streptococcus Salivarius.

Statement of the Problem: There are global efforts for introducing a new herbal antimicrobial agent with minimal side effects. There are some reports about the antimicrobial properties of Pimpinella anisum and Oregano Vulgare. Purpose: In this study, the antimicrobial properties of Pimpinella anisum and Oregano Vulgare have been assessed. Material and Method: In this experimental in vitro study, the dental plaque samples were collected from children aged 3 to 5 years old who were referred to a private dental office with diagnosis of dental caries. After determination of the bacterial colonies of Streptococcus sanguinis, Streptococcus mutans and Streptococcus salivarius, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic and methanolic extracts of Pimpinella anisum and Oregano vulgare were measured by macrodilution and microdilution methods. Results: The mean MIC and MBC of Pimpinella anisum extract and Oregano vulgare extract and their combination against Streptococcus mutans, Streptococcus sanguinis, and Streptococcus salivarius were statistically different (p< 0.001). The combination of these extracts showed the lowest MIC and MBC. Conclusion: Hydroalcoholic extracts of the Pimpinella anisum and Oregano Vulgare were effective antibacterial agent against Streptococcus mutans, Streptococcus salivarius, and Streptococcus sanguinis so the combination of these two extracts showed the highest antibacterial properties on all the bacteria evaluated.

Evaluation of Association of Oral Bacterial Profile with HBV and HCV Infection and T Lymphocyte Level in HIV-Positive Patients.

Background: This study was aimed to determine the oral bacterial profile of HIV-positive patients and their correlation with T lymphocyte and CD4 count and hepatitis B and C incidence. Methods: In this study, 73 patients who were diagnosed HIV-positive and were referred to Shiraz HIV research center for routine dental treatment were enrolled. Demographic data including sex, ethnicity, CD4+ T cell, and T lymphocyte counts were collected from their medical records. Supragingival dental plaque and samples from the dorsal of the tongue were collected by sterile swabs. These samples were transferred to the microbiology laboratory of Jahrom University of Medical Sciences. After primary biochemical test of cultured samples, assessment of bacterial biofilms was done by DNA extraction. Real-time PCR with specific primer of each bacterial species was done, and assessment of the results of real time PCR led to determination of the species of the evaluated bacteria. The correlation of bacterial prevalence with hepatitis B and C was evaluated by chi-square test. Furthermore, Mann-Whitney test was used to evaluate the association of bacterial species prevalence with CD4 and T lymphocyte level. Results: The prevalence of none of the detected bacteria had statistically significant relationship with hepatitis C, except for Peptostreptococcaceae (p value = 0.016) in the tongue plaque and Leptotrichia (p value = 0.022) in dental plaque. None of the evaluated bacteria showed any significant association with CD4 and T lymphocytes level, except for Kingella (p value = 0.025, 0.019, respectively), and also no significant correlation was reported with CD4, except for Gemella (p value = 0.021) and Campylobacter gracilis (p value = 0.029). Conclusions: The diversity of the detected bacteria was more in dental plaque, while their density was more noticeable in the tongue plaque. No significant correlation was found between the prevalence of most of the detected bacteria and CD4 level and T lymphocyte level and incidence of hepatitis B and C.

Class 1 integrons in non-clonal multidrug-resistant Acinetobacter baumannii from Iran, description of the new blaIMP-55 allele in In1243.

Infections and outbreaks caused by multidrug-resistant Acinetobacter baumannii (MDR-AB) are prevalent and have been reported worldwide over the past 20 or more years. Class 1 integron in MDR-AB plays an important role in the spread of antibiotic resistance in clinical settings. This study has been conducted to evaluate the detection of metallo-ß-lactamase, characterization of class 1 integron and determination of clonal relatedness among A. baumannii hospital isolates. Sixty-five clinical isolates of MDR-AB were recovered from two Iranian hospital's intensive care units from February to August 2013. Integrase (intI1) and blaIMP genes were detected in 70.8 % (n=46/65) and 9.23 % (n=6/65) of the isolates using PCR assay, respectively. No other metallo-ß-lactamase genes (blaVIM, blaSIM and blaNDM) were detected. PCR sequencing of integron gene cassette revealed the following arrays: blaOXA10-aacA4-blaIMP-55-cmlA5 (as a novel array was designated In1243), aacC1 and aadA1. Analysis of blaIMP gene revealed a new allele designated as blaIMP-55. Gene transfer experiment by conjugation showed the 36 kb conjugative plasmid harbouring In1243. The clonal assessment by repetitive extragenic palindromic PCR demonstrated a high-degree relatedness among the strains, but strains harbouring In1243 displayed a different repetitive extragenic palindromic PCR profile. In this study, we found that a novel class 1 integron (In1243) that encoded a new blaIMP allele resided on a transferable plasmid in non-clonal strains of MDR-AB.

Cloning and expression of quorum sensing N-acyl-homoserine synthase (LuxI) gene detected in Acinetobacter baumannii.

BACKGROUND AND OBJECTIVES: In present study we aimed to clone the luxI gene encoding N-acyl-homoserine synthase detected in clinical isolates of Acinetobacter baumannii and study its expression in Escherichia coli transformants. MATERIALS AND METHODS: Four A. baumannii hospital strains which demonstrated strong biofilm activity were selected in this investigation. The presence of luxI gene was detected using PCR technique. Purified PCR product DNA was initially cloned into pTG19 and transformed to E. coli DH5α. The gene was then recovered from agarose gel and ligated by T4 DNA ligase into pET28a expression vector using NdeI and XhoI enzymes. pET28a + luxI was transformed into E. coli BL21 (DE3). The luxI putative gene was further detected in the transformants by colony PCR. Expression of the luxI gene in the recombinant E. coli BL21 cells was studied by quantitative real time PCR (qRT-PCR) and the presence of N-acylhomoserine lactone (AHL) was checked by colorimetric assay and Fourier Transform Infra-Red (FT-IR) spectroscopy. RESULTS: We successfully cloned AHL gene from A. baumannii strain 23 to pET28a expression vector. There was four fold increases in expression of luxI in the transformants (P ≤ 0.05). It was found that, strain 23 and the transformants showed highest amount of AHL activity (OD = 1.524). The FT-IR analysis indicated stretching C=O bond of the lactone ring and primary amides (N=H) at 1764.69 cm(-1) and 1659.23 cm(-1) respectively. CONCLUSION: From above results we concluded that, luxI in A. baumannii is indeed responsible for AHL production and not regulation and pET28a vector allows efficient AHL expression in E. coli BL21 transformants.

Molecular Analysis and Expression of bap Gene in Biofilm-Forming Multi-Drug-Resistant Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is commonly resistant to nearly all antibiotics due to presence of antibiotic resistance genes and biofilm formation. In this study we determined the presence of certain antibiotic-resistance genes associated with biofilm production and the influence of low iron concentration on expression of the biofilm-associated protein gene (bap) in development of biofilm among multi-drug-resistant A. baumannii (MDRAB). METHODS: Sixty-five MDRAB isolates from clinical samples were collected. Molecular typing was carried out by random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR). Biofilm formation was assayed by the microtiter method. RESULTS: The sequence of bap was determined and deposited in the GenBank database (accession no. KR080550.1). Expression of bap in the presence of low iron was analyzed by relative quantitative real time PCR (rqRT-PCR). Nearly half of the isolates belonged to RAPD-types A and B remaining were either small clusters or singleton. The results of biofilm formation revealed that 23 (35.4%), 18 (27.7%), 13 (20%), and 11 (16.9%) of the isolates had strong, moderate, weak, and no biofilm activities, respectively. ompA and csuE genes were detected in all, while bap and blaPER-1 were detected in 43 (66%) and 42 (64%) of the isolates that showed strong and moderate biofilm activities (p ≤ 0.05), respectively. Analysis of bap expression by rqRT-PCR revealed five isolates with four-fold bap overexpression in the presence of low iron concentration (20 µM). CONCLUSION: The results suggest that bap overexpression may influence biofilm formation in presence of low iron concentration.

Effect of iron on expression of efflux pump (adeABC) and quorum sensing (luxI, luxR) genes in clinical isolates of Acinetobacter baumannii.

Resistance-nodulation-division efflux system (RND) adeABC contributes to intrinsic resistance to various drug classes in Acinetobacter baumannii. Similarly, quorum sensing (QS) plays an important role in the biofilm formation and pathogenicity of this bacterium. The aims of this study were to evaluate the influence of iron limitation on the expression of efflux pump (adeABC) genes and QS (luxI, luxR) system by relative quantitative real-time polymerase chain reaction (qRT-PCR). In addition, DNA sequence and phylogenetic relatedness of biofilm-associated protein (Bap) gene was also investigated. Sixty-five multidrug-resistant isolates of A. baumannii were recovered from ICU patients of three hospitals in Kerman, Iran. The isolates were highly resistant to at least 11 antibiotics (MIC ≥64 µg/mL); however, 87% and 89% were susceptible to colistin and tigecycline, respectively (MIC 0.05 µg/mL) (p ≤ 0.05). We detected the presence of RND efflux pump, QS, and bap genes with the frequencies of 92% (adeA), 61.5% (adeB), 84.6% (adeC), 80% (luxI), 61% (luxR), and 66% (bap), respectively. qRT-PCR analysis showed that in some isolates, expression of both adeABC and luxI/R was increased more than fourfold in the presence of low iron (20 µm), suggesting the additional regulatory role of iron on both efflux pump and QS system. Alignment and phylogenetic analysis on the strong biofilm forming isolates confirmed that the fragments amplified were indeed part of bap gene and deduced sequence was similar to A. baumannii K9B410.

Molecular Detection of Class-D OXA Carbapenemase Genes in Biofilm and Non-Biofilm Forming Clinical Isolates of Acinetobacter baumannii.

BACKGROUND: Emergence and spread of carbapenemase (bla OXA) genes in multidrug resistant Acinetobacter baumannii (MDR-AB) forming biofilm complicated treatment of the patients infected with this microorganism particularly in intensive care units (ICUs). OBJECTIVES: The current study aimed to determine the prevalence of molecular class-D OXA carbapenemase in biofilm and non-biofilm forming strains of MDR-AB. MATERIALS AND METHODS: A total of 65 strains of MDR-AB were isolated from the patients hospitalized in the ICU of two hospitals in Kerman, Iran. The isolates were identified by conventional microbiological tests as well as API 20NE assay. Antibiotic susceptibility was carried out by disk diffusion method; minimum inhibitory concentration (MIC) of carbapenems was measured by E-test. The presence of bla OXA genes among the isolates were studied by duplex-polymerase chain reaction and application of appropriate primers. Biofilm formation was detected by microtiter plate method. RESULTS: The isolates were highly resistant to ciprofloxacin, levofloxacin, piperacillin, nalidixic acid and third generation cephalosporins such as tigecycline (7%; n = 5) and colistin (13%; n = 8). Among the isolates, 77% (n = 50) exhibited high MIC (265µg/mL) for imipenem. Both the bla OXA-51 and bla OXA-23 like genes coexisted in all the isolates; while, bla OXA-24/40 like gene was only detected in 29 imipenem-resistant strains (P ≤ 0.05). The bla OXA-58 like gene was not detected among the isolated strains. Quantification of biofilm introduced 23 isolates (including bla OXA-24/40 strains) with efficient attachment to microtiter plate; while, those isolates without bla OXA-24/40, or imipenem-sensitive strains formed weak or no biofilm. CONCLUSIONS: Coexistence of the bla OXA-51, bla OXA-23 and bla OXA-24/40 like genes, along with formation of strong biofilm, in MDR-AB strains particularly with indiscriminate use of imipenem, complicated treatment of the patients infected with these bacteria in the hospitals understudy.

Iron limitation enhances acyl homoserine lactone (AHL) production and biofilm formation in clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii is an important source of infections in intensive care units (ICUs) of our hospitals in Kerman, Iran and the most frequently isolated strains produce biofilm. There is a little information about role of iron (Fe) levels on acyl homoserine lactone (AHL) production and biofilm formation in this microorganism. In the present study, we investigated the influence of iron-III limitation on AHL, siderophore, catechol and virulence factors in the biofilm forming clinical strains of A. baumannii. A total of 65 non-duplicated multidrug resistance (MDR) strains of A. baumannii were isolated from patients in ICUs of 2 hospitals in Kerman, Iran. Antibiotic susceptibility, siderophore and other iron chelators, hemolysis, cell twitching motility, capsule, gelatinase and DNase were studied. Presence of quorum sensing, LuxI and LuxR genes was detected by multiplex-PCR. AHL activity quantified by colorimetric method and the functional groups were determined by Fourier Transform Infra-Red Spectroscopy (FT-IR). Biofilm formation was detected by microtiter plate technique. All of the isolates were resistant to third generation of cephalosporins, ciprofloxacin, levofloxacin, tetracycline, whereas, 78% and 81% were resistant to amikacin and carbapenems, respectively. The siderophore activity was highest at 20 µM Fe(3+) (70%); however, it decreased to 45% as concentration of Fe(3+) increased to 80 µM. Furthermore, screening of the isolates for LuxI and LuxR genes showed that presence of both genes required in the isolates with high AHL activity. FT-IR analysis indicated C=O bond of the lactone ring and primary amides. Significantly, a higher amount of AHL (70%) was detected in the presence of low concentration of iron-III (20 µM); as iron concentration increased to 80 µM, the AHL activity was reduced to 40% (P ≤ 0.05). All the isolates exhibited twitching motility and had a capsule. No any gelatinase or DNase activity was detected. Quantification of the biofilm formation introduced 23 isolates with efficient attachment to microplate wells and strong biofilm. We found that both the AHL production and biofilm formation were regulated by iron concentration in a dose dependent manner. These findings provide evidence that iron limitation plays an important regulatory role in AHL and siderophore production resulting in strong or weak biofilm, thereby helping the organism to persist in less available micronutrient environment.