Amphiphilic peptide Mastoparan-B induces conformational changes within the AdeB efflux pump, down-regulates adeB gene expression, and restores antibiotic susceptibility in an MDR strain of Acinetobacter baumannii.

Mastoparan B (MP-B) is an amphiphilic peptide with a potent antimicrobial activity against most Gram-negative bacteria. However, there is little information available on the inhibition of the Acinetobacter baumannii resistance-nodulation-cell-division (RND) efflux pump using this antimicrobial peptide. Here, we carried out a series of in-silico experiments to find the mechanisms underlying the anti-efflux activity of MP-B using a multi-drug resistant (MDR) strain of A. baumannii (AB). According to our findings, MP-B demonstrated a potent antibacterial activity against an MDR-AB (minimum inhibitory concentration [MIC] = 1 µg/mL) followed by a 20-fold reduction in the adeB gene expression in the presence of sub-MIC of this peptide. Using Groningen Machine for Chemicals Simulation (GROMACS) via PyMOL Graphical User Interface (GUI), (we observed that, the AdeB transporter had conserved helix-turn-helix regions and a tight pore rich in Phe and Ala residues. To understand how inhibition of the AdeB is achieved, we generated 20 apo-MP-B poses using the InterPep and SiteMap tools. The high-quality model was created by homology modeling and used for docking via AutoDock/Vina to identify the MP-B binding sites. We established that the most apo-MP-B formed H-bonds to the backbone of five amino acids in the Helix-5. As a result, the dihedral angles of the involved amino acids shift by 9.0-9.6 Ǻ, causing a change in the conformation of the AdeB protein. This led to helix conformation stereoisomerization and block the AdeB activity. MP-B presumably has dual mechanisms. (1) It blocks the AdeB transporter by changing its conformation. (2) MP-B influences the adeB gene expression by binding to G-protein which laterally controls efflux regulators like MarA, RamA, SoxS, and Rob proteins.

Insights from the molecular docking analysis of colistin with the PmrA protein model from Acinetobacter baumannii.

Acinetobacter baumannii (AB) is one of the most common causes of nosocomial infections. Therefore, it is of interest to design and develop drugs against Acinetobacter baumannii. A strain of AB showing MIC 32 µg/ml against colistin was isolated from a hospital environment in Iran. Hence, we document data to glean insights from the molecular docking analysis of colistin with the PmrA protein from this bacterium.

Stereochemical Trajectories of a Two-Component Regulatory System PmrA/B in a Colistin-Resistant Acinetobacter baumannii Clinical Isolate.

Background: There is limited information on the three-dimensional (3D) prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of phosphotransferase membrane receptor A/B (PmrA/B) in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods: The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and minimum inhibitory concentration assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 × 10−50). Results: Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 ß-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three ß-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. WP_071210493.1) revealed no distortion in conformation, due to Glu→Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 ß-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21→Ser and Ser28→Arg) in the transmembrane domain were detected. Conclusion: The DNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of colistin resistance in A. baumannii.

Genetic characterization of two vancomycin-resistant Staphylococcus aureus isolates in Kerman, Iran.

AIM: The aim of this study was the genetic characterization of two clinical vancomycin-resistant Staphylococcus aureus (VRSA) isolates. MATERIALS AND METHODS: Resistance to vancomycin was determined by phenotypic method. PCR was used for detection of mecA, vanA, ermA, ermB, ermC, msrA/B, aph(2")-Ic, aph(3')-IIIa, pvl, Immune Evasion Cluster [sea, sep, chip, sak and scn] genes and biofilm operon icaABCD. On the other hand, multilocus sequence typing and agr typing methods were performed for the determination of clonal relationship and van operon was detected and sequenced. RESULTS: Vancomycin-resistant Staphylococcus aureus strain 1 (VRSA-1) was positive for vanA, ermA, ermC, aph(2")-Ic, aph(3')-IIIa, sea, sep, icaD genes, belonging to agr type I; SCCmec type III; spa type t030; and ST239. However, the genetic characterization of Vancomycin-resistant Staphylococcus aureus strain 2 (VRSA-2) revealed the presence of various types of resistance genes vanA, ermA, ermC, aph(2")-Ic, aph(3')-IIIa, sea, icaD, relating to agr type I; SCCmec type III; spa type t459; and ST239. The presence of transposon Tn1546 was determined by PCR sequencing.The Basic Local Alignment Search Tool analysis of van operon in the VRSA isolates showed 99.6% sequence homology to Tn1546 in vancomycin-resistant enterococci, indicating the vanA operon has an enterococcal origin. CONCLUSION: In conclusion, the ST239 is one of the most common clones of MRSA isolates which involved the hospital-associated infections, therefore, the emergence of VRSA isolates with ST239 increased the spread of resistance to vancomycin in the hospital settings.

Antibiotic resistance, ESBL genes, integrons, phylogenetic groups and MLVA profiles of Escherichia coli pathotypes isolated from patients with diarrhea and farm animals in south-east of Iran.

The aims of this study were to investigate the prevalence, antibiotic resistance, presence of class 1 and 2 integrons, Extended Spectrum ß-Lactamases (ESBL) genes, phylogenetic group and epidemiological relationships of EPEC, ETEC and EHEC pathotypes isolated from patients with diarrhea and farm animals in south east region of Iran. A total of 671 diarrheagenic E. coli (DEC) were collected from stool samples of 395 patients with diarrhea and 276 farm cattles and goats. Presence of EPEC, ETEC and EHEC were identified using multiplex-PCR employing primers targeted the shiga toxin (stx), intimin (eae), bundle forming pili (bfp), and enterotoxins (lt and st) genes. The highest proportion of the patients (64%) were children under age 1-15 year (p ≤ 0.05). Among the isolates, atypical EPEC was detected in 26 patients and 14 animal stool samples, while typical EPEC was found in 2 cattles. ETEC isolates were detected in stools of 13 patients and 4 EHEC was identified in 3 goats and one cattle. The isolates were checked for susceptibility to 14 antibiotics. 50% (n = 13) of EPEC and 61.5% (n =8) of ETEC showed multi-drug resistance (MDR) profiles and one EPEC was found to be extensive drug resistant (XDR). In contrast, EHEC isolates were susceptible to the majority of antimicrobial agents. The MDR isolates were positive for blaTEM and blaCTX-M ESBL genes and carried class 1 integrons. Further study on the biofilm formation indicated that, 3 out of 4 EHEC isolates showed strong biofilm, while other pathotypes had either moderate, weak or no biofilm activity. Majority of EPEC isolates were belonged to phylogenetic group B1, all except one ETEC were classified as phylogenetic group A and two EHEC were belonged to phylogroup D, respectively. A multilocus variable tandem repeat analysis (MLVA) exhibited 22 distinct patterns. In conclusion, MLVA data showed high clonal diversity. Presence of EHEC in animal origins pose public health concern in this region.

Prevalence of ß-lactamase genes, class 1 integrons, major virulence factors and clonal relationships of multidrug-resistant Pseudomonas aeruginosa isolated from hospitalized patients in southeast of Iran.

OBJECTIVES: Pseudomonas aeruginosa is one of the most important nosocomial pathogens causing a high rate of mortality among hospitalized patients. Herein, we report the prevalence of antibiotic resistance genes, class 1 integrons, major virulence genes and clonal relationship among multidrug- resistant (MDR) P. aeruginosa, isolated from four referral hospitals in the southeast of Iran. MATERIALS AND METHODS: In this study, 208 isolates of P. aeruginosa were collected from four referral hospitals in southeast of Iran. Disk diffusion method was used to determine susceptibility to 13 antibacterial agents. AmpC was detected by phenotypic method and ß-lactamase genes, virulence genes and class 1 integrons were detected by PCR. Clonal relationship of the isolates was determined by RAPD-PCR. RESULTS: All the isolates were susceptible to polymyxin-B and colistin. Overall, 40.4% of the isolates were MDR, among which resistance to third generation cephalosporins, aminoglycosides, and carbapenems was 47.5%, 32.3% and 40%, respectively. None of the isolates was positive for bla NDM-1 genes, while 84.5% and 4.8% were positive for the bla IMP-1 and bla VIM, metallo-ß-lactamase genes, respectively. Incidence of class 1 integrons was 95% and AmpC was detected in 33% of the isolates. Prevalence of exoA, exoS, exoU, pilB and nan1 were 98.8%, 44%, 26%, 8.3% and 33.3%, respectively. RAPD profiles identified four large clusters consisting of 77 isolates, and two small clusters and three singletons. CONCLUSION: The rate of MDR P. aeruginosa isolates was high in different hospitals in this region. High genetic similarity among MDR isolates suggests cross-acquisition of infection in the region.

Serogroups, subtypes and virulence factors of shiga toxin-producing Escherichia coli isolated from human, calves and goats in Kerman, Iran.

AIM: The present study was conducted to detect the occurrence, serogroups, virulence genes and phylogenetic relationship of shiga toxin-producing Escherichia coli (STEC) in human, clave and goat in Kerman (southeast of Iran). BACKGROUND: STEC have emerged as the important foodborne zoonotic pathogens causing human gastrointestinal disease and confirming the risk to public health. METHODS: A total of 671 fecal samples were collected from diarrheic patients (n=395) and healthy calves (n=156) and goats (n=120) and screened for the presence of stx gene. Furthermore, the prevalence of stx1 and stx2 variants, serotypes (O157, O145, O103, O26, O111, O91, O128, and O45), phylogenetic groups and the presence of ehxA, eae, hylA, iha and saa virulence genes were studied. RESULTS: Prevalence of STEC in human diarrheic isolates was 1.3% (5 isolates), in claves was 26.3% (41 isolates) and in goats was 27.5% (33 isolates). stx1 gene was the most prevalent variant and detected in 75 isolates. Furthermore, stx1c was the most predominant stx subtype, found in 56 isolates. The ehxA identified in 36 (45.6%) isolates, followed by iha 5 (6.3%), eaeA 4 (5.1%), hlyA 2 (2.5%) and saa 2 (2.5%). Most of the isolates belonged to phylogroup B1. Only two O26 and one O91 isolates were detected in our study. CONCLUSION: Our results show that STEC strains were widespread among healthy domestic animals in the southeast of Iran.

Effect of nano-silver, nano-copper, deconex and benzalkonium chloride on biofilm formation and expression of transcription regulatory quorum sensing gene (rh1R) in drug-resistance Pseudomonas aeruginosa burn isolates.

BACKGROUND: Biofilm forming drug-resistant Pseudomonas aeruginosa are responsible for major death in burn center of different hospitals across the globe. OBJECTIVE: The aims of this study were to evaluate the effect of nano-silver (Ag), nano-copper (Cu), and two hospital disinfectants (deconex and benzalkonium chloride) on biofilm formation and expression of transcription regulatory quorum sensing gene rh1R in P. aeruginosa burn isolates. METHODS: 28 multidrug-resistant P. aeruginosa (MDRPA) strains were isolated from patients hospitalized in the burn center of a referral hospital in Kerman, Iran. Sizes and purities of nanoparticles were checked by TEM and X-ray diffraction (XRD) analysis. The Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the nanoparticles (NPs), deconex and benzalkonium chloride were determined by broth microdilution method. Antibiofilm activities of these compounds were measured by microtiter assay. Polymerase chain reaction (PCR) was used for detection of qacEΔ1, cepA, copA and rhlR genes. Quantification of rhlR gene expression in presence and absence of the above compounds was carried out by relative quantitative real-time PCR (qRT-PCR). RESULTS: Benzalkonium chloride had a potent antimicrobial activity and inhibited growth of all the isolates at MIC 0.06±0.2mg/mL, while nano-Ag was effective at MIC 20±0.2mg/mL. Furthermore, 28.5% of the isolates showed strong, 25% moderate, 14% weak and 32% demonstrated no biofilm activity. Ag NPs exerted highest antibiofilm activity, follow by deconex and benzalkonium chloride. The qacEΔ1 was absent in this study, whereas 17.8% and 60.8% of the isolates were positive for cepA and copA genes. Benzalkonium chloride, Ag NPs and deconex increased the expression of rhlR gene 64, 2 and 7 folds, respectively. CONCLUSION: Our results suggest that, there is direct relationship between decrease in antibiofilm activity and increase in expression of the rhlR gene in the presence of benzalkonium chloride. Absence of qacEΔ1 gene may be contributed in sensitivity of the isolates to the above agents.

Role of antigen-43 on biofilm formation and horizontal antibiotic resistance gene transfer in non-O157 Shiga toxin producing Escherichia coli strains.

BACKGROUND AND OBJECTIVES: The objectives of this study were to evaluate the antibiotic resistance profiles, biofilm formation, presence of antigen 43 (Ag43) gene, and transfer of antibiotic resistance phenotype among non-O157 Shiga toxin producing Escherichia coli (STEC). MATERIALS AND METHODS: From October 2014 to November 2015 a total of 276 stool samples were collected from healthy calves, goats and 395 patients with the sign of nonbloody diarrhea and screened for presence of stx and serotype O157 genes by polymerase chain reaction (PCR) technique. Susceptibility to 14 antibiotics was determined as per CLSI guideline. Presence of Ag43 and intimin (eaeA) genes were detected by PCR. Biofilm formation was measured by microtiter plate method. Conjugation was carried out by membrane filter technique. RESULTS: We isolated 74 (93.6%) non-O157 STEC strains from 41 calves, 33 goats and 5 (6.3%) patients' stools, however, no O157 serotype was detected in our study. Resistance was observed most commonly to tobramycin (66.2%), kanamycin (48.6%), and amikacin (29.7%) and less frequently to ciprofloxacin (4.1%), amoxicillin-clavulanic acid (5.4%), and ceftriaxone (9.5%) in isolates recovered from calves and goats fecal samples, whereas, all human isolates were sensitive to ceftazidime, ciprofloxacin, tobramycin and imipenem, respectively. Furthermore, Ag43 was detected in 60 STEC isolated from animals and 5 human origins (no eaeA gene was found in this study). Biofilm formation from Ag43+ and Ag43- colonies showed 20 isolates with strong biofilm activities. Cefotaxime resistance phenotype was transferred to E. coli ATCC 25922.1 (Nalr) by conjugation at a frequency of 1.6×10-4. CONCLUSION: From the above results we concluded that, human infections with non-O157 STEC were significantly low in Kerman. Ag43 was insignificant with biofilm quantity in most cases.

Insight into stereochemistry of a new IMP allelic variant (IMP-55) metallo-ß-lactamase identified in a clinical strain of Acinetobacter baumannii.

Metallo-ß-lactamases (MBLs) such as IMPs are broad-spectrum ß-lactamases that inactivate virtually all ß-lactam antibiotics including carbapenems. In this study, we investigated the hydrolytic activity, phylogenetic relationship, three dimensional (3D) structure including zinc binding motif of a new IMP variant (IMP-55) identified in a clinical strain of Acinetobacter baumannii (AB). AB strain 56 was isolated from an adult ICU of a teaching hospital in Kerman, Iran. It exhibited MIC 32µg/ml to imipenem and showed MBL activity. Hydrolytic property of the MBL enzyme was measured phenotypically. Presence of blaIMP gene encoded by class 1 integrons was detected by PCR-sequencing. Phylogenetic tree of IMP protein was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and 3D model including zinc binding motif was predicted by bioinformatics softwares. Analysis of IMP sequence led to the identification of a novel IMP-type designated as IMP-55 (GenBank: KU299753.1; UniprotKB: A0A0S2MTX2). Impact in term of hydrolytic activity compared to the closest variants suggested efficient imipenem hydrolysis by this enzyme. Evolutionary distance matrix assessment indicated that IMP-55 protein is not closely related to other A. baumannii IMPs, however, shared 98% homology with Escherichia coli IMP-30 (UniprotKB: A0A0C5PJR0) and Pseudomonas aeruginosa IMP-1 (UniprotKB: Q19KT1). It consisted of five α-helices, ten ß-sheets and six loops. A monovalent zinc ion attached to core of enzyme via His95, His97, His157 and Cys176. Multiple amino acid sequence alignments and mutational trajectory with reported IMPs showed 4 amino acid substitutions at positions 12(Phe→Ile), 31(Asp→Glu), 172(Leu→Phe) and 185(Asn→Lys). We suggest that the pleiotropic effect of mutations due to frequent administration of imipenem is responsible for emergence of new IMP variant in our hospitals.

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.

Prevalence of mip virulence gene and PCR-base sequence typing of Legionella pneumophila from cooling water systems of two cities in Iran.

Legionella pneumophila is the primary respiratory pathogen and mostly transmitted to human through water cooling systems and cause mild to severe pneumonia with high mortality rate especially in elderly both in hospitals and community. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods. Here, we investigated the presence of L. pneumophila mip gene in water samples collected from different hospitals cooling towers, nursing homes and building/hotels water coolants from two geographical locations of Iran (Kerman and Bam cities) during summer season of 2015 by both nested and real-time PCR methods. Analysis of the 128 water samples for presence of the mip gene by nested-PCR revealed, 18 (23%) positive cases in Kerman and 7(14%) in Bam. However, when samples were tested by real-time PCR, we identified 4 more new cases of L. pneumophila in the hospitals as well as nursing homes water systems that were missed by nested-PCR. The highest rate of contamination was detected in water obtained from hospitals cooling towers in both the cities (p≤0.05). Dendrogram analysis and clonal relationship by PCR-base sequence typing (SBT) of the L. pneumophila genomic DNAs in Kerman water samples showed close clonal similarities among the isolates, in contrast, isolates identified from Bam city demonstrated two fingerprint patterns. The clones from hospital water samples were more related to the L. pneumophila serogroup- 1.

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.

Biochemical detection of N-Acyl homoserine lactone from biofilm-forming uropathogenic Escherichia coli isolated from urinary tract infection samples.

BACKGROUND: N-Acyl homoserine lactone (AHL) is found to be the main component of quorum sensing (QS) in Gram-negative bacteria and plays an important role in biofilm formation. Little information is available regarding the role of AHL in biofilm formation in Escherichia coli (E. coli). The purpose of this investigation was to biochemically detect and characterize AHL activity in biofilm-forming uropathogenic E. coli (UPEC) isolated from urine samples of the patients with urinary tract infections (UTIs) in Kerman, Iran. METHODS: Thirty-five UPEC isolates were obtained from urine samples of the patients with UTIs referred to the Afzalipoor hospital. The isolates were identified by biochemical tests. Biofilm analyses of all the isolates were performed using the microtiter plate method at OD 490nm. N-Acyl homoserine lactone was separated from cell mass supernatants by liquid-liquid extraction (LLE) and analyzed by a colorimetric method. N-Acyl homoserine lactone functional groups were identified by Fourier Transform-Infrared Spectroscopy (FT-IR). RESULTS: The biofilm formation assay identified 10 (28.57%) isolates with strong, 16 (45.71%) with moderate, and 9 (25.71%) with weak biofilm activities. The UPEC isolates with strong and weak biofilm activities were subjected to AHL analyses. It was found that isolates with the highest AHL activities also exhibited strong adherence to microplate wells (P≤0.05). Two E. coli isolates with the highest AHL activities were selected for FT-IR spectroscopy. Peaks at 1764.33, 1377.99, and 1242.90 cm(-1) correspond to the C=O bond of the lactone ring, and the N=H and C-O bonds of the acyl chain, respectively. CONCLUSION: We found that many UPEC isolates exhibited strong biofilm formation. The control of this property by AHL may contribute to the pathogenesis of the organism in UTI's.

Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80-220nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO2 was also evaluated.

Antibiotic resistance pattern among biofilm producing and non producing Proteus strains isolated from hospitalized patients; matter of hospital hygiene and antimicrobial stewardship.

A retrospective study on antimicrobial susceptibility and biofilm production were carried out for eighty eight strains of Proteus strains isolated from UTI and other hospital samples during April 2011-April 2012. The antibiotic susceptibility was carried out by Kirby-Bauer disk diffusion and MIC by E-test. Biofilm production was measured by microtiter method and confirmed by Scanning electron microscopy. Plasmids from biofilm producing isolates were detected by alkaline lysis technique. From 88 patients infected by proteus species, 58% were female and 42% were mail. The most frequent age range was 20-29 (77.39%) and the least were 60-69 years old (3.4%) (p = 0.05). Eighty one isolates were identified as P. mirabilis while, 7 identified as P. vulgaris. 67.04% [n = 59] of the isolates showed MIC range (16-32 +/- 0.05 microg mL(-1)) to ceftriaxone, 46.59% [n = 41] exhibited least MIC range to chloramphenicol (8-64 +/- 0.08 microg mL(-1)). 31% [n = 28] of the isolates also exhibited MIC range 1-4 microg mL(-1) to ciprofloxacin. 17% [n = 15] of the isolates exhibited strong biofilm while, 6% [n = 6] did not show any biofilm (p < or = 0.05). Plasmid isolation from biofilm producing isolates revealed that stains number 19, 24 and 87' that produced strong biofilm carried similar high M. Wt. plasmid. From above results it can be concluded that the majority of Proteus isolated from UTI patients were belong to P. mirabilis. Ciprofloxacin was the most effective antibiotic for treatment of the infected patients. Limited number of the isolates could produce strong biofilm that were bearing plasmids. Majority of the biofilm producing isolates were also resistance at least to 4 antibiotics routinely prescribed in our hospital.