Molecular detection of Class 1, 2, and 3 integrons in hypervirulent and classic Klebsiella pneumoniae isolates: A cross-sectional study.

Background and Aims: The "hypervirulent" variant of Klebsiella pneumoniae (hvKp) is an emerging pathogen that cause life-threatening infection. The present study was conducted to identify the prevalence of hvKp and to investigate the presence class 1, 2, and 3 integrons in these isolates. Methods: A cross-sectional study was conducted at three teaching hospitals, Ahvaz, South-west of Iran, from January 1, 2019 to December 31, 2020. Samples were collected from inpatients and included only the first samples collected from each patient. K. pneumoniae strains were isolated from different specimens using biochemical test and confirmed by targeting 16S-23S rDNA internal transcribed spacer. HvKp isolates were recovered using string test and were further characterized by detection virulence-associated genes (rmpA, iucA, and magA). Antibiotic susceptibility patterns of isolates were determined using the disc diffusion method. Isolates were screened for presence the integron genes (intI, intII, and intIII) and repetitive element sequence-based polymerase chain reaction (PCR) performed to determine strain relatedness. SPSS version 22 was used for the data analysis. Results: Seventy-one (77%) of isolates showed multidrug-resistant (MDR) phenotype. HvKP accounted for 14% (13/92) of cKp isolated from blood (46%) and urinary tract infection (38%), and the great majority of them (61.5%; 8/13) exhibited MDR phenotype. Using the PCR assay, 29 of 92 isolates (31.5%) were found to have positive results for the presence of IntI. Three of the IntI-positive strains were hvKP. Class 2 integron was present in 8/92 cKp isolates. Integron Class 2 was found to coexist with Class 1 integron in 3/8 isolates. All integron-positive isolates (IntI and/or IntII) were resistant to at least three different classes of antibiotics and showed MDR phenotype. No Class 3 integrons were detected among the isolates. Conclusion: The results of our study revealed that considering the role of integrons in facilitating the acquisition and dissemination of resistance genes among bacteria, monitoring the emergence of hvKp, emphasizing on the mechanism of antimicrobial resistance, can prevent from the spread of carbapenemase-producing hvKp strains.

Design of a novel analogue peptide with potent antibiofilm activities against Staphylococcus aureus based upon a sapecin B-derived peptide.

Nowadays, antimicrobial peptides are promising to confront the existing global crisis of antibiotic resistance. Here, a novel analogue peptide (mKLK) was designed based upon a D-form amidated sapecin B-derived peptide (KLK) by replacing two lysine residues with two tryptophan and one leucine by lysine, and inserting one alanine. The mKLK displayed superior amphipathic helixes in which the most of hydrophobic residues are confined to one face of the helix and had a higher hydrophobic moment compared with KLK. The mKLK retained its antibacterial activity and structure in human serum, suggesting its stability to proteolytic degradation. The values of MIC and MBC for mKLK were equal to those of KLK against clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA). However, mKLK showed more capability of in vitro inhibiting, eradicating, and dispersing MRSA and MSSA biofilms compared with KLK. Furthermore, a remarkable inhibitory activity of mKLK against MRSA and MSSA biofilms was seen in the murine model of catheter-associated biofilm infection. Results of this study show that mKLK not only exhibits antibacterial activity and serum stability but also a potent biofilm inhibitory activity at sub-MIC concentrations, confirming its potential therapeutic advantage for preventing biofilm-associated MRSA and MSSA infections.

In vitro and in vivo antibiofilm activity of the synthetic antimicrobial peptide WLBU2 against multiple drug resistant Pseudomonas aeruginosa strains.

BACKGROUND: The global crisis of antibiotic resistance increases the demand for the novel promising alternative drugs such as antimicrobial peptides (AMPs). Here, the antibiofilm activity of the WLBU2 peptide against Pseudomonas aeruginosa (P. aeruginosa) isolates was investigated in this study. METHODS: Two clinical MDR and carbapenem resistant P. aeruginosa (CRPA) isolates, and standard P. aeruginosa ATCC 27,853 were investigated. The MIC and MBC of WLBU2 were determined. The MBIC was determined to evaluate inhibitory activity of WLBU2 on biofilm formation and MBEC to dispersal activity on preformed biofilm. The relative expression levels of biofilm-associated genes including rhlI, rhlR, lasI and lasR were analyzed using RT-qPCR. In vivo evaluation of inhibitory effect of WLBU2 on biofilm formation was performed in the murine models of P. aeruginosa biofilm-associated subcutaneous catheter infection. RESULTS: MIC and MBC of WLBU2 for both MDR and ATCC 27,853 P. aeruginosa strains were 8 and 16 µg/mL, respectively, while both the MIC and MBC against the CR strain were 4 µg/mL. MBIC was estimated to be 64 µg/ml for all strains. MBEC against MDR and ATCC 27,853- P. aeruginosa strains was 128 µg/ml and against CRPA was 64 µg/ml. The bacterial adhesion to a static abiotic solid surface (the surface in the polypropylene microtiter wells) was significantly inhibited at 1/4× MIC in all P. aeruginosa strains and at 1/8× MIC in CRPA strain (P < 0.05). Following treatment with WLBU2 at 1/8× MIC, significant inhibition in biofilm formation was observed in all isolates (P < 0.05). Results of the colorimetric assay showed that WLBU2 at 4× MIC was able to disperse 69.7% and 81.3% of pre-formed biofilms on abiotic surface produced by MDR and standard (ATCC 27,853) P. aeruginosa, respectively (P < 0.03), while a 92.2% reduction in the CRPA biofilm was observed after treatment with 4× MIC WLBU2 (P < 0.03). The expression levels of all genes in isolates treated with 1/2 MIC of WLBU2 were down-regulated by more than four-fold compared to the untreated isolates (P < 0.05). WLBU2 significantly inhibited biofilm formation in murine catheter-associated CRPA infection model at 1/4×MIC, 1/2×MIC, and 1×MIC by 33%, 52%, and 67%, respectively. CONCLUSION: Considering relatively strong inhibitory and eradication potency of WLBU2 on the P. aeruginosa biofilms in in vitro and in vivo conditions, the peptide can be considered as a promising candidate for designing an antibiofilm drug.

Toxin-antitoxin Genes Expression in Multidrug-resistant Mycobacterium tuberculosis Isolates under Drug Exposure.

INTRODUCTION: Toxin-antitoxin systems (TAs) are highly conserved in Mycobacterium tuberculosis (Mtb). The TAs role in maintaining and disseminating drug resistance in bacterial populations has been indicated. So, we aimed to analyze the expression level of mazEF-related genes in drugsusceptible and multidrug-resistant (MDR) Mtb isolates under isoniazid (INH) and rifampin (RIF) stress. METHODS: We obtained 23 Mtb isolates, including 18 MDR and 5 susceptible isolates, from the Ahvaz Regional TB Laboratory collection. The expression levels of mazF3, mazF6, and mazF9 toxin genes, and mazE3, mazE6, and mazE9 antitoxin genes in MDR and susceptible isolates were evaluated by quantitative real-time PCR (qRT-PCR) after exposure to RIF and INH. RESULTS: The mazF3, F6, and F9 toxin genes were overexpressed in at least two MDR isolates in the presence of RIF and INH, in contrast to mazE antitoxin genes. More MDR isolates were induced to overexpress mazF genes by RIF than INH (72.2% vs. 50%). Compared to the H37Rv strain and susceptible isolates, the expression levels of mazF3,6 by RIF and mazF3,6,9 by INH were significantly upregulated in MDR isolates (p<0.05), but no remarkable difference was detected in the expression level of mazF9 genes by INH between these groups. In susceptible isolates, the expression levels of mazE3,6 by RIF and mazE3,6,9 by INH were induced and enhanced significantly compared to MDR isolates, but there was no difference between MDR and H37Rv strain. CONCLUSION: Based on the results, we propose that mazF expression under RIF/INH stress may be associated with drug resistance in Mtb in addition to mutations, and the mazE antitoxins may be related to enhanced susceptibility of Mtb to INH and RIF. Further experiments are needed to investigate the exact mechanism underlying the TA system's role in drug resistance.

Molecular evaluation of aminoglycosides resistance and biofilm formation in Klebsiella pneumoniae clinical isolates: A cross-sectional study.

Background and Aims: Resistance to antibiotics and the capability to develop biofilm as two main virulent determinants of Klebsiella pneumoniae have important role in infection persistence. The aim of the study was to evaluate the association between the prevalence of aminoglycoside resistance and virulence genes and biofilm formation capacity in K. pneumoniae strains isolated from hospitalized patients in South-West of Iran. Methods: A total of 114 non-duplicate clinical isolates of K. pneumoniae collected from Ahvaz teaching hospitals. Identification of species was performed by biochemical tests and then confirmed by polymerase chain reaction (PCR) of rpoB gene. The susceptibility to antibiotics was determined by Kirby-Bauer disk diffusion method. Biofilm formation was assessed by microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants including fimbrial genes, aminoglycoside modifying enzymes- and 16S rRNA methylase (RMTase) genes. Results: Totally, all collected strains were carbapenem resistant and showed multidrug- and extensively drug-resistance phenotype (75% and 25%, respectively). Seventy-one percent (n = 81) of isolates were non-susceptible to aminoglycosides. Among aminoglycoside antibiotics, K. pneumoniae isolates showed the highest and lowest resistance rates to tobramycin (71%) and the amikacin (25%), respectively. All biofilm producer strains were positive for the presence virulence determinants including ecpA, fimA, mrkD, and mrkA. Of 81 aminoglycosides non-susceptible isolates 33% were positive for the presence ant (2″)-Ia as the most prevalent gene followed by aac (3')-IIa and armA (27%), aac (6')-Ib (18%), and aph (3')-Ia (15%). Conclusion: K. pneumoniae isolates showed the highest and the lowest aminoglycoside resistance rates to tobramycin and amikacin, respectively. Majority of isolates were biofilm producers and there was significant association between antibiotic resistance pattern and the strength of biofilm production. The ant(2″)-Ia, aac (3')-IIa, and armA genes in aminoglycoside-resistant isolates.

Diagnostic Accuracy of Rapid Antigen Tests for COVID-19 Detection: A Systematic Review With Meta-analysis.

Introduction: Reverse transcription-polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 is time-consuming and sometimes not feasible in developing nations. Rapid antigen test (RAT) could decrease the load of diagnosis. However, the efficacy of RAT is yet to be investigated comprehensively. Thus, the current systematic review and meta-analysis were conducted to evaluate the diagnostic accuracy of RAT against RT-PCR methods as the reference standard. Methods: We searched the MEDLINE/Pubmed and Embase databases for the relevant records. The QUADAS-2 tool was used to assess the quality of the studies. Diagnostic accuracy measures [i.e., sensitivity, specificity, diagnostic odds ratio (DOR), positive likelihood ratios (PLR), negative likelihood ratios (NLR), and the area under the curve (AUC)] were pooled with a random-effects model. All statistical analyses were performed with Meta-DiSc (Version 1.4, Cochrane Colloquium, Barcelona, Spain). Results: After reviewing retrieved records, we identified 60 studies that met the inclusion criteria. The pooled sensitivity and specificity of the rapid antigen tests against the reference test (the real-time PCR) were 69% (95% CI: 68-70) and 99% (95% CI: 99-99). The PLR, NLR, DOR and the AUC estimates were found to be 72 (95% CI: 44-119), 0.30 (95% CI: 0.26-0.36), 316 (95% CI: 167-590) and 97%, respectively. Conclusion: The present study indicated that using RAT kits is primarily recommended for the early detection of patients suspected of having COVID-19, particularly in countries with limited resources and laboratory equipment. However, the negative RAT samples may need to be confirmed using molecular tests, mainly when the symptoms of COVID-19 are present.

Antibiotic resistance, biofilm production ability and genetic diversity of carbapenem-resistant Pseudomonas aeruginosa strains isolated from nosocomial infections in southwestern Iran.

BACKGROUND: This study was aimed to evaluate the antibiotic resistance, biofilm formation, and genetic diversity of carbapenem-resistant Pseudomonas aeruginosa (CRPA) strains isolated from four types of nosocomial infections (NIs) including urinary tract infection (UTI), ventilator-associated pneumonia (VAP), surgical site infection (SSI), and bloodstream infection (BSI). METHODS AND RESULTS: In total, 115 isolates of NIs-causing P. aeruginosa were collected from NIs. Antibiotic susceptibility testing (AST) was performed using disk diffusion method and minimum inhibitory concentrations. Biofilm formation was tested on 96-well polystyrene microtiter plates (MTP). CRPA isolates were genotyped using multiple-locus variable number of tandem repeat analysis (MLVA). The most resistance and susceptibility rates were observed to amikacin (70.6%) and colistin (96.1%), respectively. Colistin and meropenem were the most active antimicrobial agents in VAP, SSI, and BSI. While, colistin and cefepime were the most active in UTIs. In total, 52.2% (n = 60/115) of P. aeruginosa isolates were carbapenem resistant, of which 95.0%, 55.0%, and 5.0% were multidrug-resistant, extensively drug-resistant, and pandrug-resistant, respectively. There was a significant association between resistance to carbapenem and resistance to other antibiotics except for piperacillin/tazobactam. The biofilm production of CRPA isolates was 95.0%, of which 23.3% were strong biofilm producers. Based on MLVA, there were 34 different types of CRPA isolates classified into three main clusters and 5 sub clusters. CONCLUSION: The association of CRPA with other antibiotic resistance, the high rates of biofilm production, and the high genetic diversity of the isolates may be a warning of the need for a careful surveillance program.

Antimicrobial and anti-biofilm potencies of dermcidin-derived peptide DCD-1L against Acinetobacter baumannii: an in vivo wound healing model.

BACKGROUND: The global emergence of Acinetobacter baumannii resistance to most conventional antibiotics presents a major therapeutic challenge and necessitates the discovery of new antibacterial agents. The purpose of this study was to investigate in vitro and in vivo anti-biofilm potency of dermcidin-1L (DCD-1L) against extensively drug-resistant (XDR)-, pandrug-resistant (PDR)-, and ATCC19606-A. baumannii. METHODS: After determination of minimum inhibitory concentration (MIC) of DCD-1L, in vitro anti-adhesive and anti-biofilm activities of DCD-1L were evaluated. Cytotoxicity, hemolytic activity, and the effect of DCD-1L treatment on the expression of various biofilm-associated genes were determined. The inhibitory effect of DCD-1L on biofilm formation in the model of catheter-associated infection, as well as, histopathological examination of the burn wound sites of mice treated with DCD-1L were assessed. RESULTS: The bacterial adhesion and biofilm formation in all A. baumannii isolates were inhibited at 2 × , 4 × , and 8 × MIC of DCD-1L, while only 8 × MIC of DCD-1L was able to destroy the pre-formed biofilm in vitro. Also, reduce the expression of genes involved in biofilm formation was observed following DCD-1L treatment. DCD-1L without cytotoxic and hemolytic activities significantly reduced the biofilm formation in the model of catheter-associated infection. In vivo results showed that the count of A. baumannii in infected wounds was significantly decreased and the promotion in wound healing by the acceleration of skin re-epithelialization in mice was observed following treatment with 8 × MIC of DCD-1L. CONCLUSIONS: Results of this study demonstrated that DCD-1L can inhibit bacterial attachment and biofilm formation and prevent the onset of infection. Taking these properties together, DCD-1L appears as a promising candidate for antimicrobial and anti-biofilm drug development.

Mechanism of Action, Resistance, Synergism, and Clinical Implications of Delamanid Against Multidrug-Resistant Mycobacterium tuberculosis.

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis (MTB) remain a primary global threat to the end of tuberculosis (TB) era. Delamanid (DLM) is a nitro-dihydro-imidazooxazole derivative utilized to treat MDR-TB. DLM has distinct mechanism of action, inhibiting methoxy- and keto-mycolic acid (MA) synthesis through the F420 coenzyme mycobacteria system and generating nitrous oxide. While DLM resistance among MTB strains is uncommon, there are increasing reports in Asia and Europe, and such resistance will prolong the treatment courses of patients infected with MDR-TB. In this review, we address the antimycobacterial properties of DLM, report the global prevalence of DLM resistance, discuss the synergism of DLM with other anti-TB drugs, and evaluate the documented clinical trials to provide new insights into the clinical use of this antibiotic.

Substitution of lysine for isoleucine at the center of the nonpolar face of the antimicrobial peptide, piscidin-1, leads to an increase in the rapidity of bactericidal activity and a reduction in toxicity.

Purpose: Piscidin-1 is an effective antimicrobial peptide (AMP) against a variety of microbes. However, its toxicity has been reported as a limitation for its potential therapeutic applications. The toxicity of piscidin-1 may be related to the long nonpolar face of this AMP. Here, we investigated different piscidin-1 analogs to reach a peptide with the reduced toxicity. Material and methods: In vitro and in vivo antibacterial activity and toxicity of piscidin-1 analogs generated by replacement of isoleucine at the border (I9) or the center (I16) of the nonpolar face of piscidin-1 by alanine or lysine were investigated. Results: The results indicated that among all peptides, piscidin-1 with the highest HPLC retention time (RT) and I16K-piscidin-1 with the lowest RT had the highest and lowest cytotoxicity, respectively. Although I16K-piscidin-1 possessed the same MIC value as the parent peptide (piscidin-1) and other analogs, I16K-piscidin-1 exhibited a higher rapidity of bactericidal action at 5×MIC. The ß-galactosidase leakage and propidium iodide staining assays indicated a higher pore-forming capacity of I16K-piscidin-1 relative to the parent peptide (piscidin-1). Taken together, RT is suggested to have a direct association with the toxicity and an inverse association with the rapidity of bactericidal action and pore-forming capacity. After infection of mice with clinical colistin-resistant Acinetobacter baumannii or clinical methicillin-resistant Staphylococcus aureus strains, treatment with I16K-piscidin-1, but not piscidin-1 and other analogs, resulted in a significantly stronger bactericidal potency. Furthermore, I16K-piscidin-1 exhibited the lowest in vivo toxicity.  Conclusion: Overall, in vitro and in vivo comparison of piscidin-1 and its analogs together documented that replacement of isoleucine at the center of the nonpolar face of piscidin-1(I16) by lysine leads to not only a decrease in toxicity potential but also an increase in bactericidal potential.

Identification and Characterization of Novel Antimicrobial Peptide from Hippocampus comes by In Silico and Experimental Studies.

Antimicrobial peptides (AMPs) have attracted attentions as a novel antimicrobial agent because of their unique activity against microbes. In the present study, we described a new, previously unreported AMP, moronecidin-like peptide, from Hippocampus comes and compared its antimicrobial activity with moronecidin from hybrid striped bass. Antibacterial assay indicated that gram-positive bacteria were more sensitive to moronecidin and moronecidin-like compared with gram-negative bacteria. Furthermore, both AMPs were found to exhibit effective antifungal activity. Comparative analysis of the antimicrobial activity revealed that moronecidin-like peptide has higher activity against Acinetobacter baumannii and Staphylococcus epidermidis relative to moronecidin. Both moronecidin-like and moronecidin peptides retained their antibacterial activity in physiological pH and salt concentration. The time-killing assay showed that the AMPs completely killed A. baumannii and S. epidermidis isolates after 1 and 5 h at five- and tenfold above their corresponding MICs, respectively. Anti-biofilm assay demonstrated that peptides were able to inhibit 50% of biofilm formation at sub-MIC of 1/8 MIC. Furthermore, moronecidin-like significantly inhibited biofilm formation more than moronecidin at 1/16 MIC. Collectively, our results revealed that antimicrobial and anti-biofilm activities of moronecidin-like are comparable to moronecidin. In addition, the hemolytic and cytotoxic activities of moronecidin-like were lower than those of moronecidin, suggesting it as a potential novel therapeutic agent, and a template to design new therapeutic AMPs.

Association of virulence gene expression with colistin-resistance in Acinetobacter baumannii: analysis of genotype, antimicrobial susceptibility, and biofilm formation.

BACKGROUND: Acinetobacter baumannii causes difficult-to-treat nosocomial infections, which often lead to morbidity due to the development of antimicrobial drug resistance and expression of virulence genes. Data regarding the association of resistance to colistin, a last treatment option, and the virulence gene expression of A. baumannii is scarce. METHODS: We evaluated the MLVA genotype, antimicrobial resistance, and biofilm formation of 100 A. baumannii isolates from burn patients, and further compared the in vitro and in vivo expression of four virulence genes among five colistin-resistant A. baumannii (Cst-R-AB) isolates. Five Cst-R-AB isolates were tested; one from the present study, and four isolated previously. RESULTS: Our results showed that reduced expression of recA, along with increased in vivo expression of lpsB, dnaK, and blsA; are associated with colistin resistance among Cst-R-AB isolates. Differences in virulence gene expressions among Cst-R-AB isolates, may in part explain common discrepant in vitro vs. in vivo susceptibility data during treatment of infections caused by Cst-R-AB. CONCLUSIONS: Our findings highlight the intricate relationship between colistin-resistance and virulence among A. baumannii isolates, and underscore the importance of examining the interactions between virulence and antimicrobial resistance toward efforts to control the spread of multidrug-resistant A. baumannii (MDR-AB) isolates, and also to reduce disease severity in burn patients with MDR-AB infection.

Growth Rate and Biofilm Formation Ability of Clinical and Laboratory-Evolved Colistin-Resistant Strains of Acinetobacter baumannii.

Two different mechanisms of resistance to colistin in Acinetobacter baumannii have been described. The first involves the total loss of lipopolysaccharide (LPS) due to mutations in the lpxACD operon, which is involved in the lipid A biosynthesis pathway. The second entails the addition of ethanolamine to the lipid A of the LPS resulting from mutations in the PmrAB two-component system. To evaluate the impact of colistin resistance-associated mutations on antimicrobial resistance and virulence properties, four pairs of clinical and laboratory-evolved colistin-susceptible/colistin-resistant (ColS/ColR) A. baumannii isolates were used. Antimicrobial susceptibility, surface motility, in vitro and in vivo biofilm-forming capacity, in vitro and in vivo expression levels of biofilm-associated genes, and in vitro growth rate were analyzed in these strains. Growth rate, in vitro and in vivo biofilm formation ability, as well as expression levels of biofilm-associated gene were reduced in ColR LPS-deficient isolate (the lpxD mutant) when compared with its ColS partner, whereas there were not such differences between LPS-modified isolates (the pmrB mutants) and their parental isolates. Mutation in lpxD was accompanied by a greater reduction in minimum inhibitory concentrations of azithromycin, vancomycin, and rifampin than mutation in pmrB. Besides, loss of LPS was associated with a significant reduction in surface motility without any change in expression of type IV pili. Collectively, colistin resistance through loss of LPS causes a more considerable cost in biological features such as growth rate, motility, and biofilm formation capacity relative to LPS modification. Therefore, ColR LPS-modified strains are more likely to spread and transmit from one patient to another in hospital settings, which results in more complex treatment and control.

An investigation of electrospun Henna leaves extract-loaded chitosan based nanofibrous mats for skin tissue engineering.

Wound healing characteristics of some plant extracts have been well known for many years, and they have been utilized for such applications in traditional way. Recently electrospun nanofibrous mats showed promising properties for tissue engineering and especially for skin repair. It is expected that incorporation of plant extracts into such structures could provide higher performance and synergistic effect for biomedical and wound healing applications. The final purpose of this study is to fabricate chitosan based nanofiber mats loaded with a traditional plant extract of Lawsonia inermis (Henna) leaves to enhance the antibacterial efficacy and wound healing of the precursor nanofibers. The morphology, structure, mechanical properties and swelling and weight loss degree of the electrospun nanofibers have been investigated in this study. Antibacterial activity, cell biocompatibility evaluations and in vivo wound healing activity of the abovementioned mats were also studied. The FESEM images of Henna leaves extract-loaded nanofibers proved that homogeneous, smooth and defect free nanofibers of 64-87nm in diameter have been prepared. Presence of Henna extract in the electrospun fibers was approved by Fourier Transform Infrared spectroscopy. Incorporation of Henna extract into the nanofiber mats exhibited significant synergistic antibacterial activity against bacterial cells. It was well supported by the results of cell viability and proliferation of human foreskin fibroblast cells on the prepared scaffolds. Therefore, the results of this work showed that Henna leaves extract incorporated chitosan nonwoven mats have a great potential to be used as the biodegradable, biobased and antibacterial wound healing dressings.

Modulation of virulence in Acinetobacter baumannii cells surviving photodynamic treatment with toluidine blue.

INTRODUCTION: Widespread resistance to antimicrobial agents has led to a dearth of therapeutic choices in treating Acinetobacter baumannii infections, leading to new strategies for treatment being needed. We evaluated the effects of photodynamic therapy (PDT) as an alternative antimicrobial modality on the virulence features of cell-surviving PDT. MATERIALS AND METHODS: To determine the sublethal PDT (sPDT), a colistin-resistant, extensively drug-resistant A. baumannii (CR-XDR-AB) clinical isolate and A. baumannii and ATCC 19606 strains, photosensitized with toluidine blue O (TBO), were irradiated with light emitting diodes, following bacterial viability measurements. The biofilm formation ability, outer membrane (OM) integrity, and antimicrobial susceptibility profiles were assessed for cell-surviving PDT. The effects of sPDT on the expression of virulent genes were evaluated by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS: sPDT resulted in the reduction of the biofilm formation capacity, and its metabolic activity in strains. The OM permeability and efflux pump inhibition of the sPDT-treated CR-XDR-AB cells were increased; however, there was no significant change in OM integrity in ATCC 19606 strain after sPDT. sPDT reduced the minimum inhibitory concentrations of the most tested antimicrobials by ≥2-fold in CR-XDR-AB. lpsB, blsA, and dnaK were upregulated after the strains were treated with sPDT; however, a reduction in the expression of csuE, epsA, and abaI was observed in the treated strains after sPDT. CONCLUSION: The susceptibility of CR-XDR-AB to a range of antibiotics was enhanced following sPDT. The virulence of strains is reduced in cells surviving PDT with TBO, and this may have potential implications of PDT for the treatment of A. baumannii infections.

Wide distribution of carbapenem resistant Acinetobacter baumannii in burns patients in Iran.

Antimicrobial resistance in carbapenem non-susceptible Acinetobacter baumannii (CNSAb) is a major public health concern globally. This study determined the antibiotic resistance and molecular epidemiology of CNSAb isolates from a referral burn center in Tehran, Iran. Sixty-nine CNSAb isolates were tested for susceptibility to antimicrobial agents using the E test methodology. Multiple locus variable number tandem repeat analysis (MLVA), Multilocus sequence typing (MLST) and multiplex PCR were performed. PCR assays tested for ambler classes A, B, and D ß-lactamases. Detection of ISAba1, characterization of integrons, and biofilm formation were investigated. Fifty-three (77%) isolates revealed XDR phenotypes. High prevalence of bla OXA-23-like (88%) and bla PER-1 (54%) were detected. ISAba1 was detected upstream of bla ADC, bla OXA-23-like and bla OXA51-like genes in, 97, 42, and 26% of isolates, respectively. Thirty-one (45%) isolates were assigned to international clone (IC) variants. MLVA identified 56 distinct types with six clusters and 53 singleton genotypes. Forty previously known MLST sequence types forming 5 clonal complexes were identified. The Class 1 integron (class 1 integrons) gene was identified in 84% of the isolates. The most prevalent (33%) cassette combination was aacA4-catB8-aadA1. The IC variants were predominant in the A. baumannii lineage with the ability to form strong biofilms. The XDR-CNSAb from burned patients in Iran is resistant to various antimicrobials, including tigecycline. This study shows wide genetic diversity in CNSAb. Integrating the new Iranian A. baumannii IC variants into the epidemiologic clonal and susceptibility profile databases can help effective global control measures against the XDR-CNSAb pandemic.

The Prevalence of IS Aba 1 and IS Aba 4 in Acinetobacter baumannii Species of Different International Clone Lineages Among Patients With Burning in Tehran, Iran.

BACKGROUND: Multidrug resistant strains of Acinetobacter baumannii (MDR-AB) have emerged as alarming nosocomial pathogens among patients with burning. OBJECTIVES: The current study aimed to determine the susceptibility of A. baumannii species, carbapenems resistance patterns, and their association with IS Aba 1 and IS Aba 4 elements upstream of the bla OXA-like genes, and the distribution of international clone (IC) of A. baumannii isolates among patients with burning in Tehran, Iran. MATERIALS AND METHODS: In the current study, 62 A. baumannii species isolates from patients with burning in Tehran, Iran, in 2012 were evaluated for the antimicrobial susceptibility, genetic relationships, ICs, carbapenemase encoding genes, and insertion elements IS Aba upstream of bla OXA-like genes. RESULTS: The highest rates of susceptibility were observed with colistin (88.7%) and tigecycline (82.2%). The extensively drug-resistance and pan drug-resistance were observed in 37.1% and 8.1% of the isolates, respectively. About 98.3% of 17 genotypes categorized into three distinct clusters. Thirty-six of the 62 isolates (58%) belonged to the IC II lineage. The most prevalent acquired OXA-type carbapenemase was bla OXA-23-like (62.9%). IS Aba 1 and IS Aba 4 were detected upstream of bla OXA-23-like genes in 45.1% and 12.9% of isolates, respectively. In 32.2% of all isolates, IS Aba 1 laid upstream of bla OXA-51-like genes. The PCR results were negative for carbapenemase genes of Ambler class A and B, except bla VIM-2 . (1.6%). CONCLUSIONS: It was the first study that attempted to detect the insertion elements IS Aba and IC lineages in MDR-AB species isolated from patients with burning in Iran.

Spread of extended-spectrum ß-lactamase genes of blaOXA-10, blaPER-1 and blaCTX-M in Pseudomonas aeruginosa strains isolated from burn patients.

BACKGROUND: Pseudomonas aeruginosa is resistant to many antibiotics due to production of different classes of extended spectrum ß-lactamases (ESBLs). Prevalence of ESBLs among P. aeruginosa has been increased in recent years, demonstrate a serious health problem especially in burn units worldwide. OBJECTIVE: Present study was designed to determine the ESBL producing strains and identify the genes encoding three different ESBLs of bla PER-1, bla OXA-10 and bla CTX-M genes in P. aeruginosa isolates from burn patients. METHODS: In total 185 clinical isolates of P. aeruginosa were collected from infectious wounds of hospitalized burn patients. Antimicrobial susceptibility testing and phenotypic detection of ESBL were performed by disk diffusion method and Double disk Synergy Test (DDST). Polymerase Chain Reaction (PCR) was done for detection of bla OXA-10, bla PER-1 and bla CTX-M ESBL encoding genes. RESULTS: In total, 176 (95.13%) isolates were multidrug resistant. The DDST demonstrated 96 (51.9%) isolates as putative ESBL producers with 100% or highly resistance to ofloxacin, cephalexin, aztreonam (97.57%) and ceftriaxone (91.6%). By PCR amplification, bla PER-1, bla OXA-10 and bla CTX-M genes were detected in 52 (54.16%), 66 (68.75%) and 1 (1.04%) isolates of ESBL producers respectively. Forty-three isolates (44.79%) were simultaneously positive for both bla OXA-10 and bla PER-1 related genes. CONCLUSION: The rate of ESBL producing P. aeruginosa was notable in present study. Since there are only limited effective antibiotics against the bacterium, therefore all isolates must be investigated by antimicrobial susceptibility testing, which limits resistance development in burn units and helps the management of treatment strategy.

Inducible clindamycin resistance in clinical isolates of Staphylococcus aureus due to erm genes, Iran.

BACKGROUND AND OBJECTIVES: Resistance to macrolide can be mediated by erm and msrA genes in Staphylococcus aureus. There are the evidences that show erm genes may be causative agent of inducible or constitutive resistance. The aim of this study was to investigate the incidence of inducible clindamycin resistance and determine the most frequency of erm and msrA genes among S. aureus isolates. MATERIALS AND METHODS: In this study a total of 124 non duplicated clinical isolates of S. aureus were tested with disk diffusion method. All isolates were tested by PCR for mecA, ermA, ermB, ermC and msrA genes. RESULTS: According to PCR results, 48.4% had mecA gene and 51.6% were mecA negative. By phenotypic D-test method, 32.3% revealed inducible resistance and recorded as D and D(+). Sensitive and constitutive phenotypes were found in 54.8% and 12.9% of isolates respectively. Inducible clindamycin resistance was more prevalent in MRSA (29%) than MSSA isolates (2.4%). Among studied erm genes, the most frequency genes were ermA and ermC with 41.1% and 17.7% respectively. Three isolates of them had D phenotype, while the PCR results of erm genes were negative. All isolates were negative for ermB or msrA genes. CONCLUSION: Since S. aureus isolates with inducible resistance may mutate and change to constitutive resistance, to prevent treatment failure, we suggest that inducible resistance test be performed on erythromycin resistant/clindamycin sensitive isolates.