Genomic diversity of ß-lactamase producing Pseudomonas aeruginosa in Iran; the impact of global high-risk clones.

BACKGROUND: Hospital-acquired infections caused by multidrug-resistant Pseudomonas aeruginosa incline hospital stay and costs of treatment that resulted in an increased mortality rate. The frequency of P. aeruginosa high-risk clones producing carbapenemases was investigated in our clinical samples. METHODS: In this cross-sectional study, 155 non-repetitive P. aeruginosa isolates were included from different medical centers of Iran. Antibiotic susceptibility testing was determined, and the presence of ß-lactamases were sought by phenotypic and genotypic methods. The clonal relationship of all isolates was investigated, and multi-locus sequence typing (MLST) was used for finding the sequence types of carbapenemase-producers. RESULTS: The agent with highest percent susceptibility rate was recorded for colistin (94.9%). MOX and FOX were found both as low as 1.95% (3/155). The most frequent narrow spectrum ß-lactamase was SHV with 7.7% (12/155) followed by PER, OXA-1, and TEM with the frequency of 7.1% (11/155), 3.2% (5/155), and 1.3% (2/155), respectively. Carbapenemases were detected in 28 isolates (18%). The most frequent carbapenemase was IMP with 9% (14/155) followed by NDM, 8.4% (13/155). OXA-48 and VIM were also detected both per one isolate (0.65%). MLST of carbapenem resistant P. aeruginosa isolates revealed that ST244, ST664, ST235, and ST357 were spread in subjected clinical settings. REP-PCR uncovered high genomic diversity in our clinical setting. CONCLUSION: Clonal proliferation of ST235 strain plays a key role in the propagation of MDR pattern in P. aeruginosa. Our data showed that high-risk clones has distributed in Iran, and programs are required to limit spreading of these clones.

Relationship between antibiotic resistance with class 1 integron and SmeDEF efflux pump encoding genes in clinical isolates of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an emerging multidrug-resistant organism with an increasing frequency of hospital-acquired infections predominantly in developing countries. The purpose of this study was to determine the antibiotic resistance and frequency of the smeD, class 1 integron, and sul1 genes in clinical isolates of S. maltophilia in two Iranian provinces. From January 2020 to September 2021, 38 clinical isolates of S. maltophilia were collected from patients in hospitals in Tabriz and Sanandaj provinces of Iran. S. maltophilia isolates were confirmed by standard bacteriological tests and 16S rRNA gene PCR. Disk diffusion and the MIC test strip methods were used to determine the antibiotic resistance patterns. PCR was performed to investigate the presence of smeD, class 1 integron, and sul1 genes. The antimicrobial test for the isolated S. maltophilia showed a high level of sensitivity against most of the antibiotics used. Maximum sensitivity was recorded for ciprofloxacin (100% (38/38)) and levofloxacin 100% (38/38), followed by ceftazidime (97.36% (37/38)), trimethoprim-sulfamethoxazole (81.57% (31/38)), ticarcillin-clavulanate (60.52% (23/38)), and piperacillin-tazobactam (55.26% (21/38)). We observed a high prevalence of smeD (100% (38/38)) and class 1 integron (94.73% (36/38)) genes in the isolates, and none of the isolates carried the sul1 gene. The findings from this study indicate that resistance to trimethoprim-sulfamethoxazole was not observed, and still, trimethoprim-sulfamethoxazole is the best drug with desirable antimicrobial effect in the treatment of nosocomial infections caused by S. maltophilia strains. Despite the observation of a high number of class 1 integron, the sul1 gene was not observed, which indicates the role of this gene in high-level trimethoprim-sulfamethoxazole resistance and not having a role in low-level resistance. Based on our results, clinical microbiology laboratories need continuous surveillance of resistance rates to trimethoprim-sulfamethoxazole, because of the possibility of S. maltophilia acquiring trimethoprim-sulfamethoxazole-resistance by mobile gen elements.

Investigation of NDM-1 and OXA-48 producing carbapenem resistant Klebsiella pneumoniae ST15 in Iran.

The aim of this study was to determine the frequency of carbapenem resistant Klebsiella pneumoniae (CRKP) sequence types (STs) in Iran. Samples were collected from three university hospitals in Sanandaj, Iran, from December 2016 to March 2018. Antibiotic susceptibility testing, phenotypic and genotypic detection of carbapenemases were performed. Common K. pneumoniae capsular types were sought for all isolates. The genetic relatedness of isolates was investigated by multilocus sequence typing (MLST). Plasmids were detected by PCR-based Replicon Typing (PBRT). During the study, 67 K. pneumoniae isolates were identified. Of which, 18 (26.9%) isolates were detected as carbapenem-resistant. The most effective antibacterial agent was tigecycline (97%, 65 isolates) followed by imipenem and ertapenem (73.13%, 49 isolates). PCR showed that 13 isolates (19.4%) had blaNDM-1 gene and 5 (7.5%) harbored blaOXA-48. Examination of common capsular types showed that 2 isolates had K2 and 2 others had K54. REP-PCR revealed 10 clones and 11 singleton strains. MLST analysis of CRKP found ST15 as the most common type (13 isolates, 72.2%), but other STs were also detected namely, ST19, ST117, ST1390, and ST1594. ColE1 and IncL/M plasmids were the carriers of blaNDM-1 and blaOXA-48, respectively. The results showed that CRKP spread in our health centers. Our results, therefore, indicate a worrying trend of resistance to carbapenems in K. pneumoniae.

Prevalence of algD, pslD, pelF, Ppgl, and PAPI-1 Genes Involved in Biofilm Formation in Clinical Pseudomonas aeruginosa Strains.

Introduction: Biofilm formation is one of the main virulence factors in Pseudomonas aeruginosa infections. This study is aimed at investigating the presence of genes involved in biofilm formation in clinical P. aeruginosa isolates. Material and Methods. A cross-sectional study was conducted on 112 P. aeruginosa isolates. The biofilm formation assay was performed on all isolates. Antimicrobial resistance was determined by the disk diffusion method, and the presence of genes was detected by polymerase chain reaction. Isolates were typed with Rep-PCR. Results: The results of biofilm formation demonstrated that 85 strains (75.9%) were biofilm producers, and 27 strains (24.1%) were nonproducer isolates. Antibiotic susceptibility pattern in biofilm-positive and biofilm-negative isolates obtained from hospitalized patients showed a high rate of antibiotic resistance to amoxicillin with 95.7% and 92.3%, respectively. Based on PCR amplification results, the frequency of genes involved in biofilm formation among all isolates was as follows: algD (78.6%), pelF (70.5%), pslD (36.6%), Ppgl (0%), and PAPI-1 (77.6%). Rep-PCR typing demonstrated that 112 P. aeruginosa isolates were classified into 57 types according to 70% cut-off. The predominant type was A which contained 15 isolates. Moreover, 7 isolates were clustered in genotype B, followed by C type (6), D (4), E (4), F (4), G (4), H (3), I (3), J (3 isolates), and 12 genotypes, each containing two isolates. Also, 35 isolates were distributed in scattered patterns and showed single types. Conclusion: Study results showed significant association between biofilm formation and resistance to antibiotics such as ceftazidime and meropenem. Analysis of Rep-PCR patterns indicated that the evaluated isolates were heterogeneous, relatively.

Effect of calcium hydroxide on morphology and physicochemical properties of Enterococcus faecalis biofilm.

Calcium hydroxide Ca(OH)2 has been used as an intracanal medicament to targets microbial biofilms and avert secondary infection in the root canal system. This study evaluated the effects of this material on the morphology and physicochemical properties of an established in-vitro biofilm of Enterococcus faecalis. A biofilm of E. faecalis was grown in multichannel plates. The chemicals including Ca2+, OH-, and saturated Ca(OH)2 (ie 21.6 mM) were prepared in order to evaluate which component eradicated or amplified biofilm structure. Various biochemical and microscopic methods were used to investigate the properties of the biofilm. Biofilms treated with Ca(OH)2 absorbed more Ca2+ because of the alkaline pH of the environment and the ions affected the physicochemical properties of the E. faecalis biofilm. A denser biofilm with more cavities and a granular surface was observed in the presence of Ca2+ ions. This resulted in a decrease in the surface-to-biofilm ratio with increases in its biomass, thickness, colony size, and volume. Calcium hydroxide did not destroy E. faecalis biofilms but rather contributed to the biofilm structure. This in-vitro study sheds light on a missing link in the formation of E. faecalis biofilm in which the Ca2+ in Ca(OH)2.

Prevalence of Neisseria gonorrhoeae in Western Iran.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhoea. This bacterium infects the epithelial cells of the cervix of women and the urethra of men. However, its disease symptoms in the lower genitalia are found only in a small percentage of people. This study aimed to compare the frequency of N. gonorrhoeae genital infection among two groups of pregnant women, those with spontaneous abortions and those with normal pregnancies. This cross-sectional study was conducted in Western Iran. It included 417 women: 109 of whom had spontaneous abortions, 109 had normal deliveries, 100 were fertile, and 99 were infertile. Specific primers were used and DNA was extracted by endocervical swabs. A polymerase chain reaction test was then performed to detect N. gonorrhoeae. Data analysis was performed using the chi-squared test and t-tests. In all the above steps, a level of 5% was considered statistically significant, and the average ages in women with normal delivery, women with spontaneous abortion, fertile women, and infertile women were 27.8 ± 4.87, 29.6 ± 5.9, 32.1 ± 5.1, and 29.1 ± 6.3 years, respectively. The total frequency of N. gonorrhoeae infection was 0 (0%). The prevalence of N. gonorrhoeae infection was zero, and the disease was not associated with spontaneous abortion or infertility.

Detection of efflux pump genes in multiresistant Acinetobacter baumannii ST2 in Iran.

Acinetobacter baumannii, as a nosocomial pathogen has become a worldwide concern in recent years. In the current study, the resistance to tetracyclines and colistin were assessed in the isolates from different provinces of Iran.During the timeline of this study, a number of 270 isolates of A. baumannii were collected from tracheal aspirates, wounds, urine and blood cultures. The minimum inhibitory concentration (MIC) for tetracycline, doxycycline, minocycline, tigecycline and colistin were evaluated. Tetracycline resistance genes were assessed by PCR. The mean expression level of adeB, adeJ and adeG were assessed using semi quantitative Real-Time PCR. The clonal relationship of the isolates was evaluated by the repetitive extragenic palindromic PCR (REP-PCR), International Clonal (IC) Lineage Multiplex PCR and multilocus sequence typing (MLST) (Pasteur scheme) methods.The MIC by microdilution method showed that 87.5, 51.4, 28, 0.74 and 0% of the isolates were resistant to tetracycline, doxycycline, minocycline, tigecycline and colistin respectively. The prevalence of tetracycline resistance genes was 99.2, 99.2, 98, 86.7, 10, 3.33, 0.37, 0% for adeB, adeJ, adeG, tetB, tetA(39), tetA, tetM and tetH in tetracycline-resistant isolates. Moreover, the expression level of adeB, adeJ, adeG genes in tigecycline-nonsusceptible A. baumannii (TNAB) strain was higher compared to the tigecycline-susceptible A. baumannii (TSAB). A broad genomic diversity was revealed, but ST2 was the most prevalent ST. Our results indicated that tetracycline resistance in Iran is mediated by resistance-nodulation-cell division (RND) and tetB efflux pumps.

Ratiometric fluorescence resonance energy transfer aptasensor for highly sensitive and selective detection of Acinetobacter baumannii bacteria in urine sample using carbon dots as optical nanoprobes.

Development of sensitive and selective analytical method for accurate diagnosis of Acinetobacter baumannii (Ab) bacteria in biological samples is a challenge. Herein, we developed an ingenious ratiometric fluorescent aptasensor for sensitive and selective detection of (Ab) bacteria based on fluorescence resonance energy transfer (FRET) between ortho-phenylenediamines carbon dot (o-CD), nitrogen-doped carbon nanodots (NCND) as donor's species and graphene oxide (GO) as acceptor. NCND that assembled onto the edge of graphene oxide (GO) exhibited quenched photoluminescence emission, and with the absorption of the modified o-CD with aptamer (o-CD-ssDNA) onto the graphene oxide surface the fluorescence of o-CD was efficiently quenched. The aptamer (ssDNA) as a biorecognition element is bound with A. baumannii specifically which releases the o-CD-ssDNA from GO and the recovery of the fluorescence signal of o-CD, while the fluorescence intensity of NCND only slightly altered and acted as the reference signal in ratiometric fluorescence assay. The fluorescence intensity ratio (I550 nm/I440nm) varied from 2.0 to 10.0 with the concentration of bacteria changing from 2.0 × 103 to 4.5 × 107 cfu/mL and the low detection limit of 3.0 × 102 cfu/mL (S/N = 3). The feasibility of the developed aptasensor for selective detection of A. baumannii in urine sample with satisfactory results was also demonstrated.

Prevalence of Metallo-ß-Lactamases in Acinetobacter Baumannii in Iran: A Review and Meta-Analysis.

Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen that causes major public health concerns, especially in hospitalized patients due to acquisition of resistant genes. The aim of this study was to systematically review the published data on the prevalence and dispersion of metallo-ß-lactamases (MBLs) genes in A. baumannii in different provinces of Iran and provide an overall prevalence rate using meta-analysis. All available national and international databanks from 2011 to 2017 were searched to find published studies. Quality of studies was assessed by STROBE. Due to the fact that a significant heterogeneity was observed, the random effects model was used to combine the results. Statistical analysis was performed by comprehensive meta-analysis (CMA) V2 software. Out of 78 articles, 28 were extracted based on certain inclusion and exclusion criteria. Most of the A. baumannii isolates were obtained from intensive care unit (ICU) ward of hospitals. Based on phenotypic and molecular detection tests, pooled prevalence of all MBLs was 58%, and blaVIM, blaIMP, and blaSPM-1 genes were estimated to be at 10.5, 6, and 5%, respectively. Based on the results, further attention should be given to report MBL genes in A. baumannii based on molecular detection rather than the phenotypic one. Furthermore, more effort should be focused on ICU sections in order to avoid the distribution of resistant genes.

Diversity of aminoglycoside modifying enzymes and 16S rRNA methylases in Acinetobacter baumannii and Acinetobacter nosocomialis species in Iran; wide distribution of aadA1 and armA.

PURPOSE: Acinetobacter baumannii-calcoaceticus complex (ABC) make a great burden on health-care systems due to hospital-acquired infections and antibacterial resistance. Aminoglycoside in combination with other antibacterials used as treatment options. However, ABC species overcome this class of antibacterials in different ways. This study provides a comprehensive report on the distribution of aminoglycoside modifying enzymes (AMEs) and 16S rRNA methylase in Acinetobacter baumannii and Acinetobacter nosocomialis isolated from various provinces in Iran. METHODS: During six month of study, from eight referral centers in seven provinces across the country, Iran, 178 A. baumannii and 43 A. nosocomialis isolates were collected. The minimum inhibitory concentration of amikacin, gentamicin, netilmicin, kanamycin and tobramycin were measured by microbroth dilution method. AMEs and 16S rRNA methylase variants were sought by PCR. RESULTS: High rates of resistance were seen in all centers. MIC50 and MIC90 for all A. baumannii and A. nosocomialis isolates from different centers were > 512 mg/L. The most frequent AME was ant(3″)-Ia (aadA1) in both of A. baumannii (74.1%) and A. nosocomialis (86%). armA was detected in A. baumannii and A. nosocomialis at the frequency of 41.6% and 67.4%, respectively. rmtA, B, C, D, aac(3)-Ia (aacC1) and aac(6')-Im were not detected, neither in A. baumannii nor A. nosocomialis. Moreover, aac(6')-Ih was only found in A. baumannii isolates. The distribution of some of the ARGs was limited to a definite center. CONCLUSION: The overall high-level carriage of ARGs in Acinetobacter species may limited usage of this class of antibacterials as a treatment option.

Design of peptides interfering with iron-dependent regulator (IdeR) and evaluation of Mycobacterium tuberculosis growth inhibition.

OBJECTIVES: Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), stayed a global health thread with high mortality rate. Since TB has a long-term treatment, it leads high risk of drug resistant development, and there is an urgent to find new drugs. The aim of this study was designing new inhibitors for a new drug target, iron dependent regulator, IdeR. MATERIALS AND METHODS: Based on the interaction most populated amino acids of IdeR to the related gene operators, 50 short peptides were modeled. Bonding affinity of short peptides toward DNA were studied by docking. Top 10 best predicted bonding affinity were selected. DNA binding assay, microplate alamar blue assay, colony counting, quantitative real time- PCR (qRT-PCR) of IdeR corresponding genes, cell wall-associated mycobactin and whole-cell iron estimation were done to prove the predicted mechanism of in silico potent constructs. RESULTS: Amongst the 10 synthesized short peptide candidates, glycine-valine-proline-glycine (GVPG) and arginine-proline-arginine (RPR) inhibited Mtbin vitro at 200 µM concentration. qRT-PCR showed mbtB 58-fold over expression that resulted in Mtb growth inhibition. Cell wall-associated mycobactin and whole-cell iron estimation confirmed the results of qRT-PCR. CONCLUSION: We introduced two new lead compounds to inhibit Mtb that are promising for the development of more potent anti-tubercular therapies.

Molecular characterization and genetic relatedness of clinically Acinetobacter baumanii isolates conferring increased resistance to the first and second generations of tetracyclines in Iran.

BACKGROUND: The increasing resistance of Acinetobacter baumannii to antibiotics has recently been regarded as a notable therapeutic difficulty. Evaluating resistance rates of some A. baumannii isolates to tetracyclines had an impact on understanding the antibiotic resistance dissemination. By comparing genetic characteristics and relatedness of A. baumannii isolates, we are able to determine the transition dynamics of outbreak isolates. METHODS: A total of 72 non-duplicate isolates of A. baumannii were recovered in 2011 and 2015 and minimum inhibitory concentration (MIC) range distribution of the isolates to tetracyclines was performed by broth micro dilution (BMD) assay, and to determine the lineage relatedness of the outbreak isolates repetitive extragenic palindromic element based on polymerase chain reaction (rep-PCR) and international clonal (ICs) investigations were performed. RESULTS: Resistance rates to tetracycline, doxycycline and minocycline in 2011 were 73, 2 and 0%, while these rates in 2015 increased up to 90, 84 and 52%, respectively. The tetB existed in 100% of all the isolates of both years. tetA was not found in any of the isolates. According to the rep-PCR assays, up to 83% of all isolates clustered distinctly and only 6% of isolates had a common root. The percentage rates of IC1 decreased from 42% in 2011 to 22% in 2015, while those of IC2 increased from 28 to 36%, from 2011 to 2015. CONCLUSIONS: Our data showed that resistance to the first and second generations of tetracyclines is on the rise and the clonal transition dynamics of isolates are in progress in our hospital.

Delayed identification of Acinetobacter baumannii during an outbreak owing to disrupted blaOXA-51-like by ISAba19.

Early detection of Acinetobacter baumannii, an emerging pathogen in hospital settings, is of interest. The blaOXA-51-like family had been used as a marker to detect A. baumannii. During an infection outbreak, 14 isolates produced a 1.7-kb PCR band instead of 353 bp for this marker. This work sought the reasons behind the increased marker size. Characterisation of isolates was done by API-20NE, gyrB multiplex PCR and 16S-23S rRNA ITS sequencing. The 1.7-kb band generated and the complete blaOXA-51-like variant were sequenced to find the probable integrated element. Susceptibility testing to various antimicrobials was performed by microdilution. blaOXA-like, metallo-ß-lactamases (MBLs), the ISAba1 element and the presence of ISAba1 adjacent to blaOXA-like were sought. rep-PCR, global clonal (GC) lineage determination and multilocus sequence typing (MLST) were performed to analyse the relationship among isolates. Isolates were characterised as Acinetobacter baumannii-calcoaceticus complex by API-20NE. gyrB multiplex PCR and 16S-23S ITS sequencing verified the isolates as A. baumannii. Sequencing of the 1.7-kb band revealed ISAba19 as the disrupting element. The blaOXA-51 variant was blaOXA-66, which was elongated to 2.2 kb due to ISAba19. The blaOXA-23-like family was found in 67% of isolates. MBL genes were not detected; however, ISAba1-blaOXA-23-like was characterised in carbapenem-resistant isolates (53%; 8/15). Isolates were divided into three clusters by rep-PCR. All strains were ST2 and all but one belonged to GC II. Identification of A. baumannii based only on blaOXA-51-like is not reliable. Besides blaOXA-51-like, multiplex PCR of gyrB and rpoB could provide rapid and cost-effective results.

Elevated prevalence of multidrug-resistant Acinetobacter baumannii with extensive genetic diversity in the largest burn centre of northeast Iran.

OBJECTIVES: The emergence and spread of multidrug-resistant (MDR) Acinetobacter baumannii isolates is now frequently associated with nosocomial infections. The aim of this study was to evaluate the genetic relatedness and patterns of antimicrobial resistance amongst A. baumannii isolated from a burn centre at a teaching hospital in Iran. METHODS: A total of 54 A. baumannii isolates were collected from burn wound infections of hospitalised patients. Antimicrobial susceptibility of the isolates was determined, and genotyping analysis was performed by repetitive extragenic palindromic PCR (rep-PCR). PCR assay was performed to investigate the distribution of ß-lactamase, aminoglycoside-modifying enzyme and efflux pump genes. RESULTS: Etest results revealed that the most active antimicrobial agent was colistin (100% susceptibility), followed by tigecycline (96.3%). The blaOXA-51 and blaADC genes were detected in all of the isolates, but blaOXA-58-like was not detected. The prevalence of blaTEM, blaIMP, blaVIM, blaOXA-23-like and blaOXA-24-like genes was 64.8%, 70.4%, 70.4%, 66.7% and 68.5%, respectively. ISAba1 was detected upstream of blaOXA-23-like and blaADC in 66.7% and 77.8% of isolates, respectively. This study showed a high level of distribution of adeB (72.2%), aphA6 (81.5%), aacC1 (85.2%), aadA1 (59.3%), aadB (31.5%), tetB (70.4%) and aphA1 (29.6%) in A. baumannii strains. Based on rep-PCR analysis, four clusters (I-IV) were defined. CONCLUSIONS: The elevated prevalence of MDR A. baumannii strains in this burn centre suggests that local antibiotic prescription policies should be precisely revised. Moreover, strict infection control procedures to prevent further dissemination need to be prioritised immediately.

DNA fingerprinting and antimicrobial susceptibility pattern of clinical and environmental Acinetobacter baumannii isolates: a multicentre study.

BACKGROUND AND AIM: The aims of this study were to establish antibiotic profile and the molecular epidemiology of Acinetobacter baumannii isolates, with considering the effectiveness of control infection measures across three hospitals in the Kurdistan, west part of Iran. METHODS: Fifty-four A. baumannii isolates were collected from patients and environmental specimens. Antibiotic susceptibility patterns (Antibio-type) were evaluated for 17 different antibiotics and MIC for imipenem was done. Isolates were assessed for the presence of metallo-beta-lactamases (MBLs), class 1 and 2 integrons, and integrated gene cassettes and blaOXA-likefamilies genes. Repetitive-sequence-based PCR (REP-PCR) was done for analysing clonality and relativeness of isolates (REP-type). RESULTS: Antibiotic susceptibility patterns distinguished 11 distinct Antibio-types and REP-PCR showed three clusters with 20 subclusters, mostly belonged to two clonal subgroups, A1 and B1. blaOXA-51 and blaOXA-23 were detected in 100% (54/54) and 52% (28/54), respectively, while blaOXA-24-like and blaOXA-58 were not present in isolates. MBLs were not detected, but, however, high rate of imipenem resistance was observed (52%). MIC90 of imipenem was 16 mg/ml. Class 1 integrons were detected in 11% (6/54) of isolates followed by 24% (13/54) of class 2. Both classes of integron genes were detected in 15% (8/54) of isolates. Integrated gene cassettes were in low level (11% of class 1 harboring isolates). Two arrays of gene cassettes were revealed, dfrA5-like and dfrA17-aadA5. CONCLUSION: Infection control surveillance should be considered as a serious manner, even the superficial eradication of hospital acquired pathogens. MBL genes were not induced carbapenem resistance in studied hospital settings, but blaOXA-51 & 23 contributed in imipenem resistant. Integrons had a little share in resistance of A. baumannii isolates.

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.

Limited genetic diversity and extensive antimicrobial resistance in clinical isolates of Acinetobacter baumannii in north-east Iran.

This study determined the mechanisms and patterns of antimicrobial resistance among the isolates obtained from different wards of a teaching hospital in the city of Mashhad in north-east Iran. Between January 2012 and the end of June 2012, 36 isolates of Acinetobacter baumannii were collected from different wards of Ghaem Hospital. Antimicrobial susceptibility testing and epsilometer testing (E-test) were performed. The genetic resistance determinants of A, B and D classes of ß-lactamases, aminoglycoside modifying enzymes (AMEs), efflux pumps and ISAba1 elements were assessed by PCR. Repetitive extragenic palindromic element (REP)-PCR was performed to find the genetic relatedness of the isolates. Colistin was the most effective antibiotic of those tested, where all isolates were susceptible. E-test results revealed high rates of resistance to imipenem, ceftazidime and ciprofloxacin. The majority of isolates (97  %) were multidrug-resistant. OXA-51, OXA-23 and tetB genes were detected in all isolates, but OXA-58, IMP and tetA were not detected. The prevalence of OXA-24, bla(TEM), bla(ADC), bla(VIM) and adeB were 64, 95, 61, 64 and 86  %, respectively. ISAba1 was found to be inserted into the 5' end of OXA-23 in 35 isolates (97  %). Of the AMEs, aadA1 (89  %) was the most prevalent, followed by aphA1 (75  %). The band patterns reproduced by REP-PCR showed that 34 out of 36 isolates belonged to one clone and two singletons were identified. The results confirmed that refractory A. baumannii isolates were widely distributed and warned the hospital infection control team to exert strict measures to control the infection. An urgent surveillance system should be implemented.

Prevalence of Acinetobacter baumannii harboring ISAba1/bla OXA-23-like family in a burn center.

BACKGROUND: Refractory carbapenem resistant Acinetobacter baumannii (CRAB) isolates increase mortality and morbidity rates among patients with underlying disorders, especially in patients with burns. The aim of the current study was to understand the resistant determinants of CRAB isolates and their clonal relatedness collected from referral Burn center. METHODS: CRAB isolates were initially characterized and then antimicrobial susceptibility testing was assessed by E-test. Resistance determinants were investigated by PCR. Repetitive extragenic palindromic elements-PCR (REP-PCR) was used for clonality relatedness among isolates. RESULTS: Thirty CRAB isolates were collected during the study. Colistin was the most effective antibiotic, but, all of the isolates were resistant to carbapenems. intI1 was detected in two isolates and MBLs and gene cassettes were not detected. ISAba1, blaOXA-51, blaOXA-23 and ISAba1/blaOXA23-like were detected in all, while blaOXA-24-like were present in 73% and blaOXA-58&OXA-143 were not present in isolates. REP-PCR demonstrated three clusters, with the dominant B cluster, which contained 16 subgroups. CONCLUSION: CRAB harboring ISAba1/blaOXA-23-like family is widely disseminated in the studied Burn ward setting and the emergence of infection control measures should be regarded to limit refractory CRABs.

DNA fingerprinting and antimicrobial susceptibility pattern of clinical and environmental Acinetobacter baumannii isolates: a multicentre study.

Backgrounds and aims: The aims of this study were to establish antibiotic profile and the molecular epidemiology of Acinetobacter baumannii isolates, with considering the effectiveness of control infection measures across three hospitals in the Kurdistan, west part of Iran. Methods: Fifty-four A. baumannii isolates were collected from patients and environmental specimens. Antibiotic susceptibility patterns (Antibio-type) were evaluated for 17 different antibiotics and MIC for imipenem was done. Isolates were assessed for the presence of metallo-beta-lactamases (MBLs), class 1 and 2 integrons, and integrated gene cassettes and blaOXA-like family genes. Repetitive-sequence-based PCR (REP-PCR) was done for analysing clonality and relativeness of isolates (REP-type). Results: Antibiotic susceptibility patterns distinguished 11 distinct Antibio-types and REP-PCR showed three clusters with 20 subclusters, mostly belonged to two clonal subgroups, A1 and B1. blaOXA-51 and blaOXA-23 were detected in 100% (54/54) and 52% (28/54), respectively, while blaOXA-24-like and blaOXA-58 were not present in isolates. MBLs were not detected, but, however, high rate of imipenem resistance was observed (52%). MIC90 of imipenem was 16 µg/ml. Class 1 integrons were detected in 11% (6/54) of isolates followed by 24% (13/54) of class 2. Both classes of integron genes were detected in 15% (8/54) of isolates. Integrated gene cassettes were in low level (11% of class 1 harboring isolates). Two arrays of gene cassettes were revealed, dfrA5-like and dfrA17-aadA5. Conclusion: Infection control surveillance should be considered as a serious manner, even the superficial eradication of hospital acquired pathogens. MBL genes were not induced carbapenem resistance in studied hospital settings, but blaOXA-51 & 23 contributed in imipenem resistant. Integrons had a little share in resistance of A. baumannii isolates.