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Mahsa Ziasistani1,2, Mohammad Reza Shakibaie3,4, Davood Kalantar-Neyestanaki31Student Research Committee, Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 2Pathology and Stem Cell Research Center٫ Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 3Department of Microbiology and Virology, Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 4Research Center for Infectious Diseases and Tropical Medicine, Kerman University of Medical Sciences, Kerman, IranAim: 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(3ʹ)-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(3ʹ)-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(3ʹ)-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.Keywords: VRSA, MLST, SCCmec, spa, agr type, van operon

Somayeh Kiaei,1,2 Mohammad Moradi,2 Hossein Hosseini-Nave,2 Mahsa Ziasistani,3 Davood Kalantar-Neyestanaki2 1Department of Microbiology and Virology, Student Research Committee, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 2Department of Microbiology and Virology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 3Pathology and Stem Cell Research Center, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran Introduction: The emergence and spread of Klebsiella pneumoniae strains resistant to multiple antimicrobial agents are considered as a serious challenge for nosocomial infections. Materials and methods: In this study, 175 nonrepetitive clinical isolates of K. pneumoniae were collected from hospitalized patients in Kerman, Iran. Extended-spectrum β-lactamases (ESBLs), AmpC, and carbapenemase-producing isolates were recognized by phenotypic methods. The resistance genes including efflux pumps oqxA/oqxB, 16S rRNA methylase, ESBL, AmpC, and carbapenemase were detected by PCR-sequencing method. Molecular typing was performed by enterobacterial repetitive intergenic consensus-PCR and multilocus sequence typing methods among blaNDM-positive isolates. Results: Thirty-seven (21.14%) isolates along with sequence types (STs): ST43, ST268, ST340, ST392, ST147, and ST16 were harbored blaNDM. ST43 in 2015 and ST268 during 2016–2017 were the most frequent STs among New Delhi metallo-beta-lactamase (NDM)-positive isolates. We found the distribution of some isolates with blaNDM, blaCTX-M, blaSHV, blaOXA, blaTEM, blaCMY, rmtC, and oqxA/oqxB. Enterobacterial repetitive intergenic consensus-PCR represented seven clusters (A–G) plus four singletons among NDM-positive isolates. This study provides the first report of blaNDM-1-positve K. pneumoniae along with ST268 as well as the spread of nosocomial infections with six different STs harboring blaNDM-1 and other resistance genes in hospital settings especially neonatal intensive care unit. Conclusion: The dissemination of various clones of NDM-producing K. pneumoniae can contribute to increase the rate of their spread in health care settings. Therefore, molecular typing and detection of resistance genes have an important role in preventing and controlling infection by limiting the dissemination of multidrug-resistant isolates. Keywords: blaNDM, 16S rRNA methylase, MLST, ERIC-PCR

Background and Objectives: Staphylococcus aureus is the main Gram-positive bacteria isolated from patients with ocular infections. Herein, we describe the pattern of antibiotic resistance, presence of resistance genes including ermA, ermB, ermC, msrA, mecA and the pvl cytotoxin gene in S. aureus isolates collected from patients with external ocular infection. Materials and Methods: In this study, 8 S. aureus isolates were collected from 81 patients that suffered from eye damage. Antibacterial susceptibility of isolates was determined using the Kirby-Bauer disk diffusion method. Resistance genes including ermA, ermB, ermC, msrA, mecA and the pvl virulence gene were detected by PCR method. Staphylococcal cassette chromosome mec (SCCmec) in MRSA isolates were detected by the multiplex-PCR method. Results: Three isolates were resistant to cefoxitin which is considered MRSA. The mecA gene was identified in MRSA isolates. SCCmec type IV and the pvl gene were detected in one of the MRSA isolates that was recovered from a diabetic patient. Conclusion: The emergence of S. aureus isolates belonging to SCCmec type IV and pvl gene among patients with ocular infection is very serious; therefore, identify genetic characterization of MRSA isolates for empirical therapy and infection control is very important.

Background and Objectives: This study was designed to evaluate the activity of Quercus infectoria galls extract (QIFGE) on virulence factor production and inhibition of quorum sensing (QS) in Pseudomonas aeruginosa. Materials and Methods: Minimum inhibitory concentration (MIC) of QIFGE against 5 strains of P. aeruginosa was determined. The extract at sub-MIC was used to determine biofilm formation, level of protease LasA, LasB, swarming and twitching motility and QS using Chromobacterium violaceum CV026 as a biosensor. Effect of the extract on expression levels of lasR gene was determined by real time PCR. Results: QIFGE inhibited the QS and all other tested virulence factors compared with the control grown in the absence of the extract (P=0.001). Real time PCR showed 2 to 8-fold reduction in lasR gene expression in presence of the extracts compared with the control. QIFGE significantly inhibited the virulence factor production, had inhibitory effect on QS, and resulted in the lower expression of lasR gene. Conclusion: QIFGE showed novel inhibitory effect against QS related virulence factor production, which was unrelated to antimicrobial effect. The extract can down regulate the production of virulence factor and should be evaluated as a candidate for alternative treatment of pseudomonad infections in future.

Background and Objectives: Pseudomonas aeruginosa is responsible for devastating nosocomial infections among severely burn patients. Class C of cephalosporinase (AmpC-β-lactamases) is important cause of multiple β-lactam resistance in P. aeruginosa. The aim of this study was to detect the AmpC-β-lactamases producing isolates among carbapenem resistant P. aeruginosa isolated from burn patient. Material and Methods: a total of 100 isolates of carbapenem resistant P. aeruginosa isolates from different burn patients were investigated. Three phenotypic methods were selected for identification of the AmpC-β-lactamases producing isolates. Results: Fifty four isolates were AmpC producer as detected by AmpC disk test. Seventeen isolates were identified as AmpC producer using combined disk method. Fifty two isolates showed a twofold or threefold dilution difference between the minimum inhibitory concentration of imipenem or ceftazidime and the minimum inhibitory concentration of imipenem or ceftazidime plus cloxacillin. One isolate was identified as AmpC producer using three methods. Three isolates produced AmpC as detected by both AmpC disk test and combined disk methods and 19 isolates were found as AmpC producer using both AmpC disk test and minimum inhibitory concentration methods. Six isolates were AmpC producer as shown by the MICs of both imipenem and ceftazidime. Conclusion: According to the results of this study, AmpC- β-lactamase looks to be the main mechanism of resistance of Pseudomonas aeruginosa to cephalosporins and carbapenems in the study hospital.

Background and Objectives: The oprD mutation and AmpC overproduction are the main mechanisms of intrinsic resistance to carbapenems such as imipenem and meropenem in Pseudomonas aeruginosa. Materials and Methods: In this study, we investigated intrinsic resistance to carbapenems including mutation of oprD and AmpC overproduction in a carbapenem-resistant P. aeruginosa isolated from a burn patient by phenotypic and molecular methods. Results: In our study, the carbapenem-resistant P. aeruginosa isolate was resistant to imipenem, meropenem, cefepime, gentamicin, ceftriaxone, carbenicillin, aztreonam and ciprofloxacin but was susceptible to ceftazidime and polymyxin B. The minimum inhibitory concentrations (MICs) against imipenem, meropenem and ceftazidime were 64 μg/ml, 16 μg/ml and 2μg/ml, respectively. The isolate was ESBLs and AmpC overproducer. No carbapenemase activity was detected by Modified Hodge test (MHT). This isolate was carrying only blaOXA-10. PCR amplification and sequencing of oprD performed on isolate resulted in PCR product of 2647bp. Sequence analysis of the 2647bp product revealed insertion of a sequence of 1232 bp at position 8 in coding region of oprD. Conclusion: According to the results of this study, oprD mutation and AmpC overproduction can cause the main mechanism of resistance of P. aeruginosa to carbapenems.

Today, resistance to antibacterial agents is the most important problem facing public health. Pseudomonas aeruginosa is a common gram-negative bacterium and an important cause of nosocomial infections. Resistance to many antibiotics in strains of P. aeruginosa isolated from hospital settings such as cephalosporins and carbapenems have been recently reported. Therefore, the introduction of a new strategy to treat the infection of these organisms will be beneficial. In this study we determined the ability of cloxacillin to reduce Minimum Inhibitory Concentrations (MICs) of carbapenem-resistant P. aeruginosa to imipenem (IMI), meropenem (MEM), ceftazidime (CAZ), and cefepime (FEP). From 2015 to 2017, 61 non-duplicates of carbapenem-resistant P. aeruginosa were collected from clinical samples of hospitalized patients in Kerman, Iran. The MICs of the isolates to IMI, MEM, CAZ, and FEP with/without cloxacillin were determined by microbroth dilution method. The level of MIC of isolates to carbapenems (IMI and MEM) and cephalosporins (CAZ and FEP) ranged from 1-256 μg/mL and 4-1024 μg/mL alone and from 1-32 μg/mL and 1-512 μg/mL in combination with cloxacillin, respectively. The MIC showed a significant difference reduction after the addition of cloxacillin (P ≤ 0.05). Our results showed in vitro potentially of cloxacillin in reduction of MIC to IMI, MEM, CAZ, and FEP in multi-drug resistant P. aeruginosa, therefore combination of these antibiotics with cloxacillin could be beneficial for treatment of infections caused by multi-drug resistant P. aeruginosa.