Staphopain mediated virulence and antibiotic resistance alteration in co-infection of Staphylococcus aureus and Pseudomonas aeruginosa: an animal model.

Polymicrobial communities lead to worsen the wound infections, due to mixed biofilms, increased antibiotic resistance, and altered virulence production. Promising approaches, including enzymes, may overcome the complicated condition of polymicrobial infections. Therefore, this study aimed to investigate Staphopain A-mediated virulence and resistance alteration in an animal model of Staphylococcus aureus and Pseudomonas aeruginosa co-infection. S. aureus and P. aeruginosa were co-cultured on the L-929 cell line and wound infection in an animal model. Then, recombinant staphopain A was purified and used to treat mono- and co-infections. Following the treatment, changes in virulence factors and resistance were investigated through phenotypic methods and RT-PCR. Staphopain A resulted in a notable reduction in the viability of S. aureus and P. aeruginosa. The biofilm formed in the wound infection in both animal model and cell culture was disrupted remarkably. Moreover, the biofilm-encoding genes, quorum sensing regulating genes, and virulence factors (hemolysin and pyocyanin) controlled by QS were down-regulated in both microorganisms. Furthermore, the resistance to vancomycin and doripenem decreased following treatment with staphopain A. According to this study, staphopain A might promote wound healing and cure co-infection. It seems to be a promising agent to combine with antibiotics to overcome hard-to-cure infections.

The healing effect of Pseudomonas Quinolone Signal (PQS) with co-infection of Staphylococcus aureus and Pseudomonas aeruginosa: A preclinical animal co-infection model.

BACKGROUND: Because of the rise in antibiotic resistance and the control of pathogenicity, polymicrobial bacterial biofilms exacerbate wound infections. Since bacterial quorum sensing (QS) signals can dysregulate biofilm development, they are interesting therapeutic treatments. In this study, Pseudomonas Quinolone Signal (PQS) was used to treat an animal model of a wound that had both Staphylococcus aureus and Pseudomonas aeruginosa co-infection. METHODS: S. aureus and P. aeruginosa mono- and co-infection models were developed in vitro on the L-929 cell line and in an animal model of wound infection. Moreover, PQS was extracted and purified using liquid chromatography. Then, the mono- and co-infection models were treated by PQS in vitro and in vivo. RT-PCR analysis was used to look into changes in biofilm, QS, tissue regeneration, and apoptosis genes after the treatment. RESULTS: PQS significantly disrupted established biofilm up to 90% in both in vitro and in vivo models. Moreover, a 93% reduction in the viability of S. aureus and P. aeruginosa was detected during the 10 days of treatment in comparison to control groups. In addition, the biofilm-encoding and QS-regulating genes were down-regulated to 75% in both microorganisms. Also, fewer epithelial cells died when treated with PQS compared to control groups in both mono- and co-infection groups. CONCLUSION: According to this study, PQS may facilitate wound healing by stimulating the immune system and reducing apoptosis. It seems to be a potential medication to use in conjunction with antibiotics to treat infections that are difficult to treat.

Inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) with different multi-locus sequence types (MLST), SCCmec, and spa types.

BACKGROUND: The inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) are unclear. The main aim is to better understand of inhibitory activities of vitamins K2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutation and gene expressions. MATERIALS AND METHODS: After collecting S. aureus clinical isolates and detecting QR-MRSA, the genes encoding norA, grlA, grlB, gyrA, and gyrB were sequenced. After treating isolates by vitamin K2, isolates were prepared to measure norA, grlA, grlB, gyrA, and gyrB gene expression. The quantitative-real-time PCR was used to measure the expression of efflux pump genes. RESULTS: QR-MRSA, MDR, and XDR strains were reported in 59.4%, 73.9%, and 37.6% of isolates, respectability. SCCmecIV (36.5%) and SCCmecV (26.8%) had the highest frequency. Thirty-nine spa types were identified, t021, t044, and t267 types most prevalent in QR-MRSA isolates. ST22 and ST30 dominated the invasive, drug-resistant isolates and QR-MRSA. In 24 h incubated isolates, the most noticeable change of gene expression with vitamin K2 was that the norA, gyrA, and grlB genes were highly repressed. However, the down-regulation of grlA at 24 h after being treated by vitamin K2 was more than another gene. Further, a significant decrease was observed in QR-MRSA-treated isolates compared to un-treated isolates. In other words, norA, grlA, grlB, gyrA, and gyrB genes were less suppressed by QR-MRSA (p ≤ 0.01, p ≤ 0.05). CONCLUSION: Vitamin K2 has significant inhibitory effects on the genes responsible for resistance to fluoroquinolone antibiotics. However, a subminimum inhibitory concentration (sub-MIC) level of vitamin K2 was delayed but did not completely inhibit norA, grlA, grlB, gyrA, and gyrB genes in MRSA strains.

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-ß-lactamase (NDM) producing Pseudomonas aeruginosa.

New Delhi metallo-ß-lactamase-1 (NDM-1) producing Pseudomonas aeruginosa strain detection plays a vital role in confirming bacterial disease diagnosis and following the source of an outbreak for public health. However, the standard method for NDM-1 determination, which relies on the features of the colony of the bacteria cultured from the patient's specimen, is time-consuming and lacks accuracy and sensitivity. This study aimed to standardize a high-resolution melting curve analysis (HRMA) assay to detect NDM producing P. aeruginosa. For optimization and development of the HRMA method, a reference strain of P. aeruginosa was used. For evaluating the broad range PCR data, ABI Step One-Plus Manager Software version 3.2 and Precision Melt Analysis Software 3.02 (Applied Biosystems) were used. Based on the results, expected results were obtained for all tested strains, with high analytical sensitivity and specificity. Temperature melting analyses of the HRMA time PCR assays showed the Tm at 89.57 °C, 76.92 °C and 82.97 °C for N-1, N-2 and N-3 genes, respectively. Also, melting point temperatures of the bla VIM, bla SPM and bla SIM amplicons for isolates identified as MBL strains were 84.56 °C, 85.35 °C and 86.62 °C, respectively. The amplification results using negative control genomes as templates were negative, showing the specificity of the designed assays. Our study's data indicated that the sensitivity and specificity of the HRMA method are linked to the primer length and the fluorescent dye. We can further identify antibiotic resistance in NDMproducing P. aeruginosa by software analysis and melting curve analysis.

Detection of blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 Genes and Carbapenem-Hydrolyzing Class D ß-Lactamases (CHDLs) in Clinical Isolates of Pseudomonas aeruginosa Collected from West of Iran, Hamadan.

Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are on the rise and are a major public health problem worldwide. Pseudomonas aeruginosa is resistant to carbapenem; currently, the most effective treatment option is being increasingly reported. This study aimed to identify blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes in CHDL strains. Samples were collected from clinical specimens admitted to the hospital. Antibiotic susceptibility was determined using the disk diffusion methods. CHDL strains were detected using a phenotypic method (disk diffusion). The PCR method was used to identify blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes. Piperacillin-resistant strains (n = 9, 17.4%) had the lowest frequency, and cefoxitin-resistant strains (n = 100, 91.7%) had the highest distribution in P. aeruginosa isolates. Also, 29.35%, 12.8%, and 8.2% were multidrug-resistant, extensively drug-resistant, and pan drug-resistant, respectively. MBL-producing P. aeruginosa and KPC-producing P. aeruginosa were detected, respectively, in 47.7% and 37.6% of isolates. Biofilm formation was observed in 63.3% of P. aeruginosa isolates. The frequency of OXA genes was as follows: blaOXA-145 gene in 30 isolates (27.5%), blaOXA-224 in 24 isolates (22.0%), blaOXA-539 in 22 isolates (20.1%), and blaOXA-675 in 13 isolates (11.9%). However, 19 (17.4%) isolates carry all blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes. The antimicrobial resistance and OXA genes among biofilm former strains were significantly higher than those of nonbiofilm former strains (p < 0.05). The emergence of carbapenem-resistant isolates of P. aeruginosa has posed serious threats to the community because they exhibit multiple drug resistance, thus limiting the therapeutic options for clinicians.

Molecular epidemiology and collaboration of siderophore-based iron acquisition with surface adhesion in hypervirulent Pseudomonas aeruginosa isolates from wound infections.

Iron/siderophore uptake may play an important role in the biofilm formation and secretion of extracellular proteins in Pseudomonas aeruginosa isolates. In the present study, the role of siderophores, heme, and iron regulatory genes in the virulence of Pseudomonas aeruginosa isolates collected from wound infection was investigated. Three hundred eighty-four (384) swab samples were collected from wound infection and identified by phenotypic methods. The quantitative real-time PCR (qRT-PCR) method was evaluated for the gene expressions study. Multi-locus sequence typing (MLST) was used to screen unique sequence types (ST) and clonal complexes (CC). Fifty-five (55) P. aeruginosa isolates were detected in all swab samples. Also, 38 (69.1%) isolates formed biofilm. The prevalence of virulence factor genes was as follows: plcN (67.2%), exoY (70.9%), exoA (60.0%), phzM (58.1%), plcH (50.9%), lasB (36.3%), aprA (69.1%), lasA (34.5%), nanI (74.5%), exoU (70.9%), exoS (60.0%), exoT (63.6%) and algD (65.4%). According to qRT-PCR, genes regulating iron uptake were highly expressed in the toxigenic isolate. The highest expressions levels were observed for hemO, hasR, and pvdA genes in the biofilm-forming isolates. The MLST data confirmed a high prevalence of ST1, ST111, and ST235, with six, five, and 12 clusters, respectively. ST235 and ST1 were the most present among the biofilm-forming and toxigenic strains. Also, the nuoD gene with 54 and guaA with 19 showed the highest and lowest number of unique alleles. We demonstrated that iron/siderophore uptake is sufficient for biofilm formation and an increase in the pathogenesis of P. aeruginosa. These results suggest that the iron/siderophore uptake system may alter the MLST types of P. aeruginosa and predispose to bacterial pathogenesis in wound infections.

Antibiotic resistance alters through iron-regulating Sigma factors during the interaction of Staphylococcus aureus and Pseudomonas aeruginosa.

Iron is a limiting factor in such a condition that usually is sequestered by the host during polymicrobial infections of Pseudomonas aeruginosa and Staphylococcus aureus. This study aimed to investigate the interaction of S. aureus and P. aeruginosa, which alters iron-related sigma factors regulation and antibiotic resistance. The antibiotic resistance of P. aeruginosa and S. aureus was investigated in a L929 cell culture model. The expression level of pvdS, hasI (P. aeruginosa sigma factors), and sigS (S. aureus sigma factor) genes was determined using Quantitative Real-Time PCR. pvdS and hasI were downregulated during co-culture with S. aureus, while the susceptibility to carbapenems increased (p-value < 0.0001). Also, there was a direct significant relationship between resistance to vancomycin with sigS. Regarding the findings of the current study, iron-related sigma factors of P. aeruginosa and S. aureus play a role in induction susceptibility to various antibiotics, including carbapenems and vancomycin.

Coexistence of Virulence Factors and Efflux Pump Genes in Clinical Isolates of Pseudomonas aeruginosa: Analysis of Biofilm-Forming Strains from Iran.

BACKGROUND: Biofilm formation and efflux pumps (EPs) correlation play a critical role in the pathogenicity and antibiotic resistance of Pseudomonas aeruginosa. In this study, biofilm formation and EP's collaborative role in clinical isolates of P. aeruginosa infection were investigated. METHODS: Eighty-six (86) P. aeruginosa isolates were collected from different clinical specimens and were confirmed using different biochemical tests. The formation of biofilm was investigated by using a crystal violet assay. Also, EP genes were identified by the PCR method. RESULTS: Based on the results, gentamicin-resistant (n = 50, 66.29%) and ciprofloxacin-resistant (n = 61, 69.66%) strains were the most frequent and colistin (n = 1, 1.12%) and ceftazidime (n = 12, 7.86%) resistant strains were the least prevalent. Furthermore, 22 isolates (31.42%) were MDR, and 11 isolates (12.35%) were XDR strains. Also, 19 isolates (22.47%) were classified as strong biofilm, 29 isolates (21.34%) as moderate biofilm, and 3 (11.23%) isolates as weak biofilm producers. The distribution of the EP genes was as follows: mexA (n = 44, 34.83%), mexB (n = 33, 32.58%), oprM (n = 59, 29.21%), oprD (n = 61, 30.33%), tetA (n = 22, 25.58%), tetR (n = 19, 22.09%), and emrE (n = 21, 24.41%). However, there was a strong significant association between biofilm formation and EPs in P. aeruginosa. Conclusions. In this study, we suggested that the presence of a multidrug resistance efflux pump, MexEF-OprN, significantly reduced P. aeruginosa pathogenicity. In contrast, the presence of the MexAB-OprM and MexCD-OprJ pumps did not affect virulence.

Regulation of virulence and ß-lactamase gene expression in Staphylococcus aureus isolates: cooperation of two-component systems in bloodstream superbugs.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA)-bloodstream infections (BSI) are predominantly seen in the hospital or healthcare-associated host. Nevertheless, the interactions of virulence factor (VFs) regulators and ß-lactam resistance in MRSA-BSI are unclear. This study aims to characterize the molecular relationship of two-component systems of VFs and the expression of the ß-lactamase gene in MRSA-BSI isolates. In this study, 639 samples were collected from BSI and identified by phenotypic methods. We performed extensive molecular characterization, including SCCmec type, agr type, VFs gene profiles determinations, and MLST on isolates. Also, a quantitative real-time PCR (q-RT PCR) assay was developed for identifying the gene expressions. RESULTS: Ninety-one (91) S. aureus and 61 MRSA (67.0%) strains were detected in BSI samples. The presence of VFs and SCCmec genes in MRSA isolates were as follows: tst (31.4%), etA (18.0%), etB (8.19%), lukS-PVL (31.4%), lukF-PV (18.0%), lukE-lukD (16.3%), edin (3.2%), hla (16.3%), hlb (18.0%), hld (14.7%), hlg (22.9%), SCCmecI (16.3%), SCCmecII (22.9%), SCCmecIII (36.0%), SCCmecIV (21.3%), and SCCmecV (16.3%). Quantitative real-time PCR showed overexpression of mecRI and mecI in the toxigenic isolates. Moreover, RNAIII and sarA genes were the highest expressions of MRSA strains. The multi-locus sequence typing data confirmed a high prevalence of CC5, CC8, and CC30. However, ST30, ST22, and ST5 were the most prevalent in the resistant and toxigenic strains. CONCLUSION: We demonstrated that although regulation of ß-lactamase gene expressions is a significant contributor to resistance development, two-component systems also influence antibiotic resistance development in MRSA-BSI isolates. This indicates that resistant strains might have pathogenic potential. We also confirmed that some MLST types are more successful colonizers with a potential for MRSA-BSI.

A Comprehensive Study of the Relationship between the Production of ß-Lactamase Enzymes and Iron/Siderophore Uptake Regulatory Genes in Clinical Isolates of Acinetobacter baumannii.

BACKGROUND: The iron/siderophore uptake system (IUS) involved in the Acinetobacter baumannii pathogenicity. However, IUS's role in antibiotic resistance and the production of ß-lactamase enzymes of A. baumannii are unclear. This study aimed to investigate the relationship between the production of ß-lactamase enzymes and IUS regulatory genes in clinical isolates of A. baumannii. Methods. A. baumannii isolates were collected from clinical isolates using biochemical tests. The antibiotic resistance patterns and ß-lactamase-producing strains were identified using the disk diffusion method (DDM). Also, IUS genes were detected by the polymerase chain reaction (PCR) method. RESULTS: Seventy-two (72) A. baumannii isolates were collected from a different clinical specimen. Gentamicin-resistant strains (43%) had the highest frequency, and aztreonam-resistant strains (12.5%) had the lowest frequency. Also, the distribution of AmpC and MBL producing isolates were 27.7% and 35%, respectively. Moreover, the frequencies of basD, bauA, pld, paaE, entA, feoB, hemO, and tonB genes were as follows: 12.5%, 15.2%, 11.1%, 15.2%, 19.4%, 16.6%, 23.6%, and 6.9%. Further, a strong correlation was observed between the abundance of ß-lactamase-producing strains and IUS genes. CONCLUSIONS: Based on our knowledge from this study, the association between ß-lactamase production and IUS genes in A. baumannii plays an essential role in the emergence of drug-resistant strains.

The inhibitory effects of Staphylococcus aureus on the antibiotic susceptibility and virulence factors of Pseudomonas aeruginosa: A549 cell line model.

Pseudomonas aeruginosa and Staphylococcus aureus often lead to serious lung infections. This study aimed to investigate the role of S. aureus in the expression of the ß-lactamase enzymes and virulence factors of P. aeruginosa in the polymicrobial infections of the respiratory tract. Biofilm and planktonic co-culture of P. aeruginosa and S. aureus were performed in the A549 cell line. Then, antibiotic resistance and virulence factors of P. aeruginosa were examined, and the expression of lasR, lasI, algD, mexR, and KPC genes were determined using qPCR. S.aureus decreased ß-lactam resistance but increased resistance to tobramycin in the biofilm condition. Furthermore, S.aureus showed a positive effect on reducing resistance to meropenem, doripenem, and tobramycin (except PA-2). Altough it was demonstrated that S.aureus reduced the viability of P. aeruginosa, particularly in the biofilm state, the pathogenicity of the recovered strains of P.aeruginosa increased. Moreover, the gene expression levels for lasR/I and algD were increased in biofilm conditions. The levels of lasI were more prominent in the virulent strain than the ß-lactamase producing strain. Furthermore, the expression of KPC was increased in all strains of P. aeruginosa. According to the findings of this study, S. aureus has an inhibitory effect in polymicrobial infections by suppressing the ß-lactamase genes and viability of P. aeruginosa. Also, it cooperates with the biofilm-producing P. aeruginosa strains to increase pathogenicity and resistance to tobramycin.

Prevalence and distribution of resistance and enterotoxins/enterotoxin-like genes in different clinical isolates of coagulase-negative Staphylococcus.

BACKGROUND: Coagulase-negative staphylococcus (CoNS) is considered to be the major reservoirs for genes facilitating the evolution of S. aureus as a successful pathogen. The present study aimed to determine the occurrence of genes conferring resistance to fluoroquinolone, determining of the prevalence of insertion sequence elements IS256, IS257 and different superantigens (SAgs) among CoNS isolates obtained from various clinical sources. MATERIALS AND METHODS: The current study conducted on a total of the 91 CoNS species recovered from clinical specimens in Hamadan hospitals in western Iran in 2017-2019. The antimicrobial susceptibility testing was performed using disk diffusion method and the presence of the IS256 and IS257, genes conferring resistance to fluoroquinolone and enterotoxins/enterotoxin-like encoding genes were investigated by polymerase chain reaction (PCR) method. RESULTS: Among genes encoding classic enterotoxins, sec was the most frequent which was carried by 48.4% of the 91 isolates, followed by seb in 27.5% of the isolates. None of the CoNS isolates was found to be positive to enterotoxin-like encoding genes. In 11(12%) of all isolates that were phenotypically resistant to levofloxacin, 9 isolates (81.8%) were positive for gyrB, 8 isolates (72.7%) were positive for gyrA, 8 isolates (72.7%) harbored grlB and 7 isolates (63.6%) were found to carry grlA. The IS256 and IS257 were identified in 31.8% and 74.7% of the isolates, respectively. The results of statistical analysis showed a significant association between the occurrence of staphylococcal enterotoxins (SEs) encoding genes and antimicrobial resistance. CONCLUSION: Antimicrobial resistant determinants and SEs are co-present in clinical CoNS isolates that confer selective advantage for colonization and survival in hospital settings. The coexistence of insertion elements and antibiotic resistance indicate their role in pathogenesis and infectious diseases.

Co-harboring of mcr-1 and ß-lactamase genes in Pseudomonas aeruginosa by high-resolution melting curve analysis (HRMA): Molecular typing of superbug strains in bloodstream infections (BSI).

Background Colistin resistance in P. aeruginosa (CRPA) is due to the appearance of superbug strains. As this pathogen gains more transferrable resistance mechanisms and continues to adapt to acquire additional resistance mechanisms during antimicrobial therapy rapidly, we face the growing threat of CRPA in bloodstream infections (BSI). This study designed to evaluate the frequency of CRPA strains producing different ß-lactamases by the High-Resolution Melting Curve Analysis (HRMA) method in BSI and to characterize the different types by multilocus sequence typing (MLST). MATERIAL AND METHODS: Sixty-nine (69) P. aeruginosa isolates were collected from blood culture. MIC E-test methods examined the antimicrobial susceptibilities of the bacterial isolates. Detection of resistant strains performed by using HRMA assay. RESULTS: The strains resistant to amikacin (n = 11; 15.94%) and colistin (n = 10; 14.49%) were the least abundant and the gentamicin (n = 56; 82.6%) and ciprofloxacin (n = 67; 97.10%) resistant strains were the most frequent. Also, 39 isolates (56.52%) considered as multidrug-resistant (MDR), 20 isolates (28.98%) as extensively drug resistant (XDR), and 11 isolates (15.94%) as Pandrug Resistance (PDR). Further, 32 isolates (46.37%) considered as AmpC producer, and 28 isolates (40.57%) were considered an MBL producer. According to HRMA results, the blaSPM gene was detected in 19 isolates (27.53%), blaNDM gene in 11 isolates (15.94%), blaFOX gene in 31 isolates (44.92%), mcr-1 gene in 10 isolates (14.49%), blaACC and blaVIM genes in 27 isolates (39.13%), and blaTEM gene was reported in 20 isolates (28.98%). Furthermore, P. aeruginosa PASGNDM699, ST3340, and ST235 identified in 1.44%, 11.59% and 17.39% isolates, respectively. CONCLUSION: CRPA strains play an essential role in the spread of antibiotic resistance in BSI. Likewise, the HRMA method was sensitive and specific for the detection of superbugs. Moreover, MLST analysis of a diverse collection of P. aeruginosa from blood culture suggests that particular strains or clonal complexes are associated with antibiotic resistance profile.

Prevalence and Molecular Typing of Colistin-Resistant Pseudomonas aeruginosa (CRPA) Among ß-Lactamase-Producing Isolates: A Study Based on High-Resolution Melting Curve Analysis Method.

BACKGROUND: The frequency and production of ß-lactamase enzymes may be different in colistin-resistant Pseudomonas aeruginosa (CRPA) strains compared to susceptible strains. The purpose of this study was to investigate the relationship between colistin resistance and ß-lactamase enzymes in different Sequence Types (ST) of P. aeruginosa. METHODS: A total of 101 P. aeruginosa isolates were collected from different samples. The antimicrobial susceptibilities of the bacterial isolates were examined by disk diffusion and MIC E-test methods. Also, real-time PCR and high-resolution melting curve analysis (HRMA) assay were performed to detect the resistance genes. RESULTS: Out of the 101 P. aeruginosa isolates, four isolates (3.96%) were resistant to colistin. Also, 39 isolates (38.61%) were considered as MDR, and eight isolates (7.92%) were considered as XDR. Further, 25 (24.75%) and 26 isolates (25.74%) were produced ESBL and carbapenemase enzymes, respectively. According to HRMA results, four isolates (3.96%) were positive for pmrA, three isolates (2.97%) were positive for mcr-1, 25 isolates (24.75%) were positive for blaTEM, 24 isolates (23.76%) were positive for blaSHV, 26 isolates (25.75%) were positive for blaKPC, and 23 isolates (22.77%) were positive for blaIMP genes. Furthermore, ST108 and ST250 showed the highest distribution in P. aeruginosa isolates. Also, ST217, ST1078, and ST3340 were reported as novel types in CRPA strains. CONCLUSION: Concerns about the prevalence of CRPA strains should be taken seriously. Also, our results showed that the mcr-1 gene plays a vital role in the distribution of ESBL and KPC-producing P. aeruginosa strains.

Distribution of Class B and Class A ß-Lactamases in Clinical Strains of Pseudomonas aeruginosa: Comparison of Phenotypic Methods and High-Resolution Melting Analysis (HRMA) Assay.

BACKGROUND: There are various phenotypic methods for identifying class B and class A ß-lactamase enzymes in Pseudomonas aeruginosa. The purpose of this study was to compare the sensitivity and specificity of different phenotypic methods with HRMA assay to detect ß-lactamase-producing P. aeruginosa strains. METHODS: Eighty-eight of P. aeruginosa isolates were collected from different specimens. Conventional double-disk test (DDT) and EDTA-imipenem microbiological (EIM) were performed to detect ESBL and MBL-producing strains, respectively. Meanwhile, the Modified Hodge test and Carba-NP test were performed on all carbapenem-resistant strains. HRMA method and sensitivity and specificity of primers were determined based on the melt curve temperature range. In all comparisons, PCR was considered as the gold standard. RESULTS: Of the 402 isolates collected from different clinical specimens, 88 isolates of P. aeruginosa were identified. However, 43 strains were (48.88%) ESBL-producing, and 7 strains (7.95%) were MBL-producing. Also, using the Modified Hodge test and Carba-NP method, 11 (12.5%) and 19 (21.59%) strains were carbapenemase-producing, respectively. The results of the HRMA test revealed that genes coding for bla SHV, bla TEM, bla KPC, bla IMP, bla VIM, and bla GES were detected in 44.31%, 22.72%, 13.63%, 14.7%, 5.6%, and 2.27% of P. aeruginosa isolates. Nonetheless, for bla KPC and bla GES genes, sensitivity and specificity of the Carba-NP test were 90.47%, 94.87%, and 83.36%, 94.80%, respectively. However, sensitivity and specificity of MHT was 91.66%, 98.70%, and 77.77%, 96.42%, respectively. For bla SHV and bla TEM genes, sensitivity and specificity of DDT were 95.55%, 95.55%, and 86%, 83.50%, respectively. However, sensitivity and specificity of EMI were 77.77%, 97.59%, and 91.66%, 97.43% for bla VIM and bla IMP, respectively. CONCLUSION: The HRMA is a powerful, accurate, closed-tube, rapid method for detecting ß-lactamase genes in P. aeruginosa. The high sensitivity and specificity of this method, along with phenotypic tests, play a useful role in increasing the predictive value of clinical reports.

Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates.

BACKGROUND: High-resolution melting analysis (HRMA) is a novel molecular technique based on the real-time PCR that can be used to detect vancomycin resistance Enterococcus (VRE). The purpose of this study was to identify VRE species with HRMA in clinical isolates. RESULTS: Out of 49 Enterococcus isolates, 11 (22.44%) E. faecium isolates and 19 (38.77%) E. faecalis isolates were detected. Average melting temperatures for divIVA in E.faecalis, alanine racemase in E.faecium, and vanA in VRE strains were obtained as 79.9 ± 0.5 °C, 85.4 ± 0.5 °C, and 82.99 ± 0.5 °C, respectively. Furthermore, the data showed that the HRMA method was sensitive to detect 100 CFU/ml for the divIVA, alanine racemase, and vanA genes. Also, out of 49 Enterococcus spp., which were isolated by HRMA assay, 8 isolates (16.32%) of E. faecium and 18 isolates (36.73%) of E. faecalis were detected. The vanA gene was reported in 2 isolates (25%) of E. faecium and 9 isolates (50%) of E. faecalis. CONCLUSIONS: This study demonstrated that using the HRMA method, we can detect E. faecium, E. faecalis, and the vanA gene with high sensitivity and specificity.

Comparing the Success Rate and Side Effects of Endovenous Laser Ablation and Radiofrequency Ablation to Treat Varicose Veins in the Lower Limbs: A Randomized Clinical Trial.

Introduction: Varicosis is a condition affecting the superficial venous system of the lower limbs. Endovenous laser ablation (EVLA) is a minimally invasive method that uses different frequencies of laser for treatment. Radiofrequency (RF) has also been effectively used for vein ablation through thermal mechanisms. This study compares the success rate and side effects of EVLA and RF to treat varicose veins in the lower limbs within 12 months. Methods: In this two-arm, parallel-group, randomized controlled, single-blind study, 1090 patients with lower limb varicosis proven by Doppler ultrasonography (DUS) were randomly placed into one of the two groups: EVLA and RF ablation. In the EVLA group, we used a 980-nm diode laser in a pulse mode and in the RF group, a 7-cm ClosureFast (Covidien, USA) catheter with an RF generator. We assessed pain during the operation and 8 and 24 hours after the surgery by the numerical rating scale (NRS). The patients were followed up on day 7, then 3, 6, and 12 months postoperatively. The presence or absence of pain and then the severity of pain (according to NRS), the recurrence rate, complications, and the length of the procedure were compared. Results: There was no significant difference between the two groups in the patients' characteristics, major adverse events and pain severity during the procedure and 8 and 24 hours after the surgery. According to NRS, the mean chronic pain severity in the EVLA group was 3.99± 0.754 versus 4.50± 0.657 in the RF group, and the difference was not significant. The length of operation and the recurrence rate 12 months after the procedure were not significantly different. Conclusion: The results of our study further establish the efficacy of EVLT and RF ablation for lower limb varicosis treatment without any significant difference in the clinical outcomes and complications during a 12-months follow-up.

Relationship between biofilm gene expression with antimicrobial resistance pattern and clinical specimen type based on sequence types (STs) of methicillin-resistant S. aureus.

The ica genes in methicillin-resistant Staphylococcus aureus (MRSA) play an important role in biofilm formation. The aim of this study is to define effect of antibiotic resistance and clinical specimens to the expression of ica genes based on their sequence types (STs) and clonal complex (CC). One-hundred (100) S. aureus strain were collected from two teaching therapeutic centers in Hamedan, Iran. Then, the PCR, qPCR, and MLST were used to characterize strains. The results indicated that 29 (29%), 15 (15%), and 5 (5%) strain were strong, mediate, weak biofilm producer, respectively, and the icaA (17%) and icaC (14%) genes were the most abundant. However, two unique STs (3667, 491) in Iran were reported and ST30 and ST11 were the most abundant STs and CC30 and CC5 were observed among MRSA and MSSA strains. High activity in ica locus was observed among strains collected from wound and catheter strains. Also, expression level of icaA gene increased in all strains except ST30 and ST491. Moreover, the highest expression level was observed in CC1, CC7, and CC11. Likewise, activity of the icaC gene was only observed in CC5. Furthermore, the expression of all ica genes in CC5 was significantly correlated with the type of biofilm and the clinical sample. In this study demonstrated that the frequency distribution of STs and CCs in different strains of MRSA was higher than methicillin-sensitive strains. Also, the type of clinical specimen and expression of ica genes played an important role in this abundance.

The Clinical Utility of Analysis High Resolution Melting Curve Assay for Simultaneous Identification of Methicillin and Mupirocin Resistant in Coagulase-Negative Staphylococci.

BACKGROUND: Resistance to methicillin and mupirocin in coagulase-negative staphylococci (CoNS) is considered as one of the most important issues. Meanwhile, use of phenotypic methods in detecting the resistance to methicillin and mupirocin has a lower accuracy and speed compared to other molecular methods, such as High-Resolution Melting Analysis (HRM). In this study, HRM technique was used to identify CoNS resistance stains. METHODS: This experimental study was done on 86 isolates of CoNS strains isolated at Hamadan Hospital. Pheno-typic tests were used to identify Staphylococcus saprophyticus and S. epidermidis. Methicillin and Mupirocin resistant strains were tested using the MIC and HRM methods, and sensitivity and specificity of the primers were determined based on melting curve temperature range. In addition, data was analyzed by Applied Biosystems StepOne v 2.3 and Applied Biosystems HRM v 3.0.1 software. RESULTS: Eighty-six (86) coagulase-negative isolates were isolated from different clinical specimens. Among these, 69 isolates of S. epidermidis and 17 isolates of S. saprophyticus were identified. Of the 69 S. epidermidis isolates, 19 isolates with oxacillin MIC ≥ 0.5 µg/mL and methicillin-resistant, and 11 isolates with mupirocin MIC ≥ 32 µg/mL and resistant to mupirocin. Of the 17 S. saprophyticus isolates, three isolates with oxacillin MIC ≥ 0.5 µg/mL were also methicillin-resistant, and one isolate with mupirocin MIC ≥ 32 µg/mL was resistant to mupirocin. Melting curve analysis for mecA and mupA primers was determined 81.7 ± 0.5°C and 74 ± 0.5°C, respectively. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of HRM assay for detection of methicillin and mupirocin strains testing were 100%. CONCLUSIONS: Identification of methicillin and mupirocin resistance in CoNSs using the HRM method has high sensitivity and specificity. Molecular methods are more accurate and faster than phenotypic methods and can identify a large number of resistant isolates in a short time.