Effect of Predator Bacteria Bdellovibrio bacteriovorus on Clinical Pathogens and Biofilms.

Antimicrobial resistance has become one of the most important public health problems of our century. In addition to the spread of resistance, biofilm production also makes the treatment of infections increasingly difficult. Therefore, this study, it was aimed to investigate the effect of the predator bacterium Bdellovibrio bacteriovorus HD100 on various clinical pathogens and their biofilms. A large panel of Gram-positive and negative clinical isolates were included in the study. The double-layer agar method was used to optimize the cultivation of predatory bacteria. The effectiveness of Bdellovibrio bacteriovorus HD 100 on planktonic cells and biofilms, was determined by co-culture and crystal violet staining methods, respectively. The antibiofilm activity was also visualized via scanning electron microscopy. The predator bacteria was found effective against most of the Gram-negative isolates. But it was determined that the lowest activity among these isolates was shown to Pseudomonas aeruginosa and Acinetobacter baumannii. Although it is known that B. bacteriovorus does not predate on Gram-positive isolates, interestingly, Staphylococci species included in this study were found to be inhibited in co-culture studies. As determined in co-culture and biofilm studies, B. bacteriovorus can be used to control both bacterial growth and biofilms in most Gram-negative species. Interestingly, our data also suggest that predatory bacteria may also be effective against Gram-positive bacterial biofilms in addition to Staphylococcus aureus. Although the evaluation of different species of isolates in this study demonstrates the potential of predatory bacteria, the host specificity and the relation of prey and predator need to be demonstrated. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01071-y.

99mTc-Labeled, Colistin Encapsulated, Theranostic Liposomes for Pseudomonas aeruginosa Infection.

Infectious diseases are still the major issue not only due to antibiotic resistance but also causing deaths if not diagnosed at early-stages. Different approaches including nanosized drug delivery systems and theranostics are researched to overcome antibiotic resistance, decrease the side effects of antibiotics, improve the treatment response, and early diagnose. Therefore, in the present study, nanosized, radiolabeled with 99mTc, colistin encapsulated, neutral and cationic liposome formulations were prepared as the theranostic agent for Pseudomonas aeruginosa infections. Liposomes exhibited appropriate physicochemical properties thanks to their nano-particle size (between 173 and 217 nm), neutral zeta potential value (about - 6.5 and 2.8 mV), as well as encapsulation efficiency of about 75%. All liposome formulations were radiolabeled with over 90% efficiency, and the concentration of stannous chloride was found as 1 mg.mL-1 to obtain maximum radiolabeling efficiency. In alamar blue analysis, neutral liposome formulations were found more biocompatible compared with the cationic formulations. Neutral colistin encapsulated liposomes were found to be more effective against P. aeruginosa strain according to their time-dependent antibacterial effect, in addition to their highest bacterial binding capacity. As conclusion, theranostic, nanosized, colistin encapsulated, neutral liposome formulations were found as promising agents for the imaging and treating of P. aeruginosa infections.

Carbapenemase investigation with rapid phenotypic test (RESIST-4 O.K.N.V) and comparison with PCR in carbapenem-resistant Enterobacterales strains.

Background and Objectives: RESIST-4 O.K.N.V. assay is a lateral immunochromatocraphic test for the identification of oxacillinase (OXA)-48-like, Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-ß-lactamase (NDM), and Verona integron-encoded metallo-ß-lactamase (VIM) producing strains. It was aimed to evaluate the performance of the RESIST-4 O.K.N.V. test and to compare it with the reference method polymerase chain reaction (PCR). Also, the objective was to determine the distribution of carbapenemase types of CRE strains isolated in our hospital. Materials and Methods: Between January 2016-October 2019, 187 strains isolated from clinical samples were included in this study. Bacterial identification was done using MALDI-TOF MS. Antibiotic susceptibility tests were studied with the VITEK-2 automated system. Meropenem minimum inhibitory concentrations (MICs) were determined by the gradient test. All strains were studied with the RESIST-4 O.K.N.V. test and then the strains were selected for the PCR test. bla OXA-48, bla NDM, bla KPC, and bla VIM were investigated with PCR. K. pneumoniae NCTC® 13438 (KWIKSTIKTM, Microbiologics®, USA) was used as the positive control, E. coli ATTC® 25922 TM (Microbiologics®, USA) and three carbapenem-sensitive clinical isolates were also used as the negative control. Results: Meropenem MIC50 and MIC90 values were determined to be >32 mg/L. With PCR bla OXA-48, bla NDM, bla KPC and bla VIM were detected in 79, 63, 20, and 4 strains, respectively. bla OXA-48 and bla NDM were found together in 51 of the isolates. bla OXA-48, bla NDM, bla KPC, and bla VIM were not detected in two strains with carbapenem resistance in susceptibility tests. The sensitivity of the immunochromatographic test was 100% for OXA-48, KPC, and VIM but 84.1% for NDM. Specificity was determined as 100% for OXA-48, NDM, KPC, and VIM. Conclusion: RESIST-4 O.K.N.V. test showed high sensitivity and specificity in detecting OXA-48, KPC, NDM, and VIM type carbapenemases. However, it should be kept in mind that there may be false-negative results related to NDM.

Silencing acpP gene via antisense oligonucleotide-niosome complex in clinical Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is one of the major causes of nosocomial infections. In addition to its physiological adaptation capacity, it can develop resistance to disinfectants and antibiotics through various mechanisms. Recently, new eradication methods are gaining attention. Therefore, in this study, an LNA-2'-O-methyl hybrid antisense oligonucleotide targeting the acyl carrier protein P (acpP) gene was introduced into P. aeruginosa isolates. The design was determined through sequence analysis and prediction of the secondary structure of mRNA by software. Niosomes were used for enhancing cellular uptake. The control of the binding and transfection ability of the sequence was determined fluorometrically by labeling with 6-Fam. The effects were determined with broth microdilution method and qPCR studies. Eight different formulations were prepared. Among these, one formulation has shown to have ASO complexation ability whose composition was 312 µl Span 80 + 69.5 mg Cholesterol+ 36.4 mg CTAB+1 ml Chloroform and 5 ml dH2O. Thus this formulation was determined as the delivery system for the next stages. Significant gene inhibition was detected at the six isolates. Results of this study suggested that niosomes can be used as a delivery system for cellular uptake of ASO and could eliminate bacterial growth.

Effects of disinfectants and ciprofloxacin on quorum sensing genes and biofilm of clinical Pseudomonas aeruginosa isolates.

BACKGROUND: Pseudomonas aeruginosa is one of the most virulent bacteria and quorum sensing (QS) genes have an importance on virulence factors such as biofilm that provide resistance against disinfectants and antibiotics. OBJECTIVE: This study aimed to determine the minimum inhibitory concentrations of the disinfectants, to investigate the effects of disinfectants and ciprofloxacin on biofilm production mature biofilm of clinical P. aeruginosa isolates, and it was aimed to investigate the effects of the agents on the expression levels of several QS-related genes in the isolates. METHODS: Minimum inhibitory concentration (MIC) levels of polyhexamethylene biguanide (PHMB), chlorhexidine (CHX), quaternary ammonium compounds (QAC), glutaraldehyde (GLU) and ciprofloxacin (CIP) against clinical P. aeruginosa isolates were evaluated by microdilution method. Effects of the agents on the biofilm producing capacities of clonally unrelated nine strains were investigated by spectrophotometric method. Alterations in the expression of QS-related genes (lasI, lasR, rhlI and rhlR) were investigated by qPCR in three isolates that were CIP-susceptible and strong biofilm producer. RESULTS: According to microdilution method results, three isolates were found as resistant, one isolate was found as intermediate susceptible and five isolates were found as susceptible to CIP, and CHX (7.81-31.25 µg/mL) had the lowest MIC against P. aeruginosa. CHX inhibited biofilm production levels of eight of nine isolates, and GLU and CIP inhibited six of nine isolates in the presence of agents at MIC levels. GLU inhibited the mature biofilm levels of three of nine isolates at MIC and MIC/4 levels and four of nine isolates at MIC/2 levels. Expression levels of QS-related genes were reduced or induced in the presence of different disinfectants. CONCLUSIONS: More efforts are required to decrease the risk of ineffective and low-dose application of disinfectants and antimicrobials against bacteria. Targeting of QS-related genes may be a reasonable strategy for the inhibition of virulence factors in P. aeruginosa.

Antifungal and Antibiofilm Activities of Selective Serotonin Reuptake Inhibitors Alone and in Combination with Fluconazole.

OBJECTIVES: Candida spp. are clinically important pathogens that cause difficulties for treatment by biofilm formation. Considering antifungal resistance rates and the limitations in the discovery of new antifungals, the antifungal and antibiofilm effects of various drugs used for different therapeutic purposes are becoming more important. The goal of our study was to determine the antifungal and antibiofilm effects of the selective serotonin reuptake inhibitors (SSRIs), namely sertraline (SRT), paroxetine (PRX), and fluoxetine (FLX) alone and in combination with fluconazole (FLC) against Candida spp. MATERIALS AND METHODS: Twenty Candida spp. strains isolated from clinical samples from Ege University Hospital were identified by the Dalmau method and matrix-assisted laser desorption ionization time of flight mass spectrometry. The minimum inhibitory concentrations (MICs) of the SSRIs and FLC were detected by broth microdilution method. Synergistic interactions between the SSRIs and FLC were investigated by checkerboard assay. The antibiofilm effects of the SSRIs were determined by spectrophotometric microplate method. RESULTS: Among the isolates, five different Candida spp. (C. albicans, C. glabrata, C. krusei, C. tropicalis, and C.parapsilosis) were identified. The MICs of the SSRIs ranged between 16-512 µg/mL. While SRT showed the highest antifungal effect, the antibiofilm efficacy of FLX was higher than that of the other agents. Moreover, FLX and PRX showed a synergistic effect with FLC in 13 and 19 isolates, respectively. Four isolates were strong biofilm producers while nine isolates were moderate biofilm producers. C. parapsilosis strains showed higher biofilm production than the other species. At MIC/2 concentration, FLX and SRT alone inhibited mature biofilms in six and five isolates, respectively, while PRX caused increases biofilm formation in seven isolates. CONCLUSION: This study revealed that MIC/2 concentrations of SSRIs could have antifungal and antibiofilm effects. SRT and FLX alone or in combination with antifungals may possibly have therapeutic potential for combating fungal infections.

Investigation of the Antimicrobial Susceptibility Profile, Virulence Genes, and Epidemiologic Relationship of Clinical Salmonella Isolates.

OBJECTIVES: The objectives of this study were to investigate the epidemiologic relationship, prevalence of the beta-lactamase and virulence genes of clinical ampicillin-resistant Salmonella enterica. MATERIALS AND METHODS: In vitro ampicillin susceptibilities of 117 Salmonella enterica isolates obtained between 2011-2012 from Ege University Hospital, Bacteriology Laboratory of Medical Microbiology Department were examined using disc diffusion assays in accordance with the CLSI guidelines. The MIC levels in the ampicillin-resistant bacteria were determined using the broth microdilution method. The resistant strains were serotyped by the Public Health Institution. Epidemiologic relations of resistant strains were evaluated using ERIC-PCR. The presence of beta-lactamase genes and virulence factors were detected using PCR. RESULTS: The 117 S. enterica strains had ten isolates that were resistant to ampicillin, and the MIC range of ampicillin was found as 512-128 µg/mL. Ampicillin-resistant strains were susceptible to nalidixic acid, ciprofloxacin, cefotaxime, sulfamethoxazole/trimethoprim. Four different serotypes were identified and isolates were grouped into seven clusters. Five isolates carried blaTEM , and two carried the blaCTX-M gene. However, it was determined that blaSHV and blaPER genes did not exist in these strains. Virulence genes invA, pipD, and sopB were found in all isolates. sifA, pefA, and sopE genes were found in seven, four, and three isolates, respectively. CONCLUSION: Our data suggest that the rate of ampicillin resistance in S. enterica isolates was 8.5% in the two year period, but this ratio was generally lower than rates abroad. blaCTX-M and blaTEM genes could be responsible for ampicillin resistance. The blaSHV gene, which is highly prevalent in our country, was not found in any strains. sopB and pipD genes, which might be associated with beta-lactam resistance, were found in all strains. It is also noteworthy that the three isolates containing the sopE gene, which is associated with epidemic cases, were of the same serotypes and epidemiologic clusters.

[Comparison of phenotypic methods and polymerase chain reaction for the detection of carbapenemase production in clinical Klebsiella pneumoniae isolates]. / Klinik Klebsiella pneumoniae izolatlarinda karbapenemaz üretiminin saptanmasinda polimeraz zincir reaksiyonu ve fenotipik yöntemlerin karsilastirilmasi.

Being a member of the Enterobacteriaceae family, Klebsiella pneumoniae is an opportunistic pathogen that inhabits normal human microbiota and causes predominantly hospital-acquired infections. The emergence of K.pneumoniae isolates which are resistant particularly to the carbapenem group of antibiotics has led to an increase in hospitalization period, mortality and morbidity. Although different rates of resistance are observed between countries, regions and even healthcare facilities, there has been a rapid increase in the prevalence of carbapenem-resistant strains in the last 10 years. Fast and correct identification of carbapenem-resistant strains is important for the successful treatment of infections caused by these resistant bacteria. The objective of this study was to investigate the presence and the types of carbapenemases in carbapenem-resistant K.pneumoniae strains using "MASTDISCS™ ID carbapenemase detection disc set", a commercial product that can be used for this purpose, and "Carbapenem Inactivation Method (CIM)", a relatively new method, and compare the results of these methods by polymerase chain reaction (PCR). For this purpose, we used 54 K.pneumoniae strains isolated in 2015-2016, that were resistant to any of the ertapenem, meropenem or imipenem antibiotics. The identification of the strains was performed using VITEK MS and their antibiotic susceptibility tests were carried out using the VITEK 2 Compact® automated system. For the strains that were found resistant to carbapenems in the automated system, the minimum inhibitor concentration (MIC) values were determined by the gradient testing method according to the recommendations of "The European Committee on Antimicrobial Susceptibility Testing (EUCAST)". The blaOXA-48, blaIMP, blaNDM, blaVIM, and blaSIM genes were investigated with PCR among these isolates. Phenotypic enzyme typing was performed in the carbapenem-resistant strains using the "MASTDISCS™ ID carbapenemase detection disc set" and "Carbapenem Inactivation Method (CIM)". REP-PCR was used to reveal clonal relationship of the isolates. The 54 K.pneumoniae isolates were found as resistant to carbapenem and the MIC50 and MIC90 values of imipenem, meropenem and ertapenem were 32 µg/ml. Only 33 of the strains had blaOXA-48 and two of them had only blaNDM, the remaining 19 strains had both of these two genes. The blaIMP, blaVIM and blaSIM genes were not encountered in any of the isolates. When the isolates were assessed by the REP-PCR method, six main clones were detected. The "MASTDISCS™ ID carbapenemase detection disc set" was able to detect all the carbapenemase producing strains and it remained incapable of distinguishing OXA-48 in the strains which had both OXA-48 and metallo beta lactamase (MBL) enzymes. The CIM method showed a low rate of positivity (46.15%) in the strains containing blaOXA-48, but was found much more successful in the strains containing blaNDM with a detection rate of 85.71%. In this study, it was concluded that the Mastdiscs-ID method could be successfully used to detect the presence of blaOXA-48 which has a high prevalence in our country.