Pathogen In Nosocomial Infections Research Articles

Panel of primers for evaluation of antibiotic resistance genes with real time detection of results

Introduction. The problem of the spread of antibiotic resistance among pathogens of nosocomial infections is becoming increasingly important. In order to improve microbiological monitoring, it is advisable to use methods that allow you to quickly determine the largest number of antibiotic resistance determinants. In this regard, it seems relevant to develop PCR test systems for detecting antibiotic resistance genes, in particular, for screening hospitalized patients and identifying cases of nosocomial infection.Purpose. Develop a panel of primers for detection of antibiotic resistance genes using PCR with real-time visualization of results. Materials and methods. For the design of specific primers and probes, reference sequences from the databases of the National Center for Biotechnology Information (NCBI) were used. Primer selection and specificity assessment were performed using the NCBI PrimerBlast and Primer3 programs. Primers for PCR amplification of a number of antibiotic resistance genes were tested on strains of Klebsiella pneumoniae. These strains were isolated from the clinical material of intensive care patients with COVID-19 and whole genome sequencing of the strains was carried out with a detailed assessment of the resistome, virulome. The results of whole genome sequencing and multiplex real-time PCR were compared.Results. We have developed a set of primers for the detection of antibiotic resistance genes, including carbapenemase, using real-time multiplex PCR.Conclusion. The developed panel of primers can be used to screen Klebsiella pneumoniae isolates for the presence of resistance genes; further expansion of the spectrum of detected genes and testing of the panel on clinical material is required.

Sensitivity of microbiota of bioaerosol and surfaces of boxes for holding animals in veterinary clinics to antimicrobial drugs

The formation of antibiotic resistance in microorganisms in veterinary medicine clinics is considered a severe problem not only for veterinary medicine but also for the public because the circulating microflora contaminates the clinic's environment and the veterinary staff, patients' animals, their owners, and their homes. The work aimed to determine the sensitivity of microorganisms isolated from bioaerosol and the surfaces of boxes for keeping animals in veterinary clinics to antimicrobial drugs. Collection of washings from the inner surfaces of the boxes was carried out with the help of disposable sterile tampons of industrial production with an average area of 100 cm2. Identification of isolated microorganisms was carried out according to morphological, cultural, biochemical properties, and signs of pathogenicity, which are described in Bergey's identifier of bacteria. The sensitivity of bacteria to antibiotics was performed on the Muller-Hinton medium according to the classical Bauer-Kirbi disco-diffusion method. It was established that gram-positive bacteria isolated from bioaerosol and the surface of boxes for keeping sick animals in veterinary clinics were sensitive in 66.7–100 % of cases to antibiotics used in these clinics. At the same time, the high bactericidal activity of antimicrobial drugs of the groups: cephalosporins, fluoroquinolones, and nitrofurans was found. Since the sensitivity of Acinetobacter baumani, Enterobacter spp., Escherichia coli, and Pseudomonas aeruginosa was from 80 to 100 % of the cultures tested. In addition, antibiotics of the penicillin series, macrolides, and azalides, practically did not act on these bacteria because they naturally resisted them. Therefore, to effectively use antimicrobial drugs to treat chronic infections, it is necessary to determine the sensitivity of the isolated microflora. Therefore, based on the study results, it can be concluded that in veterinary clinics, bioaerosols, and equipment, the pathogens of nosocomial infections of gram-negative and gram-positive bacteria resistant to antimicrobial drugs may circulate, provided that antibiotics are prescribed without determining their sensitivity to antibiotics.

Acinetobacter baumannii adaptation to the host pH microenvironment is mediated by allelic variation in a single residue of BauA protein.

Acinetobacter baumannii has been listed as one of the most critical pathogens in nosocomial infections; however, the key genes and mechanisms to adapt to the host microenvironment lack in-depth understanding. In this study, a total of 76 isolates (from 8 to 12 isolates per patient, spanning 128 to 188 days) were longitudinally collected from eight patients to investigate the within-host evolution of A. baumannii. A total of 70 within-host mutations were identified, 80% of which were nonsynonymous, indicating the important role of positive selection. Several evolutionary strategies of A. baumannii to increase its potential to adapt to the host microenvironment were identified, including hypermutation and recombination. Six genes were mutated in isolates from two or more patients, including two TonB-dependent receptor genes (bauA and BJAB07104_RS00665). In particular, the siderophore receptor gene bauA was mutated in multiple isolates from four patients with three MLST types, and all mutations were at amino acid 391 in ligand-binding sites. With 391T or 391A, BauA was more strongly bound to siderophores, which promoted the iron-absorption activity of A. baumannii at acidic or neutral pH, respectively. Through the A/T mutation at site 391 of BauA, A. baumannii displayed two reversible phases to adapt to distinct pH microenvironments. In conclusion, we demonstrated the comprehensive within-host evolutionary dynamics of A. baumannii, and discovered a key mutation of BauA site 391 as a genetic switch to adapt to different pH values, which may represent a model in the pathogen evolutionary adaption of the host microenvironment.

A Library of Promoter-gfp Fusion Reporters for Studying Systematic Expression Pattern of Cyclic-di-GMP Metabolism-Related Genes in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter-gfp fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c-di-GMP metabolism-related (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter-gfp fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter-gfp fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. IMPORTANCE The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter-gfp transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.

Non-antibiotic prevention and treatment against Acinetobacter baumannii infection: Are vaccines and adjuvants effective strategies?

Acinetobacter baumannii (A. baumannii) is a Gram-negative opportunistic pathogen widely attached to the surface of medical instruments, making it one of the most common pathogens of nosocomial infection, and often leading to cross-infection and co-infection. Due to the extensive antibiotic and pan-resistance, A. baumannii infection is facing fewer treatment options in the clinic. Therefore, the prevention and treatment of A. baumannii infection have become a tricky global problem. The requirement for research and development of the new strategy is urgent. Now, non-antibiotic treatment strategies are urgently needed. This review describes the research on A. baumannii vaccines and antibacterial adjuvants, discusses the advantages and disadvantages of different candidate vaccines tested in vitro and in vivo, especially subunit protein vaccines, and shows the antibacterial efficacy of adjuvant drugs in monotherapy.

Multidrug Resistant Corynebacterium accolens Strains Identified as Pathogens of Nosocomial Infections

Multidrug Resistant Corynebacterium accolens Strains Identified as Pathogens of Nosocomial Infections

Evaluation of the effectiveness of antimicrobial stewardship program: results from a ten-year study in a multidisciplinary hospital

Objective. To evaluate the results of the implementation of antimicrobial stewardship (AMS) program in a multidisciplinary hospital. Materials and Methods. A retrospective intervention study was performed in the 600-bed multidisciplinary hospital of the Pirogov National Medical and Surgical Center (Moscow, Russia). The study included a number of sequential interventions, aimed at improving the prevention, diagnosis and treatment of infections, with an assessment of the following indicators dynamics: the structure of microorganisms isolated in the hospital, the level of resistance to antimicrobial drugs (AMD) among the pathogens of the ESKAPE group, the structure of prescribed AMD, as well as an assessment of clinical and economic consequences of antimicrobial resistance changes. For a comprehensive assessment of trend in antibiotic resistance and its connection with consumption of antibiotics, the drug resistance index (DRI) was used. DRI is based on the ratio of the level of resistance of the microorganism and the frequency of administration of AMD, potentially effective against this pathogen. The intervention started in 2013, so the data of 2012 (pre-intervention period) and 2022 were compared in this study. Results. The implementation of AMS program measures helped to reduce the relative frequency of ESKAPE group microorganisms in the structure of nosocomial infection pathogens in the hospital from 36.5% to 22% (p < 0.0001). The proportion of gram-negative (Gr-) isolates resistant to meropenem decreased from 32.4% to 10.9% (p < 0.0001). As a result of the introduction of AMS program, DDDh has been reduced almost three times: from 48.1 to 17.2 DDDh. The median duration of the AMT course in a specialized intensive care unit for the treatment of patients with nosocomial infections decreased from 12 to 8 days (p < 0.0001), the number of AMT days per 1 patient decreased from 7.7 to 4.2 (p < 0.0001). The proportion of ESKAPE pathogens in the structure of bloodstream infections in the hospital decreased from 53.1% to 26% (p < 0.0001), which led to decrease in mortality from 28.4% to 12.8%, (p = 0.012) and length of hospital stay from 31 to 22 days (p < 0.001) in this group of patients. Conclusions. The implementation of AMS program in a multidisciplinary hospital helps to reduce the frequency of inappropriate use of antimicrobial drugs and reduces the consumption of antibiotics, decrease the level of antimicrobial resistance, and in such way improves the results of treatment of patients with nosocomial infections.

Antimicrobial Effect of Lemongrass and Ginger Essential Oils Against Nosocomial Infection Pathogens

Background: According to the 2011 WHO survey on nosocomial infection, 15% patients treated as in-patient had nosocomial infection globally. The causal pathogens had resistance to general antibiotics used by clinicians, therefore it is getting harder to treat patients with these infections. The study aims to investigate the antimicrobial effects of lemongrass dan ginger essential oils against nosocomial infection pathogens.Methods: The research is an experimental research. The strain used are Pseudomonas aeruginosa ATCCâ 27853 and Acinetobacter baumannii ATCCâ BAA-747. The antimicrobial assays were done triplicate using disk diffusion method and microdilution method.Results: From disk diffusion assay, lemongrass essential oil shows zone of inhibition against both pathogens while ginger essential oils doesn’t show antimicrobial effect against both pathogens. The mean highest zone of inhibition is 11,33 mm on 100% concentration against A.baumannii. From microdilution assay, lemongrass and ginger oil show antimicrobial effect against both pathogens, with lowest MIC detected for lemongrass and ginger essential oils are <0.5?g/ml (against A.baumannii) and 32?g/ml (against P.aeruginosa and A.baumannii).Conclusion/Conclusions: Lemongrass and ginger essential oils had antimicrobial effects against nosocomial infection pathogens. Antimicrobial activity of lemongrass oil is more potent than ginger oil on both antimicrobial assay against both pathogens tested.

Carbapenem-Resistant Klebsiella pneumoniae: Diversity, Virulence, and Antimicrobial Resistance.

BackgroundKlebsiella pneumoniae (K. pneumoniae) is one of the most important pathogens in nosocomial infections. It has resistance to most antibiotics, even carbapenem, resulting in restricted therapeutic options.PurposeWe tried to assess the antimicrobial resistance and virulence fitness of carbapenem-resistant K. pneumoniae (CRKP) in addition to their phenotypic and genotypic diversity.Materials and MethodsThe conventional methods, automated Vitek-32 system, and antimicrobial susceptibility pattern were used to detect CRKP isolates. Virulence and resistance genes profiles were created by using PCR technique. The correlation analysis was done by using R-program.ResultsThe antimicrobial resistance profile for all our K. pneumoniae isolates was shocking as the MDR and CRKP were the most prominent phenotypes. Unfortunately, high degrees of heterogeneity among our CRKP isolates were recorded, as 97.5% of them were differentiated into different clusters. We found a negative correlation between the existence of virulence and antimicrobial resistance genes. In contrast to sputum and urine CRPK isolates, the blood isolates showed high antimicrobial resistance and low virulence fitness. Finally, K. pneumoniae creates several outbreaks and crises in Egypt owing to the highly heterogeneity and the wide spread of multidrug-resistant (MDR) and multi-virulent CRKP phenotypes.ConclusionOur results are significant and alarming to health organizations throughout the world for the severity and heterogeneity of K. pneumoniae infections. Therefore, the traditional method for treatment of CRKP infections must be renewed. Additionally, the treatment protocols must be well correlated with the site of infections, phenotypes, and genotypes of CRKP strains.

Study on antibacterial effect of halicin (SU3327) against Enterococcus faecalis and Enterococcus faecium.

Enterococci are important pathogens of nosocomial infections and are increasingly difficult to treat due to their intrinsic and acquired resistance to a range of antibiotics. Therefore, there is an urgent need to develop novel antibacterial agents, while drug repurposing is a promising approach to address this issue. Our study aimed to determine the antimicrobial efficacy of halicin against enterococci and found that the minimum inhibitory concentrations (MIC) of halicin against different strains of Enterococcus faecalis and Enterococcus faecium ranged from 4 to 8 μg/ml. In addition, the synergistic antibacterial effect between halicin and doxycycline (DOX) against Enterococcus was observed through the checkerboard method, and it was observed that halicin and DOX could significantly synergistically inhibit biofilm formation and eradicate preformed biofilms at sub-MICs. Moreover, the electron microscope results revealed that halicin could also disrupt the bacterial cell membrane at high concentrations. Furthermore, it is also confirmed that the combination of halicin and DOX has no significant cytotoxic effect on erythrocytes and other human-derived cells. In addition, the mouse subcutaneous model and H&E staining showed that the combination of halicin and DOX could effectively reduce the bacterial load and inflammatory infiltration without obvious side effects. In nutshell, these results demonstrate the potential of halicin in combination with DOX as a novel therapy against infections by Enterococcus.

The Effect of The Anolyte Solution on Pathogenic Microflora

An experimental study of the antimicrobial properties of the Anolyte disinfectant solution on the microorganisms Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae , and Pseudomonas aeruginosa was carried out using the «Turbidity Standard» method. The high activity of the Anolyte solution against the pathogens of nosocomial infections in vitro was shown.

Can bee propolis help us fight against methicillin-resistant Staphylococcus aureus (MRSA)?

Abstract Objectives Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen in nosocomial infections. Since the 1950s, MRSA has acquired several resistance factors including efflux pumps and drug target modifications. Some studies investigated the anti-MRSA capacity of propolis samples collected in different regions and their immunomodulatory action. The aim of this review is to gather the data published up to August 2022 about propolis action on MRSA strains and its modulatory action on phagocytes. Methods The PubMed database was used to look for articles containing the keywords ‘propolis’, ‘immunomodulation’, ‘MRSA’ and the name of each compound. As propolis contains a variety of compounds making it impossible to isolate the major bioactive components, we reviewed the main compounds found in several propolis samples and their mechanisms towards the resistance factors displayed by MRSA. Some perspectives for using propolis-based medications and the formulation of new antimicrobial/immunomodulatory agents are discussed. Key findings Propolis extracts and active compounds exert antibacterial action over MRSA strains acting on resistance factors. Moreover, propolis modulates pro-inflammatory markers in phagocytes. Conclusions Because propolis compounds may act synergistically, it’s crucial to understand how these components interact to synthesize standardized formulations and enhance their bioavailability for clinical applications to combat MRSA.

Effects of Natural Borneol on Germ Tube Formation and Preformed Biofilm Activity in Candida albicans

Candida albicans infection mainly occurs in patients with suppressed immune function and is the main pathogen of nosocomial infections. The use of natural products aimed at controlling fungal diseases is considered an interesting alternative to synthetic fungicides due to their lower adverse reactions and the lower cost of plant preparations compared to modern conventional pharmaceuticals. Natural borneol has a long history of treating ulcers and local infections in traditional Chinese medicine. In this study, we present an analysis of the in vitro effects of natural borneol on planktonic cells of C albicans in the liquid and vapor phases. We also investigated the effects of natural borneol on germ tube formation and mature biofilm activity of C albicans. We found that vapor-phase borneol (minimum inhibitory concentration [MIC] 0.4 mg/cm3) inhibited C albicans more effectively than in the liquid phase (MIC 2 mg/mL). The C albicans germ tube decreased by 99% to 60% at sub-MICs of 0.5 to 0.125 mg/mL. The inhibitory effects of 0.25, 0.5, 1, 2, and 4 mg/mL borneol on the biofilm activity were 33.7%, 48.6%, 49.9%, 52.9%, and 58.2%, respectively. Natural borneol may have potential in the treatment and prevention of C albicans infections.

Microflora of boxes for holding veterinary patients in clinics

A significant element of the prophylaxis of nosocomial infection in veterinary clinics is monitoring ambient objects, air, equipment, and instruments. In order to determine the role of boxes for keeping ill animals as a source of transmission of pathogens of nosocomial infections in veterinary clinics, we studied the microflora of surfaces of boxes and bioaerosol prior and after sanitation. For this purpose, we collected rinses from the surfaces of plastic and steel boxes, air samples prior to morning sanitation, after cleaning and wiping the surfaces with water and detergents and after disinfection. From the surfaces of the boxes for holding animals, we mostly isolated bacteria of Staphylococcus spp., Streptococcus spp., Micrococcus spp., Corynebacterium spp., Enterococcus spp. and Bacillus spp. Gram-negative species we found were bacteria of Escherichia spp., Acinetobacter spp. and Enterobacter spp. After wet cleaning and disinfection of plastic boxes, we detected species of Staphylococcus spp. and Enterococcus spp. in 5.4% of the samples, Micrococcus spp. in 8.1% and Bacillus spp. in 2.7%. Gram-negative bacteria of Enterobacter spp. were found in 2.7% of the samples. At the same time, the number of microorganisms in samples in which the bacteria were found after disinfection on the surfaces of stainless-steel boxes was 2.0 times lower than in such from the surfaces of plastic boxes. We determined that after wet disinfection of boxes’ surfaces, there occurred decrease in the microbial number in the air, equaling 3.7 times on average, compared with prior to disinfection. The basis of the air microflora after disinfection comprised species of Micrococcus spp., Corynebacterium spp. and Staphylococcus spp., which can be airborne-transmitted. Bacteria that were isolated from the boxes after disinfection (Micrococcus spp., Staphylococcus spp.) formed highly dense biofilms, which probably ensure the survival of the microbial cells, thus making the boxes a probable source of nosocomial infection.

Green Synthesis of Silver Nanoparticles Using Ocimum tenuiflorum Leaf Extract and its Antimicrobial Activity against Certain Pathogens

Nanoparticles (NPs) are particles less than 100 nm in size. Silver nanoparticles have many important chemical and biological applications. These applications drag the attention of researchers for the production of silver nanoparticles. The objective of the study is to synthesize silver nanoparticles from the green method without using any hazardous chemicals. In the present study, Ocimum tenuiflorum (Tulsi) leaf extract was used to synthesize silver nanoparticles (Ag-NPs) from silver nitrate. Moreover, Tulsi leaf extract was also exploited, both traditionally and commercially, for their antibacterial potential which is essential oil components. The synthesis of Ag-NPs was visually deduced by a change in colour from light yellow to reddish-brown. UV-Visible Spectrometry, X-ray diffraction (XRD), and Transmission Electron Microscopic (TEM) analysis were then used to characterize synthesized nanoparticles. This study also determined the effect of temperature and effect of silver nitrate concentration on nanoparticle synthesis. Synthesized silver nanoparticles showed absorbance ‘peaks’ at 420 nm with UV-visible spectrometry. X-ray diffraction studies showed that the synthesized silver nanoparticles were crystalline. The mean crystallite size was estimated using the Debye-Scherrer equation. However, TEM showed that silver nanoparticles have spherical (40 nm) shape with a narrow size distribution. These synthesized nanoparticles were used to evaluate antimicrobial activity against the most common pathogens of nosocomial infections, MRSA, E. coli, Salmonella typhi, and Klebsiella pneumoniae (ESBL). O. tenuiflorum extract showed strong potential for the synthesis of silver nanoparticles from silver nitrate and the synthesized silver nanoparticles had efficient antimicrobial properties against different pathogens including drug-resistant pathogens in vitro. These synthesized nanoparticles showed maximum ‘zones’ of inhibition against MRSA among these four organisms.

Antimicrobial Peptide-Loaded Gelatinase-Responsive Photothermal Nanogel for the Treatment of Staphylococcus aureus-Infected Wounds.

As the most common pathogen of community and nosocomial infection, the resistance of Staphylococcus aureus (S. aureus) to traditional antibiotics is still increasing with years. Although the potent antibacterial activity of antimicrobial peptides (AMPs) has been widely confirmed, the unpredictable cytotoxicity remains the biggest obstacle to their clinical application. The development of a targeted drug delivery system for S. aureus is a practical strategy to ameliorate the inherent limitations of AMPs. In this work, we constructed an AMP release nanogel (cypate-GNPs@Cy3-AMP, CGCA) of S. aureus infection microenvironment using gelatinase nanoparticles (GNPs) for toxicity control and bacterial clearance. Gelatinase present in the infected site degrades GNPs, thus releasing Cy3-AMP in situ to destroy bacterial cells. Cypate modified on the surface of GNPs supports CGCA to generate localized heat under near-infrared (NIR) laser irradiation, which together with AMPs could cause irreversible physical damage to bacteria. In addition, the encapsulation from GNPs not only effectively limited the toxicity of AMPs but also significantly promoted cell proliferation and migration in vitro. In the mouse infection model, CGCA also exhibited excellent effects of bacterial clearance and wound healing, providing a potential direction for the correct use of AMPs.

Disinfection Strategies for Carbapenem-Resistant Klebsiella pneumoniae in a Healthcare Facility.

Disinfectant resistance is evolving into a serious problem due to the long-term and extensive use of disinfectants, which brings great challenges to hospital infection control. As a notorious multidrug-resistant bacterium, carbapenem-resistant Klebsiella pneumoniae (CRKP) is one of the most common and difficult pathogens of nosocomial infection. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests of seven kinds of disinfectants (0.1% benzalkonium bromide, 4% aqueous chlorhexidine, 75% alcohol, entoiodine II, 2% glutaraldehyde, 2000 mg/L chlorine-containing disinfectants, and 3% hydrogen peroxide) were detected by the broth dilution method. Three efflux pump genes (oqxA, oqxB, and qacE∆1-sul1) were detected by PCR. The mean MIC value of aqueous chlorhexidine from the intensive care unit (ICU) (0.0034%) was significantly higher than that from non-ICUs (0.0019%) (p < 0.05). The positive rates of three efflux pump genes oqxA, oqxB and qacE∆1-sul1 were 60.9% (39/64), 17.2% (11/64) and 71.9% (46/64) in the detected CRKP isolates, respectively. This study discovered that CRKP strains demonstrated extensive resistance to clinical disinfectants and suggest that it is necessary to perform corresponding increases in the concentration of aqueous chlorhexidine and chlorine-containing disinfectants on the basis of current standards in the healthcare industry.

Molecular Detection of VEB and OXA-23 Resistance Gene in Pseudomonas Aeruginosa Isolates at Dr. Wahidin Sudirohusodo Hospital Makassar

Pseudomonas aeruginosa is a common gram-negative pathogen in nosocomial infections in immune-compromised patients. It exhibits high rates of intrinsic resistance to many classes of antibiotics, especially beta-lactam antibiotics. Production of extended-spectrum beta-lactamase (ESBL) and genes belonging to the carbapenem-hydrolyzing class D subgroup β-lactamases (CHDL) are a problem for increasing antibiotic resistance worldwide. This study aimed to identify P. aeruginosa containing the VEB and OXA-23 genes. Eighty-five clinical isolates of P. aeruginosa from various clinical samples were identified and tested for antimicrobial susceptibility using VITEK 2 compact. VEB and OXA-23 genes were detected using the Polymerase Chain Reaction (PCR) method. The PCR results revealed that 13 (15.3%) of P. aeruginosa isolates were positive OXA-23 gene, but no isolate positive for the VEB gene in P. aeruginosa isolates. The study results demonstrated the spread of the OXA-23 gene in P. aeruginosa isolates at Dr. Wahidin Sudirohusodo Hospital Makassar.

Overexpression of BIT33_RS14560 Enhances the Biofilm Formation and Virulence of Acinetobacter baumannii.

Acinetobacter baumannii, a strictly aerobic, non-lactose fermented Gram-negative bacteria, is one of the important pathogens of nosocomial infection. Major facilitator superfamily (MFS) transporter membrane proteins are a class of proteins that widely exists in microbial genomes and have been revealed to be related to biofilm formation in a variety of microorganisms. However, as one of the MFS transporter membrane proteins, little is known about the role of BIT33_RS14560 in A. baumannii. To explore the effects of BIT33_RS14560 on biofilm formation of A. baumannii, the biofilm formation abilities of 62 isolates were firstly investigated and compared with their transcript levels of BIT33_RS14560. Then, this specific gene was over-expressed in a standard A. baumannii strain (ATCC 19606) and two isolates of extensively drug-resistant A. baumannii (XDR-Ab). Bacterial virulence was observed using a Galleria mellonella infection model. High-throughput transcriptome sequencing (RNA seq) was performed on ATCC 19606 over-expressed strain and its corresponding empty plasmid control strain. Spearman’s correlation analysis indicated a significant negative correlation (R = −0.569, p = 0.000) between the △CT levels of BIT33_RS1456 and biofilm grading of A. baumannii isolates. The amount of A. baumannii biofilm was relatively high within 12–48 h. Regardless of standard or clinical strains; the biofilm biomass in the BIT33_RS14560 overexpression group was significantly higher than that in the control group ( p < 0.0001). Kaplan–Meier survival curve analysis showed that the mortality of G. mellonella was significantly higher when infected with the BIT33_RS14560 overexpression strain (χ2 = 8.462, p = 0.004). RNA-Seq showed that the mRNA expression levels of three genes annotated as OprD family outer membrane porin, glycosyltransferase family 39 protein, and glycosyltransferase family 2 protein, which were related to bacterial adhesion, biofilm formation, and virulence, were significantly upregulated when BIT33_RS14560 was over-expressed. Our findings provided new insights in identifying potential drug targets for the inhibition of biofilm formation. We also developed a practical method to construct an over-expressed vector that can stably replicate in XDR-Ab isolates.

High level of persister frequency in clinical staphylococcal isolates

BackgroundStaphylococcus aureus is a notorious human pathogen that causes often lethal systemic conditions that are mostly medical device associated biofilm infections. Similarly, coagulase negative staphylococci are emerging as leading pathogen for nosocomial infections owing to their ability to form biofilm on implanted medical equipment. Chronic in nature, these infections are difficult to treat. Such recalcitrance of these infections is caused mainly due to the presence of persister cells, which exhibit transient yet extreme tolerance to antibiotics. Despite tremendous clinical significance, there is lack of studies on persister cells formation among clinical bacterial isolates. Considering the importance of factors influencing persister formation, in this study, we evaluate the association of antibiotic tolerance with biofilm production, antibiotic stress, growth phase, specimen type, and dependency on staphylococcal species. Biofilm formation was detected among 375 clinical staphylococcal isolates by quantitative tissue culture plate method (TCP) and icaAD genes by genotypic method. The antibiotic susceptibility was determined by Kirby Bauer disc diffusion method while minimum inhibitory concentration values were obtained by agar dilution method. Persister cells were measured in the susceptible staphylococcal isolates in the presence of clinically relevant antibiotics.ResultsIn the study, 161 (43%) S. aureus and 214 (57%) coagulase negative staphylococci (CNS) were isolated from different clinical samples. TCP method detected biofilm production in 84 (52.2%) S. aureus and 90 (42.1%) CNS isolates. The genotypic method detected icaAD genes in 86 (22.9%) isolates. Majority (> 90%) of both the biofilm producers and non-producers were sensitive to chloramphenicol and tetracycline but were resistant to penicillin. Interestingly, all isolates were sensitive to vancomycin irrespective of biofilm production. While high persister frequency was observed among all staphylococci isolates in the stationary growth phase, the persister frequency in exponential growth phase was statistically high among isolates possessing icaAD genes compared to icaAD negative isolates.ConclusionThe research findings provide strong evidence that the clinical staphylococcal isolates exhibit extreme antibiotic tolerance suggesting their causal link with treatment failures. Understanding the factors influencing the formation and maintenance of persister cells are of utmost important aspect to design therapeutics and control recalcitrant bacterial infections.