Strong Biofilm Producers Research Articles

Exploration of agr types, virulence-associated genes, and biofilm formation ability in Staphylococcus aureus isolates from hemodialysis patients with vascular access infections.

Staphylococcus aureus, is a pathogen commonly encountered in both community and hospital settings. Patients receiving hemodialysis treatment face an elevated risk of vascular access infections (VAIs) particularly Staphylococcus aureus, infection. This heightened risk is attributed to the characteristics of Staphylococcus aureus, , enabling it to adhere to suitable surfaces and form biofilms, thereby rendering it resistant to external interventions and complicating treatment efforts. Therefore this study utilized PCR and microtiter dish biofilm formation assay to determine the difference in the virulence genes and biofilm formation among in our study collected of 103 Staphylococcus aureus, isolates from hemodialysis patients utilizing arteriovenous grafts (AVGs), tunneled cuffed catheters (TCCs), and arteriovenous fistulas (AVFs) during November 2013 to December 2021. Our findings revealed that both MRSA and MSSA isolates exhibited strong biofilm production capabilities. Additionally, we confirmed the presence of agr types and virulence genes through PCR analysis. The majority of the collected isolates were identified as agr type I. However, agr type II isolates displayed a higher average number of virulence genes, with MRSA isolates exhibiting a variety of virulence genes. Notably, combinations of biofilm-associated genes, such as eno-clfA-clfB-fib-icaA-icaD and eno-clfA-clfB-fib-fnbB-icaA-icaD, were prevalent among Staphylococcus aureus, isolates obtained from vascular access infections. These insights contribute to a better understanding of the molecular characteristics associated with Staphylococcus aureus, infections in hemodialysis patients and provided more targeted and effective treatment approaches.

Overexpression of lasB Gene in Klebsiella pneumoniae and its Effect on Biofilm Formation and Antibiotic Resistance

Background: Klebsiella pneumoniae is the second most frequent pathogenic bacterium in the Enterobacteriaceae family, after E. coli. It is also regarded as a major pathogen responsible for healthcare-associated infections around the world. Objective: To look at how overexpressing the elastase gene (lasB) in K. pneumoniae affects biofilm development and antibiotic resistance. Methods: 25 clinical isolates of K. pneumoniae were received from Baghdad's Chemistry Analysis Center (CAC) and re-identified using the Vitek-2 method. The microtiter plate was used to measure biofilm production with ELISA. The disc diffusion method was used in antibiotic sensitivity tests in accordance with the CLSI 2022 criteria. The PlasB plasmid was transformed into K. pneumoniae via electroporation. Results: Out of 25 isolates, 11 (44%), 11 (44%), and 3 (12%) produced strong, moderate, and weak biofilms, respectively. One strong biofilm producer (KA1) was chosen for further investigation. The lasB plasmid was successfully transformed into KA1, yielding the KA1 (plasB) isolate. KA1 (plasB) formed considerably fewer biofilms than KA1, and it was more susceptible to tetracycline, doxycycline, and amoxicillin-clavulanic acid than KA1. Furthermore, KA1 (plasB) has shown a significant decrease in ampicillin resistance and an increase in ciprofloxacin sensitivity, but no variations in susceptibility to levofloxacin, cefotaxime, piperacillin-tazobactam, amikacin, or erythromycin when compared to KA1. Conclusions: Overexpression of the elastase gene (plasB) has a major impact on biofilm development and antibiotic resistance in K. pneumoniae.

Biofilm Formation by Hospital-Acquired Resistant Bacteria Isolated from Respiratory Samples

BackgroundHospital-acquired resistant infections (HARI) are infections, which develop 48 h or more after admission to a healthcare facility. HARI pose a considerably acute challenge, due to limited treatment options. These infections are associated bacterial biofilms, which act as a physical barrier to diverse external stresses, such as desiccation, antimicrobials and biocides. We assessed the influence of multiple factors on biofilm production by HARI -associated bacteria.MethodsBacteria were isolated from samples of patients with respiratory HARI who were hospitalized during 2020–2022 in north Israel. Following antibiotic susceptibility testing by disc diffusion or broth microdilution, biofilm formation capacities of resistant bacteria (methicillin-resistant staphylococcus aureus, extended spectrum beta-lactamase-producing Escherichia coli and Klebsiela pneumonia, and multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii) was assessed using the crystalline violet staining method. Data regarding season, time to infection, bacterial species, patient age and gender, year, and medical department were collected from the patient medical records.ResultsAmong the 226 study isolates, K. pneumonia was the most prevalent (35.4%) bacteria, followed by P. aeruginosa (23.5%), and methicillin-resistant staphylococcus aureus (MRSA) (21.7%). A significantly higher rate of HARI was documented in 2022 compared to 2020–2021. The majority of isolates (63.3%) were strong biofilm producers, with K. pneumonia (50.3%) being most dominant, followed by P. aeruginosa (29.4%). Biofilm production strength was significantly affected by seasonality and hospitalization length, with strong biofilm production in autumn and in cases where hospitalization length exceeded 30 days.ConclusionBiofilm production by HARI bacteria is influenced by bacterial species, season and hospitalization length.

Antibiotic Susceptibility and Biofilm Production among Coagulase Negative Staphylococci Isolated from Clinical Samples at Tertiary Care Hospital.

Coagulase Negative Staphylococci have been widely associated with medical device implant treatment and immune-compromised patients. Despite having increasing interest in Coagulase Negative Staphylococci, few studies from Nepal have reported the association of these organisms with urinary tract infections, conjunctivitis, high vaginal swabs, and cerebrospinal fluid. This study was carried out to determine antibiotic susceptibility pattern and biofilm production among Coagulase Negative Staphylococci isolated from clinical samples at tertiary care hospital. This study was a hospital based cross-sectional study in which 3690 clinical samples were included. Isolation and identification of isolates was done following standard microbiological protocol. Coagulase Negative Staphylococci were identified phenotypically on the basis of gram staining, slide and tube coagulase test and by various sugar fermentation tests. Antibiotic susceptibility test was done following Kirby Bauer disk diffusion method (Clinical and Laboratory Standards Institute 2020). Biofilm production was determined by Tissue Culture Plate technique. A total of 113 isolates of Coagulase Negative Staphylococci were detected. Among them S. epidermidis (45.1%), S. saprophyticus (23.9%), S. haemolyticus (16.8%), S. hominis (5.3%), S. capitis (2.7%), -----S. cohini (1.8%), S. lugdunensis (1.8%) and S. sciuri (2.7%) were identified phenotypically. All isolates were found to be resistant against Ampicillin and 111 (98.2%) were sensitive against Linezolid.23.9% of CoNS were strong biofilm producers, 19.5% moderate and 56.6 % were non/weak biofilm producers. It requires susceptibility test for prescribing antibiotics against Coagulase Negative Staphylococci in hospital and the misuse of antibiotics should be prevented.

Pathogenic potential of meat-borne coagulase negative staphylococci strains from slaughterhouse to fork.

This study aimed to determine the prevalence of coagulase-negative staphylococci (CoNS) in meat processing lines for their pathogenic potential associated with biofilm formation, staphylococcal toxin genes, and antibiotic resistance in obtained isolates. Out of 270 samples, 56 isolates were identified as staphylococcal with their species level, and their antimicrobial resistance profiles were also determined with the BD Phoenix™ system. Among these, CoNS were found in 32 isolates, including S. epidermidis (22%), S. warneri (22%), S. cohnii (9%), S. schleiferi (9%), S. capitis (6%), S. haemolyticus (6%), S. lugdunensis (6%), S. chromogenes (6%), S. kloosii (3%), S. sciuri (3%), S. lentus (3%), and S. caprae (3%). Biofilm formation was observed in 78.1% of CoNS isolates, with 56% being strong biofilm producers; and the frequency of the icaA, fnbA, and fnbB genes were 43.7% and 34.3%, and 9.3% in isolates, respectively. Twenty-five (78.1%) of these strains were resistant to at least one antimicrobial agent, 20 (80%) of which exhibited multidrug resistance (MDR). Regarding genotypic analyses, 15.6%, 22.2%, 87.5%, and 9% of isolates, were positive for blaZ, ermC, tetK, and aacA-aphD, respectively. In 8 (25%) of all isolates had one or more staphylococcal toxin genes: the sed gene was the most frequent (12.5%), followed by eta (9.3%), tst-1 (6.25%), and sea (3.1%). In conclusion, this study highlights meat; and meat products might be reservoirs for the biofilm-producing MDR-CoNS, which harbored several toxin genes. Hence, it should not be ignored that CoNS may be related to foodborne outbreaks.

Antimicrobial and Antibiofilm Activity of Highly Soluble Polypyrrole Against Methicillin-Resistant Staphylococcus aureus.

As a new promising therapeutic option, the activity of highly soluble polypyrrole (Hs-PPy), a specific formulation of a conducting polymer (polypyrrole - PPy) is evaluated against bacteria of clinical importance. The purpose was to evaluate the antimicrobial activity of Hs-PPy, alone or combined with oxacillin, as well as its antibiofilm potential against methicillin-resistant Staphylococcus aureus strains. Furthermore, the in silico inhibitory mechanism in efflux pumps was also investigated. 10 clinical isolates of MRSA and two reference strains were used. Antimicrobial activity was determined by broth microdilution, and the combination effect with oxacillin was evaluated by the checkerboard assay. The biofilm formation capacity of MRSA and the interference of Hs-PPy were evaluated. The inhibitory action of Hs-PPy on the efflux pump was evaluated in silico through molecular docking. Hs-PPy showed activity against the isolates, with inhibitory action between 62.5 and 125 μg/mL and bactericidal action at 62.5 μg/mL, as well as synergism in association with oxacillin. The isolates ranged from moderate to strong biofilm producers, and Hs-PPy interfered with the formation of this structure, but not with mature biofilm. There was no in silico interaction with the efflux protein EmrD, the closest homolog to NorA. Hs-PPy interferes with biofilm formation by MRSA, has synergistic potential, and is an efflux pump inhibitor.

DETECTION OF BIOFILM PRODUCTION FOR ORAL BACTERIAL ISOLATES AND IT’S IMPACT ON DENTAL CARIES OCCURANCE

Background and aims: Microorganisms are known to be involved in the formation of biofilms. These biofilms are often seen in the oral cavity, tooth surfaces, prosthesis surfaces, attached oral mucosa, dental implants, etc. These are capable of causing dental caries or gingivitis, and most of them are also known to possess a higher ability to cause dental caries than non-biofilm-producing bacteria. This study was conducted to detect the biofilm formation in bacterial isolates from the oral cavity of patients attaining dental clinics in the Faculty of Dentistry, Sana’a University. In addition, the study examined the association between DMFT, decay, missing teeth due to caries, and filled teeth in the permanent teeth and the degree of bacteria ability to produce biofilm. Materials and Methods: Biofilm production was performed on 294 oral bacteria isolates from 100 patients who visited dental clinics run by Sana'a University's Faculty of Dentistry and private dental clinics. Biofilm-forming oral bacteria were detected by the tissue culture plate (TCA) method. The impact of biofilm production was correlated with the DMFT index of the tested patients. Results: Out of 294 isolates, biofilm formation was seen in 285 isolates (96.9%) by the TCP method. 21.4% of the iosolates showed a weak ability to produce biofilms, 72.4% showed moderate positivity, and only 3.1% showed strong positivity for biofilm production. Most S. aureus strains showed moderate and strong biofilm production (93.8% and 4.2%, respectively); S. mutans had 86.6% of strains with moderate biofilm production and 1.2% with strong biofilm production, while other streptococci had less biofilm production capacity. The DMFT index for the Yemeni patients included in the study was 5.9±2.4; there was a higher mean±SD (6.2±1.9) of DMFT for weak biofilm-producing bacteria with a difference equal to 3.5, 95% CI=1.4-4.2 (p=0.0001); for moderate biofilm-producing bacteria (6.9±2.5, difference=3.5, 95% CI=1.8-5.2, p=0.0001); and for strong-producing biofilms (mean±SD=6.2±2.9, p=0.03). Conclusion: In conclusion, a higher DMFT was observed in biofilm producers than in non-biofilm producers. The prime biofilm producers were S. aureus, S. mutans, and E. coli. Peer Review History: Received 6 December 2023; Revised 30 January 2024; Accepted 26 February; Available online 15 March 2024 Academic Editor: Dr. Asia Selman Abdullah, Pharmacy institute, University of Basrah, Iraq, asia_abdullah65@yahoo.com Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.0/10 Reviewers: Ahmad Najib, Universitas Muslim Indonesia, Makassar, Indonesia, ahmad.najib@umi.ac.id Dr. Mohsen Ali Al-Hamzi, Thamar University, Factuality of Dentistry, Yemen. mohsenalhamzi@gmail.com

Genotype–phenotype correlation of fecal Streptococcus regulator (fsr) locus with gelatinase activity and biofilm formation intensity in clinical E. faecalis isolates

BackgroundEnterococci, known for their disturbing involvement in nosocomial infections, possess a diverse set of virulence factors, regulated by multiple genes. A key virulence regulator is the fecal Streptococcus regulator (Fsr) quorum sensing system. Multiple reports describe the involvement of fsr genes in several virulence mechanisms, notably gelatinase production and biofilm formation; however, the presence of fsr genes does not necessarily predict those virulence phenotypes. This study investigates the factors affecting the relation between molecular detection of fsr genes and accurate prediction of gelatinase activity and biofilm formation intensity.MethodsOne hundred enterococcal samples were collected from patients suffering from urinary tract infections. The isolates were identified through the use of a polymerase chain reaction (PCR) technique targeting the ddl gene. Biofilm formation was quantified by the crystal violet assay, while gelatinase activity was evaluated on gelatin agar plates. PCR was used to detect the fsrA and fsrB genes, as well as the gelatinase enzyme-encoding gene (gelE).ResultsOut of the collected 100 isolates, 93% were identified as Enterococcus faecalis. The isolates formed biofilm with different intensities: 47% were strong biofilm producers, 28% moderate, and 21% weak, while only four isolates (4%) did not form biofilm. Only 14% of all isolates had detectable gelatinase activity. The fsrA and fsrB genes were detected in 26% and 28% of the tested isolates, respectively, while gelE was detected in 57% of the isolates. Whereas no association was found between biofilm formation intensity and fsr locus genes or gelatinase activity, a strong positive correlation (r = 1) was found between the detection of both fsrA and fsrB genes and the gelatinase activity.ConclusionfsrA and fsrB have a diagnostic value and may be used as biomarkers for gelatinase activity in E. faecalis.

Biofilm-producing Bacteria and Quality of Life after Endoscopic Sinus Surgery in Patients with Chronic Rhinosinusitis with Nasal Polyposis.

Chronic rhinosinusitis (CRS) is one of the most common health disorders in humans and has a major impact on health-related quality of life (HRQoL). Of the many factors contributing to the etiology of CRS, less is known about the correlation between CRS and bacterial biofilms and their impact on HRQoL. The aim of this prospective study was to investigate the relationship between biofilm-producing bacteria and patients' objective findings and HRQoL. Forty-eight patients with CRSwNP were enrolled in a 12-month prospective study. The Lund-Mackay (LM) CT and endoscopic Lund-Kennedy (LK) scores were obtained before endoscopic sinus surgery (ESS), and patients completed the HRQoL instruments: the 22-item Sinonasal Outcome Test (SNOT-22), the 36-item Short Questionnaire (SF-36), and the visual analog scale (VAS). A sinus culture was obtained at ESS, bacteria were isolated, and in vitro quantification of the biofilm was performed. The LK score and HRQoL were determined postoperatively at months 1, 3, 6, and 12. The most common bacterial isolates in patients with CRSwNP were Staphylococcus aureus (28%), coagulase-negative staphylococci (52%), and Pseudomonas aeruginosa (8%). Preoperatively, the highest LM and LK scores were found in patients with strong biofilm producers. Postoperative LK scores were significantly reduced in all patients. Postoperative VAS scores were significantly reduced from month 1 to month 12 postoperatively. Patients with strong biofilm producers had significantly worse nasal blockage, secretion, headache, facial pressure and pain, and loss of smell preoperatively, compared to patients with low biofilm producers. The most significant reduction in preoperative scores SNOT-22 and SF-36 (excluding physical functioning) was seen in patients with S. aureus and P. aeruginosa. Patients with strong biofilm producers had higher LK and LM scores preoperatively, and greater improvement in LK and HRQoL scores postoperatively. Microbiologic surveillance of all CRS patients is recommended.

تأثير دقائق اوكسيد الزنك النانوية المصنعة بايولوجيًا على النمط الظاهري والجيني للغشاء الحيوي لبكتريا Proteus mirabilis

تصنف بكتريا Proteus mirabilis كثالث مسبب لالتهاب المجاري البولية المصاحب لتركيب أنابيب القسطرة البولية. تعد الاغشية الحيوية من عوامل الضراوة اللتي تستعملها البكتريا لمقاومة المضادات الحيوية وفي ضل تطور مقاومة البكتريا للمضادات الحيوية, صار لزامًا على العلماء والباحيثن في مجال الاحياء المجهرية أيجاد بدائل ذات كفائة عالية قادرة على اختراق جدار الخلية البكتيرية وبالتالي قتلها.في السنوات الاخيرة, اكتسبت دقائق النانو المصنعة بايولوجيا والصديقة للبيئة أهتمامًا كبيرًا من قبل الباحثين لأمتلاكها خصائص مميزة كصغر حجمها المتناهي واشكالها المختلفة يمَكنهم من استعمالها كمواد مضادة للبكتيريا في مختلف المجلات الطبية والصحية. تهدف هذه الدراسة الى التحري عن قدرة دقائق اوكسيد النحاس النانوية المصنعة بايولوجيا على تثبيط تكوين الغشاء الحيوي المنتج من قبل بكتيريا Proteus mirabilis. أنتجت هذه الدقائق بأستعمال راشح بكتيري يحتوي على الانزيمات المختزلة لملح النحاس واستخدمت عدة تقنيات لتحديد الصفات الفيزيائية المظهرية لدقائق النانو المنتجة حيث استخدمت تقنية XRD لتأكيد التركيب خماسي الشكل لهذه الدقائق , بينما استعملت تقنية TEM لتحديد قطر الجزيئات المنتجة بمعدل 84.45 نانومتر. اما FESEM فقد تم استخدامه لتحديد الصفات الخارجية لسطح جزيئات النانو المنتجة , تم تأكيد النتائج بأستخدام تقنية AFM للكشف عن توزيع و وعورة جزيئات النانو.اما UV فقد سجلت ااقصى امتصاصية على الطول الموجي nm287 .تم التحري في هذه الدراسة عن فعالية دقائق اوكسيد النحاس النانوية في منع تكون الغشاء الحيوي حيث أستعمل مستحلب دقائق اوكسيد النحاس النانوي بالتركيز تحت القاتل 32 μg/ml . سجلت نتائج هذه الدراسة انخفاضا ملحوضَا في انتاج الغشاء الحيوي للبكتريا المنتجه له حيث سجلت العزلات ذات الانتاجية العالية للغشاء الحيوي ضعفأ في الانتاج بعد معاملتها بالتركيز المستعمل من دقائق اوكسيد النحاس النانوية. جاء تأثير هذه الدقائق على كل من النمط الظاهري والتعبير الجيني لجين LuxS المسؤول عن تكوين الغشاء الحيوي بأستخدام تقنية rtPCRحيث سجلت الدراسة انخفاضا في التعبير الجيني لهذا الجين بعد معاملة سلالات البكتريا قيد الدراسة بدقائق اوكسيد النحاس النانوية مقارنتاً بالعزلات غير المعاملة. من خلال نتائج هذه التجربة يمكن التوصل الى امكانية اعتماد دقائق اوكسيد الزنك النانوية كعامل مضاد للبكتيريا عن استخدامه بالتركيز المناسب.

Characteristics of Bacterial Biofilm Formation in Nasolacrimal Silicone Tubes Post-dacryocystorhinostomy.

To examine the biofilm formation characteristics of bacteria identified at the genus level in samples obtained from silicone tubes after dacryocystorhinostomy surgery. In the study involving consecutive patients who underwent dacryocystorhinostomy surgery at Ankara Bilkent City Hospital and whose silicone tubes were removed six months after surgery, between January 2023 and May 2023; the tubes were placed in glycerol-PBS (phosphate buffered saline) solution and cultured on descriptive selective media at the genus level. The biofilm-forming properties of the obtained isolates were examined in solid-air and liquid-air interphases. Salmonella Typhimurium ATCC SL1344 strain was used as the control bacterium. As a result of the analysis of the samples taken from the patients, Pseudomonas spp. was identified in three of the samples, Staphylococcus spp. in five of the samples, and Streptococcus spp. in one of the samples. Among these samples, except for the bacteria identified in samples one and five, the rest were found to be strong biofilm producers. In all strong biofilm producers, the maximum biofilm production time was determined as 72 h and the incubation temperature was 37°C. The presence of cellulose and amyloid proteins in biofilm matrix structures is identified. Swimming and swarming motilities were observed in all bacterial samples. Since biofilms are considered potential factors in the pathogenesis of infectious and inflammatory diseases, they are a subject that needs to be thoroughly investigated. In our study, although there were no clinical infections in any of the patients, biofilm formation was detected in the patient samples. The fact that the bacteria exhibited moderate to strong biofilm formation characteristics suggests that these microorganisms could be persistent infectious agents.

Biofilm Formation, Pyocyanin Production, and Antibiotic Resistance Profile of Pseudomonas aeruginosa Isolates from Wounds.

Pseudomonas aeruginosa is one of the most frequently resistant and dangerous bacteria isolated from infected wounds of patients. This study aimed to determine the prevalence of P. aeruginosa from infected wounds of patients in the Dschang District Hospital to evaluate their antibiotic susceptibility profiles and their ability to swarm and swim and correlate pyocyanin production with biofilm formation. Wound swab samples were collected and the identification of P. aeruginosa was performed using microbiological and biochemical tests. Their antimicrobial susceptibility was determined by the broth microdilution method. Swarming and swimming were determined by measuring the diameters of motility in semisolid/low-viscosity media. Furthermore, pyocyanin production and biofilm formation were evaluated spectrophotometrically using a microtiter plate. The prevalence of P. aeruginosa from infected wounds in our study population was 26%. All P. aeruginosa isolates were resistant to streptomycin and paromomycin, and the frequency of multidrug resistance (MDR) was 65.8%. All P. aeruginosa isolates showed the ability to produce biofilm and pyocyanin. Out of the 37 isolates screened, 19 including the reference strains (51.4%) were strong biofilm producers. A significant positive correlation was observed among biofilm formation, pyocyanin production, and the antibiotic resistance profile of the isolates. Findings from this study suggest that infected wounds could act as a reservoir for MDR and virulent P. aeruginosa. The presence of strong biofilm producers of P. aeruginosa in infected wounds is a serious public health concern. Therefore, surveillance programs to monitor and control MDR P. aeruginosa in these patients are required to prevent their dissemination in hospital settings.

Investigating the relationship between carbapenemase production and biofilm formation in Klebsiella pneumoniae clinical isolates

ObjectiveCarbapenemase production and biofilm formation in K. pneumoniae are crucial factors influencing the pathogenicity and antibiotic resistance of this bacterium. This study investigated the interplay between carbapenemase production and biofilm formation in K. pneumoniae clinical isolates.ResultsThe distribution of biofilm-forming ability significantly differed between carbapenemase-producing (CP-Kp) (n = 52) isolates and carbapenemase-nonproducing (CN-Kp) isolates (n = 37), suggesting a potential link between carbapenemase production and biofilm formation. All the blaNDM-1-harbouring isolates demonstrated biofilm formation, with varying levels classified as strong (33.33%), moderate (22.22%), or weak (44.45%). blaNDM-1 and blaKPC-coharbouring isolates did not exhibit strong or moderate biofilm formation. blaNDM-1 and blaOXA-48-coharbouring isolates were predominantly moderate (48.65%), followed by weak (32.43%), with none showing strong biofilm production. These findings suggest a correlation between the presence of carbapenemases and biofilm-forming ability; however, the heterogeneity in biofilm-forming abilities associated with different carbapenemase types and the absence of strong biofilm producers in the detected carbapenemase combinations prompt a closer look at the complex regulatory mechanisms governing biofilm formation in CP-Kp isolates.

Great Plasticity in a Great Pathogen: Capsular Types, Virulence Factors and Biofilm Formation in ESBL-Producing Klebsiella pneumoniae from Pediatric Infections in Uruguay.

Klebsiella pneumoniae is widely recognized as an opportunistic hospital and community pathogen. It is one of the priority microorganisms included in the ESKAPE group, and its antibiotic resistance related to extended-spectrum β-lactamases (ESBL) is a global public health concern. The multi-drug resistance (MDR) phenotype, in combination with pathogenicity factors, could enhance the ability of this pathogen to cause clinical infections. The aim of this study was to characterize pathogenicity factors and biofilm formation in ESBL-producing K. pneumoniae from pediatric clinical infections. Capsular types, virulence factors, and sequence types were characterized by PCR. Biofilm formation was determined by a semiquantitative microtiter technique. MDR phenotype and statistical analysis were performed. The K24 capsular type (27%), virulence factors related to iron uptake fyuA (35%) and kfuBC (27%), and sequence types ST14 (18%) and ST45 (18%) were the most frequently detected. Most of the strains were biofilm producers: weak (22%), moderate (22%), or strong (12%). In 62% of the strains, an MDR phenotype was detected. Strains with K24 capsular type showed an association with ST45 and the presence of fyuA; strains with kfuBC showed an association with moderate or strong biofilm production and belonging to ST14. Weak or no biofilm producers were associated with the absence of kfuBC. The MDR phenotype was associated with the main ESBL gene, blaCTX-M-15. The high plasticity of K. pneumoniae to acquire an MDR phenotype, in combination with the factors exposed in this report, could make it even more difficult to achieve a good clinical outcome with the available therapeutics.

Aeromonas spp. in Freshwater Bodies: Antimicrobial Resistance and Biofilm Assembly.

Aeromonas spp. are environmental bacteria able to infect animals and humans. Here, we aim to evaluate the role of biofilms in Aeromonas persistence in freshwater. Aeromonas were isolated from water and biofilm samples and identified by Vitek-MS and 16S rRNA sequencing. Antibiotic susceptibility profiles were determined according to EUCAST, and a crystal violet assay was used to assess biofilm assembly. MTT and the enumeration of colony-forming units were used to evaluate biofilm and planktonic Aeromonas susceptibility to chlorination, respectively. Identification at the species level was challenging, suggesting the need to improve the used methodologies. Five different Aeromonas species (A. salmonicida, A. hydrophila, A. media, A. popoffii and A. veronii) were identified from water, and one species was identified from biofilms (A. veronii). A. veronnii and A. salmonicida presented resistance to different antibiotics, whith the highest resistance rate observed for A. salmonicida (multiple antibiotic resistance index of 0.25). Of the 21 isolates, 11 were biofilm producers, and 10 of them were strong biofilm producers (SBPs). The SBPs presented increased tolerance to chlorine disinfection when compared with their planktonic counterparts. In order to elucidate the mechanisms underlying biofilm tolerance to chlorine and support the importance of preventing biofilm assembly in water reservoirs, further research is required.

Random Amplified Polymorphic DNA (RAPD)-PCR analysis of genotypic and phenotypic characteristics of MethicillinResistant <i>Staphylococcus epidermidis</i> (MRSE) strains involved in biofilm formation

Purpose: The current study aims to investigate genotypic and phenotypic aspects of methicillinresistant Staphylococcus epidermidis (MRSE) strains involved in the biofilm formation and the attendance of the icaADB gene and genotypic characterization of this gene by random amplified polymorphic DNA (RAPD) PCR.Methods: 60 Staphylococcus epidermidis strains were isolated from clinical specimens, suspected of having the bacteria, from the laboratories of Isfahan. Biofilm formation was measured by the microtiter plate method. The attendance of biofilm formation genes was studied using PCR and all isolates producing biofilm (strong and moderate) were genetically classified by RAPD-PCR.Results: 37 isolates (61.7%) were MRSE and all positive biofilm strains. The prevalence of biofilmrelated genes in the isolates was SesC (100%), SesI (45.9%), icaA (29.7%), icaB (37.8%), icaC (81.08%), icaD (70.2%), arcA (81.08%), and opp3AB (70%). PCR analysis showed that among 30 isolates of strong and medium biofilm production, 70% (21/30) positive for the icaADB gene. The Dendrograms obtained from RAPD-PCR results showed that all nine main clusters were at an 80% similarity level, and there were four isolates of a single type.Conclusion: These findings confirmed the high genotypic diversity of biofilm-producing MRSE strains and the relative diffusion of specific clones among clinical samples.

Inhibitory Effects of Biosynthesized Copper Nanoparticles on Biofilm Formation of Proteus mirabilis

This study aimed to explore whether green synthesized copper nanoparticles (CuNPs) can function as an anti-biofilm agent produced by P. mirabilis. The nanoparticles were synthesized from cells free extract of P. mirabilis. Characterization of biosynthesized copper nanoparticles was carried out to determine the chemical and physical properties of the product using atomic-force microscopy (AFM), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD) and UV-visible spectroscopy. The hexagonal structure was confirmed by XRD, size range was marked 13-19nm by TEM. FESEM was used to confirm the surface morphology. AFM analysis was used to reveal the roughness and distribution of nanoparticles. UV–visible spectra of the synthesized nanoparticles recorded a maximum peak at 380 nm. Copper nanoparticles showed remarkable biofilm inhibitory effects on wild type strains of multidrug resistant Proteus mirabilis. After incubation for 24 and 48 hours at 37oC with 128 μg/ml sub minimum inhibitory concentration (MIC) of CuO nanoparticles, strong biofilm producer strains showed weak biofilm production. Down regulation changes in LuxS expression using real time PCR technology were detected after treatment with copper nanoparticles of these strains.

ISOLATION AND IDENTIFICATION OF BIOFILM PRODUCING ENTEROCOCCUS FAECALIS FROM ROOT CANAL

This study was aimed to one of the most prevalent causes for endodontic treatment failure is the presence of Enterococcus faecalis bacterium within teeth root canals. To achieve successful treatment, it is so important to study E. faecalis behavior. The aim of study was to investigate biofilm production and antibiotic sensitivity of E. faecalis isolated from root canals. Results showed isolation of E. feacalis (65%) of samples, identified by specific gene by PCR technique. Most isolates were sensitive to Imipenem and resistant to Erythromycin, Clindamycin, Tetracycline and Trimethoprim. Strong biofilm production was detected among 29.5% of highest antibiotic resistant isolates. The results may indicate that infected root canals with E. feacalis may lead to serious complication for patients.

Genotypic characterization of hypervirulent Klebsiella pneumoniae (hvKp) in a tertiary care Indian hospital.

HypervirulentKlebsiella pneumoniae(hvKp) is an emerging pathogen and causes endophthalmitis, liver abscess, osteomyelitis, meningitis, and necrotizing soft tissue infections in both immunodeficient and healthy people. The acquisition of the antibiotic resistance genes of hvKp has become an emerging concern throughout the globe. In this study, a total of 74K. pneumoniaeisolates were collected and identified by VITEK2 andblaSHVgene amplification. Out of these, 18.91% (14/74) isolates were identified as hvKp by both phenotypic string test and genotypiciucAPCR amplification. The antibiotic susceptibility revealed that 57.14% (8/14) isolates were multidrug-resistant (MDR) and 35.71% (5/14) isolates were extremely drug-resistant (XDR). All the isolates were resistant to β-lactam, β-lactamase + inhibitor groups of antibiotics, and the least resistance to colistin. Of 14 hvKp isolates, all isolates are positive for iroB(100%),followed byiutA(92.85%),peg344(85.71%), rmpA (57.14%), and magA(21.42%) genes. Among serotypes, K1 was the most prevalent serotype 21.4% (3/14), followed by K5 14.3% (2/14). The most common carbapenemase gene was blaOXA-48 (78.57%) followed by blaNDM (14.28%) and blaKPC (14.28%) which co-carried multiple resistance genes such as blaSHV (100%), blaCTX-M (92.85%), and blaTEM (78.57%). About 92.85% (13/14) of hvKp isolates were strong biofilm producers, while one isolate (hvKp 10) was the only moderate biofilm producer. The (GTG)5-PCR molecular typing method revealed high diversity among the hvKp isolates in the tertiary care hospital. Our findings suggest that MDR-hvKp is an emerging pathogen and a challenge for clinical practice. In order to avoid hvKp strain outbreaks in hospital settings, robust infection control and effective surveillance should be implemented.

Isolation, characterization and antibiofilm efficacy of a novel Klebsiella pneumoniae phage

The multidrug-resistant (MDR) Klebsiella pneumoniae is an important pathogen that threatens public health directly with life-threatening infections. The need for the development of new effective and well tolerated alternative treatments for these infections is crucial. Therefore, the interest in phage therapy as a promising alternative is increasing. Here, a novel phage named ΦAYH was isolated from the Tigris River water, Baghdad, IRAQ near sewage of Baghdad Medical City with its specific host from the same site. Phage ΦAYH belongs to Podoviridae family in the order Caudovirales. The ΦAYH maintained stability at different temperatures (−10 to 60°C) and pH values (5–11). The phage was able to lyse 8 from 32 clinical K. pneumoniae isolates in vitro with no lytic activity against other tested species. For one-step growth, latent period was 10 min with burst size ∼64 virions/cell at multiplicity of infection (MOI) 10. The SDS-PAGE test revealed one major structural protein and different structural proteins ranging from 28 to 89 kDa in size. The phage host and 32 clinical K. pneumoniae isolates were tested for phenotypic identification and antibiotics profile by VITEK-2 system and genotypically using rpob gene. All clinical K. pneumoniae isolates showed resistance to the most antibiotics tested while phage host was resistant only to amoxicillin. Biofilm production by all clinical isolates including the host isolate was tested. These isolates showed different ability as following: 72.72% as weak, 6.06% as moderate, and 21.21% as strong biofilm producer. Together these results demonstrate that ΦAYH is a promising alternative against MDR K. pneumoniae.