The Influence of Seasonality and Antimicrobial Resistance Genes on Biofilm Formation in Hospital-Acquired Resistant Bacteria.

Background Hospital-acquired resistant infections (HARI) are difficult to manage due to limited treatment options and to their ability to further resistance stress conditions by producing biofilm. This work aimed to assess the distribution of HARI-associated bacterial species in north Israel and to investigate associations between biofilm formation and extended beta-lactamase (ESBL) genes, bacterial and patient characteristics, and hospitalization length, season and year. Methods Methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) P. aeruginosa and A. baumannii, ESBL-producing Escherichia coli (ESBL-E. coli), Klebsiela pneumoniae (ESBL-K. pneumoniae) and Proteus mirabilis (ESBL- P. mirabilis) were isolated from 569 blood, urine, wound and respiratory samples of patients with HARI hospitalized during 2020–2022 in north Israel. Biofilm-formation capacity was assessed by the crystalline violet method. ESBL genes were detected by real-time PCR. Data regarding season, time to infection, bacterial species, patient demographics, year, and hospital department, were collected from medical records. Results HARI rates were significantly lower in 2022 compared to 2020. ESBL-K. pneumoniae was the most prevalent (31.6%) bacteria. Strong biofilms were produced by 346 (60.8%), and were most common among ESBL-K. pneumoniae samples (46.2%). blaCTX−M was the most commonly detected ESBL gene (87.7%). Most strains (61.3%) carried more than one ESBL gene. Hospitalization season had a notable impact on biofilm production, with a heightened risk of infection by robust biofilm producers during spring, summer and autumn compared to winter. Furthermore, the presence of blaSHV and blaTEM genes were significantly associated with enhanced biofilm production. Bacteria harboring all three ESBL genes exhibited the highest biofilm production capacities, compared to those carrying fewer than three. Conclusions Biofilm-production intensity differs across bacterial species and seasons and is influenced by the presence of ESBL genes.

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.

Bacterial biofilm formation has been implicated in the high incidence of persistent otorrhoea after tympanostomy tube insertion. The aim of the study was to investigate whether biofilm formation on tympanostomy tubes depends on the genetic profile of methicillin-resistant Staphylococcus aureus (MRSA) strains. Capacity of biofilm formation on fluoroplastic tympanostomy tubes (TTs) was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on TTs. Tested MRSA strains were classified into SCCmec type I (36.7 %), III (23.3 %), IV (26.7 %) and V (13.3 %), agr type I (50 %), II (36.7 %) and III (13.3 %), and 5 clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on TTs was as following: 13.3 % of strains belonged to the category of no biofilm producers, 50 % to the category of weak biofilm producers and 36.7 % to moderate biofilm producers. There was a statistically significant difference between CC, SCCmec and agr types and the category of biofilm production on TTs tubes (p < 0.001): CC5, SCCmecI type and agrII type with a moderate amount of biofilm, and CC8 and agrI type with a low amount of biofilm. Biofilm formation by MRSA on TTs is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related post-tympanostomy tube otorrhea.

Methicillin-susceptible Staphylococcus aureus (MSSA) is an important nosocomial pathogen worldwide. This study aims to investigate the in vitro biofilm-forming ability of clinical MSSA isolated from various sources in the main public tertiary referral hospital in Terengganu, Malaysia and to detect the presence of biofilm-associated and regulatory genes among these isolates. A total of 104 MSSA isolates [pus (n = 75), blood (n = 24), respiratory secretions (n = 2), eye (n = 2), and urine (n = 1)] were investigated for slime production and biofilm formation using Congo red agar and crystal violet microtitre plate, respectively. Fifteen MSSA isolates with varying degrees of biofilm formation were selected for validation via a real-time cell analyser. All isolates were screened for microbial surface components recognising adhesive matrix molecules (MSCRAMM) and accessory gene regulator (agr) using polymerase chain reaction assay. A total of 76.0% (79/104) isolates produced slime layer, while all isolates developed biofilm as follows: 52.8% (55/104) strong biofilm producers, 40.4% (42/104) intermediate biofilm producers, and 6.7% (7/104) weak biofilm producers. A total of 98.1% (102/104) isolates carried at least one of the screened MSCRAMM gene(s) with the eno gene detected at the highest rate (87.5%, 91/104), while the sasG gene was significantly associated with strong biofilm production (p = 0.015). Three agr groups, 1, 2, and 3, were detected among the MSSA isolates with a predominance of agr-3 (32.7%, 34/104). In conclusion, biofilm formation varied greatly among clinical MSSA isolates, and the presence of sasG gene and agr-1 may play important role in initiating MSSA infections via biofilm formation.

Acinetobacter baumannii is a multi-drug resistant (MDR) gram-negative pathogen leading to nosocomial infections. Hospital-acquired infections due to A.baumannii occur especially in patients hospitalized in intensive care units. Important infections related to this bacterium are pneumonia, bacteremia, endocarditis, skin and soft tissue, urinary tract infections and meningitis. Human transmission is usually through the hospital environment or through medical personnel. A.baumannii isolates increases their virulence not only being multiple resistance to antibiotics but as well as the ability to form biofilm. The biofilm formation of A.baumannii isolates were mostly related with genes encoding curli fiber (csgA), the chaperone-usher fimbria (csuE) and the outer membrane (ompA). The aim of this study was to demonstrate biofilm production and virulence genes in MDR invasive A.baumannii isolates. MDR and similarity status previously known invasive A.baumannii (n= 156) isolates were included in the study. Biofilm production was determined by quantitative microplate biofilm method. Virulence genes csgA, csuE, fimH, ompA and blaPER-1 were investigated by polymerase chain reaction (PCR). It was determined that 60.3% (94/156) of all the isolates formed biofilm. Of these 94 isolates, 17 were weak, 33 were medium and 44 were strong. The mean biomass forming capacity of the isolates was found to be 2.23 ± 0.0033. Among the isolates included in the study (n= 156) the frequency of csgA, csuE, ompA, fimH and blaPER-1 genes were 71.2%, 32.1%, 21.8%, 7.1% and 3.2% respectively. The frequency of csgA, ompA, bap, csuE, fimH virulence genes were found to be 41.5%, 24.5%, 20.2% and 5.3% among biofilm positive isolates respectively. Biofilm-forming isolates were most commonly found in pulsotype II 19.1% (18/94), pulsotype IX 17.0% (16/94) and pulsotype VI 12.8% (12/94). In this study, when the distribution of virulence genes were compAred with the isolates that have weak, medium and strong biofilm, all of the studied genes were found to be more abundant in isolates with strong and medium positive biofilm production. This has shown that excluding fimH gene, csgA, csuE and ompA genes have contributed to the biofilm formation in invasive A.baumannii isolates, respectively.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) has become the leading cause of skin and soft tissue infections (SSTIs). Biofilm production further complicates patient treatment, contributing to increased bacterial persistence and antibiotic tolerance. The study aimed to explore the efficacy of different antibiotics on biofilm-producing MRSA isolated from patients with SSTI. A total of 32 MRSA strains were collected from patients with SSTI. The MIC and minimal biofilm eradication concentration (MBEC) were measured in planktonic and biofilm growth. The study showed that dalbavancin, linezolid, and vancomycin all inhibited MRSA growth at their EUCAST susceptible breakpoint. Of the MRSA strains, 87.5% (n = 28) were strong biofilm producers (SBPs), while only 12.5% (n = 4) were weak biofilm producers (WBPs). The MBEC90 values for dalbavancin were significantly lower than those of linezolid and vancomycin in all tested strains. We also found that extracellular DNA (eDNA) contributes to the initial microbial attachment and biofilm formation. The amount of eDNA differed among MRSA strains and was significantly higher in those isolates with high dalbavancin and vancomycin tolerance. Exogenously added DNA increased the MBEC90 and protection of biofilm cells from dalbavancin activity. Of note, the relative abundance of eDNA was higher in MRSA biofilms exposed to MBEC90 dalbavancin than in untreated MRSA biofilms and those exposed to sub-MIC90. Overall, dalbavancin was the most active antibiotic against MRSA biofilms at concentrations achievable in the human serum. Moreover, the evidence of a drug-related increase of eDNA and its contribution to antimicrobial drug tolerance reveals novel potential targets for antibiofilm strategies against MRSA.IMPORTANCEStaphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) worldwide. In addition, methicillin-resistant S. aureus (MRSA) is increasingly frequent in postoperative infections and responsible for a large number of hospital readmissions and deaths. Biofilm formation by S. aureus is a primary risk factor in SSTIs, due to a higher antibiotic tolerance. Our study showed that the biofilm-forming capacity varied among MRSA strains, although strong biofilm producers were significantly more abundant than weak biofilm producer strains. Notably, dalbavancin demonstrated a potent antibiofilm activity at concentrations achievable in human serum. Nevertheless, dalbavancin activity was affected by an increased concentration of extracellular DNA in the biofilm matrix. This study provides novel insight for designing more targeted therapeutic strategies against MRSA and to prevent or eradicate harmful biofilms.

The aim of this study was to investigate biofilm formation on silicone tubes by genetically diverse methicillin-resistant Staphylococcus aureus (MRSA) strains. Capacity of biofilm formation on dacryocystorhinostomy silicone tubes was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on silicone tubes. Tested MRSA strains were classified into SCCmec type I (33.3%), II (3.3%), III (20.0%), IV (26.7%) and V (16.7%), agr type I (56.7%), II (36.7%) and III (6.6%), and eight spa clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on silicone tubes was as follows: 33.3% of strains belonged to the category of low biofilm producers, and 66.7% to moderate biofilm producers. There was statistically significant correlation between spa CC and the category of biofilm production on silicone tubes (p = 0.01): CC5 and CC45 with moderate amount of biofilm, and CC8 with low amount of biofilm. A moderate amount of biofilm formed on silicone tubes correlated with agr type II MRSA strains (p = 0.008). Biofilm formation by MRSA on silicone tubes is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related ocular device infections. Genotyping and biofilm quantification may be helpful in determining when decolonisation and cohort isolation are required to prevent device-related infections.

Background: Antibiotic resistance and biofilm production have become a significant and growing threat to public and environmental health. The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Staphylococcus aureus (VRSA) in Nigeria is well documented in clinical samples. However, findings on MRSA and VRSA from hospital environmental surfaces (HES) are yet to be adequately reported. The current study determined the prevalence of MRSA and VRSA in some HES and their ability to form biofilms. Methods: A total of 60 samples obtained from some HES which included walls, door handles, toilet seats, floors, bed rails and bedside tables were assessed for Staphylococcus aureus using Blood agar and mannitol salt agar (MSA) by a standard culture-based approach. Methicillin and vancomycin susceptibility profile of the Staphylococcus aureus isolates were determined using the disc diffusion method. Biofilm production was determined using Congo red agar (CRA) assay. Results: Of the 60 samples tested, 42 (70.0%) Staphylococcus aureus were isolated, with a majority recovered from walls, bed rails and bedside tables. Among the Staphylococcus aureus isolates, 90.5% (38/42) were MRSA and all the MRSA isolates were resistant to vancomycin. Biofilm profile revealed 20 (52.6%) strong biofilm formers and 2 (5.3%) moderate biofilm formers. Conclusion: The high prevalence of MRSA, VRSA and strong biofilm forming capacity of isolates from HES is a threat to environmental health and risk of disease development in hospitalized patients.

Assessing biofilm formation by Listeria monocytogenes strains

BackgroundBiofilm formation is a major virulence factor contributing to the chronicity of infections. To date few studies have evaluated biofilm formation in infecting isolates of patients including both Gram-positive and Gram-negative multidrug-resistant (MDR) species in the context of numerous types of infectious syndromes. Herein, we investigated the biofilm forming capacity in a large collection of single patient infecting isolates and compared the relationship between biofilm formation to various strain characteristics.MethodsThe biofilm-forming capacity of 205 randomly sampled clinical isolates from patients, collected from various anatomical sites, admitted for treatment at Brooke Army Medical Center (BAMC) from 2004–2011, including methicillin-resistant/methicillin susceptible Staphylococcus aureus (MRSA/MSSA) (n=23), Acinetobacter baumannii (n=53), Pseudomonas aeruginosa (n=36), Klebsiella pneumoniae (n=54), and Escherichia coli (n=39), were evaluated for biofilm formation using the high-throughput microtiter plate assay and scanning electron microscopy (SEM). Relationships between biofilm formation to clonal type, site of isolate collection, and MDR phenotype were evaluated. Furthermore, in patients with relapsing infections, serial strains were assessed for their ability to form biofilms in vitro.ResultsOf the 205 clinical isolates tested, 126 strains (61.4%) were observed to form biofilms in vitro at levels greater than or equal to the Staphylococcus epidermidis, positive biofilm producing strain, with P. aeruginosa and S. aureus having the greatest number of biofilm producing strains. Biofilm formation was significantly associated with specific clonal types, the site of isolate collection, and strains positive for biofilm formation were more frequently observed to be MDR. In patients with relapsing infections, the majority of serial isolates recovered from these individuals were observed to be strong biofilm producers in vitro.ConclusionsThis study is the first to evaluate biofilm formation in a large collection of infecting clinical isolates representing diverse types of infections. Our results demonstrate: (1) biofilm formation is a heterogeneous property amongst clinical strains which is associated with certain clonal types, (2) biofilm forming strains are more frequently isolated from non-fluid tissues, in particular bone and soft tissues, (3) MDR pathogens are more often biofilm formers, and (4) strains from patients with persistent infections are positive for biofilm formation.

Siphonocholin isolated from red sea sponge Siphonochalina siphonella attenuates quorum sensing controlled virulence and biofilm formation

Inhibitory effect of naringin, naringenin, and crocin on biofilm formation and lecA gene expression in Pseudomonas aeruginosa clinical isolates.

Background: Staphylococcus aureus including methicillin resistant S. aureus (MRSA) is one of the most effective biofilm-forming organisms, biofilm contribute in protecting the microorganism from host defenses and prevent the effective penetration of antimicrobial agents. Biofilm formation is considered as an important contributing factor for the initiation and establishment of chronic infection by S. aureus and known as a major obstacle in the treatment of S. aureus infections is their ability to develop resistance to antimicrobials.&#x0D; Aims : To screen clinical Staphylococcus aureus including MRSA isolates for their biofilm forming abilities and their association with antimicrobial resistance.&#x0D; Methods: A total of 196 clinical isolates of S. aureus were obtained from different sample sources using standard microbiological techniques from three major hospitals in Gaza strip. Biofilm formation of these isolates was determined by tissue culture plate (TCP) method and tube adherence method (TM). Antimicrobial susceptibility test was performed using the modified Kirby–Bauer disk diffusion method as per Clinical and Laboratory Standards Institute guidelines. MRSA was detected using the cefoxitin disk test.&#x0D; Results: Biofilm formation was observed in 174 (88.8 %) and in 145 (74.0%) isolates of S. aureus via TCP method and TM, respectively. The highest resistance percent was for penicillin (92.9%), followed by cefoxitin (80.6%) and oxacillin (67.9%), while the lowest resistance percent was for linezolid and ceftaroline (1%). Among the 196 isolates 71.4% (N=140) were classified as MDR with a MAR index (≥ 0.2).&#x0D; A total of 158 isolates (80.6%) were identified as MRSA distributed as 90.4%, 79.4% and 70.9% from Al-Shifa, Al-Nasser pediatrics and Al-Aqsa hospitals respectively. Large proportions (82.1%) of biofilm producers were identified as MRSA. Biofilm-producing MRSA exhibited a higher percent (90.5%) when compared with the biofilm non-producer MRSA (9.5%). Importantly, 89.2% of biofilm-producing S. aureus were multidrug resistant.&#x0D; Conclusions: S. aureus isolates possessed high biofilm-forming ability. Biofilm-producing strains have very high tendency to exhibit antimicrobial resistance, multidrug resistance and methicillin resistance. Regular surveillance of biofilm formation by S. aureus and their antimicrobial resistance profile may lead more success in treating S. aureus infections.&#x0D; &#x0D; Keywords: Biofilm, MDR, MRSA, TCP, TM, Gaza strip, Palestine.

Assessment of biofilm formation, antibiotic resistance patterns, and the prevalence of adhesion-related genes in clinical Staphylococcus aureus isolates

The present study was planned to detect the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among bacteria obtained from retail goat meat. Fermenting and non-fermenting bacterial isolates (n=57), recovered from 61 goat meat samples, were identified by 16S rRNA gene sequencing. Antimicrobial susceptibility of isolates was tested by the broth dilution method using ceftazidime, cefotaxime, meropenem and imipenem. Plasmids were isolated and tested for their physical characters. Plasmids were subjected to screening of carbapenemase, ESBL and intI1 gene. Conjugation assay was performed using blaNDM -positive isolates as the donor, and Escherichia coli HB101 as the recipient. Isolates showed the high rates of resistance to ceftazidime (77·2%), cefotaxime (70·2%), meropenem (22·8%) and imipenem (17·5%). They showed variability in number and size (~1 to >20kb) of plasmids. Among all, 1, 4, 13 and 31 isolates showed the blaKPC , blaNDM , blaSHV and blaTEM genes, respectively. The blaKPC-2 gene was observed in one E. coli isolate. The blaNDM-1 gene was detected in Stenotrophomonas maltophilia (n=2), Acinetobacter baumannii (n=1) and Ochrobactrum anthropi (n=1) isolates. These isolates co-harboured the blaTEM and blaSHV genes. The intI1 gene was detected in 22 (38·6%) isolates, and 16 of these isolates showed the carbapenemase and/or ESBL genes. The conjugative movement of blaNDM gene could not be proved after three repetitive mating experiments. The presence of genes encoding carbapenemases and ESBLs in bacteria from goat meat poses public health risks.