Phenotypic and Genotypic Characterization of Antibiotic Resistance in Staphylococcus aureus Isolated from Different Sources

Background: Staphylococcus aureus is a bacterial pathogen that can cause a wide range of nosocomial infections. Nasal colonization by S. aureus plays an important role both in the epidemiology and pathogenesis of infection. Objectives: This study aimed at detecting the biofilm-forming capacity of clinical isolates and detection of icaA and agr genes. Methods: A total of 150 clinical specimens was collected from patients in different hospitals in Baghdad. The clinical samples included wounds, abscess, sputum, and ear infections. The suspected isolates were cultured for one day at 37 °C on mannitol salt agar in an aerobic environment. Results: The results showed that of 150 samples, 44 isolates were S. aureus (29.3%), of wounds samples, 22 isolates (45.83%) were S. aureus, 13 (37.14%) were from abscess, 7 (17.95%) from sputum, and 2 isolates (7.14%) from ear samples. This study found that most isolates formed biofilm, but the levels of biofilm were distributed across three ranges. The results also indicated that 47.7% of the isolates produced a strong biofilm, as well as 38.6 and 13.6% produced moderate and weak biofilms, respectively. The present molecular results showed that S. aureus from different samples were 13 (59.1%), 4 (30.77%), 3 (42.85%), 0 (0%) from wounds, abscess, sputum, ear, respectively, were positive for agr gene. While the results showed 18 (81.8%), 10 (76.9%), 5 (71.4%), 1 (50%), respectively, were positive for icaA gene. Conclusions: Most S. aureus isolates isolated from wound were biofilm positive. These isolates bore icaA and agr genes in a high quantity.

Virulence Factors in Staphylococcus Associated with Small Ruminant Mastitis: Biofilm Production and Antimicrobial Resistance Genes

Small ruminant mastitis is a serious problem, mainly caused by Staphylococcus spp. Different virulence factors affect mastitis pathogenesis. The aim of this study was to investigate virulence factors genes for biofilm production and antimicrobial resistance to β-lactams and tetracyclines in 137 staphylococcal isolates from goats (86) and sheep (51). The presence of coa, nuc, bap, icaA, icaD, blaZ, mecA, mecC, tetK, and tetM genes was investigated. The nuc gene was detected in all S. aureus isolates and in some coagulase-negative staphylococci (CNS). None of the S. aureus isolates carried the bap gene, while 8 out of 18 CNS harbored this gene. The icaA gene was detected in S. aureus and S. warneri, while icaD only in S. aureus. None of the isolates carrying the bap gene harbored the ica genes. None of the biofilm-associated genes were detected in 14 isolates (six S. aureus and eight CNS). An association was found between Staphylococcus species and resistance to some antibiotics and between antimicrobial resistance and animal species. Nine penicillin-susceptible isolates exhibited the blaZ gene, questioning the reliability of susceptibility testing. Most S. aureus isolates were susceptible to tetracycline, and no cefazolin or gentamycin resistance was detected. These should replace other currently used antimicrobials.

The Correlation between icaA and icaD Genes with Biofilm Formation Staphylococcus epidermidis In Vitro

This study was conducted to identify the presence of icaA and icaD genes in S. epidermidis and to analyze the relationship between the presence of icaA and icaD genes with the ability of in vitro biofilm formation in S. epidermidis. S. epidermidis isolates from patients and healthy people were collected and PCR was examined to detect icaA and icaD genes. which then continued to examine the ability of biofilm formation by the method of Congo Red Agar. The results of this genotypic and phenotypic examination were then tested for correlation with statistical tests using SPSS 23.0. A total of 40 S. epidermidis isolates were collected, consisting of 20 clinical isolates and 20 isolates of normal flora. The icaA gene was positive in 5 isolates (12.5%), and 8 isolates (20%) were positive for the icaD gene, 3 isolates with icaA and icaD were both positive. One hundred percent of isolates with icaA or icaD positively formed biofilms, but there were 15 isolates (42.9%) who did not have the icaA gene but showed the ability to form biofilms, while 12 isolates (37.5%) who did not have the icaD gene also formed biofilms. Fifty percent of S. epidermidis isolates showed the ability to form biofilms at CRA. The Fisher Exact test showed a significant relationship between the icaA gene and the ability of biofilm formation (p=0.047 (p<0.05)) as well as the icaD gene (p=0.03 (p<0.05)). The icaA and icaD genes have a significant relationship to biofilm formation in S. epidermidis. There was another mechanism in the formation of biofilms that are not dependent on the ica gene.

Detection of icaA Gene Expression in Clinical Biofilm-Producing Staphylococcus Aureus Isolates

The pathogenicity resulting from Staphylococcus aureus infection has remarkable importance as one of the community-associated bacterial infections, due to the virulent ability of these bacteria to produce biofilms. This study was designed to detect biofilm production in clinical isolates from samples of wounds and urinary tract infections. The expression levels of the icaA gene that is responsible of slime layer production in biofilms was compared in isolates with different biofilm producing capabilities. Fifty seven samples that included 32 samples from urine and 25 samples from wounds were collected from Alwasti Hospital, Al-Kindi Teaching Hospital, and Alzahraa Clinic, Baghdad, Iraq. The bacteria was identified according to biochemical tests, API20 strip test, and PCR assay. The results of 16S rRNA PCR detection revealed that nine isolates were identified as S. aureus. The biofilm assay showed that 46.15% of the isolates were strong biofilm producers, 46.15% had moderate ability to produce biofilm, and 7.70% were weak producers. Quantitative PCR assay was carried out on three isolates with different biofilm-producing abilities. The results demonstrated that the strong biofilm-producing isolates had significantly higher (P ≤0.01) gene expression level (6.508) compared with the moderate (1.624) and the weak (1.231) isolates.

Staphylococcus spp. Isolated from Bovine Subclinical Mastitis in Different Regions of Brazil: Molecular Typing and Biofilm Gene Expression Analysis by RT-qPCR

Bovine mastitis is mainly caused by bacteria of the genus Staphylococcus spp., which possess different virulence factors, including the capacity for biofilm formation that provides enhanced protection against the action of immune system components and serves as a barrier against the penetration of antimicrobial agents. This study aimed to characterize 181 Staphylococcus spp. Strains—including Staphylococcusaureus and coagulase-negative staphylococci (CoNS) isolated from bovine subclinical mastitis in six Brazilian states—by molecular methods. RT-qPCR was used to verify the expression of genes of the ica operon—mainly responsible for biofilm formation—as well as bap and bhp. Chromosome similarity among the isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The icaA gene was detected in 79 (43.6%) isolates, icaB in 24 (13.2%), icaC in 57 (31.4%), and icaD in 127 (70.1%). The bap gene was identified in 66 (36.4%) isolates, while the bhp gene was found in nine (4.9%). RT-qPCR confirmed the expression of the icaA gene in 60 (75.9%) isolates, of icaB in six (25%), of icaC in 26 (45.6%), and of icaD in 80 (63%). Clonal typing of the isolates by PFGE permitted the identification of eight Staphylococcusaureus clusters that simultaneously included ≥3 strains, with a similarity of ≥80%. Regarding the other species studied, three clusters were observed for Staphylococcuschromogenes and four clusters for Staphylococcusepidermidis. Only one cluster each was identified for Staphylococcussaprophyticus and Staphylococcussimulans, while the other species did not form any cluster. With respect to MLST, ST126 and ST1 were the prevalent sequence types in S. aureus, while in S.epidermidis all sequence types were different. These results reveal strains with the same evolutionary origin as other isolates, which might cause infections in humans and animals, suggesting their ability to spread between these species.

Detection of Biofilm Formation by Different Bacterial Isolates of Contact Lens

Background: Biofilms are groups of microorganisms that collect to each other and with different surfaces by adherence mechanisms. These are formed of cells and extracellular matrix manufactured by these cells. There may be a great problem in some situations e.g. on medical implants and resistance against antibiotics. Objective: The objective of this study is to determine biofilm forming power of bacteria isolated from the conjunctiva, contact lens and the lens storage case by both phenotypic and genotypic detection methods. Methodology: Samples were taken from (36) persons in the period from January 2020 to June 2020 at Ophthalmology Department, Tanta University Hospitals, all the samples were transported to the Medical Microbiology & Immunology Department, Tanta University where bacterial strains were isolated. The biofilm formation phenotypic detection was performed by both tube method and Congo red agar method. The biofilm-forming genes of coagulase negative Staphylococcus (CoNS) and Staphylococcus aureus (ica A) and that of P. aeruginosa (psl A), were detected by PCR. Results: The (216) samples (swabs & discarded lenses) gave rise to a total number of (247) bacterial isolates. By using tube method; (52.3%) were moderately positive, (31.5%) strongly positive and (16.2%) negative for biofilm formation while after using the Congo red agar method; (35.3%) were moderately positive, (38.4%) strongly positive and (26.3%) negative for biofilm formation. Regarding the Staphylococcus aureus isolates, two (50%) of these were containing (icaA) gene. Regarding the (21) CoNS isolates, three (14.3%) contained (icaA) gene. Although all of the Pseudomonas isolates didn't contain pslA (1119 bp) gene, these were positive for biofilm production by phenotypic methods. Conclusion: The majority of the isolates had the capacity to form biofilms. Both tube and Congo red agar methods showed clear significant correlation and detected a high number of biofilm-producing strains. The absence of genes responsible for biofilm formation did not exclude the phenotypic biofilm production by these bacteria which is a common state.

Evaluate the Effect of Zinc Oxide and Silver Nanoparticles on Biofilm and icaA Gene Expression in Methicillin-Resistant Staphylococcus aureus Isolated From Burn Wound Infection

Methicillin-resistant Staphylococcus aureus is the cause of nosocomial and community-acquired infections. This study aimed to evaluate the effect of zinc oxide and silver nanoparticles (ZnO-Ag NPs) on biofilms formation and icaA gene expression in methicillin-resistant S. aureus (MRSA). In this study, three standard strains (ATCC 43300, 25923, and 29913) and a clinical isolate are included. The minimum inhibitory concentration (MIC) of nanoparticles was determined by microdilution broth method. The antibacterial effects of ZnO-Ag NPs either alone or in combination with each other were compared with vancomycin (as the control group). The effect of MIC and sub-MIC concentrations of ZnO-Ag NPs on biofilm formation was determined by the microtiter plate method. The expression level of the icaA gene was assessed by real-time PCR LightCycler® 96 software (Version 1.1.0.1320, Roche, Germany). technique. All experiments were repeated three times. Data were analyzed using SPSS software through ANOVA and t-test. The P-value of less than .05 was considered as statistically significant. The average MICs of ZnO, Ag, and ZnO-Ag NPs compounds were 393.2, 179.8, and 60.8 μg/ml, respectively. The compound of ZnO-Ag NPs had a synergistic effect against all isolates. ZnO-Ag NPs decreased the biofilm formation rate at MIC and sub-MIC concentrations (P &lt; .001). Sub-MIC ZnO-Ag NPs concentration significantly reduced the icaA gene expression in S. aureus strains (P &lt; .03). The sub-MIC concentration of ZnO-Ag NPs reduced biofilm formation rate and icaA gene expression in Staphylococcus aureus strains compared with vancomycin. It can be used to cover medical devices after examining more clinical isolates to prevent bacterial colonization.

The Correlation between icaA and icaD Genes with Biofilm Formation Staphylococcus epidermidis In Vitro

This study was conducted to identify the presence of icaA and icaD genes in S. epidermidis and to analyze the relationship between the presence of icaA and icaD genes with the ability of in vitro biofilm formation in S. epidermidis. S. epidermidis isolates from patients and healthy people were collected and PCR was examined to detect icaA and icaD genes. which then continued to examine the ability of biofilm formation by the method of Congo Red Agar. The results of this genotypic and phenotypic examination were then tested for correlation with statistical tests using SPSS 23.0. A total of 40 S. epidermidis isolates were collected, consisting of 20 clinical isolates and 20 isolates of normal flora. The icaA gene was positive in 5 isolates (12.5%), and 8 isolates (20%) were positive for the icaD gene, 3 isolates with icaA and icaD were both positive. One hundred percent of isolates with icaA or icaD positively formed biofilms, but there were 15 isolates (42.9%) who did not have the icaA gene but showed the ability to form biofilms, while 12 isolates (37.5%) who did not have the icaD gene also formed biofilms. Fifty percent of S. epidermidis isolates showed the ability to form biofilms at CRA. The Fisher Exact test showed a significant relationship between the icaA gene and the ability of biofilm formation (p=0.047 (p<0.05)) as well as the icaD gene (p=0.03 (p<0.05)). The icaA and icaD genes have a significant relationship to biofilm formation in S. epidermidis. There was another mechanism in the formation of biofilms that are not dependent on the ica gene.

Association of intercellular adhesion gene A with biofilm formation in staphylococci isolates from patients with conjunctivitis

ABSTRACT BACKGROUND: There is a great negative impact of biofilm-mediated infection on patient health which necessitates the use of reliable methods for detecting biofilm producers. AIMS: This study was done to determine biofilm-producing ability and the presence of intercellular adhesion gene A in clinical staphylococcal isolates and to assess the reliability of two phenotypic methods used for biofilm detection. MATERIALS AND METHODS: Fifty staphylococcal strains were isolated from 100 conjunctival swabs from patients attended the Ophthalmology Outpatient Department of the Research Institute of Ophthalmology. Two phenotypic methods were used for detection of biofilm production; qualitative congo red agar (CRA); and quantitative microtiter plate. Polymerase chain reaction was used to determine the presence of icaA gene. RESULTS: In Staph aureus, 60% were positive biofilm forming and 40% were negative biofilm forming by both phenotypic methods. All positive biofilm-forming isolates were positive for icaA gene production. In coagulase negative staph, 50% were positive biofilm forming and 50% were negative biofilm forming by both phenotypic methods. All positive biofilm-forming strains were positive for icaA gene. All negative cases by CRA and microtiter plate methods were negative for icaA gene except two isolates. All staphylococcal isolates were subjected to antibiotic susceptibility test to correlate biofilm formation with multidrug resistance in staph. CONCLUSION: There is high significant correlation between icaA gene presence and biofilm forming ability; however, the biofilm-forming ability of some isolates in the absence of icaA gene highlights the importance of further genetic investigations of ica-independent biofilm formation mechanisms.