Congo Red Agar Plate Research Articles

Antimicrobial Resistance Profiles, Virulence Determinants, and Biofilm Formation in Enterococci Isolated from Rhesus Macaques (Macaca mulatta): A Potential Threat for Wildlife in Bangladesh?

Enterococci are commensal bacteria that inhabit the digestive tracts of animals and humans. The transmission of antibiotic-resistant genes through human-animal contact poses a potential public health risk worldwide, as zoonoses from wildlife reservoirs can occur on every continent. The purpose of this study was to detect Enterococcus spp. in rhesus macaques (Macaca mulatta) and to investigate their resistance patterns, virulence profiles, and biofilm-forming ability. Conventional screening of rectal swabs (n = 67) from macaques was followed by polymerase chain reaction (PCR). The biofilm-forming enterococci were determined using the Congo red agar plate assay. Using the disk diffusion test (DDT), antibiogram profiles were determined, followed by resistance and virulence genes identification by PCR. PCR for bacterial species confirmation revealed that 65.7% (44/67) and 22.4% (15/67) of the samples tested positive for E. faecalis and E. faecium, respectively. All the isolated enterococci were biofilm formers. In the DDT, enterococcal isolates exhibited high to moderate resistance to penicillin, rifampin, ampicillin, erythromycin, vancomycin, and linezolid. In the PCR assays, the resistance gene blaTEM was detected in 61.4% (27/44) of E. faecalis and 60% (9/15) of E. faecium isolates. Interestingly, 88.63 % (39/44) of E. faecalis and 100% (15/15) of E. faecium isolates were phenotypically multidrug-resistant. Virulence genes (agg, fsrA, fsrB, fsrC, gelE, sprE, pil, and ace) were more frequent in E. faecalis compared to E. faecium; however, isolates of both Enterococcus spp. were found negative for the cyl gene. As far as we know, the present study has detected, for the first time in Bangladesh, the presence of virulence genes in MDR biofilm-forming enterococci isolated from rhesus macaques. The findings of this study suggest employing epidemiological surveillance along with the one-health approach to monitor these pathogens in wild animals in Bangladesh, which will aid in preventing their potential transmission to humans.

Designing Alginate/Chitosan Nanoparticles Containing Echinacea angustifolia: A Novel Candidate For Combating Multidrug-resistant Staphylococcus aureus.

Nanoparticles (NPs) may help treat multidrug-resistant Staphylococcus aureus (MDR). This study prepared and evaluated chitosan/alginate-encapsulated Echinacea angustifolia extract against MDR strains. Evaluating synthesized NPs with SEM, DLS, and FTIR. Congo red agar and colorimetric plate techniques examined isolate biofilm formation. NP antibacterial power was assessed using well diffusion. Real-time PCR assessed biofilm-forming genes. MTT assessed the synthesized NPs' toxicity. According to DLS measurements, spherical E. angustifolia NPs had a diameter of 335.3 ± 1.43 nm. The PDI was 0.681, and the entrapment effectiveness (EE%) of the E. angustifolia extract reached 83.45 %. Synthesized NPs were most antimicrobial. S. aureus resistant to several treatments was 80 percent of 100 clinical samples. Biofilm production was linked to MDR in all strains. The ALG/CS-encapsulated extract had a 4 to 32-fold lower MIC than the free extract, which had no bactericidal action. They also significantly decreased the expression of genes involved in biofilm formation. E. angustifolia-encapsulated ALG/CS decreased IcaD, IcaA, and IcaC gene expression in all MDR strains (***p<0.001). Free extract, free NPs, and E. angustifolia-NPs had 57.5%, 85.5%, and 90.0% cell viability at 256 μg/ml. These discoveries could assist generate stable plant extracts by releasing natural-derived substances under controlled conditions.

Prevalence Study of Biofilm Marker ica D Gene among Coagulase Positive Staphylococcus of Mastitic Origin

Bovine Mastitis is an inflammation of the mammary gland, usually due to microbial infection, which causes economic loss to dairy producer every year. Staphylococcus is one of the most common pathogens responsible for contagious mastitis in bovines. The aim of this study was to identify coagulase positive Staphylococci isolated from bovine subclinical mastitis. In vitro biofilm forming ability of the isolates, an important virulence factor of mastitogen and prevalence of biofilm marker icaD gene among the isolates. In the present study, 70 isolates were biochemically identified as S. aureus. Biochemically characterized strains of S. aureus were screened for biofilm formation. Phenotypic detection of biofilm formation of S.aureus were done by two methods, namely, Congo Red Agar (CRA) method and Tube Method (TM). The frequency of biofilm formation in S. aureus strains was 28.57 per cent on CRA plates while 71.42 per cent were biofilm non-producers. The frequency of biofilm formation in S. aureus strains was 38.57 per cent in total as shown by tube method while 61.42 per cent were biofilm non-producers. Employing PCR, molecular detection technique of icaD gene was carried out. In this study, 72.85 per cent S. aureus strains were found to be positive for icaD gene which produced an amplicon of 381bp.

Virulence Factors of Clinical and Fecal Isolates of Enterococci Species

Enterococci species are known commensals of the gastrointestinal flora; however, in recent years, they have emerged as important nosocomial pathogens that possess many virulence factors that are attributed to the pathogenesis of diseases caused by them. The study evaluated and compared the virulence factors of Enterococci isolated from fecal and clinical samples. From the obtained isolates, the clinical enterococcal isolates produced 35%, 20%, and 50%, and fecal isolates produced 23%, 13%, and 13% gelatinase, hemolysin, and biofilm, respectively. Biofilm production determined by the Congo Red agar, tube, and microtiter plate methods was 23%, 39%, and 49%, respectively. The sensitivity of the Congo Red agar and tube method compared to the microtiter plate method was 27% and 46%, respectively, whereas the specificity of both tests was 79%. This study showed that biofilm production plays a significant role in the pathogenesis of diseases caused by Enterococci. Detection of biofilm production using the microtiter plate method is more sensitive and specific than the Congo Red agar and tube method.

Detection of biofilm formation in multi-drug resistant Staphylococcus aureus among clinical isolates in Zaria metropolis, Kaduna, Nigeria

Antimicrobial resistance are associated with several virulence factors such as biofilm. The biofilm formation ability of S. aureus complicates effective therapeutic management of infections. In this research, we intend to observe biofilm formation in S. aureus using two in vitro phenotypic methods: tissue culture plate and Congo red agar as well as biofilm associated genes Ica A B &amp; D and 16SrRNA. A total of 150 presumptive coagulase positive staphylococcal isolates from all specimens (blood, urine, high vaginal swab, wound swab, ear swab, endocervical swab) submitted to the microbiology laboratory of the selected hospitals were collected. Seventy-three (73) or 48.7% of the isolates were identified as coagulase positive Staphylococcus using microbiology standard methods (tube coagulase test, growth on mannitol salt agar &amp; Dnase agar). Using microgen staph identification kit, the isolates were further characterized into various Staph spp. Disc agar diffusion method was used for eleven (11) antibiotics susceptibility test while vancomycin screening agar and BMD (Broth Micro dilution) MIC test was used for vancomycin susceptibility evaluation. Indirect observation of biofilm was performed using congo red agar (CRA) and Microtitre plate assay method. PCR and sequencing were conducted on the isolates to molecularly detect the presence of 16SrRNA, IcaA, IcaB, IcaD genes. Susceptibility of S aureus (21%) isolates tested against 12 different categories of antibiotics shows 4(27%) not multidrug resistant (MDR), 7(47%) were MDR and 4 (27%) were extensively drug resistant (XDR). High percentage (74%) of Multiple antibiotic resistant index at ≥0.3 suggested that the isolates originated from an environment where antibiotics are often used. Biofilm assessment of MDR S aureus isolates revealed that 9(82%) and 11 (100%) of the isolates were biofilm formers using Congo Red Agar method and Microtiter plate assay respectively. All isolates amplified with 16SrRNA primer signifying all are S. aureus, 100% of the isolates amplified with IcaA, 90% isolates amplified with IcaD, and 50% isolates amplified with IcaB. We can conclude that there is high prevalence of Biofilm forming S. aureus strains in the hospitals studied. This result should be born in mind by clinicians while prescribing medication for S. aureus positive conditions. Hospital managements should be proactive in preventing the preservation of the biofilm formers in their hospital environment through adequate sanitary and disinfection protocols. It is also necessary for clinician to note that nitrofurantoin, linezolid and vancomycin were most effective against the biofilm producing S. aureus and microtitre plate assay method was observed to be most reliable assay in the study of biofilm.

MOLECULAR IDENTIFICATION OF ROSEOMONAS MUCOSA AND DETERMINATION THE SOME OF ITS VIRULENCE FACTORS

This study was aimed to characterize Roseomonas mucosa isolate using molecular genetics and determination some bacterial virulence factors such as capsular, biofilm formation and antimicrobial susceptibility. By using Polymerase chain reaction (PCR) technique, the molecular characterization of the R. mucosa was done by using 16S rRNA gene sequence and phylogenetic analysis. Antimicrobial susceptibility test was carried out by Kirby-Bauer disk diffusion method. Capsular detection was examined using nigrosin pigment. While Congo red agar and microtiter plate using enzyme-linked immunosorbent assay (ELISA) reader methods are used to determined bacterial isolate capability to produce a biofilm. The molecular characterization shows the bacterium that isolated from catheter of a dialysis Iraqi patient is belong to Roseomonas mucosa. The isolated R. mucosa strain showed resistant to Amoxicillin, methicillin, carbapenemes, bacitracin and Sulfamethoxazole. While it was susceptible to tobramycin, levofloxacin, colisitin sulphate, doxycycline, azithromycin and cephalexin. The results show R. mucosa was encapsulated. Furthermore, it has the ability to form weak biofilm. The findings suggest that in spite of this genus seems to have an overall little pathogenic potential for humans, Roseomonas sp. in particular, R. mucosa may be substantial pathogens in individuals with underlying medical complications.

The phenotypes and genotypes associated with biofilm formation among methicillin-susceptible Staphylococcus aureus (MSSA) isolates collected from a tertiary hospital in Terengganu, Malaysia.

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.

Chitosan/silver nanocomposites enhanced the biofilm eradication in biofilm forming Gram positive S. aureus

Biofilm is the important virulence factors in bacteria to develop multi drug resistant effect against existing bacteria word wide. The eradication of biofilm is the important research recent years due to the increased mortality rate. For this, the current research was focused on inhibition of biofilm producing bacteria S. aureus using chitosan/silver nanocomposites by various invitro experiments. The result of anti-biofilm activity was shown 26 mm zone of inhibition against gram positive S. aureus after treatment with chitosan/silver nanocomposites combination at 24 h incubation. In addition, the biofilm adherent ability in 24-well polystyrene well plate result was exhibited 96 % biofilm degradation and viewed by after crystal violet stain wash. Importantly, biofilm survival rate was decreased to 97 % at 400 µg/mL concentrations, and it confirmed by liquid maintenance method. Furthermore, the complete exopolysaccharide damages were observed at 400 µg/mL concentrations, and confirmed by congo red agar assay. In congo red agar plate, the black color was absent in the biofilm forming S. aureus after 24 h incubation. It conveyed that the synthesized Chitosan/silver nanocomposites was very efficient nanomaterial to eradicate the biofilm formation in gram positive bacteria at concentration dependent manner.

Phenotypic and Genotypic Identification of Typical and Atypical Staphylococcus aureus Isolated From Bovine Milk Samples

This study aimed to determine the phenotypic and genotypic identification and differentiation of typical coagulase positive Staphylococcus aureus (CPSA) and atypical coagulase negative Staphylococcus aureus (CNSA) from other Staphylococci isolated form bovine milk. Out of 111 milk samples, 67 Staphylococcus strains were isolated, phenotypically, based on resistance to Acriflavine; Staphylococcus aureus (S. aureus) was detected in 44 isolates of them (30 CPSA strains and 14 CNSA strains). Twenty five from CPSA and 5 from CNSA isolates were positive for slime production on Congo red agar plates. Genotypically, the all five tested typical Staphylococcus aureus (CPSA) as well as three atypical Staphylococcus aureus (CNSA) strains encoded all investigated genes (nuc, spa-x and clfA), while the other two atypical CNSA strains, one encoded only nuc gene and the other encoded both nuc and clfA genes. Acriflavine sensitivity test must be included in the routine of phenotypic identification S. aureus as the gold method together with tube coagulase test. PCR analysis is most important confirmation method by detection of nuc gene. Genotypically both typical and atypical S. aureus isolates are virulent. Typical and atypical S. aureus isolated from isolated from bovine milk samples sold in dairy shops were with higher percentage than subclinical mastitic milk samples threating public health hazard. Attention must be paid toward detection and identification of atypical tube coagulase negative S. aureus strains.

Biosynthesis of Gold Nanoparticles and Its Effect against Pseudomonas aeruginosa.

Antimicrobial resistance has posed a serious health concern worldwide, which is mainly due to the excessive use of antibiotics. In this study, gold nanoparticles synthesized from the plant Tinospora cordifolia were used against multidrug-resistant Pseudomonas aeruginosa. The active components involved in the reduction and stabilization of gold nanoparticles were revealed by gas chromatography-mass spectrophotometry(GC-MS) of the stem extract of Tinospora cordifolia. Gold nanoparticles (TG-AuNPs) were effective against P. aeruginosa at different concentrations (50,100, and 150 µg/mL). TG-AuNPs effectively reduced the pyocyanin level by 63.1% in PAO1 and by 68.7% in clinical isolates at 150 µg/mL; similarly, swarming and swimming motilities decreased by 53.1% and 53.8% for PAO1 and 66.6% and 52.8% in clinical isolates, respectively. Biofilm production was also reduced, and at a maximum concentration of 150 µg/mL of TG-AuNPs a 59.09% reduction inPAO1 and 64.7% reduction in clinical isolates were observed. Lower concentrations of TG-AuNPs (100 and 50 µg/mL) also reduced the pyocyanin, biofilm, swarming, and swimming. Phenotypically, the downregulation of exopolysaccharide secretion from P. aeruginosa due to TG-AuNPs was observed on Congo red agar plates.

Biofilm formation, antibiotic resistance and genotyping of Shiga toxin-producing Escherichia coli isolated from retail chicken meats

ABSTRACT 1. The Shiga toxin-producing Escherichia coli (STEC) is a hazardous zoonotic agent for chicken meat consumers. This study determined the serogroups and evaluated the virulence genes, antibiotic resistance, biofilm-forming profiles and genetic relationships of STEC isolates in chicken meat. 2. A total of 100 samples belonging to dressed-whole chicken and different parts of the chicken (wing, breast, thigh, drumstick) were collected between September and November 2019 from different retail markets in Kayseri, Türkiye. 3. Phenotypic (identification, disc diffusion test, Congo red agar and microtitre plate tests) and molecular tests (identification, serogrouping, virulence factors, biofilm, antibiotic susceptibility, 16S rRNA sequencing and enterobacterial repetitive intergenic consensus-PCR for typing of the isolates) were carried out. 4. E. coli was isolated from 35% of the samples and 35% of the samples harboured at least one STEC. Among 35 STEC isolates, 3 (8.5%), 6 (17.1%), 2 (5.7%) and 3 (8.5%) were found to be positive for fliCH2, fliCH8, fliCH11, fliCH19 genes, respectively. Out of 35 STEC positive isolates, 4 (11.4%) were identified as E. coli O157, from which 2 (5.7%) were E. coli O157:H7. E. coli O157 was detected in two (10%), one (5%), one (5%) of the thigh, drumstick and whole chicken samples, respectively. 5. Biofilm-forming ability was reported in 33 (94.2%) of 35 E. coli isolates, whilst the biofilm-associated genes detected among 35 STEC isolates included csgA (88.5%), fimH (88.5%), bcsA (85.7%), agn43 (14.2%) and papC (8.5%). The STEC strains showed resistance against ampicillin (88.5%) and erythromycin (88.5%), followed by tetracycline (74.2%) and gentamicin (25.7%). However, the distribution of isolates harbouring bla CMY, ere(A), tet(A) and aac(3)-IV antibiotic resistance genes was found to be 17.1%, 11.4%, 85.7% and 5.7%, respectively. 6. ERIC-PCR showed that E. coli strains obtained from different parts and whole of chicken samples had genetic diversities. ERIC-PCR patterns grouped strains of 35 STEC into eight clusters designated A-H, with 73% similarity. Proper hygiene measures and staff training are essential for public health during poultry processing and in retail stores to control STEC.

Incidence of biofilm formation among MRSA and MSSA clinical isolates from hospitalized patients in Israel.

AimTo assess the biofilm‐producing capacities of Staphylococcus aureus strains isolated from hospitalized patients in Israel.Methods and ResultsA total of 16 S. aureus (80 MRSA and 83 MSSA) from screening (nasal swab) and clinical samples (blood and wounds) were characterized. Biofilm‐producing capacities were determined using two different biofilm detection assays: Congo Red agar (CRA) and microtiter plate (MtP). In addition, a real‐time PCR analysis was performed to detect the presence of biofilm‐associated genes (icaA and icaD) and mecA gene. The two assays showed similar biofilm production pattern (28.2% agreement). MRSA strains tended to be greater biofilm‐producers than MSSA strains. The presence of mecA was associated with biofilm production (p = 0.030). Additionally, bacteria isolated from blood samples produced less biofilm compared to those from other sources. Finally, no association was found between icaA and icaD presence and biofilm production.ConclusionThis study supports earlier assumptions that biofilm formation depends strongly on environmental conditions.Significance and Impact of StudyThis study significantly improved our knowledge on the biofilm production capacity of S. aureus strains in Israel. Moreover, it revealed an association between the mecA gene and biofilm production. Finally, this study underscores the importance of further research to evaluate risk factors for biofilm production.

Isolation and molecular identification of cellulose/hemicellulose degrading bacteria from agricultural compost and determination of their hydrolytic potential

Nine cellulolytic bacteria were isolated from agricultural residues compost on selective culture medium amended with cellulose (filter paper) as the sole carbon source. Five of the nine isolates (CX2, CX4, CX5, CX7 and CX9) were able to degrade both cellulose (CMC) and xylan in congo red agar plates. The five isolates were able to depolymerize the agricultural wastes, rice straw and sugarcane bagasse. CMC-ase and xylanase production by the five isolates were determined on rice straw and sugarcane bagasse. CX2 and CX9 exhibited the highest CMC-ase and xylanase production on both rice straw and sugarcane bagasse. Among the isolated strains, CX9 produced the highest CMC-ase activity (71 pKat ml−1) on rice straw, whereas CX9 produced the highest xylanase activity (7.83 nKat ml−1) on sugarcane bagasse. In general, higher CMC-ase and xylanase production was obtained on rice straw compared with sugarcane bagasse. According to the sequences of the 16 s rRNA genes, the five isolates were identified as: Paracoccus kondratievae strain GB (CX2 and CX4), Paracoccus communis (CX5), Bacillus australimaris (CX7) and Bacillus pumilus (CX9). The sequences of the 16S rRNA genes of five isolates were submitted to the gene bank.

Phenotypic and Genotypic Detection of Biofilm-Forming Staphylococcus aureus from Different Food Sources in Bangladesh.

Simple SummaryBiofilm formation by Staphylococcus aureus in foods poses a potential concern for public health and food safety. Therefore, the present study was conducted to detect biofilm-producing S. aureus from foods and human hand swabs using phenotypic and genotypic assays. In this study, S. aureus was detected in 23.81% (100/420) of samples, and among them, 89 and 97 of the isolates were biofilm producers by qualitative and quantitative tests, respectively. At least one biofilm-forming gene was detected in 21 S. aureus isolates, of which four isolates harbored all five adhesion genes (icaA, icaB, icaC, icaD, and bap). In addition, the occurrence of adhesion genes in S. aureus isolates showed a strong significant correlation among themselves. This is the first report on detecting biofilm-forming S. aureus from foods and hand swabs in Bangladesh using the molecular technique. The findings from this study indicate a significant public health risk and suggest the necessity of maintaining food hygiene practices at every step of the food chain to prevent and control S. aureus foodborne illness.Staphylococcus aureus is a major foodborne pathogen. The ability of S. aureus to produce biofilm is a significant virulence factor, triggering its persistence in hostile environments. In this study, we screened a total of 420 different food samples and human hand swabs to detect S. aureus and to determine their biofilm formation ability. Samples analyzed were meat, milk, eggs, fish, fast foods, and hand swabs. S. aureus were detected by culturing, staining, biochemical, and PCR. Biofilm formation ability was determined by Congo Red Agar (CRA) plate and Crystal Violet Microtiter Plate (CVMP) tests. The icaA, icaB, icaC, icaD, and bap genes involved in the synthesis of biofilm-forming intracellular adhesion compounds were detected by PCR. About 23.81% (100/420; 95% CI: 14.17–29.98%) of the samples harbored S. aureus, as revealed by detection of the nuc gene. The CRA plate test revealed 20% of S. aureus isolates as strong biofilm producers and 69% and 11% as intermediate and non-biofilm producers, respectively. By the CVMP staining method, 20%, 77%, and 3% of the isolates were found to be strong, intermediate, and non-biofilm producers. Furthermore, 21% of S. aureus isolates carried at least one biofilm-forming gene, where icaA, icaB, icaC, icaD, and bap genes were detected in 15%, 20%, 7%, 20%, and 10% of the S. aureus isolates, respectively. Bivariate analysis showed highly significant correlations (p < 0.001) between any of the two adhesion genes of S. aureus isolates. To the best of our knowledge, this is the first study in Bangladesh describing the detection of biofilm-forming S. aureus from foods and hand swabs using molecular-based evidence. Our findings suggest that food samples should be deemed a potential reservoir of biofilm-forming S. aureus, which indicates a potential public health significance.

Green synthesis and characterization of silver nanoparticles using Eugenia roxburghii DC. extract and activity against biofilm-producing bacteria

The green synthesis of silver nanoparticles (AgNPs) and their applications have attracted many researchers as the AgNPs are used effectively in targeting specific tissues and pathogenic microorganisms. The purpose of this study is to synthesize and characterize silver nanoparticles from fully expanded leaves of Eugenia roxburghii DC., as well as to test their effectiveness in inhibiting biofilm production. In this study, at 0.1 mM concentration of silver nitrate (AgNO3), stable AgNPs were synthesized and authenticated by monitoring the color change of the solution from yellow to brown, which was confirmed with spectrophotometric detection of optical density. The crystalline nature of these AgNPs was detected through an X-Ray Diffraction (XRD) pattern. AgNPs were characterized through a high-resolution transmission electron microscope (HR-TEM) to study the morphology and size of the nanoparticles (NPs). A new biological approach was undertaken through the Congo Red Agar (CRA) plate assay by using the synthesized AgNPs against biofilm production. The AgNPs effectively inhibit biofilm formation and the biofilm-producing bacterial colonies. This could be a significant achievement in contending with many dynamic pathogens.

No Correlation between Biofilm-Forming Capacity and Antibiotic Resistance in Environmental Staphylococcus spp.: In Vitro Results.

The production of biofilms is a critical factor in facilitating the survival of Staphylococcus spp. in vivo and in protecting against various environmental noxa. The possible relationship between the antibiotic-resistant phenotype and biofilm-forming capacity has raised considerable interest. The purpose of the study was to assess the interdependence between biofilm-forming capacity and the antibiotic-resistant phenotype in 299 Staphylococcus spp. (S. aureus n = 143, non-aureus staphylococci [NAS] n = 156) of environmental origin. Antimicrobial susceptibility testing and detection of methicillin resistance (MR) was performed. The capacity of isolates to produce biofilms was assessed using Congo red agar (CRA) plates and a crystal violet microtiter-plate-based (CV-MTP) method. MR was identified in 46.9% of S. aureus and 53.8% of NAS isolates (p > 0.05), with resistance to most commonly used drugs being significantly higher in MR isolates compared to methicillin-susceptible isolates. Resistance rates were highest for clindamycin (57.9%), erythromycin (52.2%) and trimethoprim-sulfamethoxazole (51.1%), while susceptibility was retained for most last-resort drugs. Based on the CRA plates, biofilm was produced by 30.8% of S. aureus and 44.9% of NAS (p = 0.014), while based on the CV-MTP method, 51.7% of S. aureus and 62.8% of NAS were identified as strong biofilm producers, respectively (mean OD570 values: S. aureus: 0.779±0.471 vs. NAS: 1.053±0.551; p < 0.001). No significant differences in biofilm formation were observed based on MR (susceptible: 0.824 ± 0.325 vs. resistant: 0.896 ± 0.367; p = 0.101). However, pronounced differences in biofilm formation were identified based on rifampicin susceptibility (S: 0.784 ± 0.281 vs. R: 1.239 ± 0.286; p = 0.011). The mechanistic understanding of the mechanisms Staphylococcus spp. use to withstand harsh environmental and in vivo conditions is crucial to appropriately address the therapy and eradication of these pathogens.

Biofilm-forming ability and methicillin resistance among Staphylococci isolated from various surfaces in community settings

Biofilm-forming ability and sensitivity to antibiotic methicillin of staphylococci outside the healthcare facilities is not sufficiently explored. The aim of this study was to examine the biofilm-forming capacity of staphylococci on various metal, plastic and leather surfaces in our living and working environment and the association between their biofilm production and methicillin resistance. Detection of methicillin resistance in staphylococci isolates were examined by cefoxitin disc diffusion test. Production of bacterial biofilms was investigated by two phenotypic methods: crystal violet (CV) tube-adherence method and Congo red agar (CRA) plate method. A total of 88 staphylococci isolates, 47 (53.41%) of S. aureus and 41 (46.59 %) of S. epidermidis strains were recovered from 100 collected samples. Among them, 35 methicillin-resistant strains (20 MRSA and 15 MRSE) were detected. Applying the CV tube adherence method, 21 (39.62%) MRSA and MRSE isolates were positive for biofilm production, while using the CRA method 15 (44.12%) MRSA and MRSE strains were detected as biofilm producers. Statistical analysis did not find association between biofilm production ability and methicillin resistance in both applied methods for a significance level of 0.05. The study shows that different objects and surfaces contaminated with biofilm-forming staphylococci, including MRSA and MRSE, may serve as environmental source of infection for long periods of time.

Antibiofilm activity of berberine and capsaicin in combination with quinolones against Staphylococcus aureus from bovine mastitis

Antimicrobial resistance which causes failure of antibiotic therapy has become a serious global issue nowadays. Bacteria develop resistance towards an antibacterial agent via many mechanisms. Biofilm formation by bacteria poses severe threat to the treatment of infections. The combination of plant molecules with antibiotics can combat antimicrobial resistance. In the present study, antibiofilm activity of two plant molecules berberine and capsaicin was investigated against Staphylococcus aureus isolates from bovine mastitis cases. Antibiotic sensitivity testing revealed that berberine and capsaicin when combined with quinolones increased the diameter of zone of inhibition in a dose dependent manner. In vitro biofilm assay using congo red agar plate method revealed the antibiofilm activity of berberine and capsaicin in combination with quinolone antibiotics. The study concludes that the combination of phytoconstituents with antibiotics can alleviate resistance mechanisms in bacteria.

Extended-spectrum β-lactamase Enzymes (ESBLs) and Slime Production of some Gram-negative Bacilli Isolated from Human, Animal and Environmental Sources in Port Harcourt, Nigeria

Aim: To determine extended-spectrum β-lactamase enzymes (ESBLS) and slime production of some Gram-negative Bacilli isolated from human, animal and environmental sources in Port Harcourt, Nigeria&#x0D; Study design: Cross-sectional study.&#x0D; Place and Duration of Study: University of Port Harcourt Teaching Hospital, Trans-Amadi and Rumuodomaya Slaughters and Bob-D Ventures Poultry Farms, all in Port Harcourt, Nigeria, between July and December, 2019.&#x0D; Methodology: This study was conducted at Port Harcourt and Obio/Akpor local government areas of Rivers State, Nigeria from 2019-2020. Hospital wastewater was taken from different sections at the two University Teaching Hospitals- University of Port Harcourt and Rivers State University Teaching Hospitals. Abattoir effluent water samples were taken at different sites from Trans-Amadi and Rumuodumaya Abattoirs. Chicken cloaca samples as well as Hand swab samples of Butchers were collected at the two Abattoirs. All samples were processed following standard procedures and identified organisms were assessed for susceptibilities to different antibiotics following Kirby-Bauer disk diffusion and Microbroth dilution methods. Isolates from the susceptibility testing with zone diameters found to be resistant to the Cephalosporins were suspected to be ESBL Positive (β-lactamase positive isolates). All isolates were also subjected to detection of slime production by Congo red agar plate method. Data were analyzed using SPSS version 22.0. Percentages and Chi square were used to summarize the data and p values less than 0.05 were considered significant.&#x0D; Results: The results showed that out of the 224 isolates, 38 (17.0%) were ESBL-producing. ESBL-producers were more frequently isolated from Hospital Wastewaters (26.3%), followed by isolates from Poultry dung samples 12(24.0%) and Abattoir Effluent waters (15.0%). No ESBL-producing bacteria was recovered from the Butchers’ Hand swabs. Again, of the 38 ESBL-producing organisms, E. coli accounted for 24 (63.2%), followed by Klebsiella spp. 8 (21.1%), while Pseudomonas sp. recorded 6 (15.8%). Of the 224 isolates, 98 (43.8) were slime-producing, while 23 (60.5%) of the 38 ESBL-producing isolates produced slime.&#x0D; Conclusion: ESBL and slime producing organisms (mostly E. coli) were more frequently isolated from Hospital Wastewaters compared to other sample areas.

Clonal groups of extended-spectrum β-lactamase and biofilm producing uropathogenic Escherichia coli in Iran

ABSTRACT Pathogenicity of a bacterium is affected by the social characteristics of the population and environmental factors. The ability of biofilm formation among β-lactamase-producing uropathogenic Escherichia coli (UPEC) could facilitate the exchange of antibiotic-resistance genes, which resulted in widespread dissemination of antibacterial drug resistance. We investigated the prevalence of biofilm and β-lactamase producing UPECs among patients with urinary tract infection (UTI) in two cities with different demographics and climates in Iran. A total of 265 E. coli was isolated from patients with UTIs from two referral hospitals (n = 191) and two outpatient clinics (n = 74) in Isfahan and Zahedan, Iran. Production of curli and cellulose, and, biofilm formation was investigated using Congo red agar and microtiter plate methods, respectively. Biofilm producing (BFP) isolates (n = 107) were further characterized using rep-PCR, antimicrobial susceptibility testing and extended-spectrum β-lactamase (ESBL)/AmpC phenotypic production. Isolates were also screened for the presence of carbapenemase, ESBL and AmpC genes using multiplex PCR. High diversity was found among BFP strains in both cities, with 58% strains producing ESBL and 21% producing AmpC. ESBL (98%), AmpC (50%) and carbapenemase genes (40%) were identified in BFP strains with ESBL-positive phenotype, respectively. The prevalence of BFP strains, antibiotic resistance and β-lactamase genes in Zahedan, a low socioeconomic city with a warm climate, was significantly higher than that of Isfahan. High prevalence of biofilm and β-lactamase producing UPEC strains among strains from Zahedan suggests that socioeconomic status and environmental factors might have a role in pathogenicity of the strains.