Non-biofilm Producers Research Articles

Quinolone resistance phenotype and genetic characterization of Salmonella enterica serovar Pullorum isolates in China, during 2011 to 2016

BackgroundPullorum disease, caused by Salmonella enterica serovar Pullorum (S. Pullorum), is one of the most important bacterial infections in the poultry industry in developing countries, including China. To examine the prevalence and characteristics of S. Pullorum, the Multilocus Sequence Typing (MLST) genotypes, fluoroquinolones resistance, and biofilm-forming abilities of S. Pullorum isolates were investigated, collected from 2011 to 2016 in China.ResultsThirty S. Pullorum isolates collected from 2011 to 2016 were analyzed. Quinolones susceptibility testing showed that 90% of the isolates were resistant to the first generation of quinolines nalidixic acid, but the resistance rates to different fluoroquinolones agents were lower than 13.3%; for some there was even no resistance. Multilocus sequence typing (MLST) showed that ST-92 was the dominating genotype, accounting for 90.0% of all S. pullorum strains. The remaining three isolates were of the new reported sequence type ST-2151. Interestingly, the Asp87Gly substitution in quinolone resistance-determining regions (QRDR) of GyrA was only observed in the three strains of ST-2151, suggesting a potential correlation between Asp87Gly substitution and sequence type (p < 0.05). However, Asp87Gly substitution could not confer the resistant to ofloxacin and ciprofloxacin of these isolates. The plasmid-mediated quinolone resistance (PMQR) gene was not found in any of the tested isolates. Furthermore, an assay measuring biofilm-forming abilities showed that 46.7% of the isolates were non-biofilm producers, while 53.3% could form very weak biofilms, which might explain the relatively lower resistance to fluoroquinolones.ConclusionsWe reported a high resistance rate to the first generation of quinolines nalidixic acid and relatively low resistance rates to fluoroquinolones in S. Pullorum isolates. In addition, weak biofilm-forming abilities were found, which might be an important reason of the low fluoroquinolones resistance rates of S. Pullorum isolates. ST-92 was the dominating genotype demonstrated by MLST, and the new sequence type ST-2151 showed a potential correlation with Asp87Gly substitution in QRDR of GyrA. We believe the characterization of these S. Pullorum isolates will be helpful to develop prevention and control strategies.

Vancomycin Resistant Staphylococcus aureus Reported from Tertiary Care Hospital in Nepal

Objectives: The study was conducted to assess the rate of Methicillin-resistant Staphylococcus aureus (MRSA) among patients and healthcare personnel at Manmohan Memorial College and Teaching Hospital, Kathmandu, Nepal and to evaluate the minimum inhibitory concentration of Vancomycin to MRSA isolates.Methods: A total of 1433 different clinical specimens from patients and 33 nasal swabs from healthcare personnel were subjected to bacteriological investigation following standard protocol. S. aureus were isolated and identified by using standard Microbiological tools. Those isolates were subjected to Antimicrobial susceptibility testing using modified Kirby-Bauer’s disc diffusion method following CLSI guidelines.Results: The rate of S. aureus carriage was found to be 65 (18.9%) in the samples from clinical patients and 24 (72.7%) in the samples from healthcare personnel. The rate of MRSA was found to be 57(85.1%) in patients and 24 (100%) in healthcare personnel. The high distribution of MRSA was found in female of age group 21-30 years (patients: 10.4%; healthcare personnel: 70.8%). Amikacin was found to be most effective antimicrobial. All S. aureus isolates were found to be multidrug resistant (100%). On performing D-test, 10 (17.5%) and 22 (38.6%) of MRSA from clinical specimens showed inducible and constitutive Clindamycin resistance respectively. Whereas, 11 (45.8%) and 4 (16.7%) of MRSA from nasal swabs were found to be inducible and constitutive Clindamycin resistance respectively. Upon performing minimum inhibitory concentration (MIC) test for clinical isolates, 3.5% (2) of MRSA were found to be Vancomycin resistant (VRSA), 54.4% (31) were Vancomycin intermediate (VISA) and 42.1% (24) were found to be Vancomycin sensitive (VSSA). All of the nasal swab MRSA isolates were found sensitive to Vancomycin. Congo red agar method was done for biofilm production. For clinical isolates, 32 (47.8%) were found to be strong, 6 (8.9%) moderate and 29 (43.3%) were non biofilm producer. For nasal swab isolates, 66.7% (16) and 33.3% (8) were found as strong and non-biofilm producer respectively.Conclusion: This study reported the case of VRSA which hasn’t been reported in Nepal. Though present study showed that Vancomycin remains the main choice of treatment of MRSA infection. Therefore, to preserve its value, use of vancomycin should be limited only to those cases where there are clearly needed.

Relationships between Virulence Factors and Antimicrobial Resistance among Escherichia coli Isolated from Urinary Tract Infections and Commensal Isolates in Tehran, Iran.

ObjectivesUropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs). Here, we determined whether sensitivity to antibiotics was related to the prevalence of iron scavenging genes, or to biofilm and hemolysis formation.MethodsA total of 110 UPEC and 30 E coli isolates were collected from the urine of UTI patients and feces of healthy individuals without UTI, respectively. The presence of iron receptor genes and phenotypic properties were evaluated by polymerase chain reaction and phenotypic methods, respectively. Susceptibility to routine antibiotics was evaluated using the disc diffusion method.ResultsThe prevalence of iron scavenging genes ranged from 21.8% (ireA) to 84.5% (chuA) in the UPEC. Resistance to ceftazidime and cefotaxime was significantly correlated with the presence of fyuA and iutA iron genes. Biofilm production was significantly associated with the prevalence of fyuA and hma iron genes. A higher degree of antibiotic resistance was exhibited by isolates that produced biofilms than by their non-biofilm producing counterparts.ConclusionOur study clearly indicates that biofilm production is associated with antibiotic resistance, and that iron receptors and hemolysin production also contribute to reduced antibiotic sensitivity. These results further our understanding of the role that these virulence factors play during UPEC pathogenesis, which in turn may be valuable for the development of novel treatment strategies against UTIs.

A steam-based method to investigate biofilm

Biofilm has become a major topic of interest in medical, food, industrial, and environmental bacteriology. To be relevant, investigation of biofilm behavior requires effective and reliable techniques. We present herein a simple and robust method, adapted from the microplate technique, in which steam is used as a soft washing method to preserve biofilm integrity and to improve reproducibility of biofilm quantification. The kinetics of steam washing indicated that the method is adapted to remove both planktonic bacteria and excess crystal violet (CV) staining for S. aureus, S. epidermidis, S. carnosus, P. aeruginosa, and E. coli biofilm. Confocal laser scanning microscopy confirmed that steam washing preserved the integrity of the biofilm better than pipette-based washing. We also investigated the measurement of the turbidity of biofilm resuspended in phosphate-buffered saline (PBS) as an alternative to staining with CV. This approach allows the discrimination of biofilm producer strains from non-biofilm producer strains in a way similar to CV staining, and subsequently permits quantification of viable bacteria present in biofilm by culture enumeration from the same well. Biofilm quantification using steam washing and PBS turbidity reduced the technical time needed, and data were highly reproducible.

Detection of Biofilm Formation in Pseudomonas aeruginosa Isolates from Clinical Specimens

The ability of a microorganism to develop biofilm is considered as a marker of clinically relevant infection. Infections caused by P. aeruginosa are difficult to treat as the majority of isolates exhibit high level of innate resistance to many antibiotics and tendency to form biofilms. The study was undertaken to investigate biofilm-forming capabilities of P. aeruginosa isolated from clinical specimens by two main different methods tissue culture plate (TCP) method, and congo red agar (CRA) method. Further, to investigate the antimicrobial resistance profile among biofilm producing isolates. A total of 300 specimens were collected from patients. Ninety six isolates of P. aeruginosa were obtained from various clinical samples. We applied Vitek-2 automated system as a panel of antimicrobial agents. TCP method and CRA method assay was chosen to detect the biofilm formation. Out of the 96 isolates, the results by TCP method and CRA method were 84(87.5%) and 76(79.1%) isolates respectively. TCP method was the most sensitive method for detection of biofilm production. TCP method detected 78 as strong, 6 as moderate and 12 as weak or non- biofilm producers. By CRA method, 65,11and 20 of isolates were strong, moderate and weak or non-biofilm producers, respectively. The antibiotic resistance pattern of P. aeruginosa was found higher in biofilm producers than in biofilm non-producers.TCP method was considered as the gold standard and reliable method for detection of biofilm formation. We conclude that there has been a positive association between drug resistance and biofilm formation of P. aeruginosa.

Catheter-Associated Urinary Tract Infection and Obstinate Biofilm Producers.

Background Biofilms, or colonies of uropathogen growing on the surface of indwelling medical devices, can inflict obstinate or recurring infection, thought-provoking antimicrobial therapy. Methods This prospective analysis included 105 urine samples from catheterized patients receiving intensive care. Ensuing phenotypic identification, antibiotic sensitivity test was performed by modified Kirby–Bauer disc diffusion method following CLSI guidelines; MDR isolates were identified according to the combined guidelines of the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC). Biofilm-forming uropathogens were detected by the tissue culture plate (TCA) method. Results The predominant uropathogen in catheter-associated UTIs (CAUTIs) was Escherichia coli 57%, followed by Klebsiella pneumonia 15%, Pseudomonas aeruginosa 12%, Staphylococcus aureus 8%, Enterobacter spp. 3%, Enterococcus faecalis, Acinetobacter spp., and Proteus mirabilis 1.5%, of which 46% isolates were biofilm producers. Prime biofilm producers were Escherichia coli 33%, followed by Klebsiella pneumoniae 30%, Pseudomonas aeruginosa 20%, Staphylococcus aureus 10%, Acinetobacter, and Enterobacter 3.33%. Multidrug resistance associated with biofilm producers were greater than biofilm nonproducers. The Gram-negative biofilm producers found 96.15%, 80.76%, 73.07%, 53.84%, 53.84%, 46.15%, 19.23%, and 11.5% resistant to amoxyclave, ceftazidime, tetracycline, gentamicin, meropenem, nitrofurantoin, amikacin, imipenem, and fosfomycin, respectively. Gram-positive biofilm producers, however, were found 100% resistant to tetracycline, cloxacillin, and amoxyclave: 66.67% resistant to ampicillin while 33.33% resistant to gentamicin, ciprofloxacin, and nitrofurantoin. Conclusion High antimicrobial resistance was observed in biofilm producers than non-biofilm producers. Of recommended antimicrobial therapies for CAUTIs, ampicillin and amoxicillin-clavulanate were the least active antibiotics, whereas piperacillin/tazobactam and imipenem were found as the most effectual for gram-negative biofilm producer. Likewise, amoxicillin-clavulanate and tetracycline were the least active antibiotics, whereas vancomycin, fosfomycin, piperacillin-tazobactam, and meropenem were found as the most effective antibiotic for Gram-positive biofilm producer. In the limelight, the activity fosfomycin was commendable against both Gram-positive and Gram-negative biofilm producers.

Detection of biofilm formation by Escherichia coli with its antibiogram profile

Background: In urinary tract infections, an important role is played by bacterial biofilms which are responsible for persistence infections together with the antimicrobial resistance. Higher resistance can be seen in biofilm forming uropathogens in comparison with free-floating bacteria. So, the present study was performed with a goal to find the prevalence of biofilm formation and also the antimicrobial resistant pattern of uropathogens.Methods: A descriptive method was conducted at Modern Technical College, Sanepa, Lalitpur in samples isolated from UTI suspected patients. The overall duration of this study was approximately 3 months. Total of 50 isolated E. coli was tested for biofilm formation and antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion method on Mueller Hinton agar as per CLSI guidelines.Results: From the 50 isolates of E. coli, 32 were biofilm producers (3 strong and 29 moderate) and 18 were weak/non-biofilm producers. Among the biofilm producers, cefotaxime was more resistant in 20 of the isolates followed by ceftriaxone in 16 and amoxyclav in 13, whereas amikacin was least resistant in 2 of the isolates.Conclusions: Among the isolated E. coli, biofilm-forming isolates showed higher antimicrobial resistance as compared to the non-biofilm producer. Thus, uropathogen should be routinely screened for biofilm formation.

Biofilm production and its correlation with antibiotic resistance pattern among clinical isolates of Pseudomonas aeruginosa in a tertiary care hospital in north-east India

Background: Pseudomonas aeruginosa is an ubiquitous pathogen capable of surviving in a variety of environmental conditions. It is increasingly gaining importance as a multidrug resistant nosocomial pathogen. Biofilm acts as a barrier, reducing the penetration of these drugs and consequently, preventing them from exercising their actions. The aim of this study is to isolate and identify Pseudomonas aeruginosa from various clinical specimen and to find out their production of biofilms and their correlation with antibiotic susceptibility pattern.Methods: All Pseudomonas aeruginosa over a period of 1 year were isolated and identified from clinical specimens and antibiotic susceptibility test was done following standard operative procedures. Biofilm detection was done by Congo Red Agar method (CRA).Results: 134 isolates of Pseudomonas aeruginosa was isolated. Maximum isolates were isolated from sputum samples 55 (41%) and most were from wards 68 (51%) giving a probability of increased healthcare associated infections. Biofilm production by the isolates was seen in 39 (29%). All the biofilm producing isolates shows more resistant pattern in comparison to non-biofilm producers. 69% of Imipenem and 82% of Meropenem resistant isolates produce biofilm. All the P. aeruginosa including MDR and biofilm forming strains were sensitive to Colistin.Conclusions: Resistance to antimicrobial agents is the most important feature of biofilm infections. Ability of P. aeruginosa to form biofilms renders antibiotic treatments inefficient and therefore promotes chronic infectious diseases. As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate.

Biofilm testing of microbiota: An essential step during corneal scrap examination in Egyptian acanthamoebic keratitis cases

PurposeTo detect co-infections in the culture-proven acanthamoebic keratitis (AK) cases, and to test the capability of biofilm formation in the isolated microbiota. The clinical findings, habit of wearing contact lens and in-vitro antibiotic resistance were analyzed further according to the biofilm formation capability. MethodsAfter clinical examination, corneal scraps and swabs were taken from 240 clinically suspected AK cases, for Acanthamoeba and microbiological cultures. In cases of keratoplasty, trimmed corneal tissue was collected and sent for histopathological examination. Scanning electron microscopy was done for some samples. Biofilm formation capability was investigated using a tissue culture plate method. Antibiotic resistance pattern was determined using a modified-Kirby-Bauer disc diffusion method. ResultsIn 102 AK culture proven cases, 11 had no co-infection, 74 had a single co-infection and 17 had double co-infections. Enterobactericae and Aspergillus were the commonest bacterial and fungal isolates, respectively. Regarding the biofilm formation, 64.7% of Enterobactericae, 50% of Pseudomonas aeuroginosa, 43.75% of Staph aureus, 76.92% of Streptococcus pneumoniae, 28.57% of Corynebacterium, 60% of α-haemolytic streptococci, 40% of Acinetobacter, 100% of Candida and 77.8% Aspergillus isolates were biofilm producers. Severe manifestations were more frequently reported in cases co-infected with biofilm producers than with non-biofilm producers. Generally, high percentages of the biofilm forming bacterial isolates were sensitive to antibiotics in-vitro. ConclusionsRoutine investigations for co-infection and biofilm formation in addition to Acanthamoeba culture are strongly recommended in suspected AK cases. Co-infection with biofilm producers may precipitate extrinsic in-vivo drug resistance despite of the in-vitro sensitivity. Designing a biofilm-dissolving topical drug is highly recommended to enhance the response to the standard therapeutic regimen especially in the resistant AK cases.

Appraisal of Biofilm Formation in Diabetic Foot Infections by Comparing Phenotypic Methods With the Ultrastructural Analysis

Diabetic patients are more prone to the development of foot ulcers, because their underlying tissues are exposed to colonization by various pathogenic organisms. Hence, biofilm formation plays a vital role in disease progression by antibiotic resistance to the pathogen found in foot infections. The present study has demonstrated the correlation of biofilm assay with the clinical characteristics of diabetic foot infection. The clinical characteristics such as the ulcer duration, size, nature, and grade were associated with biofilm production. Our results suggest that as the size of the ulcer with poor glycemic control increased, the organism was more likely to be positive for biofilm formation. A high-degree of antibiotic resistance was exhibited by the biofilm-producing gram-positive isolates for erythromycin and gram-negative isolates for cefpodoxime. Comparisons of biofilm production using 3 different conventional methods were performed. The strong producers with the tube adherence method were able to produce biofilm using the cover slip assay method, and the weak producers in tube adherence method had difficulty in producing biofilm using the other 2 methods, indicating that the tube adherence method is the best method for assessing biofilm formation. The strong production of biofilm with the conventional method was further confirmed by scanning electron microscopy analysis, because bacteria attached as a distinct layer of biofilm. Thus, the high degree of antibiotic resistance was exhibited by biofilm producers compared with nonbiofilm producers. The tube adherence and cover slip assay were found to be the better method for biofilm evaluation.

In vitro biofilm formation by Staphylococcus aureus isolated from wounds of hospital-admitted patients and their association with antimicrobial resistance.

IntroductionStaphylococcus aureus including methicillin-resistant S. aureus (MRSA) has the propensity to form biofilms, and causes significant mortality and morbidity in the patients with wounds. Our aim was to study the in vitro biofilm-forming ability of S. aureus isolated from wounds of hospitalized patients and their association with antimicrobial resistance.Materials and methodsForty-three clinical isolates of S. aureus were obtained from 150 pus samples using standard microbiological techniques. Biofilm formation in these isolates was detected 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.ResultsBiofilm formation was observed in 30 (69.8%) and 28 (65.1%) isolates of S. aureus via TCP method and TM, respectively. Biofilm-producing S. aureus exhibited a higher incidence of antimicrobial resistance when compared with the biofilm nonproducers (P<0.05). Importantly, 86.7% of biofilm-producing S. aureus were multidrug resistant (MDR), whereas all the biofilm nonproducers were non-MDR (P<0.05). Large proportions (43.3%) of biofilm producers were identified as MRSA; however, none of the biofilm nonproducers were found to be MRSA (P<0.05).ConclusionBoth the in vitro methods showed that S. aureus isolated from wound infection of hospitalized patients have high degree of 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 to the early treatment of the wound infection.

High Level of Biofilm Formation and Virulence Factors in Enterococci Species Isolated From Clinical and Normal Flora Samples

Abstract Enterococci are gram-positive bacteria, and they are members of normal intestinal microflora of humans as well as animals. However, recently, they evolved from common avirulent commensal bacteria into a multidrug-resistant health care–associated pathogen. In this study, 147 isolates were collected from clinical and healthy people. The presence of 7 genes including ace, ebp, efaA, ebrB, ecbA, fsrB, and sgrA were investigated by polymerase chain reaction method. Finally, for detection of biofilm formation, Congo red agar and microliter plate methods were used. Our results showed that ace (94.6%), efaA (94.6%), and ebp (92.5%) were present almost in all of the isolates, followed by fsrB (53.7%), sgrA (36%), ecbA (33.3%), and ebrB (13.6%). By Congo red method, 38% of our isolates formed black colonies on Congo red agar (biofilm producer), whereas others (62%) had red colonies (nonbiofilm producer). Finally, by using microliter plate method, 23.8% of isolates were able to form biofilm. These results indicate that normal flora isolates can constitute as a reservoir for virulent Enterococcus isolates.

Biofilm production by Staphylococcal isolates from chronic osteomyelitis and orthopedic device-related infections in a tertiary care centre, Imphal

Background: Microorganisms growing in a biofilm are associated with chronic, recurrent infections, and health problems for patients with indwelling devices which are difficult to treat with antimicrobial agents. Staphylococcus species are the most common microorganisms isolated from these patients. This study was conducted to detect biofilm production in Staphylococcal isolates from orthopedic device-related infections (ODRI) and chronic osteomyelitis (CO) and correlation of antimicrobial susceptibility pattern between biofilm producers and nonbiofilm producers. Materials and Methods: A cross-sectional study was carried out at the Department of Microbiology in a tertiary care center after the Institutional Ethics Committee approval, from October 2014 to September 2016. Detection of biofilm was performed by Congo Red Agar (CRA), tube method (TM), and tissue culture plate (TCP) methods. Antimicrobial susceptibility test was performed by Kirby–Bauer disk diffusion method, and minimum inhibitory concentration for oxacillin and vancomycin was determined according to the Clinical Laboratory Standard Institute guidelines. Results: A total of 51 clinical isolates, consisting of 45 Staphylococcus aureus and 6 coagulase-negative Staphylococci (CoNS), were isolated from 47 CO and 4 ODRI patients of which 32 were methicillin-resistant S. aureus and 2 MRCONS. Biofilm production was detected in 22 isolates by TCP, 18 by TM, and 13 by CRA methods. Fifty percent ODRI isolates and 42.5% CO isolates were biofilm producers. The biofilm producers have lower antimicrobial sensitivity pattern in all except vancomycin. Conclusion: Biofilm producers were detected from 43.1% of total isolates with higher antimicrobial resistance pattern.

Genetic Diversity, Antimicrobial Susceptibility, and Biofilm Formation of Cronobacter spp. Recovered from Spices and Cereals.

Cronobacter species are important food-borne opportunistic pathogens which have been implicated in the cause of necrotizing enterocolitis, sepsis, and meningitis in neonates and infants. However, these bacteria are routinely found in foodstuffs, clinical specimens, and environmental samples. This study investigated the genetic diversity, antimicrobial susceptibility, and biofilm formation of Cronobacter isolates (n = 40) recovered from spices and cereals in China during 2014–2015. Based on the fusA sequencing analysis, we found that the majority (23/40, 57.5%) of Cronobacter isolates in spices and cereals were C. sakazakii, while the remaining strains were C. dublinensis (6/40, 15.0%), C. malonaticus (5/40, 12.5%), C. turicensis (4/40, 10.0%), and C. universalis (2/40, 5.0%). Multilocus sequence typing (MLST) analysis produced 30 sequence types (STs) among the 40 Cronobacter isolates, with 5 STs (ST4, ST13, ST50, ST129, and ST158) related to neonatal meningitis. The pattern of the overall ST distribution was diverse; in particular, it was revealed that ST148 was the predominant ST, presenting 12.5% within the whole population. MLST assigned 12 isolates to 7 different clonal complexes (CCs), 4, 13, 16, 17, 72, 129, and 143, respectively. The results of O-antigen serotyping indicated that C. sakazakii serotype O1 and O2 were the most two prevalent serotypes. The antimicrobial susceptibility testing showed that the 40 Cronobacter isolates were susceptible to most of the antibiotics tested except for ceftriaxone, meropenem, and aztreona. Of the 40 Cronobacter strains tested, 13 (32.5%) were assessed as weak bioflim producers, one (2.5%) was a moderate biofilm producer, one (2.5%) was strong biofilm producer, and the others (62.5%) were non-biofilm producers. MLST and O-antigen serotyping have indicated that Cronobacter strains recovered from spices and cereals were genetically diverse. Isolates of clinical origin, particularly the C. sakazakii ST4 neonatal meningitic pathovar, have been identified from spices and cereals. Moreover, antimicrobial resistance of Cronobacter strains was observed, which may imply a potential public health risk. Therefore, the surveillance of Cronobacter spp. in spices and cereals should be strengthened to improve epidemiological understandings of Cronobacter infections.

Association between Extracellular Material and Biofilm Formation in Response to Sodium Hypochlorite by Clinical Isolates of Enterococcus faecalis

IntroductionExtracellular material (ECM) surrounding Enterococcus faecalis may play a role in increasing resistance to environmental stresses. Our aim was to determine ECM levels in response to subminimal inhibitory concentrations of sodium hypochlorite (sub-MIC/NaOCl) or anaerobic growth and determine the impact on biofilm development. MethodsFrom 37 E. faecalis clinical strains, 19 were selected according to their biofilm-producing ability by using a crystal violet biofilm assay: 10 strong, 4 intermediate, and 5 non-biofilm producers. Biofilm assays were subsequently performed on all strains when subjected to sub-MIC/NaOCl. All strains were evaluated for ECM production under aerobic and anaerobic conditions and with sub-MIC/NaOCl. ECM production was assessed by using scanning electron microscopy. Double-blinded independent assessors were used to score levels of ECM production. The esp gene was detected by using polymerase chain reaction. Gelatinase activity was determined by using Todd-Hewitt and gelatin agar. ResultsIn aerobic conditions, ECM was expressed in all strains. In the presence of sub-MIC/NaOCl, of the 10 strong biofilm producers, 5 increased their ECM production, and 4 showed increased biofilm growth. Two strains had less ECM production and showed decreased biofilm growth. One isolate demonstrated no observable changes. Most non-biofilm producers demonstrated no observable differences in ECM production, although 1 strain increased biofilm growth. ECM production in anaerobic conditions was highly variable. The esp gene (n = 15) and gelatinase activity (n = 7) were evident among the isolates. ConclusionsClonal diversity among strains of E. faecalis suggests that some strong biofilm producers can upregulate ECM production and increase biofilm growth in response to sub-MIC/NaOCl.

The Possible Influence of Non-synonymous Point Mutations within the FimA Adhesin of Non-typhoidal Salmonella (NTS) Isolates in the Process of Host Adaptation.

Non-typhoidal Salmonella (NTS) remains a global pathogen that affects a wide range of animal species. We analyzed a large number of NTS isolates of different host origins, including Salmonella Heidelberg (n = 80, avian), S. Dublin (50, bovine), S. Typhimurium var 5- (n = 40, porcine), S. 4,5,12,:i:- (n = 40, porcine), S. Cerro (n = 16, bovine), and S. Montevideo (n = 14, bovine), using virulence profiling of the bcfC, mgtC, ssaC, invE, pefA, stn, sopB, and siiE virulence-associated genes, a biofilm production assay, pulsed field gel electrophoresis, and the full-length sequencing of the fimA (adhesin) and iroN (receptor) genes. We determined a key amino acid substitution, A169 (i.e., threonine changed to alanine at position 169), in the FimA protein that changed ligand affinity of FimA toward N-acetyl-D-glucosamine. This finding clearly indicates the important role of non-synonymous single nucleotide polymorphism (nsSNPs) in adhesin functionality that may impact the host tropism of NTS. This nsSNP was found in S. Heidelberg and S. Cerro isolates. Although this was not the case for the IroN receptor, the phylogeny of this receptor and different host origins of NTS isolates were positively correlated, suggesting existence of specific host immune selective pressures on this unique receptor in S. enterica. We found that pefA, a gene encoding major fimbrial subunit, was the most-segregative virulence factor. It was associated with S. Heidelberg, S. Typhimurium var 5- and S. 4,5,12,:i:- but not with the rest of NTS strains. Further, we observed a significantly higher frequency of non-biofilm producers among NTS strains that do not carry pefA (42.5%) compared to S. Heidelberg (2.5%) and S. Typhimurium var 5- (7.5%) and S. 4,5,12,:i:- (0%). This study provides new insights into the host adaptation of avian and mammalian NTS isolates that are based on the bacterial antigens FimA and IroN as well as the interrelationships between host adaptation, overall genetic relatedness, and virulence potential in these NTS isolates.

Evaluation of the biofilm formation capacity of Pasteurella multocida strains isolated from cases of fowl cholera and swine lungs and its relationship with pathogenicity

ABSTRACT: Pasteurella multocida is a Gram-negative bacillus that causes economic losses due to the development of respiratory diseases in several animal species. Among the mechanisms of virulence, the formation of biofilms is an important factor for bacterial survival in hostile environments. Studies of biofilm formation by P. multocida are needed because P. multocida is an important pathogen involved in respiratory infections. However, in contrast to other microorganisms, few studies of biofilm formation have examined P. multocida. Studies comparing the pathogenicity of microbial strains as a function of their biofilm production capacity are also rare. Consequently, the aim of this study was to evaluate the biofilm formation capacity of 94 P. multocida strains isolated from cases of fowl cholera and from swine lungs on polystyrene plates. The associations of the biofilm formation capacity with the pathogenicity index (PI) in vivo and with the presence of four genes (screened by PCR) of the tad locus (tadB, tadD, tadE and tadG), described as adhesion markers, were also determined. Strains from both animal origins were able to form biofilms. However, most of the specimens (52.13%) were classified as weak producers, and more than 40% of the strains of P. multocida (40.42%) did not produce biofilms. There was no significant difference (p&gt;0.05) in the degree of biofilm production between the two sources of isolation. Of the analyzed strains, 56.52% contained all four genes (tadB, tadD, tadE and tadG). The PI arithmetic mean of the strains classified as non-biofilm producers was significantly different (p&lt;0.05) from the PI of moderate-producer strains. The PI of specimens classified as weak biofilm producers also differed significantly (p&lt;0.05) from that of the moderate-producer strains. The results indicate that even though the P. multocida strains isolated from cases of fowl cholera and swine lungs formed biofilms on polystyrene surfaces, adhesion was usually weak. The genes tadB, tadD, tadE and tadG were not significantly associated (p&gt;0.05) with the production of biofilms and with the origin of a given strain. Finally, low virulence strains may suggest a higher biofilm formation capacity on polystyrene plates.

Biofilm production by uropathogens causing catheter associated urinary tract infection

Background: The microbial populations within urinary catheter frequently develop as biofilms, directly attaching to the surface of catheters. Bacteria in biofilm are protected from antimicrobial chemotherapy as well as host defence mechanisms, establishing chronic persistent infections, septicemia and death if not treated. Material and Method: The present study, includes 200 patients, catheterized for >48 hours at CIMS, Bilaspur. Urine samples were collected and inoculated in nutrient agar, blood agar and MacConkey agar plates and identification done as per standard procedure. This study was conducted to detect biofilm formation ability of uropathogens by two different methods (Tube and Congo red agar method) and compare their antibiotic sensitivity by using Kirby-Bauer disc diffusion method. Results: Out of 200 urine samples significant bacteriuria were detected in 148(74%) of samples and no growth found in 52(26%) samples and 14 samples showed growth of 2 microorganisms. A total no. of 162 microorganisms were isolated from 200 urine samples. Among these 162 isolates E. Coli was 29.62%, Pseudomonas aeruginosa 11.72%, Klebsiella sp 18.51%, Citrobactor sp 7.40%, Staphylococcus aureus 3.08%, and Coagulase negative Staphylococci 11%. Among these 162 isolates, a total of 91(56.17%) isolates showed biofilm production. Percentage of biofilm formation was highest in P. aeruginosa (63.15%). Conclusion: Tube test method was found to be more reliable method. The in vitro antibiotic susceptibility pattern of biofilm producing organisms showed less sensitivity as compared to non-biofilm producing organisms.

The Role of Quorum Sensing Genes (Las and Rhl) in Biofilm Forming Pseudomonas Aeruginosa Isolates

Background: Quorum sensing (QS) is a bacterial cell-cell communication process that involves the production, detection, and response to extracellular signaling molecules called autoinducers (AIs). Processes controlled by QS include bioluminescence, sporulation, competence, antibiotic production, biofilm formation, and virulence factor secretion. Pseudomonas aeruginosa (P. aeruginosa) produces multiple virulence factors and causes different types of infections. Objectives: to determine if deficiency within the QS system decreases the ability of P. aeruginosa to establish biofilm formation and infections in humans or not. Methodology: This was achieved through determination of the levels of P. aeruginosa autoinducers as well as the presence of QS-controlled genes (lasI, lasR, rhlI and rhlR) within the biofilm-forming clinical samples. Results: We collected 186 P. aeruginosa isolates, 89.3% were significantly biofilm producer strains. About 66% were strong, 28% were moderate and 15% were weak biofilm producer clinical isolates. Autoinducers evaluation revealed that, 8-clinical isolates (CIs) for 3OC 12 -HSL and/or C 4 -HSL concentration were deficient. The amount of 3OC 12 -HSL produced by 8-CIs was significantly lower than that produced by PAO1. While 6-CIs produced no C 4 -HSL and 2-CIs produced detectable levels of C 4 -HSL. Polymerase chain reaction (PCR) analysis of autoinducers deficient CIs revealed that, two isolates contained all four QS genes (lasI, lasR, rhlI and rhlR) and were moderate-biofilm producers while three isolates lacked all QS genes and were non-biofilm producer strains. Analysis of remaining autoinducers deficient CIs revealed that, they were weak-biofilm producer strains and they had intact las system in two strains and lacked lasR in third one while they were completely lacked the rhl system. Patients had autoinducers deficient CIs were isolated from cardiac catheterization, tracheostomy tubes, sputum, and wound infections. Conclusion: Naturally occurring QS-deficient strains of P. aeruginosa were capable of causing different infections despite the loss of optimum gene. QS-deficient genes affected the severity of biofilm formation. So the development of efficient cell-to-cell signaling blockers may reduce P. aeruginosa biofilm formation on medical devices.

Bacterial biofilm-based catheter-associated urinary tract infections: Causative pathogens and antibiotic resistance

We sought to determine the incidence of bacterial biofilm-based catheter-associated urinary tract infections, identify variables affecting biofilm formation, and identify etiologic bacterial pathogens and antibiotic-resistance patterns associated with biofilm-based catheter-associated urinary tract infections (CAUTIs) in our setup. Patients who developed at least 2 symptoms of urinary tract infection after at least 2 days of indwelling urinary catheters were included. Urine was collected aseptically from catheter tubing and processed per standard microbiologic practices. Bacterial pathogens were identified on the basis of gram staining, colony morphology, and biochemical reactions. The detection of the biofilm was done using the tube adherence method. Drug susceptibility testing was done using the Kirby-Bauer disc diffusion method. Biofilm was detected in 73.4% isolates, whereas 26.6% of isolates were nonbiofilm producers. Mean duration of catheterization after which biofilm was detected was 5.01 ± 1.31days. A latex catheter was used in 69.5% of patients, whereas a silicone catheter was used in 30.4% of patients. Escherichia coli was found to be the most common pathogen isolated (52.3%), whereas Enterobacter cloacae exhibited the highest biofilm production (87.5%) among isolated pathogens. Among biofilm producers, the highest resistance was observed with ampicillin (100%). Fosfomycin exhibited the lowest resistance (17.2%). Significant association with biofilm was detected for gender, duration of catheterization, and type of catheter. Biofilm-based CAUTI is an emerging problem. E coli was the most frequent isolate. High antibiotic resistance was observed in biofilm-producing strains. Using the variables affecting biofilm formation, tailored intervention strategies can be implemented to reduce biofilm-based CAUTIs.