Prevalence and Characterization of Monophasic Salmonella Serovar 1,4,[5],12:i:- of Food Origin in China

Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, which has recently been recognized as an emerging cause of infection worldwide. This bacterium has also ranked among the four most frequent serovars causing human salmonellosis in China. However, there are no reports on its contamination in Chinese food. Serotyping, polymerase chain reaction, antibiotic resistance, virulotyping, and multilocus sequence typing (MLST) assays were used to investigate the prevalence of this serological variant in food products in China, and to determine phenotypic and genotypic difference of monophasic isolates and Salmonella Typhimurium isolated over the same period. Salmonella 1,4,[5],12:i:- was prevalent in various food sources, including beef, pork, chicken, and pigeon. The study also confirmed the high prevalence (53.8%) of resistance to ampicillin, streptomycin, sulfonamides, and tetracycline in Salmonella 1,4,[5],12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,[5],12:i:- isolates in our study were different from Salmonella Typhimurium isolates by the absence of three plasmid-borne genes (spvC, pefA, and rck) and the presence of gipA in all isolates. All Salmonella 1,4,[5],12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,[5],12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,[5],12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,[5],12:i:- clones from Spain, America, and Italy, which provided some new evidence on the independent evolution of the multiple successful monophasic clones from Salmonella Typhimurium ancestors. This study is the first report of Salmonella 1,4,[5],12:i:- in food products from China. The data are more comprehensive and representative, providing valuable information for epidemiological studies, risk management, and public health strategies.     

Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

Background

In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.

Methods

We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali.

Principal Findings

We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.

Conclusion

We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.     

Characteristics of Salmonella enterica Serovar 4,[5],12:i:- as a Monophasic Variant of Serovar Typhimurium

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- (S. 4,[5]12:i:-) is believed to be a monophasic variant of S. enterica serovar Typhimurium (S. Typhimurium). This study was conducted to corroborate this hypothesis and to identify the molecular and phenotypic characteristics of the S. 4,[5]12:i:- isolates in Japan. A total of 51 S. 4,[5]12:i:- isolates derived from humans, cattle, swine, chickens, birds, meat (pork), and river water in 15 prefectures in Japan between 2000 and 2010 were analyzed. All the S. 4,[5],12:i:- isolates were identified as S. Typhimurium by two different polymerase chain reactions (PCR) for identification of S. Typhimurium. Of the 51 S. 4,[5],12:i:- isolates, 39 (76.5%) harbored a 94-kb virulence plasmid, which is known to be specific for S. Typhimurium. These data suggest that the S. 4,[5],12:i:- isolates are monophasic variants of S. Typhimurium. The flagellar phase variation is induced by three adjacent genes (fljA, fljB, and hin) in the chromosome. The results of PCR mapping of this region and comparative genomic hybridization analysis suggested that the deletion of the fljAB operon and its flanking region was the major genetic basis of the monophasic phenotype of S. 4,[5],12:i:-. The fljAB operon and hin gene were detectable in eight of the S. 4,[5],12:i:- isolates with common amino acid substitutions of A46T in FljA and R140L in Hin. The introduction of these mutations into S. Typhimurium isolates led to the loss of selectability of isolates expressing the phase 2 H antigen. These data suggested that a point mutation was the genetic basis, at least in part, of the S. 4,[5],12:i:- isolates. The results of phenotypic analysis suggested that the S. 4,[5],12:i:- isolates in Japan consist of multiple distinct clones. This is the first detailed characterization of the S. 4,[5],12:i:- isolates derived from various sources across Japan.     

Biofilm Production by Candida: Comparison of Bloodstream Isolates with Cervical Isolates

The present study was undertaken to investigate biofilm formation among the clinical Candida isolates from blood and cervical swabs. A total of 16 Candida blood isolates from neonates and 21 cervical isolates from pregnant women with vulvovaginitis were included in the study. Each isolate was identified to species level by various phenotypic tests. Biofilm formation was detected by colorimetric method. C. glabrata and C. albicans were the major isolates from blood and cervical swab respectively. The biofilm formation was found in 14 (87.5 %) blood isolates and only in 4 (19.1 %) cervical isolates.     

A Widely Used In Vitro Biofilm Assay Has Questionable Clinical Significance for Enterococcal Endocarditis

Biofilm formation may play an important role in the pathogenesis of infections caused by Enterococcus faecalis, including endocarditis. Most biofilm studies use a polystyrene dish assay to quantify biofilm biomass. However, recent studies of E. faecalis strains in tissue and animal models suggest that polystyrene dish results need to be interpreted with caution. We evaluated 158 clinical E. faecalis isolates using a polystyrene dish assay and found variation in biofilm formation, with many isolates forming little biofilm even when different types of media were used. However, all tested clinical isolates were able to form biofilms on porcine heart valve explants. Dextrose-enhanced biofilm formation in the polystyrene dish assay was found in 6/12 (50%) of clinical isolates tested and may explain some, but not all of the differences between the polystyrene dish assay and the heart valve assay. These findings suggest that in studies assessing the clinical relevance of enterococcal biofilm-forming ability, ex vivo biofilm formation on a relevant tissue surface may be warranted to validate results of in vitro assays.     

Biochemical detection of N-Acyl homoserine lactone from biofilm-forming uropathogenic Escherichia coli isolated from urinary tract infection samples

Background:

N-Acyl homoserine lactone (AHL) is found to be the main component of quorum sensing (QS) in Gram-negative bacteria and plays an important role in biofilm formation. Little information is available regarding the role of AHL in biofilm formation in Escherichia coli (E. coli). The purpose of this investigation was to biochemically detect and characterize AHL activity in biofilm-forming uropathogenic E. coli (UPEC) isolated from urine samples of the patients with urinary tract infections (UTIs) in Kerman, Iran.

Methods:

Thirty-five UPEC isolates were obtained from urine samples of the patients with UTIs referred to the Afzalipoor hospital. The isolates were identified by biochemical tests. Biofilm analyses of all the isolates were performed using the microtiter plate method at OD 490nm. N-Acyl homoserine lactone was separated from cell mass supernatants by liquid-liquid extraction (LLE) and analyzed by a colorimetric method. N-Acyl homoserine lactone functional groups were identified by Fourier Transform-Infrared Spectroscopy (FT-IR).

Results:

The biofilm formation assay identified 10 (28.57%) isolates with strong, 16 (45.71%) with moderate, and 9 (25.71%) with weak biofilm activities. The UPEC isolates with strong and weak biofilm activities were subjected to AHL analyses. It was found that isolates with the highest AHL activities also exhibited strong adherence to microplate wells (P≤0.05). Two E. coli isolates with the highest AHL activities were selected for FT-IR spectroscopy. Peaks at 1764.33, 1377.99, and 1242.90 cm^-1 correspond to the C=O bond of the lactone ring, and the N=H and C-O bonds of the acyl chain, respectively.

Conclusion:

We found that many UPEC isolates exhibited strong biofilm formation. The control of this property by AHL may contribute to the pathogenesis of the organism in UTI’s.Key Words: Biofilm, FT-IR, N-acylhomoserine lactone, Uropathogenic Escherichia coli     

Pork Contaminated with Salmonella enterica Serovar 4,[5],12:i:?, an Emerging Health Risk for Humans

Salmonella enterica subsp. enterica serovar 4,[5],12:i:− is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes bla_TEM1-like, strA-strB, tet(B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,[5],12:i:− and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,[5],12:i:− strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.Salmonella enterica subsp. enterica serovar Typhimurium is a ubiquitous serovar that usually induces gastroenteritis in a broad range of unrelated host species. Following the White-Kauffmann-Le Minor scheme, the seroformula for S. enterica serovar Typhimurium is 4,[5],12:i:1,2 (14). Salmonella serotyping is based on antigenic variability of lipopolysaccharides (O antigen) and flagellar proteins (H1 and H2 antigens).In the mid-1990s a monophasic S. enterica serovar with the seroformula 4,[5],12:i:− started to emerge in Europe (10). Initial characterization of isolates from pig samples in Spain in 1997 demonstrated that this serovar in comparison with S. enterica serovar Typhimurium (4,[5],12:i:1,2) lacked the fljB gene encoding the structural subunit of the phase two flagellar (H2) antigen (11). The predominant phage type was U302. Another DNA microarray-based typing study indicated that the monophasic serovar had a gene repertoire highly similar to that of S. enterica serovar Typhimurium, indicating a close genetic relatedness between the serovars (13). Similarly, multi-locus sequence typing showed that S. enterica serovar 4,[5],12:i:− and S. enterica serovar Typhimurium represent a highly clonal group (23).Within the last years S. enterica serovar 4,[5],12:i:− has increasingly been implicated in human disease worldwide (1, 10, 24, 25). Recently, larger outbreaks caused by this serovar have been reported from Luxembourg and the United States (5, 19). A European Union (EU) baseline survey on the prevalence of Salmonella in slaughter-age pigs in 2006 to 2007 revealed that the monophasic serovar was isolated from pigs in 9 of 25 participating member states (12). At the EU level, S. enterica serovar 4,[5],12:i:− was the fourth most prevalent serovar in slaughter-age pigs. In Germany it was the second most prevalent serovar after S. enterica serovar Typhimurium (12). Between 1999 and 2008 the proportion of S. enterica serovar 4,[5],12:i:− isolates among all S. enterica isolates received by the German National Reference Laboratory for Salmonella increased from 0.1% to 8.3% (305 isolates in 2008), with the most remarkable increase between 2006 and 2007. Most of these strains (48% on average between 2006 and 2008) were isolated from pigs, followed by cattle (13%), poultry (5%), and other isolates sporadically found in the environment, wildlife, and reptiles. Remarkably, the annual proportion of the monophasic serovar among all S. enterica serovar 4,[5],12:i:− and S. enterica serovar Typhimurium isolates increased from 0.3% to 32.7% in the same decade. Interestingly, the number of S. enterica serovar 4,[5],12:i:− strains isolated from humans and sent on voluntary basis to the National Reference Centre for Salmonella and other Enterics increased from 0.1% in 1999 to 14.0% (456 isolates) in 2008. Likewise, the proportion of the monophasic serovar among all S. enterica serovar 4,[5],12:i:− and S. enterica serovar Typhimurium isolates increased from 0.3% to 42.8% in the same time because of declining numbers of S. enterica serovar Typhimurium isolates.In the present study a collection of S. enterica serovar 4,[5],12:i:− strains isolated from pigs, pork, and humans in Germany during the years 2006 and 2007 was examined using phenotypic and molecular methods. The aim of the analyses was to gain a better understanding of the clonality of the serovar and of the ability of its subtypes to be transmitted to humans via pigs and pork. Additionally, the genetic relatedness as well as the pathogenicity and antimicrobial resistance gene repertoire of S. enterica serovar 4,[5],12:i:− was compared with selected S. enterica serovar Typhimurium strains representing corresponding phage types in order to estimate the potential health risk for humans.     

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides,proteins, eDNA and their correlation with biofilm mass

Abstract

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R² = 0.882), while the content of proteins turned out to be weakly (R² = 0.465) and that of eDNA very weakly associated (R² = 0.202) to the biofilm mass.     

Population Structure of Salmonella enterica Serovar 4,[5],12:b:? Strains and Likely Sources of Human Infection

Salmonella enterica serovar 4,[5],12:b:− is a monophasic serovar not able to express the second-phase flagellar antigen (H2 antigen). In Germany, the serovar is occasionally isolated from poultry, reptiles, fish, food, and humans. In this study, a selection of 67 epidemiologically unrelated Salmonella enterica serovar 4,[5],12:b:− strains isolated in Germany between 2000 and 2011 from the environment, animal, food, and humans was investigated by phenotypic and genotypic methods to better understand the population structure and to identify potential sources of human infections. Strains of this monophasic serovar were highly diverse. Within the 67 strains analyzed, we identified 52 different pulsed-field gel electrophoresis XbaI profiles, 12 different multilocus sequence types (STs), and 18 different pathogenicity array types. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was in good agreement with grouping by MLST. S. enterica serovar 4,[5],12:b:− is distributed across multiple unrelated eBurst groups and consequently is highly polyphyletic. Two sequence types (ST88 and ST127) were linked to S. enterica serovar Paratyphi B (d-tartrate positive), two single-locus variants of ST1583 were linked to S. enterica serovar Abony, and one sequence type (ST1484) was associated with S. enterica serovar Mygdal, a recently defined, new serovar. From the characterization of clinical isolates and those of nonhuman origin, it can be concluded that the potential sources of sporadic human infections with S. enterica serovar 4,[5],12:b:− most likely are mushrooms, shellfish/fish, and poultry.     

Targeting Acyl Homoserine Lactones (AHLs) by the quorum quenching bacterial strains to control biofilm formation in Pseudomonas aeruginosa

Navigating novel biological strategies to mitigate bacterial biofilms have great worth to combat bacterial infections. Bacterial infections caused by the biofilm forming bacteria are 1000 times more resistant to antibiotics than the planktonic bacteria. Among the known bacterial infections, more than 70% involve biofilms which severely complicates treatment options. Biofilm formation is mainly regulated by the Quorum sensing (QS) mechanism. Interference with the QS system by the quorum quenching (QQ) enzyme is a potent strategy to mitigate biofilm. In this study, bacterial strains with QQ activity were identified and their anti-biofilm potential was investigated against the Multidrug Resistant (MDR) Pseudomonas aeruginosa. A Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136-based bioassays were used to confirm the degradation of different Acyl Homoserine Lactones (AHLs) by QQ isolates. The 16S rRNA gene sequencing of the isolated strains identified them as Bacillus cereus strain QSP03, B. subtilis strain QSP10, Pseudomonas putida strain QQ3 and P. aeruginosa strain QSP01. Biofilm mitigation potential of QQ isolates was tested against MDR P. aeruginosa and the results suggested that 50% biofilm reduction was observed by QQ3 and QSP01 strains, and around 60% reduction by QSP10 and QSP03 bacterial isolates. The presence of AHL degrading enzymes, lactonases and acylases, was confirmed by PCR based screening and sequencing of the already annotated genes aiiA, pvdQ and quiP. Altogether, these results exhibit that QQ bacterial strains or their products could be useful to control biofilm formation in P.aeruginosa.     

Heparin-benzyl alcohol enhancement of biofilms formation and antifungal susceptibility of vaginal Candida species isolated from pregnant and nonpregnant Saudi women

Biofilm formation by Candida species is a major contribute to their pathogenic potential.The aim of this study was to determine in vitro effects of EDTA, cycloheximide, and heparin-benzyl alcohol preservative on C. albicans (126) and non-albicans (31)vaginal yeast isolates biofilm formations and their susceptibility against three antifungal Etest strips. Results of the crystal violet-assay, indicated that biofilms formation were most commonly observed [100%] for C. kefyr, C. utilis, C. famata, and Rhodotorula mucilaginosa, followed by C. glabrata [70%], C. tropicalis [50%], C. albicans [29%], Saccharomyces cerevisiae [0.0%]. EDTA (0.3mg/ml) significantly inhibited biofilm formation in both C. albicans and non-albicans isolates (P=0.0001) presumably due to chelation of necessary metal cations for the process-completion. In contrast, heparin (-benzyl alcohol preservative) stimulated biofilm formation in all tested isolates, but not at significant level (P=0.567). Conversely, cycloheximide significantly (P=0.0001) inhibited biofilm formation in all C. albicans strains(126) and its effect was even 3 fold more pronounced than EDTA inhibition, probably due to its attenuation of proteins (enzymes) and/or complex molecules necessary for biofilm formation. Results also showed that all nonalbicans yeasts isolates were susceptible to 5-flucytosine (MIC₅₀, 0.016 µg/ml; MIC₉₀, 0.064 µg/ml), but 14% of C. albicans isolates were resistant (MIC₅₀, 0.064 µg/ml; MIC₉₀ >32 µg/ml). The MIC₅₀ value of amphotricin B for all C. albicans and non-albicans isolates was at a narrow range of 0.023 µg /ml, and the MIC₉₀ values were 0.047 µg/ml and 0.064 µg/ml respectively, thereby confirming its efficacy as a first line empiric- treatment of Candida spp infections.     

Staphylococcus epidermidis in Orthopedic Device Infections: The Role of Bacterial Internalization in Human Osteoblasts and Biofilm Formation

Background

Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms.

Materials and Methods

Orthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method.

Results

No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/−631 and 347+/−431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups.

Conclusion

Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.     

The Impact of spgM,rpfF, rmlA Gene Distribution on Biofilm Formation in Stenotrophomonas maltophilia

Background

Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression.

Methods

The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression.

Results

Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains.

Conclusion

Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.     

Biofilm Production Potential of Salmonella Serovars Isolated from Chickens in North West Province,South Africa

Bacterial biofilms have recently gained considerable interest in the food production and medical industries due to their ability to resist destruction by disinfectants and other antimicrobials. Biofilms are extracellular polymer matrices that may enhance the survival of pathogens even when exposed to environmental stress. The effect of incubation temperatures (25°C, 37°C, and 40°C) and Salmonella serotype on biofilm-forming potentials was evaluated. Previously typed Salmonella serotypes (55) isolated from the gut of chickens were accessed for biofilms formation using a standard assay. Salmonella Typhimurium ATCC 14028^TM and Salmonella Enteritidis ATCC 13076^TM (positive controls), Escherichia coli (internal control) and un-inoculated Luria Bertani (LB) broth (negative control) were used. The isolates formed no biofilm (11.86–13.56%), weak (11.86–45.76%), moderate (18.64–20.34%), strong biofilms (23.73–54.24%) across the various temperatures investigated. Serotypes, Salmonella Heidelberg and Salmonella Weltevreden were the strongest biofilm formers at temperatures (25°C, 37°C, and 40°C, respectively). The potential of a large proportion (80%) of Salmonella serotypes to form biofilms increased with increasing incubation temperatures but decreased at 40°C. Findings indicate that average temperature favours biofilm formation by Salmonella serotypes. However, the influence of incubation temperature on biofilm formation was greater when compared to serotype. A positive correlation exists between Salmonella biofilm formed at 25°C, 37°C and 40°C (p ≥ 0.01). The ability of Salmonella species to form biofilms at 25°C and 37°C suggests that these serotypes may present severe challenges to food-processing and hospital facilities.Key words: Salmonella, biofilm, biofilm production potential, crystal violet microtitre     

Molecular characterization of clinical and environmental Pseudomonas aeruginosa isolated in a burn center

In burn centers, Pseudomonas aeruginosa acts as a major cause of nosocomial infections. Therefore, this study aimed to characterize molecularly P. aeruginosa isolates collected from environmental samples and burn patients. A total of 78 strains (including 58 clinical and 20 environmental isolates) of the P. aeruginosa were collected from Beasat hospital of Hamadan, west of Iran, and was identified using API 20NE. The disk diffusion method according to the CLSI was applied for determination of the antimicrobial resistance. Moreover, the microtiter plate test was used for the quantification of Biofilm formation. The genomic features of the isolated strains was evaluated using Pulsed Field Gel Electrophoresis (PFGE). We found that 94.8% of clinical and 80% environmental isolates were capable of forming biofilm. The rate of MDR in clinical and environmental isolates was 51.7% and 40%, respectively. A significant relationship was observed between biofilm formation capability and multiple drug resistance (p < 0.05). PFGE typing showed 11 different clusters with two major clusters A with 30 (38.5%) and B with 14 (17.9%) members, containing up to 56.4% of all isolates. There was no relationship between biofilm formation ability and antibiotic resistance patterns with PFGE patterns. According to the results, the clonal spread of environmental P. aeruginosa isolates is associated with clinical isolates, and both environmental and clinical isolates are attributed to a high prevalence of the antibiotic resistance and biofilm formation ability. This study highlighted that the prevention programs should be implemented in the hospital environment to control the spread of P. aeruginosa in burn units.     

Prevalence of Virulence Genes and Antibiotic Resistance Pattern in Enterococcus Faecalis Isolated from Urinary Tract Infection in Shahrekord,Iran

Background:This study aims to specify the antimicrobial resistance pattern and virulence genes of Enterococcus faecalis isolated from urinary tract infections in Shahrekord, Iran. Methods:Urine samples of 1000 people suspected of having urinary tract infections referred to Shahrekord medical diagnostic laboratories were examined. Biofilm assays were performed by microtiter plate test through reading the OD490. Polymerase Chain Reaction (PCR) was applied to study the virulence factors.Results:Enterococcus faecalis was detected in 60 samples. After performing microbiological tests, all samples were positive in the molecular analysis. Strong, moderate and weak biofilm reactions reported 66.67%, 25%, and 8.33% respectively. The most resistance reported to cotrimoxazole, vancomycin and amikacin and the lowest resistance to nitrofurantoin (8.33%) was reported. Statistical analysis with Fisher''s exact test showed a statistically significant relationship between biofilm production and resistance to cotrimoxazole, vancomycin and cefotaxime. Prevalence of efe A, ace, gel E, esp, cyl M, agg, cyl A and cyl B in strong biofilm formation isolates was reported 100%, 87.5%, 82%, 62.5%, 55%, 37.5% 25% and 22.5% respectively. There was a significant relationship between the frequency of efa A and strong biofilm reaction.Conclusion:The presence of E. faecalis strains resistant to co-trimoxazole and vancomycin and present of some virulence factors is alarming the researchers. Since antibiotic resistance genes are probably transmitted among enterococci, and Staphylococci, controlling infections made by enterococci as well as the appropriate administration of antibiotics could treat the nosocomial infections effectively.Key Words: Antibiotic Resistance, Enterococcus faecalis, Urinary Tract Infection, Virulence genes     

IscR Controls Iron-Dependent Biofilm Formation in Escherichia coli by Regulating Type I Fimbria Expression

Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation in Escherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions in E. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae in E. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.     

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Antibiotic Susceptibility of Biofilm Cells and Molecular Characterisation of Staphylococcus hominis Isolates from Blood

Objectives

We aimed to characterise the staphylococcal cassette chromosome mec (SCCmec) type, genetic relatedness, biofilm formation and composition, icaADBC genes detection, icaD expression, and antibiotic susceptibility of planktonic and biofilm cells of Staphylococcus hominis isolates from blood.

Methods

The study included 67 S. hominis blood isolates. Methicillin resistance was evaluated with the cefoxitin disk test. mecA gene and SCCmec were detected by multiplex PCR. Genetic relatedness was determined by pulsed-field gel electrophoresis. Biofilm formation and composition were evaluated by staining with crystal violet and by detachment assay, respectively; and the biofilm index (BI) was determined. Detection and expression of icaADBC genes were performed by multiplex PCR and real-time PCR, respectively. Antibiotic susceptibilities of planktonic cells (minimum inhibitory concentration, MIC) and biofilm cells (minimum biofilm eradication concentration, MBEC) were determined by the broth dilution method.

Results

Eighty-five percent (57/67) of isolates were methicillin resistant and mecA positive. Of the mecA-positive isolates, 66.7% (38/57) carried a new putative SCCmec type. Four clones were detected, with two to five isolates each. Among all isolates, 91% (61/67) were categorised as strong biofilm producers. Biofilm biomass composition was heterogeneous (polysaccharides, proteins and DNA). All isolates presented the icaD gene, and 6.66% (1/15) isolates expressed icaD. This isolate presented the five genes of ica operon. Higher BI and MBEC values than the MIC values were observed for amikacin, vancomycin, linezolid, oxacillin, ciprofloxacin, and chloramphenicol.

Conclusions

S. hominis isolates were highly resistant to methicillin and other antimicrobials. Most of the detected SCCmec types were different than those described for S. aureus. Isolates indicated low clonality. The results indicate that S. hominis is a strong biofilm producer with an extracellular matrix with similar composition of proteins, DNA and N-acetylglucosamine; and presents high frequency and low expression of icaD gene. Biofilm production is associated with increased antibiotic resistance.     

Biofilm formation by five species of Candida on three clinical materials

Most recalcitrant infections are associated with colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial. Fungi can form biofilms on biomaterials commonly used in clinical practice (intravascular catheters, dentures, heart valves, implanted devices, contact lenses and other devices) and are associated with infections.A variety of in vitro models using different substrates/devices have been described. These models have been used to investigate the effect of different variables, including flow, growth time, nutrients and physiological conditions on fungal biofilm formation, morphology and architecture.The purpose of our study is to analyze biofilm formation capacity by 84 strains of Candida spp. (23 C. albicans, 23 C. parapsilosis, 16 C. tropicalis, 17 C. glabrata and 5 C. krusei) on three materials used in medical devices and its quantification using a method based on viable cell count.Under the conditions of our study, all assayed Candida strains have been able to form biofilms. All species showed greater biofilm formation capacity on Teflon™, with the exception of C. glabrata which displayed higher biofilm formation capacity on PVC. Biofilm formation by Candida spp. varies depending on the type of material on which it grows and on the species and strain of Candida.The method we propose could be of great use to deepen scientific knowledge on this subject of remarkable clinical significance, considering the absence of standard biofilm formation and quantification techniques on the catheters and the level of difficulty associated to those available.