Genetic diversity and virulence characteristics of biofilm-producing uropathogenic Escherichia coli / Diversidad genética y características de virulencia de Escherichia coli uropatógena productora de biopelículas

Uropathogenic E. coli (UPEC) strains exhibit different levels of biofilm formation that help adhesion of the bacteria to uroepithelial cells. We investigated the genetic diversity and virulence-associated genes (VAGs) of biofilm-producing UPEC. A collection of 107 biofilm-producing (BFP) UPEC strains isolated from patients with UTI in Iran were divided into three groups of strong, moderate, and weak BFPs after a quantitative microtiter plate assay, and the involvement of curli and cellulose in adhesion of the strains to T24 cell line was confirmed by the construction of csgD and yedQ mutants of two representative UPEC strains. BFP strains were tested for their genetic diversity, phylogenetic groups, and the presence of 15 VAGs. A significant decrease in adhesion of csgD and yedQ mutant strains confirmed the role of biofilm production in adhesion to uroepithelial cells. A high diversity was found among all three groups of strong (Di = 0.998), moderate (Di = 0.998), and weak (Di = 0.988) BFPs with majority of the strains belonging to phylogroups B2 (44.9%) and A (24.3%). Strong BFP strains carried significantly higher level papEF, hlyA, and iutA than other BFP groups. In contrast, the presence of fimH, focG, sfaS, set-1, and cvaC was more pronounced among weak BFP strains. There exists a high genetic diversity among the BFP strains with different VGA profiles. However, the high prevalence of phylogroup A among BFP strains suggests the fitness of commensal E. coli strains to cause UTI in this country.(AU)

Genetic diversity and virulence characteristics of biofilm-producing uropathogenic Escherichia coli.

Uropathogenic E. coli (UPEC) strains exhibit different levels of biofilm formation that help adhesion of the bacteria to uroepithelial cells. We investigated the genetic diversity and virulence-associated genes (VAGs) of biofilm-producing UPEC. A collection of 107 biofilm-producing (BFP) UPEC strains isolated from patients with UTI in Iran were divided into three groups of strong, moderate, and weak BFPs after a quantitative microtiter plate assay, and the involvement of curli and cellulose in adhesion of the strains to T24 cell line was confirmed by the construction of csgD and yedQ mutants of two representative UPEC strains. BFP strains were tested for their genetic diversity, phylogenetic groups, and the presence of 15 VAGs. A significant decrease in adhesion of csgD and yedQ mutant strains confirmed the role of biofilm production in adhesion to uroepithelial cells. A high diversity was found among all three groups of strong (Di = 0.998), moderate (Di = 0.998), and weak (Di = 0.988) BFPs with majority of the strains belonging to phylogroups B2 (44.9%) and A (24.3%). Strong BFP strains carried significantly higher level papEF, hlyA, and iutA than other BFP groups. In contrast, the presence of fimH, focG, sfaS, set-1, and cvaC was more pronounced among weak BFP strains. There exists a high genetic diversity among the BFP strains with different VGA profiles. However, the high prevalence of phylogroup A among BFP strains suggests the fitness of commensal E. coli strains to cause UTI in this country.

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

Pathogenicity of a bacterium is affected by the social characteristics of the population and environmental factors. The ability of biofilm formation among ß-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 ß-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 ß-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 ß-lactamase genes in Zahedan, a low socioeconomic city with a warm climate, was significantly higher than that of Isfahan. High prevalence of biofilm and ß-lactamase producing UPEC strains among strains from Zahedan suggests that socioeconomic status and environmental factors might have a role in pathogenicity of the strains.

Characterization of methicillin-resistant Staphylococcus aureus strains in sewage treatment plants in Tehran, Iran.

Multidrug-resistant Staphylococcus aureus strains have been commonly found in hospitals and communities causing wide ranges of infections among humans and animals. Typing of these strains is a key factor to reveal their clonal dissemination in different regions. We investigated the prevalence and dissemination of different clonal groups of S. aureus with resistance phenotype to multiple antibiotics in two sewage treatment plants (STPs) in Tehran, Iran over four sampling occasions. A total of 576 S. aureus were isolated from the inlet, sludge and outlet. Of these, 80 were identified as methicillin-resistant S. aureus (MRSA) and were further characterized using a combination of Phene Plate (PhP) typing, staphylococcal cassette chromosome mec (SCCmec), ccr types, prophage and antibiotic-resistant profiling. In all, eight common type (CT) and 13 single PhP type were identified in both STPs, with one major CT accounting for 38.8% of the MRSA strains. These strains belonged to three prophage patterns and five prophage types with SCCmec type III being the predominant type. Resistance to 11 out of the 17 antibiotics tested was significantly (P < 0.0059) higher among the MRSA isolates than methicillin-sensitive S. aureus (MSSA) strains. The persistence of the strains in samples collected from the outlet of both STPs was 31.9% for MRSA and 23.1% for MSSA. These data indicated that while the sewage treatment process, in general, is still useful for removing most MRSA populations, some strains with SCCmec type III may have a better ability to survive the STP process.

Biofilm production among methicillin resistant Staphylococcus aureus strains isolated from catheterized patients with urinary tract infection.

Between June 2011 and May 2014, we isolated a total of 419 Staphylococcus aureus strains from catheterized patients with UTI in a referral hospital in Tehran. Of these, 108 were identified as methicillin resistant (MRSA) based on their phenotypic resistance to oxacillin and the presence of mecA gene. The MRSA isolates were tested for their clonality using a combination of PFGE, prophage typing, SCCmec and ccr typing and examined for their biofilm formation as well as their resistance against 17 antibiotics. In all, 15 common pulsotypes consisted of 105 isolates and 3 single types were identified among the MRSA strains of which, 97% carried SCCmec type III and type 3 ccr. Eighty three (77%) strains were positive for biofilm formation and also carried icaA and icaD genes. Moreover, agr group III and its related tst gene were detected in 81% and 77% of biofilm producing strains, respectively 105 of the 108 MRSA were multidrug resistant with 82.4% being resistant to more than 10 antibiotics. Strains with SCCmec type IV and type 2 ccr, contained SGA and SGL prophage types, were positive for pvl gene and belonged to single PFGE types. This study highlights the important role of biofilm formation and virulence factors of MRSA strains in catheterized patients.

Evaluation of antibacterial efficiency of chitosan and chitosan nanoparticles on cariogenic streptococci: an in vitro study.

BACKGROUND AND OBJECTIVES: The most prevalent and worldwide oral disease is dental caries that affects a significant proportion of the world population. There are some classical approaches for control, prevention and treatment of this pathologic condition; however, the results are still not completely successful. Therefore new methods are needed for better management of this important challenge. Chitosan is a natural and non-toxic polysaccharide with many biological applications, particularly as an antimicrobial agent. Chitosan nanoparticle is a bioactive and environment friendly material with unique physicochemical properties. The aim of the present study was to investigate the antimicrobial effect of chitosan and nano-chitosan on the most important cariogenic streptococci. MATERIALS AND METHODS: For evaluation of antimicrobial effect of chitosan and nano-chitosan against oral streptococci broth micro-dilution method was carried out for four bacterial species; Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis and Streptococcus salivarius. Also the effect of these materials on adhesion of above bacteria was evaluated. One-way ANOVA and post hoc Tukey test were used for statistical analysis. RESULTS: The MICs of chitosan for S. mutans, S. sanguis, S. salivarius and S. sobrinus were 1.25, 1.25, 0.625 and 0.625 mg/mL, respectively. The MIC of chitosan nanoparticle for S. mutans, S. salivarius and S. sobrinus was 0.625 mg/mL and for S. sanguis was 0.312 mg/mL. Chitosan and chitosan nanoparticles at a concentration of 5 mg/mL also reduced biofilm formation of S. mutans up to 92.5% and 93.4%, respectively. CONCLUSION: The results of this study supported the use of chitosan and chitosan nanoparticles as antimicrobial agents against cariogenic Streptococci.

Characterization of methicillin resistant Staphylococcus aureus strains among inpatients and outpatients in a referral hospital in Tehran, Iran.

Methicillin resistant Staphylococcus aureus is one of the most common causes of a variety of infections ranging from wound infections to urinary tract infections (UTI) in hospital and community. In this study during 3 years we characterized the antibiotic resistance patterns of 491 hospital acquired MRSA and community associated MRSA strains by the guidelines of clinical and laboratory standard institute. A combination of high resolution PhP typing method and SCCmec typing were used for clonal dissemination of isolates. Among all 491 MRSA strains, diverse PhP types consisting of 29 common types (CTs) and 4 single types (STs) and also 2 different SCCmec types (III and IVa) were detected. In addition, 18 CTs were common among CA- and HA-MRSA strains and the presence of all 4 STs was limited to HA-MRSA strains. All isolates were resistant to penicillin and high level resistance was observed against ciprofloxacin, erythromycin, tobramycin and kanamycin and the rate of resistance to most of the antibiotic tested among HA-MRSA was significantly higher than CA-MRSA isolates. Moreover, all isolates showed susceptibility to linezolid, vancomycin and quinupristin-dalfopristin and very low resistance to fusidic acid, nitrofurantoin and chloramphenicol were detected. Our findings illustrated the increasing rate of clonal dissemination and persistence of highly antibiotic resistant CA-MRSA strains in Tehran hospitals, and also indicated the important role of the hospitals as the reservoir of MRSA strains.

Characterization of Resistance to Aminoglycosides in Methicillin-Resistant Staphylococcus aureus Strains Isolated From a Tertiary Care Hospital in Tehran, Iran.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens which can cause a broad spectrum of infections. OBJECTIVES: The current study aimed to describe the frequency and antibiotic susceptibility patterns of clonal groups of gentamicin-resistant strains of MRSA isolated from a tertiary care hospital in Tehran, Iran. MATERIALS AND METHODS: A total of 301 S. aureus isolates were collected during January to November 2012. All of the isolates were identified at the species level and typed using the Phene-Plate (PhP) system. The antibiotic susceptibility patterns of the MRSA strains and the presence of different aminoglycoside resistance genes were determined. RESULTS: Of the 301 S. aureus isolates, 90 (29.9%) strains were confirmed as MRSA, and they showed high resistance to penicillin, ciprofloxacin, kanamycin, tobramycin, erythromycin, and tetracycline. On the other hand, 43 of the 90 strains (47.8%) were resistant to gentamicin. Aac (6')-Ie + aph (2''), ant (4')-Ia, aph (3')-IIIa, and ant (6)-Ia were detected in 65.6%, 42.2%, 20%, and 47.8% of the gentamicin-resistant strains, respectively. Diverse PhP types consisting of seven common types and four single types were identified among the strains. CONCLUSIONS: Our results illustrated the presence of clonal groups of highly gentamicin-resistant strains of MRSA in hospitals in Tehran. The PhP typing method provided useful information for both clonal dissemination and determining the epidemiological links of the clonal groups of the MRSA strains.

Identification of Genomic Species of Acinetobacter Isolated from Burns of ICU Patients.

BACKGROUND: The worldwide emergence of multi-drug resistant (MDR) bacteria in recent years has caused many problems for hospitals and patients, especially intensive care unit patients. Among these clinically important MDR bacteria are Acinetobacter baumannii complex species (A. baumannii, Acinetobacter genomic species 3 and Acinetobacter genomic species 13TU) that cause a wide range of infections. METHODS: The sequencing and bioinformatics analysis of a part of the Zone 1 of rpoB gene was performed for species identification of Acinetobacter isolates obtained from ICU patients with infected burns hospitalized in a hospital in Isfahan, Iran, over a 9-month period. Antibiotic sensitivity of Acinetobacter isolates was investigated using the disk diffusion method and different classes of antibiotics including amikacin, cefotaxime, ceftriaxone, ciprofloxacin, imipenem and piperacillin. RESULTS: Acinetobacter spp. were isolated from 10 of 80 (12.5%) investigated patients. All of the 10 Acinetobacter isolates were identified as Acinetobacter baumannii and multi-drug resistant according to antibiotic susceptibility tests. CONCLUSION: Of the Acinetobacter baumannii complex members, only A. baumannii species was identified among the isolates obtained from patients with infected burns in an Isfahan hospital over a 9-month period.

Biochemical Fingerprinting of Methicillin-Resistant Staphylococcus aureus Isolated From Sewage and Hospital in Iran.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is known as a common pathogen in nosocomial and community-acquired infections. Sewage acts as an environmental reservoir and may have a significant role in development and dissemination of antibiotic resistance. OBJECTIVES: This study was undertaken to determine the epidemiological relatedness between the MRSA isolated from sewage and human infections. MATERIALS AND METHODS: Samples were collected from a referral hospital and also a sewage treatment plant in Tehran, Iran, during 2010. All the MRSA isolates were identified at the species level and typed using Phene plate (PhP) system and SCCmec typing. Antibiotic susceptibility tests were also performed. RESULTS: Of the 1142 isolates, 200 MRSA strains from the sewage (n = 100) and the clinic (n = 100) were isolated. Distinct PhP types, consisting of 16 common types and 13 single types, and also 3 different staphylococcal cassette chromosome mec (SCCmec) types (III, IVa and IVc) were found amongst the MRSA isolated from the two different sources. The results of antibiotic susceptibility testing showed an increased resistance to penicillin, ciprofloxacin, erythromycin, clindamycin and tetracycline. In addition, none of the isolates showed resistance to vancomycin, quinupristin -dalfopristin and linezolid. CONCLUSIONS: The presence of common PhP types and also SCCmec type III, as an indicator for hospital strains, among the isolates, may indicate an epidemiological link between clinical and sewage MRSA isolates in Tehran.

Isolation and Biochemical Fingerprinting of Vancomycin-Resistant Enterococcus faecium From Meat, Chicken and Cheese.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens and food chain has been considered as an assumed source for dissemination of VRE to human. OBJECTIVES: The presence of VRE isolates from food samples and typing of these isolates with Phene plate, a biochemical fingerprinting method, were investigated. MATERIALS AND METHODS: Thirty samples of meat, chicken and cheese were analyzed for VRE during 2010. Antibiotic susceptibility tests and minimum inhibitory concentration (MIC) were also examined. VRE isolates were typed with the Phene plate system (PhPlate), a biochemical fingerprinting method. RESULTS: A total of 70 VRE isolates were obtained and identified as Enterococcus faecium by species-specific PCR. All the isolates carried vanA, while none of them harbored vanB. The VRE isolates included 35, 27, and 8 isolates from meat, chicken and cheese, respectively. Typing with the PhPlate revealed a diversity index of 0.78 for E. faecium, containing 10 common and four single types. The results of antibiotic susceptibility and MIC tests showed an increased resistance to ciprofloxacin, erythromycin, ampicillin and gentamicin, to which, 100%, 100%, 100%, and 95% of VRE isolates were resistant, respectively. Only 5% of the isolates were resistant to chloramphenicol and the MIC of the isolates for vancomycin and teicoplanin was ≥ 256 µg/mL and for gentamicin-resistant isolates it was 1024 µg/mL. Conventional and molecular identification tests exhibited that all the isolates were E. faecium carrying vanA. None of the isolates harbored vanB. CONCLUSIONS: The results showed that enterococci are common contaminants in food. Indeed, this study indicates a high prevalence of multidrug-resistant enterococci in food of animal origin in Iran. Isolating some persisting enterococcal isolates revealed that continuous surveillance of antimicrobial resistance in enterococci from food is essential.

Determination of Vancomycin Resistant Enterococcus faecium Diversity in Tehran Sewage Using Plasmid Profile, Biochemical Fingerprinting and Antibiotic Resistance.

BACKGROUND: Sewage treatment plants are considered to be the hotspots for antibiotic resistance transfer among bacterial species. Many fecal bacteria including Enterococci circulate and are exposed to antibiotic residues in this environment. Being as one of the most common cause of nosocomial infections, special concerns have risen worldwide about the rate and characteristics of Enterococci (especially, isolates with high resistance against glycopeptides) which are available in raw sewages. OBJECTIVES: Study on the vancomycin Resistant E. faecium diversity in Tehran sewage by plasmid profile, biochemical fingerprinting and antibiotic resistance. MATERIALS AND METHODS: Forty isolates recovered from an urban sewage treatment plant were studied during 2009- 2010. The antibiotic resistance of isolates against 7 antibiotics was examined by disk diffusion method. Extraction of plasmid DNA was performed and identification of van genotype (vanA and vanB) was done by PCR. Biochemical fingerprinting was done by the use of Phene-Plate system (PhP). RESULTS: All isolates were found to be resistant to erythromycin, ampicillin and ciprofloxacin. The PCR analyses showed that all E. faecium isolates harbored vanA gene and 5 (13%) isolates harbored vanA and vanB concomitantly. By plasmid profiling the VRE isolates differentiated into 11 types. PhP showed that VRE isolates were grouped into 23 biochemical types. CONCLUSIONS: The combination of plasmid profiling and PhP techniques revealed the presence of diverse population of VRE in sewage treatment plant in Tehran. Furthermore, the results showed that the PhP technique is a reliable method in determining the VRE clonal diversity.

Characteristics of hospital- and community-acquired meticillin-resistant Staphylococcus aureus in Tehran, Iran.

Staphylococcus aureus is a leading cause of hospital-acquired (HA) and community-acquired (CA) infections worldwide. Recently, S. aureus strains resistant to meticillin (MRSA) have become established within both communities. We isolated 314 isolates of MRSA from hospitalized patients in a referral hospital (HA isolates) and 268 isolates from its outpatient clinic (CA isolates) in Tehran, Iran, between February 2008 and December 2010. These isolates were tested for their susceptibility to 17 antibiotics and typed using the PhPlate system. The diversity in the structures of staphylococcal cassette chromosome mec (SCCmec) elements and ccr types was also detected using a multiplex-PCR assay and isolates were examined for the presence of different classes of prophages. Whilst all isolates were resistant to penicillin, the HA isolates were significantly more resistant to all other antibiotics tested than the CA isolates. Isolates carrying only SCCmec type III and ccr type 3 were dominant (91 %), but 20 % of the CA isolates belonging to less prevalent types carried only SCCmec types IVa, c and ccr type 2. These isolates also carried pvl genes and contained SGA prophage type. Our results indicate that whilst the dominant clonal groups of HA- and CA-MRSA belong to SCCmec type III and carry ccr type 3 genes, several distinct but less prevalent types of CA-MRSA carrying SCCmec type IVa, c and type 2 ccr are also found in Tehran. These strains carry pvl genes and the SGA prophage type, a characteristic that might be used as a marker for detection of CA-MRSA in this country.

Prophage and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus strains in Iran.

A total of 114 isolates of methicillin-resistant Staphylococcus aureus (MRSA) were collected from hospitals in Tehran, Iran. A multiplex PCR was designed to examine the presence of six different prophage classes. The results showed high diversity of bacteriophages, with four different prophage types and eight prophage patterns. An important S. aureus phage coding for several virulence factors, Φ-77-like phage, was detected in 97 % of the isolates. We found a high rate of resistance of MRSA isolates to penicillin, ciprofloxacin, tobramycin and kanamycin. This is the first study showing high prevalence and diverse bacteriophage populations in MRSA strains in Iranian hospitals.

Epidemiological link between wastewater and human vancomycin-resistant Enterococcus faecium isolates.

We investigated the prevalence of vancomycin-resistant enterococci (VRE) isolated from wastewater (n = 593) and clinical (n = 450) samples, and the genetic linkage between the isolates was compared. Out of the total samples, 38 Enterococcus faecium (3.6%) from sewage (n = 19) and clinical (n = 19) isolates were found to be highly resistant to vancomycin. The majority of the VRE isolates from the two sources showed distinct phenotyping and genotyping patterns. At the same time, one common pulsed-field gel electrophoresis pattern was found among the VRE obtained from wastewater and human clinical isolates, suggestive of an epidemiological link.

Distribution of enterococcal species and detection of vancomycin resistance genes by multiplex PCR in Tehran sewage.

BACKGROUND: Enterococci are important because of their role as the leading cause of nosocomial infections which have a significant role in the dissemination and persistence of antimicrobial resistance genes. METHODS: In this study, we determined the distribution of enterococcal species in the sewage treatment plants in Iran. Furthermore, we improved a rapid and specific PCR method using primers (sodA and ddl genes) for identification of enterococci spp. RESULTS AND CONCLUSION: A total number of 712 enterococci spp. were isolated and the results showed that 56%, 24%, 12%, 4%, 2%, 1% and 1% isolates were E. faecium, E. hirae, E. faecalis, E. gallinarum, E. casseliflavus, E. mundtii and other enterococcal spp., respectively. The use of species-specific PCR was in agreement with the biochemical tests. Furthermore, multiplex PCR was developed to study the presence of vancomycin resistant genes in E. faecium or E. faecalis. The multiplex PCR appeared to be a useful, rapid and specific method for detecting and discriminating genotypes for vancomycin-resistant Enterococcus.