Carrying a 112 bp-segment in Helicobacter pylori dupA may associate with increased risk of duodenal ulcer.

The discovery of Helicobacter pylori in 1983 challenged researchers around the world to identify this pathogen's major virulence factors. The main rationale for this kind of research was to identify a biomarker associated with specific diseases following H. pylori colonization. Among different investigated virulence factors, duodenal ulcer promoting gene A (dupA) has been found to be associated with duodenal ulcer (DU), but its effect was different in various geographical regions. To determine the prevalence of dupA, we applied both classic primer pairs and our newly developed primers producing a highly conserved segment in PCR method. In our survey, 143 (47%) H. pylori isolates were obtained from 304H. pylori-colonized individuals [age range of 19-92; 113 (37%) males with the mean age of 50 and 191 (63%) females with the mean age of 49]. The presence of the dupA gene was investigated by using the different specific primers. The prevalence of the 112 bp segment isolated from H. pylori strains recovered from DU, GU and atrophy groups were significantly higher (81%, p value = .002, 64%, p = .065, 68% and p = .047 38%, respectively) than our control group, where the prevalence of the 112 bp segment was only 38%. Interestingly, a significant relationship was observed between the occurrence of DU and the presence of the 112 bp segment [p = .002; OR: 6.98; (95% CI: 1.94-25.00)]. Taken as a whole, we believe the 112 bp region of H. pylori dupA may serve as the first detected biomarker for the early detection of DU in patients admitted to hospitals.

A survey on chimeric UreB229-561-HpaA protein targeting Helicobacter pylori: Computational and in vitro urease activity valuation.

Helicobacter pylori (H. pylori) as microaerophilic, Gram-negative bacterium colonize the human gastric milieu, where it impetuses chronic disorders. Vaccination is a complementary plan, along with antibiotic therapy, for clearance of H. pylori. Today, Computer based tools are essential for the evaluation, design, and experiment for novel chimeric targets for immunological administration. The purpose of this experiment was immunoinformatic analysis of UreB and HpaA molecules in a fusion arrangement and also, construction and expression of recombinant protein containing chimeric sequences. The targets sequences were screened by using of standard in silico tools and immunoinformatic web servers. The high-resolution 3D models of the protein were created and were validated; indeed, the B-and T-cell restricted epitopes were mapped on the chimeric protein. The recombinant protein in frame of the expression vector pET28a were expressed and purified successfully. The urease activity and immunoblotting were performed in vitro condition. This study confirmed that the engineered protein as a highly conserved, hydrophilic, non-allergenic contained remarkable B-cell and T-cell epitopes. It was magnificently attained; chimeric UreB229-561-HpaA could provoke both humoral and cellular immunity. The immunoblotting was shown that the chimeric protein could be detected by serum of immunized animal and H.pylori positive patients. In this study, several antigenic patches from UreB and HpaA were identified that could be an efficient immune system activator. The in vitro analysis of our chimeric molecule confirmed its urease activity. It also confirmed that the chimeric protein could be detected by serum of immunized animal and H.pylori positive patients.

Expression and purification of recombinant TAT-BoNT/A(1-448) under denaturing and native conditions.

Botulinum toxin type A can temporarily inhibit muscle contraction. Currently, physicians administer this toxin as a bio-drug in treatment of some muscle contraction disorders. TAT-BoNT/A(1-448) is a functional recombinant protein derived from botulinum toxin light chain. Unlike the full length botulinum toxin, TAT-BoNT/A(1-448) is a self-permeable molecule which can pass through bio-surfaces so can be used as a topical therapeutic agent without injection. To maintain the functionality of TAT-BoNT/A(1-448), it is necessary to restore its normal folding upon expression and purification. In this study, we have investigated and optimized expression conditions for this novel recombinant protein. Under denaturing condition (1 mM IPTG, at 37°C), the chimeric protein was produced as inclusion body and required to be purified using denaturing agents (e.g. urea). Yet, lower incubation temperature (18°C) and less IPTG concentration (0.5 mM) induce a protein under native condition. In such condition, about 60% of the chimeric protein was expressed in soluble form.

Genotyping and characterization of CTX-M-15 -producing Klebsiella pneumoniae isolated from an Iranian hospital.

The aims were to describe the genetic characterization of blaCTX-M-1 group gene in Klebsiella pneumoniae and to investigate the relationship between isolates by MLVA and PFGE. We analyzed 36 CTX-M group 1-ESBL producing K. pneumoniae. rmpA and wcaG virulence genes were identified by PCR. The genetic environment of blaCTX-M-1 was analyzed by PCR and sequencing. Plasmid replicons were determined using PCR-based replicon typing. The isolates were typed by MLVA and PFGE. All blaCTX-M-1 were blaCTX-M-15. The wcaG and rmpA were detected in 1 and 2 isolates, respectively. IncF were the most frequently detected replicons (63.88%). In all isolates, ISEcp1 was found upstream and orf477 downstream of blaCTX-M-15, IS26 was found in two isolates. MLVA identified 20 MLVA types, whereas PFGE identified 25 different profiles. The dissemination of CTX-M-15 in our isolates was due to the clonal spread of isolates and to the genetic transfer of mobile elements among unrelated strains.

TAT-BoNT/A(1₋448), a novel fusion protein as a therapeutic agent: analysis of transcutaneous delivery and enzyme activity.

Botulinum neurotoxin type A (BoNT/A) has been used as an injectable therapeutic agent for the treatment of some abnormal muscle contractions. In this study, TAT(47-57) peptide, a cell-penetrating peptide, was fused with the catalytic domain of BoNT/A for therapeutic purposes. HeLa and BE(2)-C cell lines were treated separately with purified TAT-BoNT/A(1-448) recombinant protein, and transduction of protein was analyzed by western blotting. Also, transcutaneous delivery through mouse skin surface was evaluated by immunohistochemistry. The in vitro catalytic activity of TAT-BoNT/A(1-448) was evaluated by HPLC. The presence of recombinant protein was detected in both of the cell lines as well as mouse skin cryosections after 60 and 120 min of incubation. The concentration of intracellular proteins was increased over time. HPLC analysis showed that this fusion protein has a biological activity 1.5 times as much as the full-length BoNT/A(1-448) protein. TAT-BoNT/A(1-448) fusion protein is biologically active and can transmit through living cells in vitro and in vivo successfully and more effectively compared with BoNT/A(1-448) protein as control.

Preparation and Evaluation of a New Lipopolysaccharide-based Conjugate as a Vaccine Candidate for Brucellosis.

OBJECTIVES: Development of an efficacious vaccine against brucellosis has been a challenge for scientists for many years. At present, there is no licensed vaccine against human brucellosis. To overcome this problem, currently, antigenic determinants of Brucella cell wall such as Lipopolysaccharide (LPS) are considered as potential candidates to develop subunit vaccines. METHODS: In this study, Brucella abortus LPS was used for conjugation to Neisseria meningitidis serogroup B outer membrane vesicle (OMV) as carrier protein using carbodiimide and adipic acid-mediated coupling and linking, respectively. Groups of eight BALB/c mice were injected subcutaneously with 10 µg LPS alone, combined LPS + OMV and conjugated LPS-OMV on 0 days, 14 days, 28 days and 42 days. Anti-LPS IgG was measured in serum. RESULTS: The yield of LPS to OMV in LPS-OMV conjugate was 46.55%, on the basis of carbohydrate content. The ratio for LPS to OMV was 4.07. The LPS-OMV conjugate was the most immunogenic compound that stimulated following the first injection with increased IgG titer of ∼5-fold and ∼1.3-fold higher than that produced against LPS and LPS in noncovalent complex to OMV (LPS + OMV), respectively. The highest anti-LPS IgG titer was detected 2 weeks after the third injection (Day 42) of LPS-OMV conjugate. The conjugated compound elicited higher titers of IgG than LPS + OMV, that showed a 100-120-fold rise of anti-LPS IgG in mice. CONCLUSION: These results indicate that our conjugated LPS-OMV can be used as a brucellosis vaccine, but further investigation is required.

Protective efficacy of recombinant exotoxin A--flagellin fusion protein against Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa is an opportunistic bacterium that causes serious nosocomial infection in immunocompromised patients. The aim of this study was to prepare a fusion protein consisting of exotoxin A (ExoA) and flagellin (Fla) from P. aeruginosa and to evaluate its potential as a vaccine candidate against P. aeruginosa infection. The genes encoding for ExoA and Fla proteins were cloned in-frame and expressed in Escherichia coli. The recombinant ExoA-Fla fusion protein was purified by Ni-NTA affinity chromatography. Mice were immunized subcutaneously with ExoA, Fla, and ExoA-Fla fusion proteins, and the humoral immune response was evaluated by ELISA method. The immunized and control group mice were challenged with a 2× LD50 (7.5 × 10(7) CFU) of P. aeruginosa for the protection assay. The results indicated that vaccination with Fla, ExoA, and ExoA-Fla fusion proteins produced a significant amount of specific immunoglobulin G antibodies. Immunization of mice with ExoA-Fla fusion protein showed significant protection against intraperitoneal challenge with 7.5 × 10(7) CFU (2× LD50) P. aeruginosa. Results of this study suggest that recombinant ExoA-Fla fusion protein is a highly immunogenic protective protein showing promise as a vaccine candidate against P. aeruginosa.

Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection.

The aim of this study was to investigate the phylogenetic background and to assess hlyD (involved in the secretion of haemolysin A) and intI1 (encoding a class 1 integrase) in Escherichia coli isolates derived from urinary and fecal specimens. A total of 200 E. coli isolates was collected from patients presenting with urinary tract infection (UTI) during September 2009 to September 2010 and screened for hlyD and intI1 genes by polymerase chain reaction (PCR). Phylogenetic analysis showed that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D) and that uropathogenic E. coli (UPEC) isolates mainly belong to groups B2 (54%) and D (34%) whereas group A (44%) and D (26%) are predominant among commensal E. coli isolates. In this study, hlyD was present in 26% of UPEC and 2% of commensal E. coli isolates. However, hemolytic activity was detected for 42% of UPEC and 6% of commensal E. coli isolates (p < 0.05). intI1 gene was more frequently expressed in UPEC (24%) in comparison with commensal E. coli isolates (12%). Resistance to aztreonam, co-trimoxazole and cefpodoxime were frequently found among UPEC isolates whereas commensal E. coli isolates were commonly resistant to co-trimoxazole, nalidixic acid and cefotaxime. Concluding, a considerable difference between UPEC and commensal E. coli isolates was observed regarding their phylogenetic groups, presence of class 1 integron and hlyD gene, hemolysin activity and resistance pattern. The detection of class 1 integrons and hlyD gene was higher among UPEC compared with commensal E. coli isolates. These findings may contribute for a better understanding of the factors involved in the pathogenesis of UPEC.

High rate of aminoglycoside resistance in CTX-M-15 producing Klebsiella pneumoniae isolates in Tehran, Iran.

OBJECTIVE: To determine the prevalence of extended-spectrum beta lactamases (ESBLs), the bla(CTX-M) genes, and aminoglycoside modifying enzymes genes in clinical isolates of Klebsiella pneumoniae (K. pneumoniae). METHODS: We collected 200 nonduplicate clinical isolates of K. pneumoniae in hospitals in Tehran, Iran. We determined antibacterial susceptibility and confirmed ESBL production via the disk diffusion and minimum inhibitory concentration (MIC) methods. We identified bla(CTX-M) and aminoglycoside modifying enzymes genes via polymerase chain reaction (PCR). RESULTS: We detected 72 (36.0%) ESBL-positive K. pneumoniae, in which the bla(CTX-M-15) was dominant (62.5%). A total of 54.0% of isolates were resistant to at least 1 tested aminoglycoside; also, we detected aac(6')-Ib in 42.5% of isolates and aac(3)-IIa in 35.1% of them. We observed a high rate of aminoglycoside-resistant genes (71.0%) among bla(CTX-M-15)-carrying isolates. CONCLUSION: We report that CTX-M-15 is the dominant type of CTX-M, which associates with entities that have high aminoglycoside resistance. Continuous surveillance and monitoring of this entity are needed because the codissemination of multiple drug-resistant genes with K. pneumoniae may become a serious therapeutic problem.

Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection

The aim of this study was to investigate the phylogenetic background and to assess hlyD (involved in the secretion of haemolysin A) and intll (encoding a class 1 integrase) in Escherichia coli isolates derived from urinary and fecal specimens. A total of 200 E. coli isolates was collected from patients presenting with urinary tract infection (UTI) during September 2009 to September 2010 and screened for hlyD and intll genes by polymerase chain reaction (PCR). Phylogenetic analysis showed that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D) and that uropathogenic E. coli (UPEC) isolates mainly belong to groups B2 (54%) and D (34%) whereas group A (44%) and D (26%) are predominant among commensal E. coli isolates. In this study, hlyD was present in 26% of UPEC and 2% of commensal E. coli isolates. However, hemolytic activity was detected for 42% of UPEC and 6% of commensal E. coli isolates (p < 0.05). intll gene was more frequently expressed in UPEC (24%) in comparison with commensal E. coli isolates (12%). Resistance to aztreonam, co-trimoxazole and cefpodoxime were frequently found among UPEC isolates whereas commensal E. coli isolates were commonly resistant to co-trimoxazole, nalidixic acid and cefotaxime. Concluding, a considerable difference between UPEC and commensal E. coli isolates was observed regarding their phylogenetic groups, presence of class 1 integron and hlyD gene, hemolysin activity and resistance pattern. The detection of class 1 integrons and hlyD gene was higher among UPEC compared with commensal E. coli isolates. These findings may contribute for a better understanding of the factors involved in the pathogenesis of UPEC.

In Vitro Activity of Tigecycline and Colistin against clinical isolates of Acinetobacter baumannii in Hospitals in Tehran and Bandar-Abbas, Iran.

BACKGROUND: The Acinetobacter species, particularly A. baumannii, has emerged as one of the main causes of nosocomial infections in recent years. The high prevalence of drug resistance in A. baumannii limits the therapeutic options for treating infections caused by these bacteria. The objective of this study was to determine the in vitro activity of Tigecycline and Colistin against clinical isolates of A. baumannii in Tehran and Bandar Abbas, Iran. METHODS: This study was conducted from March 2009 to November 2010 at three hospitals in Tehran and Bandar Abbas, Iran, using 165 Acinetobacter species isolated from clinical specimens. All isolates were subjected to PCR to detect bla OXA-51-like genes that are unique to Acinetobacter baumannii. Isolates that gave a band for the bla OXA-51-like genes were identified as A. baumannii. Anti-microbial susceptibility tests were performed for Tigecycline, Colistin, and other antibiotics. RESULTS: Sensitivity rates to Colistin and Polymyxin-B were 100%. Resistance rates for Tigecycline were 4.2% in Tehran and 8.8% in Bandar-Abbas according to Jones criteria, whereas, according to U.S. FDA criteria, the resistance rates were 20.8% and 17.6%, respectively. CONCLUSIONS: New alternative drugs are needed for the treatment of drug resistant A. baumannii. Although Colistin appears to be a good choice, adverse reactions have limited its usage. Tigecycline is effective against A. baumannii isolates, and it shows promise for solving the problem.

Genetic characterization of a vancomycin-resistant Staphylococcus aureus isolate from the respiratory tract of a patient in a university hospital in northeastern Iran.

Emergence of vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains has led to global concerns about treatments for staphylococcal infections. These strains are currently rare even though there is an upward trend in their reported incidence. Therefore, appropriate screening and epidemiological evaluation of VRSA strains can affect future global health care policies. Isolates of Staphylococcus aureus were obtained from various clinical samples and were then evaluated with agar screening, disk diffusion, and MIC methods to determine resistance to vancomycin and methicillin. After confirmation of the isolated VRSA strain, genetic analysis was performed by evaluating mecA and vanA gene presence, SCCmec, agr, and spa types, and toxin profiles. Multilocus sequence typing (MLST) and plasmid analysis were also performed. The VRSA strain was resistant to oxacillin (MIC of 128 µg/ml) and vancomycin (MIC of 512 µg/ml). Disk diffusion antimicrobial susceptibility tests showed resistance to oxacillin, vancomycin, levofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, clindamycin, rifampin, and tetracycline. The isolate was susceptible to minocycline and gentamicin. PCRs were positive for the mecA and vanA genes. Other genetic characteristics include SCCmec type III, agr I, spa type t037, and sequence type (ST) 1283. The plasmid profile shows five plasmids with a size of ~1.7 kb to >10 kb. The isolated VRSA strain was obtained from a critically ill hospitalized patient. Genetic analysis of this strain suggested that the strain was a methicillin-resistant S. aureus (MRSA) clone endemic in Asia that underwent some genetic changes, such as mutation in the gmk gene and acquisition of the vanA gene.

Recombinant outer membrane secretin PilQ(406-770) as a vaccine candidate for serogroup B Neisseria meningitidis.

Secretin PilQ is an antigenically conserved outer membrane protein which is present on most meningococci. This protein naturally expressed at high levels and is essential for meningococcal pilus expression at the cell surface. A 1095 bp fragment of C-terminal of secretin pilQ from serogroup B Neisseria meningitidis was cloned into prokaryotic expression vector pET-28a. Recombinant protein was overexpressed with IPTG and affinity-purified by Ni-NTA agarose. BALB/c mice were immunized subcutaneously with purified rPilQ(406-770) mixed with Freund's adjuvant. Serum antibody responses to serogroups A and B N. meningitidis whole cells or purified rPilQ(406-770) and functional activity of antibodies were determined by ELISA and SBA, respectively. The output of rPilQ(406-770) was approximately 50% of the total bacterial proteins. Serum IgG responses were significantly increased in immunized group with PilQ(406-770) mixed with Freund's adjuvant in comparison with control groups. Antisera produced against rPilQ(406-770) demonstrated strong surface reactivity to serogroups A and B N. meningitidis tested by whole-cell ELISA. Surface reactivity to serogroup B N. meningitidis was higher than serogroup A. The sera from PilQ(406-770) immunized animals were strongly bactericidal against serogroups A and B. These results suggest that rPilQ(406-770) is a potential vaccine candidate for serogroup B N. meningitidis.

The prevalence of aminoglycoside-modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in Pseudomonas aeruginosa.

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6')-II (36%) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a significant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B.

INTRODUCTION: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA. METHODOLOGY: A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porA plasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification. RESULTS: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle. CONCLUSION: This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.

Cloning and Expression of S1 Subunit of Pertussis Toxin in Escherichia coli.

Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human (whooping cough). Pertussis toxin (PT) is the main component of current acellular pertussis vaccine and the S1 (subunit1) is the main immunogenic part of it. Thus, S1 has been the target of many studies as a potent candidate of acellular vaccine against Bordetella pertussis, lacking the side effects of whole cell based ones. S1 gene was amplified and inserted in three expression vectors including pET-14b, pET-22b(+) and pAED4. The possibility and level of expression of these constructs were investigated in BL21 (DE3) strain of Escherichia coli (E.coli) as expression host. The highest expression was in pET-22b(+)-S1. Best expression achieved 6 hr post induction with 0.2 mM IPTG in LB broth containing ampicillin, at 30°C with shaking (250 rpm). Recombinant S1 protein was observed in two distinct separated proteins with 28 and 31 kDa estimated molecular weight. In spite of toxicity of PT and S1 in the E.coli, considerable amount of S1 was expressed in E.coli. Two rS1 bands were detected on SDS-PAGE. Both were confirmed as S1 in western blot with specific monoclonal and polyclonal antibodies against pertussis toxin. Appearance of two distinct bands could be the result of leader peptidase activity or nonspecific peptidase from E.coli on recombinant S1. As the recombinant S1 is a suitable antigen for studies as a candidate acellular vaccine or development of ELISA for detection of Bordetella pertussis, further studies are underway.

The prevalence of aminoglycoside-modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43 percent, tobramycin 38 percent, and amikacin 24 percent. Of the genes examined, aac (6')-II (36 percent) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

Association of Ureaplasma urealyticum infection with varicocele-related infertility.

BACKGROUND: The role of seminal colonization of Ureaplasma urealyticum in varicocele-related infertility was investigated. METHODOLOGY: Semen samples were obtained from infertile patients with or without varicocele and healthy controls and were subjected to routine semen analysis and PCR. DNA was extracted by Cadieux method and analyzed by PCR protocol with species-specific primers for U. urealyticum (urease gene). RESULTS: U. urealyticum was detected by PCR in 23 of 146 (15.75%) semen specimens from infertile patients and in 3 of 100(3%) healthy men (P<0.001). Infertile patients with varicocele had higher U. urealyticum colonization [17/81(20.98%)] than those without varicocele [6/65(9.23%), P=0.086] or healthy controls [3/100 (3%), P<0.001].The percentage of sperm cells with motility, volume of semen fluid, concentration of sperm cells, and sperm cell with normal morphology were significantly decreased in infertile men (P<0.001). In the group of varicocele patients with PCR positive for U. urealyticum the volume, count and morphology of semen samples were lower than those in the varicocele patients with PCR negative results, but the differences were not significant (P>0.05). CONCLUSION: Although the colonization of U. urealyticum does not affect the semen quality, the high prevalence of this microorganism in varicocele patients may be an additional negative factor affecting varicocele status and worsening reproductive potential.

Influence of type 1 piliation on chemotaxis and intracellular killing of uropathogenic E. coli by human polymorphonuclear leukocyts.

Uropathogenic Escherichia coli (UPEC) that cause urinary tract infections bind to target cells via several distinct paris of adhesins and receptors. In this study we determine the role of type 1 pili in interaction of UPEC with human neutrophils. Type 1 piliated and unpiliated strains (obtained by growth at a pilus-restrictive temperature) of UPEC were used for determining the effect of this adhesin on migration of neutrophils towards bacteria in Boyden chamber. The lectinophagocytosis and intracellular killing of bacteria with purified human neutrophils were estimated by counting of the number of viable bacteria in 45 min. The results indicate that type 1 piliated UPEC stimulated significantly greater chemotaxis than did unpiliated bacteria and bacteria in which the piliation was suppressed. Phagocytosis of type 1 piliated UPEC occurred in the direct and opsonin-independent manner. In contrast, unpiliated bacteria failed to bind to PMN.