Increased Risk of Infertility in Women Infected with Human Papillomavirus.

Background: Among several causes of infertility, urogenital infections seem to be influencing factors. The effect of bacterial or viral sexually transmitted infections (STIs) on human fertility is not well understood. The aim of this study was to determine the frequency of STIs in cervical samples of infertile and fertile women and study the relationship between these agents and infertility. Methods: In this case-control study, cytobrush was used for collecting of cervical sample from each infertile and fertile woman (n=95) who attended Research and Clinical Centers for Infertility in Kerman, Iran. PCR and real-time PCR methods were used to detect the presence of bacterial (genital Ureaplasma species, genital Mycoplasma species, Chlamydia trachomatis (C. trachomatis), and Gardnerella vaginalis) and viral (herpes simplex virus, human papillomavirus (HPV), and Epstein-Barr virus) agents, respectively. Fisher's exact test and the logistic regression with the significance level of ≤5% were used for statistical analyses. Results: In general, 78.94% and 14.73% of specimens were positive for one or more studied microorganisms, respectively. Among studied agents, only the infection with HPV was significantly different between infertile and fertile groups (p=0.005) which may enhance the likelihood of female infertility (OR=5.30, 95% CI:1.47-19.11, p< 0.05). After adjusting for age, irregular menstrual cycle, abnormal vaginal discharge, and ectopic pregnancy, the odds ratio of infertility in HPV-infected women increased (OR=7.02, 95% CI:1.52-32.3, p<0.05). Conclusion: Since HPV infection is asymptomatic, periodic screening of women in reproductive age especially infertile couples is recommended for early diagnosis and prevention of infection progression and cross contamination.

Determination of efflux activity in Enterococci by Hoechst accumulation assay and the role of zinc oxide nanoparticles in inhibition of this activity.

BACKGROUND: Contribution of efflux pumps in development of antimicrobial resistance has been largely addressed in Gram negative and to a much lesser extent in Gram positive bacteria. Measuring accumulation of Hoechst (H) dye is known as a safe and rapid method for monitoring efflux activity in bacteria. Antimicrobial effects of metal nanoparticles have been attributed in part to inhibition of efflux pumps. This study aimed to first determine efflux activity in enterococci by Hoechst accumulation assay, and to second characterize the role of zinc oxide nanoparticles (ZnONPs) in inhibition of these pumps. RESULTS: Increased accumulation of Hoechst dye showed more potential of ZnONPs in efflux inhibition compared with CCCP. H33258 represented more suitability for accumulation studies in enterococci. Two to six-fold reduction in minimum inhibitory concentration (MIC) values of antimicrobial agents in the presence of ZnONPs was observed. CONCLUSIONS: Efflux activity in enterococcal strains can be measured by H33258 accumulation assay. Application of ZnONPs as an efflux inhibitor, may rejuvenate the use of conventional antimicrobial agents against these bacteria.

Comparative study of Staphylococcus aureus from burn patients and healthcare workers in a burn center, Yazd, Iran.

Healthcare workers (HCWs) are proposed as the potential source of transmission of Staphylococcus aureus to hospitalized patients, especially in burn units. This study aimed to investigate S. aureus from burn wound infections and those from the nose of HCWs in terms of antibiotic resistance, the presence of Panton-Valentine leucocidin-encoding gene (pvl) and the arginine catabolic mobile element (ACME), and the ability for biofilm formation. Also, the genetic diversity of isolates was assessed using staphylococcal protein A (spa) typing and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, regarding the studied factors, significant differences were found neither between isolates from patients and HCWs nor between methicillin-resistant and methicillin-susceptible isolates (except for multidrug resistance which was significantly higher in MRSA). The most frequent SCCmec types were type I and III. ACME-arcA was only detected in isolates from patients and similarly the presence of ACME-opp3 was the most prevalent in this group. The presence of common clonal complexes among patient isolates and more importantly between isolates from patients and HCWs is warning. The high prevalence of virulence factors, both in MRSA and MSSA, emphasizes the importance of MSSA in burn centers. Finding no significant difference in the presence of virulence-associated factors between isolates from patients and HCWs demonstrates the need to take HCWs into account as important reservoirs.

Reduced Susceptibility to Biocides among Enterococci from Clinical and Non-Clinical Sources.

BACKGROUND: Wide use of biocidal agents such as benzalkonium chloride (BCC) and chlorhexidine digluconate (CHX) in hospitals and non-hospital environments, has raised concerns over the emergence of non-susceptible strains. Efflux pumps are of known main mechanisms in biocide tolerance which have been rarely addressed in enterococci - members of gut microbiota which can cause serious problems particularly in hospitalized patients. The purpose of this study was to investigate the susceptibility of enterococci from different sources (clinical and fecal isolates) toward BCC and CHX, and its correlation with efflux associated genes. Also, possible link between biocide tolerance and antibiotic resistance was examined. MATERIALS AND METHODS: One hundred and four enterococcus isolates including clinical (n = 54) and fecal isolates (n = 50) were studied for susceptibility toward BCC, CHX, ciprofloxacin, gentamicin and vancomycin. Twelve efflux associated genes were investigated by polymerase chain reaction assay. RESULTS: In clinical isolates, reduced susceptibility to CHX and resistance to gentamicin and ciprofloxacin were significantly higher than fecal isolates. Vancomycin resistance was associated with increasing minimum inhibitory concentration of CHX. Among all investigated genes, only three ones, efrA, efrB and emeA were detected which were significantly associated with reduced susceptibility to CHX and were more frequent among clinical isolates. Also, high level resistance to gentamicin was significantly associated with the presence of efrA/B as well as with reduced susceptibility to CHX. CONCLUSION: As expected, reduced susceptibility to CHX, was significantly higher in clinical isolates. However, the presence of a vancomycin-resistant enterococci among fecal isolates of healthy people which showed resistance/tolerance to studied antimicrobial agents, was unexpected and highlights the need to investigate other non-hospital environments to avoid dissemination of antimicrobial resistance. Correlation between reduced susceptibility to CHX and high level resistance to gentamicin, substantiates monitoring of biocide tolerance particularly in the healthcare settings to control the establishment of antimicrobial resistant strains.

High-Level Resistance to Erythromycin and Tetracycline and Dissemination of Resistance Determinants among Clinical Enterococci in Iran.

OBJECTIVES: The purpose of this study was to investigate the distribution pattern of genes responsible for erythromycin and tetracycline resistance and their association with resistance phenotypes in enterococcus isolates. MATERIALS AND METHODS: Eighty-six Enterococcus faecalis and 26 E. faecium isolates were collected from 2 hospitals in Kerman, Iran. Minimum inhibitory concentration of erythromycin and tetra-cycline was determined and then genes encoding resistance to erythromycin - erm (A-C), mef, and msr - and tetracycline - tet (M), tet (O), tet (S), tet (K), and tet (L) - were investigated. RESULTS: In all resistant isolates (n = 72, 64%), high-level resistance to both tested antibiotics was found. The most prevalent erm gene was erm (B) (77.7%), followed by erm (A) (15.2%) and erm (C) (8.3%). Genes mediating erythromycin efflux were detected in 70.8% (mef) and 9.7% (msr) of resistant isolates. Regarding tetracycline, tet (M) was detected at the highest rate (50%), followed by tet (O) (31%) and tet (S) (11%). Export of tetracycline was found in 31% (tet (K)) and 12% (tet (L)) of isolates. CONCLUSION: A high prevalence of high-level resistance to both erythromycin and tetracycline was documented. Alterations at the ribosomal level was more frequently detected in erythromycin and tetracycline resistance than efflux systems. Concurrent resistance mechanisms were more involved in resistance to erythromycin than tetracycline.

Bacteriospermia and its association with seminal fluid parameters and infertility in infertile men, Kerman, Iran: A cross-sectional study.

Background: The role of genital Ureaplasma species, genital Mycoplasma (M) species, and Chlamydia (C.) trachomatis, the most prevalent sexually transmitted bacteria, in male infertility are still not clear. Different reports about the impact of these bacteria on semen quality are controversial. Objective: This study was proposed to determine the frequency of bacteriospermia in men and investigate the relationship between the presence of these bacteria and semen quality using molecular assay. Materials and Methods: In this cross-sectional study, 200 semen samples obtained from men attending the research and clinical centers for fertility in Kerman, Iran, between July and December 2019 were analyzed for semen volume, progressive motility, non-progressive motility, total progressive motility, and viability according to the World Health Organization guidelines. The polymerase chain reaction was used for the detection of related bacteria. Results: The mean values of volume, progressive motility, non-progressive motility, total progressive motility, and viability were significantly lower in infertile men (p < 0.001). Statistically significant correlations were observed between the presence of M. genitalium and progressive sperm motility, M. hominis and semen volume, Ureaplasma parvum and the sperm normal form, and C. trachomatis and the sperm progressive motility and viability. Logistic regression analysis showed that M. genitalium (OR = 8.06, p < 0.001) and C. trachomatis (OR = 16, p = 0.01) were significantly associated with male infertility. Conclusion: During the infertility assessment, clinicians should consider of role C. trachomatis and M. genitalium in male infertility. Screening test particularly for asymptomatic individuals is recommended.

Clonal dissemination of high-level gentamicin-resistant isolates of Enterococcus faecalis within a university hospital in southeastern Iran.

BACKGROUND: Combination of a cell wall-active antibiotic with an aminoglycoside confers a synergistic effect in the treatment of some severe enterococcal infections. Unfortunately, with the emergence of enterococci with high-level resistance to aminoglycosides, particularly to gentamicin, the efficacy of the synergistic combinations has decreased. In this study, high-level gentamicin-resistant (HLGR) isolates of enterococci and the diversity of the genes encoding aminoglycoside-modifying enzymes (AMEs) as well as putative clonal dissemination of HLGR isolates were investigated in a university hospital in southeastern Iran. METHODS: The minimum inhibitory concentration of gentamicin was determined and HLGR isolates were investigated for AME genes. Genetic similarity between isolates was analyzed using repetitive extragenic palindromic (rep)-Polymerase Chain Reaction (PCR) assay. RESULTS: Of 150 Enterococcus isolates, 62 isolates including Enterococcus faecalis (n = 46) and E. faecium (n = 16) were identified as HLGR. The most prevalent AME genes in both species were as follows: aph(3')-IIIa (n = 44), aac(6')-Ie-aph(2')-Ia (n = 36), and ant(4')-Ia (n = 15). The rep-PCR analysis showed clonality among E. faecalis isolates, so that 27 isolates were grouped in seven clusters representing similarity greater than 95%. CONCLUSIONS: No link between AME determinants and clusters was found. Clonal spread of HLGR isolates of E. faecalis was found within our hospital. More rigorous recommendations are required to avoid dissemination of such resistant microorganisms in the hospital setting.

Spa gene-based molecular typing of nasal methicillin-susceptible staphylococcus aureus from patients and health-care workers in a dialysis center in southeast Iran.

Staphylococcus aureus is an important infectious agent in hemodialysis patients. This study was conducted to determine the frequency of S. aureus nasal carriage in hemodialysis patients (HD) and health-care workers (HCW) at the main dialysis center of Bam city, located in southeast of Iran. In this cross-sectional study, a total of 52 nasal swabs were obtained from health-care workers and hemodialysis patients to detect methicillin-sensitive S. aureus (MSSA) isolates. The resistance to different antibacterial agents was determined by disk diffusion method. Also, Panton-Valentine Leukocidin (PVL) - encoding gene as well as Staphylococcal protein A (spa) type were determined. The nasal carriage rate of S. aureus was found to be 24.4% and 18.8% in patients on hemodialysis and health-care workers, respectively. Among identified isolates, no methicillin-resistant S. aureus (MRSA) was found. Only two MSSA isolates (16.7%) were resistant to trimethoprim/sulfamethoxazole. One isolate (8.6%) was positive for pvl gene. Moreover, 8 spa types were found. According to BURP analysis, six out of the 12 S. aureus isolates (50%) belonged to the same clone, indicating a prevalence of a major clone among MSSA in carriage, including patients and HCW. Mupirocin is still the appropriate drug for reducing nasal colonization in our setting. Accumulation of isolates from patients and staff in one spa clonal complex is alarming for the necessity of more serious infection control in this center. Therefore, it is necessary to screen patients and health-care workers as a health priority, in order to prevent cross transmission.

Co-Incidence of Type II Topoisomerase Mutations and Efflux Expression in High Fluoroquinolone Resistant Enterococcus faecalis Isolated from Urinary Tract Infections.

INTRODUCTION: Enterococcus faecalis is one of the most common pathogens in urinary tract infections (UTIs). Fluoroquinolones have been frequently used to treat E. faecalis UTIs, and the emergence of fluoroquinolone-resistant E. faecalis strains has recently been reported in several countries. This study aimed to elucidate the mechanisms involved in fluoroquinolone resistance in clinical E. faecalis isolates by analyzing mutations in quinolone- resistance-determining regions (QRDRs) of gyrA and parC and investigating the role of some efflux pumps. METHODS: In total, 70 clinical E. faecalis isolates collected from UTIs were identified by phenotypic and genotypic methods. Antimicrobial susceptibility testing was performed and multidrug-resistant (including ciprofloxacin resistant) isolates were studied for minimum inhibitory concentrations to ciprofloxacin, levofloxacin, and ofloxacin. In the following, mutations in QRDRs of gyrA and parC and expression of EfrA, EfrB, and EmeA efflux pumps were investigated in 20 high-level ciprofloxacin resistant and two ciprofloxacin susceptible isolates. RESULTS: High-level resistance to ciprofloxacin was detected in 97.5% of isolates. Sequencing of QRDRs revealed that 65% and 75% of isolates carried mutations in gyrA and parC, respectively. The presence of efflux genes was detected in all studied isolates, but expression of efrA, emeA, and efrB was demonstrated in 50%, 40%, and 30% of resistant isolates, respectively. Neither QRDR mutation nor the expression of efflux genes showed any significant association with MIC. CONCLUSION: Co-incidence of mutation and efflux gene expression in more than half of isolates (13/20) suggests that both mechanisms may play a role in fluoroquinolone resistance. The other unknown mechanisms including different efflux pumps and probably other QRDRs mutations may contribute to fluoroquinolone resistance in E. faecalis.

Amino acid substitution mutations and mRNA expression levels of the pbp5 gene in clinical Enterococcus faecium isolates conferring high level ampicillin resistance.

In this study, clinical ampicillin-resistant Enterococcus faecium isolates with minimum inhibitory concentrations (MICs) for ampicillin in the ranges from 128 to ˃512 µg/mL (n = 17) and two ampicillin-susceptible isolates (MIC 1 µg/mL) were investigated. No ß-lactamase production was detected in these isolates. Alterations in the C-terminal part of pbp5 and levels of pbp5 mRNA expression were investigated by sequencing and quantitative real-time qRT-PCR, respectively. Sequencing analysis revealed five different pbp5 alleles (A to E) having differences in 18 amino acid positions spanning from residue 426 to 642. Allele A (V-462 â†’ A, H-470 â†’ Q, M-485 â†’ A, N-496 â†’ K, A-499 â†’ T, E-525 â†’ D, N-546 â†’ T, A-558 â†’ T, G-582 â†’ S, E-629 â†’ V, K-632 â†’ Q, and P-642 â†’ L) was the most frequent allele. The presence of just two susceptible isolates in allele E suggests a possible correlation between amino acid patterns and MIC, even if there is no discernible correlation with specific single amino acid differences. Also, these were the only isolates that showed much lower expression of class B penicillin-binding protein 5 (PBP5) compared to isolates with MIC of 128 or greater. Thus, ampicillin MICs were correlated with PBP5 expression.

In vitro activity of linezolid alone and combined with other antibiotics against clinical enterococcal isolates.

BACKGROUND: The increasing incidence of antimicrobial resistance has led to research on finding new antimicrobial agents or identifying drug combinations with synergistic effects. Enterococcal infections, particularly those associated with vancomycin-resistant enterococci (VREs), are therapeutic problems. Linezolid (LZD), an oxazolidinone antibiotic, shows good activity against Gram-positive bacteria including enterococci. To avoid the emergence of linezolid-resistant subpopulations and achieve enhanced activity or bactericidal effect, the use of combined therapy has been considered. METHODS: The in vitro activity of LZD in combination with five different antibiotics was evaluated using a microdilution checkerboard method and time-kill study against 12 clinical enterococcus isolates. RESULTS: With the checkerboard method, LZD plus doxycycline (DX) had the highest frequency among all synergistic combinations. This combination and the one of LZD plus ceftriaxone (CRO) were the most frequent effective combinations against VREs. Time-kill studies using selected synergistic combinations-LZD + DX and LZD + CRO-showed an indifferent interaction. One tested combination of LZD + rifampicin showed antagonism. CONCLUSIONS: Antagonistic interactions in combinations containing LZD are rare. LZD + DX and LZD + CRO may be beneficial in the treatment of VREs. However, more time-kill studies as well as in vivo experiments are required.

Prevalence of meningococcal carriage among male university students living in dormitories in Kerman, southeast of Iran.

Neisseria meningitidis is an important causative agent of bacterial meningitis. The nasopharynx is the only known reservoir of this organism. Although the relationship between carriage and invasive disease is not completely understood, asymptomatic meningococcal carriers are considered as the most important sources for causing strains of disease. Living in closed and overcrowded places such as university dormitories can increase the carriage rate and meningococcal disease. This cross-sectional study was conducted to determine the prevalence of N. meningitidis carriers among male students living in three dormitories affiliated with Kerman University of Medical Sciences (Kerman, Iran). Nasopharyngeal swab was taken from all participants recruited in the study. Conventional microbiological tests were performed for isolation and detection of the organism. The amplification of crgA gene was used to confirm the identity of isolates. Molecular serogrouping was used to detect the six most frequent serotypes. The overall carriage rate was 6.8% (23/335). The capsular type of these isolates was in determinate (56.5%) or of serogroup C (43.5%). Multivariate logistic regression analysis revealed that cigarette smoking was significantly associated with meningococcal carriage (OR = 5.02; p = 0.01). Additionally, using univariate regression analysis, a significant association was found between water pipe smoking and carriage (p = 0.018). The rate of meningococcal carriage among male students in the studied population was lower as compared to other high-risk group (freshmen conscripts) in Iran. University students should be aware of the consequences of cigarette and water pipe smoking as risk factors in meningococcal carriage.

Distribution of Ebp pili among clinical and fecal isolates of Enterococcus faecalis and evaluation for human platelet activation.

Although Enterococcus faecalis is known as normal flora in colon, it is also amongst the most common causative agents of infective endocarditis (IE). Platelet activation resulting from adherence to platelets is an essential step in the pathogenesis of IE. One of the factors proposed in adhesion is endocarditis- and biofilm- associated pili encoded by ebp operon. The aim of this study was to investigate ebp in isolates from different origins and analyze the potential of isolates to activate human platelets of different donors. The ebp distribution was investigated in E. faecalis from different origin infections (n = 103) and fecal flora (n = 20). Then, selected isolates from blood (n = 5), urine (n = 2), and fecal flora (n = 3) were analyzed by flow cytometry assay for the ability to activate platelets of four different donors. No statistically significant difference was found for the ebp presence between infective and fecal isolates. Also, it was found that the ability for platelet activation is independent of the bacterial origin. However, significant difference was found in platelet activation between different donors. The results suggest that the presence or absence of ebp is not a critical factor for platelet activation by E. faecalis isolates. However, host factors seem to contribute in this activity.

Virulence Genes, Antibiotic Resistance and Capsule Locus Polymorphisms in Enterococcus faecalis isolated from Canals of Root-Filled Teeth with Periapical Lesions.

Frequent isolation of Enterococcus faecalis from root canal treated teeth with apical periodontitis, has proposed the role of this organism in endodontic treatment failures. Different factors have been suggested in the pathogenicity of this organism. In this study, 22 E. faecalis isolates from canals of root-filled teeth were identified, and phenotypic and genotypic characteristics were investigated. No resistance to vancomycin and gentamicin was noted, and most isolates (91%) were susceptible to ampicillin. Biofilm formation was detected in 73% of the isolates and may be considered as the most important virulence factor involved in the pathogenesis of these isolates.

Survey for Correlation between Biofilm Formation and Virulence Determinants in a Collection of Pathogenic and Fecal Enterococcus faecalis Isolates.

BACKGROUND: Enterococcus faecalis is an opportunistic pathogen that causes most of the enterococcal infections. Among the different factors implicated in the pathogenesis of these organisms, biofilm formation and antibiotic resistance are the most important. The ability for biofilm formation has been attributed to the presence of some virulence genes. However, no definite correlation has been found. This study aimed to detect biofilm formation and antibiotic resistance patterns in E. faecalis isolates collected from clinical and fecal samples, and to investigate possible correlation between some virulence genes (esp, cyl, gelE) and biofilm formation. MATERIALS AND METHODS: A collection of 123 E. faecalis isolates were investigated for antibiotic resistance and production of hemolysin, gelatinase, and biofilm using phenotypic methods. The esp, gelE and cyl genes were detected using polymerase chain reaction. RESULTS: Thirty-eight pathogenic isolates (37%) were positive for biofilm formation. Additionally, the gelE, esp, and cyl genes were detected in 74 (71.8%), 79 (76.7%) and 42 (40.8%) isolates, respectively. In the fecal samples, 18 (90%) isolates were biofilm producers and 11 (55%), 17 (85%) and 8 (40%) isolates were positive for gelE, esp, and cyl, respectively. There were significant differences in biofilm production between pathogenic and fecal isolates (P <0.001). Multidrug resistance (MDR) was found among 32% (n = 33) and 15% (n = 3) of the clinical and fecal isolates, respectively. However, no significant difference was seen between MDR and biofilm formation. Five pathogenic and two fecal isolates were negative for all investigated genes while they were they were biofilm producers. In contrast, 22 pathogenic isolates and 1 fecal isolate were positive for the tested genes, but did not form any biofilm. No significant differences were observed between biofilm formation and the presence of the esp, gelE and cyl genes in the pathogenic and fecal isolates (P >0.05). CONCLUSION: The presence of the esp, gelE and cyl genes might not be determining factors for biofilm formation in enterococci and other mechanisms might be involved in this process.

Recombinant flagellin-PAL fusion protein of Legionella pneumophila induced cell-mediated and protective immunity against bacteremia in BALB/c mice.

We report a new recombinant fusion protein composed of full-length Legionella pneumophila flagellin A and peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing protective immunity against L. pneumophila. The recombinant protein was over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+) expression vector (pET28a-flaA-pal) and purified by Ni2+ exchange chromatography. Immunological properties of rFLA-PAL were assessed in a mouse model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase in serum antibody concentration against each of its protein portions. Furthermore, a strong activation of both innate and adaptive cell-mediated immunity was observed as indicated by antigen-specific splenocyte proliferation, IFN-γ and IL-12 production, and early production of TNF-α in the serum and in splenocyte cultures which were separately assessed against PAL and FLA. BALB/c mice were challenged with a lethal dose of L. pneumophila intravenously. In a 10-days follow-up after intravenous lethal challenge with L. pneumophila, a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for those immunized with a sublethal dose of L. pneumophila. Based on the potent immune responses observed in mice immunized with rFLA-PAL, this recombinant fusion protein could be a potential vaccine candidate against the intracellular pathogen L. pneumophila.

Molecular characterization of nasal methicillin resistant Staphylococcus aureus isolates from workers of an automaker company in southeast Iran.

Colonization of methicillin resistant Staphylococccus aureus (MRSA) can occur more commonly in healthy people who live in close together or are in close physical contact with each other. Having knowledge about the molecular characteristics of these strains provides considerable discernment into the epidemiology of this important microorganism. A total of 806 nasal swabs were collected from healthy workers of an automaker company in the southeast of Iran and were analyzed to detect MRSA isolates. Multilocus sequence typing (MLST), spa typing, and detection of staphylococcal cassette chromosome mec (SCCmec) were performed. The presence of genes encoding Panton-Valentine Leukocidin (PVL) and Arginine Catabolic Mobile Element (ACME) were also investigated. Carriage rate of S. aureus was 20%. Among 10 identified MRSA, no acme was found while high prevalence of pvl (60%) was of great concern. Seven different spa types including five new ones were identified. The most frequent sequence type was the novel one; ST 3373 (n = 3), followed by each of ST22, ST88, ST859 (n = 2) and ST1955 (n = 1). MRSA isolates were clustered into two main clonal complexes; CC22 (n = 6) and CC88 (n = 4). Low genetic diversity with the dominance of CC22, SCCmecIV was found. Distribution of previously found hospital-associated MRSA was demonstrated among our isolates.

Protection against Legionnaire's Disease: Recombinant Flagellin A of Legionella pneumophila Can Induce Protective Immunity against Bacteremia in a BALB/c Murine Model.

To investigate the immunoprotective effects of the recombinant type A flagellin (FLA), the flaA gene of Legionella pneumophila serogroup 1 strain Paris was cloned into pET28a(+). Recombinant FLA (rFLA) was overexpressed in E. coli BL21 (DE3) and purified by Ni2+ exchange chromatography. Female BALB/c aged 6-8 weeks were immunized with 20 µg of rFLA. Nonimmunized mice along with mice inoculated with a sublethal dose of live L. pneumophila intravenously were considered as negative and positive controls, respectively. A significant serum antibody response was observed in female BALB/c mice immunized with rFLA. Production of IFN-γ and IL-12, and TNF-α in the serum and the splenocyte cultures, and antigen-specific splenocyte proliferation suggested a strong innate and adaptive cell-mediated immunity response in rFLA-immunized mice. Intravenous lethal challenge with L. pneumophila serogroup 1 (strain Paris) showed that 60% of mice immunized with rFLA survived in a 10-day follow-up survey. These results show that rFLA from L. pneumophila can elicit strong innate and adaptive immune responses and suggest the possibility of a long-term immunity against lethal challenge with L. pneumophila.

Prevalence of mip virulence gene and PCR-base sequence typing of Legionella pneumophila from cooling water systems of two cities in Iran.

Legionella pneumophila is the primary respiratory pathogen and mostly transmitted to human through water cooling systems and cause mild to severe pneumonia with high mortality rate especially in elderly both in hospitals and community. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods. Here, we investigated the presence of L. pneumophila mip gene in water samples collected from different hospitals cooling towers, nursing homes and building/hotels water coolants from two geographical locations of Iran (Kerman and Bam cities) during summer season of 2015 by both nested and real-time PCR methods. Analysis of the 128 water samples for presence of the mip gene by nested-PCR revealed, 18 (23%) positive cases in Kerman and 7(14%) in Bam. However, when samples were tested by real-time PCR, we identified 4 more new cases of L. pneumophila in the hospitals as well as nursing homes water systems that were missed by nested-PCR. The highest rate of contamination was detected in water obtained from hospitals cooling towers in both the cities (p≤0.05). Dendrogram analysis and clonal relationship by PCR-base sequence typing (SBT) of the L. pneumophila genomic DNAs in Kerman water samples showed close clonal similarities among the isolates, in contrast, isolates identified from Bam city demonstrated two fingerprint patterns. The clones from hospital water samples were more related to the L. pneumophila serogroup- 1.