Sonia Pérez

Cloning, Nucleotide Sequencing, and Analysis of the AcrAB-TolC Efflux Pump of Enterobacter cloacae and Determination of Its Involvement in Antibiotic Resistance in a Clinical Isolate

ABSTRACT Enterobacter cloacae is an emerging clinical pathogen that may be responsible for nosocomial infections. Management of these infections is often difficult, owing to the high frequency of strains that are resistant to disinfectants and antimicrobial agents in the clinical setting. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role as a mechanism of antimicrobial resistance in gram-negative pathogens. In the present study, we cloned and sequenced the genes encoding an AcrAcB-TolC-like efflux pump from an E. cloacae clinical isolate (isolate EcDC64) showing a broad antibiotic resistance profile. Sequence analysis showed that the acrR, acrA, acrB, and tolC genes encode proteins that display 79.8%, 84%, 88%, and 82% amino acid identities with the respective homologues of Enterobacter aerogenes and are arranged in a similar pattern. Deletion of the acrA gene to yield an AcrA-deficient EcDC64 mutant (EcΔacrA) showed the involvement of AcrAB-TolC in multidrug resistance in E. cloacae. However, experiments with an efflux pump inhibitor suggested that additional efflux systems also play a role in antibiotic resistance. Investigation of several unrelated isolates of E. cloacae by PCR analysis revealed that the AcrAB system is apparently ubiquitous in this species.

Enterococcus faecium resistente a glucopeptidos en un hospital del norte de Espana. Caracterizacion molecular y epidemiologia clinica

INTRODUCTION Glycopeptide resistance in Enterococcus spp. is a clinical problem because of the rapid dissemination of these microorganisms, possible transfer of vancomycin resistance to more virulent pathogens, such as Staphylococcus aureus, and the limited therapeutic possibilities for the infections they cause. In this study, 10 strains of vancomycin-resistant Enterococcus faecium isolated from 10 different patients in our hospital during 2004 to 2005 were characterized. METHODS PCR was used to analyze the gene implicated in glycopeptide resistance. Molecular analysis of clinical isolates was performed by pulsed-field gel electrophoresis (PFGE). The presence of genes previously associated with epidemicity/virulence (esp, hyl, asa1, gel, cyl) was also studied. Risk factors were determined in a case-control study. RESULTS The most commonly detected gene was vanA. PFGE analysis revealed 5 different genotypes (A-E), with a predominance of genotype A (n=3) and B (n=3). The esp (surface protein) and hyl (hyaluronidase) genes were detected in all strains, with the exception of genotypes B and D. The purK1 allele, associated with clonal complex 17 was demonstrated in all strains. CONCLUSION Prior administration of cephalosporins, aminoglycosides and vancomycin alone or in combination was significantly associated with colonization/infection by vancomycin-resistant Enterococcus faecium.

Evaluation of the Vitek 2 AST-P559 Card for Detection of Oxacillin Resistance in Staphylococcus aureus

Staphylococcus aureus strains with resistance to methicillin or oxacillin (MRSA) represent one of the main nosocomial pathogens at present. MRSA infections are clearly associated with higher mortality and economic cost than those caused by methicillin-susceptible S. aureus (1). In Spain, the prevalence of methicillin resistance among S. aureus isolates has increased since the 1990s and in some cases has now reached levels higher than 30% (2). Rapid and accurate detection of MRSA strains in hospital as well as community settings is imperative. The performance of cefoxitin either as a disk or as a supplement in agar medium for the detection of MRSA has been confirmed extensively (6, 7). Also, cefoxitin-based screening broth proved to be more sensitive and rapid than oxacillin-based broth (3). Recently, a new Vitek card (AST P549) for determining susceptibility of S. aureus to oxacillin has been reported (5). The Vitek 2 AST-P559 card (bioMerieux) incorporates specific antimicrobial agents to test susceptibility and also includes the determination of oxacillin MICs as well as screening for cefoxitin (6 μg/ml). The main advantage of the card is its promptness in detecting methicillin resistance, as it is possible to interpret the results of a cefoxitin screen after 4 h of card inoculation. However, final confirmation is done after 12 h upon reading the oxacillin MICs as well as the cefoxitin screen well and according to the manufacturer′s instructions. The Vitek 2 AST-P559 card cannot be manually read, so interpretive information must be derived from the software interpretation. A total of 301 S. aureus strains were evaluated (51 mecA negative and 250 mecA positive as determined by PCR). S. aureus ATCC 29213 was used as a negative control. Molecular typing of the X region of the spa gene was done with the 250 mecA-positive isolates (4), and these were then grouped into a spa clonal complex (BURST.Ridom StaphType software), with 4 types more prevalent than the others (t02, 23.6%; t18, 22%; t67, 17.6%; and t12, 16.3%). Overall results are shown in Table ​Table1.1. Three out of 250 isolates yielded a negative result by the cefoxitin screening methods as well as yielding an oxacillin MIC of ≤2. However, when these isolates were preincubated for 24 h in BBL CHROMagar MRSA medium (BD Diagnostics, Sparks, MD), which includes 6 μg/ml of cefoxitin, and the assay was repeated, they yielded a positive result with both antibiotics, therefore suggesting that the existence of subpopulations heteroresistant to oxacillin may cause a false-negative result with the Vitek 2 card. All mecA-positive isolates yielded a positive result with the cefoxitin screening (and also yielded an oxacillin MIC of ≥4 μg/ml) and showed positive PBP 2a agglutination with the Slidex MRSA detection kit (bioMerieux). Regarding specificity, all mecA-negative S. aureus strains were negative by the cefoxitin screening method and also yielded an oxacillin MIC of ≤2 μg/ml. However, PBP 2a agglutination yielded three false-positive results (96% specificity). TABLE 1. Number of isolates yielding results corresponding to the different tests with the Vitek 2 AST-P559 card Considering the results from these three isolates at preincubation time with the cefoxitin agar plate (false negative), the overall results of this study with the AST-P559 card showed sensitivity and specificity of 98.8% and 100%, respectively. The positive and negative predictive values were 100% and 94%, respectively. However, the values for all parameters increased to 100% when the three strains were analyzed after induction of the mecA gene product with cefoxitin. The main advantage of the AST-P559 card is the incorporation of two tests to provide simultaneous determination of oxacillin MICs as well as cefoxitin screening. Several studies (6, 7) have shown that cefoxitin is a better predictor of the presence of mecA than oxacillin, and, indeed, Velasco et al. (7) reported an increase of 2% in sensitivity to detect mecA when the cefoxitin screening test was used in addition to the oxacillin MIC. For these reasons, the simultaneous presence of two tests (oxacillin MIC and cefoxitin screening) in the same card may offer a noticeable advantage for detection of some mecA-negative S. aureus isolates which are identified by cefoxitin screening rather than oxacillin MIC. In addition, information about susceptibility to antibiotics against gram-positive microorganisms is provided by this card, which offers an undoubted advantage with respect to chromogenic agars, agglutination latex, or molecular techniques, since they only detect the marker of resistance to oxacillin. In addition to that, the possibility of diagnosis of an MRSA infection in 4 h (by the software interpretation of the cefoxitin screening) will make the use of this automated method very attractive for clinical microbiology laboratories worldwide.

Lifestyle factors and oncogenic papillomavirus infection in a high-risk male population.

Background High risk human papillomavirus (HR-HPV) infection in males is a health issue with implications for HPV-related lesions in their partners. The identification of risk factors for male infection may improve our understanding of HR-HPV transmission and prevention. The aim of this study was to evaluate the relationships between lifestyle, genital warts and HR-HPV infection. The study was focused on men with an increased risk of HR-HPV infection: male sexual partners of women diagnosed with high-grade squamous intraepithelial cervical lesions. Methods Men were enrolled and prospectively recruited within the first six months after diagnosis of cervical lesions in their female partners (n = 175, 2013–2016). Epidemiological and sexual behaviour data were obtained. The presence of genital warts was established by visual inspection. Detection and genotyping of HR-HPV infection in genital samples were performed with a Linear Array HPV Genotyping Test. All HR-HPV positive men were offered a follow-up exam at 12 months. SPSS version 19 was used for statistical analysis. Results and discussion The prevalence of HR-HPV infection in men was 45.1% (79/175). Genital warts were observed in 10.3% (18/175) of the subjects. Detection of genital warts (OR 3.5, p = 0.015), smoking habits (OR 2.3, p = 0.006) and sexual debut before 16 years old (OR 2, p = 0.035) were associated with an increased risk for HR-HPV infection (univariate analysis). This association was also observed for genital warts and smoking status in a multivariate analysis. The same genotype was found after one year in 71.4% (20/28) of subjects. Conclusions The presence of genital warts and smoking habits seem to be associated with a higher risk of HR-HPV infection in males. Earlier sexual debut may increase this risk. Extensive knowledge of the natural history of HR-HPV infection in males is an absolute requirement for the design and implementation of prevention strategies for the general population as well as for specific populations such as couples after treatment for high-grade cervical lesions.

Prevalence and concordance of high-risk papillomavirus infection in male sexual partners of women diagnosed with high grade cervical lesions.

BACKGROUND Little is known about the characteristics of high-risk papillomavirus (HR-HPV) infection in men. The aims of this cross-sectional study were: (a) to investigate HR-HPV prevalence and genotype distribution in men, sexual partners of women presenting with high-grade cervical intraepithelial neoplasia (HG-CIN), according to epidemiological characteristics, and (b) to assess type-specific concordance between partners. METHODS A total of 125 men were recruited within the first 6 months after HG-CIN diagnosis of their partner. Samples from the coronal sulcus, glans penis shaft, and scrotum were tested with linear array HPV genotyping assay (Roche Diagnostics, Mannheim, Germany). Type-specific concordance within 120 couples was studied. Epidemiological factors were evaluated by multivariate logistic regression analysis. SPSS 19 (IBM, Chicago, USA). RESULTS The prevalence of HR-HPV infection in males was 50.4% (63/125). HPV16/53/52/51/66/31 were the most frequent genotypes (24/10.4/9.6/8.8/8/7.2%, respectively). Current smoking was associated with an increased risk for HR-HPV infection in men (38.2% (21/55) vs 60% (42/70), OR 2.4, p=0.025). Among 60 infected couples, 62% shared at least one genotype: 41.7% couples were concordantly HPV16 positive and 18.3% were HPV16 negative (kappa value: 0.21). The proportion of women with the same genotype as their male partner was higher than the proportion of men sharing the same genotype as their female partner: 58.7% (37/63) vs 30.8% (37/120), p<0.0001. CONCLUSIONS Sexual partners of women with HG-CIN are a significant reservoir and vector of HPV infection, a fact that could contribute to making viral clearance more difficult to achieve in their partners after treatment of their HG-CIN lesions.

Correction: Lifestyle factors and oncogenic papillomavirus infection in a high-risk male population

[This corrects the article DOI: 10.1371/journal.pone.0184492.].

Direct, indirect and total effectiveness of bivalent HPV vaccine in women in Galicia, Spain

Bivalent human papillomavirus (HPV) vaccine was incorporated into the childhood vaccination calendar in Galicia, Spain in 2008. The objectives of this study were to estimate direct, indirect and total effectiveness of HPV vaccine and to identify sexual habits changes in the post-vaccination period in Galicia, Spain.Endocervical scrapings of 745 women attending 7 Health Areas of the Galician Public Health Service were collected in the post-vaccination period, from 2014–2017. Two groups were studied: women born between 1989 and 1993 (n = 397) and women born in 1994 or later (n = 348). Twelve high-risk human papillomavirus (HR-HPV) genotypes were detected by Cobas® 4800 HPV test (Roche Diagnostics, Mannheim, Germany). The Linear Array® HPV Genotyping Test (Roche Diagnostics) was used for HR-HPV genotype detection other than HPV 16/18. Information about sexual habits was collected by a self-filled questionnaire. Post-vaccination data were compared to previously published pre-vaccination data obtained between 2008 and 2010 in Galicia from women of the same age (18–26 years old, n = 523). The Stata 14.2 software was employed for statistical analyses.Data from 392 unvaccinated and 353 vaccinated women were compared. For unvaccinated and vaccinated women, HPV 16/18 prevalence was 9.2% and 0.8%, respectively, and HPV 31/33/45 prevalence was 8.4% and 1.1%, respectively. Direct, indirect and total effectiveness of the HPV vaccine were (%, 95% CI): 94 (72−99), 30 (-11−56) and 95 (79−99), respectively, for HPV 16/18 and 83 (46−94), -10 (-88−33) and 84 (54−94), respectively, for HPV 31/33/45. The number of women with first intercourse before 17 years old and 3 or more sexual partners along life was higher in the post-vaccination period (p < 0.05). A positive impact of bivalent HPV vaccine was observed, both on direct and cross protection. Sexual habits could have changed in the post-vaccination period.

Original articlePrevalence and concordance of high-risk papillomavirus infection in male sexual partners of women diagnosed with high grade cervical lesionsPrevalencia y concordancia de infección por papilomavirus de alto riesgo en las parejas sexuales masculinas de mujeres diagnosticadas de lesiones cervicales de alto grado

Background Little is known about the characteristics of high-risk papillomavirus (HR-HPV) infection in men. The aims of this cross-sectional study were: (a) to investigate HR-HPV prevalence and genotype distribution in men, sexual partners of women presenting with high-grade cervical intraepithelial neoplasia (HG-CIN), according to epidemiological characteristics, and (b) to assess type-specific concordance between partners.

Diagnosis and Prevalence of High-Risk Human Papillomavirus Infection in Heterosexual Men

A better understanding of human papillomavirus (HPV) infection in men is an essen‐ tial component of prevention programs aimed to reduce cervical cancer and other HPVrelated diseases. A screening test capable of detecting asymptomatic/subclinical genital HPV infection in men at a reasonable price and causing minimal discomfort to the patient would be very valuable. The following chapter focuses on acetowhite test usefulness in the detection of asymptomatic/subclinical genital high-risk (HR) HPV infection in highrisk men populations, HR-HPV prevalence in sexually active healthy male partners of women diagnosed of high-grade cervical intraepithelial neoplasia and genotypespecific concordance between partners, addressing the preventive strategies that would reduce HPV infection in men. We present data from 125 men, sexual partners of women with preneoplastic cervical lesions. Prevalence of HR-HPV infection in male was high (50, 24% HPV16) and genotype concordance within the 60 infected couples was remarkable (62% shared at least one genotype). Acetowhite (AW) test was positive in 27% patients, showing low sensitivity for the identification of HR-HPV infection but allowed the diagnosis of subclinical HPV-related lesions in more than 10%. Current smoking and genital warts were associated with an increased risk of HR-HPV infection in men (OR: 2.4 and 5.6, respectively).