Molecular Epidemiology of Multidrug-Resistant Uropathogenic Escherichia coli O25b Strains Associated with Complicated Urinary Tract Infection in Children.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) has increased the incidence of urinary tract infection (UTI). It is the cause of more than 80% of community-acquired cystitis cases and more than 70% of uncomplicated acute pyelonephritis cases. AIM: The present study describes the molecular epidemiology of UPEC O25b clinical strains based on their resistance profiles, virulence genes, and genetic diversity. METHODS: Resistance profiles were identified using the Kirby-Bauer method, including the phenotypic production of extended-spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs). The UPEC serogroups, phylogenetic groups, virulence genes, and integrons were determined via multiplex PCR. Genetic diversity was established using pulsed-field gel electrophoresis (PFGE), and sequence type (ST) was determined via multilocus sequence typing (MLST). RESULTS: UPEC strains (n = 126) from hospitalized children with complicated UTIs (cUTIs) were identified as O25b, of which 41.27% were multidrug resistant (MDR) and 15.87% were extensively drug resistant (XDR). The O25b strains harbored the fimH (95.23%), csgA (91.26%), papGII (80.95%), chuA (95.23%), iutD (88.09%), satA (84.92%), and intl1 (47.61%) genes. Moreover, 64.28% were producers of ESBLs and had high genetic diversity. ST131 (63.63%) was associated primarily with phylogenetic group B2, and ST69 (100%) was associated primarily with phylogenetic group D. CONCLUSION: UPEC O25b/ST131 harbors a wide genetic diversity of virulence and resistance genes, which contribute to cUTIs in pediatrics.

Control of Methicillin-Resistant Staphylococcus aureus Strains Associated With a Hospital Outbreak Involving Contamination From Anesthesia Equipment Using UV-C.

Methicillin-resistant Staphylococcus aureus (MRSA) is considered an opportunistic pathogen in humans and is mainly associated with healthcare-associated infections (HCAIs). This bacterium colonizes the skin and mucous membranes of healthy people and causes frequent hospital outbreaks. The aim of this study was to perform molecular typing of the staphylococcal cassette chromosome mec (SCCmec) and agr loci as wells as to establish the pulsotypes and clonal complexes (CCs) for MRSA and methicillin-sensitive S. aureus (MSSA) outbreaks associated with the operating room (OR) at a pediatric hospital. Twenty-five clinical strains of S. aureus (19 MRSA and 6 MSSA strains) were recovered from the outbreak (patients, anesthesia equipment, and nasopharyngeal exudates from external service anesthesia technicians). These clinical S. aureus strains were mainly resistant to benzylpenicillin (100%) and erythromycin (84%) and were susceptible to vancomycin and nitrofurantoin. The SCCmec type II was amplified in 84% of the S. aureus strains, and the most frequent type of the agr locus was agrII, which was amplified in 72% of the strains; however, the agrI and agrIII genes were mainly detected in MSSA strains. A pulsed-field gel electrophoresis (PFGE) analysis grouped the 25 strains into 16 pulsotypes (P), the most frequent of which was P1, including 10 MRSA strains related to the anesthesia equipment, external service anesthesia technicians, and hospitalized patients. Multilocus sequence typing (MLST) identified 15 sequence types (STs) distributed in nine CCs. The most prevalent ST was ST1011, belonging to CC5, which was associated with the SCCmec type II and agrII type. We postulate that the external service anesthesia technicians were MRSA carriers and that these strains were indirectly transmitted from the contaminated anesthesia equipment that was inappropriately disinfected. Finally, the MRSA outbreak was controlled when the anesthesia equipment disinfection was improved and hand hygiene was reinforced.

Molecular Epidemiology, Antibiotic Resistance, and Virulence Traits of Stenotrophomonas maltophilia Strains Associated With an Outbreak in a Mexican Tertiary Care Hospital.

Stenotrophomonas maltophilia, an emerging opportunistic pathogen, is widely distributed in the environment the resistance mechanisms, and virulence factors of this bacterium facilitate its dissemination in hospitals. This study aimed to characterize the molecular epidemiology of S. maltophilia strains associated with an outbreak in the Children's Hospital of México Federico Gómez (HIMFG). Twenty-one clinical S. maltophilia strains were recovered from cultures of blood and urine samples from 10 pediatric patients at the emergency department, and nine environmental S. maltophilia strains recovered from faucets in the same area were also included. Two of the 10 patients were related with health care-associated infections (HCAIs), and the other eight patients (8/10) were infected with environmental S. maltophilia strains. The outbreak was controlled by monthly disinfection of the faucets in the emergency department. Typing using pulsed-field gel electrophoresis (PFGE) showed a 52% genetic diversity with seven pulsotypes denoted P1-P7 among all S. maltophilia strains. Three pulsotypes (P2, P3, and P7) were identified among both the clinical and environmental S. maltophilia strains and associated with two type sequences (STs), namely, ST304 and ST24. Moreover, 80% (24/30) of the strains exhibited resistance mainly to tetracycline, 76.66% (23/30) to trimethoprim-sulfamethoxazole, and 23.33% (7/30) to the extended-spectrum ß-lactamase (ESBL) phenotype. The main resistance genes identified by multiplex PCR were sul1 in 100% (30/30), qnr in 86.66% (26/30), and intl1 in 80% (24/30) of the samples, respectively. Furthermore, the pilU, hlylII, and rmlA genes were identified in 96.6% (29/30), 90% (27/30), and 83.33% (25/30) of the samples, respectively. Additionally, 76.66% (23/30) of the S. maltophilia strains exhibited high swimming motility, 46.66% (14/30) showed moderate biofilm formation capacity, 43.33% (13/30) displayed moderate twitching motility, and 20% (6/30) exhibited high adherence. The clinical S. maltophilia strains isolated from blood most strongly adhered to HTB-9 cells. In conclusion, the molecular epidemiology and some of the features such as resistance, and virulence genes associated with colonization patterns are pathogenic attributes that can promote S. maltophilia dissemination, persistence, and facilitate the outbreak that occurred in the HIMFG. This study supports the need for faucet disinfection as a control strategy for clinical outbreaks.

Uropathogenic Escherichia coli strains harboring tosA gene were associated to high virulence genes and a multidrug-resistant profile.

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to ß-lactam and non-ß-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.

Everybody hands-on to avoid ESKAPE: effect of sustained hand hygiene compliance on healthcare-associated infections and multidrug resistance in a paediatric hospital.

PURPOSE: Hand hygiene is the most important strategy for preventing healthcare-associated infections (HCAIs); however, the impact of hand hygiene in middle-income countries has been poorly described. In this work, we describe the impact of the programme 'Let's Go for 100' on hand hygiene adherence, HCAIs rates and multidrug-resistant (MDR) bacteria, including the molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains. METHODOLOGY: A multimodal, hospital-wide hand hygiene programme was implemented from 2013. 'Let's Go for 100' involved all healthcare workers and encompassed education, awareness, visual reminders, feedback and innovative strategies. Monthly hand hygiene monitoring and active HCAI surveillance were performed in every ward. Molecular typing of MRSA was analysed by pulsed-field gel electrophoresis (PFGE).Results/Key findings. Hand hygiene adherence increased from 34.9 % during the baseline period to 80.6 % in the last 3 months of this study. The HCAI rate decreased from 7.54 to 6.46/1000 patient-days (P=0.004). The central line-associated bloodstream infection (CLABSIs) rate fell from 4.84 to 3.66/1000 central line-days (P=0.05). Negative correlations between hand hygiene and HCAIs rates were identified. The attack rate of MDR-ESKAPE group bloodstream infections decreased from 0.54 to 0.20/100 discharges (P=0.024). MRSA pulsotypes that were prevalent during the baseline period were no longer detected after the 5th quarter, although new strains were identified. CONCLUSIONS: A multimodal hand hygiene programme in a paediatric hospital in a middle-income country was effective in improving adherence and reducing HCAIs, CLABSIs and MDR-ESKAPE bloodstream infections. Sustaining hand hygiene adherence at a level of >60 % for one year limited MRSA clonal transmission.

Cost-effective procedure for Streptococcus pyogenes immobilized DNA preparation and miniPFGE subtyping.

INTRODUCTION: Group A streptococci (GAS) is responsible of several human diseases ranging from mild infection to severe invasive toxin-mediated disease and post-infectious sequelae. Accordingly, a GAS surveillance program based on molecular techniques is advisable for its epidemiological control. Pulsed-field gel electrophoresis (PFGE) is the gold standard for GAS molecular subtyping, but a major disadvantage is the length of the procedure, which takes 1-3 days of work, minimum. The aim of this study was to develop a rapid and cost-effective procedure for PFGE subtyping of GAS isolates. METHODOLOGY: Different incubation times of GAS, immobilized in agarose miniplugs, in solutions containing lysozyme and/or mutanolysine followed by solutions with urea instead of proteinase K, were assayed. DNA was restricted with SmaI and the fingerprints were obtained in clamped homogeneous electric field (CHEF) chambers and minichambers. The modified procedure was used to subtype 22 GAS isolates. RESULTS: Intact DNA molecules of GAS immobilized in agarose miniplugs were prepared incubating the cells, in situ, with a solution containing lysozyme for 4 hours, followed by the incubation in a non-enzymatic solution with urea for 2 hours. SmaIDNA macrorestriction fragments were well resolved in 5 hours and 14 minutes by electrophoresis in a CHEF minichamber at 10 V/cm. This procedure for GAS DNA preparation was useful for fingerprinting GAS strains in the format of CHEF Mapper (BioRad). CONCLUSIONS: The procedure took 13 hours for GAS strains subtyping. Both sample preparation and electrophoresis in CHEF minichamber represent an economic alternative for performing massive epidemiological studies of this human pathogen.

Resistencia a linezolid en Staphylococcus aureus resistente a meticilina y enterococos con elevada resistencia a aminoglucósidos en un hospital pediátrico de tercer nivel / Resistance to linezolid in methicillin-resistant Staphylococcus aureus and Enterococcus with high-level resistance to aminoglycosides in a third-level pediatric hospital

Introducción: El surgimiento de resistencia a oxazolidinonas en Staphylococcus aureus resistentes a meticilina (SAMR) y Enterococcus spp con elevada resistencia a aminoglucósidos (EERA), aún cuando no han sido expuestos al antibiótico, es una de las principales razones para el control en el uso clínico de estos antibióticos. Métodos: Se estudiaron 95 cepas de SAMR y EERA, las cuales fueron aisladas de enero 2003 a diciembre 2007 en el Hospital Infantil de México Federico Gómez; se identificaron por pruebas convencionales. Se evaluó la susceptibilidad a diversos antimicrobianos incluyendo linezolid de acuerdo al Instituto de Estándares Clínicos y de Laboratorio (CLSI). Se comprobó la elevada resistencia a aminoglucósidos al amplificar los genes aac(6')-le, aph(2")-la y ant(6') en enterococos y el tipo de cásete cromosomal estafilocócico mee (SCCmec) asociado a la resistencia a meticilina en S. aureus, por técnicas moleculares previamente descritas. Resultados: Todas las cepas de SAMR mostraron el SCCmec tipo II. El 100% de los enterococos con fenotipo EERA mostraron genes asociados con los niveles elevados de resistencia a aminoglucósidos. El 12% (6/50) de EERA presentó valores intermedios a linezolid (concentración inhibitoria mínima (CIM) de 4 μg/mL) y sólo una cepa fue resistente (CIM 128 μg/mL); un aislamiento fue resistente a vancomicina fenotipo y genotipo van A, pero sensible a linezolid. El 2.2% (1/45) de los SAMR fue resistente a linezolid (CIM 8 μg/mL). Conclusión: Linezolid es una opción terapéutica de gran valor clínico. Sin embargo, son necesarios monitoreos continuos para conocer el riesgo de surgimiento de cepas resistentes y establecer lineamientos en el uso apropiado del antibiótico.

Background: The emergence of resistance to the oxazolidinones by methicillin-resistant Staphylococcus aureus (MRSA) and high-level aminoglycoside-resistant (HLRA) Enterococcus spp not exposed is one of the main reasons for control of the clinical use of these antibiotics. Methods: We studied 95 strains of MRSA and HLAR, which were isolated from January 2003 to December 2007 at the Hospital Infantil de México Federico Gómez. The strains were identified by conventional tests. Antimicrobial susceptibility was evaluated for several antimicrobial agents including linezolid according to the Clinical and Laboratory Standards Institute (CLSI). The high resistance to aminoglycosides was tested by amplification of genes aac (6')-/e, aph (2")-and ant (6') in enterococci. Staphylococcal cassette chromosomal mec (SCCmec) associated with MRSA was identified by molecular techniques described previously. Results: All MRSA strains showed SCCmec type II, and 100% of enterococci strains with phenotype HLAR showed genes associated with high-level aminoglycoside resistance; 12% of HLAR enterococci strains showed intermediate values to linezolid (MIC 4 μg/mL) and only one strain was resistant (MIC 128 μg/mL). Of the MRSA strains, 2.2% were resistant to linezolid (MIC 8 μg/mL). Conclusion: Linezolid is a clinically valuable option as a form of therapy. However, continuous surveillance is necessary to determine the emergent risk of resistance strains and to establish guidelines for appropriate use.