High-Yield Expression and Purification of Scygonadin, an Antimicrobial Peptide, Using the Small Metal-Binding Protein SmbP.

(1) Background: Producing active antimicrobial peptides with disulfide bonds in bacterial strains is challenging. The cytoplasm of Escherichia coli has a reducing environment, which is not favorable to the formation of disulfide bonds. Additionally, E. coli may express proteins as insoluble aggregates known as inclusion bodies and have proteolytic systems that can degrade recombinant peptides. Using E. coli strains like SHuffle and tagging the peptides with fusion proteins is a common strategy to overcome these difficulties. Still, the larger size of carrier proteins can affect the final yield of recombinant peptides. Therefore, a small fusion protein that can be purified using affinity chromatography may be an ideal strategy for producing antimicrobial peptides in E. coli. (2) Methods: In this study, we investigated the use of the small metal-binding protein SmbP as a fusion partner for expressing and purifying the antimicrobial peptide scygonadin in E. coli. Two constructs were designed: a monomer and a tandem repeat; both were tagged with SmbP at the N-terminus. The constructs were expressed in E. coli SHuffle T7 and purified using immobilized metal-affinity chromatography. Finally, their antimicrobial activity was determined against Staphylococcus aureus. (3) Results: SmbP is a remarkable fusion partner for purifying both scygonadin constructs, yielding around 20 mg for the monomer and 30 mg for the tandem repeat per 1 mL of IMAC column, reaching 95% purity. Both protein constructs demonstrated antimicrobial activity against S. aureus at MICs of 4 µM and 40 µM, respectively. (4) Conclusions: This study demonstrates the potential of SmbP for producing active peptides for therapeutic applications. The two scygonadin constructs in this work showed promising antimicrobial activity against S. aureus, suggesting they could be potential candidates for developing new antimicrobial drugs.

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides.

An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) Escherichia coli strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against Staphylococcus aureus, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae bacteria clinical isolates at a lower minimum inhibition concentration level (10-33 µM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50-100 µM). KEY POINTS: • The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker. • The peptide was expressed with the carrier proteins SmbP and CusF3H+. • The hybrid peptide shows antibacterial potency against clinical bacterial isolates.

Sexually Transmitted Infections in Male Patients with Urethritis.

(1) Background: Sexually Transmitted Infections (STIs) in men are a significant public health problem due to the consequences they can have, such as chronic diseases, infertility, cancer, and even death. This study aimed to determine the frequency of microorganisms associated with STIs in men with urethritis attending urology consultations, and to explore their clinical correlations. (2) Methods: A population that attended the urology consultation of the University Hospital "Dr. José E. González" was studied. Written consent was obtained, and interviews and clinical history were conducted about specific risk factors identifying signs and symptoms associated with any genitourinary condition; after that, urine samples were collected. Identification of C. trachomatis, N. gonorrhoeae, U. urealyticum, and M. genitalium was based on amplifying species-specific DNA fragments. (3) Results: A total of 200 patients were included. The mean age was 55 years (20-95). According to the interviews, only 32.5% (n = 65) had received prior sex education. Additionally, 75% (n = 150) do not usually use any protection during sexual intercourse. Regarding clinical factors, 69.4% (n = 138) presented burning or pain when urinating. Molecular analysis revealed the presence of C. trachomatis to be 9.5% (n = 19), with U. urealyticum at 13% (n = 26), and M. genitalium at 2% (n = 4). (4) Conclusions: This is the most extensive molecular epidemiological study of the frequency of STIs in men in Mexico in third-level care and its association with different risk factors. As reported globally, a similar frequency of C. trachomatis, U. urealyticum, and M. genitalium was detected.

Case Report: Molecular Characterization of Bloodstream Infections due to Carbapenem-Resistant Acinetobacter baumannii: A Pediatric Case Series.

Acinetobacter baumannii poses a significant threat to public health due to the high rate of multidrug-resistant strains. However, information on the molecular characterization of carbapenem-resistant Acinetobacter baumannii (CRAB) bloodstream infections in children is scarce. This study aimed to describe the molecular characterization of carbapenem-resistant A. baumannii infections in children from a hospital in Mexico. A retrospective study was conducted during the period 2017-2022. Clinical and demographic data were collected from the clinical records. Mass spectrometry was used for the identification of the strains. To confirm A. baumannii strains, a polymerase chain reaction (PCR) method was applied using a gyrB sequence. The carbapenemase-encoding resistance genes were detected by PCR. Six cases of CRAB were documented, including five in neonates. The median intensive care unit stay was 20 days, and all cases had an invasive medical device. Half of the patients had at least one medical condition. A high prevalence of coresistance was observed in most of the antibiotic groups. Three of the six strains coharbored carbapenemase genes: blaOXA-51, blaOXA-24, and blaIMP. Mortality was reported in two neonate patients. The present study shows a high rate of coharboring blaOXA-51, blaOXA-24, and blaIMP-1, which has a direct impact on therapeutic decisions. Implementation of antimicrobial stewardship programs is urgent to stop the spread of this microorganism.

Carbapenem-Resistant Acinetobacter baumannii Infection in Children From a Third-Level Hospital in Mexico: Clinical Characteristics and Molecular Epidemiology.

BACKGROUND: Acinetobacter baumannii has emerged as a threat to public health due to the high prevalence of multidrug-resistant isolates. Information regarding the clinical and molecular characterization of carbapenem-resistant A. baumannii (CRAB) infections in children is scarce. Our study aimed to describe the clinical and molecular characteristics of CRAB infections in children from a third-level center in Mexico. METHODS: Consecutive cases of CRAB infections were documented during 2017-2022. Clinical and demographic data were collected from clinical records. Mass spectrometry was used for the identification of the isolates. The identification of A. baumannii strains was confirmed by conducting a polymerase chain reaction (PCR) assay targeting the gyrB sequence. In addition, the carbapenemase-encoding resistance genes were detected by PCR. RESULTS: Twenty-one cases of CRAB infections were documented: 76% female and 62% were neonates. The median hospital length of stay at the time of positive culture was 37 days (interquartile range, 13-54). Sixty-four percent of the isolates were recovered from bronchial secretions. A co-resistance rate greater than 60% was observed for most groups of antibiotics. All carbapenem-resistant isolates carried blaOXA-24 genes. BlaIMP genes were detected in half of the cases, with all strains co-harboring blaOXA-24 genes. CONCLUSIONS: The present study demonstrated a high proportion of CRAB infections in the neonatal population, a high prevalence of co-resistance to antibiotics, and a high rate of isolates carrying blaOXA-24 and blaIMP genes. CRAB is a significant concern due to the mortality rate and the lack of therapeutic alternatives; implementing infection prevention and control programs is urgent to stop the spread of carbapenem-resistant A. baumannii.

Molecular epidemiology and drug resistance of Mycobacterium tuberculosis in a tertiary care hospital in northeastern Mexico.

INTRODUCTION: Tuberculosis (TB) is a re-emerging disease considered a public health concern. In the present study, we analyzed the epidemiology and drug resistance of Mycobacterium tuberculosis strains isolated from patients with pulmonary TB. METHODOLOGY: Mycobacterium tuberculosis isolates (n = 190) were obtained from patients with pulmonary TB admitted to Dr. José Eleuterio González University Hospital (UH). Each M. tuberculosis isolate was analyzed by spoligotyping (spacer oligonucleotide typing) and MIRU-VNTR (Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat). Drug resistance was evaluated using the Anyplex™ II MTB/MDR/XDR assay. RESULTS: The predominant spoligotypes observed were X1 (SIT 119, n = 46), T1 (SIT 53, n = 40), H3 (SIT 50, n = 13), Beijing (SIT 1, n = 11), and EAI2-Manila (SIT 19, n = 8). MIRU-VNTR analysis showed that the locus QUB-26 had the highest allelic variability. The observed drug resistance included monoresistance to rifampicin (2.6%; n = 5), isoniazid (3.2%; n = 6), and fluoroquinolones (1.6%; n = 3) as well as multidrug resistance (5.3%; n = 10). All of the Beijing strains were susceptible. Regarding comorbidities, 13.7% (26/190) of the patients were co-infected with TB and HIV (TB+HIV+), and 31.6% (55/190) had TB along with diabetes (TB + diabetes). CONCLUSIONS: The most prevalent lineages were X1 (SIT 119; 24.3%) and T1 (SIT 53; 21%). An alarming proportion (12.6%) of M. tuberculosis isolates presented drug resistance. To effectively manage TB, continuous surveillance of regional strain dissemination, drug resistance profiles, and TB-associated comorbidities is crucial.

The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli.

We have recently shown that SmbP, the small metal-binding protein of Nitrosomonas europaea, can be employed as a fusion protein to express and purify recombinant proteins and peptides in Escherichia coli. SmbP increases solubility, allows simple, one-step purification through affinity chromatography, and provides superior final yields due to its low molecular weight. In this work, we report for the first time the use of SmbP to produce a recombinant peptide with anticancer activity: the antitumor-analgesic peptide (BmK-AGAP), a neurotoxin isolated from the venom of the Chinese scorpion Buthus martensii Karsch. This peptide was expressed in Escherichia coli SHuffle for correct, cytoplasmic, disulfide bond formation and tagged with SmbP at the N-terminus to improve its solubility and allow purification using immobilized metal affinity chromatography. SmbP_BmK-AGAP was found in the soluble fraction of the cell lysate. After purification and removal of SmbP by digestion with enterokinase, 1.8 mg of pure and highly active rBmK-AGAP was obtained per liter of cell culture. rBmK-AGAP exhibited antiproliferative activity on the MCF-7 cancer cell line, with a half-maximal inhibitory concentration value of 7.24 µM. Based on these results, we considered SmbP to be a suitable carrier protein for the production of recombinant, biologically active BmK-AGAP. We propose that SmbP should be an attractive fusion protein for the expression and purification of additional recombinant proteins or peptides that display anticancer activities.

The Small Metal-Binding Protein SmbP Simplifies the Recombinant Expression and Purification of the Antimicrobial Peptide LL-37.

(1) Background: The cathelicidin peptide LL-37 is a prominent molecule with many biological activities, including antimicrobial. Due to its importance, here, we describe the production of LL-37 tagged with SmbP, a relatively new carrier protein that improves the production of recombinant proteins and peptides in Escherichia coli. We present an alternative method for the rapid expression, purification, and antimicrobial evaluation of LL-37, that involves only one purification step. (2) Methods: A DNA construct of SmbP_LL-37 was transformed into E. coli BL21(DE3); after overnight expression, the protein was purified directly from the cell lysate using immobilized metal-affinity chromatography. SmbP_LL-37 was treated with Enterokinase to obtain the free LL-37 peptide. The antimicrobial activity of both SmbP_LL-37 and free LL-37 was determined using the colony forming unit assay method. (3) Results: SmbP_LL-37 was observed in the soluble fraction of the cell lysate; after purification with IMAC, protein gel electrophoresis, and analysis by ImageJ, it showed 90% purity. A total of 3.6 mg of SmbP_LL-37 was produced from one liter of cell culture. SmbP_LL-37 and free LL-37 both showed inhibition activity against Staphylococcus aureus and Escherichia coli. (4) Conclusions: The SmbP fusion protein is a valuable tool for producing biologically-active LL-37 peptide. The production method described here should be of interest for the expression and purification of additional cationic peptides, since it cuts the purification time considerably prior to determination of antimicrobial activity.

[Meta-analysis of diagnostic tests for the detection of COVID-19]. / Metaanálisis de pruebas diagnósticas para la detección de COVID-19.

BACKGROUND: COVID-19 disease emerged at the end of 2019 and it started to be considered a pandemic at the beginning of 2020. The reference methodology for the COVID-19 diagnosis is the RT-qPCR. OBJECTIVE: It was assessed the sensitivity and specificity of molecular test kits found on the list of tests of the Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE, Institute for Epidemiologic Diagnosis and Reference). MATERIAL AND METHODS: By using the PubMed and the Google Scholar platforms, a search was carried out for articles, out of which 14 were selected, retrieving the number of samples analyzed and their status (positive or negative), according to the molecular reference method, the name of the test kit used, as well as the characteristics and results obtained, expressed as true positives, false negatives, true negatives and false positives. Sensitivity and specificity of 8 kits were calculated, with compilations of the OpenEpi, version 3.01, statistical calculators, by using the Wilson score with a 95% confidence interval. RESULTS: The sensitivity and specificity results for the kits were greater than 90%. CONCLUSIONS: Tests evaluated allow the detection of presence or absence of virus, but they can be affected by conditions during the stages of the method.

INTRODUCCIÓN: la enfermedad COVID-19 surgió a finales de 2019 y se empezó a considerar pandemia a principios de 2020. La metodología de referencia para su diagnóstico es la prueba RT-qPCR. OBJETIVO: se evalúo la sensibilidad y la especificidad de kits de pruebas moleculares que se encuentran en la lista de pruebas del Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE). MATERIAL Y MÉTODOS: a partir del uso de las plataformas PubMed y Google Scholar se realizó una búsqueda de artículos, de los cuales se seleccionaron 14 y se recuperó el número de muestras analizadas y su estado (positivo o negativo) según el método molecular de referencia, el nombre del kit de prueba utilizado, así como las características y los resultados obtenidos, expresados como verdaderos positivos, falsos negativos, verdaderos negativos y falsos positivos. Se calculó la sensibilidad y la especificidad de ocho kits con la compilación de calculadoras estadísticas OpenEpi versión 3.01, mediante el método de puntos de Wilson con un intervalo de confianza de 95%. RESULTADOS: los kits evaluados tuvieron una sensibilidad y una especificidad superior al 90%. CONCLUSIONES: las pruebas permiten detectar la presencia o ausencia del virus, pero pueden verse afectadas por condiciones durante las etapas del método.

Expression and purification of the antimicrobial peptide Bin1b in Escherichia coli tagged with the fusion proteins CusF3H+ and SmbP.

We have previously shown that the small metal-binding proteins CusF3H+ and SmbP can be used as fusion proteins for the expression and purification of recombinant proteins in Escherichia coli. Because of their small size, both around 10 kDa, they are suitable for the production of peptides to avoid meager yields after the final purification step of tag removal. Bin1b is a beta-defensin found in the epididymis of rats that has shown to have antimicrobial activity. Previous methodologies used to express this antimicrobial peptide in E. coli involve the expression of the peptide as inclusion bodies followed by in vitro refolding or the supplementation of the proteins necessary for proper folding of the peptide in the cytoplasm via a second plasmid. Here, we developed a methodology that forgoes these approaches and instead uses the fusion proteins CusF3H+ or SmbP and the E. coli strain SHuffle to obtain a soluble recombinant protein that contains the mature Bin1b peptide. The recombinant protein is purified using IMAC chromatography and is subsequently cleaved with enterokinase to separate the fusion protein from Bin1b. The purified peptide displays antimicrobial activity against E. coli, as previously shown. Furthermore, we also tested its antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and found that Bin1b is also capable of inhibiting the growth of this bacterium. In conclusion, we developed a practical methodology for the expression and purification of the bioactive Bin1b peptide in E. coli using the fusion proteins CusF3H+ and SmbP. This approach could be further applied for the production of more biologically active peptides.

Expression and Purification of the VpDef Defensin in Escherichia coli using the Small Metal-Binding Proteins CusF3H+ and SmbP.

BACKGROUND: The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. OBJECTIVES: Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6.9 kDa) in Escherichia coli. METHODS: The recombinant VpDef (rVpDef) peptide was expressed as a translational fusion with CusF3H+ and SmbP in Escherichia coli SHuffle under different small-scale culture conditions. The best conditions were applied to 1-liter cultures, with subsequent purification of the recombinant protein through IMAC chromatography. The recombinant protein was digested using enterokinase to liberate the peptide from the fusion protein, and a second IMAC chromatography step removed the fusion protein. The purified peptide was tested against two Gram-positive and two Gram-negative bacteria. RESULTS: The use either of CusF3H+ or of SmbP results in recombinant proteins that are found in the soluble fraction of the bacterial lysate; these recombinant proteins are easily purified through IMAC chromatography, and rVpDef is readily separated following enterokinase treatment. The purified rVpDef peptide exhibits antimicrobial properties against both Gram-positive and Gram-negative. CONCLUSION: Use of the fusion proteins CusF3H+ and SmbP results in production of a soluble recombinant protein containing the antimicrobial peptide rVpDef that is correctly folded and that retains its antimicrobial properties once purified.

Norovirus Is the Most Frequent Cause of Diarrhea in Hospitalized Patients in Monterrey, Mexico.

Little information is available regarding the pathogens that cause diarrhea in hospitalized patients who also have various clinical problems. The purpose of this study was to determine the presence of pathogens in fecal samples of hospitalized patients all suffering diarrhea in addition to other problems in Mexico. Diarrheic stools from 240 patients were obtained in a third-level hospital in Monterrey, Mexico. PCR was used for the detection of Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., Aeromonas spp., Clostridioides difficile, and norovirus GI and GII. The presence of trophozoites, cysts of protozoa, eggs, and/or helminth larvae was determined by microscopic observation. Of the 240 patients analyzed, 40.4% presented at least one of the pathogens analyzed. Norovirus was the pathogen most frequently found (28.6%), followed by bacteria (11.7%), and parasites (8.3%). The majority of co-infections were parasites + norovirus, and bacteria + norovirus. Norovirus was detected mainly in children aged 0 to 10 years (9/15, 60%). Patients aged 0-20 years did not present co-infections. Entamoeba coli and Entamoeba histolytica were the most common parasites, (8/240), and Salmonella was the most prevalent bacteria (10/240). This information can help design specific strategies useful for hospitalized people with a compromised status.

Bacteremia due to Weeksella virosa in a pediatric patient with embryonal rhabdomyosarcoma.

Background: Weeksella virosa is one of the two species of the genus Weeksella. Clinical disease due to this bacterium in humans is rare, for which only nine cases have been reported in the literature. Case report: A 4-year-old male patient was diagnosed with a left orbit rhabdomyosarcoma Stage III and was admitted to a northeast third level referral center in Mexico. Aerobic, non-pigmented, Gram-negative rod was isolated from a blood culture. W. virosa was identified by Sensititre™ ARIS. This organism has been described in cases of spontaneous bacterial peritonitis, sepsis, pneumonia, ventriculitis, and urinary tract infection. Conclusions: Clinicians should consider the diagnosis of W. virosa bacteremia in cases involving immunocompromised patients with oral lesions, although it is infrequent. To the best of our knowledge, this is the first clinical report of W. virosa bacteremia described in an immunocompromised pediatric patient.

Introducción: Weeksella virosa es una de las dos especies del género Weeksella. En los humanos, los reportes de infección por este microorganismo son raros. Solo se han reportado nueve casos en la literatura. Caso clínico: Paciente masculino de 4 años con diagnóstico de rabdomiosarcoma embrionario de órbita izquierda en estadio III, que fue atendido en un hospital de tercer nivel en el noreste de México. Se realizó un hemocultivo, a partir del cual se aisló un bacilo gramnegativo aerobio, no pigmentado. Se identificó W. virosa mediante Sensititre™ ARIS. Este microorganismo ha sido descrito en casos de peritonitis bacteriana espontánea, sepsis, neumonía, ventriculitis e infección del tracto urinario. Conclusiones: Aunque los casos de bacteriemia por W. virosa son raros, los clínicos deben considerar este agente en pacientes inmunocomprometidos con lesiones orales. Hasta donde se conoce, este es el primer caso que se describe de bacteriemia por W. virosa en un paciente pediátrico inmunocomprometido.

Bacteremia due to Weeksella virosa in a pediatric patient with embryonal rhabdomyosarcoma / Bacteriemia por Weeksella virosa en un paciente pediátrico con rabdomiosarcoma embrionario

Abstract Background: Weeksella virosa is one of the two species of the genus Weeksella. Clinical disease due to this bacterium in humans is rare, for which only nine cases have been reported in the literature. Case report: A 4-year-old male patient was diagnosed with a left orbit rhabdomyosarcoma Stage III and was admitted to a northeast third level referral center in Mexico. Aerobic, non-pigmented, Gram-negative rod was isolated from a blood culture. W. virosa was identified by Sensititre™ ARIS. This organism has been described in cases of spontaneous bacterial peritonitis, sepsis, pneumonia, ventriculitis, and urinary tract infection. Conclusions: Clinicians should consider the diagnosis of W. virosa bacteremia in cases involving immunocompromised patients with oral lesions, although it is infrequent. To the best of our knowledge, this is the first clinical report of W. virosa bacteremia described in an immunocompromised pediatric patient.

Resumen Introducción: Weeksella virosa es una de las dos especies del género Weeksella. En los humanos, los reportes de infección por este microorganismo son raros. Solo se han reportado nueve casos en la literatura. Caso clínico: Paciente masculino de 4 años con diagnóstico de rabdomiosarcoma embrionario de órbita izquierda en estadio III, que fue atendido en un hospital de tercer nivel en el noreste de México. Se realizó un hemocultivo, a partir del cual se aisló un bacilo gramnegativo aerobio, no pigmentado. Se identificó W. virosa mediante Sensititre™ ARIS. Este microorganismo ha sido descrito en casos de peritonitis bacteriana espontánea, sepsis, neumonía, ventriculitis e infección del tracto urinario. Conclusiones: Aunque los casos de bacteriemia por W. virosa son raros, los clínicos deben considerar este agente en pacientes inmunocomprometidos con lesiones orales. Hasta donde se conoce, este es el primer caso que se describe de bacteriemia por W. virosa en un paciente pediátrico inmunocomprometido.

Serological screening in donors in Mexico: advances and technology / Tamizaje serológico en donadores de México: avances y tecnología

The residual risk of transfusion-related infections has decreased dramatically in countries that have routinely implemented serological screening. Most of the donation in Mexico is from replacement practice, a risk factor for positive serology. In Mexico, the altruistic donation is only 2.7%. The heterogeneity of technical factors, regional factors and internal policies of each center influences the variability of data on the prevalence of positive screening, as well as the prevalence of confirmed cases. The main advantage of nucleic acid technology is the detection of donors in the period of serological window or occult infections, being occult hepatitis reports in Mexican donors from 1 to 3.4%. The limitation of available technology, the scope of the clinic and perspectives, invites us to improve technology and health policies in the interest of transfusion safety.

El riesgo residual de las infecciones relacionadas a la transfusión ha disminuido drásticamente en los países que han implementado rutinariamente el tamizaje serológico. La mayor parte de la donación en México es de reposición, factor de riesgo para serología positiva, y en donde la donación altruista es de apenas 2.7%. La heterogeneidad de factores técnicos, regionales y políticas internas de cada centro influyen en la variabilidad de datos en prevalencia del tamizaje positivo, así como en la prevalencia de casos confirmados. La principal ventaja de la tecnología de ácidos nucleicos es la detección de donadores en periodo de ventana serológico o de infecciones ocultas, con reportes de hepatitis ocultas en donadores de 1-3.4% en México. Las limitantes de la tecnología disponible, el alcance de la clínica y de las perspectivas, nos invita a la mejora tecnológica y de las políticas sanitarias en aras de la seguridad transfusional.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

Microbiology of genus Raoultella, clinical features and difficulties in its diagnosis / Microbiología del género Raoultella, características clínicas y dificultades para su diagnóstico

The genus Raoultella, which belongs to the Enterobacteriaceae family, encompasses gramnegative, oxidase-negative, aerobic, encapsulated and motionless bacilli. Currently, four species of this genus have been described: Raoultella terrigena (R. terrigena), Raoultella planticola (R. planticola), Raoultella ornithinolytica (R. ornithinolytica) and Raoultella electrica (R. electrica), out of which R. planticola and R. ornithinolytica are the most important because of their probable association as pathogenic agents. The incidence of this genus has been underestimated, given that detecting it is really difficult, since it is misidentified as Klebsiella spp. by using manual and automated conventional phenotypic methods. The rapid and correct detection has been increasing since the advent of mass spectrometers (MALDI-TOF MS), which have been used for the differentiation of Klebsiella spp. and Raoultella spp. Currently 80 cases of bacteremia secondary to Raoultella spp. have been identified with different sites of primary infection, which is why it is important to emphasize proper detection in hospitals and healthcare facilities.

El género Raoultella, perteneciente a la familia Enterobacteriaceae, engloba bacilos gram negativos, oxidasa negativos, aeróbicos, encapsulados e inmóviles. Actualmente se han descrito cuatro especies de este género: Raoultella terrigena (R. terrigena), Raoultella planticola (R. planticola), Raoultella ornithinolytica (R. ornithinolytica) y Raoultella electrica (R. electrica), entre las que la R. planticola y la R. ornithinolytica son las más importantes por su probable asociación como agentes patógenos. Se ha subestimado la incidencia del género por la dificultad que presenta su caracterización al ser mal identificado como Klebsiella spp., al usar métodos fenotípicos manuales y automatizados convencionales. La rápida y correcta detección de Raoultella spp. ha ido aumentando desde la llegada de espectrómetros de masas (MALDI-TOF MS), útiles en la diferenciación entre Klebsiella spp. y Raoultella spp. Se han identificado 80 casos de bacteremia por Raoultella spp., con infección primaria en diversos sitios, por lo que es importante hacer énfasis en su correcta detección en los hospitales y centros de atención médica.

Frequency and genotypes of Chlamydia trachomatis in patients attending the obstetrics and gynecology clinics in Jalisco, Mexico and correlation with sociodemographic, behavioral, and biological factors.

BACKGROUND: Chlamydia trachomatis is the causative agent of the most common bacterial sexually transmitted infection worldwide. The aim of this study was to investigate the frequency and genotypes of C. trachomatis in patients attending an obstetrics and gynecology clinic in Jalisco, Mexico and correlates them with sociodemographic, behavioral, and biological factors. METHODS: C. trachomatis detection was performed in endocervical samples from 662 patients by direct fluorescence assay (DFA) and two PCR assays that amplified the phospholipase D endonuclease superfamily protein (PLDESP) and OmpA genes. Positive samples were genotyped using PCR-restriction fragment length polymorphism assays. Sociodemographic, behavioral, and biological data were collected. RESULTS: The mean age of the study population was 31 (range, 14-78) years. C. trachomatis positivity was detected by DFA in 16.7% (n = 111), PLDESP gene amplification in 14.2% (n = 94), and OmpA gene amplification in 14.5% (n = 96) of the population. Eight C. trachomatis genotypes were detected: E (39.6%), F (29.2%), D (15.6%), K (6.3%), L2 (3.1%), G, J, and I (2.1% each). C. trachomatis infection was associated with age, marital status, pregnancy, and hormonal contraceptive use (all p = 0.01); intrauterine device use and previous premature birth (both p = 0.03); and infection during pregnancy, previous ectopic pregnancy, pelvic inflammatory disease (PID), and green vaginal discharge (all p = 0.04). C. trachomatis genotype K was more likely to be detected in women histories of ≥2 sexual partners, genotype F was more likely in pregnant women, genotype L2 was more likely in women with PID, genotype D was more likely in women who had had infection during previous pregnancies, and genotype E was more likely in those with previous ectopic pregnancies and green vaginal discharge (all p = 0.01). CONCLUSIONS: The frequency of C. trachomatis in our population was higher than previously reported worldwide, but within the range reported for Mexico. Genotype E was detected most frequently in the study population. Infection by C. trachomatis and C. trachomatis genotypes K, F, D, and E was strongly associated with multiple sociodemographic, behavioral, and biological factors. C. trachomatis genotype L2 was detected in women with PID.

Sexually transmitted pathogens, coinfections and risk factors in patients attending obstetrics and gynecology clinics in Jalisco, Mexico.

OBJECTIVE: To determine the frequency of nine sexually transmitted pathogens, coinfections and risk factors in patients attending obstetrics and gynecology clinics in Jalisco, Mexico. MATERIALS AND METHODS: Samples from 662 patients attending obstetrics and gynecology clinics were analyzed. Treponema pallidum, HIV, and HCV were detected by serology. HPV was detected by Polimerase Chain Reaction (PCR), and its genotype was determined by Restriction Fragment Length Polymorphism (RFLP). Trichomonas vaginalis, HSV-1, HSV-2, Mycoplasma genitalium, Neisseria gonorrhoeae and T. pallidum were detected by multiplex PCR. RESULTS: By serology, HIV frequency was 6.8%, T. pallidum was 2.26%, and HCV was 0.15%. By PCR, HPV frequency was 13.9%, (more frequent genotype was 16, 33.7%), followed by T. vaginalis (14.2%), HSV-1 (8.5%), M. genitalium (2,41%), N. gonorrhoeae (2.11%), HSV-2 (1.8%), and T. pallidum (1.05%). Patients infected with T. vaginalis were more likely to have multiple coinfections (p = 0.01). CONCLUSION: The frequency of HPV, HVS-1, HSV-2, M. genitalium and T. vaginalis was lower than that reported. However, a high frequency of HIV, T. pallidum, and N. gonorrhoeae was detected.

Sexually transmitted pathogens, coinfections and risk factors in patients attending obstetrics and gynecology clinics in Jalisco, Mexico / Patógenos de transmisión sexual, coinfecciones y factores de riesgo en pacientes que asisten a clínicas de obstetricia y ginecología en Jalisco, México

Abstract: Objective: To determine the frequency of nine sexually transmitted pathogens, coinfections and risk factors in patients attending obstetrics and gynecology clinics in Jalisco, Mexico. Materials and methods: Samples from 662 patients attending obstetrics and gynecology clinics were analyzed. Treponema pallidum, HIV, and HCV were detected by serology. HPV was detected by Polimerase Chain Reaction (PCR), and its genotype was determined by Restriction Fragment Length Polymorphism (RFLP). Trichomonas vaginalis, HSV-1, HSV-2, Mycoplasma genitalium, Neisseria gonorrhoeae and T. pallidum were detected by multiplex PCR. Results: By serology, HIV frequency was 6.8%, T. pallidum was 2.26%, and HCV was 0.15%. By PCR, HPV frequency was 13.9%, (more frequent genotype was 16, 33.7%), followed by T. vaginalis (14.2%), HSV-1 (8.5%), M. genitalium (2,41%), N. gonorrhoeae (2.11%), HSV-2 (1.8%), and T. pallidum (1.05%). Patients infected with T. vaginalis were more likely to have multiple coinfections (p = 0.01). Conclusion: The frequency of HPV, HVS-1, HSV-2, M. genitalium and T. vaginalis was lower than that reported. However, a high frequency of HIV, T. pallidum, and N. gonorrhoeae was detected.

Resumen: Objetivo: Determinar la frecuencia de nueve patógenos de transmisión sexual, coinfecciones y factores de riesgo en pacientes que acudieron a una consulta de ginecología y obstetricia en Jalisco, México. Material y métodos: Se analizaron muestras de 662 pacientes que asistieron a la consulta de ginecología y obstetricia. Se detectaron Treponema pallidum, VIH y VHC mediante serología. Se detectó VPH por Reacción de Cadena de Polimerasa (PCR) y sus genotipos se detectaron por Polimorfismos de Longitud de Fragmentos de Restricción (RFLP). Se detectaron Trichomonas vaginalis, VHS-1,VHS-2, Mycoplasma genitalium, Neisseria gonorrhoeae y T. pallidum por PCR múltiple. Resultados: Por serología, la frecuencia deVIH fue 6.8%, de T. pallidum fue 2.26% y deVHC fue 0.15%. Por PCR, la frecuencia más alta fue deVPH (13.9%, el genotipo más frecuente fue el 16, 33.7%), seguida deT. vaginalis (14.2%), VHS-1 (8.5%), M. genitalium (2.41%), N. gonorrhoeae (2.11%), VHS-2 (1.8%) y T. pallidum (1.05%). Los pacientes infectados con T. vaginalis presentaron más probabilidades de tener múltiples coinfecciones (p = 0.01). Conclusiones: La frecuencia de infección por VPH, VHS-1,VHS-2, M.genitalium y T. vaginalis fue menor a lo reportado. Sin embargo, se detectó una alta frecuencia de VIH, T. pallidum, y N. gonorrhoeae.