Comprehensive comparative analysis of the periodontal pathogen Porphyromonas gingivalis: exploring the pan-genome, the reconstruction of the gene regulatory network and genome-scale metabolic network.

Porphyromonas gingivalis is a nonmotile, obligate anaerobic, Gram-negative bacterium known for its association with periodontal disease and its involvement in systemic diseases such as atherosclerosis, cardiovascular disease, colon cancer, and Alzheimer's disease. This bacterium produces several virulence factors, including capsules, fimbriae, lipopolysaccharides, proteolytic enzymes, and hemagglutinins. A comparative genomic analysis revealed the open pangenome of P. gingivalis and identified complete type IV secretion systems in strain KCOM2805 and almost complete type VI secretion systems in strains KCOM2798 and ATCC49417, which is a new discovery as previous studies did not find the proteins involved in secretion systems IV and VI. Conservation of some virulence factors between different strains was observed, regardless of their genetic diversity and origin. In addition, we performed for the first time a reconstruction analysis of the gene regulatory network, identifying transcription factors and proteins involved in the regulatory mechanisms of bacterial pathogenesis. In particular, QseB regulates the expression of hemagglutinin and arginine deaminase, while Rex may suppress the release of gingipain through interactions with PorV and the formatum/nitrate transporter. Our study highlights the central role of conserved virulence factors and regulatory pathways, particularly QseB and Rex, in P. gingivalis and provides insights into potential therapeutic targets.

The CRISPR-Cas system in clinical strains of Acinetobacter baumannii: an in-silico analysis.

Acinetobacter baumannii is a relevant bacterium due to its high-resistance profile. It is well known that antimicrobial resistance is primarily linked to mutations and the acquisition of external genomic material, such as plasmids or phages, to which the Clustered Regularly Interspaced Short Palindromic Repeats associated with Cas proteins, or CRISPR-Cas, system is related. It is known that the system can influence the acquisition of foreign genetic material and play a role in various physiological pathways. In this study, we conducted an in-silico analysis using 91 fully assembled genomes of clinical strains obtained from the NCBI database. Among the analyzed genomes, the I-F1 subtype of the CRISPR-Cas system was detected showcasing variations in architecture and phylogeny. Using bioinformatic tools, we determined the presence, distribution, and specific characteristics of the CRISPR-Cas system. We found a possible association of the system with resistance genes but not with virulence determinants. Analysis of the system's components, including spacer sequences, suggests its potential role in protecting against phage infections, highlighting its protective function.

Analysis of Rocky Mountain spotted fever cases in Northern Mexico reveals genetic variability of Rickettsia rickettsii and the different distribution of genotypes.

Rickettsioses have been reported in parts of Mexico since the last century, with Rocky Mountain spotted fever (RMSF) being one of the most prevalent in northern states. Unfortunately, fatality rates for RMSF in Mexico are higher than in other countries, like the USA. The reason for this difference in fatality rates is currently unknown and could be associated with a genotype of the bacterium, but no comparative molecular typing has been conducted in Mexico to date. The purpose of this study was to analyze 47 RMSF samples with different outcomes from several states in northern Mexico to know the genetic variability of Rickettsia rickettsii, as well as to reconstruct its phylogeny, for which the following intergenic regions were sequenced: RR0155-rpmB, cspA-ksgA, RR1240-tlc5, and Spo0J-abc T1, as well as the following partial genes: ompA, ompB, and gltA. We identified 8 genotypes with different distribution and prevalence among the states analyzed, as well as a different association with case outcome; these genotypes were clustered in 2 clades and 5 lineages were revealed, some of them probably exclusive from Mexico.

Evasion of Antimicrobial Activity in Acinetobacter baumannii by Target Site Modifications: An Effective Resistance Mechanism.

Acinetobacter baumannii is a Gram-negative bacillus that causes multiple infections that can become severe, mainly in hospitalized patients. Its high ability to persist on abiotic surfaces and to resist stressors, together with its high genomic plasticity, make it a remarkable pathogen. Currently, the isolation of strains with high antimicrobial resistance profiles has gained relevance, which complicates patient treatment and prognosis. This resistance capacity is generated by various mechanisms, including the modification of the target site where antimicrobial action is directed. This mechanism is mainly generated by genetic mutations and contributes to resistance against a wide variety of antimicrobials, such as ß-lactams, macrolides, fluoroquinolones, aminoglycosides, among others, including polymyxin resistance, which includes colistin, a rescue antimicrobial used in the treatment of multidrug-resistant strains of A. baumannii and other Gram-negative bacteria. Therefore, the aim of this review is to provide a detailed and up-to-date description of antimicrobial resistance mediated by the target site modification in A. baumannii, as well as to detail the therapeutic options available to fight infections caused by this bacterium.

Colistin Resistance in Aeromonas spp.

The increase in the use of antimicrobials such as colistin for the treatment of infectious diseases has led to the appearance of Aeromonas strains resistant to this drug. However, resistance to colistin not only occurs in the clinical area but has also been determined in Aeromonas isolates from the environment or animals, which has been determined by the detection of mcr genes that confer a resistance mechanism to colistin. The variants mcr-1, mcr-3, and mcr-5 have been detected in the genus Aeromonas in animal, environmental, and human fluids samples. In this article, an overview of the resistance to colistin in Aeromonas is shown, as well as the generalities of this molecule and the recommended methods to determine colistin resistance to be used in some of the genus Aeromonas.

The outer membrane vesicles: Secretion system type zero.

Gram-negative bacteria have mechanisms through which they can colonize and survive in different environments, such as the secretion systems types (1-6) that have been widely studied and characterized. Nowadays, some authors have proposed extracellular structures, such as the outer membrane vesicles (OMVs), to be considered as an additional and independent secretion system. The OMVs are spherical particles of 50-250 nm in diameter; they originate in the outer membrane, and therefore they have a very similar composition to the latter. These particles can transport an important variety of biomolecules: enzymes, toxins, antigenic determinants and even nucleic acids. Thus, it is of great interest to collect data describing the advantages of the transport of biomolecules through the OMVs and, thus, determine their role as a potential secretion system.

Mutational landscape and intra-host diversity of human papillomavirus type 16 long control region and E6 variants in cervical samples.

Human papillomavirus genotype 16 (HPV16) is the most frequent high-risk HPV (HR-HPV) identified in cervical precursor lesions and cervical cancer (CC) worldwide. The oncogenic potential of HPV16 is partly dependent on the lineage involved in the infection and the presence of clinically relevant mutations. In this report, we present the distribution of HR-HPV and the mutational profile and intra-host variability of HPV16 lineages, based on analysis of the long control region (LCR) and the E6 gene in samples with normal cytology (n = 39), squamous intraepithelial lesions (n = 25), and CC (n = 39). HR-HPV genotyping was performed using multiplex real-time PCR. HPV16 lineage assignments and mutation frequencies were determined by conventional PCR and Sanger DNA sequencing, and intra-patient viral populations were analyzed using next-generation sequencing (NGS). The most frequent HR-HPV type was HPV16, followed by HPV31 and HPV18. The frequency of HPV16 sublineages was A1/A2 > D2 > D3 and B1. Moreover, the most frequent mutations, both in samples from this study and in the available sequences from Mexican isolates in the GenBank database were LCR-G7518A, which is involved in carcinogenesis, and E6-T350G (producing L83V), associated with persistence of infection. Otherwise, deep sequencing revealed high conservation of viral lineages and mutations, independently of the stages studied. In conclusion, the high frequency and stability of these molecular markers, as well as the circulating viral lineages, could be related to the incidence of CC associated with HPV16. Hence, they deserve a broader analysis to determine the risk of specific populations for progression of the disease.

Utility of high-throughput DNA sequencing in the study of the human papillomaviruses.

The Papillomaviridae family is probably the most diverse group of viruses that affect vertebrates. The study of the relationship between infection by certain types of human papillomavirus (HPV) and the development of neoplastic epithelial lesions is of particular interest because of the high prevalence of HPV-related carcinomas in populations of developing countries. To understand the mechanisms of infection and their association with different clinical manifestations, molecular tools play an important role in the description of new types of HPV, the characterization of effector properties of the viral factors, the specific diagnosis and monitoring of HPV types, and the alteration patterns at genetic level in the host. Technological advances in the field of DNA sequencing have led to the development of different next-generation sequencing systems, allowing obtaining a large amount of data and broadening the applications to study viral diseases. In this review, we summarize the main approaches and their perspectives where the use of massively parallel sequencing has been proved as a useful tool in the research of the HPV infection.

Adherence of Ornithobacterium rhinotracheale to chicken embryo lung cells as a pathogenic mechanism.

Ornithobacterium rhinotracheale is a bacterium that causes respiratory disease in birds and it has been isolated in countries with a large poultry production, including Mexico. The pathogenicity mechanisms of this bacterium have not been completely elucidated yet. The capacity of the bacterium to adhere to epithelial cells of chicken in vitro has been evidenced, and since this bacterium has been isolated from the lungs and air sacs of several avian species, the aim of this study was to determine if this bacterium can adhere to chicken lung cells. We used five O. rhinotracheale reference serovars (A-E) that were in contact with primary lung cells cultured from a 19-day-old chicken embryo. O. rhinotracheale adherence was evaluated through optical and transmission electron microscopies. The results revealed that O. rhinotracheale is capable of adhering to chicken embryo lung cells within 3 h of incubation with a diffuse adherence pattern. The adherence percentages of the chicken embryo lung cells were 51-96% according to the serovar of the bacterium. Relative adherence was from 4 to 8 bacteria per cell. Transmission electron microscope data revealed intracellular bacteria inside a vacuole in less than 3 h of incubation.

Design, Synthesis, and Evaluation of Alkyl-Quinoxalin-2(1H)-One Derivatives as Anti-Quorum Sensing Molecules, Inhibiting Biofilm Formation in Aeromonas caviae Sch3.

With the increasing antibiotic resistance of bacterial strains, alternative methods for infection control are in high demand. Quorum sensing (QS) is the bacterial communication system based on small molecules. QS is enables bacterial biofilm formation and pathogenic development. The interruption of QS has become a target for drug discovery, but remains in the early experimental phase. In this study, we synthesized a set of six compounds based on a scaffold (alkyl-quinoxalin-2(1H)-one), new in the anti-QS of Gram-negative bacteria Aeromonas caviae Sch3. By quantifying biofilm formation, we were able to monitor the effect of these compounds from concentrations of 1 to 100 µM. Significant reduction in biofilm formation was achieved by 3-hexylylquinoxalin-2(1H)-one (11), 3-hexylylquinoxalin-2(1H)-one-6-carboxylic acid (12), and 3-heptylylquinoxalin-2(1H)-one-6-carboxylic acid (14), ranging from 11% to 59% inhibition of the biofilm. This pilot study contributes to the development of anti-QS compounds to overcome the clinical challenge of resistant bacteria strains.

Aetiology and Significance of Hospital-Acquired Infections in Mexico.

Hospital-acquired infections (HAIs) are infections that develop in the hospital environment and can be acquired by a patient or hospital staff. They are complications that combine diverse risk factors that make an individual susceptible and are frequently caused by endogenous and exogenous bacterial agents. The most commonly studied etiological agents are bacteria and fungi, with the former representing the most common etiological agents reported to the Hospital Epidemiological Surveillance Network (RHOVE) between 2007 and 2012. Among these agents were Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, coagulase-negative Staphylococci (CNS), Enterococcus spp., and Streptococcus pneumoniae. Although obligate anaerobic bacteria are also etiological agents of HAIs, clinical laboratories do not usually perform bacteriological tests to isolate and identify these bacteria. As a result, patients are at a greater risk of not surviving an infection and the epidemiology of this bacterial group is unknown. An important problem associated with HAIs is bacterial multiple drug resistance, which not only increases morbidity and mortality but also the cost of inpatient care. The aim of this review is to provide current information to healthcare professionals on the status of HAIs in Mexico with an emphasis on the etiology, diagnosis, and antimicrobial resistance.

Conditions that induce biofilm production by Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacillus that causes respiratory disease in birds, and directly affects the poultry industry. The mechanisms behind these infections are not completely known. Currently, its capacity to form biofilms on inert surfaces has been reported; however, the conditions for biofilm development have not been described yet. The present work was aimed at identifying the conditions that enhance in vitro biofilm formation and development by ORT. For this, serovars A-E were analysed to assess their ability to induce biofilm development on 96-well flat-bottom polystyrene microtitre plates under diverse conditions: temperature, incubation time, and CO2 concentration. The results obtained showed not only that all serovars have the ability to produce in vitro biofilms, but also that the optimal conditions for biofilm density were 40°C after 72 h at an elevated CO2 concentration. In conclusion, ORT biofilm formation depends on the environmental conditions and may contribute to the persistence of this microorganism.

Lactobacillus species isolated from vaginal secretions of healthy and bacterial vaginosis-intermediate Mexican women: a prospective study.

BACKGROUND: Lactobacillus jensenii, L. iners, L. crispatus and L. gasseri are the most frequently occurring lactobacilli in the vagina. However, the native species vary widely according to the studied population. The present study was performed to genetically determine the identity of Lactobacillus strains present in the vaginal discharge of healthy and bacterial vaginosis (BV) intermediate Mexican women. METHODS: In a prospective study, 31 strains preliminarily identified as Lactobacillus species were isolated from 21 samples collected from 105 non-pregnant Mexican women. The samples were classified into groups according to the Nugent score criteria proposed for detection of BV: normal (N), intermediate (I) and bacterial vaginosis (BV). We examined the isolates using culture-based methods as well as molecular analysis of the V1-V3 regions of the 16S rRNA gene. Enterobacterial repetitive intergenic consensus (ERIC) sequence analysis was performed to reject clones. RESULTS: Clinical isolates (25/31) were classified into four groups based on sequencing and analysis of the 16S rRNA gene: L. acidophilus (14/25), L. reuteri (6/25), L. casei (4/25) and L. buchneri (1/25). The remaining six isolates were presumptively identified as Enterococcus species. Within the L. acidophilus group, L. gasseri was the most frequently isolated species, followed by L. jensenii and L. crispatus. L. fermentum, L. rhamnosus and L. brevis were also isolated, and were placed in the L. reuteri, L. casei and L. buchneri groups, respectively. ERIC profile analysis showed intraspecific variability amongst the L. gasseri and L. fermentum species. CONCLUSIONS: These findings agree with previous studies showing that L. crispatus, L. gasseri and L. jensenii are consistently present in the healthy vaginal ecosystem. Additional species or phylotypes were detected in the vaginal microbiota of the non-pregnant Mexican (Hispanic-mestizo) population, and thus, these results further our understanding of vaginal lactobacilli colonisation and richness in this particular population.

ESKAPE and Beyond: The Burden of Coinfections in the COVID-19 Pandemic.

The ESKAPE group constitute a threat to public health, since these microorganisms are associated with severe infections in hospitals and have a direct relationship with high mortality rates. The presence of these bacteria in hospitals had a direct impact on the incidence of healthcare-associated coinfections in the SARS-CoV-2 pandemic. In recent years, these pathogens have shown resistance to multiple antibiotic families. The presence of high-risk clones within this group of bacteria contributes to the spread of resistance mechanisms worldwide. In the pandemic, these pathogens were implicated in coinfections in severely ill COVID-19 patients. The aim of this review is to describe the main microorganisms of the ESKAPE group involved in coinfections in COVID-19 patients, addressing mainly antimicrobial resistance mechanisms, epidemiology, and high-risk clones.

Genomic Analysis of Multidrug-Resistant Escherichia coli Strains Isolated in Tamaulipas, Mexico.

The global spread of antimicrobial resistance genes (ARGs) is a major public health concern. Mobile genetic elements (MGEs) are the main drivers of this spread by horizontal gene transfer (HGT). Escherichia coli is widespread in various environments and serves as an indicator for monitoring antimicrobial resistance (AMR). Therefore, the objective of this work was to evaluate the whole genome of multidrug-resistant E. coli strains isolated from human clinical, animal, and environmental sources. Four E. coli strains previously isolated from human urine (n = 2), retail meat (n = 1), and water from the Rio Grande River (n = 1) collected in northern Tamaulipas, Mexico, were analyzed. E. coli strains were evaluated for antimicrobial susceptibility, followed by whole genome sequencing and bioinformatic analysis. Several ARGs were detected, including blaCTX-M-15, blaOXA-1, blaTEM-1B, blaCMY-2, qnrB, catB3, sul2, and sul3. Additionally, plasmid replicons (IncFIA, IncFIB, IncFII, IncY, IncR, and Col) and intact prophages were also found. Insertion sequences (ISs) were structurally linked with resistance and virulence genes. Finally, these findings indicate that E. coli strains have a large repertoire of resistance determinants, highlighting a high pathogenic potential and the need to monitor them.

Disinfection efficacy of ozone on ESKAPE bacteria biofilms: Potential use in difficult-to-access medical devices.

BACKGROUND: Medical devices can be reservoirs of multidrug-resistant bacteria that may be involved in the acquisition of infections since bacteria with the ability to form biofilms that are difficult to eradicate, mainly in mechanical ventilators. The aim of this work was to evaluate the efficacy of O3 against biofilms of bacteria ESKAPE group through disinfection studies. METHODS: The formation of biofilms of ESKAPE group bacteria was induced in vitro. O3 was injected at different exposure times at a constant dose of 600 mg/h. The recovery of surviving bacteria after O3 treatment was assessed by bacterial counts and biofilm disruption was analyzed. Finally, the viability and integrity of biofilms after O3 treatment was determined by confocal laser scanning microscopy (CLSM). RESULTS: O3 showed bactericidal activity on biofilms from 12 min/7.68 ppm for A. baumannii and C. freundii. P. aeruginosa, K. pneumoniae and S. aureus were killed after 15 min/9.60 ppm. Correlation analyses showed inversely proportional relationships between the variables "disruption versus O3". CLSM revealed that death was time-dependent of biofilms upon O3 exposure. Orthogonal plane analysis showed that bacteria located in the outer region of the biofilms were the ones that initially suffered damage from O3 exposure. CONCLUSIONS: Our findings suggest that this method could be an alternative for the disinfection in mechanical ventilators colonized by bacteria biofilm forming.

ESKAPE bacteria characterization reveals the presence of Acinetobacter baumannii and Pseudomonas aeruginosa outbreaks in COVID-19/VAP patients.

INTRODUCTION: A decrease of detection of outbreaks by multidrug-resistant bacteria in critical areas has been reduced due to COVID-19 pandemic. Therefore, molecular epidemiological surveillance should be a primary tool to reveal associations not evident by classical epidemiology. The aim of this work was to demonstrate the presence of hidden outbreaks in the first wave of the COVID-19 pandemic and to associate their possible origin. METHODS: A population of 96 COVID-19 patients was included in the study (April to June 2020) from Hospital Juárez de México. Genetic identification and antimicrobial susceptibility testing of VAP causative agents isolated from COVID-19 patients was performed. Resistance phenotypes were confirmed by PCR. Clonal association of isolates was performed by analysis of intergenic regions obtained. Finally, the association of clonal cases of VAP patients was performed by timelines. RESULTS: ESKAPE and non-ESKAPE bacteria were identified as causative agents of VAP. ESKAPE bacteria were classified as MDR and XDR. Only A. baumannii and P. aeruginosa were identified as clonally distributed in 13 COVID-19/VAP patients. Time analysis showed that cross-transmission existed between patients and care areas. CONCLUSIONS: Acinetobacter baumannii and Pseudomonas aeruginosa were involved in outbreaks non-detected in COVID-19/VAP patients in the first wave of COVID-19 pandemic.

Typing of Candida spp. from Colonized COVID-19 Patients Reveal Virulent Genetic Backgrounds and Clonal Dispersion.

Advances in the knowledge of the pathogenesis of SARS-CoV-2 allowed the survival of COVID-19 patients in intensive care units. However, due to the clinical characteristics of severe patients, they resulted in the appearance of colonization events. Therefore, we speculate that strains of Candida spp. isolated from COVID-19 patients have virulent genetic and phenotypic backgrounds involved in clinical worsening of patients. The aim of this work was to virutype Candida spp. strains isolated from colonized COVID-19 patients, analyze their genomic diversity, and establish clonal dispersion in care areas. The virulent potential of Candida spp. strains isolated from colonized COVID-19 patients was determined through adhesion tests and the search for genes involved with adherence and invasion. Clonal association was done by analysis of intergenic spacer regions. Six species of Candida were involved as colonizing pathogens in COVID-19 patients. The genotype analysis revealed the presence of adherent and invasive backgrounds. The distribution of clones was identified in the COVID-19 care areas, where C. albicans was the predominant species. Evidence shows that Candida spp. have the necessary genetic tools to be able colonize the lungs, and could be a possible causal agent of coinfections in COVID-19 patients. The detection of dispersion of opportunistic pathogens can be unnoticed by classical epidemiology. Epidemiological surveillance against opportunistic fungal pathogens in COVID-19 patients is an immediate need, since the findings presented demonstrate the potential virulence of Candida spp.

Epidemiological Overview of Urogenital Gonorrhea in Mexico (2003-2020).

In Mexico, urogenital gonorrhea (UG) is one of the main sexually transmitted diseases notifiable by health systems around the world. Epidemiological data on sexually transmitted infections (STIs) in Mexico indicated that UG was "under control" until 2017. However, international epidemiological reports indicate the increase in incidence due to several factors, including an increase during the first year of the COVID-19 pandemic. These factors suggest that this phenomenon may occur in developing countries, including Mexico. Therefore, the aim of this study was to analyze national surveillance data on UG from 2003-2019 and the first year of the COVID-19 pandemic. An epidemiological study of cases and incidence of UG (2003-2020) was performed in the annual reports issued by the General Directorate Epidemiology in Mexico. Cases and incidence were classified and analyzed by year, sex, age group, and seasons (by temperature). Distribution of UG was carried out using heat maps for the whole country. Ultimately, a seasonal and correlation analysis was performed for UG cases versus temperature. The results showed that the distribution of cases and incidence by sex showed that there was no variation over 14 years. From 2016 onward, a significant increase in UG was observed before the pandemic. During the first year of the pandemic, a significant increase was observed in females aged 24-44 years. A heterogeneous distribution of UG was identified; however, border states were ranked among the top states with elevated incidences and cases. Lastly, the occurrence of UG was associated with temperature, related to summer. The information presented is intended to be useful to promote prevention and to contribute to visualize the distribution of UG over the last 18 years for decision making, and to show one of the consequences of the collapse of epidemiological surveillance of UG during the first year of the COVID-19 pandemic.

A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.