Verónica-Iranzú Martínez-Santos

The aim of this study was to determine biofilm production and motility in Bacillus pacificus B630 and Bacillus cereus ATCC 14579, and to perform a comparative genome analysis using bioinformatic tools to understand the differences between the two strains. Biofilm production was performed in glass tubes stained with safranin; motility was determined on soft agar. Bioinformatic analysis was performed using genomic information from both strains, including the identification of orthologous genes, the similarity between genes of the eps1 and sipW-tasA-calY operons, and the SipW and TasA model prediction. B. pacificus B630 produces a greater amount of biofilm on glass than B. cereus ATCC 14579 (p < 0.01). Furthermore, B. pacificus B630 shows lower motility than B. cereus ATCC 14579 (p < 0.001). B. pacificus B630 contains 45 unshared genes, whereas B. cereus ATCC 14579 has 27 unshared genes. Differences in similarity were observed between the genes of the eps1 and sipW-tasA-calY operons. These differences between SipW and TasA may affect protein structural predictions. In SipW, the differences may affect the C-terminal region. In TasA, the number of B-sheets differed between the two proteins, and amino acid substitutions were found in regions of high protein aggregation. Genomic differences in genes associated with biofilm production may explain differences in biofilm production between the strains studied.

Spontaneous fermentation is central to ancestral mezcal production, yet the bacterial and fungal communities associated with Agave cupreata must remain insufficiently characterized. This exploratory study described community composition and predicted functional profiles at three stages of a traditional fermentation defined by sugar depletion: 10 °Brix (0 h), 5 °Brix (72 h), and 2 °Brix (144 h). Ambient temperature ranged from 21.7 to 23.7 °C, must temperature from 28.5 to 29.9 °C, pH from 4.6 to 5.2, and relative humidity from 51.3% to 58.7%. At each stage, material collected from two wooden vats and three vertical positions was pooled into one composite sample. Bacterial 16S rRNA gene and fungal ITS1 amplicons were sequenced and analyzed using QIIME 2. At 10 °Brix, five dominant bacterial genera accounted for more than 93% of relative abundance. Lactic and acetic acid bacteria represented more than 91% at 5 °Brix, while Pediococcus, Lactiplantibacillus, and Loigolactobacillus were prominent at 2 °Brix. Seven fungal genera represented more than 98% of the initial profile, whereas Saccharomyces, Debaryomyces, and Maudiozyma accounted for more than 98% at 2 °Brix. PICRUSt2 predicted pathways related to carbohydrate degradation and fermentation, whereas FUNGuild assignments were predominantly saprotrophic at the later stages. Because one pooled sample was analyzed per stage, these patterns are descriptive and require validation in replicated fermentations. Despite these limitations, this study provides baseline information on the microbial communities associated with ancestral A. cupreata fermentation and identifies taxa and predicted functions that merit future isolation and experimental validation.

In this study, we evaluated the biofilm-forming capacity of the B. pacificus B630 strain on table eggshells and its behavior in the presence of egg components, in comparison with B. cereus ATCC 14579. Strain B630, previously characterized as nhe⁺ and cytK⁺ and as a strong biofilm producer on glass, was confirmed as motile and positive for protease and phospholipase production. In static assays on disinfected eggshell pieces, B630 formed significantly more biofilm than ATCC 14579, while both strains exhibited comparable numbers of vegetative cells and spores embedded in the biofilm. Scanning electron microscopy and Fourier transform infrared (FT-IR) analysis revealed a dense extracellular matrix, altered eggshell crystal morphology, and a reduction in calcite-associated bands in biofilm-positive shells. In brain–heart infusion (BHI) broth supplemented with egg white, growth and spore germination of ATCC 14579 were strongly inhibited, whereas B630 displayed markedly higher tolerance. In an eggshell contamination model with an initial inoculum of 1 × 10⁵ colony-forming units, B630 persisted on the shell for at least 15 days at room temperature, while neither strain was recovered from egg white or yolk. These findings indicate that B. pacificus B630 combines robust biofilm formation with enhanced tolerance to egg white, favoring prolonged persistence on eggshells and underscoring the potential role of highly biofilm-forming B. cereus s.l. strains in table egg contamination. The persistence of strains of the B. cereus s.l. group in the eggshell may compromise the safety of the product.

Enteropathogenic Escherichia coli (EPEC) is a common cause of childhood diarrhea in developing countries. Extensive studies have established the role of virulence factors, such as the type 3 secretion system encoded by the locus of enterocyte effacement (LEE), in intestinal colonization and the central mechanisms that control their expression. However, the role of the second messenger c-di-GMP in virulence gene regulation in EPEC remains poorly understood, despite the extensive repertoire of genes encoding enzymes involved in its synthesis and degradation. In this work, we examined the regulation and role of DgcZ, a highly conserved diguanylate cyclase in commensal and pathogenic E. coli strains, in LEE gene regulation in the prototype EPEC strain E2348/69. Using reporter fusions of the dgcZ upstream regulatory region to the cat reporter gene, we first compared dgcZ promoter activity. A DgcZ deletion, partially lacking the CZB domain (DgcZ-∆NT), was constructed. Unexpectedly, the EPEC type 3 secretion system remains active when this deletion is introduced into EPEC growing under non-inducing conditions (LB medium). Whether this phenotype is caused by a protein-protein interaction between DgcZ lacking the CZB domain (DgcZ-∆NT) and an unknown effector remains to be determined.

Background: Urinary tract infections (UTIs) are very common worldwide. According to their symptomatology, these infections are classified as pyelonephritis, cystitis, or asymptomatic bacteriuria (AB). Approximately 75–95% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which is an extraintestinal bacterium that possesses virulence factors for bacterial adherence and invasion in the urinary tract. In addition, UPEC possesses type 6 secretion systems (T6SS) as virulence mechanisms that can participate in bacterial competition and in bacterial pathogenicity. UPEC UMN026 carries three genes, namely, ECUMN_0231, ECUMN_0232, and ECUMN_0233, which encode three uncharacterized proteins related to the T6SS that are conserved in strains from phylogroups B2 and D and have been proposed as biomarkers of UTIs. Aim: To analyze the frequency of the ECUMN_0231, ECUMN_0232, ECUMN_0233, and vgrG genes in UTI isolates, as well as their expression in Luria Bertani (LB) medium and urine; to determine whether these genes are related to UTI symptoms or bacterial competence and to identify functional domains on the putative proteins. Methods: The frequency of the ECUMN and vgrG genes in 99 clinical isolates from UPEC was determined by endpoint PCR. The relationship between gene presence and UTI symptomatology was determined using the chi² test, with p < 0.05 considered to indicate statistical significance. The expression of the three ECUMN genes and vgrG was analyzed by RT-PCR. The antibacterial activity of strain UMN026 was determined by bacterial competence assays. The identification of functional domains and the docking were performed using bioinformatic tools. Results: The ECUMN genes are conserved in 33.3% of clinical isolates from patients with symptomatic and asymptomatic UTIs and have no relationship with UTI symptomatology. Of the ECUMN⁺ isolates, only five (15.15%, 5/33) had the three ECUMN and vgrG genes. These genes were expressed in LB broth and urine in UPEC UMN026 but not in all the clinical isolates. Strain UMN026 had antibacterial activity against UPEC clinical isolate 4014 (ECUMN⁻) and E. faecalis but not against isolate 4012 (ECUMN⁺). Bioinformatics analysis suggested that the ECUMN genes encode a chaperone/effector/immunity system. Conclusions: The ECUMN genes are conserved in clinical isolates from symptomatic and asymptomatic patients and are not related to UTI symptoms. However, these genes encode a putative chaperone/effector/immunity system that seems to be involved in the antibacterial activity of strain UMN026.

Background: In recent years Staphylococcus epidermidis has been considered an important and frequent causative agent of health care-associated infections (HAIs), increasing the costs of hospitalization, morbidity, and mortality. Antibiotic resistance and biofilm formation are the most important obstacles in the treatment of infections caused by this microorganism. The aim of this work was to determine the most prevalent STs, as well as the antibiotic resistance profile and biofilm formation of S. epidermidis clinical isolates obtained from hospitalized patients in two hospitals in Acapulco, Guerrero in two time periods. Methods: Twenty methicillin-resistant S. epidermidis strains isolated from patients with bacteremia in two hospitals in two time periods were analyzed. Identification and antibiotic susceptibility were performed using the Vitek automated system. Molecular confirmation of the identification and methicillin resistance was performed by duplex PCR of the mecA and nuc genes. Biofilm production was analyzed, and the clonal origin was determined by multilocus sequence typing (MLST). Results: We identified 14 antibiotic resistance profiles as well as 13 sequence types (ST), including the new ST761. We also found that ST2 and ST23 were the most prevalent and, together with ST59, were found in both time periods. Seventeen of our clinical isolates were multidrug-resistant, but all of them were sensitive to linezolid and vancomycin, and this was not related to biofilm production. Additionally, we standardized a duplex PCR to identify methicillin-resistant S. epidermidis strains. In conclusion, S. epidermidis STs 2, 23, and 59 were found in both time periods. This study is the first report of S. epidermidis ST761. The clinical isolates obtained in this work showed a high multidrug resistance that is apparently not related to biofilm production.

Background: Helicobacter pylori detection in asymptomatic children with suspected infection or with symptoms that suggest gastric pathology is problematic, since most of the methods depend on the endoscopic study, an invasive and expensive method. Non-invasive methods can be a feasible alternative but must be validated. The purpose of this study was to evaluate the concordance between H. pylori DNA detection in saliva and dental plaque by PCR, with antigen detection in stool by immunochromatography, among asymptomatic children in the state of Guerrero, Mexico. Methods: Dental plaque, saliva, and stool samples were obtained from 171 children between 6 and 12 years old. H. pylori detection in saliva and dental plaque was performed by PCR using specific primers for the 16S rRNA gene, while the detection in stool samples was performed by immunochromatography using the CerTest kit. Results: We found an overall H. pylori prevalence of 59.6% (102/171). Of the H. pylori positive children 18% (20/111) were positive in saliva samples, 28.1% (34/121) in dental plaque samples, and 50.4% (71/141) in stool samples. A higher prevalence was found in girls (64.7%, p = 0.002). Although some of the children declared some dyspeptic symptoms, these were no related to H. pylori. In conclusion, we found a high prevalence of H. pylori in asymptomatic children and the highest proportion was detected by stool antigen test, which was the most feasible method to detect H. pylori infection.

Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa in humans. One of the main virulence factors of H. pylori is the cag pathogenicity island (cagPAI), which encodes a type 4-secretion system (T4SS) and the cytotoxin CagA. Translocation of CagA through the T4SS triggers host-signaling pathways. One of the T4SS proteins is CagL, which is necessary for CagA translocation. CagL is a 26-kDa protein that contains a hypervariable motif, which spans residues 58 to 62. Several polymorphisms in this region have been associated with different disease outcomes, e.g. in Mexico, N58 is associated with a higher risk of gastric cancer. The aim of this work is to analyze the sequence of the hypervariable motif (residues 58 to 62) of clinical isolates from Mexican patients with chronic gastritis, and to correlate these polymorphisms with the vacA genotype. Of the 164 biopsies analyzed, only 30.5% (50/164) were positive for H. pylori. Thirty-six of the 50 clinical isolates (72%) were cagA positive, and 40 (80%) had the most virulent vacA genotype (s1/m1). Of the cagA positive strains, 94.4% were vacA s1/m1. All the cagA+ strains contained the cagL gene. The most prevalent sequence in the polymorphic region (residues 58–62) was DKMGE (75.8%, 25/33), followed by NKMGQ and NEIGQ (6.1%, 2/33), and DEIGQ, NKMGE, DKIGE, and DKIGK (3%, 1/33). Regarding polymorphisms in positions 58 and 59, the most common were D58/K59 (81.8%, 27/33), followed by N58/K59 (9.1%, 3/33), and D58/E59 (3%, 1/33). Only two isolates (6.1%) contained residues N58/E59, which correspond to those found in H. pylori strain ATCC 26695. 92.6% of the clinical isolates having polymorphism D58/K59 had the genotype vacA s1/m1, considered to be the most virulent, while 7.4% had the genotypes vacA s1/m2 and s2/m2. In Mexican patients, CagL polymorphisms D58, K59, M60, E62, K122, and I134 are more common in patients with chronic gastritis.

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Escherichia coli is the main cause of urinary tract infections (UTI) in ambulatory patients, especially strains belonging to the B2 phylogenetic group and ST131 clonal group. Antibiotic treatment is usually administered empirically; however, it is not always effective due to bacterial multidrug resistance and the production of extended spectrum β-lactamases (ESBLs). The aim of this study was to characterize E. coli clinical isolates from patients with UTI in a community of the State of Guerrero, Mexico. From January to August 2014, 134 clinical isolates of E. coli were recovered. Strain identification and antibiotic susceptibility were performed using the Vitek automated system. Phylogenetic and O25b-ST13 groups were determined by multiple PCR. Identification of the blaCTX-M, blaTEM, and blaSHV genes was performed by conventional PCR. We found that over 50% of the isolates were resistant to betalactams and quinolones, while 0 to 33% were resistant to aminoglycosides and nitrofurans, and 56.49% of the strains were ESBL producers. B2 phylogenetic group was the most predominant (43%) compared to the other groups. The prevalence of bla genes was: blaCTX-M 64.3%, blaSHV 41.4%, and blaTEM 54.3%. These results show a high percentage (55%) of multidrug-resistant strains isolated from UTI patients from the community in the city of Chilpancingo, Guerrero, Mexico. [Int Microbiol 19(4): 209-215 (2016)].