Several microorganisms produce nosocomial infections (NIs), among which Pseudomonas aeruginosa stands out as an opportunist pathogen with the capacity to develop multiresistance to first-choice antibiotics. From 2007 to 2013, forty-six NIs produced by P. aeruginosa were detected at a pediatric tertiary care hospital in Mexico with a significant mortality rate (17.39%). All isolates (n = 58/46 patients) were characterized by evaluating their response to several antibiotics as panresistant (PDR), extensively resistant (XDR), multiresistant (MDR) or sensitive (S). In addition, all isolates were typified through multilocus sequencing of seven genes: acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. Furthermore, to establish the genetic relationships among these isolates, we carried out a phylogenetic inference analysis using maximum likelihood to construct a phylogenetic network. To assess evolutionary parameters, recombination was evaluated using the PHI test, and the ratio of nonsynonymous to synonymous substitutions was determined. Two of the strains were PDR (ST1725); 42 were XDR; four were MDR; and ten were S. Twenty-one new sequence types were detected. Thirty-three strains exhibited novel sequence type ST1725. The ratio of nonsynonym to synonym substitutions was 1:1 considering all genes. Phylogenetic analysis showed that the genetic relationship of the PDR, XDR and MDR strains was mainly clonal; however, the PHI test and the phylogenetic network suggest that recombination events occurred to produce a non-clonal population. This study aimed not only to determine the genetic diversity of clinical P. aeruginosa but also to provide a warning regarding the identification and spreading of clone ST1725, its ability to cause outbreaks with high mortality rates, and to remain in the hospital environment for over seven years. These characteristics highlight the need to identify clonal outbreaks, especially where high resistance to most antibiotics is observed, and control measures are needed. This study also represents the first report of the PDR ST1725.

Microbiomes are defined as complex microbial communities, which are mainly composed of bacteria, fungi, and viruses residing in diverse regions of the human body. The human stomach consists of a unique and heterogeneous habitat of microbial communities owing to its anatomical and functional characteristics, that allow the optimal growth of characteristic bacteria in this environment. Gastric dysbiosis, which is defined as compositional and functional alterations of the gastric microbiota, can be induced by multiple environmental factors, such as age, diet, multiple antibiotic therapies, proton pump inhibitor abuse, H. pylori status, among others. Although H. pylori colonization has been reported across the world, chronic H. pylori infection may lead to serious consequences; therefore, the infection must be treated. Multiple antibiotic therapy improvements are not always successful because of the lack of adherence to the prescribed antibiotic treatment. However, the abuse of eradication treatments can generate gastric dysbiotic states. Dysbiosis of the gastric microenvironment induces microbial resilience, due to the loss of relevant commensal bacteria and simultaneous colonization by other pathobiont bacteria, which can generate metabolic and physiological changes or even initiate and develop other gastric disorders by non-H. pylori bacteria. This systematic review opens a discussion on the effects of multiple environmental factors on gastric microbial communities.

Recent multidrug resistance in Pseudomonas aeruginosa has favoured the adaptation and dissemination of worldwide high-risk strains. In June 2018, 15 P. aeruginosa strains isolated from patients and a contaminated multi-dose meropenem vial were characterized to assess their association to an outbreak in a Mexican paediatric hospital. The strains were characterized by antibiotic susceptibility profiling, virulence factors’ production, and biofilm formation. The clonal relationship among isolates was determined with pulse-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) sequencing. Repressor genes for the MexAB-OprM efflux pump were sequenced for haplotype identification. Of the strains, 60% were profiled as extensively drug-resistant (XDR), 33% as multidrug-resistant (MDR), and 6.6% were classified as sensitive (S). All strains presented intermediate resistance to colistin, and 80% were sensitive to aztreonam. Pyoverdine was the most produced virulence factor. The PFGE technique was performed for the identification of the outbreak, revealing eight strains with the same electrophoretic pattern. ST235 and ten new sequence types (STs) were identified, all closely related to ST233. ST3241 predominated in 26.66% of the strains. Twenty-five synonymous and seventeen nonsynonymous substitutions were identified in the regulatory genes of the MexAB-OprM efflux pump, and nalC was the most variable gene. Six different haplotypes were identified. Strains from the outbreak were metallo-β-lactamases and phylogenetically related to the high-risk clone ST233.

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BACKGROUND The high prevalence and persistence of Helicobacter pylori ( H. pylori ) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340 hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor. AIM To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric (AGS) cells. METHODS Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0, 3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via , I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling. RESULTS HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells. Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells. Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori . Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found; modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage. CONCLUSION The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.

Background: Pseudomonas aeruginosa is a major opportunistic pathogen involved in healthcare-associated infections with high mortality rates. This bacterium exhibits elevated resistance to a wide range of antibiotics, resulting in part from the overexpression of efflux pumps, among which MexAB-OprM stands out as constitutive. Antibiotic resistance in clinical isolates is associated with mutations in the mexR, nalC, and nalD repressors that modulate the expression of this efflux pump. This study identifies point mutations in the mexR, nalC, and nalD genes and investigates their associations with antibiotic resistance and sequence type in clinical and epidemiologically high-risk clones of P. aeruginosa. Results: A total of 91 P. aeruginosa strains isolated at a pediatric hospital in Mexico (2007–2015) were classified according to their resistance to antibiotics. The strains were typed by multilocus sequencing of 7 genes. The MexAB-OprM efflux pump phenotype was determined using the minimal inhibitory concentration for the reporter antibiotic carbenicillin in the presence/absence of the efflux pump inhibitor Phe-Arg-β-naphthylamine. Sequencing of the mexR, nalC, and nalD genes to identify mutations was performed. Genetic relationship among the strains was evaluated by a phylogenetic inference analysis using maximum likelihood to construct a phylogenetic network. The relationship between variables was determined by a principal component analysis. STs revealed six main complexes. Mutations in the mexR, nalC, and nalD genes revealed 27 different haplotypes. Pan-drug and extensive drug resistant profiles were associated with specific STs with haplotypes 1 (ST1725, endemic clone), 8, 12 (ST233, epidemiologically high-risk clone), and 5 [related to dead when compared to ST1725 and ST233 (RRR 23.34; p=0.009 and RRR 32.01; p=0.025)], however the resistance in these strains was not mainly attributed to the MexAB-OprM phenotype. Strains with the same haplotype and resistant profile showed different pump behavior.Conclusions: A significant relationship between ST and resistant profiles was observed; on one hand, the mexR-nalC-nalD haplotypes were not related to the MexAB-OprM efflux pump phenotypic behavior. On the other hand, the relationship between mexR-nalC-nalD haplotypes and phylogenetically related ST, suggest mutations in these repressors are highly maintained within these STs.