Gut colonization and subsequent infection of neonates caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

The gut microbiota harbors diverse bacteria considered reservoirs for antimicrobial resistance genes. The global emergence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) significantly contributes to healthcare-associated infections (HAIs). We investigated the presence of ESBL-producing Escherichia coli (ESBL-PEco) and ESBL-producing Klebsiella pneumoniae (ESBL-PKpn) in neonatal patients' guts. Furthermore, we identified the factors contributing to the transition towards ESBL-PEco and ESBL-PKpn-associated healthcare-associated infections (HAIs). The study was conducted from August 2019 to February 2020, in a Neonatal Intensive Care Unit of the Hospital Infantil de México Federico Gómez. Rectal samples were obtained upon admission, on a weekly basis for a month, and then biweekly until discharge from the neonatology ward. Clinical data, culture results, and infection information were gathered. We conducted antimicrobial tests, multiplex PCR assay, and pulsed-field gel electrophoresis (PFGE) to determine the antimicrobial resistance profile and genetic relationships. A comparison between the group's controls and cases was performed using the Wilcoxon and Student t-tests. Of the 61 patients enrolled, 47 were included, and 203 rectal samples were collected, identifying 242 isolates. In 41/47 (87%) patients, colonization was due to ESBL-PEco or ESBL-PKpn. And nine of them developed HAIs (22%, 9/41). ESBL-PEco resistance to cephalosporins ranged from 25.4% to 100%, while ESBL-PKpn resistance varied from 3% to 99%, and both bacteria were susceptible to carbapenems, tigecillin, and colistin. The prevalent bla CTX-M-group-1 gene accounted for 77.2% in ESBL-PEco and 82.2% in ESBL-PKpn, followed by bla TEM 50% and bla OXA-1 43.8% in ESBL-PEco and bla TEM 80.2% and bla SHV 76.2% in ESBL-PKpn. Analysis of clonality revealed identical colonizing and infection isolates in only seven patients. Significant risk factors included hospital stay duration, duration of antibiotic treatment, and invasive device usage. Our findings suggest high ESBL-PEco and ESBL-PKpn rates of colonization often lead to infection in neonates. Attention should be paid to patients with ESBL-PE.

HP0953 - hypothetical virulence factor overexpresion and localization during Helicobacter pylori infection of gastric epithelium.

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.

New Variants of Pseudomonas aeruginosa High-Risk Clone ST233 Associated with an Outbreak in a Mexican Paediatric Hospital.

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-ß-lactamases and phylogenetically related to the high-risk clone ST233.

Nucleotide substitutions in the mexR, nalC and nalD regulator genes of the MexAB-OprM efflux pump are maintained in Pseudomonas aeruginosa genetic lineages.

Pseudomonas aeruginosa has different resistant mechanisms including the constitutive MexAB-OprM efflux pump. Single nucleotide polymorphisms (SNPs) in the mexR, nalC, and nalD repressors of this efflux pump can contribute to antimicrobial resistance; however, it is unknown whether these changes are mainly related to genetic lineages or environmental pressure. This study identifies SNPs in the mexR, nalC, and nalD genes in clinical and environmental isolates of P. aeruginosa (including high-risk clones). Ninety-one P. aeruginosa strains were classified according to their resistance to antibiotics, typified by multilocus sequencing, and mexR, nalC, and nalD genes sequenced for SNPs identification. The mexAB-oprM transcript expression was determined. The 96.7% of the strains were classified as multidrug resistant. Eight strains produced serine carbapenemases, and 11 strains metallo-ß-lactamases. Twenty-three new STs and high-risk clones ST111 and ST233 were identified. SNPs in the mexR, nalC, and nalD genes revealed 27 different haplotypes (patterns). Sixty-two mutational changes were identified, 13 non-synonymous. Haplotype 1 was the most frequent (n = 40), and mainly identified in strains ST1725 (33/40), with 57.5% pan drug resistant strains, 36.5% extensive drug resistant and two strains exhibiting serin-carbapenemases. Haplotype 12 (n = 9) was identified in ST233 and phylogenetically related STs, with 100% of the strains exhibiting XDR and 90% producing metallo-ß-lactamases. Haplotype 5 was highly associated with XDR and related to dead when compared to ST1725 and ST233 (RRR 23.34; p = 0.009 and RRR 32.01; p = 0.025). A significant relationship between the mexR-nalC-nalD haplotypes and phylogenetically related STs was observed, suggesting mutational changes in these repressors are highly maintained within genetic lineages. In addition, phylogenetically related STs showed similar resistant profiles; however, the resistance was (likely or partly) attributed to the MexAB-OprM efflux pump in 56% of the strains (only 45.05% showed mexA overtranscription), in the remaining strains the resistance could be attributed to carbapenemases or mechanisms including other pumps, since same SNPs in the repressor genes gave rise to different resistance profiles.

Genomic characterization of two bacteriophages (vB_EcoS-phiEc3 and vB_EcoS-phiEc4) belonging to the genus Kagunavirus with lytic activity against uropathogenic Escherichia coli.

In this study, the genomes of two lytic bacteriophages, vB_EcoS-phiEc3 and vB_EcoS-phiEc4, were sequenced and characterized using bioinformatics approaches. Whole-genome analysis showed that both phages belonged to the Kagunavirus genus, Guernseyvirinae subfamily and Siphoviridae family. Moreover, their genomes had 45, 288 bp and 44,540 bp, and G + C content of 48.42% and 50.04%, respectively. The genome of vB_EcoS-phiEc3 harbored 80 protein coding sequences (CDSs), whereas vB_EcoS-phiEc4 harbored 75 CDSs. Among them, 50 CDSs in vB_EcoS-phiEc3 and 44 CDSs in vB_EcoS-phiEc4 were considered as functional genes. Their lytic activity against multidrug-resistant uropathogenic Escherichia coli (UPEC) strains, as well as the absence of antibiotic resistance genes, lysogenic and virulence genes, enable vB_EcoS-phiEc3 and vB_EcoS-phiEc4 as a safe therapy option against UPEC infections.

Role of Helicobacter pylori and Other Environmental Factors in the Development of Gastric Dysbiosis.

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.

Identification of extensive drug resistant Pseudomonas aeruginosa strains: New clone ST1725 and high-risk clone ST233.

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.