Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Pink colored breast milk! Serratia marcescens infection, series of 4 cases.

BACKGROUND: Pink coloration of breast milk is uncommon and it´s associated with colonization by Serratia marcescens, which is most frequently isolated in intensive care settings. Misinterpretation of the pink coloration may lead to premature cessation of breastfeeding. The objective is to present four cases of pink discoloration. METHODS: Two retrospective and two prospective cases of pink discoloration in breast milk are described, which were reported to the lead author. RESULTS: Four healthy mother-infant pairs with documented pink discoloration are presented. S. marcescens was isolated from breast milk samples. All four infants were asymptomatic and underwent enterobacteria cultures. The mothers received outpatient antibiotic treatment, and two infants received treatment as well. Subsequent cultures yielded negative results, and the pink discoloration ceased. All mothers successfully resumed breastfeeding. CONCLUSIONS: There are very few reported cases of pink breast milk in the global literature. Colonization by S. marcescens is not an indication for discontinuation of breastfeeding.

INTRODUCCIÓN: La coloración rosa de la leche materna es poco frecuente y está asociada a colonización por Serratia marcescens. Se aísla con mayor frecuencia en entornos de cuidados intensivos. La desinformación por la coloración rosa puede conducir a una terminación prematura de la lactancia. El objetivo es presentar cuatro casos de coloración rosa de la leche materna. MÉTODOS: Se describen dos casos retrospectivos y dos prospectivos de presentación de leche materna de color rosa. Los casos fueron reportados a la autora principal. RESULTADOS: Se presentan cuatro binomios sanos con reporte de coloración rosa. Se aisló S. marcescens en una muestra de leche materna. Los cuatro lactantes eran asintomáticos y tuvieron cultivos para la enterobacteria. Las madres fueron tratadas con antibiótico ambulatorio. Dos lactantes recibieron tratamiento. Todos los cultivos posteriores fueron negativos y la coloración rosa cesó. Todos reanudaron la lactancia materna de forma exitosa. CONCLUSIONES: Existen muy pocos casos de leche de color rosa reportados en la literatura mundial. La colonización por S. marcescens no es una indicación de suspensión de la lactancia.

Rapid antibiotic susceptibility testing (RAST) of blood cultures in enterobacteria with inducible chromosomal AmpC-type ß-lactamase.

INTRODUCTION: Early and adequate treatment of bloodstream infections decreases patient morbidity and mortality. The objective is to develop a preliminary method for rapid antibiotic susceptibility testing (RAST) in enterobacteria with inducible chromosomal AmpC. METHODS: RAST was performed directly on spiked blood cultures of 49 enterobacteria with inducible chromosomal AmpC. Results were read at 4, 6 and 8h of incubation. Commercial broth microdilution was considered the reference method. Disks of 10 antibiotics were evaluated. RESULTS: The proportion of readable tests at 4h was 85%. All RAST could be read at 6 and 8h. For most antibiotics, the S or R result at 4, 6 and 8h was greater than 80% after tentative breakpoints were established and Area of Technical Uncertainty was defined. CONCLUSIONS: This preliminary method seems to be of practical use, although it should be extended to adjust the breakpoints and differentiate them by species.

Infectious etiology and indicators of malabsorption or intestinal injury in childhood diarrhea. / Etiología infecciosa e indicadores de malabsorción o daño intestinal en diarrea infantil

INTRODUCTION: The multifactorial etiology of gastroenteritis emphasizes the need for different laboratory methods to identify or exclude infectious agents and evaluate the severity of diarrheal disease. OBJECTIVE: To diagnose the infectious etiology in diarrheic children and to evaluate some fecal markers associated with intestinal integrity. MATERIALS AND METHODS: The study group comprised 45 children with diarrheal disease, tested for enteropathogens and malabsorption markers, and 76 children whose feces were used for fat evaluation by the traditional and acid steatocrit tests. RESULTS: We observed acute diarrhea in 80% of the children and persistent diarrhea in 20%. Of the diarrheic individuals analyzed, 40% were positive for enteropathogens, with rotavirus (13.3%) and Giardia duodenalis (11.1%) the most frequently diagnosed. Among the infected patients, occult blood was more evident in those carrying pathogenic bacteria (40%) and enteroviruses (40%), while steatorrhea was observed in infections by the protozoa G. duodenalis (35.7%). Children with diarrhea excreted significantly more lipids in feces than non-diarrheic children, as determined by the traditional (p<0.0003) and acid steatocrit (p<0.0001) methods. Moreover, the acid steatocrit method detected 16.7% more fecal fat than the traditional method. CONCLUSIONS: Childhood diarrhea can lead to increasingly severe nutrient deficiencies. Steatorrhea is the hallmark of malabsorption, and a stool test, such as the acid steatocrit, can be routinely used as a laboratory tool for the semi-quantitative evaluation of fat malabsorption in diarrheic children.

Introducción. La etiología multifactorial de la gastroenteritis enfatiza la necesidad de usar diferentes métodos de laboratorio para identificar o excluir agentes infecciosos y evaluar la gravedad de la enfermedad diarreica. Objetivo. Diagnosticar la etiología infecciosa de la diarrea en niños y evaluar algunos marcadores fecales asociados con la integridad intestinal. Materiales y métodos. Se estudiaron 45 niños con enfermedad diarreica, en los cuales se evaluaron la presencia de enteropatógenos y los marcadores de malabsorción. Se analizaron las muestras fecales de 76 niños, mediante las pruebas de esteatocrito tradicional y esteatocrito ácido, para la cuantificación de la grasa. Resultados. Se observó diarrea aguda en el 80 % de los niños y diarrea persistente en el 20 %. De los individuos con diarrea, el 40 % fue positivo para enteropatógenos; los más diagnosticados fueron rotavirus (13,3 %) y Giardia duodenalis (11,1 %). Entre los pacientes infectados, la sangre oculta fue más evidente en aquellos portadores de bacterias patógenas (40 %) o enterovirus (40%), mientras que la esteatorrea se observó en infecciones por el protozoo G. duodenalis (35,7 %). Los niños con diarrea excretaron significativamente más lípidos en las heces que aquellos sin diarrea, según lo determinado por los métodos de esteatocrito tradicional (p<0,0003) y esteatocrito ácido (p<0,0001). Conclusiones. La diarrea infantil puede provocar deficiencias graves de nutrientes. La esteatorrea es distintiva de la malabsorción intestinal y puede detectarse mediante la estimación del esteatocrito ácido. Esta prueba podría utilizarse de forma rutinaria como una herramienta de laboratorio para la evaluación semicuantitativa de la malabsorción de grasas en niños con diarrea.

Genetic diversity of KPC-2-producing Klebsiella pneumoniae complex from aquatic ecosystems.

During the COVID-19 pandemic, the occurrence of carbapenem-resistant Klebsiella pneumoniae increased in human clinical settings worldwide. Impacted by this increase, international high-risk clones harboring carbapenemase-encoding genes have been circulating in different sources, including the environment. The blaKPC gene is the most commonly disseminated carbapenemase-encoding gene worldwide, whose transmission is carried out by different mobile genetic elements. In this study, blaKPC-2-positive Klebsiella pneumoniae complex strains were isolated from different anthropogenically affected aquatic ecosystems and characterized using phenotypic, molecular, and genomic methods. K. pneumoniae complex strains exhibited multidrug-resistant and extensively drug-resistant profiles, spotlighting the resistance to carbapenems, ceftazidime-avibactam, colistin, and tigecycline, which are recognized as last-line antimicrobial treatment options. Molecular analysis showed the presence of several antimicrobial resistance, virulence, and metal tolerance genes. In-depth analysis showed that the blaKPC-2 gene was associated with three different Tn4401 isoforms (i.e., Tn4401a, Tn4401b, and Tn4401i) and NTEKPC elements. Different plasmid replicons were detected and a conjugative IncN-pST15 plasmid harboring the blaKPC-2 gene associated with Tn4401i was highlighted. K. pneumoniae complex strains belonging to international high-risk (e.g., ST11 and ST340) and unusual clones (e.g., ST323, ST526, and ST4216) previously linked to clinical settings. In this context, some clones were reported for the first time in the environmental sector. Therefore, these findings evidence the occurrence of carbapenemase-producing K. pneumoniae complex strains in aquatic ecosystems and contribute to the monitoring of carbapenem resistance worldwide.

Exploring Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli in Food-Producing Animals and Animal-Derived Foods.

Antimicrobials serve as crucial treatments in both veterinary and human medicine, aiding in the control and prevention of infectious diseases. However, their misuse or overuse has led to the emergence of antimicrobial resistance, posing a significant threat to public health. This review focuses on extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in animals and their associated food products, which contribute to the proliferation of antimicrobial-resistant strains. Recent research has highlighted the presence of ESBL-producing E. coli in animals and animal-derived foods, with some studies indicating genetic similarities between these isolates and those found in human infections. This underscores the urgent need to address antimicrobial resistance as a pressing public health issue. More comprehensive studies are required to understand the evolving landscape of ESBLs and to develop strategic public health policies grounded in the One Health approach, aiming to control and mitigate their prevalence effectively.

Detection of Escherichia coli O157H7 and Campylobacter jejuni in Bovine Carcasses in Two Slaughterhouses in Bio-Bío District, Chile.

Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.

Assessing the Microbial Quality of Shrimp (Xiphonaeus kroyeri) and Mussels (Perna perna) Illegally Sold in the Vitória Region, Brazil, and Investigating the Antimicrobial Resistance of Escherichia coli Isolates.

The consumption of seafood is crucial for food security, but poor hygiene along the food production chain can result in low microbiological quality, posing significant risks for public health and seafood quality. Thus, this study aimed to assess the microbiological quality and antimicrobial sensitivity of E. coli from 69 samples of illegally marketed shrimp and mussels in the Vitória Region, Brazil. These foods exhibited poor microbiological quality due to high counts of mesophilic, psychrotrophic, and enterobacteria microorganisms. While this issue is widespread in this area, shrimp samples displayed higher microbial counts compared to mussels, and fresh mussels had elevated counts of enterobacteria compared to frozen ones. Among the 10 E. coli isolates, none carried the genes blaCTX-M-1, blaCTX-M-2, blaCTX-M-3, blaCTX-M-15, mcr-1, mcr-2, mcr-3, mcr-4, and tet, associated with antibiotic resistance. Phenotypical resistance to tetracycline and fosfomycin was not observed in any isolate, while only 20% demonstrated resistance to ciprofloxacin. Regarding ampicillin and amoxicillin with clavulanic acid, 60% of isolates were resistant, 10% showed intermediate susceptibility, and 30% were sensitive. One isolate was considered simultaneously resistant to ß-lactams and quinolones, and none were conserved as ESBL producers. These findings highlight the inherent risks to local public health that arise from consuming improperly prepared seafood in this area.

Unraveling the diversity and dissemination dynamics of antimicrobial resistance genes in Enterobacteriaceae plasmids across diverse ecosystems.

AIM: The objective of this study was to investigate the antimicrobial resistance genes (ARGs) in plasmids of Enterobacteriaceae from soil, sewage, and feces of food-producing animals and humans. METHODS AND RESULTS: The plasmid sequences were obtained from the NCBI database. For the identification of ARG, comprehensive antibiotic resistance database (CARD), and ResFinder were used. Gene conservation and evolution were investigated using DnaSP v.6. The transfer potential of the plasmids was evaluated using oriTfinder and a MOB-based phylogenetic tree was reconstructed using Fastree. We identified a total of 1064 ARGs in all plasmids analyzed, conferring resistance to 15 groups of antibiotics, mostly aminoglycosides, beta-lactams, and sulfonamides. The greatest number of ARGs per plasmid was found in enterobacteria from chicken feces. Plasmids from Escherichia coli carrying multiple ARGs were found in all ecosystems. Some of the most abundant genes were shared among all ecosystems, including aph(6)-Id, aph(3'')-Ib, tet(A), and sul2. A high level of sequence conservation was found among these genes, and tet(A) and sul2 are under positive selective pressure. Approximately 62% of the plasmids carrying at least one ARG were potentially transferable. Phylogenetic analysis indicated a potential co-evolution of Enterobacteriaceae plasmids in nature. CONCLUSION: The high abundance of Enterobacteriaceae plasmids from diverse ecosystems carrying ARGs reveals their widespread distribution and importance.

Genomic analysis of a cAmpC (CMY-41)-producing Citrobacter freundii ST64 isolated from patient.

Antibiotic resistance in Citrobacter freundii is a public health concern. This study evaluated the closed genome of a C. freundii isolated from the stool of a hospitalized patient initially related to a Salmonella outbreak. Confirmation of the isolate was determined by whole-genome sequencing. Nanopore sequencing was performed using a MinION with a Flongle flow cell. Assembly using SPAdes and Unicycler yielded a closed genome annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline. Genomic analyses employed MLST 2.0, ResFinder4.1, PlasmidFinder2.1, and VFanalyzer. Phylogenetic comparison utilized the Center for Food Safety and Applied Nutrition (CFSAN)-single nucleotide polymorphism pipeline and Genetic Algorithm for Rapid Likelihood Inference. Antimicrobial susceptibility was tested by broth microdilution following Clinical and Laboratory Standards Institute criteria. Multi-locus sequence type in silico analysis assigned the C. freundii as sequence type 64 and the blaCMY-41 gene was detected in resistome investigation. The susceptibility to antibiotics, determined using Sensititre® plates, revealed resistance to aztreonam, colistin, cefoxitin, amoxicillin/clavulanic acid, sulfisoxazole, ampicillin, and streptomycin. The genetic relatedness of the C. freundii CFSAN077772 with publicly available C. freundii genomes revealed a close relationship to a C. freundii SRR1186659, isolated in 2009 from human stool in Tanzania. In addition, C. freundii CFSAN077772 is nested in the same cluster with C. freundii clinical strains isolated in Denmark, Mexico, Myanmar, and Canada, suggesting a successful intercontinental spread.

The Inhibitory Activity of Lactobacillus plantarum Supernatant against Enterobacteria, Campylobacter, and Tumor Cells.

Cell-free supernatant of Lactobacillus plantarum exhibit a strong antimicrobial effect against a number of pathogenic enterobacteria (E. coli, Shigella flexneri, Salmonella typhimurium, Proteus mirabilis, and Campylobacter jejuni). The degree of growth inhibition in broth culture reached a high level for all tested bacteria. The highest rates were noted for P. mirabilis (by 13 times) and the lowest for S. flexneri (by 5 times) and C. jejuni (by 4.5 times). Significant antiproliferative effect of the supernatant on cells of tumor-derived epithelial cell lines was shown. The highest degree of inhibition (by 22 times) was observed for HT-29 cells (colon carcinoma). Thus, inclusion of probiotics in traditional treatment schemes can increase the effectiveness of antibacterial and antitumor drug therapy.

The Emergence of Carbapenem- and Colistin-Resistant Enterobacteria in Senegal.

Antibiotic resistance is a public health problem. The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a concern, particularly in Senegal. (1) Methods: Between January 2019 and July 2022, 240 isolates of enterobacteria resistant to third-generation cephalosporins and imipenem from biological samples from Fann Hospital (Dakar) and Hôpital Paix (Ziguinchor) were selected. The isolates were identified by MALDI-TOF mass spectrometry, and susceptibility tests were performed by the disk diffusion method. Antibiotic-resistance genes for class A beta-lactamases, carbapenemases, and plasmid resistance to colistin resistance (mcr-1-8) were screened by RT-PCR. (2) Results: The 240 enterobacteria were composed of: Escherichia coli (60.83%), Klebsiella pneumoniae (21.67%), Enterobacter cloacae (13.75%), Citrobacter freundii (2.08%), Serratia marcescens (0.83%), Klebsiella aerogenes (0.42%), and Proteus mirabilis (0.42%). Class A beta-lactamase genes were found in 229 isolates (70.41% blaTEM, 37.5% blaSHV, 83.75% blaCTX-A, and 0.42% blaCTX-B). The carbapenemase genes blaOXA-48 and blaNDM were found in 25 isolates, including 14 isolates with blaOXA-48, 13 isolates with blaNDM, and 2 isolates with both genes simultaneously. The mcr-8 gene was found in one isolate of E. cloacae. (3) Conclusions: The epidemiology of antibiotic-resistance genes in enterobacteria in Senegal shows the emergence of CPEs. This phenomenon is worrying, and rigorous surveillance is necessary to avoid further spread.

Resistance and Biofilm Production Profile of Potential Isolated from Kpètè-Kpètè Used to Produce Traditional Fermented Beer.

This study aimed to characterize the pathogenicity of bacteria isolated from the starter of two traditional beers produced and consumed in Benin. After standard microbial identification, species were identified by specific biochemical tests such as catalase, coagulase, and API 20 E. Antibiotic sensitivity was tested according to the French Society of Microbiology Antibiogram Committee. The crystal violet microplate technique evaluated the biofilm production and conventional PCR was used to identify genes encoding virulence and macrolide resistance. According to our data, the traditional starter known as kpètè-kpètè that is used to produce beer is contaminated by Enterobacteriaceae and staphylococci species. Thus, 28.43% of the isolated bacteria were coagulase-negative staphylococci (CNS), and 10.93% coagulase-positive staphylococci (CPS). Six species such as Klebsiella terrigena (1.38%), Enterobacter aerogens (4.14%), Providencia rettgeri (5.51%), Chryseomonas luteola (6.89%), Serratia rubidae (15.16%), and Enterobacter cloacae (27.56%) were identified among Enterobacteriaceae. Those bacterial strains are multi-resistant to conventional antibiotics. The hight capability of produced biofilms was recorded with Enterobacter aerogens, Klebsiella terrigena (100%), Providencia rettgeri (75%), and Staphylococcus spp (60%). Enterobacter cloacae (4%) and coagulase-negative Staphylococcus (5.55%) harbor the macrolide resistance gene. For other strains, these genes were not detected. Foods contaminated with bacteria resistant to antibiotics and carrying a virulence gene could constitute a potential public health problem. There is a need to increase awareness campaigns on hygiene rules in preparing and selling these traditional beers.

Effect of butyrate-producing enterobacteria on advanced hepatocellular carcinoma treatment with atezolizumab and bevacizumab.

AIM: Multiple studies have revealed the correlation between gut microbiome and the response to checkpoint inhibitors (CPIs) in patients with cancer, and oral administration of butyrate-producing enterobacteria has been reported to enhance the efficacy of CPIs. However, the effects of enterobacteria on patients with hepatocellular carcinoma (HCC) are not well understood. METHODS: In this retrospective multicenter study, we enrolled 747 patients with advanced HCC, treated with atezolizumab and bevacizumab combination therapy. Tumor response, survival, and adverse effects were compared between 99 patients who ingested drugs containing butyric acid-producing enterobacteria (butyric acid group) and the remaining patients (control group). RESULTS: Objective response and disease control rates in butyric acid group (29.7% and 77.8%, respectively) were higher than those in the control group (26.4% and 72.7%, respectively). However, the differences were not statistically significant (p = 0.543 and p = 0.222, respectively). No difference in median survival time was observed between the two groups (20.0 months and 21.4 months, respectively; p = 0.789), even after matching the backgrounds of the patients with propensity scores (p = 0.714). No adverse effects occurred upon the administration of butyrate-producing bacteria. However, proteinuria (41.4% vs. 30.9%; p = 0.041), fever (17.2% vs. 10.2%, p = 0.036), and diarrhea (15.2% vs. 6.2%; p = 0.001) occurred more frequently in the butyric acid group. CONCLUSION: Butyrate-producing bacteria does not enhance the efficacy of atezolizumab-bevacizumab combination therapy in patients with HCC.

Trends in Antibiotic-Resistant Bacteria Isolated from Screening Clinical Samples in a Tertiary Care Hospital over the 2018-2022 Period.

To assess the putative impact of the COVID-19 pandemic on multidrug-resistant (MDR) bacteria recovered from routine screening samples and, more globally, the trends in time to first positive screening sample and carriage duration of those bacteria in patients admitted to a tertiary hospital, data from laboratory results were retrospectively mined over the 2018-2022 period. No significant differences could be found in the number of positive patients or MDR isolates per year, time to positive screening, or carriage duration. Extended-spectrum beta-lactamase producers were dominant throughout the studied period but their relative proportion decreased over time as well as that of meticillin-resistant Staphylococcus aureus. Meanwhile, carbapenemase-producing enterobacteria (CPE) proportion increased. Among the 212 CPE isolates, Klebsiella pneumoniae and Escherichia coli were the more frequent species but, beginning in 2020, a significant rise in Enterobacter cloacae complex and Citrobacter freundii occurred. OXA48 was identified as the leading carbapenemase and, in 2020, a peak in VIM-producing enterobacteria linked to an outbreak of E. cloacae complex during the COVID-19 pandemic was singled out. Finally, a worrisome rise in isolates producing multiple carbapenemases (NDM/VIM and mostly NDM/OXA48) was highlighted, especially in 2022, which could lead to therapeutic dead-ends if their dissemination is not controlled.

Antibacterial MccM as the Major Microcin in Escherichia coli Nissle 1917 against Pathogenic Enterobacteria.

Probiotic Escherichia coli Nissle 1917 (EcN) possesses excellent antibacterial effects on pathogenic enterobacteria. The microcins MccM and MccH47 produced in EcN played critical roles, but they are understudied and poorly characterized, and the individual antibacterial mechanisms are still unclear. In this study, three EcN mutants (ΔmcmA, ΔmchB, and ΔmcmAΔmchB) were constructed and compared with wild-type EcN (EcN wt) to test for inhibitory effects on the growth of Escherichia coli O157: H7, Salmonella enterica (SE), and Salmonella typhimurium (ST). The antibacterial effects on O157: H7 were not affected by the knockout of mcmA (MccM) and mchB (MccH47) in EcN. However, the antibacterial effect on Salmonella declined sharply in EcN mutants ΔmcmA. The overexpressed mcmA gene in EcN::mcmA showed more efficient antibacterial activity on Salmonella than that of EcN wt. Furthermore, the EcN::mcmA strain significantly reduced the abilities of adhesion and invasion of Salmonella to intestinal epithelial cells, decreasing the invasion ability of ST by 56.31% (62.57 times more than that of EcN wt) while reducing the adhesion ability of ST by 50.14% (2.41 times more than that of EcN wt). In addition, the supernatant of EcN::mcmA culture significantly decreased the mRNA expression and secretion of IL-1ß, TNF-α, and IL-6 on macrophages induced by LPS. The EcN::mcmA strain generated twice as much orange halo as EcN wt by CAS agar diffusion assay by producing more siderophores. MccM was more closely related to the activity of EcN against Salmonella, and MccM-overproducing EcN inhibited Salmonella growth by producing more siderophores-MccM to compete for iron, which was critical to pathogen growth. Based on the above, EcN::mcmA can be developed as engineered probiotics to fight against pathogenic enterobacteria colonization in the gut.

The distribution of fitness effects of plasmid pOXA-48 in clinical enterobacteria.

Antimicrobial resistance (AMR) in bacteria is a major public health problem. The main route for AMR acquisition in clinically important bacteria is the horizontal transfer of plasmids carrying resistance genes. AMR plasmids allow bacteria to survive antibiotics, but they also entail physiological alterations in the host cell. Multiple studies over the last few years have indicated that these alterations can translate into a fitness cost when antibiotics are absent. However, due to technical limitations, most of these studies are based on analysing new associations between plasmids and bacteria generated in vitro, and we know very little about the effects of plasmids in their native bacterial hosts. In this study, we used a CRISPR-Cas9-tool to selectively cure plasmids from clinical enterobacteria to overcome this limitation. Using this approach, we were able to study the fitness effects of the carbapenem resistance plasmid pOXA-48 in 35 pOXA-48-carrying isolates recovered from hospitalized patients. Our results revealed that pOXA-48 produces variable effects across the collection of wild-type enterobacterial strains naturally carrying the plasmid, ranging from fitness costs to fitness benefits. Importantly, the plasmid was only associated with a significant fitness reduction in four out of 35 clones, and produced no significant changes in fitness in the great majority of isolates. Our results suggest that plasmids produce neutral fitness effects in most native bacterial hosts, helping to explain the great prevalence of plasmids in natural microbial communities.

Factors influencing somatic cell counts and bacterial contamination in unpasteurized milk obtained from water buffalo in Bangladesh.

Little has been published on the factors influencing the safety and quality of milk derived from water buffalo in Bangladesh. This study aims to describe the milk hygiene parameters and milk chain characteristics of unpasteurized raw milk sold to consumers in order to improve milk hygiene. A quantitative study design evaluated somatic cell counts, total bacterial counts, and specific gram-negative (Enterobacteria) and gram-positive (staphylococci) pathogens in 377 aseptically collected milk samples. Samples were collected at multiple nodes along the buffalo milk value chain: 122 bulk tank milk samples were collected at the farm level, 109 milk samples at the middlemen level, and 111 milk samples at the milk collection centers. In addition, 35 samples were taken from various milk products at the retail level. It was found that progressively increasing somatic cell counts and bacterial counts, including potential pathogens, occurred along the milk chain. A seasonal increase in spring was found, varying based on the farming system (semi-intensive versus intensive). Other factors included water purity and cleanliness of containers, mixing buffalo and cow's milk, and the location of the water buffalo milk producer (coastal or river basin). This study demonstrated how improving udder health and milk hygiene along the water buffalo milk value chain would increase the safety and quality of water buffalo milk in the study area.

[Urinary tract infections including pyelonephritis]. / Harnwegsinfektionen einschließlich Pyelonephritis.

Urinary tract infections (UTIs) are among the most common bacterial infections. The clinical phenotypes of UTIs are heterogeneous, ranging from rather benign uncomplicated infections to complicated UTIs and pyelonephritis to severe urosepsis. There has been a sharp increase in the incidence of severe UTIs, whilst the incidence of sepsis in general is declining. The classifications of UTIs partially differ between clinical and regulatory claims. Experience has also been gained over the last few years to define the appropriate endpoints that are used in clinical studies. Above all, patient-centered evaluation strategies of the endpoints were developed, in order to be able to identify the advantages of new antibiotics compared to traditional antibiotics. The development of new antibiotics for UTIs is essential, since multidrug-resistant enterobacteria, which belong to the typical bacterial spectrum of UTIs, are frequently associated with mortality from infections. In recent years, several new antibiotics/combinations that are particularly effective against multi-resistant gram-negative pathogens have been investigated for the treatment of UTIs.

Low-diversity microbiota and an increased metabolism of arginine and aromatic amino acids: a hallmark of hepatic encephalopathy in western Mexican patients with alcohol-associated cirrhosis.

AIMS: To evaluate the composition and functions of the gut microbiota in patients with decompensated alcohol-associated cirrhosis, with and without hepatic encephalopathy (HE). METHODS AND RESULTS: Faecal samples from 31 inpatients (20 with HE, 11 without HE), and from 18 age-balanced healthy controls (HC), were included. Microbial composition was determined by 16S rRNA amplicon sequencing and analysed using QIIME2. Metabolic pathways were inferred by PICRUSt2, and short-chain fatty acids (SCFAs) quantification was performed by gas chromatography. The gut microbiota of patients with HE was characterized by a diminished α-diversity, compared to no-HE (P < 0.01) and HC (P < 0.001) groups; ß-diversity also differed between HE vs no-HE patients (P < 0.05), and between HE vs HC (P < 0.001). In patients with HE, Escherichia/Shigella, Burkholderiales and Lactobacillales taxa predominated. In contrast, patients without HE were characterized by Veillonella and Bacteroides. Reduced levels of faecal SCFAs in both groups correlated with a depletion of beneficial taxa, such as Ruminococcus or Faecalibacterium. PICRUSt2 analysis showed both an enhanced catabolism of arginine through ammonia-producing pathways and chorismate biosynthesis in HE patients, a key precursor of aromatic amino acids. CONCLUSIONS: The gut microbiota of HE patients exhibits a proinflammatory dysbiotic profile, plus metabolic pathways that produce potentially neurotoxic byproducts.