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.

Salmonella Heidelberg side-step gene loss of respiratory requirements in chicken infection model.

Among the important recent observations involving anaerobic respiration was that an electron acceptor produced as a result of an inflammatory response to Salmonella Typhimurium generates a growth advantage over the competing microbiota in the lumen. In this regard, anaerobically, salmonellae can oxidize thiosulphate (S2O32-) converting it into tetrathionate (S4O62-), the process by which it is encoded by ttr gene cluster (ttrSRttrBCA). Another important pathway under aerobic or anaerobic conditions is the 1,2-propanediol-utilization mediated by the pdu gene cluster that promotes Salmonella expansion during colitis. Therefore, we sought to compare in this study, whether Salmonella Heidelberg strains lacking the ttrA, ttrApduA, and ttrACBSR genes experience a disadvantage during cecal colonization in broiler chicks. In contrast to expectations, we found that the gene loss in S. Heidelberg potentially confers an increase in fitness in the chicken infection model. These data argue that S. Heidelberg may trigger an alternative pathway involving the use of an alternative electron acceptor, conferring a growth advantage for S. Heidelberg in chicks.

Genomic features of mecA-positive methicillin-resistant Mammaliicoccus sciuri causing fatal infections in pets admitted to a veterinary intensive care unit.

Methicillin-resistant staphylococci have become leading cause of infectious diseases in humans and animals, being categorized as high priority pathogens by the World Health Organization. Although methicillin-resistant Staphylococcus sciuri (recently moved to Mammaliicoccus sciuri) has been widely reported in companion animals, there is scarce information regarding their clinical impact and genomic features. Herein, we reported the occurrence and genomic characteristics of methicillin-resistant M. sciuri recovered from fatal infections in pets admitted to an intensive care unit of a veterinary hospital, in Brazil. Two M. sciuri strains were isolated from bronchoalveolar lavage samples collected from dog (strain SS01) and cat (strain SS02) presenting with sepsis and acute respiratory distress syndrome. Both isolates displayed a multidrug-resistant profile, whereas whole-genome sequencing analysis confirmed the presence of the mecA gene, along to genetic determinant conferring resistance to macrolides, streptogramins, aminoglycosides, and trimethoprim. For both strains, the mec and crr gene complex shared high identity (≥97%) with analogue sequences from a M. sciuri isolated from a human wound infection, in the Czech Republic. Strains were assigned to the sequence type ST52 and the novel ST74. Phylogenomic analysis revealed a broad host range association of these strains with several hosts and sources, including humans, animals, food, and the environment through different years and geographic locations. Our findings demonstrate that infections caused by mecA-positive M. sciuri strains can be a serious threat for veterinary intensive care patients and the medical staff, with additional implications for One Health approaches.

Whole-genome sequencing analysis and CRISPR genotyping of rare antibiotic-resistant Salmonella enterica serovars isolated from food and related sources.

For decades, Salmonella Typhimurium and Salmonella Enteritidis have prevailed in several countries as agents of salmonellosis outbreaks. In Brazil, the largest exporter of poultry meat, relatively little attention has been paid to infrequent serovars. Here, we report the emergence and characterization of rare serovars isolated from food and related sources collected between 2014 and 2016 in Brazil. Twenty-two Salmonella enterica isolates were analyzed through the use of whole-genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats (CRISPR) genotyping. These isolates were classified into 10 infrequent serovars, including S. Abony, S. Isangi, S. Rochdale, S. Saphra, S. Orion, S. Ouakam, S. Grumpensis, S. Carrau, S. Abaetetuba, and S. Idikan. The presence of six antimicrobial resistance (AMR) genes, qnrB19, blaCMY-2, tetA, aac(6')-Iaa, sul2 and fosA7, which encode resistance to quinolones, third-generation cephalosporin, tetracycline, aminoglycoside, sulfonamide and fosfomycin, respectively, were confirmed by WGS. All S. Isangi harbored qnrB19 with conserved genomic context across strains, while S. Abony harbored blaCMY-2. Twelve (54.5%) strains displayed chromosomal mutations in parC (Thr57→Ser). Most serovars were classified as independent lineages, except S. Abony and S. Abaetetuba, which phylogenetically nested with Salmonella strains from different countries. CRISPR analysis revealed that the spacer content was strongly correlated with serovar and multi-locus sequence type for all strains, independently confirming the observed phylogenetic patterns, and highlighting the value of CRISPR-based genotyping for Salmonella. These findings add valuable information to the epidemiology of S. enterica in Brazil, where the emergency of antibiotic-resistant Salmonella continues to evolve.

Genomic insights of Klebsiella pneumoniae isolated from a native Amazonian fish reveal wide resistome against heavy metals, disinfectants, and clinically relevant antibiotics.

A multidrug-resistant CTX-M-15-producing Klebsiella pneumoniae (KpP1 strain) was isolated from a native Amazonian fish (Brachyplatystoma filamentosum) at the Brazilian Amazon. The strain was identified by MALDI-TOF. The genome was extracted, purified and a Nextera DNA Flex library was prepared and sequenced by Illumina platform. The sequenced genome was de novo assembled using Unicycler and in silico prediction accomplished by curated bioinformatics tools. The size of the genome is 5.6 Mb with 5715 genes. Whole-genome sequencing analysis revealed the presence of wide resistome, with genes conferring resistance to clinically relevant antibiotics, heavy metals and disinfectants. The KpP1 strain was assigned to the sequence type ST3827, KL111 (wzi113) and O3b locus. Native freshwater fish sold in wet markets of the Amazonian region could be an important vehicle for transmission of multidrug-resistant bacteria to humans. This study may give genomic insights on the spread of critical-priority WHO pathogens in a One Health context.

Genome-based diagnostic of MDR Escherichia coli ONT:H19 ST10955 causing human gastrointestinal infection.

This study focuses on the genomic characterization of a multidrug-resistant Escherichia coli strain responsible for a severe gastrointestinal infection in a 33-year-old male. The patient initially received sulfamethoxazole/trimethoprim treatment, which proved ineffective. Fecal culture confirmed the presence of E. coli displaying a MDR profile to ampicillin, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, and tetracycline. Serotyping identified the strain as ONT:H19. Virulence analysis indicated a highly virulent profile with numerous virulence markers. Plasmid analysis uncovered various plasmids carrying both antimicrobial resistance and virulence genes. MLST assigned the strain to ST10955. Phylogenomic analysis revealed similarity to an older Brazilian isolate, suggesting the persistence of a common lineage with evolving antimicrobial resistance. This report highlights the first identification of a multidrug-resistant ST10955 E. coli strain with a wide resistome and virulence potential, emphasizing the importance of ongoing surveillance of E. coli strains due to their potential for severe infections, resistance development, and virulence.

Pre-Harvest Food Safety Challenges in Food-Animal Production in Low- and Middle-Income Countries.

Food safety remains a significant global public health concern, with the risk of unsafe food varying worldwide. The economies of several low- and middle-income countries (LMICs) heavily rely on livestock, posing a challenge to ensuring the production of safe food. This review discusses our understanding of pre-harvest critical issues related to food safety in LMICs, specifically focusing on animal-derived food. In LMICs, food safety regulations are weak and inadequately enforced, primarily concentrating on the formal market despite a substantial portion of the food sector being dominated by informal markets. Key critical issues at the farm level include animal health, a low level of good agriculture practices, and the misuse of antimicrobials. Effectively addressing foodborne diseases requires a comprehensive One Health framework. Unfortunately, the application of the One Health approach to tackle food safety issues is notably limited in LMICs. In conclusion, considering that most animal-source foods from LMICs are marketed through informal channels, food safety legislation and policies need to account for this context. Interventions aimed at reducing foodborne bacterial pathogens at the farm level should be scalable, and there should be strong advocacy for the proper implementation of pre-harvest interventions through a One Health approach.

Genomic insights of Lactiplantibacillus plantarum CNPC024: a potential probiotic strain producing immune-boosting tryptophan-derived metabolites.

Probiogenomics can provide important insights bout probiotic candidate bacteria. This study aimed to perform an in-depth genomic characterization of the probiotic candidate Lactiplantibacillus plantarum CNPC024 to investigate its probiosis mechanisms, identify metabolic pathways that might benefit the host, and improve the safety assessment for this strain to be effectively used as a probiotic. After whole-genome sequencing in Illumina MiSeq platform, the de novo genome assembly resulted in a 3.2 Mb draft genome. According to the Average Nucleotide Identity (ANI) analysis with 46 randomly validated probiotic LAB belonging to the Lactobacillaceae family, the strain showed a 99% nucleotide identity with other L. plantarum probiotic species. We identified a set of determinants conferring tolerance to bile salts and low pH conditions, as well as temperature, oxidative and osmotic stressors via the glutathione-glutaredoxin system (Grxs). As a ß­galactosidase­producing strain, it has the potential to be used in fermented dairy products for lactose-intolerant individuals. There were no significant hits for transferable antibiotic-resistance genes. We also identified gene clusters associated with production of bacteriocins (plantaricins E, F and K). Lastly, we detected metabolic pathways associated with the production of tryptophan-derived metabolites that could potentially modulate the host's immune responses.

Virulent shiga toxin-producing Escherichia coli (STEC) O157:H7 ST11 isolated from ground beef in Brazil.

In this study, a total of 248 ground beef samples were analyzed for the presence of Shiga toxin-producing Escherichia coli (STEC). Out of these samples, only one (0.4%) tested positive for STEC. Further analysis using PCR confirmed the presence of all tested genes associated with STEC, including stx1, stx2, eae, ehx, uid, rfbO157, and fliCH7 in this isolate. Interestingly, no STEC strains were detected in the remaining 100 beef cut samples or the 100 chicken cut samples, indicating the absence of detectable STEC contamination in those specific samples. The isolated strain exhibited significant cytotoxic activity in Vero cells, indicating its ability to produce cytotoxic Shiga toxins. To further investigate the strain, whole-genome sequencing (WGS) analyses were performed. The resistome analysis revealed the absence of acquired antimicrobial resistance genes, indicating a pan-susceptible phenotype. However, this strain presented chromosomal mutations in gyrA, gyrB, parC, parE, pmrA, pmrB, and folP. Plasmid analysis identified the presence of two plasmids, namely IncFIB(AP001918) and IncFII. The multi-locus sequence typing (MLST) identified the strain as belonging to sequence type (ST) 11, which is associated with E. coli O157:H7 strains. The virulome analysis confirmed the presence of several canonical virulence markers, including stx1, stx2, eae-g01-gamma, ehxA, stx1a-O157, and stx2a-O157. Overall, this study identified for the first time a rare occurrence of STEC contamination in ground beef, with the isolated strain belonging to the highly virulent O157:H7 serotype. These findings contribute to our understanding of STEC prevalence and characteristics in food samples, highlighting the importance of effective food safety measures to prevent potential health risks associated with STEC contamination.

Whole genome sequence datasets of Salmonella enterica serovar Saintpaul ST50 and serovar Worthington ST592 strains isolated from raw milk in Brazil.

Herein we report the draft genome sequences of Salmonella enterica subsp. enterica serovars Saintpaul ST50 and Worthington ST592 isolated from raw milk samples in Northeastern Brazil. The 4,696,281 bp S. Saintpaul ST50 genome contained 4,628 genes in 33 contigs, while S. Worthington ST592 genome was 4,890,415 bp in length, comprising 4,951 genes in 46 contigs. S. Worthington ST592 carried a conserved Col(pHAD28) plasmid which contains the antimicrobial resistance determinants tet(C), acc(6')-Iaa, and a nonsynonymous point mutation in ParC (p.T57S). The data could support further evolutionary and epidemiologic studies involving Salmonella organisms.

Unravelling the role of anaerobic metabolism (pta-ackA) and virulence (misL and ssa) genes in Salmonella Heidelberg shedding using chicken infection model.

The mechanism of colonisation of the chicken intestine by Salmonella remains poorly understood, while the severity of infections vary enormously depending on the serovar and the age of the bird. Several metabolism and virulence genes have been identified in Salmonella Heidelberg; however, information on their roles in infection, particularly in the chicken infection model, remains scarce. In the present publication, we investigated three Salmonella Heidelberg mutants containing deletions in misL, ssa, and pta-ackA genes by using signature-tagged mutagenesis. We found that mutations in these genes of S. Heidelberg result in an increase in fitness in the chicken model. The exception was perhaps the pta-ackA mutant where colonisation was slightly reduced (2, 7, 14, and 21 days post-infection) although some birds were still excreting at the end of the experiment. Our results suggest that for intestinal colonisation of the chicken caecum, substrate-level phosphorylation is likely to be more important than the MisL outer membrane protein or even the secretion system apparatus. These findings validate previous work that demonstrated the contribution of ackA and pta mutants to virulence in chickens, suggesting that the anaerobic metabolism genes such as pta-ackA could be a promising mitigation strategy to reduce S. Heidelberg virulence.

Absence of Shiga toxin-producing Escherichia coli (STEC) in organic leafy greens from the metropolitan region of São Paulo, Brazil.

Shiga toxin-producing Escherichia coli (STEC) is an important pathogen with public health implications, including its potential association with vegetables. In this study, we investigated the presence of STEC in vegetables obtained from organic producers located in São Paulo city, Brazil. As part of a routine surveillance study conducted over (years of isolation), a total of 200 samples of organic vegetables were screened using biochemical and PCR methods. Among the vegetable samples tested, 30 (15%) were positive for non-Shiga toxin-producing E. coli. While no STEC was detected in the organic vegetables in this study, the presence of non-STEC in vegetables raises concerns about the lack of proper hygiene practices during vegetable handling. This contamination represents a public health risk, particularly considering that these isolates can still be pathogenic, and vegetables are often consumed raw. To address this important issue, continuous monitoring of these farms is recommended to ensure the quality and safety of organic vegetables produced for both domestic consumption and exportation.

Phylogenomic Analysis of CTX-M-15-Positive Escherichia coli from Companion Animal Reveals Intercontinental Dissemination of ST90 Within a One Health Framework.

The global dissemination of extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli has been considered a critical issue within a One Health framework. The aim of this study was to perform a genomic investigation of an ESBL-producing E. coli strain belonging to the globally spread sequence type/clonal complex ST90/CC23, isolated from gastrointestinal tract of a dog, in Brazil. Besides CTX-M-15 ESBL, this E. coli isolate carried mutations conferring resistance to human and veterinary fluoroquinolones (GyrA [Ser83Leu, Asp87Asn], ParC [Ser80Ile] and ParE [Ser458Ala]), and resistance determinants to disinfectants and pesticides. Noteworthy, phylogenomic analysis revealed that this multidrug E. coli strain clustered with ST90 lineages isolated from human, dog, and livestock in Brazil. The phylogenetic tree also revealed that this E. coli strain shares a common ancestor with isolates from the United States, Russia, Germany, and China, highlighting the potential global spreading of this clone. In summary, we report genomic data of CTX-M-15-positive E.coli ST90 colonizing a pet. Colonization of companion animals by critical resistant pathogens highlights the need for close monitoring to better understand the epidemiology and genetic factors contributing for successful adaptation of global clones at the human-animal interface.

Salmonella enterica serovars in absence of ttrA and pduA genes enhance the cell immune response during chick infections.

Salmonella spp. is one of the major foodborne pathogens responsible for causing economic losses to the poultry industry and bringing consequences for public health as well. Both the pathogen survival ability in the intestinal environment during inflammation as well as their relationship with the host immune system, play a key role during infections in poultry. The objective of this study was to quantify the presence of the macrophages and CD4+/CD8+ cells populations using the immunohistochemistry technique, in commercial lineages of chickens experimentally infected by wild-type and mutant strains of Salmonella Enteritidis and Salmonella Typhimurium lacking ttrA and pduA genes. Salmonella Enteritidis ∆ttrA∆pduA triggered a higher percentage of the stained area than the wild-type, with exception of light laying hens. Salmonella Typhimurium wild-type strain and Salmonella Typhimurium ∆ttrA∆pduA infections lead to a similar pattern in which, at 1 and 14 dpi, the caecal tonsils and ileum of birds showed a more expressive stained area compared to 3 and 7 dpi. In all lineages studied, prominent infiltration of macrophages in comparison with CD4+ and CD8+ cells was observed. Overall, animals infected by the mutant strain displayed a positively stained area higher than the wild-type. Deletions in both ttrA and pduA genes resulted in a more intense infiltration of macrophages and CD4+ and CD8+ cells in the host birds, suggesting no pathogen attenuation, even in different strains of Salmonella.

Clustered Regularly Interspaced Short Palindromic Repeats Genotyping of Multidrug-Resistant Salmonella Heidelberg Strains Isolated From the Poultry Production Chain Across Brazil.

Salmonella enterica subsp. enterica serovar Heidelberg has been associated with a broad host range, such as poultry, dairy calves, swine, wild birds, environment, and humans. The continuous evolution of S. Heidelberg raises a public health concern since there is a global dispersal of lineages harboring a wide resistome and virulome on a global scale. Here, we characterized the resistome, phylogenetic structure and clustered regularly interspaced short palindromic repeats (CRISPR) array composition of 81 S. Heidelberg strains isolated from broiler farms (n = 16), transport and lairage (n = 5), slaughterhouse (n = 22), and retail market (n = 38) of the poultry production chain in Brazil, between 2015 and 2016 using high-resolution approaches including whole-genome sequencing (WGS) and WGS-derived CRISPR genotyping. More than 91% of the S. Heidelberg strains were multidrug-resistant. The total antimicrobial resistance (AMR) gene abundances did not vary significantly across regions and sources suggesting the widespread distribution of antibiotic-resistant strains from farm to market. The highest AMR gene abundance was observed for fosA7, aac(6')-Iaa, sul2, tet(A), gyrA, and parC for 100% of the isolates, followed by 88.8% for bla CMY-2. The ß-lactam resistance was essentially driven by the presence of the plasmid-mediated AmpC (pAmpC) bla CMY-2 gene, given the isolates which did not carry this gene were susceptible to cefoxitin (FOX). Most S. Heidelberg strains were classified within international lineages, which were phylogenetically nested with Salmonella strains from European countries; while CRISPR genotyping analysis revealed that the spacer content was overall highly conserved, but distributed into 13 distinct groups. In summary, our findings underscore the potential role of S. Heidelberg as a key pathogen disseminated from farm to fork in Brazil and reinforce the importance of CRISPR-based genotyping for salmonellae. Hence, we emphasized the need for continuous mitigation programs to monitor the dissemination of this high-priority pathogen.

HPMCAS-Coated Alginate Microparticles Loaded with Ctx(Ile21)-Ha as a Promising Antimicrobial Agent against Salmonella Enteritidis in a Chicken Infection Model.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) in poultry is most often transmitted by the fecal-oral route, which can be attributed to high population density. Upon encountering the innate immune response in a host, the pathogen triggers a stress response and virulence factors to help it survive in the host. The aim of this study was to evaluate the effect of hypromellose acetate/succinate (HPMCAS)-coated alginate microparticles containing the Ctx(Ile21)-Ha antimicrobial peptide (AMP) on both intestinal colonization and systemic infection of laying hens challenged with S. Enteritidis. The applied AMP microsystem reduced the bacterial load of S. Enteritidis in the liver, with a statistical significance between groups A (control, no Ctx(Ile21)-Ha peptide) and B (2.5 mg of Ctx(Ile21)-Ha/kg) at 2 days postinfection (dpi), potentially indicating the effectiveness of Ctx(Ile21)-Ha in the first stage of infection by S. Enteritidis. In addition, the results showed a significant decrease in the S. Enteritidis counts in the spleen and cecal content at 5 dpi; remarkably, no S. Enteritidis counts were observed in livers at 5, 7, and 14 dpi, regardless of the Ctx(Ile21)-Ha dosage (p-value <0.0001). Using the Chi-square test, the effect of AMP microparticles on S. Enteritidis fecal excretion was also evaluated, and a significantly lower bacterial excretion was observed over 21 days in groups B and C, in comparison with the untreated control (p-value <0.05). In summary, the use of HPMCAS-Ctx(Ile21)-Ha peptide microcapsules in laying hens drastically reduced the systemic infection of S. Enteritidis, mainly in the liver, indicating a potential for application as a feed additive against this pathogen.

Correction: Class 1 integron-borne cassettes harboring blaCARB-2 gene in multidrug-resistant and virulent Salmonella Typhimurium ST19 strains recovered from clinical human stool samples, United States.

[This corrects the article DOI: 10.1371/journal.pone.0240978.].

Genomic investigation of antimicrobial resistance determinants and virulence factors in Salmonella enterica serovars isolated from contaminated food and human stool samples in Brazil.

This study investigated the antimicrobial resistance determinants, virulence factors and identified serovars in 37 Salmonella enterica strains isolated from human stool and contaminated foods linked to outbreaks that occurred in Brazil over 7 years using whole genome sequencing (WGS). Phylogenetic analysis of selected serovars (S. Typhimurium, S. Infantis, S. London, and S. Johannesburg) was performed. Ten distinct serovars were identified and, 51% of the tested strains (n = 19) showed disagreement with the previous conventional serotyping. The antimicrobial resistance (AMR) determinants or plasmids varied among the strains. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac (6')-laa, aph (3″)-lb, aph (6)-ld, aadA1 and aadA2], sulfonamides (sul1), trimethoprin (dfrA8), fosfomycin (fosA7) and tetracyclines (tetA, tetB, tetC), as well as point mutations in parC (T57S) and gyrA (S83F). Plasmidome showed the presence of IncHI2, IncHI2A, IncFIB (S), IncFII (S), IncI1 and p0111 plasmids. Eight Salmonella pathogenicity islands and up to 102 stress and/or virulence genes were identified in the evaluated genomes. Virulence genes of K88 fimbrial adhesin were first reported in S. enterica (S. Pomona, S. Bredeney and S. Mbandaka strains). pilW gene was first identified in S. Pomona. Phylogenetic analysis showed that some serovars circulated in Brazil for decades, primarily within the poultry production chain. Findings highlighted the virulence and AMR determinants in strains that may lead to recurring food outbreaks.