Members Of Enterobacteriaceae Research Articles

First Report of Brown Spot on White Button Mushroom (Agaricus bisporus) Caused by Cedecea neteri in Iran.

In autumn 2018, cap discoloration and browning symptoms (up to 20% incidence) were observed on commercially grown white button mushrooms (Agaricus bisporus) in two distinct farms located in Shiraz and Marvdasht Counties in Southern Iran. Symptomatic caps (13 and six caps from Shiraz and Marvdasht Counties, respectively) were characterized by visible brown discoloration with no blotch symptoms, bacterial sticky mass and cap wilting. Isolation of bacterial strains from infected cap tissues was performed on yeast-extract peptone glucose agar (YPGA) according to Hamidizade et al. (2020). The resulted bacterial colonies were oyster-white in color, non-fluorescent, domed convex circular with smooth margins 1-2 mm in diameter. A total of six bacterial strains (Shiraz: Ir1002, Ir1003, Ir1004, Ir1005, Ir1007 and Marvdasht: Ir1027) were isolated from distinct mushroom samples. Standard biochemical and phenotypic analyses (Schaad et al. 2001) showed that the bacterial strains were Gram and oxidase negative, catalase positive and facultatively anaerobic, while no capsule or endospore was observed. All strains were positive in urease production, arginine dihydrolase, hydrolysis of tween 80, and utilization of sucrose and D-sorbitol, while they were negative in amylase, cellulose, lecithinase, pectinase, and protease production as well as casein hydrolysis. Based on these phenotypic characteristics, the strains were supposed to be members of Enterobacteriaceae. They also did not induce hypersensitive reaction (HR) on tobacco (Nicotiana tabacum cv. Turkish) leaves nor did they produce tolaasin when streaked side-by-side with "Pseudomonas reactans" strains on King B medium (Osdaghi et al. 2019). Pathogenicity of the strains was evaluated (repeated twice) on fresh caps of white button mushroom using cut-cap method (Hamidizade et al. 2020). Reference strains of Pseudomonas tolaasii (CFBP 8707) and Mycetocola spp. (CFBP 8708) were used as positive controls, while sterile distilled water was used as a negative control. Brown discoloration appeared 24-36 hours post inoculation on cap surfaces while control caps remained asymptomatic. Koch's postulates were accomplished by re-isolation and identification of bacterial strains from the symptomatic caps using colony morphology and Gram staining. For molecular identification, all initial as well as re-isolated strains were subjected to amplification and sequencing of 16S rDNA and gyrB (Yamamoto and Harayama 1995; Hamidizade et al. 2020). Obtained nucleotide sequences were deposited into NCBI GenBank (16S: MZ298620 to MZ298625; gyrB: MZ313184 to MZ313189). BLAST search using the 16S rDNA and gyrB sequences showed that the strains isolated in this study had 97-99% sequence similarity to the reference strains of Cedecea neteri. Phylogenetic analyses also confirmed close relationship of bacterial strains from this study to C. neteri strains. Pure cultures of representative strains Ir1004 (CFBP 8900) and Ir1027 (CFBP 8896) are deposited in CIRM-CFBP culture collection. This is the first report of C. neteri causing brown spot disease on button mushroom in Iran, while the bacterium has previously been reported to cause soft rot on Pholiota nameko (Yan et al. 2018), and yellow sticky disease on Flammulina velutipes (Yan et al. 2019) in China. Further comprehensive investigations will shed a light on the economic impact of the brown spot disease on mushroom industry in Iran.

First identification of bla NDM-5 producing Escherichia coli from neonates and a HIV infected adult in Tanzania.

Introduction. Carbapenem-resistant members of the family Enterobacteriaceae are emerging as a global public-health threat and cause substantial challenges in clinical practice. Gap Statement. There is a need for increased and continued genomic surveillance of antimicrobial resistance genes globally in order to detect outbreaks and dissemination of clinically important resistance genes and their associated mobile genetic elements in human pathogens. Aim. To describe the resistance mechanisms of carbapenem-resistant Escherichia coli . Methods. Rectal swabs from neonates and newly diagnosed human immunodeficiency virus (HIV) infected adults were collected between April 2017 and May 2018 and screened for faecal carriage of carbapenamases and OXA-48 producing members of the family Enterobacteriaceae. Bacterial isolates were identified using matrix assisted laser desorption ionization time of flight mass spectrometry. Antimicrobial susceptibility testing was performed by E-test. Whole genomes of carbapenem-resistant E. coli were investigated using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads. Results. Three carbapenem-resistant E. coli were detected, two from neonates and one from an HIV infected adult. All three isolates carried bla NDM-5. Two E. coli from neonates belonged to ST167 and bla NDM-5 co-existed with bla CTX-M-15 and bla OXA-01, and all were carried on IncFIA type plasmids. The E. coli from the HIV infected adult belonged to ST2083, and carried bla NDM-5 on an IncX3 type plasmid and bla CMY-42 on an IncI type plasmid. All bla NDM-5 carrying plasmids contained conjugation related genes. In addition, E. coli from the HIV infected adult carried three more plasmid types; IncFIA, IncFIB and Col(BS512). One E. coli from a neonate also carried one extra plasmid Col(BS512). All three E. coli harboured resistance genes to fluoroquinolone, aminoglycosides, sulfamethoxazole, trimethoprim, macrolides and tetracycline, carried on the IncFIA type plasmid. Furthermore, E. coli from the neonates carried a chloramphenicol resistance gene (catB3), also on the IncFIA plasmid. All three isolates were susceptible to colistin. Conclusion. This is the first report, to our knowledge, from Tanzania detecting bla NDM-5 producing E. coli. The carbapenemase gene was carried on an IncFIA and IncX3 type plasmids. Our findings highlight the urgent need for a robust antimicrobial resistance (AMR) surveillance system to monitor and rapidly report on the incidence and spread of emerging resistant bacteria in Tanzania.

IncHI1A plasmids potentially facilitate horizontal flow of antibiotic resistance genes to pathogens in microbial communities of urban residential sewage.

Horizontal gene transfer via plasmids is important for the dissemination of antibiotic resistance genes among medically relevant pathogens. Specifically, the transfer of IncHI1A plasmids is believed to facilitate the spread of antibiotic resistance genes, such as carbapenemases, within the clinically important family Enterobacteriaceae. The microbial community of urban wastewater treatment plants has been shown to be highly permissive towards conjugal transfer of IncP1 plasmids. Here, we tracked the transfer of the P1 plasmid pB10 and the clinically relevant HI1A plasmid R27 in the microbial communities present in urban residential sewage entering full-scale wastewater treatment plants. We found that both plasmids readily transferred to these communities and that strains in the sewage were able to further disseminate them. Furthermore, R27has a broad potential host range, but a low host divergence. Interestingly, although the majority of R27 transfer events were to members of Enterobacteriaceae, we found a subset of transfer events to other families, even other phyla. This indicates that HI1A plasmids facilitate horizontal gene transfer both within Enterobacteriaceae, but also across families of, in particular, Gammaproteobacteria, such as Moraxellaceae, Pseudomonadaceae and Shewanellaceae. pB10 displayed a similar potential host range to R27. In contrast to R27, pB10had a high host divergence. By culture enrichment of the transconjugant communities, we show that sewage strains of Enterobacteriaceae and Aeromonadaceae can stably maintain R27 and pB10, respectively. Our results suggest that dissemination in the urban residual water system of HI1A plasmids may result in an accelerated acquisition of antibiotic resistance genes among pathogens.

Comparison of Carbapenem Resistance Detected by the BD Phoenix Automated System in Enterobacteriaceae Isolates with E-Test Method

Objective: Automatic identification and antimicrobial susceptibility systems are frequently used to identify clinical isolates in hospitalized patients, but mistakes in these systems can lead to potentially devastating treatment failures for patients. Therefore, the ‘‘Centers for Disease Control and Prevention (CDC)’’ recommends confirming all Carbapenem-resistant and low-susceptibility isolates with a different method. The aim of this study is to compare the Carbapenem susceptibility results of isolates reported as Carbapenem-resistant Enterobacteriaceae according to the BD Phoenix 100 automated system with the E-test method. Materials and Methods: The study included 70 strains of Carbapenem-resistant Enterobacteriaceae members which were isolated and grown from several types of clinical samples in the Medical Microbiology Laboratory. Conventional methods (Gram stain, negative oxidase test) and the BD Phoenix 100 automated system were used to identify the isolates. The susceptibility of all strains to imipenem, ertapenem and meropenem was investigated by E-test method. Automated system results and E-test results were compared. Results: The frequency distribution of all isolated bacterial strains comprised K. pneumoniae in 56 (80%) of the samples included in the study. The automated system test results were correlated with the results of the E-test at a rate of 96.1 % for the imipenem-resistant strains, 84.3% for the meropenem-resistant strains, 84.1% for the ertapenem-resistant strains Conclusions: Automated systems are frequently used in microbiology laboratories to identify isolates. However, automated systems can show a high error rate against some antimicrobials. For this reason, comparing the results of automated system test results with tests such as E-test is very important to prevent both treatment failures and inappropriate antibiotic use that may occur on a patient basis.

Faecal carriage of multi-drug resistant Enterobacteriaceae in hospitalized children at University Teaching Hospital Sylvanus Olympio of Lomé, Togo

Background: High prevalence of infections and associated antibiotherapy may put children at increased risk for development of multidrug-resistance (MDR), mostly to bacterial infections. The objective of this study therefore was to determine the prevalence of gastrointestinal carriage of MDR Enterobacteriaceae among hospitalized children in the Paediatric department of Sylvanus Olympio University Hospital, Lomé, Togo.Methodology: A descriptive cross-sectional study was carried out on randomly selected hospitalized children in the Paediatric wards of the hospital from November 03 to November 10, 2020, after obtaining informed consent from their parents/guardians. Rectal swabs and stool samples were collected from each participant and cultured for isolation of members of the family Enterobacteriaceae on Hektoen enteric agar containing 4 µg/L cefotaxime, which was incubated aerobically at 37oC for 24 hours. The isolates were identified using in-house biochemical tests. Antibiotic susceptibility test (AST) of each isolate to a panel of antibiotics including ertapenem and imipenem was done by the disc diffusion method and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints version 2020 V.1.1. ESBL production was detected by the double-disc synergy test of amoxicillin and clavulanic acid, and resistance to carbapenem was inferred by resistance to ertapenem and imipenem discs in the AST. Multi-drug resistance (MDR) was defined as resistance to at least three families of antibiotics. Statistical analysis of data was carried out on Excel 2010 and EPI INFO 7.2 and p value < 0.05 was considered to be statistical significance. Results: A total of 70 hospitalized children during the study period were randomly recruited with an average age of 4 years 3 days and a range of 1 day to 18 years. The male gender was predominant (54.3%) with a M: F ratio of 1.2. Samples were culture positive in 85.7% (60/70) and a total of 72 species of Enterobacteriaceae were isolated in 93.3% (56/60) of these cultures. Escherichia coli was the most frequently isolated species(56.9%, 41/72). More than 90% (65/72) of the isolates were resistant to ampicillin, 58.3% (42/72) to third generation cephalosporins, 59.7% (43/72) to fourth generation cephalosporins, 43% (31/72) to amino-glycosides, 55.6% (40/72) were multi-drug resistant, 48.6% (35/72) were ESBL-producing strains, and 6.9% (5/72) were carbapenem resistant. Eighty-three percent (29/35) of ESBL-producing and all the carbapenem resistant isolates (5/5) were recovered from children on antibiotic therapy. The prevalence of ESBL-producing phenotypes among culture-positive children on antibiotic treatment (72.5%, 29/40) was significantly higher than among culture-positive children not on antibiotic treatment (20.0%, 4/20), indicating that antibiotic therapy was significantly associated with carriage of MDR isolates (OR=10.545, 95% CI=2.882-38.590, p=0.0002). Conclusion: The high faecal carriage rate of MDR Enterobacteriaceae, which are all ESBL-producing strains, in this study is worrying. There is urgent need to develop measures to monitor and limit the spread of these MDR organisms in children and the community in Togo.

Isolation of a multidrug-resistant Escherichia coli pathotype Stx2:Cnf1:Cnf2:Eae as a potential cause of hemorrhagic diarrhea and secondary septicemia in a dog.

Escherichia coli is a member of the family Enterobacteriaceae and is a commensal in the intestine of many animals as well as humans. Most strains are of low virulence. A dog developed vomiting and hemorrhagic diarrhea after surgery and died despite treatment. Postmortem examination revealed hemorrhagic gastroenteritis and colitis. A multidrug-resistant E. coli, with virulence factors Shiga-toxin-producing gene, stx2, eae gene, and cytotoxic necrotic factors CNF-1 and CNF-2, was isolated from internal organs. E. coli can easily acquire new genes for virulence factors and antimicrobial resistance as demonstrated by this isolate with characteristics of both enterohemorrhagic E. coli and necrotoxigenic E. coli. In addition, the isolate was resistant to all beta-lactam antibiotics tested, as well as to enrofloxacin by a disk diffusion methodology. Broth-based minimum inhibitory concentration analysis confirmed resistance to amoxicillin (>32 μg/mL), enrofloxacin (>32 μg/mL), fosfomycin (>128 μg/mL), and neomycin (>32 μg/mL). The discovery of such strains is a cause for concern given that E. coli can be shared by companion animals and their human owners.

Antimicrobial and therapeutic effect of probiotics in cases of experimental purulent wounds

Probiotics based on bacteria of the genus Bacillus with a multifactorial mechanism of action are considered as a possible alternative to antibiotics in the treatment of purulent wounds. The aim of the study was to determine the antimicrobial and therapeutic effect of the Arederma probiotic preparation containing probiotic strains of the genus Bacillus in an experimental model of a purulent wound in animals. The antimicrobial efficacy of the probiotic against test strains and clinical isolates of pathogenic and opportunistic microorganisms was studied using the method of delayed antagonism. Staphylococcus aureus ATCC 6538 and Streptococcus pyogenes K-7 were used to model a purulent wound. From the surface of the wounds, bacteria of Staphylococcus, Streptococcus, Enterobacteriaceae, Pseudomonas genera and Enterobacteriaceae family were sown on appropriate selective media for the cultivation and enumeration of different groups of microorganisms by generally accepted microbiological research methods. The formation of a purulent wound in rabbits caused by mechanical skin damage and subsequent double infection with Staphylococcus aureus ATCC 6538 and Streptococcus pyogenes K-7 strains was accompanied with a pronounced inflammatory process, necrosis, the formation of purulent exudate and general intoxication. Representatives of the genera Staphylococcus and Streptococcus, microscopic fungi and, to a lesser extent, members of the family Enterobacteriaceae and Pseudomonas were found on the surface of purulent wounds, which confirmed the development of the infectious-inflammatory process. Treatment of purulent wounds with a suspension of probiotic preparation once a day for 4 days led to their faster healing (gradual attenuation of the inflammatory process, reduction of edema and discharge, as well as their disappearance) compared with untreated purulent wounds (control). Representatives of the Staphylococcus and Streptococcus genera, as well as microscopic fungi, presented in purulent wounds treated with probiotic preparation in much smaller numbers than in the control, and bacteria of the Pseudomonas genus and the Enterobacteriaceae family were not detected at all. The effective antimicrobial effect of this probiotic preparation against opportunistic and pathogenic microorganisms was confirmed by in vitro studies. Therefore, the Arederma probiotic preparation showed an effective therapeutic and antimicrobial effect in the experimental model of a purulent wound in animals, so it can be recommended for further preclinical and clinical studies.

1216. Presence of the Narrow-Spectrum OXA-1 Beta-lactamase Enzyme Is Associated with Elevated Piperacillin-Tazobactam MIC Values Among ESBL-producing Escherichia coli Clinical Isolates (CANWARD, 2007-2018)

BackgroundThe clinical outcome of patients with bacteremia due to an extended-spectrum beta-lactamase (ESBL)-producing member of the family Enterobacteriaceae who are treated with piperacillin-tazobactam appears to depend, at least in part, on the piperacillin-tazobactam MIC. The purpose of this study was to determine whether there is any association between the MIC of piperacillin-tazobactam and presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Escherichia coli.Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2007 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection.ResultsIn total, 671 ESBL-producing E. coli were identified as part of the CANWARD study. The majority of isolates (92.0%; 617/671) harbored a CTX-M ESBL enzyme. CTX-M-15 (62.3%; 418/671), CTX-M-27 (13.9%; 93/671), and CTX-M-14 (13.4%; 90/671) were the most common variants identified. The narrow spectrum OXA-1 beta-lactamase enzyme was present in 42.6% (286/671) of isolates. OXA-1 was detected in 66.3% (277/418) of isolates with a CTX-M-15 ESBL enzyme versus only 3.6% (9/253) of isolates with other ESBL enzyme types. The piperacillin-tazobactam MIC50 and MIC90 values were 8 µg/mL and 32 µg/mL for isolates that possessed the OXA-1 enzyme versus 2 μg/mL and 8 µg/mL for those that did not. The percentage of ESBL-producing E. coli isolates that were inhibited by a piperacillin-tazobactam MIC of ≤8 μg/mL was 68.5% for isolates that were OXA-1 positive and 93.8% for isolates that were OXA-1 negative.ConclusionThe MIC50 and MIC90 values of piperacillin-tazobactam among ESBL-producing E. coli were higher for the subset of isolates that harbored a narrow spectrum OXA-1 beta-lactamase enzyme relative to the subset that did not. This association was primarily observed among ESBL-producers with the CTX-M-15 enzyme variant. OXA-1 was infrequently detected among isolates with other ESBL enzyme types.Disclosures George Zhanel, PhD, AVIR (Grant/Research Support)Iterum (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Sandoz (Grant/Research Support)Sunovion (Grant/Research Support)Venatorx (Grant/Research Support)Verity (Grant/Research Support)

Beta-lactam antibiotics and extended spectrum beta-lactamases

Extended spectrum beta-lactamases (ESBLs) are enzymes produced by bacteria, mostly members of the family Enterobacteriaceae commonly Escherichia coli and Klebsiella pneumoniae. ESBLs hydrolyze the beta-lactam ring of beta-lactam antibiotics making these antibiotics ineffective therefore rendering the bacteria resistance against beta-lactam antibiotics. The global upsurge of ESBLs producing bacteria causing both hospital and community acquired infections mostly urinary tract infections, pneumonia and bloodstream infections, threatens the effectiveness of infectious diseases treatment. ESBL families; TEM, SHV and CTX-M are globally disseminated and frequently detected in clinical isolates as well as colonization and contamination isolates. Various laboratory detection methods of ESBLs producing Gram negative bacteria are available. These methods; phenotypic methods, automated methods and molecular-based methods are varying in sensitivity and specificity, need of technical expertise, and rapidness. Therefore, they should be clearly understood before being employed for routine or research use for detection of ESBLs production among Enterobacteriaceae. In addition, understanding the mode of action and mechanisms of resistance to beta-lactam antibiotics, and the epidemiology of ESBLs producing bacteria is of paramount.

Comparative genomic identification and characterization of npcRNA homologs in Proteus vulgaris

Proteus vulgaris is a rod-shaped Gram-negative bacterium known to be the member of Enterobacteriaceae that is able to cause disease in human being. Generally, non-protein-coding RNAs (npcRNAs) do not code for proteins, but they play a vital role in gene regulation at the RNA level including pathogenicity. The present study aims at elucidating homologous npcRNAs from other bacteria in Proteus vulgaris. A comparative genomic analysis was carried out to identify npcRNA homolog of other Enterobacteriaceae pathogens in Proteus vulgaris. A total of 231 npcRNAs previously reported in Salmonella typhi, Salmonella typhimurium and Escherichia coli were screened using BLASTn tool against Proteus vulgaris genome. Interestingly, 33 npcRNAs are homologs to Proteus vulgaris. Northern blot analysis of 6 out of 33 npcRNA candidates confirmed their expression and showed that most of them are differentially expressed during lag, exponential and stationary growth phases. This study is the first approach of identification and characterization of npcRNAs in P. vulgaris. Hence, this could be a pioneer study to further validate the regulatory functions of these npcRNAs to fill the gaps in understanding of the pathogenicity of P. vulgaris.

Dry and Rainy Seasons Significantly Alter the Gut Microbiome Composition and Reveal a Key Enterococcus sp. (Lactobacillales: Enterococcaceae) Core Component in Spodoptera frugiperda (Lepidoptera: Noctuidae) Corn Strain From Northwestern Colombia.

Spodoptera frugiperda is a polyphagous pest of several crops of economic importance. Nowadays, the insect is broadly distributed in America and, recently, in Africa, Asia, and Australia. The species has diverged into corn and rice strains. The role of the gut microbiota in insect physiology is relevant due to its participation in crucial functions. However, knowledge of seasonal variations that alter the gut microbiome in pests is limited. Gut microbiome composition between the dry and rainy seasons was analyzed with cultured and uncultured approaches in S. frugiperda corn strain larvae collected at Northwest Colombia, as seasonal microbiome changes might fluctuate due to environmental changes. On the basis of culture-dependent methods, results show well-defined microbiota with bacterial isolates belonging to Enterococcus, Klebsiella (Enterobacteriales: Enterobacteriaceae), Enterobacter (Enterobacterales: Enterobacteriaceae), and Bacillus (Bacillales: Bacillaceae) genera. The community composition displayed a low bacterial diversity across all samples. The core community detected with uncultured methods was composed of Enterococcus, Erysipelatoclostridium (Erysipelotrichales: Erysipelotrichaceae), Rasltonia (Burkholderiales: Burkholderiaceae), and Rhizobium (Hyphomicrobiales: Rhizobiaceae) genera, and Enterobacteriaceae family members. Significant differences in microbiome diversity were observed between the two seasons. The relative abundance of Erysipelatoclostridium was high in the dry season, while in the phylotype ZOR0006 (Erysipelotrichales: Erysipelotrichaceae) and Tyzzerella (Lachnospirales: Lachnospiraceae) genus, the relative abundance was high in the rainy season. The overall low gut bacterial diversity observed in the S. frugiperda corn strain suggests a strong presence of antagonist activity as a selection factor possibly arising from the host, the dominant bacterial types, or the material ingested. Targeting the stability and predominance of this core microbiome could be an additional alternative to pest control strategies, particularly in this moth.

The treasure inside barley seeds: microbial diversity and plant beneficial bacteria

BackgroundBacteria associated with plants can enhance the plants’ growth and resistance against phytopathogens. Today, growers aim to reduce the use of mineral fertilizers and pesticides. Since phytopathogens cause severe yield losses in crop production systems, biological alternatives gain more attention. Plant and also seed endophytes have the potential to influence the plant, especially seed-borne bacteria may express their beneficiary impact at initial plant developmental stages. In the current study, we assessed the endophytic seed microbiome of seven genetically diverse barley accessions by 16S rRNA gene amplicon sequencing and verified the in vitro plant beneficial potential of isolated seed endophytes. Furthermore, we investigated the impact of the barley genotype and its seed microbiome on the rhizosphere microbiome at an early growth stage by 16S rRNA gene amplicon sequencing.ResultsThe plant genotype displayed a significant impact on the microbiota in both barley seed and rhizosphere. Consequently, the microbial alpha- and beta-diversity of the endophytic seed microbiome was highly influenced by the genotype. Interestingly, no correlation was observed between the endophytic seed microbiome and the single nucleotide polymorphisms of the seven genotypes. Unclassified members of Enterobacteriaceae were by far most dominant. Other abundant genera in the seed microbiome belonged to Curtobacterium, Paenibacillus, Pantoea, Sanguibacter and Saccharibacillus. Endophytes isolated from barley seeds were affiliated to dominant genera of the core seed microbiome, based on their 16S rRNA gene sequence. Most of these endophytic isolates produced in vitro plant beneficial secondary metabolites known to induce plant resistance.ConclusionAlthough barley accessions representing high genetic diversity displayed a genotype-dependent endophytic seed microbiome, a core seed microbiome with high relative abundances was identified. Endophytic isolates were affiliated to members of the core seed microbiome and many of them showed plant beneficial properties. We propose therefore that new breeding strategies should consider genotypes with high abundance of beneficial microbes.

Molecular Mechanisms of Colistin Resistance in Klebsiella pneumoniae in a Tertiary Care Teaching Hospital.

BackgroundOver the last two decades, the prevalence of colistin resistance among the members of Enterobacteriaceae has been increasing, particularly among Klebsiella pneumoniae isolates; this limits the potential use of colistin and leads to worsened clinical outcomes.MethodsWe investigated the prevalence and genetic characteristics of colistin-resistant K. pneumoniae (COLR-KP) in clinical isolates using genomic sequencing.ResultsIn total, 53 K. pneumoniae isolates (4.5%, 53/1,171) were confirmed as COLR-KP, of which eight isolates carried mobile colistin-resistant (mcr) gene. Although the overall prevalence rate (0.7%, 8/1,171) of mcr-like genes in clinical K. pneumoniae remained relatively low, the presence of mcr (15.1%, 8/53) among the COLR-KP isolates indicated that the mobile resistance gene was already widespread among K. pneumoniae isolates in hospital setting. We randomly selected 13 COLR-KP isolates (four mcr-bearing and nine non-mcr-bearing isolates) for whole-genome sequencing, including two pandrug-resistant and four sequence type 11 (ST11) isolates. Phylogenetic analysis revealed that all COLR-KP isolates were genetically diverse. Among the four mcr-bearing isolates, three (KP4, KP18, and KP30) were positive for mcr-1 and one (KP23) for mcr-8; none of the other mcr genes were detected. The mcr-1 in the KP4 and KP30 isolates were located in an IncX4 plasmid (approximately 33 kb) and could be successfully transferred to Escherichia coli J53AZR. In contrast, for the mcr-8-bearing plasmid in KP23 (IncFII), colistin resistance could not be transferred by conjugation. The mcr-1-producing isolate KP18 coexists a novel plasmid-carried tigecycline resistance gene tmexCD1-toprJ1. The most common chromosomal mutation associated with colistin resistance was a T246A amino acid substitution in PmrB, which was identified in most COLR-KP isolates (11/13, 84.6%). All ST11 isolates additionally had an R256G amino acid substitution. Critical virulence factors associated with hypervirulent K. pneumoniae were detected in four COLR-KP isolates; these virulence factors included aerobactin, salmochelin, and yersiniabactin.ConclusionWe found that mcr-bearing COLR-KP emerged in our hospital and was growing at an increasing rate. Simultaneous emergence of hypervirulence and colistin–tigecycline–carbapenem resistance in the epidemic clone ST11 K. pneumoniae was also observed; this highlights the significance of active and continuous surveillance.

Plant-microbe-microbe interactions influence the faba bean nodule colonization by diverse endophytic bacteria.

Legume root nodules harbor rhizobia and other non-nodulating endophytes known as nodule-associated bacteria (NAB) whose role in the legume symbiosis is still unknown. We analysed the genetic diversity of 34 NAB isolates obtained from the root nodules of faba bean grown under various soil conditions in Egypt using 16S rRNA and concatenated sequences of three housekeeping genes. All isolates were identified as members of the family Enterobacteriaceae belonging to the genera Klebsiella, Enterobacter and Raoultella. We identified nine enterobacterial genospecies, most of which have not been previously reported as NAB. All isolated strains harbored nifH gene sequences and most of them possessed plant growth-promoting (PGP) traits. Upon co-inoculation with an N2 fixing rhizobium (Rlv NGB-FR128), two strains (Enterobacter sichanensis NGB-FR97 and Klebsiella variicola NGB-FR116) significantly increased nodulation, growth and N-uptake of faba bean plants over the single treatments or the uninoculated control. The presence of these enterobacteria in nodules was significantly affected by the host plant genotype, symbiotic rhizobium genotype and endophyte genotype, indicating that the nodule colonization process is regulated by plant-microbe-microbe interactions. This study emphasizes the importance of nodule-associated enterobacteria and suggests their potential role in improving the effectiveness of rhizobial inoculants.

Comprehensive Analysis of Bacterial Community Structure and Diversity in Sichuan Dark Tea (Camellia sinensis).

Bacteria and fungi present during pile-fermentation of Sichuan dark tea play a key role in the development of its aesthetic properties, such as color, taste, and fragrance. In our previous study, high-throughput sequencing of dark tea during fermentation revealed Aspergillus was abundant, but scarce knowledge is available about bacterial communities during pile-fermentation. In this study, we rigorously explored bacterial diversity in Sichuan dark tea at each specific stage of piling. Analysis of cluster data revealed 2,948 operational taxonomic units, which were divided into 42 phyla, 98 classes, 247 orders, 461 families, 1,052 genera, and 1,888 species. Certain members of the family Enterobacteriaceae were dominant at early stages of fermentation YC, W1, and W2; Pseudomonas at middle stage W3; and the highest bacterial diversity was observed at the final quality-determining stage W4. Noticeably, probiotics, such as Bacillus, Lactobacillus, Bifidobacterium, and Saccharopolyspora were also significantly higher at the quality-determining stage W4. Our findings might help in precise bacterial inoculation for probiotic food production by increasing the health benefits of Sichuan dark tea. This research also falls under the umbrella of the “Establish Good Health and Well-Being” Sustainable Development Goals of the United Nations Organization.

English

This study aimed to determine the prevalence of extended spectrum beta-lactamase producing (ESBL) and multidrug resistant Escherichia coli and Klebsiella pneumoniae. A cross-sectional study was conducted in Ouagadougou in two poultry farms and two slaughterhouses. 375 cloacal swabs and 46 environment samples were collected and members of Enterobacteriaceae were isolated on EMB agar containing 4 µg/L of cefotaxime. E. coli and K. pneumoniae were identified using biochemical tests and ESBL production was confirmed by the double-disc synergy test. Antibiotic susceptibility was determined by the disc diffusion method. Prevalence of faecal ESBL producing E. coli and K. pneumoniae was 12.11% (95% CI = 9.3-15.6). In sampling sites, the prevalence were 5.15% in Farm A, 2.22% in Farm B, 17.50% in slaughterhouse C, 20.59% in slaughterhouse D and 19.57% in environment. E. coli (n = 43) and K. pneumoniae (n = 13) were frequently identified. ESBL-producing E. coli and K. pneumoniae MDR was 89.29% (95% CI = 78.5–95.0). Resistance to aminoglycosides was 6.25% in poultry and 10.00% in slaughterhouse, fluoroquinolones 32.5% in slaughterhouse, sulfonamides 100% in poultry and 82.50% slaughterhouse, tetracycline 100% in poultry and 95.0% in slaughterhouse. This study showed that antimicrobial resistance in poultry in Ouagadougou portends a serious problem.   Key words: Escherichia coli, Klebsiella pneumoniae, extended-spectrum β-lactamase (ESBL), poultry, farms, slaughterhouses, Ouagadougou.

Whole blood resuscitation restores intestinal perfusion and influences gut microbiome diversity.

Gut dysbiosis, an imbalance in the gut microbiome, occurs after trauma, which may be ameliorated with transfusion. We hypothesized that gut hypoperfusion following trauma causes dysbiosis and that whole blood (WB) resuscitation mitigates these effects. Anesthetized rats underwent sham (S; laparotomy only, n = 6); multiple injuries (T; laparotomy, liver and skeletal muscle crush injuries, and femur fracture, n = 5); multiple injuries and 40% hemorrhage (H; n = 7); and multiple injuries, hemorrhage, and WB resuscitation (R; n = 7), which was given as 20% estimated blood volume from donor rats 1 hour posttrauma. Baseline cecal mesenteric tissue oxygen (O2) concentration was measured following laparotomy and at 1 hour and 2 hours posttrauma. Fecal samples were collected preinjury and at euthanasia (2 hours). 16S rRNA sequencing was performed on purified DNA, and diversity and phylogeny were analyzed with QIIME (Knight Lab, La Jolla, CA; Caporaso Lab, Flagstaff, AZ) using the Greengenes 16S rRNA database (operational taxonomic units; 97% similarity). α and β diversities were estimated using observed species metrics. Permutational analysis of variance was performed for overall significance. In H rats, an average decline of 36% ± 3.6% was seen in the mesenteric O2 concentration at 1 hour without improvement by 2 hours postinjury, which was reversed following resuscitation at 2 hours postinjury (4.1% ± 3.1% difference from baseline). There was no change in tissue O2 concentration in the S or T rats. β Diversity differed among groups for all measured indices except Bray-Curtis, with the spatial median of the S and R rats more similar compared with S and H rats (p < 0.05). While there was no difference in α diversity found among the groups, indices were significantly correlated with mesenteric O2 concentration. Members of the family Enterobacteriaceae were significantly enriched in only 2 hours. Mesenteric perfusion after trauma and hemorrhage is restored with WB resuscitation, which influences β diversity of the gut microbiome. Whole blood resuscitation may also mitigate the effects of hemorrhage on intestinal dysbiosis, thereby influencing outcomes.

Distribution and molecular characterization of ESBL, pAmpC β-lactamases, and non-β-lactam encoding genes in Enterobacteriaceae isolated from hospital wastewater in Eastern Cape Province, South Africa.

Globally, there is an increasing occurrence of multidrug-resistant (MDR) Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs) and/or plasmid-encoded AmpC (pAmpC) β-lactamases in clinical and environmental settings of significant concern. Therefore, we aimed to evaluate the occurrence of ESBL/pAmpC genetic determinants, and some essential non-β-lactam genetic determinants in the MDR phenotypic antimicrobial resistance in Enterobacteriaceae isolates recovered from hospital wastewater. We collected samples from two hospitals in Amathole and Chris Hani District Municipalities in the Eastern Cape Province, South Africa, within October and November 2017. Using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), we identified a total of 44 presumptive Enterobacteriaceae isolates. From this, 31 were identified as the targeted Enterobacteriaceae members. Thirty-six percent of these belonged to Klebsiella oxytoca, while 29% were Klebsiella pneumoniae. The other identified isolates included Citrobacter freundii and Escherichia coli (10%), Enterobacter asburiae (6%), Enterobacter amnigenus, Enterobacter hormaechei, and Enterobacter kobei (3%). We established the antibiotic susceptibility profiles of these identified bacterial isolates against a panel of 18 selected antibiotics belonging to 11 classes were established following established guidelines by the Clinical and Laboratory Standard Institute. All the bacterial species exhibited resistance phenotypically against at least four antibiotic classes and were classified as MDR. Notably, all the bacterial species displayed resistance against cefotaxime, ampicillin, nalidixic acid, and trimethoprim-sulfamethoxazole. The generated multiple antibiotic resistance indices ranged between 0.5 to 1.0, with the highest value seen in one K. oxytoca isolated. Molecular characterization via the Polymerase Chain Reaction uncovered various ESBLs, pAmpCs, and other non-β-lactam encoding genes. Of the phenotypically resistant isolates screened for each class of antibiotics, the ESBLs detected were blaCTX-M group (including groups 1, 2, and 9) [51.6% (16/31)], blaTEM [32.3% (10/31)], blaOXA-1-like [19.4% (6/31)], blaSHV [12.9% (4/31)], blaPER [6.5% (2/31)], blaVEB [3.2% (1/31)], blaOXA-48-like and blaVIM [15.4% (2/13)], and blaIMP [7.7% (1/13)]. The pAmpC resistance determinants detected were blaCIT [12.9% (4/31)], blaFOX [9.7% (3/31)], blaEBC [6.5% (2/31)], and blaDHA [3.2% (1/31)]. The frequencies of the non-β-lactam genes detected were catII [79.2% (19/24)], tetA [46.7% (14/30)], sulI and sulII [35.5% (11/31)], tetB [23.3% (7/30)], aadA [12.9% (4/31)], tetC [10% (3/30)], and tetD [3.3% (1/30)]. These results indicate that hospital wastewater is laden with potentially pathogenic MDR Enterobacteriaceae with various antibiotic resistance genes that can be spread to humans throughout the food chain, provided the wastewaters are not properly treated before eventual discharge into the environment.

Microbiological quality aspects of ready-to-eat foods with focus on antibiotic resistance and biofilm formation abilities of foodborne bacteria

AbstractIn this study, microbiological quality of 93 ready-to-eat food products was determined by enumeration of total aerobic bacteria and members of Enterobacteriaceae. Presence of Staphylococcus aureus and Listeria monocytogenes in the products were also investigated. Aerobic colony counts were between 1.9 × 102–3.4 × 108 CFU g−1 for 84.9% and Enterobacteriaceae counts were between 2 × 102 and 6.7 × 106 CFU g−1 for 43% of the samples. S. aureus was detected in 7.5% of the samples, but L. monocytogenes was not detected in any sample. 72.9% of the Enterobacteriaceae isolates showed resistance to at least one antibiotic tested, and 5 among the S. aureus isolates were found to be resistant to penicillin G and 2 against methicillin. Four S. aureus isolates produced only Staphylococcal enterotoxin A and 1 isolate produced both Staphylococcal enterotoxin A and B. Biofilm experiments revealed biofilm formation on polystyrene for 95.8%, 87.5%, and 91.6% of Enterobacteriaceae isolates at 4 °C, 22 °C, and 37 °C, respectively, whereas all S. aureus isolates formed biofilm at all temperatures.

Prolonged SARS-CoV-2 RNA virus shedding and lymphopenia are hallmarks of COVID-19 in cancer patients with poor prognosis

Patients with cancer are at higher risk of severe coronavirus infectious disease 2019 (COVID-19), but the mechanisms underlying virus–host interactions during cancer therapies remain elusive. When comparing nasopharyngeal swabs from cancer and noncancer patients for RT-qPCR cycle thresholds measuring acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in 1063 patients (58% with cancer), we found that malignant disease favors the magnitude and duration of viral RNA shedding concomitant with prolonged serum elevations of type 1 IFN that anticorrelated with anti-RBD IgG antibodies. Cancer patients with a prolonged SARS-CoV-2 RNA detection exhibited the typical immunopathology of severe COVID-19 at the early phase of infection including circulation of immature neutrophils, depletion of nonconventional monocytes, and a general lymphopenia that, however, was accompanied by a rise in plasmablasts, activated follicular T-helper cells, and non-naive Granzyme B+FasL+, EomeshighTCF-1high, PD-1+CD8+ Tc1 cells. Virus-induced lymphopenia worsened cancer-associated lymphocyte loss, and low lymphocyte counts correlated with chronic SARS-CoV-2 RNA shedding, COVID-19 severity, and a higher risk of cancer-related death in the first and second surge of the pandemic. Lymphocyte loss correlated with significant changes in metabolites from the polyamine and biliary salt pathways as well as increased blood DNA from Enterobacteriaceae and Micrococcaceae gut family members in long-term viral carriers. We surmise that cancer therapies may exacerbate the paradoxical association between lymphopenia and COVID-19-related immunopathology, and that the prevention of COVID-19-induced lymphocyte loss may reduce cancer-associated death.