Global scenario of the RmtE pan-aminoglycoside-resistance mechanism: emergence of the rmtE4 gene in South America associated with a hospital-related IncL plasmid.

Antimicrobial resistance (AMR) mechanisms, especially those conferring resistance to critically important antibiotics, are a great concern for public health. 16S rRNA methyltransferases (16S-RMTases) abolish the effectiveness of most clinically used aminoglycosides, but some of them are considered sporadic, such as RmtE. The main goals of this work were the genomic analysis of bacteria producing 16S-RMTases from a 'One Health' perspective in Venezuela, and the study of the epidemiological and evolutionary scenario of RmtE variants and their related mobile genetic elements (MGEs) worldwide. A total of 21 samples were collected in 2014 from different animal and environmental sources in the Cumaná region (Venezuela). Highly aminoglycoside-resistant Enterobacteriaceae isolates were selected, identified and screened for 16S-RMTase genes. Illumina and Nanopore whole-genome sequencing data were combined to obtain hybrid assemblies and analyse their sequence type, resistome, plasmidome and pan-genome. Genomic collections of rmtE variants and their associated MGEs were generated to perform epidemiological and phylogenetic analyses. A single 16S-RMTase, the novel RmtE4, was identified in five Klebsiella isolates from wastewater samples of Cumaná. This variant possessed three amino acid modifications with respect to RmtE1-3 (Asn152Asp, Val216Ile and Lys267Ile), representing the most genetic distant among all known and novel variants described in this work, and the second most prevalent. rmtE variants were globally spread, and their geographical distribution was determined by the associated MGEs and the carrying bacterial species. Thus, rmtE4 was found to be confined to Klebsiella isolates from South America, where it was closely related to ISVsa3 and an uncommon IncL plasmid related with hospital environments. This work uncovered the global scenario of RmtE and the existence of RmtE4, which could potentially emerge from South America. Surveillance and control measures should be developed based on these findings in order to prevent the dissemination of this AMR mechanism and preserve public health worldwide.

Detection of microorganisms with antibacterial activities from different industrial wastes and GC-MS analysis of crude microbial extract / Detecção de microrganismos com atividades antibacterianas de diferentes resíduos industriais e análise por GC-MS do extrato microbiano bruto

Many soil microorganisms' i.e., bacteria and fungi produce secondary metabolites called antibiotics. These are used for the treatment of some of the bacterial, fungal and protozoal diseases of humans. There is a need for isolation of a broad spectrum of antibiotics from microorganisms due to the emergence of antibiotic resistance. In the present study two antibiotic producing bacteria Klebsiella pneumoniae and Bacillus cereus were isolated from pharmaceutical and poultry feed industry of Hattar, Haripur Pakistan. Total 10 waste samples were collected from different industries (Marble, Ghee, Soap, Mineral, Steel, Poultry Feed, Pharmaceutical, Qarshi, Cosmetic and Glass). Thirty-three bacterial strains were isolated from industrial wastes of these ten different industries. Fourteen out of thirty-three bacterial strains exhibited antimicrobial activities against at least one of the test microbes considered in this study including Escherchia coli, Staphylococcus aureus and Salmonella typhi. The bacteria were isolated by standard serial dilution spread plate technique. Morphological characterization of the isolates was done by Gram staining. Nine bacterial isolates out of fourteen were initially identified as B. cereus and five as K. pneumoniae through biochemical characterization. The antibacterial activities were tested by well diffusion method. Maximum number of antibiotic producing bacteria were isolated from pharmaceutical and poultry feed industry based on the results of primary screening, the most potential isolates S9, S19, S20, S22 and S23 were selected for secondary screening. The maximum activity against E. coli and S. aureus was recorded by bacterial isolate S19 i.e zones of inhibition of 6.5mm and 9mm while S20 showed 7.5mm and 6mm zones respectively. Molecular identification was carried out on the basis of 16S rRNA sequence [...].

Muitos microrganismos do solo, ou seja, bactérias e fungos produzem metabólitos secundários chamados antibióticos. Eles são usados para tratamento de algumas doenças bacterianas, fúngicas e protozoárias em humanos. Há necessidade de isolamento de um amplo espectro de antibióticos de microrganismos devido ao surgimento de resistência aos antibióticos. No presente estudo, duas bactérias produtoras de antibióticos, Klebsiella pneumoniae e Bacillus cereus, foram isoladas da indústria farmacêutica e de ração avícola de Hattar, Haripur, Paquistão. Um total de 10 amostras de resíduos foi coletado de diferentes indústrias (mármore, ghee, sabão, mineral, aço, ração para aves, farmacêutica, Qarshi, cosmética e vidro). Trinta e três cepas bacterianas foram isoladas de resíduos industriais dessas dez diferentes indústrias. Quatorze das 33 cepas bacterianas exibiram atividades antimicrobianas contra pelo menos um dos micróbios de teste considerados neste estudo, incluindo Escherchia coli, Staphylococcus aureus e Salmonella typhi. As bactérias foram isoladas pela técnica de placa de diluição em série padrão. A caracterização morfológica dos isolados foi feita por coloração de gram. Nove isolados bacterianos de 14 foram inicialmente identificados como B. cereus e cinco como K. pneumoniae por meio de caracterização bioquímica. As atividades antibacterianas foram testadas pelo método de difusão em poço. O número máximo de bactérias produtoras de antibióticos foi isolado da indústria farmacêutica e de ração avícola com base nos resultados da triagem primária, os isolados mais potenciais S9, S19, S20, S22 e S23 foram selecionados para a triagem secundária. A atividade máxima contra E. coli e S. aureus foi registrada pelo isolado bacteriano S19, ou seja, zonas de inibição de 6,5 mm e 9 mm, enquanto S20 mostrou zonas de 7,5 mm e 6 mm, respectivamente. A identificação molecular foi realizada com base na análise da sequência 16S [...].

Community Structure of Nitrifying and Denitrifying Bacteria from Effluents Discharged into Lake Victoria, Kenya.

An active microbial community of nitrifying and denitrifying bacteria is needed for efficient utilization of nitrogenous compounds from wastewater. In this study, we explored the bacterial community diversity and structure within rivers, treated and untreated wastewater treatment plants (WWTPs) discharging into Lake Victoria. Water samples were collected from rivers and WWTPs that drain into Lake Victoria. Physicochemical analysis was done to determine the level of nutrients or pollutant loading in the samples. Total community DNA was extracted, followed by Illumina high throughput sequencing to determine the total microbial community and abundance. Enrichment and isolation were then done to recover potential nitrifiers and denitrifiers. Physicochemical analysis pointed to high levels total nitrogen and ammonia in both treated and untreated WWTPs as compared to the samples from the lake and rivers. A total of 1,763 operational taxonomic units (OTUs) spread across 26 bacterial phyla were observed with the most dominant phylum being Proteobacteria. We observed a decreasing trend in diversity from the lake, rivers to WWTPs. The genus Planktothrix constituted 19% of the sequence reads in sample J2 collected from the lagoon. All the isolates recovered in this study were affiliated to three genera: Pseudomonas, Klebsiella and Enterobacter in the phylum Proteobacteria. A combination of metagenomic analysis and a culture-dependent approach helped us understand the relative abundance as well as potential nitrifiers and denitrifiers present in different samples. The recovered isolates could be used for in situ removal of nitrogenous compounds from contaminated wastewater.

Effects of early-life antibiotics on the developing infant gut microbiome and resistome: a randomized trial.

Broad-spectrum antibiotics for suspected early-onset neonatal sepsis (sEONS) may have pronounced effects on gut microbiome development and selection of antimicrobial resistance when administered in the first week of life, during the assembly phase of the neonatal microbiome. Here, 147 infants born at ≥36 weeks of gestational age, requiring broad-spectrum antibiotics for treatment of sEONS in their first week of life were randomized 1:1:1 to receive three commonly prescribed intravenous antibiotic combinations, namely penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime (ZEBRA study, Trial Register NL4882). Average antibiotic treatment duration was 48 hours. A subset of 80 non-antibiotic treated infants from a healthy birth cohort served as controls (MUIS study, Trial Register NL3821). Rectal swabs and/or faeces were collected before and immediately after treatment, and at 1, 4 and 12 months of life. Microbiota were characterized by 16S rRNA-based sequencing and a panel of 31 antimicrobial resistance genes was tested using targeted qPCR. Confirmatory shotgun metagenomic sequencing was executed on a subset of samples. The overall gut microbial community composition and antimicrobial resistance gene profile majorly shift directly following treatment (R2 = 9.5%, adjusted p-value = 0.001 and R2 = 7.5%, adjusted p-value = 0.001, respectively) and normalize over 12 months (R2 = 1.1%, adjusted p-value = 0.03 and R2 = 0.6%, adjusted p-value = 0.23, respectively). We find a decreased abundance of Bifidobacterium spp. and increased abundance of Klebsiella and Enterococcus spp. in the antibiotic treated infants compared to controls. Amoxicillin + cefotaxime shows the largest effects on both microbial community composition and antimicrobial resistance gene profile, whereas penicillin + gentamicin exhibits the least effects. These data suggest that the choice of empirical antibiotics is relevant for adverse ecological side-effects.

Evaluation of Rapid Immunochromatographic Tests for the Direct Detection of Extended Spectrum Beta-Lactamases and Carbapenemases in Enterobacterales Isolated from Positive Blood Cultures.

NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech) are two rapid in vitro immunochromatographic assays that are widely used for the detection of the most common extended spectrum beta-lactamases (ESBL) and carbapenemases in Enterobacterales. ESBL and carbapenemases are leading causes of morbidity and mortality worldwide and their rapid detection from positive blood cultures is crucial for early initiation of effective antimicrobial therapy in bloodstream infections (BSI) involving antibiotic-resistant organisms. In this study, we developed a rapid workflow for positive blood cultures for direct identification of Enterobacterales by MALDI-TOF mass-spectrometry, followed by detection of ESBL and carbapenemases using NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech). The workflow was evaluated using Enterobacterales isolates (n = 114), primarily Klebsiella species (n = 50) and Escherichia coli (n = 40). Compared to the standard testing approach in our institution using BD Phoenix, our new testing approach demonstrates 100% sensitivity and specificity for organism identification and detection of ESBL and carbapenemases. Implementation of a rapid workflow in diagnostic microbiology laboratories will enable more effective antimicrobial management of patients with BSI due to ESBL- and carbapenemase-producing Enterobacterales. IMPORTANCE The incidence of bloodstream infections (BSI) with extended spectrum beta-lactamase (ESBL) producing and carbapenemase producing Enterobacterales (CPE) is increasing at an alarming rate, for which only limited therapeutic options remain available. Rapid identification of these bacteria along with their antibiotic resistance mechanisms in positive blood cultures with Gram-negative bacteria will allow for early initiation of effective therapy and limit the overuse of broad-spectrum antibiotics in BSI (1). In this study we evaluated a combined approach of testing positive blood cultures directly, using MALDI-TOF MS followed by rapid immunochromatographic tests, for the detection of ESBLs and CPEs. Our approach demonstrates 100% sensitivity and specificity for the identification of Enterobacterales and detection of ESBLs and CPEs in positive blood culture with a turnaround time (TAT) of ≤60 min compared to a TAT of 48 h required by conventional culture and susceptibility testing methods.

Novel strains of Klebsiella africana and Klebsiella pneumoniae in Australian fruit bats (Pteropus poliocephalus).

Over the past decade human associated multidrug resistant (MDR) and hypervirulent Klebsiella pneumoniae lineages have been increasingly detected in wildlife. This study investigated the occurrence of K. pneumoniae species complex (KpSC) in grey-headed flying foxes (GHFF), an Australian fruit bat. Thirty-nine KpSC isolates were cultured from 275 GHFF faecal samples (14.2%), comprising K. pneumoniae (n = 30), Klebsiella africana (n = 8) and Klebsiella variicola subsp. variicola (n = 1). The majority (79.5%) of isolates belonged to novel sequence types (ST), including two novel K. africana STs. This is the first report of K. africana outside of Africa and in a non-human host. A minority (15.4%) of GHFF KpSC isolates shared STs with human clinical K. pneumoniae strains, of which, none belonged to MDR clonal lineages that cause frequent nosocomial outbreaks, and no isolates were characterised as hypervirulent. The occurrence of KpSC isolates carrying acquired antimicrobial resistance genes in GHFF was low (1.1%), with three K. pneumoniae isolates harbouring both fluoroquinolone and trimethoprim resistance genes. This study indicates that GHFF are not reservoirs for MDR and hypervirulent KpSC strains, but they do carry novel K. africana lineages. Health risks associated with KpSC carriage by GHFF are deemed low for the public and GHFF.

A new mutation in mgrb mediating polymyxin resistance in Klebsiella variicola.

Polymyxin resistance is a public health concern - present in humans, animals and the environment - caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola (K. variicola). This study performed microbiological and genomic characterisation of three polymyxin-resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil. The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing using Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial-resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50. This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.

Isolation and identification of microorganisms associated with automated teller machines on Federal Polytechnic Ede campus.

Automated Teller Machines (ATM) are visited everyday by millions of people. This machine is accessible to the general public irrespective of class, age or race. The contact point of all ATM machines is the hand which on their own are 'vaults' of microorganisms. An elaborate survey was taken for complete assessment of possible microbial contamination in the Federal Polytechnic Ede campus. Selected ATM machines on campus were used as case study to characterize, identify and determine the degree of bacterial contamination of microorganisms and their potential as reservoir of microbes. Swabs were collected from each ATM screen, buttons, floor, user's hand, and exposure of plates. After collection of the samples, they were plated in nutrient agar. The results showed the presence of increased bacterial count subsequently, most pathogens on characterization revealed the genus of the particular organisms E. coli, Pseudomonas, Staphylococcus aureus, Klebsiella, Micrococcus, Salmonella and Serratia. The study showed the potential hazard inherent in ATM machine usage and draws attention to our level of hand hygiene compliance.

Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive Klebsiella variicola in trauma patients.

Recent reclassification of the Klebsiella genus to include Klebsiella variicola, and its association with bacteremia and mortality, has raised concerns. We examined Klebsiella spp. infections among battlefield trauma patients, including occurrence of invasive K. variicola disease. Klebsiella isolates collected from 51 wounded military personnel (2009-2014) through the Trauma Infectious Disease Outcomes Study were examined using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis. K. variicola isolates were evaluated for hypermucoviscosity phenotype by the string test. Patients were severely injured, largely from blast injuries, and all received antibiotics prior to Klebsiella isolation. Multidrug-resistant Klebsiella isolates were identified in 23 (45%) patients; however, there were no significant differences when patients with and without multidrug-resistant Klebsiella were compared. A total of 237 isolates initially identified as K. pneumoniae were analyzed, with 141 clinical isolates associated with infections (remaining were colonizing isolates collected through surveillance groin swabs). Using PCR sequencing, 221 (93%) isolates were confirmed as K. pneumoniae, 10 (4%) were K. variicola, and 6 (3%) were K. quasipneumoniae. Five K. variicola isolates were associated with infections. Compared to K. pneumoniae, infecting K. variicola isolates were more likely to be from blood (4/5 versus 24/134, p = 0.04), and less likely to be multidrug-resistant (0/5 versus 99/134, p<0.01). No K. variicola isolates demonstrated the hypermucoviscosity phenotype. Although K. variicola isolates were frequently isolated from bloodstream infections, they were less likely to be multidrug-resistant. Further work is needed to facilitate diagnosis of K. variicola and clarify its clinical significance in larger prospective studies.

Rapid detection of fosfomycin resistance in Escherichia coli and Klebsiella spp. strains isolated from urinary tract infections.

This study evaluates whether the rapid fosfomycin resistance (fosfomycin NP) method can be used for detecting fosfomycin resistance in routine laboratory work. Results from the disk diffusion and rapid fosfomycin NP methods were compared with the reference agar dilution method for Escherichia coli and Klebsiella spp. strains isolated from urinary tract infections. The study included 57 E. coli and 48 Klebsiella spp. isolates from urinary tract infections. The reference agar dilution and disk diffusion methods were performed in accordance with EUCAST recommendations, and the results were evaluated according to EUCAST V.10.0. The method developed by Nordmann et al. was used for rapid detection of fosfomycin resistance (Nordmann, P., Poirel, L., Mueller, L., 2019. Rapid Detection of Fosfomycin Resistance in Escherichia coli. J Clin Microbiol. 57(1), e01531-18. doi:https://doi.org/10.1128/JCM.01531-18). The acceptable categorical agreement (CA ≥ 90%) and the rates of major error (ME <3%) and very major error (VME < 3%) of the two methods were compared with the reference method according to the criteria of ISO 20776-1. Fosfomycin resistance was detected in 15.8% of E. coli and 75% of Klebsiella spp. isolates using the reference method. Disk diffusion method showed CA 89.5%, ME 12.5% in E. coli isolates, and CA 75%, ME 100% in Klebsiella spp. isolates. No VME was detected in both methods. The rapid fosfomycin NP method resulted in CA 96.4%, ME 0.0%, VME 22.2% in E. coli isolates, and CA 77.3%, ME 81.8%, and VME 3% in Klebsiella spp. isolates. We believe the results from both of disk diffusion assay and rapid fosfomycin NP for the E. coli and Klebsiella spp. isolates are incompatible with the reference method and should not be used as an alternative to the agar dilution method.

Klebsiella sp.-related infectious spondylitis in a bearded dragon (Pogona vitticeps).

BACKGROUND: Spondylitis is an inflammation of the vertebrae that leads to a destructive process with exuberant new bone formation. Osteomyelitis can produce a distortion of the bone architecture, degenerative joint changes and ankyloses of adjacent vertebrae. In reptiles, intervertebral discs are absent, so the term discospondylitis is not used. In lizards, vertebral lesions have not been well studied. The present paper describes the first case of Klebsiella sp.-related spondylitis in a pet lizard (Pogona vitticeps). CASE PRESENTATION: A 2-year-old, female bearded dragon (Pogona vitticeps) was presented for clinical examination due to a decreased activity level, decreased appetite and constipation. Blood tests showed no remarkable alterations. The haemogram showed normal parameters with relative lymphocytosis, although the absolute number of lymphocytes did not differ from the reference values. A computed tomography scan revealed a mixed osteolytic-proliferative bone lesion diffusing to the first and last tracts of the pre-sacral vertebrae together. A small amount of material obtained from the spinal swelling was sampled with an aseptic technique for bacterial culture, which was positive for Klebsiella sp. The antibiogram revealed sensitivity to enrofloxacin, marbofloxacin, and chloramphenicol and intermediate sensitivity to gentamicin. Complete return to spontaneous feeding was achieved 15 days after the beginning of antibiotic and anti-inflammatory therapy. CONCLUSIONS: In veterinary medicine, spondylitis represents a well-known disease in small companion animals. In mammals, the most common aetiologic agents are fungi and bacteria. Antibiotic therapy was set based on the antibiogram, and marbofloxacin was chosen at a dosage of 10 mg/kg subcutaneously (SC) once per day (SID). After only 7 days of antibiotic therapy, the clinical condition improved significantly; the patient started feeding and drinking spontaneously and gained weight. This case should remind clinicians of the importance of always performing antibiograms before choosing any antibiotic therapy. Considering reptiles, there have been few papers about spinal diseases, mostly regarding snakes and a few about Iguana iguana. Relative to other species of saurians, the literature remains lacking.

Klebsiella spp. cause severe and fatal disease in Mozambican children: antimicrobial resistance profile and molecular characterization.

BACKGROUND: Klebsiella spp. are important pathogens associated with bacteremia among admitted children and is among the leading cause of death in children < 5 years in postmortem studies, supporting a larger role than previously considered in childhood mortality. Herein, we compared the antimicrobial susceptibility, mechanisms of resistance, and the virulence profile of Klebsiella spp. from admitted and postmortem children. METHODS: Antimicrobial susceptibility and virulence factors of Klebsiella spp. recovered from blood samples collected upon admission to the hospital (n = 88) and postmortem blood (n = 23) from children < 5 years were assessed by disk diffusion and multiplex PCR. RESULTS: Klebsiella isolates from postmortem blood were likely to be ceftriaxone resistant (69.6%, 16/23 vs. 48.9%, 43/88, p = 0.045) or extended-spectrum ß-lactamase (ESBL) producers (60.9%, 14/23 vs. 25%, 22/88, p = 0.001) compared to those from admitted children. blaCTX-M-15 was the most frequent ESBL gene: 65.3%, 9/14 in postmortem isolates and 22.7% (5/22) from admitted children. We found higher frequency of genes associated with hypermucoviscosity phenotype and invasin in postmortem isolates than those from admitted children: rmpA (30.4%; 7/23 vs. 9.1%, 8/88, p = 0.011), wzi-K1 (34.7%; 8/23 vs. 8%; 7/88, p = 0.002) and traT (60.8%; 14/23 vs. 10.2%; 9/88, p < 0.0001), respectively. Additionally, serine protease auto-transporters of Enterobacteriaceae were detected from 1.8% (pic) to 12.6% (pet) among all isolates. Klebsiella case fatality rate was 30.7% (23/75). CONCLUSION: Multidrug resistant Klebsiella spp. harboring genes associated with hypermucoviscosity phenotype has emerged in Mozambique causing invasive fatal disease in children; highlighting the urgent need for prompt diagnosis, appropriate treatment and effective preventive measures for infection control.

Antimicrobial resistance of Escherichia coli and Klebsiella spp. conventionally sampled from factory-farmed chickens - clinical submissions.

INTRODUCTION: The article discusses the antimicrobial resistance of poultry-isolated bacteria in the Wielkopolska region of Poland. MATERIAL AND METHODS: From August 2014 - June 2016, antibiotic resistance screening tests were performed involving 4,496 samples of Escherichia coli and 84 samples of Klebsiella spp., and the following antibiotics: amoxicillin, amoxicillin with clavulanic acid, colistin, doxycycline, enrofloxacin, florfenicol, neomycin, norfloxacin, spectinomycin, and trimethoprim with sulfamethoxazole. The research used broth the microdilution method and CLSI standards. RESULTS: During the investigation period of 22 months a growing percentage of E. coli isolates showed antibiotic resistance to amoxicillin, amoxicillin with clavulanic acid, colistin, enrofloxacin, neomycin, norfloxacin, spectinomycin, and trimethoprim with sulfamethoxazole. Resistance to doxycycline and florfenicol decreased. The most efficient antibiotics against E. coli were colistin (84.64 %), neomycin (80.62 %), and amoxicillin with clavulanic acid (73.05 %). Klebsiella samples were the most susceptible to neomycin (85.71 %), colistin (84.52 %), and trimethoprim with sulfamethoxazole (73.81 %). CONCLUSIONS: Antibiotic resistance of pathogenic micro-organisms, such as Escherichia coli and Klebsiella spp., is a serious problem both for poultry producers and for public health protection. Low efficiency of numerous antibiotic groups forces reflection on limiting the use of medicines in food-producing animals.

Colonization with selected antibiotic resistant bacteria among a cohort of Sri Lankan university students.

BACKGROUND: Antibiotic Resistance is an imminent global public health threat. Antibiotic resistance emerged in healthcare settings and has now moved on to the community settings. This study was conducted to identify the rates of asymptomatic colonization with selected antibiotic resistant organisms, (Methicillin Resistant Staphylococcus aureus (MRSA), Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli and Klebsiella spp and carbapenem resistant E.coli and Klebsiella spp) - among a group of university students in Sri Lanka. Identification of genetic determinants of MRSA and ESBL was an additional objective of the study. METHODS: A self - collected nasal swab and a peri-rectal swab collected after passing stools were obtained. Routine microbiological methods were used for the isolation S.aureus from the nasal swab and E.coli and Klebsiella species from the peri-rectal swab. Antibiotic sensitivity testing was performed as recommended by clinical and laboratory standard institute (CLSI). Three (3) genes that are responsible for ESBL production; blaCTX-M, blaSHV, and blaTEM were tested using previously described primers and PCR procedures. Identification of MecA and PVL genes attributed to MRSA was also done with PCR. RESULTS: A total of 322 participants between 21 and 28 years were recruited representing 5 different faculties of study. Seventy one (22.0%) were colonized with S.aureus and 14 among them with MRSA, making the MRSA colonization rate of 4.3%. Forty five (15%) of the participants were colonized with an ESBL producing E.coli or Klebsiella spp. No one was colonized with carbapenem resistant E.coli or Klebsiella species. Of the 45 ESBL producers the commonest genetic determinant identified was blaCTX-M (n = 36), while 16 isolates had blaTEM and 7 had blaSHV. Similarly, of the 14 isolates identified as MRSA, 3 (21.4%) were found to be PVL positive while 11 (78.6%) were MecA positive. CONCLUSIONS: A high rate of colonization with ESBL producing E.coli and Klebsiella species was noted in our study group.

Klebsiella MALDI TypeR: a web-based tool for Klebsiella identification based on MALDI-TOF mass spectrometry.

Klebsiella pathogens affect human and animal health and are widely distributed in the environment. Among these, the Klebsiella pneumoniae species complex, which includes seven phylogroups, is an important cause of community and hospital infections. The Klebsiella oxytoca species complex also causes hospital infections and antibiotic-associated haemorrhagic colitis. The unsuitability of currently used clinical microbiology methods to distinguish species within each of these species complexes leads to high rates of misidentifications that are masking the true clinical significance and potential epidemiological specificities of individual species. We developed a web-based tool, Klebsiella MALDI TypeR, a platform-independent and user-friendly application that enables uploading MALDI-TOF mass spectrometry data in order to identify Klebsiella isolates at the species complex and phylogroup levels. The tool, available at https://maldityper.pasteur.fr/, leverages a database of previously identified biomarkers that are specific for species complexes, individual phylogroups, or related phylogroups. We obtained 84%-100% identification accuracy depending on phylogroup. Identification results are obtained in a few seconds from batches of uploaded spectral data. Klebsiella MALDI TypeR enables fast and reliable identification of Klebsiella strains that are often misidentified with standard microbiological methods. This web-based identification tool may be extended in the future to other human bacterial pathogens.

Isolation and identification of naphthalene degrading bacteria and their degradation characteristics under rainwater environment in heavily polluted areas.

This study is screened for naphthalene degrading strains from a heavily polluted area with high naphthalene concentration in the rainwater for the effective removal of naphthalene from rainwater. Recently, naphthalene biodegradation has been achieved in water. However, the influences of organics and inorganics in the rainwater on the biodegradation of naphthalene remains unclear. The naphthalene degrading strain Klebsiella sp. (WJ-1) was identified from sewage sludge. The effects of temperature, pH, inoculum size, and rotation speed on the degradation ability of WJ-1 were studied. The results showed that the naphthalene degradation rates of WJ-1 in rainwater were higher than those in aqueous solution at different experimental conditions. The optimal conditions were 30 °C, 10% inoculum size, pH 7.0, and a rotation speed of 150 rpm. The substances in rainwater might be important co-metabolites of naphthalene degradation. Based on intermediate metabolites detected by gas chromatography-mass spectrometer (GC-MS), the naphthalene biodegradation pathway was identified, as being similar to the phthalic acid pathway. These results suggest WJ-1 as a good candidate for the efficient bioremediation of naphthalene from rainwater in heavily polluted areas.

Revisiting the epidemiology of bloodstream infections and healthcare-associated episodes: results from a multicentre prospective cohort in Spain (PRO-BAC Study).

The epidemiology of bloodstream infections (BSIs) is dynamic as it depends on microbiological, host and healthcare system factors. The aim of this study was to update the information regarding the epidemiology of BSIs in Spain considering the type of acquisition. An observational, prospective cohort study in 26 Spanish hospitals from October 2016 through March 2017 including all episodes of BSI in adults was performed. Bivariate analyses stratified by type of acquisition were performed. Multivariate analyses were performed by logistic regression. Overall, 6345 BSI episodes were included; 2510 (39.8%) were community-acquired (CA), 1661 (26.3%) were healthcare-associated (HCA) and 2056 (32.6%) hospital-acquired (HA). The 30-day mortality rates were 11.6%, 19.5% and 22.0%, respectively. The median age of patients was 71 years (interquartile range 60-81 years) and 3656 (58.3%; 95% confidence interval 57.1-59.6%) occurred in males. The proportions according to patient sex varied according to age strata. Escherichia coli (43.8%), Klebsiella spp. (8.9%), Staphylococcus aureus (8.9%) and coagulase-negative staphylococci (7.4%) were the most frequent pathogens. Multivariate analyses confirmed important differences between CA and HCA episodes, but also between HCA and HA episodes, in demographics, underlying conditions and aetiology. In conclusion, we have updated the epidemiological information regarding patients' profiles, underlying conditions, frequency of acquisition types and aetiological agents of BSI in Spain. HCA is confirmed as a distinct type of acquisition.

Genomic characterization and computational phenotyping of nitrogen-fixing bacteria isolated from Colombian sugarcane fields.

Previous studies have shown the sugarcane microbiome harbors diverse plant growth promoting microorganisms, including nitrogen-fixing bacteria (diazotrophs), which can serve as biofertilizers. The genomes of 22 diazotrophs from Colombian sugarcane fields were sequenced to investigate potential biofertilizers. A genome-enabled computational phenotyping approach was developed to prioritize sugarcane associated diazotrophs according to their potential as biofertilizers. This method selects isolates that have potential for nitrogen fixation and other plant growth promoting (PGP) phenotypes while showing low risk for virulence and antibiotic resistance. Intact nitrogenase (nif) genes and operons were found in 18 of the isolates. Isolates also encode phosphate solubilization and siderophore production operons, and other PGP genes. The majority of sugarcane isolates showed uniformly low predicted virulence and antibiotic resistance compared to clinical isolates. Six strains with the highest overall genotype scores were experimentally evaluated for nitrogen fixation, phosphate solubilization, and the production of siderophores, gibberellic acid, and indole acetic acid. Results from the biochemical assays were consistent and validated computational phenotype predictions. A genotypic and phenotypic threshold was observed that separated strains by their potential for PGP versus predicted pathogenicity. Our results indicate that computational phenotyping is a promising tool for the assessment of bacteria detected in agricultural ecosystems.

Screening a Strain of Klebsiella sp. O852 and the Optimization of Fermentation Conditions for Trans-Dihydrocarvone Production.

Flavors and fragrances have high commercial value in the food, cosmetic, chemical and pharmaceutical industries. It is interesting to investigate the isolation and characterization of new microorganisms with the ability to produce flavor compounds. In this study, a new strain of Klebsiella sp. O852 (accession number CCTCC M2020509) was isolated from decayed navel orange (Citrus sinensis (L.) Osbeck), which was proved to be capable of converting limonene to trans-dihydrocarvone. Besides, the optimization of various reaction parameters to enhance the trans-dihydrocarvone production in shake flask was performed for Klebsiella sp. O852. The results showed that the yield of trans-dihydrocarvone reached up to 1 058 mg/L when Klebsiella sp. O852 was incubated using LB-M medium for 4 h at 36 °C and 150 rpm, and the biotransformation process was monitored for 36 h after adding 1680 mg/L limonene/ethanol (final ethanol concentration of 0.8% (v/v)). The content of trans-dihydrocarvone increased 16 times after optimization. This study provided a basis and reference for producing trans-dihydrocarvone by biotransformation.