Colistin heteroresistance in Citrobacter freundii clinical isolates from Republic of Korea.

We investigated colistin heteroresistance in Citrobacter freundii isolates from Korean hospitals. Using population analysis profiling (PAP), we detected colistin heteroresistance in 31.3% of isolates. Among these, ST217 was the most prevalent clone (58.5%), particularly within colistin-heteroresistant isolates (80.0%). Interestingly, the second most common clone, ST248, was not found in heteroresistant isolates. We identified amino acid changes in PhoQ, PmrA, and PmrB, along with mRNA overexpression in pmrB and arnD. Colistin monotherapy showed no efficacy, but a combination of colistin and ciprofloxacin successfully eradicated all five isolates, even at 0.5 × minimum inhibitory concentrations. This study underscores the high prevalence of colistin heteroresistance in C. freundii isolates, limiting the effectiveness of colistin monotherapy. Combining colistin with ciprofloxacin may offer a viable treatment option for C. freundii infections.

FDH/Hases-S-chain mediated electron redistributing in Citrobacter freundii JH@FeS during degradation of sulfamethoxazole and nitrate.

Considering the negligent degradation of sulfamethoxazole (SMX) by Citrobacter freundii JH, the incorporation of bio-FeS could initiate the SMX biodegradation to 0.0444 (S-FeS), and further to 0.0564 mg L-1 mg-1 protein d-1 (SN-FeS) when coexisted with nitrate. Electrochemical (LSV, I-t, DPV, EIS and EDC) and respiratory inhibition experiments clarified that the bio-FeS could greatly switch/redistribute electron transmembrane-transfer from intracellular to extracellular mainly via FDH/Hases-S-chain, as revealed by the significant increase of ipa-FDH/Hases/ipa-FC-Cyts and ipc-FDH/Hases/ipc-FC-Cyts (from 1.09 and 1.07 (SN-native) to 1.50 and 3.58 (SN-FeS)), while nitrate (linear fitting with NADH (R2 = 0.9903)) mainly intensified CoQ-L-chain related INET from Complex I to CoQ to compensate for the electronic competition with SMX. SN-FeS system detoxified the SMX on microbial metabolism (such as membrane rupture and oxidative stress induction) with high SOD activity (737.93 U gFW-1). Structural equation modeling indicated that bio-FeS up-regulated PMF-mediated ATP synthesis (PPMF-ATPs from 0.12 (SN-native) to 0.74 (SN-FeS)) and PMF-mediated NADH (PPMF-NADH from -0.72 (SN-native) to 0.63 (SN-FeS)), and the nitrate addition intensified this positive feedback. Overall, this study provides a new perspective for bionanoparticles via electron transfer/redistribution to detoxify and launch the antibiotics biodegradation in ecological environment.

In-vitro assessment of a novel plant rhizobacterium, Citrobacter freundii, for degrading and biocontrol of food mycotoxin deoxynivalenol.

Deoxynivalenol (DON) is one of the most harmful and well-known toxins present in food and animal feed throughout the world. Citrobacter freundii (C. freundii-ON077584), a novel DON-degrading strain, was isolated from rice root-linked soil samples. The degrading properties, including DON concentrations, incubation pH, incubation temperatures, bacterial concentrations, and acid treatment effect on degradation, were evaluated. At pH 7 and an incubation temperature of 37 °C, C. freundii demonstrated the capability to degrade more than 90% of DON. The degraded products of DON were identified as 3-keto-DON and DOM-1, which were confirmed by High Performance Liquid Chromatography (HPLC) and Ultra-Performance Liquid Chromatography hyphenated with Tandem Mass Spectrometry (UPLC-MS/MS) analyses. The mechanism of DON degradation into 3-keto-DON and DOM-1 by this bacterial strain will be further explored to identify and purify novel degrading enzymes that can be cloned to the microorganism and added to the animal feed to degrade the DON in the digestion tract.

Characterization and whole-genome sequencing of an extreme arsenic-tolerant Citrobacter freundii SRS1 strain isolated from Savar area in Bangladesh.

Citrobacter freundii SRS1, gram-negative bacteria, were isolated from Savar, Bangladesh. The strain could tolerate up to 80 mmol L-1 sodium arsenite, 400 mmol L-1 sodium arsenate, 5 mmol L-1 manganese sulfate, 3 mmol L-1 lead nitrate, 2.5 mmol L-1 cobalt chloride, 2.5 mmol L-1 cadmium acetate, and 2.5 mmol L-1 chromium chloride. The whole-genome sequencing revealed that the genome size of C. freundii SRS1 is estimated to be 5.4 Mb long, and the G + C content is 51.7%. The genome of C. freundii SRS1 contains arsA, arsB, arsC, arsD, arsH, arsR, and acr3 genes for arsenic resistance; czcA, czcD, cbiN, and cbiM genes for cobalt resistance; chrA and chrB genes for chromium resistance; mntH, sitA, sitB, sitC, and sitD genes for manganese resistance; and zntA gene for lead and cadmium resistance. This novel acr3 gene has never previously been reported in any C. freundii strain except SRS1. A set of 130 completely sequenced strains of C. freundii was selected for phylogenomic analysis. The phylogenetic tree showed that the SRS1 strain is closely related to the C. freundii 62 strain. Further analyses of the genes involved in metal and metalloid resistance might facilitate identifying the mechanisms and pathways involved in high metal resistance in the C. freundii SRS1 strain.

M379A Mutant Tyrosine Phenol-lyase from Citrobacter freundii Has Altered Conformational Dynamics.

The M379A mutant of Citrobacter freundii tyrosine phenol-lyase (TPL) has been prepared. M379A TPL is a robust catalyst to prepare a number of tyrosines substituted at the 3-position with bulky groups that cannot be made with wild type TPL. The three dimensional structures of M379A TPL complexed with L-methionine and 3-bromo-DL-phenylalanine have been determined by X-ray crystallography. Methionine is bound as a quinonoid complex in a closed active site in 3 of 4 chains of homotetrameric M379A TPL. M379A TPL reacts with L-methionine about 8-fold slower than wild type TPL. The temperature dependence shows that the slower reaction is due to less positive activation entropy. The structure of the M379A TPL complex of 3-bromo-DL-phenylalanine has a quinonoid complex in two subunits, with an open active site conformation. The effects of the M379A mutation on TPL suggest that the mutant enzyme has altered the conformational dynamics of the active site.

Characteristics and growth kinetics of biomass of Citrobacter freundii strains PYI-2 and Citrobacter portucalensis strain YPI-2 during the biodegradation of Ibuprofen.

Ibuprofen (IBU) is the third most commonly used analgesic drug in the world. It enters the water system as a result of human excretion-based wastewater discharges. Hence, it attracts the attention of environmentalists for its ecological fate and degradation behavior. In this study, the two IBU degrading bacterial strains, Citrobacter freundii strain PYI-2 (MT039504) and Citrobacter portucalensis strain YPI-2 (MN744335), were isolated from industrial wastewater samples using an enrichment culture method, identified, and characterized. Physiological and batch culture degradation studies have indicated that these strains involved in IBU degradation and the intermediates produced during the process were analyzed. These strains degrade IBU in the batch culture. The optimum pH was reported for degradation of the PYI2 strain (6.9) and YPI2 strain (5.8), and the optimum temperatures were 42°C and 32°C, respectively. Biomass kinetic analysis of these strains was performed based on physical parameters (temperature, pH, and rpm) and confirmed by the experimental study. As indicated in the GC-MS chromatogram peaks, viz., hydroxyibuprofen, 2-(4-hydroxyphenylpropionic acid), 1,4-hydroquinone, and 2-hydroxy-1,4-quinol various intermediates compounds of degradation pathway were observed. Finally, through the GC-MS data, the metabolic pathway for degradation was predicted. In the study, it was confirmed that Citrobacter freundii strain PYI-2 and Citrobacter portucalensis strain YPI-2 exhibit metabolic potential for the biodegradation of IBU and can be further deployed in bioremediation.

Isolation of Dibutyl Phthalate-Degrading Bacteria and Its Coculture with Citrobacter freundii CD-9 to Degrade Fenvalerate.

Continued fenvalerate use has caused serious environmental pollution and requires large-scale remediation. Dibutyl phthalate (DBP) was discovered in fenvalerate metabolites degraded by Citrobacter freundii CD-9. Coculturing is an effective method for bioremediation, but few studies have analyzed the degradation pathways and potential mechanisms of cocultures. Here, a DBP-degrading strain (BDBP 071) was isolated from soil contaminated with pyrethroid pesticides (PPs) and identified as Stenotrophomonas acidaminiphila. The optimum conditions for DBP degradation were determined by response surface methodology (RSM) analysis to be 30.9 mg/l DBP concentration, pH 7.5, at a culture temperature of 37.2°C. Under the optimized conditions, approximately 88% of DBP was degraded within 48 h and five metabolites were detected. Coculturing C. freundii CD-9 and S. acidaminiphila BDBP 071 promoted fenvalerate degradation. When CD-9 was cultured for 16 h before adding BDBP 071, the strain inoculation ratio was 5:5 (v/v), fenvalerate concentration was 75.0 mg/l, fenvalerate was degraded to 84.37 ± 1.25%, and DBP level was reduced by 5.21 mg/l. In addition, 12 fenvalerate metabolites were identified and a pathway for fenvalerate degradation by the cocultured strains was proposed. These results provide theoretical data for further exploration of the mechanisms used by this coculture system to degrade fenvalerate and DBP, and also offer a promising method for effective bioremediation of PPs and their related metabolites in polluted environments.

Enhanced Light-Driven Hydrogen Production by Self-Photosensitized Biohybrid Systems.

Storage of solar energy as hydrogen provides a platform towards decarbonizing our economy. One emerging strategy for the production of solar fuels is to use photocatalytic biohybrid systems that combine the high catalytic activity of non-photosynthetic microorganisms with the high light-harvesting efficiency of metal semiconductor nanoparticles. However, few such systems have been tested for H2 production. We investigated light-driven H2 production by three novel organisms, Desulfovibrio desulfuricans, Citrobacter freundii, and Shewanella oneidensis, self-photosensitized with cadmium sulfide nanoparticles, and compared their performance to Escherichia coli. All biohybrid systems produced H2 from light, with D. desulfuricans-CdS demonstrating the best activity overall and outperforming the other microbial systems even in the absence of a mediator. With this system, H2 was continuously produced for more than 10 days with a specific rate of 36 µmol gdcw-1 h-1 . High apparent quantum yields of 23 % and 4 % were obtained, with and without methyl viologen, respectively, exceeding values previously reported.

Biosynthesis of Silver Nanoparticles from Citrobacter freundii as Antibiofilm Agents with their Cytotoxic Effects on Human Cells.

BACKGROUND: Nanomaterials have recently been identified for their potential benefits in the areas of medicine and pharmaceuticals. Among these nanomaterials, silver nanoparticles (Ag-NPs) have been widely utilized in the fields of diagnostics, antimicrobials, and catalysis. OBJECTIVE: To investigate the potential utility of Citrobacter freundii in the synthesis of silver Nanoparticles (Ag-NPs), and to determine the antimicrobial activities of the Ag-NPs produced. METHODS: Aqueous Ag+ ions were reduced when exposed to C. freundii extract and sunlight, leading to the formation of Ag-NPs. Qualitative microanalysis for the synthesized Ag-NPs was done using UVvis spectrometry, Energy Dispersive X-ray analysis (EDX), and scanning and transmission electron microscopy. The hydrodynamic size and stability of the particles were detected using Dynamic Light Scattering (DLS) analysis. The Ag-NPs' anti-planktonic and anti-biofilm activities against Staphylococcus aureus and Pseudomonas aeruginosa, which are two important skin and wound pathogens, were investigated. The cytotoxicity on human dermal fibroblast cell line was also determined. RESULTS: Ag-NPs were spherical with a size range between 15 to 30 nm. Furthermore, Ag-NPs displayed potent bactericidal activities against both S. aureus and P. aeruginosa and showed noticeable anti-biofilm activity against S. aureus biofilms. Ag-NPs induced minor cytotoxic effects on human cells as indicated by a reduction in cell viability, a disruption of plasma membrane integrity, and apoptosis induction. CONCLUSION: Ag-NPs generated in this study might be a future potential alternative to be used as antimicrobial agents in pharmaceutical applications for wound and skin related infections.

Electron transfer involved in bio-Pd (0) synthesis by Citrobacter freundii at different growth phases.

Gram-negative Citrobacter freundii with high Pd (II) reduction capacity was isolated from electroplating wastewater, and the electron transfer involved in Pd (II) bio-reduction by C. freundii JH was investigated in phosphate buffer saline solution with sodium formate as sole electron donor under anaerobic condition. FTIR spectra indicated that hydroxyl and amine groups on cell wall participated Pd (II) bio-sorption. TEM, XRD, XPS results confirmed that Pd (0) nanoparticles (NPs) could be bio-synthesized intra/extracellularly. Meanwhile, pH turn-over were observed owing to the reduction of cytochrome c (c-Cyt) in bio-reduction process. EPR spectra indicated that free radicals (OH) was generated from high concentration Pd (II), which would cause seriously damage to cell. Despite of the lower tolerance to Pd (II), the cells at logarithmic phase exhibited higher Pd (II) reduction capacity (72.21%) than that at stationary phase (56.21%), which might be related to the relatively stronger proton motive force (PMF) created by the substrate oxidation and the electron transfer, as evidenced by electrochemical experiments (CV, DPV, amperometric I-t curves) and protein denaturalization experiments. Additionally, c-Cyt and riboflavin were confirmed to be important participants in electron transfer. Finally, a putative synthesis mechanism of Pd (0)-NPs was deduced. This study contributed to further understanding the electron transfer in Pd (II) reduction, and provided more information for the bio-synthetic of metal nanoparticles.

Prevalence and characterization of ß-lactamase-producing Enterobacteriaceae in healthy human carriers.

Presence of extended-spectrum ß-lactamase (ESBL-E), AmpC-producing and carbapenemase-producing (CPE) Enterobacteriaceae has been observed not only in the clinical environment, but also in the out-of-hospital environment. The objective of this study was to isolate and characterize strains of ESBL, AmpC, and CPE present in feces of healthy carriers in Navarra (n = 125). Despite the fact that no CPE strains were isolated, 16% and 11.2% of the studied population were ESBL-E and AmpC carriers, respectively. No significant differences were found by gender or age; young people (5-18 years old) showed the highest ESBL-E prevalence (31.8%). The isolates corresponded to E. coli (57.1%), Enterobacter spp. (28.6%), and Citrobacter freundii (14.3%), and all strains showed multidrug-resistant profiles. High resistance against cephalosporins, penicillins, and monobactams, and sensitivity to carbapenems, quinolones, and aminoglycosides were observed. With respect to ESBL producers, 52.4% were CTX-M-type (19.0% CTX-M-14, 9.5% CTX-M-1, and 28.6% CTX-M-15) and 47.6% were TEM-type (38.1% TEM-171). These results confirm the extensive dissemination of these resistances among a healthy population and pose the need to implement control measures and strategies according to the One Health approach in order to prevent the increase of severe and untreatable infections in a not far future.

Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium.

Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

LMB-1 producing Citrobacter freundii from Argentina, a novel player in the field of MBLs.

Carbapenemase-producing Enterobacterales expressing OXA-48, KPC, NDM, VIM or IMP enzymes are increasingly reported worldwide. We have characterized LMB-1, a novel metallo-ß-lactamase (MBL) of Ambler class B3 from Citrobacter freundii 164 (Cf164) clinical isolate from Buenos Aires, Argentina. Cf164 displayed reduced susceptibility to carbapenems but gave inconsistent results with carbapenemase confirmatory tests, indicating the presence of a weak carbapenemase. Analysis of whole-genome sequencing (WGS) of Cf164 using Resfinder revealed four ß-lactamase genes coding for CTX-M-8, PER-2, TEM-1 and CMY-150, a novel chromosomally-encoded CMY variant. Kinetic parameters of purified CMY-150 did not reveal any carbapenemase activity. However, CMY-150 conferred higher minimum inhibitory concentrations (MICs) to E. coli for ceftazidime and aztreonam compared with CMY-2. The in-house-developed ß-lactamase search software (ResMiner) in WGS data revealed a novel subclass B3 MBL named LMB-1. LMB-1 conferred resistance to penicillins and expanded-spectrum cephalosporins and reduced susceptibility to carbapenems in E. coli. The blaLMB-1 gene was located on a 176-kb IncA/C2 plasmid. LMB-1 shared 99% amino acid sequence identity with the MBL encoded in the chromosome of Rheinheimera pacifica, it's likely progenitor. Despite repeated attempts, LMB-1 could not be purified, thus only specific activities could indicate hydrolysis of carbapenems. Here we report on CMY-150, a novel CMY-2 variant that confers increased ceftazidime and aztreonam MICs to E. coli and the first description of LMB-1 in Argentina. This work underlines the need for several carbapenemase-producing Enterobacteriaceae (CPE) confirmatory tests, as this novel enzyme might have been missed using only one.

Cytokine profiles of umbilical cord blood mononuclear cells upon in vitro stimulation with lipopolysaccharides of different vaginal gram-negative bacteria.

Inflammatory immune responses induced by lipopolysaccharides (LPS) of gram-negative bacteria play an important role in the pathogenesis of preterm labor and delivery, and in neonatal disorders. To better characterize LPS-induced inflammatory response, we determined the cytokine profile of umbilical cord blood mononuclear cells (UBMC) stimulated with LPS of seven vaginal gram-negative bacteria commonly found in pregnant women with preterm labor and preterm rupture of membrane. UBMC from ten newborns of healthy volunteer mothers were stimulated with purified LPS of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter calcoaceticus, Citrobacter freundii, and Pseudomonas aeruginosa. UBMC supernatants were tested for the presence of secreted pro-inflammatory cytokines (IL-6, IL-1ß, TNF), anti-inflammatory cytokine (IL-10), TH1-type cytokines (IL-12, IFN-γ), and chemokines (IL-8, MIP-1α, MIP-1ß, MCP-1) by Luminex technology. The ten cytokines were differentially induced by the LPS variants. LPS of E. coli and E. aerogenes showed the strongest stimulatory activity and P. aeruginosa the lowest. Interestingly, the ability of UBMC to respond to LPS varied greatly among donors, suggesting a strong individual heterogeneity in LPS-triggered inflammatory response.

Don't overlook the little guy: An evaluation of the frequency of small plasmids co-conjugating with larger carbapenemase gene containing plasmids.

As the spread of antimicrobial resistance (AMR) genes becomes an increasing global threat, improved understanding of mobile genetic elements which contribute to the spread of antimicrobial resistance genes, becomes more critical. We created transconjugants from the mating of three chromosomally isogenic Klebsiella pneumoniae carbapenemase (blaKPC) positive Citrobacter freundii isolates with a laboratory strain of Escherichia coli and evaluated the movement of small cryptic plasmids (SCPs), p3223 and p1916, when larger blaKPC-plasmids were transferred. In all of the 143 transconjugants, multiple plasmids, both large and small, transferred with each mating. When two blaKPC-plasmids were present in the host, frequently (87%; 98/113) both would be transferred during mating. p3223 is found in a wide range of bacterial hosts that harbor AMR genes; p1916 has been identified in only a limited number of publicly available sequences to date. From our evaluation, there is still much to learn about SCPs, and the high rate of co-transfer of multiple plasmids from real-world carbapenemase-producing Enterobacteriales.

Citrobacter freundii fitness during bloodstream infection.

Sepsis resulting from microbial colonization of the bloodstream is a serious health concern associated with high mortality rates. The objective of this study was to define the physiologic requirements of Citrobacter freundii in the bloodstream as a model for bacteremia caused by opportunistic Gram-negative pathogens. A genetic screen in a murine host identified 177 genes that contributed significantly to fitness, the majority of which were broadly classified as having metabolic or cellular maintenance functions. Among the pathways examined, the Tat protein secretion system conferred the single largest fitness contribution during competition infections and a putative Tat-secreted protein, SufI, was also identified as a fitness factor. Additional work was focused on identifying relevant metabolic pathways for bacteria in the bloodstream environment. Mutations that eliminated the use of glucose or mannitol as carbon sources in vitro resulted in loss of fitness in the murine model and similar results were obtained upon disruption of the cysteine biosynthetic pathway. Finally, the conservation of identified fitness factors was compared within a cohort of Citrobacter bloodstream isolates and between Citrobacter and Serratia marcescens, the results of which suggest the presence of conserved strategies for bacterial survival and replication in the bloodstream environment.

Multidrug-resistant Citrobacter freundii ST139 co-producing NDM-1 and CMY-152 from China.

The emergence of carbapenemase-producing Citrobacter freundii poses a significant threat to public health worldwide. Here, we reported a C. freundii strain CWH001 which was resistant to all tested antimicrobials except tetracycline. Whole genome sequencing and analysis were performed. The strain, which belonged to a new sequence type ST139, showed close relationship with other foreign C. freundii strains through phylogenetic analysis. A novel variant of the intrinsic blaCMY gene located on the chromosome was identified and designated as blaCMY-152. Coexistence of blaNDM-1 with qnrS1 was found on a conjugative IncN plasmid, which had a backbone appearing in various plasmids. Other class A ESBL genes (blaVEB-3 and blaTEM-1) were also detected on two different novel plasmids. The emergence of multidrug-resistant C. freundii is of major concern, causing great challenges to the treatment of clinical infections. Great efforts need to be taken for the specific surveillance of this opportunistic pathogen.

Epidemiology of Carbapenem-Resistant Enterobacteriaceae Infections: Report from the China CRE Network.

Carbapenem-resistant Enterobacteriaceae (CRE) infection is highly endemic in China, but estimates of the infection burden are lacking. We established the incidence of CRE infection from a multicenter study that covered 25 tertiary hospitals in 14 provinces. CRE cases defined as carbapenem-nonsusceptible Citrobacter freundii, Escherichia coli, Enterobacter cloacae, or Klebsiella pneumoniae infections during January to December 2015 were collected and reviewed from medical records. Antimicrobial susceptibility testing and carbapenemase gene identification were performed. Among 664 CRE cases, most were caused by K. pneumoniae (73.9%), followed by E. coli (16.6%) and E. cloacae (7.1%). The overall CRE infection incidence per 10,000 discharges was 4.0 and differed significantly by region, with the highest in Jiangsu (14.97) and the lowest in Qinghai (0.34). Underlying comorbidities were found in 83.8% of patients; the median patient age was 62 years (range, 45 to 74 years), and 450 (67.8%) patients were male. Lower respiratory tract infections (65.4%) were the most common, followed by urinary tract infection (16.6%), intra-abdominal infection (7.7%), and bacteremia (7.7%). The overall hospital mortality rate was 33.5%. All isolates showed nonsusceptibility to carbapenems and cephalosporins. The susceptibility rate of polymyxin B was >90%. Tigecycline demonstrated a higher susceptibility rate against E. coli than against K. pneumoniae (90.9% versus 40.2%). Of 155 clinical isolates analyzed, 89% produced carbapenemases, with a majority of isolates producing KPC (50%) or NDM (33.5%)-type beta-lactamases among K. pneumoniae and E. coli The incidence of CRE infection in China was 4.0 per 10,000 discharges. The patient-based disease burden in tertiary hospitals in China is severe, suggesting an urgent need to enhance infection control.

Transmission modes of a pesticide-degrading symbiont of the oriental fruit fly Bactrocera dorsalis (Hendel).

Symbionts are associated with many insects and play several multifunctional roles in insect-microorganism mutualistic relationships. The trichlorphon-degrading symbiont Citrobacter freundii (CF-BD) of the oriental fruit fly Bactrocera dorsalis was recently discovered; however, its intraspecies transmission pathway among flies remains unknown. Here, we use fluorescence in situ hybridization (FISH), PCR detection, and a series of ingenious experiments to reveal that CF-BD was aggregated in rectal pads associated with the female ovipositor, and the CF-BD symbiont was vertically transmitted via egg surface contamination. Although CF-BD was not detected in ovaries, it was found in deposited eggs. In addition, CF-BD was readily acquired horizontally between larvae or adults via oral uptake, although it was not transferred via mating behavior. Surface sterilization of eggs had a negative effect on the insects, which exhibited a lower body weight and a sharp decrease in fecundity, suggesting important biological roles of CF-BD in the fitness of the host insects. Our findings may also help to explain the high pesticide resistance levels of B. dorsalis. Furthermore, identifying a clear transmission pathway of this organophosphorus-degrading symbiont will be useful for pesticide resistance management and future pest control technologies.

Molecular and microbiological report of a hospital outbreak of NDM-1-carrying Enterobacteriaceae in Mexico.

OBJECTIVES: To characterize the microbiological, molecular and epidemiological data of an outbreak of carbapenem-resistant Enterobacteriaceae (CRE) in a tertiary-care hospital in Mexico. METHODS: From September 2014 to July 2015, all CRE clinical isolates recovered during an outbreak in the Hospital Civil "Fray Antonio Alcalde" in Jalisco, Mexico were screened for antimicrobial susceptibility, carbapenemase production, carbapenemase-encoding genes, and plasmid profiles. Horizontal transfer of imipenem resistance; and clonal diversity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST); as well as biofilm production and the presence of 14 virulence genes were analyzed in selected isolates. RESULTS: Fifty-two carbapenem-resistant isolates corresponding to 5 species were detected, i.e., Klebsiella pneumoniae (n = 46), Enterobacter cloacae (n = 3), Escherichia coli (n = 1), Providencia rettgeri (n = 1) and Citrobacter freundii (n = 1) with carbapenemase encoding genes blaNDM-1 (n = 48), blaVIM (n = 3), blaIMP (n = 1) and blaKPC (n = 1) detected in these isolates. The blaNDM-1 gene was detected in plasmids from 130- to 170-kb in K. pneumoniae (n = 46); E. cloacae (n = 3), E. coli (n = 1) and P. rettgeri (n = 1). The transfer of plasmids harboring the blaNDM-1 gene was obtained in eight transconjugants. One plasmid restriction pattern was detected, with the blaNDM-1 identified in different restriction fragments. Predominant clone A of K. pneumoniae isolates archived 28/46 (60%) isolates and belongs to ST392. Besides, ST307, ST309, ST846, ST2399, and ST2400 were detected for K. pneumoniae; as well as E. cloacae ST182 and E. coli ST10. The fimA and uge genes were more likely to be identified in K. pneumoniae carbapenem-susceptible isolates (p = <0.001) and biofilm production was more liable to be observed in carbapenem-resistant isolates (p = <0.05). CONCLUSIONS: Four Enterobacteriaceae species harboring the blaNDM-1 gene were detected in a nosocomial outbreak in Mexico; horizontal transfer and strain transmission were demonstrated for the blaNDM-1 gene. Given the variation in the size of the plasmid harboring blaNDM-1, complex rearrangements must also be occurring.