Trends and molecular characteristics of carbapenemase-producing Enterobacteriaceae in Japanese hospital from 2006 to 2015.

BACKGROUND: The increasing number of carbapenemase-producing Enterobacteriaceae (CPE) has become a global problem. Most carbapenemases detected in Japan are imipenemase, which is an imipenem-degrading enzyme with low ability; thus, CPE could have been overlooked. Therefore, this study aimed to detect and analyze CPE, without overlooking CPE showing the low minimum inhibitory concentration phenotype. METHODS: CPE screening was conducted on 531 ceftazidime-resistant Enterobacteriaceae isolated from Kitasato University Hospital during 2006-2015. We confirmed the presence of the carbapenemase genes (blaIMP, blaVIM, blaKPC, blaNDM, and blaOXA-48) by multiplex polymerase chain reaction. The detected CPE strains were analyzed by antimicrobial susceptibility testing, multilocus sequence typing, conjugal experiments, replicon typing, and plasmid profiling by restriction enzyme treatment. RESULTS: The CPE detection rate in Kitasato University Hospital within the past 10 years was 0.0003% (nine CPE strains). These nine CPE strains were identified to harbor 8 blaIMP-1 or 1 blaNDM-5. The CPE strains consisted of five species including Klebsiella pneumoniae and Citrobacter freundii. Six of eight blaIMP-1 were coded by IncHI2 plasmid, and the other two were coded by IncA/C plasmid. Plasmid profiling revealed that K. pneumoniae and C. freundii isolated from the same patient harbored the same plasmid. CONCLUSION: The CPE detection rate in this study was significantly lower than those previously reported in Japan. In one case, IncA/C plasmid transmission through different bacterial species within the body was speculated. Although the number of CPE detected was low, these results indicated that the resistance plasmid could spread to other bacterial species.

First Identification of ß-Lactamases in Antibiotic-Resistant Escherichia coli, Citrobacter freundii, and Aeromonas spp. Isolated in Stream Macroinvertebrates in a Central Italian Region.

Antibiotic-resistant bacteria (ARB) are widespread in nature and represent a serious public and environmental problem. In the present study, we report for the first time the presence of bacterial ß-lactamases in two macroinvertebrate species with different feeding traits. The class A ß-lactamases, SHV-1 and TEM-1, were found in Citrobacter freundii isolated from Gammarus elvirae and Escherichia coli from water samples, respectively. The metallo-ß-lactamase CphA was found in Aeromonas veronii and Aeromonas hydrophila strains isolated from the predator Dina lineata. The presence of a large plasmid was ascertained only in E. coli strains isolated from water. In all strains studied, an integrase I typical of class I integrin was found. In contaminated freshwater habitats, ARB and antibiotic resistance genes could be disseminated through trophic links with important ecological implications. Transmission through the food chain may contribute to spreading and transferring antibiotic resistance not only in freshwater ecosystems but also outside the aquatic habitat.

First data on microflora of loggerhead sea turtle (Caretta caretta) nests from the coastlines of Sicily.

Caretta caretta is threatened by many dangers in the Mediterranean basin, but most are human-related. The purposes of this research were: (i) to investigate microflora in samples from six loggerhead sea turtle nests located on the Sicilian coast and (ii) to understand microbial diversity associated with nests, with particular attention to bacteria and fungi involved in failed hatchings. During the 2016 and 2018 summers, 456 eggs and seven dead hatchling from six nests were collected. We performed bacteriological and mycological analyses on 88 egg samples and seven dead hatchlings, allowing us to isolate: Fusarium spp. (80.6%), Aeromonas hydrophila (55.6%), Aspergillus spp. (27.2%) and Citrobacter freundii (9%). Two Fusarium species were identified by microscopy and were confirmed by PCR and internal transcribed spacer sequencing. Statistical analyses showed significant differences between nests and the presence/absence of microflora, whereas no significant differences were observed between eggs and nests. This is the first report that catalogues microflora from C . caretta nests/eggs in the Mediterranean Sea and provides key information on potential pathogens that may affect hatching success. Moreover, our results suggest the need for wider investigations over extensive areas to identify other microflora, and to better understand hatching failures and mortality related to microbial contamination in this important turtle species.

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.

Optimizing locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH) procedure for bacterial detection.

Despite the successful application of LNA/2'OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2'OMe hybridization step-hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2'OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2'OMe and 64% of GC content, should be use in initial optimization of new LNA/2'OMe-FISH protocols.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae from animal origin and wastewater in Tunisia: first detection of O25b-B23-CTX-M-27-ST131 Escherichia coli and CTX-M-15/OXA-204-producing Citrobacter freundii from wastewater.

OBJECTIVES: This study aimed to isolate and characterise extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) isolates from animals and wastewater in Tunisia. METHODS: ESBL-E from wastewater (n=123 samples), faeces of healthy animals (poultry, sheep, goats and calves) (n=140) and raw milk from healthy cows (n=42) and goats (n=20) were investigated. Antimicrobial susceptibility was determined according to CLSI recommendations. The blaTEM, blaSHV, blaCTX-M and blaOXA-48 genes were analysed by PCR and sequencing. Phylogenetic groups were determined by PCR for Escherichia coli isolates. The clonality of E. coli and Klebsiella pneumoniae isolates was determined by XbaI-PFGE and MLST. RESULTS: A total of 81 E. coli, 20 K. pneumoniae, 4 Enterobacter cloacae, 1 Citrobacter freundii and 1 Citrobacter braakii were isolated. The blaCTX-M-1 and blaCTX-M-15 genes were predominant in E. coli and K. pneumoniae isolates. E. cloacae and C. braakii isolates harboured the blaSHV-12 gene. The C. freundii isolated from wastewater carried blaCTX-M-15, blaTEM-1 and blaOXA-204. E. coli isolates belonged to phylogroups A (37), B1 (25), B2 (7) and D (12). Seventy-eight E. coli isolates were typeable by PFGE and were classified into 34 pulsotypes. The K. pneumoniae isolates belonged to 11 pulsotypes. The E. coli isolates belonged to sequence types ST131, ST224, ST162, ST845, ST5204, ST69, ST141 and ST10. The K. pneumoniae isolates belonged to ST405, ST147, ST564, ST307, ST152, ST45, ST661 and ST1564. CONCLUSION: This is the first report of O25b-B23-CTX-M-27-ST131 E. coli isolates and of C. freundii carrying blaCTX-M-15, blaTEM-1 and blaOXA-204 in Tunisia.

Use of MALDI-TOF mass spectrometry to detect nosocomial outbreaks of Serratia marcescens and Citrobacter freundii.

MALDI-TOF mass spectrometry (MS) may be used as a rapid typing method for nosocomial pathogens. Here, we evaluated MALDI-TOF MS for discrimination of hospital outbreak-related clusters of Serratia marcescens and carbapenemase-producing Citrobacter freundii. Thirty-three S. marcescens isolates collected from neonatal intensive care unit (NICU) patients, and 23 C. freundii isolates including VIM-positive isolates from a hospital colonization outbreak were measured by Vitek MS. Consensus spectra of each isolate were clustered using SARAMIS software. Genotyping was performed by whole-genome sequencing (WGS). First, a set of 21 S. marcescens isolates from 2014 with seven genotypes including three monoclonal clusters was used for the evaluation of MALDI-TOF typing. MS clustering was largely in agreement with genotyping results when the similarity cut-off for clonal identity was set on 90%. MALDI-TOF cluster analysis was then investigated for the surveillance of S. marcescens in the NICU in 2017 and demonstrated the introduction of new strains into the hospital and nosocomial transmissions. MS analysis of the C. freundii outbreak in 2016 revealed a monoclonal cluster of VIM-positive isolates and the separation of epidemiologically non-related VIM-positive and negative isolates. Two additional VIM-positive Citrobacter isolates from food samples were closely related to the large monoclonal cluster. WGS confirmed the MS results. MALDI-TOF MS may be used as a first-line typing tool for S. marcescens and C. freundii to detect transmission events in the hospital because isolates of an identical WGS type were grouped into the same MS cluster.

Molecular characterization of plasmid-encoded Tripoli MBL 1 (TMB-1) in Enterobacteriaceae.

Objectives: Available commercial tools (molecular methods or immunochromatographic assays) usually allow the detection of the five most prevalent carbapenemases (KPC, NDM, VIM, IMP and OXA-48-like), but miss minor carbapenemases. Here, we characterize two enterobacterial isolates with reduced susceptibility to carbapenems and negative for the most commonly encountered carbapenemase genes. Methods: Enterobacter hormaechei and Citrobacter freundii isolates were recovered from a bile sample and rectal screening, respectively. Both isolates were investigated by WGS. Resistance genes were detected using ResFinder. The blaTMB-1-harbouring plasmid was reconstructed using CLC genomic workbench 10.0 and was annotated using the RAST tool. Transfer frequency was determined by conjugation experiments using the laboratory strain Escherichia coli J53. Results: The two isolates were resistant to broad-spectrum cephalosporins and carbapenems. WGS revealed the presence of blaTMB-1, which has previously only been described in non-fermenters. blaTMB-1 was located within an ISKpn19-based composite class 1 transposon. Comparative genomics revealed that this structure was carried on a conjugative IncN-type plasmid within an integration hotspot. Conjugation experiments revealed high transfer frequencies of ∼1 × 10-3. Conclusions: To the best of our knowledge, this study corresponds to the first report of Tripoli MBL 1-producing Enterobacteriaceae. Despite always being described as likely to be chromosomally located in non-fermenters, the blaTMB-1 gene is now found to be carried by a conjugative plasmid among Enterobacteriaceae, raising concern about the possible dissemination of this carbapenemase. The blaTMB-1 gene should now be suspected when PCRs targeting the main carbapenemases remain negative.

New insights into Citrobacter freundii sepsis in neonates.

BACKGROUND: The aim of this study was to investigate the clinical features of neonatal sepsis caused by Citrobacter freundii as well as the current status and treatment strategy for multi-drug resistance of infection with this bacterium. METHODS: Nine newborns were diagnosed with C. freundii sepsis between January 2014 and December 2017. We collated and analyzed a range of data for these nine patients, including general information, laboratory tests during infection, blood culture and treatment. RESULTS: One of the patients died after only 7 h of infection. In the remaining eight cases, three patients developed meningitis, although none had brain abscess. A reduction of white blood cells (WBC) was detected <24 h after the start of infection, compared with at 48-72 h, when WBC count had increased and platelets progressively decreased. In all nine cases the infection was susceptible to tigecycline and was resistant to cephalosporins, carbapenems, and quinolones. In eight cases the infection was susceptible to co-trimoxazole and in the other case it was susceptible to amikacin. Of the eight patients who were cured, three received meropenem, two received ceftriaxone, one received amikacin, and two received tigecycline. CONCLUSION: Reduction in WBC could take place in the early stages of C. freundii infection in newborns. The incidence of brain abscess was not high, but multi-drug resistance was common. Some non-sensitive drugs can also treat C. freundii sepsis effectively.

Analyses of a Ceftazidime-Avibactam-Resistant Citrobacter freundii Isolate Carrying blaKPC-2 Reveals a Heterogenous Population and Reversible Genotype.

A blaKPC-2-carrying Citrobacter freundii isolate developed ceftazidime-avibactam resistance during treatment with this agent. The initial and follow-up isolates exhibited ceftazidime-avibactam MICs of 4 and 64 µg/ml, respectively. Overexpression of AcrAB-TolC and porin alterations were detected in both isolates, but no other resistance mechanism was observed. After passaging the initial clinical isolate in ceftazidime-avibactam at a fixed concentration of 4 µg/ml and a 4:1 ratio, resistance to all ß-lactams was noted, and a percentage of the blaKPC-2 sequencing reads had mutations leading to the alterations D176Y (blaKPC-2-D176Y [78%]) or R164S plus P147L (blaKPC-2-R164S + P147L [82%]). Further investigation of the follow-up isolate showed that 11% of the blaKPC-2 reads had mutations leading to D179Y substitution (blaKPC-2-D179Y). In the absence of selective pressure, ceftazidime-avibactam MICs of the passaged and follow-up isolates revealed that 7 or 8 out of 20 screened colonies reverted to susceptible and possessed blaKPC-2 wild-type sequences. Recombinant plasmids carrying the blaKPC-2 alterations observed were transformed in Escherichia coli, and MIC values for ceftazidime ± avibactam were elevated. Lower MICs for ceftriaxone, cefepime, aztreonam, meropenem, and imipenem for the mutated KPC-2-producing isolates were observed compared to those of the isolates producing a wild-type KPC-2. Avibactam at a fixed concentration of 4 µg/ml restored the activity of all ß-lactams tested for the recombinant strains. The heterogenous population of wild-type and mutated blaKPC-2 and the reversibility of the genotypes observed suggest a significant challenge for managing KPC-producing isolates that develop ceftazidime-avibactam resistance during therapy.IMPORTANCE The development of ceftazidime-avibactam resistance among KPC-producing isolates during treatment with this agent has been reported. Usually isolates that become resistant have a mutated blaKPC gene that confers resistance to ceftazidime-avibactam and susceptibility to meropenem. We report a Citrobacter freundii isolate that developed ceftazidime-avibactam resistance due to mutations within the coding region of the blaKPC-2 Ω-loop previously reported; however, in this case, only 11% of the whole-genome sequencing reads had mutations, making this alteration difficult to detect and the treatment of these isolates more challenging. In addition to blaKPC, the initial and the follow-up patient isolates displayed hyperexpression of the AcrAB-TolC efflux system and disruption of the outer membrane protein (OMP) OmpF, which contribute to carbapenem resistance. Experiments performed to confirm our findings included generating mutant isolates from the initial patient isolate, passaging the isolates for purity in drug-free medium, resulting in a reversible phenotype, and cloning the mutations to demonstrate the resistance conferred.

Citrobacter freundii infection in silver catfish (Rhamdia quelen): Hematological and histological alterations.

Citrobacter freundii is a fish pathogen known for its ability to cause injury and high mortality. There have been no studies reporting the effect of this bacterium on hematological parameters and internal organ histology in silver catfish (Rhamdia quelen). Therefore, the aim of this study is to evaluate the hematological and histopathological effects of an experimentally induced C. freundii infection in silver catfish. Twenty fish were divided into healthy and infected groups. The fish of the infected group were inoculated intramuscularly with 100 µL of bacterial suspension (6.4 × 108 CFU mL-1), while healthy control animals received 100 µL of sterile saline. On day 18 post-infection, blood and tissues (cephalic kidneys, livers, and spleens) were collected for histological analysis. The infected animals presented high mortality, as well as hematological and histological changes. In relation to hematology, the infected fish presented aregenerative anemia, protein loss, leukopenia with neutropenia, lymphocytosis, and leukoblastosis. Regarding histology, there was liver degeneration, decrease in the amount of renal hematopoietic tissue, and the presence of melanomacrophage centers (MMCs) in the spleen and cephalic kidney of infected fish. In summary, these alterations may contribute to disease pathophysiology, contributing to high mortality of affected fish.

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.

Genetic Diversity, Multidrug Resistance, and Virulence of Citrobacter freundii From Diarrheal Patients and Healthy Individuals.

Objectives:Citrobacter freundii is a frequent cause of nosocomial infections and a known cause of diarrheal infections, and has increasingly become multidrug resistant (MDR). In this study, we aimed to determine the genetic diversity, the antimicrobial resistance profiles and in vitro virulence properties of C. freundii from diarrheal patients and healthy individuals. Methods: 82 C. freundii isolates were obtained from human diarrheal outpatients and healthy individuals. Multilocus Sequence Typing (MLST) of seven housekeeping genes was performed. Antimicrobial susceptibility testing was carried out using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Adhesion and cytotoxicity to HEp-2 cells were assessed. PCR and sequencing were used to identify blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, and qepA genes. Results: The 82 C. freundii isolates were divided into 76 sequence types (STs) with 65 STs being novel, displaying high genetic diversity. Phylogenetic analysis divided the 82 isolates into 5 clusters. All 82 isolates were sensitive to imipenem (IPM), but resistant to one or more other 16 antibiotics tested. Twenty-six isolates (31.7%) were multidrug resistant to three or more antibiotic classes out of the 10 distinct antibiotic classes tested. Five MDR isolates, all of which were isolated from 2014, harbored one or more of the resistance genes, blaTEM-1, blaCTX-M-9, aac(6')-Ib-cr, qnrS1, qnrB9, and qnrB13. All 11 qnrB-carrying C. freundii isolates belonged to cluster 1, and one C. freundii isolate carried a new qnrB gene (qnrB92). Six isolates showed strong cytotoxicity to HEp-2 cells, one of which was multidrug resistant. Conclusions:C. freundii isolates from human diarrheal outpatients and healthy individuals were diverse with variation in sequence types, antibiotic resistance profiles and virulence properties.

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.

Identification of CFE-2, a new plasmid-encoded AmpC ß-lactamase from a clinical isolate of Citrobacter freundii.

A clinical isolate of Citrobacter freundii (JA99) obtained from a bile culture of a Taiwanese patient was found to produce a plasmid-encoded ß-lactamase conferring resistance to oxyimino-cephalosporins and cephamycins. Resistance arising from production of the ß-lactamase could be transferred by conjugation with an IncW plasmid (pJA99) into Escherichia coli J53. The substrate and inhibition profiles of this enzyme resembled that of an AmpC ß-lactamase. The resistance gene of pJA99, cloned and expressed in E. coli DH5α, was shown to contain an open reading frame showing 92% amino acid identity with the plasmid-encoded enzyme CFE-1 of E. coli KU6400. DNA sequence analysis also identified a gene upstream of ampC in pJA99 whose sequence was 95.0% identical to the ampR gene from E. coli KU6400. In addition, orf1, the fumarate operon (frdABCD), blc, lolB and repB surrounding the ampR-ampC genes in C. freundii were identified. This DNA fragment was absent in other Citrobacter spp. Therefore, we describe a new plasmid-encoded AmpC ß-lactamase, named CFE-2. This study highlights the emergence of broad-spectrum cephalosporin resistance in C. freundii owing to a new type of AmpC ß-lactamase.

Coexistence of NDM-1-producing Escherichia coli and Citrobacter freundii in the same patient.

OBJECTIVES: Although blaNDM-1 has been widely detected in various Enterobacteriaceae clinical isolates, multiple blaNDM-1 colonisations within the same patient remain rare. The aim of the study was to describe a patient with respiratory tract colonisation with NDM-1-producing Escherichia coli and Citrobacter freundii during hospitalisation in China. METHODS: Two carbapenem-resistant isolates were analysed. Antimicrobial susceptibility testing, metallo-ß-lactamase production, conjugation assay, plasmid analysis and molecular typing were performed. RESULTS: The two clinical isolates carried the blaNDM-1 gene and showed resistance to carbapenems. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) revealed that E. coli NB753 harboured a plasmid of ca. 80kb, and C. freundii NB865 harboured three plasmids (ca. 70, 80 and 135kb). Southern blot and Inc replicon typing further demonstrated that both isolates carried the blaNDM-1 gene on a self-transferrable IncX3 plasmid. Moreover, the two NDM-producing plasmids were conjugative and the transconjugants showed increased resistance to carbapenems. CONCLUSION: An NDM-1-encoding plasmid harboured by various clinical isolates in a single patient is worrying considering that this plasmid may be widespread in our hospital. Furthermore, the threat of carbapenemase-producing bacterial epidemics should be closely monitored. However, a limitation of this study was the extremely small sample size.