A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses.

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

Genetic characterization of KHM-1 metallo-ß-lactamase-producing Enterobacterales isolates from inpatient sources in Osaka, Japan.

OBJECTIVES: KHM-1-metallo-ß-lactamase-producing Enterobacterales strains, of which only a few have been found, were isolated from four inpatients in Osaka, Japan during 2016 to 2020. We compared whole genomes of the four KHM-1-producing isolates, including one Enterobacter hormaechei subsp. hoffmannii, one Escherichia coli, and two Citrobacter freundii. METHODS: These isolates were characterized by whole-genome sequencing, comparative analysis of blaKHM-1-encoding plasmids with earlier reported plasmids, and antimicrobial susceptibility tests. RESULTS: Multilocus sequence typing classified the E. hormaechei subsp. hoffmannii isolate to ST78, the E. coli isolate to ST354, and the two C. freundii isolates to ST95. These isolates harboured various antimicrobial resistance genes aside from blaKHM-1 on their chromosomes and plasmids. In all four isolates, blaKHM-1 was located on 137 kbp to 213 kbp plasmids of IncC replicon type. Although there were common resistance genes such as blaKHM-1-ISEc68, class I integron cassette, and fosG, the four blaKHM-1-encoding plasmids were distinguishable into two lineages based on differences of the resistance gene components and their surrounding regions. CONCLUSION: Because no epidemiological contact was observed among the inpatients, the blaKHM-1-encoding IncC plasmids might have spread horizontally to multiple bacterial species through repeated recombination and insertion.

Molecular characterization of multidrug resistant Enterobacterales strains isolated from liver and kidney transplant recipients in Spain.

The objective of this study was to analyse the mechanisms of resistance to carbapenems and other extended-spectrum-ß-lactams and to determine the genetic relatedness of multidrug-resistant Enterobacterales (MDR-E) causing colonization or infection in solid-organ transplantation (SOT) recipients. Prospective cohort study in kidney (n = 142), liver (n = 98) or kidney/pancreas (n = 7) transplant recipients between 2014 and 2018 in seven Spanish hospitals. We included 531 MDR-E isolates from rectal swabs obtained before transplantation and weekly for 4-6 weeks after the procedure and 10 MDR-E from clinical samples related to an infection. Overall, 46.2% Escherichia coli, 35.3% Klebsiella pneumoniae, 6.5% Enterobacter cloacae, 6.3% Citrobacter freundii and 5.7% other species were isolated. The number of patients with MDR-E colonization post-transplantation (176; 71.3%) was 2.5-fold the number of patients colonized pre-transplantation (71; 28.7%). Extended-spectrum ß-lactamases (ESBLs) and carbapenemases were detected in 78.0% and 21.1% of MDR-E isolates respectively. In nine of the 247 (3.6%) transplant patients, the microorganism causing an infection was the same strain previously cultured from surveillance rectal swabs. In our study we have observed a low rate of MDR-E infection in colonized patients 4-6 weeks post-transplantation. E. coli producing blaCTX-M-G1 and K. pneumoniae harbouring blaOXA-48 alone or with blaCTX-M-G1 were the most prevalent MDR-E colonization strains in SOT recipients.

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.

Synthesis and in vitro activity of asymmetric indole-based bisamidine compounds against Gram-positive and Gram-negative pathogens.

A series of new asymmetric bisamidine was designed, synthesized, and tested for their in-vitro antibacterial activity using a range of Gram-positive and Gram-negative pathogens. Most compounds demonstrated powerful antibacterial activity, and interestingly, some displayed better activity against several Gram-negative strains than the lead compound 1. The most potent bisamidine 8l exhibited 4-fold more potent activity against E. coli, K. pneumonia, P. aeruginosa, and C. freundii than compound 1. Especially 8l exhibited a powerful activity against K. pneumonia secreting NDM-1 enzyme with a minimum inhibitory concentration (MIC) of 2 µg/mL, while levofloxacin and vancomycin displayed resistance, with MICs > 128 µg/mL.

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.

Susceptibility evolution to antibiotics of Enterobacter cloacae, Morganella morganii, Klebsiella aerogenes and Citrobacter freundii involved in urinary tract infections: an 11-year epidemiological surveillance study. / Evolución de la sensibilidad a los antibióticos de Enterobacter cloacae, Morganella morganii, Klebsiella aerogenes y Citrobacter freundii causantes de infecciones del tracto urinario: un estudio de vigilancia epidemiológica de 11 años.

INTRODUCTION: The objective of this study was to analyse the susceptibility to antibiotic of Citrobacter freundii, Klebsiella aerogenes, Enterobacter cloacae, Serratia marcescens, Providencia stuartii and Morganella morganii (CESPM group), detected in urine cultures. METHODS: Between 2006 and 2016 we analyzed CESPM group Enterobacteria isolated from urine cultures from both primary health-care centers and Hospital Virgen de las Nieves (Granada). We studied the susceptibility to aminoglycosides, fosfomycin, nitrofurantoin, quinolones, piperacillin/tazobactam, cefepime, imipenem and trimethoprim/sulfamethoxazole following CLSI interpretation criteria. RESULTS: A total of 736 isolates were studied: 30.57% E. cloacae, 23.50% M. morganii, 20.38% K. aerogenes, 10.32% C. freundii, 8.83% S. marcescens and 6.38% P. stuartii. A significant decrease in the antibiotic susceptibility was observed. Gentamicin, ciprofloxacin, imipenem and cefepime showed susceptibility over 80%. CONCLUSIONS: E. cloacae, M. morganii and K. aerogenes were the most common isolates. Cefepime and imipenem are still a good empiric therapeutic alternative given its activity in vitro.

Genomic characterization of Citrobacter freundii strains coproducing OXA-48 and VIM-1 carbapenemase enzymes isolated in leukemic patient in Spain.

Background: The emergence of carbapenemase-producing (CP) Citrobacter freundii poses a significant threat to public health, especially in high-risk populations. In this study, whole genome sequencing was used to characterize the carbapenem resistance mechanism of three C. freundii clinical isolates recovered from fecal samples of patients with acute leukemia (AL) from Spain. Materials and methods: Twelve fecal samples, collected between 2013 and 2015 from 9 patients with AL, were screened for the presence of CP strains by selecting them on MacConkey agar supplemented with ertapenem (0.5 mg/L). Bacteria were identified by MALDI-TOF mass spectrometry and were phenotypically characterized. Whole genome sequencing of C. freundii isolates was performed using the MinION and MiSeq Illumina sequencers. Bioinformatic analysis was performed in order to identify the molecular support of carbapenem resistance and to study the genetic environment of carbapenem resistance encoding genes. Results: Three carbapenem-resistant C. freundii strains (imipenem MIC≥32 mg/L) corresponding to three different AL patients were isolated. Positive modified Carba NP test results suggested carbapenemase production. The genomes of each C. freundii tested were assembled into a single chromosomal contig and plasmids contig. In all the strains, the carbapenem resistance was due to the coproduction of OXA-48 and VIM-1 enzymes encoded by genes located on chromosome and on an IncHI2 plasmid, respectively. According to the MLST and the SNPs analysis, all strains belonged to the same clone ST169. Conclusion: We report in our study, the intestinal carrying of C. freundii clone ST169 coproducing OXA-48 and VIM-1 identified in leukemic patients.

Induction of plasmid-mediated AmpC ß-lactamase DHA-1 by piperacillin/tazobactam and other ß-lactams in Enterobacteriaceae.

Chromosomal AmpC ß-lactamase induction by several types of ß-lactams has been reported, but not enough data are available on DHA-1 ß-lactamase, a plasmid-mediated AmpC ß-lactamase. Therefore, we evaluated the DHA-1 ß-lactamase induction by various antibiotics including piperacillin/tazobactam (PIP/TZB) in this study. Six strains (Enterobacter cloacae 2 strains, Citrobacter freundii 1 strain, Serratia marcescens 2 strain, and Morganella morganii 1 strain) possessing chromosomal inducible AmpC ß-lactamase were used as controls. Four strains (Escherichia coli 2 strains, Klebsiella pneumoniae 1 strain, and C. koseri 1 strain) possessing DHA-1 ß-lactamase were used. The ß-lactamase activities were determined by a spectrophotometer using nitrocefin. ß-lactamase induction by PIP, PIP/TZB was not observed in any strains and ß-lactamase induction by third- and fourth-generation cephems was not observed in most strains. The induction ratios of the chromosomal AmpC ß-lactamase in the reference group by PIP/TZB were <1.51, and those of the DHA-1 ß-lactamase were <1.36, except for K. pneumoniae Rkp2004 (2.22). The ß-lactamase induction by first- and second-generation cephems, flomoxef, and carbapenem differed in each strain. Cefmetazole (CMZ) strongly induced ß-lactamase. This study demonstrated that the induction of DHA-1 ß-lactamase was similar to that of chromosomal AmpC using various Enterobacteriaceae, although the induction of ß-lactamase in both groups by PIP/TZB was low. We also reported that the induction of PIP/TZB, a ß-lactamase inhibitor combination antibiotic, against various AmpC-producing Enterobacteriaceae, including DHA-1 producers, was low.

Interplay among IncA and blaKPC-Carrying Plasmids in Citrobacter freundii.

We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, blaKPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, blaKPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying blaKPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria.

Genetic background of CTX-M-15-producing Enterobacter hormaechei ST114 and Citrobacter freundii ST265 co-infecting a free-living green turtle (Chelonia mydas).

CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae have become identified in marine ecosystem constituting a serious ecological issue. In this respect, although contamination of coastal waters and seafood, and even colonization of seabirds and fishes have been increasingly reported, molecular data are lacking to elucidate the clinical impact of ESBL producers in infected marine animals. In this study, using a genomic approach, we have analysed the genetic background of CTX-M-15-producing Enterobacter hormaechei (belonging to the international human clone ST114) and Citrobacter freundii (ST265) co-infecting a free-living green turtle (Chelonia mydas) suffering from septic arthritis, which progressed to generalized coelomitis and death. Wide resistome of these pathogens contributed to treatment failure and death of the animal.

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.

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.

Comparative genomics analysis of pTEM-2262, an MDR plasmid from Citrobacter freundii, harboring two unclassified replicons.

AIM: To genetically characterize the multidrug-resistance (MDR) plasmid pTEM-2262 that could not be classified into any known incompatibility group from the clinical Citrobacter freundii isolate 2262. MATERIALS & METHODS: The repA or repB deletion mutants of pTEM-2262 were constructed using the scarless Cas9-assisted recombineering system. Comparative genomic analysis of pTEM-2262 and the other four previously sequenced plasmids belonging to the same incompatibility group were performed. RESULTS: pTEM-2262, a conjugative plasmid, harbored two unclassified replicons, repA and repB, while repB was not essential for pTEM-2262 replication. In five analyzed plasmids, their conserved backbones primarily integrated massive accessory modules at two 'hotspots' that were located between orf597 and orf504, and between orf393 and orf405. All the antibiotic resistance genes of pTEM-2262 were clustered in the MDR region with a complex mosaic structure. CONCLUSION: This study thoroughly investigates the detailed structure and genomic comparison of this unknown incompatibility group for the first time.

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.

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.

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.