Freundii Isolates Research Articles

Investigation of Citrobacter freundii clinical isolates in a Chinese hospital during 2020-2022 revealed genomic characterization of an extremely drug-resistant C. freundii ST257 clinical strain GMU8049 co-carrying blaNDM-1 and a novel blaCMY variant.

The emergence of multidrug-resistant Citrobacter freundii poses a significant threat to public health. C. freundii isolates were collected from clinical patients in a Chinese hospital during 2020-2022. An unusual strain, GMU8049, was not susceptible to any of the antibiotics tested, including the novel β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam. Whole-genome sequencing (WGS) revealed that GMU8049 harbors a circular chromosome belonging to the rare ST257 and an IncX3 resistance plasmid. Genomic analysis revealed the coexistence of two β-lactamase genes, including plasmid-mediated blaNDM-1 and chromosomal blaCMY encoding a novel CMY variant, combined with an outer membrane porin deficiency, which may account for the extreme resistance to β-lactams. Conjugation experiment confirmed that the blaNDM-1 resistance gene located on pGMU8049 could be successfully transferred to Escherichia coli EC600. The novel CMY variant had an amino acid substitution at position 106 (N106S) compared to the closely related CMY-51. Additionally, a GMU8049-specific truncation in an OmpK37 variant that produces a premature stop codon. Moreover, a variety of chromosome-located efflux pump coding genes and virulence-related genes were also identified. Analysis of strain GMU8049 in the context of other C. freundii strains reveals an open pan-genome and the presence of mobile genetic elements that can mediate horizontal gene transfer of antimicrobial resistance and virulence genes. Our work provides comprehensive insights into the genetic mechanisms of highly resistant C. freundii, highlighting the importance of genomic surveillance of this opportunistic pathogen as a high-risk population for emerging resistance and pathogenicity.IMPORTANCEEmerging pathogens exhibiting multi-, extremely, and pan-drug resistance are a major concern for hospitalized patients and the healthcare community due to limited antimicrobial treatment options and the potential for spread. Genomic technologies have enabled clinical surveillance of emerging pathogens and modeling of the evolution and transmission of antimicrobial resistance and virulence. Here, we report the genomic characterization of an extremely drug-resistant ST257 Citrobacter freundii clinical isolate. Genomic analysis of GMU8049 with a rare ST type and unusual phenotypes can provide information on how this extremely resistant clinical isolate has evolved, including the acquisition of blaNDM-1 via the IncX3 plasmid and accumulation through chromosomal mutations leading to a novel CMY variant and deficiency of the outer membrane porin OmpK37. Our work highlights that the emergence of extremely resistant C. freundii poses a significant challenge to the treatment of clinical infections. Therefore, great efforts must be made to specifically monitor this opportunistic pathogen.

First identification and genomic features of multidrug-resistant Citrobacter freundii ST669 strain isolated from a domesticated duck in Bangladesh

ObjectivesCitrobacter freundii is a prevalent source of nosocomial infections and a well-known cause of diarrheal diseases. In recent years, it has also become increasingly resistant to various antimicrobials. In this study, we screened and characterized a multidrug-resistant (MDR) C. freundii isolate obtained from a domesticated diseased duck to better understand the genetic features, molecular epidemiology, and underlying factors linked to the antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs) of the isolate. MethodsThe C. freundii BAU_TM8 strain was isolated using culturing, staining, biochemical, polymerase chain reaction, and Matrix-assisted laser desorption/ionization-time of flight methods. The MDR properties of the strain were determined by a disk diffusion test. The genomic sequence of C. freundii BAU_TM8 was performed using the Illumina NextSeq2000 platform. The ARGs, VFGs, and genomic functional characteristics of the C. freundii BAU_TM8 strain were identified using several open-source databases. ResultsThe sequence type of this strain was ST669, and the pathogenicity index of the strain was 0.919. Moreover, the strain had an estimated genome length of 5,797,806 bp, harboring 62 contigs, a G + C content of 54.32 %, and five contig L50s with an N50 value of 443,947 bp. Using phylogenetic analysis, this strain was closely related to two strains isolated from human and environmental samples in the USA and China despite huge geographical distances. The C. freundii BAU_TM8 strain consisted of 40 AGRs encoding resistance to 19 antimicrobial categories, e.g., fluoroquinolones, macrolides, folate pathway antagonists, aminoglycosides, tetracyclines, cephalosporins, and others. According to the phenotypic assay and genome sequence, the sensitivity and specificity of resistance profiles of the strain were 100 % and 20 %, respectively. Moreover, the virulence factor database detected 66 VFGs in this strain. This strain contained 1581 subsystems, having 33 % subsystem coverage and 2275 genes encoding amino acid derivatives, carbohydrate metabolism, protein metabolism, cofactors, vitamins, prosthetic groups, pigments, respiration, motility and chemotaxis, stress response, DNA metabolism, nucleosides and nucleotides, and others. ConclusionsTo the best of our knowledge, this is the first WGS report of C. freundii from a domesticated duck in Bangladesh. The ubiquitous occurrence of ARGs and VFGs in the C. freundii BAU_TM8 strain detected in this study highlights the growing concern about antimicrobial resistance in humans, animals, and environments.

Coexistence of a novel NDM-1-encoding MDR plasmid and an IMP-4-encoding IncN-IncU hybrid plasmid in a clinical isolate of Citrobacter freundii BC73.

To investigate the genetic characteristics and transmission mechanism of the NDM-1-, IMP-4-, and SHV-12-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii BC73. C. freundii BC73 was isolated from a urine specimen of a urological patient diagnosed with bladder cancer at a Chinese teaching hospital. Antimicrobial susceptibility testing was carried out using DL-120E susceptibility cards and DL-96A system. Whole genome sequencing (WGS) of the isolate was performed using the Illumina and Oxford Nanopore platforms to analyze the genetic context of drug resistance genes and plasmid characteristics. The phylogenetic tree was constructed and visualized by KSNP3.0 software and iTOL5.0 online database. C. freundii isolate BC73 co-carrying bla NDM-1, bla IMP-4 and bla SHV-12 were multidrug-resistant. bla NDM-1 and bla IMP-4 were located on a novel IncFIB-like plasmid, pCFBC1, and an IncN-IncU hybrid plasmid, pCFBC2, respectively. The transferability of bla NDM-1 and bla IMP-4 from C. freundii BC73 to E. coli J53 was successfully demonstrated. The genetic context of the bla NDM-1 and bla IMP-4 genes were ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble MBL-bla NDM-1-∆ISAba125-IS3000 and intI1-bla IMP-4-Kl.pn.13-mobC-IS6100, respectively. Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) were identified and numerous antimicrobial resistance genes were discovered on it. To our knowledge, our study represents the first characterization of a ST22 C. freundii isolate co-harboring bla NDM-1, bla IMP-4, and bla SHV-12, obtained from a urine sample. The dissemination of this MDR isolate should be of close concern in future clinical surveillance.

Epidemiological Characteristics of Shiga Toxin-Producing Escherichia coli Responsible for Infections in the Polish Pediatric Population.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children and the elderly. Stool samples were collected from 180 children hospitalized in five pediatric centers in Poland in 2018-2022. Direct stx1/stx2 gene detection by PCR in feces and E. coli isolates was performed. Antibiotic susceptibility was tested according to EUCAST v.12. Randomly selected isolates were serotyped with O157 antiserum and genotyped by pulsed-field gel electrophoresis (PFGE). A total of 44 E. coli isolates were confirmed as STEC by PCR. Among them, 84.4% were positive for stx2, and equally 6,8% for only stx1 and both stx1 and stx2 genes. The stx1 gene was also found in one Citrobacter freundii isolate. E. coli serotype O157 was present in 97.6% of the isolates. STEC infections most often occurred between June-October with a peak in July and August (51%). The highest, 77.8% of STEC isolates were found in the 1-5 years old group. No extended-spectrum β-lactamases (ESBL) were found. Resistance only to amoxicillin/clavulanic acid (24.4%), piperacillin/tazobactam (3%), cefotaxime (6%), gentamicin (6%), ciprofloxacin (3%), azithromycin (3%), trimethoprim/sulfamethoxazole (24,2%) was detected. PFGE analysis showed 18 PFGE types with no clonal distribution. Eight isolates with A, B, and C PFGE types showed genetic relatedness in the type with no detection of transmission way of distribution. STEC strains pose a serious threat to human health, therefore demographic and epidemiological characteristics are crucial for their surveillance.

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.

In Vitro Antimicrobial Activity of Five Newly Approved Antibiotics against Carbapenemase-Producing Enterobacteria-A Pilot Study in Bulgaria.

To solve the problem with pan-drug resistant and extensively drug-resistant Gram-negative microbes, newly approved drugs such as ceftazidime/avibactam, cefiderocol, plazomicin, meropenem/vaborbactam, and eravacycline have been introduced in practice. The aim of the present study was to collect carbapenemase-producing clinical Enterobacterales isolates, to characterize their carbapenemase genes and clonal relatedness, and to detect their susceptibility to commonly used antimicrobials and the above-mentioned newly approved antibiotics. Sixty-four carbapenemase producers were collected in a period of one year from four Bulgarian hospitals, mainly including Klebsiella pneumoniae (89% of the isolates) and also single Proteus mirabilis, Providencia stuartii and Citrobacter freundii isolates. The main genotype was blaNDM-1 (in 61%), followed by blaKPC-2 (23%), blaVIM-1 (7.8%) and blaOXA-48 (7.8%). Many isolates showed the presence of ESBL (blaCTX-M-15/-3 in 76.6%) and AmpC (blaCMY-4 in 37.5% or blaCMY-99 in 7.8% of isolates). The most common MLST type was K. pneumoniae ST11 (57.8%), followed by ST340 (12.5%), ST258 (6.3%) and ST101 (6.3%). The isolates were highly resistant to standard-group antibiotics, except they were susceptible to tigecycline (83.1%), colistin (79.7%), fosfomycin (32.8%), and aminoglycosides (20.3-35.9%). Among the newly approved compounds, plazomicin (90.6%) and eravacycline (76.3%) showed the best activity. Susceptibility to ceftazidime/avibactam and meropenem/vaborbactam was 34.4% and 27.6%, respectively. For cefiderocol, a large discrepancy was observed between the percentages of susceptible isolates according to EUCAST susceptibility breakpoints (37.5%) and those of CLSI (71.8%), detected by the disk diffusion method. This study is the first report to show patterns of susceptibility to five newly approved antibiotics among molecularly characterized isolates in Bulgaria. The data may contribute to both the improvement of treatment of individual patients and the choice of infection control strategy and antibiotic policy.

Identification and characterization of a highly virulent Citrobacter freundii isolate and its activation on immune responses in largemouth bass (Micropterus salmoides)

Citrobacter freundii, a common pathogen of freshwater fish, causes significant commercial losses to the global fish farming industry. In the present study, a highly pathogenic C. freundii strain was isolated and identified from largemouth bass (Micropterus salmoides). The pathogenicity and antibiotic sensitivity of the C. freundii strain were evaluated, and the histopathology and host immune response of largemouth bass infected with C. freundii were investigated. The results showed that C. freundii was the pathogen causing disease outbreaks in largemouth bass, and the infected fish showed typical signs of acute hemorrhages and visceral enlargement. Antimicrobial susceptibility testing showed that the C. freundii strain was resistant to Kanamycin, Medimycin, Clindamycin, Penicillin, Oxacillin, Ampicillin, Cephalexin, Cefazolin, Cefradine and Vancomycin. Histopathological analysis showed different pathological changes in major tissues of diseased fish. In addition, humoral immune factors such as superoxide dismutase (SOD), catalase (CAT) and lysozyme (LZM) were used as serum indicators to evaluate the immune response of largemouth bass after infection. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression pattern of immune-related genes (CXCR1, IL-8, IRF7, IgM, CD40, IFN-γ, IL-1β, Hep1, and Hep2) in liver, spleen, and head kidney tissues, which demonstrated a strong immune response induced by C. freundii infection in largemouth bass. The present study provides insights into the pathogenic mechanism of C. freundii and immune response in largemouth bass, promoting the prevention and treatment of diseases caused by C. freundii infection.

The Prevalence of Extended Spectrum Beta Lactamase Producing Citrrobacter Freundii and Pprovidencia Stuartii from Medical Laboratories in Parts of Lagos State, Nigeria

Citrobacter freundii and Providencia stuartii are members of the family Enterobacteriaceae which are emerging as important Extended Spectrum Beta Lactamase (ESBL) producing pathogens. This study was aimed to determine the prevalence of ESBL and carbapenemase production in C. freundii and P. stuartii. from laboratories in the northwest of Lagos. A total of 134 isolates were obtained from laboratories. Isolate identification, antibiotics susceptibility testing, and phenotypic detection of ESBL production were done using standardized procedures. Multiplex polymerase chain reaction (PCR) was performed on ESBL producing isolates to detect blaTEM, blaSHV, blaCTX-M, blaKPC, blaVIM, blaIMP and blaOXA. Out of 134 isolates 15 (11.2%) were Citrobacter freundii and only 2 (1.5%) were of the Providencia genus both of which were Providencia rettgeri and Providencia stuartii. A total of 9 (60%) tested positive for the production of Extended Spectrum Beta Lactamase (ESBL) enzyme while only Providencia stuartii of the Providencia genus also produced ESBL. Also, 8 (47%) of the initial Citrobacter and Providencia isolates produced carbapenemase Molecular characterization showed that 4 (44.4%) and 1 (100%) of the Citrobacter freundii and Providencia stuartii isolates respectively, displayed the presence of CTX-M gene while 2 (20%) of all ESBL producing isolates also possessed SHV gene including 1 (11.1%) of ESBL producing Citrobacter freundii and 1 (100%) of ESBL producing Providencia stuartii. Antibiotic susceptibility testing showed that Citrobacter freundii displayed the highest resistance to Ceftazidime and Cefotaxime at 71.4% and the least resistance to Amoxicillin-Clavulanate at 28.6%. Providencia stuartii also displayed absolute resistance (100%), to cefotaxime and ciprofloxacin and absolute susceptibility (100%) to Ceftazidime. Also, Citrobacter freundii showed the same resistance patterns with 57.1% resistance to Gentamicin, Cefuroxime and Ceftriaxone antibiotics while Providencia stuartii showed varied resistance patterns with absolute resistance (100%), absolute susceptibility (100%) and absolute resistance (100%) to Gentamicin, Cefuroxime and Ceftriaxone antibiotics respectively. In conclusion, the Multiple Antibiotic Resistance Index values of both bacterial genera showed that 70.6% were from High-Risk sources indicating that the majority of bacteria analysed were from environments where they have been subjected to a high burden of antibiotic exposure.

Complete-genome sequencing and comparative genomic characterization of blaNDM-5 carrying Citrobacter freundii isolates from a patient with multiple infections

BackgroundThe emergence and wide spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a growing threat to global public health. However, clinically derived carbapenemase-producing Citrobacter causing multiple infections has rarely been investigated. Here we first report the isolation and comparative genomics of two blaNDM-5 carrying Citrobacter freundii (C. freundii) isolates from a patient with bloodstream and urinary tract infections.ResultsAntimicrobial susceptibility testing showed that both blaNDM-5 carrying C. freundii isolates were multidrug-resistant. Positive modified carbapenem inactivation method (mCIM) and EDTA-carbapenem inactivation method (eCIM) results suggested metallo-carbapenemase production. PCR and sequencing confirmed that both metallo-carbapenemase producers were blaNDM-5 positive. Genotyping and comparative genomics analyses revealed that both isolates exhibited a high level of genetic similarity. Plasmid analysis confirmed that the blaNDM-5 resistance gene is located on IncX3 plasmid with a length of 46,161 bp, and could successfully be transferred to the recipient Escherichia coli EC600 strain. A conserved structure sequence (ISAba125-IS5-blaNDM-5-trpF-IS26-umuD-ISKox3) was found in the upstream and downstream of the blaNDM-5 gene.ConclusionsThe data presented in this study showed that the conjugative blaNDM-5 plasmid possesses a certain ability to horizontal transfer. The dissemination of NDM-5-producing C. freundii isolates should be of close concern in future clinical surveillance. To our knowledge, this is the first study to characterize C. freundii strains carrying the blaNDM-5 gene from one single patient with multiple infections.

Identification of Citrobacter freundii from Skin Wounds in Goldfish (Carassius auratus) and Determination of Antibacterial Susceptibility

Citrobacter freundii is a zoonotic pathogen that causes skin injuries and high mortality rates in fish. This study aims to determine the presence of C. freundii and antibiotic susceptibility in 50 goldfish (Carassius auratus) skin wounds with local bleeding between the ages of 8–18 months in three regions (A, B, and C) located in the West of Turkey. C. freundii (22/50; 44%) strains were isolated from wound swab samples by phenotypic and genotypic methods. C. freundii isolates were positive for H2S, mannitol, and indole, and were negative for the gas formation and urea. All C. freundii isolates showed 96% similarity to C. freundii strain BAO2749 according to the Sanger sequence method. Isolates were also resistant to ampicillin at 57.09% and susceptible to cefoperazone and sulfamethoxazole-trimethoprim by 100%. Statistical analyses were evaluated at a confidence interval of 95%. A p value < 0.05 was considered statistically significant. In conclusion, C. freundii isolates can play a significant role in the pathophysiology of infections in goldfish (Carassius auratus). It can also cause commercial losses in businesses. For this reason, it will be beneficial to control C. freundii pathogenicity in aquarium fish with prophylactic applications to improve product quality and treat diseases in ornamental fish farming.

Coexistence of blaIMP-4, blaNDM-1 and blaOXA-1 in blaKPC-2-producing Citrobacter freundii of clinical origin in China.

To explore the genetic characteristics of the IMP-4, NDM-1, OXA-1, and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii wang9. MALDI-TOF MS was used for species identification. PCR and Sanger sequencing analysis were used to identify resistance genes. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). We performed whole genome sequencing (WGS) of the strains and analyzed the resulting data for drug resistance genes and plasmids. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL. Citrobacter freundii carrying blaKPC-2, blaIMP-4, blaOXA-1, and blaNDM-1 are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The blaIMP-4 coexists with the blaNDM-1 and the blaOXA-1 on a novel transferable plasmid variant pwang9-1, located on the integron In1337, transposon TnAS3, and integron In2054, respectively. The gene cassette sequence of integron In1337 is IntI1-blaIMP-4-qacG2-aacA4'-catB3Δ, while the gene cassette sequence of In2054 is IntI1-aacA4cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1. The blaNDM-1 is located on the transposon TnAS3, and its sequence is IS91-sul-ISAba14-aph (3')-VI-IS30-blaNDM-1-ble-trpF-dsbD-IS91. The blaKPC-2 is located on the transposon Tn2 of plasmid pwang9-1, and its sequence is klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. Phylogenetic analysis showed that most of the 34°C. freundii isolates from China were divided into three clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of C. freundii from environmental samples from Zhejiang. We found C. freundii carrying blaIMP-4, blaNDM-1, blaOXA-1, and blaKPC-2 for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that blaIMP-4, blaOXA-1, and blaNDM-1 coexisted on a new transferable hybrid plasmid that carried many drug resistance genes and insertion sequences. The plasmid may capture more resistance genes, raising our concern about the emergence of new resistance strains.

Genomic epidemiology of nosocomial carbapenemase-producing Citrobacter freundii in sewerage systems in the Helsinki metropolitan area, Finland.

Multi-drug resistance is emerging in Citrobacter freundii, which is the third most common carbapenemase-producing (CP) Enterobacteriaceae in humans in Finland due to recent outbreaks. The objective of this study was to determine if wastewater surveillance (WWS) could detect CP C. freundii strains causing infections in humans. Selective culturing was used to isolate CP C. freundii from the hospital environment, hospital wastewater, and untreated municipal wastewater in Helsinki, Finland, between 2019 and 2022. Species were identified using MALDI-TOF, and presumptive CP C. freundii isolates were subjected to antimicrobial susceptibility testing and further characterized by whole genome sequencing. A genomic comparison was conducted to compare isolates collected from the hospital environment, untreated municipal wastewater, and a selection of isolates from human specimens from two hospitals in the same city. We also examined the persistence of CP C. freundii in the hospital environment and the impact of our attempts to eradicate it. Overall, 27 blaKPC - 2-carrying C. freundii were detected in the hospital environment (ST18; n = 23 and ST8; n = 4), while 13 blaKPC - 2-carrying C. freundii (ST8) and five blaVIM - 1-carrying (ST421) C. freundii were identified in untreated municipal wastewater. CP C. freundii was not identified in hospital wastewater. We found three clusters (cluster distance threshold ≤ 10 allelic difference) after comparing the recovered isolates and a selection of isolates from human specimens. The first cluster consisted of ST18 isolates from the hospital environment (n = 23) and human specimens (n = 4), the second consisted of ST8 isolates from the hospital environment (n = 4), untreated municipal wastewater (n = 6), and human specimens (n = 2), and the third consisted of ST421 isolates from the untreated municipal wastewater (n = 5). Our results support previous studies suggesting that the hospital environment could act as a source of transmission of CP C. freundii in clinical settings. Furthermore, the eradication of CP Enterobacteriaceae from the hospital environment is challenging. Our findings also showed that CP C. freundii is persistent throughout the sewerage system and demonstrate the potential of WWS for detecting CP C. freundii.

Phenotypic and Genotypic Characteristics of Antimicrobial Resistance in Citrobacter freundii Isolated from Domestic Ducks (Anas platyrhynchos domesticus) in Bangladesh.

Antimicrobial resistance (AMR) in Citrobacter freundii poses a serious challenge as this species is one of the sources of nosocomial infection and causes diarrheal infections in humans. Ducks could be the potential source of multidrug-resistant (MDR) C. freundii; however, AMR profiles in C. freundii from non-human sources in Bangladesh have remained elusive. This study aimed to detect C. freundii in domestic ducks (Anas platyrhynchos domesticus) in Bangladesh and to determine their phenotypic and genotypic antibiotic susceptibility patterns. A total of 150 cloacal swabs of diseased domestic ducks were screened using culturing, staining, biochemical, polymerase chain reaction (PCR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to detect C. freundii. Phenotypic and genotypic antibiotic susceptibility patterns were done by the disk diffusion method and PCR, respectively. In total, 16.67% (25/150) of the samples were positive for C. freundii. C. freundii isolates showed a range of 20% to 96% resistance to cefotaxime, gentamicin, levofloxacin, ciprofloxacin, cotrimoxazole, tetracycline, ampicillin, and cephalexin. More than 60% of the isolates were phenotypically MDR, and the index of multiple antibiotic resistance ranged from 0.07 to 0.79. Genes encoding resistance to beta-lactams [blaTEM-1-88% (22/25), blaCMY-2-56% (14/25), blaCMY-9-8% (2/25), and blaCTX-M-14-20% (5/25)], sulfonamides [sul1-52% (13/25), sul2-24% (6/25)], tetracyclines [tetA-32% (8/25) and tetB-4% (1/25)], aminoglycosides [aacC4-16% (4/25)], and fluoroquinolones [qnrA-4% (1/25), qnrB-12% (3/25), and qnrS-4% (1/25)] were detected in the isolated C. freundii. To the best of our knowledge, this is the first study in Bangladesh to detect MDR C. freundii with their associated resistance genes from duck samples. We suggest addressing the burden of diseases in ducks and humans and associated AMR issues using the One Health approach.

Phenotypic and genotypic discrepancies for carbapenemase-producing Citrobacter freundii in multiple isolates from a single patient

BackgroundCarbapenemase-producing gram-negative organisms continue to be a significant healthcare concern and a therapeutic challenge. Members of the genus Citrobacter have emerged as increasingly multidrug resistant and versatile healthcare-associated pathogens. In this study we investigated five KPC-producing Citrobacter freundii isolates, from the same patient, that presented unusual phenotypic characteristics including false susceptibility to carbapenems detection by culture-based methods.MethodsThe isolates were tested for antimicrobial susceptibility using broth microdilution and disk diffusion. Production of serine carbapenemase was confirmed with the mCIM (modified carbapenem inactivation method) test. Genotypes were determined by PCR and whole genome sequencing analysis.ResultsThe five isolates were susceptible to meropenem by broth microdilution and presented varying colonial morphologies and levels of susceptibility to carbapenems by multiple phenotypic methods, despite being positive for carbapenemase production by mCIM and positive for blaKPC by PCR. Whole genome sequence analysis showed that three of the five highly related isolates harbor an additional gene cassette, including blaCARB-2, ant(2''), aadA2, dfrA19, catB3, cmlA1, mph(E), msr(E), and qnrA1. The presence of these genes explains the difference in phenotypes observed.ConclusionFailure to detect and completely eradicate the carbapenemase-producing C. freundii in the urine with ertapenem therapy, likely due to the presence of a heterogeneous population, resulted in the phenotypic and genotypic adaptations of the organism as it disseminated to the bloodstream and kidneys. The fact that carbapenemase-producing C. freundii can elude detection by phenotypic methods and can so easily acquire and transfer resistance gene cassettes is of concern.

Molecular analysis of clinical Citrobacter spp. isolates: Acquisition of the Yersinia high-pathogenicity island mediated by ICEkp in C. freundii.

Studies on Citrobacter spp. are limited, hindering our understanding of its species evolution and medical relevance. A total of 164 clinical Citrobacter spp. isolates were collected from 2017 to 2020 and identified by VITEK MALDI-TOF MS or VITEK-2 Gram-Negative Identification Card. All isolates were further analyzed by whole-genome sequencing using a HiSeq sequencer. All sequences were processed using different modules of the PGCGAP integrated package: Prokka and fastANI were used for annotation and average nucleotide identification (ANI), respectively. Antibiotic resistance and virulence genes were identified by searching CARD, ResFinder, and VFDB databases, respectively. Strains were identified using Ribosomal Multi-locus Sequence Typing (rMLST) classification based on 53 ribosome protein subunits (rps). The evolutionary relationship was analyzed using kSNP3 and visualized by iTOL editor v1_1. Genetic environments were compared by BLAST and visualized by Easyfig 2.2.5. The pathogenicity of some Citrobacter freundii isolates was confirmed by Galleria mellonella larvae infection test. A total of 14 species of Citrobacter spp. were identified from 164 isolates. However, 27 and 11 isolates were incorrectly identified as C. freundii and Citrobacter braakii by MALDI-TOF MS, respectively. In addition, MS also failed to identify Citrobacter portucalensis. The virulence genes mainly encoded proteins related to flagella and iron uptake systems. Citrobacter koseri isolates (n = 28) contained two iron uptake systems, coding yersiniabactin and aerobactin, respectively. C. braakii isolates (n = 32), like Salmonella, carried Vi capsule polysaccharide synthesis genes. The yersiniabactin gene clusters identified in five C. freundii isolates are located on various ICEkp elements and have not been reported previously. Moreover, ICEkp-carrying C. freundii showed diverse pathogenic features. Conventional methods have significant defects in identifying Citrobacter spp. ICEkp-like elements-mediated acquirement of the Yersinia high-pathogenicity island was identified for the first time in C. freundii.

Molecular detection and phylogenetic analysis of Citrobacter freundii isolates from broilers in AL- Diwaniyah province of Iraq

Because of the medicinal and economic importance of Citrobacter freundii, as well as the fact that there are only a few local studies on it, the purpose of this study was to isolate and identify this important bacterial species from chicken using differential media with comparable biochemical properties. There is a severe concern about the presence of extended-spectrum beta-lactamase (ESBL) creating bacteria in animal husbandry, which could be transmitted to humans. C. freundii was detected through the use of specific associated genes that enable this bacterium to resist the effects of antibiotics, making it highly resistant. For detection of this bacteria and recognition of distinct types of resistance genes, polymerase chain reaction was used. Further confirmation, purified PCR product was subjected to sequencing. It showed 99.80% homology with Switzerland isolates available in the NCBI database. Forty two isolates of C. freundii were isolated from (450) fecal sample collected from different region of AL- Diwaniyah province.

High prevalence of Panton-Valentine Leucocidin (PVL) toxin carrying MRSA and multidrug resistant gram negative bacteria in late onset neonatal sepsis indicate nosocomial spread in a Pakistani tertiary care hospital

BackgroundNeonatal sepsis has high incidence with significant mortality and morbidity rates in Pakistan. We investigated common etiological patterns of neonatal sepsis at a tertiary care setup. Methods90 pus and blood, gram negative and gram positive bacterial isolates were analyzed for virulence and antibiotic resistance gene profiling using PCR and disc diffusion methods. ResultsStaphylococcus aureus showed strong association with neonatal sepsis (43 %) followed by Citrobacter freundii (21 %), Pseudomonas aeruginosa (13 %), Escherichia coli (15 %) and Salmonella enterica (8 %). Molecular typing of E. coli isolates depicted high prevalence of the virulent F and B2 phylogroups, with 4 hypervirulent phylogroup G isolates. 76.9 % S. aureus isolates showed presence of Luk-PV, encoding for Panton-valentine leucocidin (PVL) toxin with majority also carrying MecA gene and classified as methicillin resistant S. aureus (MRSA). ecpA, papC, fimH and traT virulence genes were detected in E. coli and Salmonella isolates. 47 % Citrobacter freundii isolates carried the shiga like toxin SltII B. Antimicrobial resistance profiling depicted common resistance to cephalosporins, beta lactams and fluoroquinolones. ConclusionPresence of PVL carrying MRSA and multidrug resistant gram negative bacteria, all isolated from late onset sepsis neonates indicate a predominant nosocomial transmission pattern which may complicate management of the disease in NICU setups

Multidrug resistance-encoding gene in Citrobacter freundii isolated from healthy laying chicken in Blitar District, Indonesia

Background and Aim: The increasing prevalence of resistance (MDR) of Enterobacteriaceae in Indonesia has caused concern regarding human health. Citrobacter freundii reportedly targets the gastrointestinal tract of animals and is a common cause of foodborne diseases associated with diarrhea, peritonitis, meningitis, brain abscess, bacteremia, and urinary tract infection. This study aimed to estimate the prevalence of MDR and the presence of Class 1 integron-encoding genes in C. freundii isolates obtained from cloacal swabs of healthy laying chickens in Blitar district, Indonesia. Materials and Methods: One hundred and sixty-five cloacal swab samples were collected from 33 farms in Blitar over a period of 4 months. Standard microbiological techniques such as bacterial culture in MacConkey agar, Simmons citrate agar, and triple sugar iron agar and biochemical tests such as the indole test were performed to identify the isolates. The antibiotic sensitivity patterns of C. freundii isolates were determined by the disk diffusion method, and MDR-encoding genes (Class 1 integron) were detected by polymerase chain reaction (PCR). Results: Out of 165 cloacal swab samples, 7 (4.24%) were positive for C. freundii. Citrobacter freundii was highly resistant to erythromycin (71.43%) and moderately to streptomycin, tetracycline, and trimethoprim-sulfamethoxazole (all 42.86%); however, it showed low resistance to ampicillin (28.57%). All isolates were found to exhibit MDR. Only 1 (14.29%) of the seven C. freundii isolates harbored a Class 1 integron gene. This study revealed that Class 1 integron-encoding genes have a low prevalence in C. freundii isolated from healthy laying chickens in Blitar, Indonesia. Conclusion: Poultry animals can play a role in the transmission of resistance genes to humans due to the MDR of Enterobacteriaceae, including C. freundii in the intestines.

Antimicrobial Resistance, Phenotypic Characteristics, and Biofilm Production in Citrobacter freundii Isolates Obtained from Urinary Tract Infections

Objective To evaluate the link between phenotypic traits, antimicrobial resistance, and biofilm-producing capacity of urinary isolates of Citrobacter freundii ( C. freundii). Methods Both pan-antibiotic-susceptible and -resistant C. freundii isolates ( n = 120) obtained from laboratory-confirmed urinary tract infections were analyzed for their link between antimicrobial resistance, phenotypic characteristics, and biofilm production. Results Of the total C. freundii isolates ( n = 120), 30% (37/120) of them formed large colonies. Among the total large colonies produced ( n = 37), they were present in 21.62%, 10.81%, 13.5%, 16.2%, 21.62%, and 16.21% in the control group, CIP-group, FOS-group, COT-group, Gent-group, and ESBL groups, respectively. Compared to the pan-susceptible isolates, the occurrence of large-sized-colony-forming strains was relatively reduced in most of the drug-resistant groups. The overall distribution of mucoid colonies produced ( n = 54) includes 9.25%, 18.51%, 16.66%, 18.51%, 20.3%, and 16.66% in the control group, CIP-group, FOS-group, COT-group, Gent-group, and ESBL groups, respectively. Of the total isolates that produced biofilm ( n = 51), 11.76% of isolates showed biofilm formation in the control group. Alternatively, the rate was found to be 15.68%, 11.76%, 25.49%, 19.6%, and 15.68% in the CIP-group, FOS-group, SXT-group, Gen-group, and ESBL-groups, respectively. Conclusion This study correlates the association between phenotypic characteristics, antimicrobial resistance, and biofilm production, the three main characteristics of C. freundii.

PROFILING OF ANTIBIOTIC RESISTANT BACTERIA ISOLATED FROM POULTRY LITTER OF COMMERCIAL FARMS IN KHULNA DISTRICT, BANGLADESH

Microbial resistance to antibiotics has become a global threat that interferes with the interaction among humans, the environment, and microbes. Our study sought to detect antibiotic-resistant bacteria in light of the growing interest in using antibiotics in the poultry sector. We collected 24 poultry liter samples from 04 different upazilas of Khulna district (Batiaghata, Dumuria, Paikgachha and Koyra). Initially, 09 bacterial isolates were selected and among them, 77.78% bacteria were found gram positive. Subsequently, to characterize these bacteria, a toal of 10 biochemical tests (methyl red test, MacConkey agar test, indole test, catalase test, triple sugar iron test, mannitol salt agar test, oxidase test, Voges-Proskauer test, citrate test and nitrate test) were carried out in this experiment. Moreover, our study also isolated and amplified bacterial DNA for Sanger sequencing and finally disclosed 09 different antibiotic resistant bacterial species, namely Priestia aryabhattai, Bacillus cereus, Priestia megaterium, Lysinibacillus macrolides, Rossellomorea aquimaris, Mammaliicoccus sciuri, Bacillus wiedmannii, Escherichia coli and Citrobacter freundii. Further, disk diffusion assays were performed following CLSI guidelines and it unveiled the susceptibility and resistant properties of isolates against commonly used 08 antibiotics (penicillin 10U, tetracycline 20µg, nitrofuran 300µg, clindamycin 2µg, azithromycin 15µg, quinolones 5µg, tetracycline 30µg and penicillin 30µg) in poultry farms. All of the bacterial isolates were found resistant to at least one of the antibiotics except C. freundii (isolate 9). Most of the isolates (66.67%) were resistant to nitrofuran, whereas all of them were susceptible to penicillin. Lastly, this study also made an effort to understand the evolutionary relationships of the identified species through a phylogenetic tree. Thus, the findings of this study will help farmers and common people to better understand the risk of developing antibiotic-resistant bacteria as a result of excessive antibiotic usage.