Clinical Isolates Of Citrobacter Freundii Research Articles

Characterization of GMB-1, a novel metallo-β-lactamase (MBL) found in three different Enterobacterales species.

To identify novel carbapenem resistance mechanisms and their potential to spread among clinical isolates. Four clinical isolates of Citrobacter freundii, Serratia marcescens and Raoultella planticola (n = 2) from one hospital in Central Germany were sent to the German National Reference Centre for Multidrug-resistant Gram-negative Bacteria for carbapenemase detection. Phenotypic tests indicated the presence of a metallo-β-lactamase (MBL), but PCR for various MBL genes could not identify any. Using WGS data, a putative bla gene was identified. Its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain, with subsequent MIC determination by broth microdilution, as well as by in vitro hydrolysis assays using purified enzyme. WGS indicated the presence of a putative β-lactamase with 48% amino acid identity to the subclass B1 MBL SPM-1. MIC studies confirmed that the novel enzyme formed a functional MBL, which was therefore designated as GMB-1 (German MBL). In vitro hydrolysis assays showed a lack of activity not only against aztreonam but also against ertapenem. WGS revealed that in all three species the blaGMB-1 gene was located on the chromosome as part of a genetic island with multiple ISs. The finding of GMB-1 once again shows that novel carbapenemases continue to emerge and make their way into clinically relevant species. The occurrence of GMB-1 in three different species demonstrates the extraordinary mobility of such genetic islands and their potential to spread carbapenemase genes into diverse genetic environments.

Plasmid Carrying blaCTX-M-15, blaPER-1, and blaTEM-1 Genes in Citrobacter spp. From Regional Hospital in Mexico

Introduction:Citrobacter spp. is an opportunistic bacteria that have been recognized as significant pathogens in patients with underlying diseases or immunocompromised status. The aim of this study was to identify extended-spectrum β-lactamases in clinical isolates of Citrobacter spp.Methods:This cross-sectional study was conducted at Hospital Central “Dr. Ignacio Morones Prieto” in San Luis Potosi, Mexico. Nineteen isolates of Citrobacter spp. were obtained from clinical specimens between April to December 2015. Four isolates were resistant to third-generation cephalosporins. The presence of genes encoding ESBL (blaCTX-M-15, blaTEM-1, blaVEB-1, blaSHV, and blaPER-1) was analyzed by PCR. For this purpose, plasmid DNA was extracted and horizontally transferred to recipient E. coli Top 10.Results:blaCTX-M-15 and blaVEB-1 genes were detected in Citrobacter freundii and Citrobacter sedlakii, whereas blaPER-1 gene was identified in 1 isolate of Citrobacter freundii. In contrast, blaSHV gene was not detected in any isolate. One strain carried blaCTX-M-15, blaTEM-1, blaVEB-1, and blaPER-1 genes, most in a 275-kb plasmid.Conclusion:This study shows the presence of different types of ESBL in clinical isolates of Citrobacter freundii and Citrobacter sedlakii, which confer resistance to broad-spectrum β-lactams. The plasmid identified in this study harboring ESBL genes could play an important role in the dissemination of antibiotic resistance.

Microbial, phytochemical, toxicity analyses and antibacterial activity against multidrug resistant bacteria of some traditional remedies sold in Buea Southwest Cameroon

BackgroundTraditional medicine remedies are commonly used for treatment of diverse ailments including bacterial infections. The activity against resistant bacteria and safety of some remedies sold as anti-infective treatments in market places in Buea, Southwest Cameroon were investigated as potential alternative treatment to counter increasing antibiotic resistance.MethodsTen remedies were purchased, their components documented and microbial load estimated. Methanol extracts of the remedies were tested for antibacterial activity by disc diffusion and microdilution. Cytotoxicity was evaluated on monkey kidney epithelial cells (LLC-MK2) while acute oral toxicity was done in BALB/c mice for the bactericidal extract. Extracts were further analysed using phytochemical tests.ResultsAll the remedies had microbial loads above the acceptable limit of 105 CFU/g. The highest activity was produced by extracts of four remedies (TP 1, 2, 4, 6a, 6b) against all clinical isolates among which three were active against four control strains. Zones of inhibition ranged from 8 to 27 mm. Two of the four extracts produced zones ≥20 mm against multidrug resistant clinical isolates of Citrobacter freundii and Escherichia coli but were less active compared to Gentamycin positive control (P < 0.0001–0.0014). The most active extracts also recorded minimum inhibitory concentrations of 1 to 4 mg/mL. One of them (TP2) was bactericidal against a clinical isolate of methicillin–resistant Staphylococcus aureus with a minimum bactericidal concentration of 8 mg/mL. Extracts of six remedies did not show cytotoxicity and no mortality or adverse effect was recorded in the acute oral toxicity test. Phytochemical screening showed the most active extracts contained relatively high amounts of alkaloids and flavonoids.ConclusionOnly four of the eight remedies tested showed activity against multidrug resistant bacteria suggesting some of these remedies may not be effective against bacterial infections. Production and handling methods should be improved and the product quality controlled to ensure biosecurity. The remedies which were both active and non-toxic should be further investigated including in vivo experiments to assess their efficacy.

Identification of CFE-2, a new plasmid-encoded AmpC β-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 β-lactamase conferring resistance to oxyimino-cephalosporins and cephamycins. Resistance arising from production of the β-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 β-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 β-lactamase, named CFE-2. This study highlights the emergence of broad-spectrum cephalosporin resistance in C. freundii owing to a new type of AmpC β-lactamase.

High Prevalence of &lt;i&gt;qnr&lt;/i&gt; and &lt;i&gt;aac(6′)-Ib-cr&lt;/i&gt; Genes in Both Water-Borne Environmental Bacteria and Clinical Isolates of &lt;i&gt;Citrobacter freundii&lt;/i&gt; in China

We investigated the prevalence of qnr and aac(6′)-Ib-cr genes in water-borne environmental bacteria and in clinical isolates of Enterobacteriaceae, as well as the subtypes of qnr. Environmental bacteria were isolated from surface water samples obtained from 10 different locations in Hangzhou City, and clinical isolates of Citrobacter freundii were isolated from several hospitals in four cities in China. qnrA, qnrB, qnrS, and aac(6′)-Ib-cr genes were screened using PCR, and the genotypes were analyzed by DNA sequencing. Ten of the 78 Gram-negative bacilli isolated from water samples were C. freundii and 80% of these isolates carried the qnrB gene. qnrS1 and aac(6′)-Ib-cr genes were detected in two Escherichia coli isolates and qnrS2 was detected in one species, Aeromonas punctata. The qnr and aac(6′)-Ib-cr genes were present in 75 (72.8%) and 12 (11.6%) of 103 clinical isolates of C. freundii, respectively. Of the clinical C. freundii isolates with the qnr gene, 65 isolates (63.1%) carried qnrB, but only three (2.9%) and one (1.0%) carried qnrA1 and qnrS2, respectively, while five isolates carried both qnrA1 and qnrB, and one isolate carried both qnrS1 and qnrB. The qnrB9 gene was the dominant qnrB subtype, followed by qnrB8 and qnrB6. Southern hybridization studies indicated that the qnr genes are located on different plasmids. Plasmids isolated from both environmental and clinical C. freundii isolates appeared to be homogenous.

Prevalence of qnr and aac（ 6＇）- I b-cr genes in water-borne environmental bacteria and clinical isolates of Citrobacter freundii in China

Objective To＇investigate the prevalence of qnr and aac（6＇）- I b-cr genes in waterborne environmental bacteria and clinical isolates of Citrobacter freundii, and the subtypes of qnr gene. Methods Environmental bacteria were isolated from surface water samples obtained from 10 distinct locations in Hangzhou city, and clinical isolates of C. freundii were isolated from several hospitals of 4 cities in China. Minimum inhibitory concentrations （MICs） of ciprofloxacin, levofloxacin and nalidixic acid were determined by agar dilution method, qnrA, qnrB, qnrS and aac （6＇）- I b-cr genes were screened by PCR, and the genotypes were analyzed by DNA sequencing. Results Seventy-eight gram negative bacilli （ including 33 Enterobacteriaceae, 21 Aeromonas spp. , 10 Acinetobacter spp. , 10 Pseudomonas spp. , 2 Alcaligenes spp. , and 2 Plesiomonas spp. ） were isolated from water samples. Among these isolates, 8 of 10 C. freundii were positive for qnrB gene. qnrS1 and aac（6＇）- I b-cr were detected in two distinct Escherichia coli, and qnrS2 was detected in a Aeromonas punctata qnr and aac（6＇）- I b-cr genes were present in 75 （72.8%） and 12 （11.6%） of 103 clinical isolates of C. freundii, respectively. Three （2.9%） C. freundii isolates were positive for qnrA1 gene, 65 （63.1%） qnrB, 1 （1.0%） qnrS2, 5 （4.8%） were positive for both qnrA1 and qnrB, and 1 was positive for both qnrS1 and qnrB. Within the subtypes of qnrB gene, qnrB9 predominated, followed by qnrB8 and qnrB6. Conclusion It was the first isolate of Aeromonas spp. harboring qnrS2 gene outside Europe. The prevalence of qnrB in water-borne environmental and clinical isolates of C. freundii was particularly high and qnrB9, qnrB8 and qnrB6 were the most common subtypes, aac（6＇）- I b-cr gene widely spread in clinical isolates of C. freundii. Key words: Environment; Bacteria; Quinolone resistance; qnr; aac（6＇）- I b-cr

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii.

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.

Worldwide Disseminated armA Aminoglycoside Resistance Methylase Gene Is Borne by Composite Transposon Tn 1548

The armA (aminoglycoside resistance methylase) gene, which confers resistance to 4,6-disubstituted deoxystreptamines and fortimicin, was initially found in Klebsiella pneumoniae BM4536 on IncL/M plasmid pIP1204 of ca. 90 kb which also encodes the extended-spectrum beta-lactamase CTX-M-3. Thirty-four enterobacteria from various countries that were likely to produce a CTX-M enzyme since they were more resistant to cefotaxime than to ceftazidime were studied. The armA gene was detected in 12 clinical isolates of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, K. pneumoniae, Salmonella enterica, and Shigella flexneri, in which it was always associated with bla(CTX-M-3) on an IncL/M plasmid. Conjugation, analysis of DNA sequences, PCR mapping, and plasmid conduction experiments indicated that the armA gene was part of composite transposon Tn1548 together with genes ant3"9, sul1, and dfrXII, which are responsible for resistance to streptomycin-spectinomycin, sulfonamides, and trimethoprim, respectively. The 16.6-kb genetic element was flanked by two copies of IS6 and migrated by replicative transposition. This observation accounts for the presence of armA on self-transferable plasmids of various incompatibility groups and its worldwide dissemination. It thus appears that posttranscriptional modification of 16S rRNA confers high-level resistance to all the clinically available aminoglycosides except streptomycin in gram-negative human and animal pathogens.

Prevalence of Derepressed AmpC Mutants and Extended-Spectrum β-Lactamase Producers among Clinical Isolates of Citrobacter freundii , Enterobacter spp., and Serratia marcescens in Korea: Dissemination of CTX-M-3, TEM-52, and SHV-12

The resistance mechanism of extended-spectrum cephalosporins in clinical isolates of Citrobacter freundii, Enterobacter spp., and Serratia marcescens was studied. Of 152 isolates, 45 isolates (29.6%) were derepressed AmpC mutants and 39 isolates (25.7%) produced extended-spectrum beta-lactamase (ESBLs). The most prevalent ESBLs were CTX-M enzymes, followed by TEM-52 and SHV-12.

Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii

Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii

Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii.

The presence of gyrA, gyrB and/or parC mutations, quinolone uptake, outer membrane protein profiles and epidemiological relationship were studied in 12 clinical isolates of Citrobacter freundii. No alterations were observed in the gyrB gene of any of the strains, or gyrA or parC of the four quinolone-susceptible strains (nalidixic acid MIC of 2-4 mg/L, and a ciprofloxacin MIC of 0.006-0.06 mg/L). The quinolone-resistant strains were classified into two groups: one group (group A) composed of strains resistant to nalidixic acid but not to ciprofloxacin and another (group B) including those resistant to both antibiotics with a mutation at codon 83 of the gyrA gene (Thr-->Ile), but no alteration in either parC or gyrB genes. In group B, three of the four resistant isolates, with a nalidixic acid MIC > 1024 mg/L and ciprofloxacin MIC of 8-32 mg/L, showed concomitant mutations at codons 83 and 87 of the gyrA gene (Thr-->Ile and Asp-->Tyr, respectively) as well as a single mutation in codon 80 of the parC gene (Ser-->Ile). The fourth isolate did not possess the mutation at codon 87 of gyrA. Two strains belong to the same clone and, although they had the same type of mutations in the gyrA and parC genes, showed different MICs of ciprofloxacin. This difference was related to an efflux pump mechanism. Mutations in the gyrA and parC genes play the main role in quinolone resistance development in Citrobacter freundii, although other factors such as overexpression of efflux pumps can play a complementary role and thus modulate the final quinolone MIC.

GyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae.

Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 microg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.

Clinical isolate of Citrobacter freundii highly resistant to new quinolones.

About 30% of 150 recent clinical isolates of Citrobacter freundii were resistant to greater than or equal to 3.13 micrograms of norfloxacin and ofloxacin per ml. Study of one quinolone-resistant strain for which the norfloxacin MIC was 100 micrograms/ml suggested that resistance was associated with both an altered A subunit of DNA gyrase and reduction in drug uptake accompanied by a decrease in an outer membrane protein.

DNA sequence of haemolysin genes fromCitrobacter freundii

The DNA sequence of a 2572-bp region containing genes encoding a hemolytic protein (citrolysin) cloned from an extra-intestinal clinical isolate of Citrobacter freundii, was determined. There were two open reading frames present on the same DNA strand, encoding predicted polypeptides of a molecular mass of 70.4 and 15.7 kDa. Based on the DNA sequence, a single putative promoter region is outlined, and a potential Shine-Dalgarno sequence precedes each of the two genes. However, structures typical of those involved in the rho-independent termination of transcription could not be detected between or within the two genes. The codon usage pattern and the low guanine-plus-cytosine content may suggest a non-Citrobacter origin for this hemolytic activity. An assay indicated the possible non-excretory nature of the C. freundii hemolysin, when expressed in E. coli.