Citrobacter Isolates Research Articles

Extended spectrum AmpC and metallo-beta-lactamases in Serratia and Citrobacter spp. in a disc approximation assay

This study aimed to develop a novel model for detection of extended spectrum beta-lactamase (ESBL), AmpC and metallo-beta-lactamase (MBL) producing Serratia and Citrobacter species using cefoperazone sulbactam as well as other inducer-substrate combinations in a disc approximation assay. In the absence of molecular tools in developing countries, we attempted to standardize simple phenotypic techniques for detection of beta-lactamases to allow effective patient care in our countries. These techniques have been scarcely used in Serratia and Citrobacter spp., which are emerging as significant pathogens in our region. Clinical isolates of Serratia and Citrobacter were tested for ESBL production. Cefoperazone (CP)/cefoperazone sulbactam (CPS), piperacillin (PIP)/piperacillin-tazobactam (TZP) and ceftazidime (CAZ)/ceftazidime-clavulanic acid (CAZ-CLAV) combinations were compared for their ability to detect ESBL producers phenotypically. Multi-drug resistant strains were further tested for detection of inducible/derepressed AmpC mutants by a disc approximation assay. Isolates were screened for MBL production by Imipenem (IMI). MBL production was confirmed using Ethylenediaminetetraacetic acid (EDTA) in a double disc synergy assay and Hodge test. Minimal inhibitory concentration (MIC) was performed for CP, CPS and IMI by agar dilution method for all isolates of Serratia and Citrobacter spp. Thirty-three percent of isolates of Serratia spp. and 35.4% of Citrobacter spp. were ESBL producers. CPS was a more sensitive inducer of ESBL than TZP and CAZ/CAZ-CLAV. AmpC producers were detected in 25.6% of the isolates of Serratia spp. (40% inducible and 60% derepressed mutants) and in 35.4% of the isolates of Citrobacter spp. (33% inducible and 66% derepressed mutants). Six isolates (four class B and two class A) of Serratia and eight isolates (seven class B and one class A) of Citrobacter spp. were MBL producers. Multiple mechanisms co-existed in eight isolates of Serratia and 15 isolates of Citrobacter spp. CPS was more effective in identifying ESBLs and inducible AmpC producers as well as type 1 carbapenemases than TZP and CAZ-CLAV were able to identify inducible AmpC producers. The high prevalence of ESBL, AmpC, and MBL in Serratia and Citrobacter species in this study suggests that detection of these by phenotypic methods in the absence of more specific molecular tests should be actively considered in not only developing countries but also in the developed world as this approach can lead to timely and appropriate antibiotic treatment. CPS may be advised due to the triple advantage of detection of all three types of beta-lactamases.

Extended spectrum AmpC and metallo-beta-lactamases in Serratia and Citrobacter spp. in a disc approximation assay

This study aimed to develop a novel model for detection of extended spectrum beta-lactamase (ESBL), AmpC and metallo-beta-lactamase (MBL) producing Serratia and Citrobacter species using cefoperazone sulbactam as well as other inducer-substrate combinations in a disc approximation assay. In the absence of molecular tools in developing countries, we attempted to standardize simple phenotypic techniques for detection of beta-lactamases to allow effective patient care in our countries. These techniques have been scarcely used in Serratia and Citrobacter spp., which are emerging as significant pathogens in our region. Clinical isolates of Serratia and Citrobacter were tested for ESBL production. Cefoperazone (CP)/cefoperazone sulbactam (CPS), piperacillin (PIP)/piperacillin-tazobactam (TZP) and ceftazidime (CAZ)/ceftazidime-clavulanic acid (CAZ-CLAV) combinations were compared for their ability to detect ESBL producers phenotypically. Multi-drug resistant strains were further tested for detection of inducible/derepressed AmpC mutants by a disc approximation assay. Isolates were screened for MBL production by Imipenem (IMI). MBL production was confirmed using Ethylenediaminetetraacetic acid (EDTA) in a double disc synergy assay and Hodge test. Minimal inhibitory concentration (MIC) was performed for CP, CPS and IMI by agar dilution method for all isolates of Serratia and Citrobacter spp. Thirty-three percent of isolates of Serratia spp. and 35.4% of Citrobacter spp. were ESBL producers. CPS was a more sensitive inducer of ESBL than TZP and CAZ/CAZ-CLAV. AmpC producers were detected in 25.6% of the isolates of Serratia spp. (40% inducible and 60% derepressed mutants) and in 35.4% of the isolates of Citrobacter spp. (33% inducible and 66% derepressed mutants). Six isolates (four class B and two class A) of Serratia and eight isolates (seven class B and one class A) of Citrobacter spp. were MBL producers. Multiple mechanisms co-existed in eight isolates of Serratia and 15 isolates of Citrobacter spp. CPS was more effective in identifying ESBLs and inducible AmpC producers as well as type 1 carbapenemases than TZP and CAZ-CLAV were able to identify inducible AmpC producers. The high prevalence of ESBL, AmpC, and MBL in Serratia and Citrobacter species in this study suggests that detection of these by phenotypic methods in the absence of more specific molecular tests should be actively considered in not only developing countries but also in the developed world as this approach can lead to timely and appropriate antibiotic treatment. CPS may be advised due to the triple advantage of detection of all three types of beta-lactamases.

Detection of the Klebsiella pneumoniae Carbapenemase Type 2 Carbapenem-Hydrolyzing Enzyme in Clinical Isolates of Citrobacter freundii and K. oxytoca Carrying a Common Plasmid

The Klebsiella pneumoniae carbapenemase (KPC) was detected in carbapenem-resistant isolates of Citrobacter freundii and Klebsiella oxytoca recovered from different patients in a Michigan hospital. Restriction analysis and hybridization with a KPC-specific probe showed the bla(KPC-2) genes of these two genera of the family Enterobacteriaceae are carried on a common plasmid.

Profile of microorganisms and antimicrobial resistance at a tertiary care referral burn centre in Iran: Emergence of Citrobacter freundii as a common microorganism

Infection and antimicrobial resistance are important issues in severe burn. The aims of this prospective study were to investigate the profile of microorganisms and resistance to antimicrobial agents in a tertiary referral burn centre; 113 people aged >10 years, with partial- or full-thickness burns, were included in the study. A total of 733 samples including 275 swabs, 164 tissue biopsies, 258 urine samples, 26 blood samples and 10 sputum samples were collected, from which 124 microorganisms were isolated. Pseudomonas aeruginosa and Citrobacter spp were the most prevalent isolates (57.3% and 35.5%, respectively); 95.5% of Citrobacter. isolates were Citrobacter freundii. Antibiogram results obtained from 15 antimicrobial agents demonstrated that imipenem was the most effective agent against P. aeruginosa, followed by ciprofloxacin and piperacillin/tazobactam (67.9%, 43% and 37.5% sensitive, respectively). At least 60% of all Citrobacter isolates were sensitive to various antimicrobial agents, the highest sensitivity being obtained by ceftazidime and cefepime (81.6% and 78.4%, respectively). Sensitivity of P. aeruginosa isolates to the agents of each antimicrobial class was significantly different ( p < 0.001). The incidence of C. freundii and the resistance of P. aeruginosa to anti-pseudomonas agents were exceptionally high.

Isolation and characterization of tetracycline-resistant Citrobacter spp. from catfish

Fifty-two tetracycline-resistant Citrobacter spp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 38 of the 52 citrobacters were Citrobacter freundii, 7 were C. amalonaticus and 7 were C. braakii. All isolates were resistant to multiple antibiotics. Polymerase chain reaction (PCR) protocols were developed to detect the presence of 3 tetracycline-resistance genes ( tetA, tetB and tetG) from Citrobacter isolates. Oligonucleotide primers specifically targeting a 967-bp region of tetB successfully amplified the PCR amplicons from 32 38 (85.0%) of C. freundii strains, 5 7 (71.0%) of C. amalonaticus and 4 7 (57%) from C. braakii. Oligonucleotide primers specific for the detection of tetA gene amplified the 417-bp PCR amplicons from 7 38 (18.0%) of tetracycline-resistant C. freundii only. The assay failed to amplify tetA genes from C. brakii or C. amalonaticus. Plasmids (2.0–16.0 kb) were isolated from 14 of the 38 strains of C. freundii. Strains of C. amalonaticus and C. brakii did not contain any plasmids. Dendrogram analysis of the SpeI pulsed field gel electrophoresis (PFGE) results identified 23 distinct macrorestriction patterns (mrps) among the 36 strains of C. freundii, 3 distinct mrps among the 7 strains of C. braakii and 4 unique mrps among the 7 strains of C. amalonaticus. Our results indicate that citrobacters from catfish could serve as reservoirs of tetracycline-resistance determinants.

Phenotypic and phylogenic groups to evaluate the diversity of Citrobacter isolates from activated biomass of effluent treatment plants

The diversity of Citrobacter isolated from effluent treatment plants (ETPs) was studied using three different parameters. Thirty Citrobacter strains were isolated from different ETPs treating wastewaters generated at various industries. All the isolates were characterized based on biochemical tests, antibiotic assay/functional analysis, and phylogenetic analysis. Results demonstrated that the pattern of grouping varied based on the selected criteria for analysis. Species that clustered together by biochemical analysis were found to vary by functional and 16S rDNA analysis and vice versa. This suggests that multiple methods approach needs to be carried out to understand the microbial diversity. Bacteria in effluent treatment plants are exposed to diverse categories of pollutants. Salicylate is a key intermediate formed during biodegradation of several aromatic compounds, a scenario expected in ETPs. Hence, the Citrobacter isolates were screened for their capability to utilize salicylate. In future studies, these isolates can be incorporated in a bioremediation program.

Isolation of Salmonella Bacteriophages from Swine Effluent Lagoons

Bacteriophages (phages) associated with Salmonella were collected from nine swine manure lagoons in Mississippi. Phages were isolated by an enrichment protocol or directly from effluent. For enrichment, chloroform-treated samples were filtered (0.22 mum) and selectively enriched by adding a cocktail of Salmonella strains in trypticase soy broth. After overnight incubation at 35 degrees C, chloroform was added and samples stored at 5 degrees C. Enriched samples were tested by double agar layer (DAL) plaque assay against individual Salmonella isolates. Phage titers of 2.9 x 10(8) to 2.1 x 10(9) plaque forming units (pfu) per mL were produced, but estimation of phage titers in lagoons was not possible. For direct isolation, effluent was clarified by centrifugation, filtered (0.22 microm), and used in DAL plaque assays to select single-plaque isolates for 15 Salmonella strains. Plaque counts varied among Salmonella strains and lagoons. The most sensitive strain for direct phage recovery was ATCC 13311. Phage titers estimated by direct isolation with ATCC 13311 ranged among lagoons from 12 to 148 pfu per mL. In limited host range tests, 66 isolates recovered by the enrichment protocol produced plaques only on Enteritidis and Typhimurium strains of Salmonella and none produced plaques on lagoon isolates of Citrobacter, Escherichia, Proteus, Providencia, or Serratia. Electron microscopy (EM) showed purified enrichment isolates had Podoviridae morphology (tailless 50-nm icosahedral heads with tail spikes). Electron microscopy of clarified concentrated effluent showed 5.5:1 tailless to tailed phages. The isolated phages have potential as typing reagents, specific indicators, and biocontrol agents of Salmonella.

Distribution of a transposon-like element carrying blaCMY-2 among Salmonella and other Enterobacteriaceae

The dissemination of cephamycin resistance in Enterobacteriaceae and its correlation with a transposon-like DNA element consisting of a specific tnpA-bla(CMY-2)-blc-sugE structure were investigated. A total of 140 enterobacterial isolates belonging to 17 species (10 genera) of Enterobacteriaceae phenotypically characterized as putative AmpC-producers were evaluated. The isolates were examined by PCR analysis, DNA-DNA hybridization and nucleotide sequencing. The bla(CMY-2)-carrying element was detected in 34 isolates from 10 species (9 genera), including all 14 Salmonella and 4 Shigella isolates as well as 7 of the 10 Escherichia coli isolates tested. The remaining 9 isolates were from 112 isolates of the other 14 species tested. The genetic structure of the bla(CMY-2)-carrying element was identical in 29 isolates, while in 3 E. coli and 2 Citrobacter isolates an additional insertion sequence IS1 was found inserted at various nucleotide positions close to the 3' end, either within or downstream, of tnpA. In 12 of the 14 representative isolates examined, the bla(CMY-2)-carrying element was found inserted in the finQ gene of various-sized plasmids with highly conserved 8 bp direct repeats flanking the junction regions. Among the other 106 non-CMY-2-producing isolates, plasmid-mediated ampC genes were found only in one isolate of Enterobacter aerogenes which carried a bla(DHA-1)-like gene. bla(CMY-2) is the most prevalent plasmid-mediated ampC gene among Enterobacteriaceae. All the bla(CMY-2) genes identified in the present study were associated with a specific transposon-like element that may be responsible for the spread of bla(CMY-2) among Enterobacteriaceae.

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.

Type 1 pili of Citrobacter freundii mediate invasion into host cells.

Citrobacter freundii, originally designated Salmonella ballerup, are Gram-negative, motile rod-shaped bacteria of the family Enterobacteriaceae. They are wide spread in nature and can be found in the environment in soil and water as well as in foodstuffs. Citrobacter freundii was also isolated from a wide variety of animals like household pets, birds, cattle and fish. Although C. freundii is often considered a commensal of the human intestinal flora, this organism may cause urinary tract infections, diarrhea and severe cases of gastritis, wound infections and nosocomial infections like pneumonia and rarely meningitis in newborns. Several probable virulence factors have been identified in C. freundii. Citrobacter isolates are often multidrug resistant. In addition, some of the corresponding antibiotic resistance genes (e.g. tetracycline, chloramphenicol, streptomycin) are located on transferable plasmids. The production of toxins has also been reported for C. freundii strains. Isolates from humans and beef samples were positive for the production of Shiga(-like) toxin II. This toxin is much likely involved in enteropathogenicity caused by C. freundii. Futhermore, strains from patients with diarrhea produced a heat-stable enterotoxin identical to the 18-amino-acid Escherichia coli heat-stable enterotoxin STIa. Another probable virulence factor is a capsule, which is closely related to the Vi capsule of Salmonella typhi. Finally, invasion ability for several cell lines has been observed. Invasiveness in vivo would provide C. freundii with several advantages. Intracellular localization would result in protection from the host’s (humoral) immune system, from the action of many antibiotics and could therefore prolonge the infection time. Invasion ability could also

Evaluation of the in vitro antimicrobial activity of cefepime compared to other broad-spectrum β-lactams tested against recent clinical isolates from 10 Chinese hospitals

A surveillance study was initiated in China in 1998 in which 10 medical centers participated. The susceptibility profiles of 996 commonly occurring pathogens belonging to 10 different species groups were tested by the Etest (AB BIODISK, Solna, Sweden) against six broad-spectrum β-lactam antimicrobial agents (cefepime, ceftazidime, ceftriaxone, imipenem, cefopera-zone/sulbactam and piperacillin or oxacillin). Quality control was closely monitored and cefepime- and/or imipenem -resistant Enterobacteriaceae were referred to the reference laboratory (University of Iowa College of Medicine, Iowa City, IA) for confirmation. The isolates of Citrobacter spp. and Enterobacter spp. were generally inhibited by imipenem (100% susceptible) and cefepime (89–94%), but were more resistant to the other drugs tested (≤74% susceptible). The indole-positive Proteus spp. and Serratia spp. isolates were >94% susceptible to all tested β-lactams except piperacillin. Organisms capable of producing extended spectrum β-lactamases (ESBLs), which included Klebsiella spp. and Escherichia coli, were most susceptible to imipenem (100%) and cefepime (>90%). Among the non-enteric Gram-negative bacilli, all drugs were marginally active against Pseudomonas aeruginosa (MIC 90s, 32- >256 ug/mL) and the Acinetobacter spp. were rather resistant to all the compounds, except imipenem (96% susceptible). All strains of Staphylococcus spp. were susceptible to the tested antimicrobials except for ceftazidime, which had a low potency (MIC 90, 12–16 μg/mL) against Chinese isolates with MICs that fell into the intermediate category. Cefepime, the fourth-generation cephalosporin, showed a very broad spectrum of activity against Gram-negative pathogens as well as oxacillin-susceptible Staphylococcus spp. that was comparable with imipenem (widest spectrum) and superior to the other tested β-lactams overall. Continued monitoring of clinical strains in China seems necessary to guide chemotherapy.

Citrobacter urinary tract infections in children

Citrobacter species have been described as the etiologic agents in cases of bacteremia, meningitis, diarrhea and brain abscess, but little is known of their role as a cause of urinary tract infections in children. The purpose of this study was to define the role of Citrobacter species in pediatric urinary tract infections. The project consisted of a retrospective chart review of microbiologic and medical records of patients younger than 18 years of age with urine cultures positive for Citrobacter species during a 3-year period. Thirty-four patients with 37 infections were included in the review. The average patient age was 6.9 years (range, 1 month to 18 years) and 71% were female. Fifty-six percent of the patients had urinary tract/renal anomalies or neurologic impairment and 26% represented nosocomial infections. Thirty-seven percent of patients were asymptomatic at the time of diagnosis, whereas 63% complained of at least one of the following findings: gastrointestinal symptoms; dysuria; fever; incontinence; penile/vaginal discharge; frequency; flank pain; and hematuria. Twenty-six of the isolates were Citrobacter freundii and 11 were Citrobacter koseri. Blood cultures were obtained in 9 patients and all were negative for Citrobacter isolates. Although it is uncommon Citrobacter can cause urinary tract infections in the pediatric population, which occur more frequently in children with underlying medical conditions. It appears that treatment similar to that of other gram-negative enteric organisms is the most prudent approach to these children until more information can be gathered.

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.

Differentiation and susceptibility of Citrobacter isolates from patients in a university hospital.

OBJECTIVE: Recently a publication of Brenner et al. introduced 11 genetically distinct species within the genus Citrobacter. These newly recognized Citrobacter species can be classified by means of their biochemical characteristics. The aim of this study was to examine the distribution and susceptibility of Citrobacter isolates in our patient population. METHODS: A total of 126 samples---containing a Citrobacter species---was collected from 116 hospitalized patients during a 6-month period. Organisms were identified according to standard procedures. Antimicrobial susceptibility testing was performed by agar dilution on Mueller-Hinton agar, and interpretation was based on NCCLS criteria. RESULTS: C. freundii was the most common organism isolated (n=59), followed by C. braakii (n=25) and C. koseri (n=23). The urinary tract and the respiratory tract were found to be the predominant sites of colonization or infection, accounting for 45% and 32% of all isolates respectively. It appeared that young children (<12 months old) and the elderly were most at risk of acquiring Citrobacter. Two-thirds of all specimens contained other organisms in addition to Citrobacter. Most Citrobacter isolates were related with a predisposing factor. Species-related differences were found in the susceptibility pattern. CONCLUSIONS: These findings suggest that citrobacteria are important opportunistic pathogens contributing to colonization or infection in our hospital population.

Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im [corrected] associated with a sulI-type integron.

The amikacin resistance gene aac(6')-Im [corrected] from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron. The sequence of this aac(6')-Im [corrected] gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C. Tenover, D. Filpula, K.L. Phillips, and J. J. Plorde, J. Bacteriol. 170:471-473, 1988). By using PCR, the aac(6')-Im [corrected] gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli. PCR mapping of the aac(6')-Im [corrected] gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.

Iron utilization studies in Citrobacter species.

Seventy-one strains of Citrobacter were screened for iron scavenging mechanisms by biologic and chemical assays. Essentially all citrobacteria (70/71) were found to elaborate enterobactin-like siderophores by both biologic and chemical assays, however only c. koseri (C. diversus) was found to produce aerobactin. The concentration of ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) required to inhibit the growth of individual Citrobacter strains by depleting free iron ranged from 250 micrograms/ml to 100 micrograms/ml. Iron utilization studies of selected citrobacter isolates indicated that hemin and hematin could reverse the effects of iron limitation on growth under iron-stressed conditions (1000 micrograms/ml of EDDA). Two C. koseri strains grown under iron-restricted conditions showed similar changes in their whole cell protein profiles including induction of high molecular mass proteins (72-83 kDa) which may play a role in iron acquisition under iron-stressed conditions. The collective results support an additional virulence-associated mechanism for C. koseri strains which may help explain the greater pathogenic potential this group has for causing serious extraintestinal disease in humans.

Iron utilization studies in Citrobacter species

Seventy-one strains of Citrobacter were screened for iron scavenging mechanisms by biologic and chemical assays. Essentially all citrobacteria ( 70 71 ) were found to elaborate enterobactin-like siderophores by both biologic and chemical assays, however only C. koseri (C. diversus) was found to produce aerobactin. The concentration of ethylenediamine di( o-hydroxyphenylacetic acid) (EDDA) required to inhibit the growth of individual Citrobacter strains by depleting free iron ranged from 250 μg/ml to 1000 μg/ml. Iron utilization studies of selected Citrobacter isolates indicated that hemin and hematin could reverse the effects of iron limitation on growth under iron-stressed conditions (1000 μg/ml of EDDA). Two C. koseri strains grown under iron-restricted conditions showed similar changes in their whole cell protein profiles including induction of high molecular mass proteins (72–83 kDa) which may play a role in iron acquisition under iron-stressed conditions. The collective results support an additional virulence-associated mechanism for C. koseri strains which may help explain the greater pathogenic potential this group has for causing serious extraintestinal disease in humans.

Heterogeneity at the -lactamase structural gene ampC amongst Citrobacter spp. assessed by polymerase chain reaction analysis: potential for typing at a molecular level

Considerable biochemical diversity and polynucleotide sequence variation have been reported amongst strains of Citrobacter spp. However, sequence heterogeneity has not been investigated at gene loci of clinical relevance. In this study, sequence heterogeneity in the beta-lactamase structural gene, ampC, amongst 91 clinical isolates of Citrobacter spp. that showed resistance to various third-generation cephalosporins was investigated. Variation was examined by high-stringency polymerase chain reactions (PCR) with primers homologous to the known ampC sequences of C. freundii strains OS60 and I113, and C. diversus NF85. If an isolate contained an ampC gene homologous to one of these three characterised ampC genes, a single PCR band of a predictable size was generated with the appropriate primer set; 50 (60%) of isolates gave a PCR product of the expected size with the OS60 primer set and nine (10%) gave a product with the I113 primer set. All these 59 isolates were identified as C. freundii by API-20E strips. Six isolates (7%) gave a product with the C. diversus NF85 primer set but only four of these were identified as C. diversus in API-20E tests; the other two isolates were identified as C. freundii. Of the 91 isolates, 28 (31%), were identified as either C. freundii or C. diversus, but gave no PCR product with any primer set tested. Five of these showed no homology to any of the reference strain ampC PCR products in hybridisation tests. Nevertheless, all showed beta-lactamase activity. Overall, this method allowed the identification of novel ampC gene loci, which may serve as a basis for the identification of Citrobacter spp. rapidly at a molecular level.

Biochemical identification of citrobacteria in the clinical laboratory

We biochemically identified 235 Citrobacter strains to the species level on the basis of the recently proposed taxonomic changes of Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993). Citrobacter isolates were initially identified as C. koseri or as members of the C. freundii complex or C. amalonaticus group on the basis of indole production, formation of H2S, malonate utilization, and acid production from D-arabitol and adonitol. On the basis of the results of these tests, 68% of the Citrobacter strains were identified as members of the C. freundii complex, 25% were C. koseri, and 8% were members of the C. amalonaticus group. By using a 15-test system recently proposed by Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993) to help identify new species in the C. freundii complex and C. amalonaticus group, 81% of the C. freundii complex strains and 100% of the C. amalonaticus strains could be definitively assigned to one of the previously established or recently designated species or hybridization groups of the genus Citrobacter. Within the C. freundii complex, C. freundii predominated overall (37%), followed by C. youngae (24%), C. braakii (13%), and C. werkmanii (6%). Only one strain each of C. sedlakii and Citrobacter DNA group 11 was identified in this study. Among C. amalonaticus complex members, all were identified as C. amalonaticus with the singular exception of one fecal isolate of C. farmeri. C. freundii and C. koseri were the two Citrobacter species most commonly (80 of 93 [86%]) isolated from extraintestinal sources (genitourinary tract, wounds, blood).