cfxA Gene Research Articles

Characterization of mobile genetic elements in multidrug-resistant Bacteroides fragilis isolates from different hospitals in the Netherlands

ObjectivesFive human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the Netherlands between 2014 and 2020 and sent to the anaerobic reference laboratory for full characterization. MethodsIsolates were recovered from a variety of clinical specimens from patients with unrelated backgrounds. Long- and short-read sequencing was performed, followed by a hybrid assembly to study the presence of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs). ResultsA cfxA gene was present on a transposon (Tn) similar to Tn4555 in two isolates. In two isolates a novel Tn was present with the cfxA gene. Four isolates harbored a nimE gene, located on a pBFS01_2 plasmid. One isolate contained a novel plasmid carrying a nimA gene with IS1168. The tetQ gene was present on novel conjugative transposons (CTns) belonging to the CTnDOT family. Two isolates harbored a novel plasmid with tetQ. Other ARGs in these isolates, but not on an MGE, were: cfiA, ermF, mef(EN2), and sul2. ARGs harboured differed between isolates and corresponded with the observed phenotypic resistance. ConclusionsNovel CTns, Tns, and plasmids were encountered in the five MDR B. fragilis isolates, complementing our knowledge on MDR and horizontal gene transfer in anaerobic bacteria.

Identification of cfxA gene variants and susceptibility patterns in β-lactamase-producing Prevotella strains

ObjectivesAntimicrobial-resistant isolates of Prevotella species, especially those resistant to β-lactams, have become increasingly common. Here, we aimed to elucidate the underlying mechanisms contributing to the emergence and spread of antimicrobial resistance in Prevotella species. MethodsPrevotella species were isolated from a variety of clinical specimens. β-lactamase production was determined using nitrocefin discs, and the determination of minimum inhibitory concentration (MIC) to ten antimicrobials was done by the agar dilution method. Four resistance genes (cfxA, tetQ, ermF, and nim) and cfxA-flanking regions were detected using polymerase chain reaction. cfxA and the flanking regions were sequenced, and a phylogenetic tree was constructed based on CfxA amino acid sequences using the UPGMA method. ResultsAmong the 45 Prevotella isolates identified, 35 (77.8%) produced β-lactamases and had the cfxA genes. The tetQ, ermF, and nim genes were detected in 53.3%, 17.8%, and 0% of the 45 isolates, respectively. Among the 33 sequenced cfxA alleles, cfxA2 (45.5%) was the most frequent, followed by cfxA3 (42.4%) and a novel variant (cfxA7, 12.1%). The novel CfxA7 β-lactamase had a novel L155F substitution not previously reported in CfxA variants. The MICs of all β-lactam agents tested, excluding cefmetazole and meropenem, were lower among cfxA7-positive isolates than in cfxA2-and cfxA3-positive isolates. ConclusionsDifferences in MICs of penicillins and cephalosporins may be due to amino acid substitutions in the CfxA variants, CfxA2, CfxA3, and CfxA7, among Prevotella isolates. Possession of cfxA-mobA, tetQ, and ermF may increase the risks of the emergence and spread of multidrug-resistant Prevotella species.

Antibiotic resistance and the presence of bla CfxA and bla CSP genes in β-lactamase-producing clinical Capnocytophaga isolates from a university hospital in Japan.

Introduction . Capnocytophaga species are common inhabitants of the oral cavity and can be responsible for systemic diseases in immunocompromised patients with granulocytopenia. Furthermore, it has been reported that some clinical isolates of Capnocytophaga species produce extended-spectrum β-lactamases (ESBLs).Gap statement. Information is lacking about the types of β-lactamase genes possessed by Capnocytophaga spp. and the antimicrobial susceptibility of Capnocytophaga spp. possessing each β-lactamase gene.Aim. The aim of this study was to investigate the presence of β-lactamase genes in clinical strains of β-lactamase-producing Capnocytophaga species isolated from clinical samples acquired at Shinshu University Hospital and examine the antimicrobial susceptibility of those strains.Methodology. The β-lactamase-producing Capnocytophaga species (n=49) were obtained from clinical specimens. PCR assays were used to detect bla CfxA, bla CSP, bla TEM, bla CepA/CblA and transposon Tn4555 genes. Southern hybridization assays were used to detect bla CfxA and bla CSP. The minimum inhibitory concentration of some β-lactams was determined using the E-test method.Results. PCR analysis indicated that the bla CfxA gene was present in 15 (30.6 %) and the bla CSP gene in 35 (69.3 %) of the 49 Capnocytophaga strains investigated, . Both bla CfxA and bla CSP genes were detected in a Capnocytophaga gingivalis strain. The PCR results were confirmed by Southern hybridization assays. Transposon Tn4555 was only detected in Capnocytophaga spp. harbouring the bla CfxA gene. All the β-lactamase-producing Capnocytophaga isolates were susceptible to ceftazidime-clavulanic acid, cefoxitin and imipenem. In contrast, most of the isolates were resistant to amoxicillin.Conclusions. The clinical isolates of Capnocytophaga spp. showed a high prevalence of the bla CSP gene in Japan. The presence of the bla CSP gene was distributed in Capnocytophaga sputigena as well as other Capnocytophaga spp. These results seem to suggest the dissemination of bla CfxA and bla CSP β-lactamase genes among Capnocytophaga species.

Antibiotic Resistance in Bacteroides and Prevotella to β-Lactams, Lincosamide and Nitroimidazole: A 20 Year Survey in Lagos, Nigeria

The propensity to develop resistance to antibiotics has accounted for the predominance of Bacteroides and Prevotella in infections due to anaerobic bacteria. The observed differences in resistance pattern across geographical boundaries underscored the timeliness for this study to review data on antibiotic susceptibility and resistance markers amongst Bacteroides and Prevotella to β-lactams, lincosamide and nitroimidazole from selected hospitals in Lagos, Nigeria from 1992-2011. Bacteroides, mostly B. fragilis were the sole anaerobic gram negative bacilli in peritonitis following lower abdominal surgery and bloodstream infections, while Prevotella bivia and P. melaninogenica were more frequent in periodontal abscess, pelvic inflammatory disease and chronic suppurative otitis media. The MIC range and MIC90 of the anaerobes for the antibiotics does not indicate changes in group resistance to antibiotics though isolated cases were recorded. While clear cut patterns were not established for other species, B. fragilis resistance increased for amoxicillin-clavulanic acid from 18.2% in 1992-1996 to 31.4% in 2006-2011 and from 27.3% in 1992-1996 to 34.3% in 2006-2011 for cefoxitin. In contrast, decrease susceptibility was obtained against clindamycin (54.5% in 1992-1996 to 22.9% in 2006-2011). Similarly, 22.7% of B. fragilis strains have the cepA and/or cfxA gene in 1992-1996 compared to 32.1% in 2006-2011. While not all isolates with cepA and/or cfxA genes were resistant to the β-lactams, all isolates carrying the ermF or nim genes were resistant to the lincosamide (clindamycin) or nitroimidazole (metronidazole) respectively. The presence of antibiotics resistance genes can be used in tracking resistance amongst species of Bacteroides and Prevotella. With emerging new technologies, gene screening may prove more effective and reliable for therapeutic decisions for anaerobic bacterial infections especially since the genes can be screened from the infective exudates. This will eliminate the need for culture and antibiotic susceptibility testing for this group of fastidious microorganisms.

Detection of beta-lactamase production in clinical Prevotella species by MALDI-TOF MS method

Penicillins, can be used in treatment of infections due to Prevotella species if they are susceptible to penicillin. Early and accurate preliminary detection of β-lactamase-producing isolates is crucial for treatment of infection. The aim of this study was to determine β-lactamase-producing Prevotella species by MALDI-TOF MS and screen them for the presence of cfxA gene, responsible for β-lactamase production. A total of 500 clinically relevant Prevotella isolates, collected from 13 countries for the previous European antibiotic resistance surveillance study, were tested. Susceptibility testing was performed against ampicillin and ampicillin/sulbactam by Etest methodology. EUCAST guidelines were used for susceptibility interpretations; the isolates with MIC value ≤ 0.5 for ampicillin were considered susceptible and >2 resistant. All Prevotella isolates, were tested for detection of β-lactamase activity by MALDI-TOF MS (Vitek® MS Research Use Only) system and the presence of the cfxA gene by PCR method. The susceptibility levels of the isolates to ampicillin/sulbactam and ampicillin were 99.6% and 43.4%, respectively. A total 59% of isolates presented β-lactamase activity and 60.8% were cfxA gene positive. Both these tests were positive for isolates in the resistant category. Additionally, >95% of the isolates (n = 65) which ampicillin MIC values ranged from >0.5 μg/mL to 2 μg/ml displayed β-lactamase activity. We also found that the MALDI-TOF MS-based β-lactamase assay delivers results in 2 h. We found a high concordance between the MALDI-TOF MS β-lactamase results in terms of cfxA β-lactamase gene presence. MALDI-TOF MS may serve as a simple and efficient alternative method of the existing phenotypic and PCR-based methods.

Fate of pirlimycin and antibiotic resistance genes in dairy manure slurries in response to temperature and pH adjustment

Quantifying the fate of antibiotics and antibiotic resistance genes (ARGs) in response to physicochemical factors during storage of manure slurries will aid in efforts to reduce the spread of resistance when manure is land-applied. The objectives of this study were to determine the effects of temperature (10, 35, and 55 °C) and initial pH (5, 7, 9, and 12) on the removal of pirlimycin and prevalence of ARGs during storage of dairy manure slurries. We collected and homogenized feces and urine from five lactating dairy cows treated with pirlimycin and prepared slurries by mixing manure and sterile water. Aliquots (200 mL) of slurry were transferred and incubated in 400 mL glass beakers under different temperatures (10, 35, and 55 °C) or initial pH (5, 7, 9, and 12). Pirlimycin concentration and abundances of 16S rRNA, mefA, tet(W), and cfxA as indicators of total bacteria and ARGs corresponding to macrolide, tetracycline, and β-lactam resistance, respectively, were analyzed during manure incubation. The thermophilic environment (55 °C) increased the deconjugation and removal of pirlimycin, while the acidic shock at pH 5 increased deconjugation but inhibited removal of pirlimycin, suggesting that the chemical stability of pirlimycin could be affected by temperature and pH. The thermophilic environment decreased mefA relative abundance on day 7 and 28 (P = 0.02 and 0.04), which indicates that the bacteria that encoded mefA gene were not thermotolerant. Although mefA relative abundance was greater at the pH 9 shock than the rest of pH treatments on day 7 (P = 0.04), no significant pH effect was observed on day 28. The tet(W) abundance under initial pH 12 shock was less than other pH shocks on day 28 (P = 0.01), while no temperature effect was observed on day 28. There was no significant temperature and initial pH effect on cfxA abundance at any time point during incubation, implying that the bacteria that carrying cfxA gene are relatively insensitive to these environmental factors. Overall, directly raising temperature and pH can facilitate pirlimycin removal and decrease mefA and tet(W) relative abundances during storage of manure slurries.

623. Dynamic Nature of the Gut Resistome Among Infants in Singapore

BackgroundThe gut microbiome harbors antibiotic resistance genes (ARGs), known as the resistome, that has the potential to spread and contribute to the global crisis of antibacterial resistance. Little is known about the genomic traits of the infant resistome, especially in areas with high endemic antibacterial resistance.MethodsWe analyzed ARGs among a subset of infants from the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) birth cohort. The subset included 75 mostly term, healthy Singaporean infants born from November 2009 to May 2011. Stool samples collected at Week 3 (W3), months 3 (M3), 6 (M6) and 12 (M12) were analyzed using shotgun metagenomics. Sequencing reads were assembled into contigs using MEGAHIT. ARGs were identified using ResFinder 2.1. Demographic, perinatal factors, pre- and postnatal antibiotic exposure were collected.ResultsOne hundred and eighty-eight stool samples from 75 infants were studied. Of the 169 ARGs detected, the four commonest ARGs were blaZ, fosA, tet(M) and mef(A), conferring resistance to β-lactams, fosfomycin, tetracyclines, and macrolides respectively. The number of ARGs per infant increased over time (median: W3 = 18.0, M12 = 22.0, P < 0.05). At W3, 118 ARGs were detected among 28 infants. The most prevalent ARGs were fos(A) and blaZ (both 96.4%) at W3. Among the 22 infants who had samples at W3 and M12, only six of 118 ARGs detected at W3 were also present at M12. These were mef(A), msr(D), tet(W), erm(B), tet(M) and fosA, conferring macrolide and tetracycline resistance. Their prevalence among at M12 was 100%, 93.3%, 90.9%, 84.6%, 68.8% and 52.4%, respectively. ARGs were not associated with gender, race, delivery mode, peripartum or postnatal antibiotics in infancy. Of note, longitudinal analysis showed that only the cfxA gene, which confers β-lactam resistance, was more prevalent in infants whose mothers received antibiotics in pregnancy than those whose mothers did not (adjusted P < 0.05).ConclusionIn regions with high endemic antimicrobial resistance such as Singapore, the infant gut harbors a diversity of ARGs as early as 3 weeks of age. Few ARGs persisted through infancy, implying the dynamic nature of the infant resistome. The lack of association of ARGs with most clinical variables evaluated here suggests that other unrecognized factors may contribute to the plasticity of ARGs in the infant gut resistome.Disclosures R. Banerjee, Accelerate Diagnostics, Biomerieux, BioFire: Grant Investigator, Research grant and Research support.

Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes.

ABSTRACTPurpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics.Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM) and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases.Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the β-lactam antibiotics.Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure.

CfxA expression in oral clinical Capnocytophaga isolates

Capnocytophaga spp. are commensal bacteria involved in oral and systemic diseases, with a variable susceptibility to beta-lactams. The cfxA gene expression level was assessed using quantitative RT-PCR, and reasons of the observed misexpression were discussed, as insertion of foreign genetic material, contributing to dissemination and evolution of antibiotic resistance genes.

Function of glucose catabolic pathways in hydrogen production from glucose in Rhodobacter sphaeroides 6016

Purple non-sulfur (PNS) bacteria can convert volatile fatty acids into hydrogen with a high substrate conversion efficiency. However, when PNS bacteria utilize sugars as a carbon source, such as glucose and sucrose, the substrate conversion efficiency is relatively low. In order to investigate the contributions of the glucose catabolic pathways in Rhodobacter sphaeroides 6016 to its hydrogen production, the cfxA gene from the Embden–Meyerhof–Parnas (EMP) pathway, edd from the Entner–Doudoroff (ED) pathway, and kdg from the semi-phosphorylative ED bypass were knocked out to construct the mutant strains edd−, cfxA−, and kdg−, respectively. Additionally, two of these three genes were knocked out to construct the mutant strains kdg−edd−, kdg−cfxA−, and cfxA−edd−. Hydrogen productions by these mutant strains were compared to that of the wild type strain 6016 using 25 mM glucose as a carbon source. Compared to 6016, variations in hydrogen production and growth were detected in the edd mutant strains (kdg−edd−, cfxA−edd−, and edd−), while no obvious changes were detected in the others. Notably, the kdg−edd− mutant did not produce hydrogen, and its maximum growth was 70% less than that of R. sphaeroides 6016. These results indicate that the ED pathway and semi-phosphorylative ED bypass have a governing impact on cell growth and hydrogen production from glucose in R. sphaeroides 6016. The potential synergistic function of the ED pathway and semi-phosphorylative ED bypass and the reasons for the low hydrogen yield from sugar carbon sources in R. sphaeroides 6016 are discussed.

Antimicrobial Resistance and Prevalence of Resistance Genes of Obligate Anaerobes Isolated From Periodontal Abscesses

This study attempts to determine the antimicrobial resistance profiles of obligate anaerobic bacteria that were isolated from a periodontal abscess and to evaluate the prevalence of resistance genes in these bacteria. Forty-one periodontal abscess samples were cultivated on selective and non-selective culture media to isolate the oral anaerobes. Their antibiotic susceptibilities to clindamycin, doxycycline, amoxicillin, imipenem, cefradine, cefixime, roxithromycin, and metronidazole were determined using the agar dilution method, and polymerase chain reaction assays were performed to detect the presence of the ermF, tetQ, nim, and cfxA drug resistance genes. A total of 60 different bacterial colonies was isolated and identified. All of the isolates were sensitive to imipenem. Of the strains, 6.7%, 13.3%, 16.7%, and 25% were resistant to doxycycline, metronidazole, cefixime, and amoxicillin, respectively. The resistance rate for both clindamycin and roxithromycin was 31.7%. Approximately 60.7% of the strains had the ermF gene, and 53.3% of the amoxicillin-resistant strains were found to have the cfxA gene. Two nim genes that were found in eight metronidazole-resistant strains were identified as nimB. In the present study, the Prevotella species are the most frequently isolated obligate anaerobes from periodontal abscesses. The current results show their alarmingly high resistance rate against clindamycin and roxithromycin; thus, the use of these antibiotics is unacceptable for the empirical therapy of periodontal abscesses. A brief prevalence of four resistance genes in the anaerobic bacteria that were isolated was also demonstrated.

The prevalence of antibiotic resistance genes in Bacteroides fragilis group strains isolated in different European countries

From the 2008–2009 European Bacteroides antibiotic resistance survey, we selected 161 strains for detection of antibiotic resistance genes (cepA, cfxA, cfiA, nim, ermB, ermF, ermG, linA, mefA, msrSA, tetM, tetQ, tetX, tetX1, tet36 and bexA). To facilitate the throughput, the genes were detected by Real-Time PCR. The presence of the genes was correlated with the known MIC data of the strains for the appropriate antibiotics. For the β-lactams, the cepA gene was found in 70.8% of the tested strains (all resistant to ampicillin), but its presence did not correlate with the ampicillin MIC values. The cepA gene occurred at different frequencies among Bacteroides fragilis and non-fragilis Bacteroides strains. The cfxA gene was not a major factor in determining cefoxitin resistance and it was found with higher prevalence in non-fragilis Bacteroides strains than in B. fragilis. Among the five possible clindamycin resistance genes, ermF was the most common and had the highest effect on clindamycin resistance after linA. The ermG-mefA-msrSA combination was found in a set of strains and their linked occurrence implied that they were harbored by the conjugative transposon CTnGERM1. All strains tested were susceptible to metronidazole and none of them harbored nim genes. TetQ was prevalent among both the B. fragilis and non-fragilis Bacteroides strains (78.9 and 84.8%, respectively) and no gene could be clearly linked to tigecycline resistance other than tetQ. BexA, which codes for the fluoroquinolone efflux pump, was found in 7.5% of strains and occurred at different frequencies among B. fragilis and non-fragilis Bacteroides strains, but was represented only in a minor proportion of moxifloxacin-resistant strains.

Molecular analysis of the effector mechanisms of cefoxitin resistance among Bacteroides strains

The characterization of Bacteroides strains with regard to the cfxA gene, the MTn4555 mobilizable transposon, the role of penicillin-binding proteins (PBPs) and heterogeneous cefoxitin resistance. Eighty-four randomly selected and 11 heterogeneously or highly cefoxitin-resistant Bacteroides isolates were included. Agar dilution and Etest methods were used for the determination of cefoxitin MICs. PCR experiments and nucleotide sequencing were used to detect the cfxA gene and the molecular features of MTn4555. Cefoxitin-binding experiments to determine its affinity (IC(50)) for PBPs and cefoxitinase assays were also applied. Southern blotting was used to determine the copy number of the cfxA genes. Sixteen strains from the random collection proved to be positive for cfxA, and the MIC distribution for the cfxA-negative and -positive strains did not display a clear separation. The majority of the cfxA-positive strains in this collection harboured a 1.2 kb common region at the 3' end of MTn4555. This region encoded an open reading frame that exhibited homology to abortive phage infection proteins (AbiD). The cfxA genes were transferable only at low frequencies in conjugation experiments. In PBP affinity studies, the PBP-A and PBP3 species were largely insensitive to cefoxitin, whereas the other PBP species were affected at very low concentrations. Seven of the heterogeneously resistant strains were positive for cfxA and most of them had mutations in the regulatory regions of cfxA. Major and minor roles for Bacteroides fragilis PBPs and the CfxA cefoxitinase, respectively, were inferred. The role of the newly recognized abiD may be to control the copy number of cfxA.

Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections

To identify genetic determinants that determine beta-lactamase expression in Bacteroides strains isolated from human infections. Beta-lactam susceptibility and beta-lactamase enzyme expression were characterized in selected strains. Beta-lactamase genes and surrounding regions were analysed by PCR, inverse PCR and Southern hybridization. High resistance to penicillins and cephalosporins was found among most isolated strains, in which all known beta-lactamase genes from Bacteroides are represented, but differences were found in their expression of enzyme activity. In contrast to the cepA gene, ubiquitously found but frequently inactive, or cfiA, which only confers carbapenem resistance in two strains, the detection of high beta-lactamase expression correlates closely with the presence of cfxA genes. This genetic determinant shares variability of upstream regulatory elements, including sequence tags from Tn4555, Tn4351 and IS614B, and polymorphisms of encoded amino acid sequences at positions G(57)C and Y(259)C, which might determine enzyme expression characteristics. The main determinant for beta-lactamase expression in Bacteroides strains is the cfxA gene, in which IS614B integration upstream of the coding sequence represents a molecular marker for higher levels of enzyme activity.

Association Between the cfxA Gene and Transposon Tn4555 in Bacteroides distasonis Strains and Other Bacteroides Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of beta-lactamases is the most important resistance mechanism including cephalosporin resistance to beta-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum beta-lactamase responsible for widespread resistance to cefoxitin and other beta-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.

Characterization of plasmid pOR1 from Ornithobacterium rhinotracheale and construction of a shuttle plasmid.

The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A β-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 μg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other β-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.

Identification of a circular intermediate in the transfer and transposition of Tn4555, a mobilizable transposon from Bacteroides spp

Transmissible cefoxitin (FX) resistance in Bacteroides vulgatus CLA341 was associated with the 12.5-kb, mobilizable transposon, Tn4555, which encoded the beta-lactamase gene cfxA. Transfer occurred by a conjugation-like mechanism, was stimulated by growth of donor cells with tetracycline (TC), and required the presence of a Bacteroides chromosomal Tcr element. Transconjugants resistant to either FX, TC, or both drugs were obtained, but only Fxr Tcr isolates could act as donors of Fxr in subsequent matings. Transfer of Fxr could be restored in Fxr Tcs strains by the introduction of a conjugal Tcr element from Bacteroides fragilis V479-1. A covalently closed circular DNA form of Tn4555 was observed in donor cells by Southern hybridization, and the levels of this circular transposon increased significantly in cells grown with TC. Both the cfxA gene and the Tn4555 mobilization region hybridized to the circular DNA, suggesting that this was a structurally intact transposon unit. Circular transposon DNA purified by CsCl-ethidium bromide density gradient centrifugation was used to transform Tcs B. fragilis 638, and Fxr transformants were obtained. Both the circular form and the integrated Tn4555 were observed in transformants, but the circular form was present at less than one copy per chromosomal equivalent. Examination of genomic DNA from Fxr transformants and transconjugants revealed that Tn4555 could insert at a wide variety of chromosomal sites. Multiple transposon insertions were present in many of the transconjugants, indicating that there was no specific barrier to the introduction of a second transposon copy.

Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides

In Rhodobacter sphaeroides, many of the structural genes encoding enzymes of the Calvin cycle are duplicated and grouped within two separate clusters. In this study, the nucleotide sequence of a 5627-base pair region of DNA that contains the form I Calvin cycle gene cluster has been determined. The five open reading frames are arranged in the order, fbpA prkA cfxA rbcL rbcS and are tightly linked and oriented in the same direction. The results of insertional mutagenesis studies suggest the genes are organized within an operon. Consistent with this proposal, the cfxA gene has been tentatively identified as a gene encoding the Calvin cycle enzyme, aldolase. Measurement of the activities of various Calvin cycle enzymes in the insertion mutants showed that inactivation of genes within one CO2 fixation cluster affected expression of genes within the second cluster, revealing a complex regulatory network.