Characterization of MLS(b) resistance among Staphylococcus aureus and Staphylococcus epidermidis isolates carrying different SCCmec types

This work characterizes MLS(b) resistance in 39 methicillin-resistant Staphylococcus aureus (MRSA) and 32 Staphylococcus epidermidis (MRSE) isolates. Of 21 erm(A) gene encoding MRSA isolates, 71.4% carried SCCmecIII, whereas of 12 isolates carrying the erm(C) gene, 83.3% carried SCCmecIV. Among the 25 MRSE isolates positive for the erm(C) gene, 80% had SCCmecIV or nontypeable cassettes. Isolates carrying these genes had MIC(90) ≥ 256 μg/mL to erythromycin and clindamycin. The msr(A) gene was associated with a low MIC(90) to these drugs. The erm(A) gene was associated with SCCmecIII in MRSA isolates, whereas the erm(C) gene was associated with SCCmecIV in both MRSA and MRSE isolates.     

Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals

Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.     

The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity

The bacA gene, the overexpression of which results in bacitracin resistance, was inactivated and shown to be non-essential for growth of Escherichia coli. It was proposed earlier that the bacA gene product may confer resistance to the antibiotic by phosphorylation of undecaprenol (Cain, B. D., Norton, P. J., Eubanks, W., Nick, H. S., and Allen, C. M. (1983) J. Bacteriol. 175, 3784-3789). In the present work, this extremely hydrophobic membrane protein was overproduced and purified to near homogeneity. The analysis of its catalytic properties clearly demonstrated that the purified BacA protein exhibited undecaprenyl pyrophosphate phosphatase activity but not undecaprenol phosphokinase activity. This finding was perfectly consistent with the mechanism of action of bacitracin that consists in the sequestration of undecaprenyl pyrophosphate, the BacA enzyme substrate. The level of undecaprenyl pyrophosphate phosphatase was increased by 280-fold in cells carrying bacA on a multicopy expression plasmid. It was decreased by approximately 75% but was not completely abolished in a bacA disruption mutant, suggesting that BacA is the main E. coli undecaprenyl pyrophosphate phosphatase but that other protein(s) exhibiting such an activity should exist to account for the residual activity and viability of the mutant strain. This is the first gene encoding undecaprenyl pyrophosphate phosphatase identified to date. Considering its newly identified function, we propose to rename the bacA gene uppP.     

A newly isolated Streptomyces sp. CS392 producing three antimicrobial compounds

With the aim of isolating new microbes capable of producing strong antimicrobial substances, strain CS392 was screened from 700 soil isolates preserved in our laboratory. The strain was related to genus Streptomyces based on various characteristics. Three highly active antimicrobial compounds, C1, C2 and C3, produced by the strain were purified by solvent extraction followed by silica gel column chromatography. These compounds were highly active against various Gram-positive resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococcus (VRE). Among three, C3 was the most active against MRSA and VRSA with minimal inhibitory concentration (MIC) of 2 μg/ml while C2 and C3 had MIC values of 4 μg/ml for the strains. In case of Bacillus subtilis ATCC6633, C1 and C3 were more effective with MIC values of 0.5 μg/ml than C2 with MIC of 2 μg/ml. Those antibiotics were variably active (MIC of 4-32 μg/ml) against Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Mycobacterium smegmatis ATCC 9341 and VRE.     

Regulation of the Bacillus subtilis bcrC bacitracin resistance gene by two extracytoplasmic function sigma factors

Bacitracin resistance is normally conferred by either of two major mechanisms, the BcrABC transporter, which pumps out bacitracin, or BacA, an undecaprenol kinase that provides C(55)-isoprenyl phosphate by de novo synthesis. We demonstrate that the Bacillus subtilis bcrC (ywoA) gene, encoding a putative bacitracin transport permease, is an important bacitracin resistance determinant. A bcrC mutant strain had an eightfold-higher sensitivity to bacitracin. Expression of bcrC initiated from a single promoter site that could be recognized by either of two extracytoplasmic function (ECF) sigma factors, sigma(X) or sigma(M). Bacitracin induced expression of bcrC, and this induction was dependent on sigma(M) but not on sigma(X). Under inducing conditions, expression was primarily dependent on sigma(M). As a consequence, a sigM mutant was fourfold more sensitive to bacitracin, while the sigX mutant was only slightly sensitive. A sigX sigM double mutant was similar to a bcrC mutant in sensitivity. These results support the suggestion that one function of B. subtilis ECF sigma factors is to coordinate antibiotic stress responses.     

In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics

Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves. All were resistant to 64 μg ml⁻¹ of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC₉₀ 0.25 μg ml⁻¹), avilamycin (MIC₉₀ 0.5 μg ml⁻¹) and salinomycin (MIC₉₀≤ 0.12 μg ml⁻¹). Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated. Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl. perfringens isolates from the same animal host species.     

Molecular typing and antimicrobial susceptibility of Clostridium perfringens from broiler chickens

Clostridium perfringens (Cp) causes necrotic enteritis disease in commercial poultry. Antimicrobials are used to control and treat this disease and sometimes clinical outbreaks do not respond well to certain treatments. This study was designed to isolate Cp from clinical cases, type these isolates by multiplex PCR, and determine their antimicrobial susceptibility by micro-dilution method. A total of 67 Cp isolates were obtained from 155 broiler chicken flocks. All isolates were classified as type A and non-enterotoxin producers. Lincomycin, erythromycins, and tilmicosin showed very high minimal inhibitory concentration (MIC) 50 of ≥256 μg/ml. However, tylosin, amoxicillin, ampicillin, penicillin, florfenicol, danofloxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline had variable MIC?? of 64, 0.5, 1, 1, 8, 4, 8, 4, 8, 0.5 μg/ml, respectively. It is recommended that Cp infections in Jordan be treated with either penicillins or tetracyclines especially amoxicillin and oxytetracycline.     

YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis

The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.     

Prevalence, development, and molecular mechanisms of bacteriocin resistance in Campylobacter

Bacteriocins (BCNs) are antimicrobial peptides produced by bacteria with narrow or broad spectra of antimicrobial activity. Recently, several unique anti-Campylobacter BCNs have been identified from commensal bacteria isolated from chicken intestines. These BCNs dramatically reduced C. jejuni colonization in poultry and are being directed toward on-farm control of Campylobacter. However, no information concerning prevalence, development, and mechanisms of BCN resistance in Campylobacter exists. In this study, susceptibilities of 137 C. jejuni isolates and 20 C. coli isolates to the anti-Campylobacter BCNs OR-7 and E-760 were examined. Only one C. coli strain displayed resistance to the BCNs (MIC, 64 μg/ml), while others were susceptible, with MICs ranging from 0.25 to 4 μg/ml. The C. coli mutants resistant to BCN OR-7 also were obtained by in vitro selection, but all displayed only low-level resistance to OR-7 (MIC, 8 to 16 μg/ml). The acquired BCN resistance in C. coli could be transferred at intra- and interspecies levels among Campylobacter strains by biphasic natural transformation. Genomic examination of the OR-7-resistant mutants by using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic resistance and acquired resistance to the BCNs. Altogether, this study represents the first report of and a major step forward in understanding BCN resistance in Campylobacter, which will facilitate the development of effective BCN-based strategies to reduce the Campylobacter loads in poultry.     

长沙地区临床分离金黄色葡萄球菌的耐药监测

目的了解长沙地区临床分离金黄色葡萄球菌（以下简称金葡菌）对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林的耐药水平。方法收集长沙地区11家医院2009年11月至2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌（MRSA）,琼脂稀释法检测头孢西丁和苯唑西林的最低抑菌浓度（MIC）。结果在被检测的24种药物中,敏感率〉50%的药物为9种,未发现对万古霉素、替考拉宁和利奈唑胺耐药菌株;耐药率〉50%的抗菌药物有11种,其中以青霉素和氨苄西林的耐药率最高（均为97.1%）。MRSA的分离率达54.5%,且对常用的16种抗菌药物的耐药率均显著高于甲氧西林敏感金黄色葡萄球菌（MSSA）。279株金葡菌中,β-内酰胺酶阳性250株（89.6%）;红霉素耐药而克林霉素敏感或中介的30株中,D试验阳性22株（73.3%）。苯唑西林（OXA）和头孢西丁（FOX）MIC范围分别为0.125～〉256μg/mL和2～〉256μg/mL,苯唑西林的MIC50和MIC90分别为128μg/mL和256μg/mL,头孢西丁的MIC50和MIC90分别为64μg/mL和256μg/mL。结论长沙地区临床分离金葡菌对常用抗菌药物呈多重耐药;MRSA不仅分离率高,而且对甲氧西林呈高水平耐药。     

R-Plasmids in Pasteurella multocida.

Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 r_k^?m_k^?. Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm³, while the density of pSR11 was 1.709 g/cm³.     

代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌 高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素,目前作为兽药广泛应用于畜禽养殖领域。前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素。文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,研究l-半胱氨酸供给模块强化对杆菌肽合成的影响。首先,构建了l-半胱氨酸合成酶基因cysK强化表达菌株,杆菌肽效价相比于对照菌株提高了9.47%。接着,为提高l-半胱氨酸合成前体供给,对l-丝氨酸乙酰转移酶基因cysE和硫代硫酸盐/硫酸盐胞内转运蛋白基因cysP进行强化,杆菌肽产量分别提高了7.23%和8.52%。随后,结果表明转运蛋白TcyP负责从胞外向胞内转运胱氨酸,强化表达TcyP后胞内l-半胱氨酸浓度和杆菌肽效价分别提高了29.19%和7.79%。通过组合代谢工程育种,在整合表达了基因cysK基础上,利用强启动子PbacA分别替换基因cysP、cysE和tcyP原始启动子,得到工程菌株CYS4 (DW2::cysK-PbacA(cysP)- PbacA(cysE)-PbacA(tcyP)),杆菌肽效价达到910.02 U/mL,相比于出发菌株DW2 (747.71 U/mL) 提高了21.10%。最后,通过3 L发酵罐小试实验,进一步证实了强化l-半胱氨酸有利于杆菌肽合成。研究表明,强化胞内l-半胱氨酸供给水平是提高地衣芽胞杆菌中杆菌肽产量的有效策略,为杆菌肽工业生产提供了一株具有良好应用前景的菌株。     

Identification of the Clostridium perfringens genes involved in the adaptive response to oxidative stress

Clostridium perfringens is a ubiquitous gram-positive pathogen that is present in the air, soil, animals, and humans. Although C. perfringens is strictly anaerobic, vegetative and stationary cells can survive in a growth-arrested stage in the presence of oxygen and/or low concentrations of superoxide and hydroxyl radicals. Indeed, it possesses an adaptive response to oxidative stress, which can be activated in both aerobic and anaerobic conditions. To identify the genes involved in this oxidative stress response, C. perfringens strain 13 mutants were generated by Tn916 insertional mutagenesis and screened for resistance or sensitivity to various oxidative stresses. Three of the 12 sensitive mutants examined harbored an independently inserted single copy of the transposon in the same operon as two genes orthologous to the ydaD and ycdF genes of Bacillus subtilis, which encode a putative NADPH dehydrogenase. Complementation experiments and knockout experiments demonstrated that these genes are both required for efficient resistance to oxidative stress in C. perfringens and are probably responsible for the production of NADPH, which is required for maintenance of the intracellular redox balance in growth-arrested cells. Other Tn916 disrupted genes were also shown to play important roles in the oxidative stress response. This is the first time that some of these genes (e.g., a gene encoding an ATP-dependent RNA helicase, the beta-glucuronidase gene, and the gene encoding the atypical iron sulfur prismane protein) have been shown to be involved in the oxidative response.     

Natural transformation-mediated transfer of erythromycin resistance in Campylobacter coli strains from turkeys and swine

Erythromycin resistance in Campylobacter coli from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli strains from turkeys than in swine-derived strains (P < 0.01). The frequency of transformation to erythromycin resistance was 10(-5) to 10(-6) for turkey-derived strains but 10(-7) or less for C. coli from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 microg/ml), as did the C. coli donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 microg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals.     

Complete genome sequence of highly multidrug-resistant Pseudomonas aeruginosa NCGM2.S1, a representative strain of a cluster endemic to Japan

We report the completely annotated genome sequence of Pseudomonas aeruginosa NCGM2.S1, a representative strain of a cluster endemic to Japan with a high level of resistance to carbapenem (MIC ≥ 128 μg/ml), amikacin (MIC ≥ 128 μg/ml), and fluoroquinolone (MIC ≥ 128 μg/ml).     

Natural Transformation-Mediated Transfer of Erythromycin Resistance in Campylobacter coli Strains from Turkeys and Swine

Erythromycin resistance in Campylobacter coli from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli strains from turkeys than in swine-derived strains (P < 0.01). The frequency of transformation to erythromycin resistance was 10⁻⁵ to 10⁻⁶ for turkey-derived strains but 10⁻⁷ or less for C. coli from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 μg/ml), as did the C. coli donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 μg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals.     

Molecular analysis of the phosphate-specific transport (pst) operon ofPseudomonas aeruginosa

Mithramycin is an antitumor antibiotic synthesized byStreptomyces argillaceus. This producer strain is highly resistant in vivo to mithramycin (MIC 100 µg/ml) but sensitive to the related drugs chromomycin and olivomycin (MIC 10 µg/ml). From a genomic library ofS. argillaceus DNA two cosmid clones were isolated which confer a high level of resistance to mithramycin onS. albus. The resistance genes were mapped by subcloning to a 3.9-kbPstI-PvuII fragment. DNA sequence analysis of this fragment revealed one incomplete and three complete open reading frames. Subcloning experiments demonstrated that resistance to mithramycin is mediated by the genesmtrA andmtrB. ThemtrA gene can potentially encode an ATP-binding protein of the ABC transporter superfamily, containing one nucleotide-binding domain and showing similarity with other ABC transporters involved in resistance to daunorubicin, oleandomycin and tetronasin in their respective producer strains. ThemtrB gene codes for an integral membrane protein with six putative transmembrane helices. A mithramycin-sensitive mutant was generated in a gene replacement experiment by disrupting themtrA gene, thus demonstrating that the system encoded by themtrAB genes is essential for conferring resistance to mithramycin inS. argillaceus.     

Non-radioactive PCR-SSCP with a single PCR step for detection of inhibitor resistant beta-lactamases in Escherichia coli

A method based on PCR-SSCP has been developed to detect presumptive Inhibitor-Resistant TEM (IRT) beta-lactamases in Escherichia coli. The capacity of this technique to differentiate genes from 11 control strains encoding IRT beta-lactamases was evaluated with PCR products digested with RsaI. All the bla(TEM) genes studied could be distinguished by their electrophoretic mobilities. Applied to 29 epidemiologically unrelated clinical isolates of E. coli resistant to amoxicillin-clavulanate (MIC, > or =32 microg/ml), the electrophoretic mobilities of the digested bla(TEM) PCR products were identical to those of the reference bla(TEM-1A) (6 strains) and bla(TEM-1B) (18 strains) genes. The remaining five bla(TEM) PCR products displayed SSCP profiles different from those of the reference bla(TEM) genes and their nucleotide sequence identified them as bla(TEM-1C) in one strain, bla(TEM-30/IRT-2) in two strains, bla(TEM-37/IRT-8) in one strain, and bla(TEM-40/IRT-11) in one isolate. Overexpression of the wild-type bla(TEM-1) gene, as detected by high-level resistance to beta-lactams and enzyme assay, accounted for resistance in the 24 E. coli containing bla(TEM-1). We report a simple one PCR step SSCP that can be used in epidemiological studies for rapid preliminary detection of IRT beta-lactamases; identification should be confirmed by sequence data.