Prospects for using DNA probes for rapid diagnosis of antibiotic resistance in clinical strains of enteric bacteria

Aminoglucoside resistance patterns of clinical strains of enteric bacteria isolated from inpatients of Moscow clinics were determined. APH(3')-I and AAC(3)-II were shown to be the most frequent. The aphA1 and aacC2 genes encoding the enzymes were cloned from the R plasmid of the transconjugant of the E. coli clinical strains. DNA probes based on the determined nucleotide sequences of the cloned genes were constructed and used in DNA-DNA hybridization experiments. The results on the occurrence of APH(3')-I and AAC(3)-II in the strains tested were confirmed by the DNA-DNA hybridization. Prospects for developing a set of DNA probes for rapid diagnosis of antibiotic resistance are discussed.     

医院内感染的病原菌分布及其耐药谱的分析

本文统计了我院１９９２年８月—１９９４年７月两车间医院内感染病原菌的分离结果,分离出病原菌２５种７５８株,其中革兰氏阳性菌１６３株（占２１．５１％）,革兰氏阴性菌３７８株（占４９．８７％）,真菌２１７株（占２８．６２％）。院感病原菌依次为,白色念珠菌、绿脓假单胞菌表、葡菌、肺炎克雷伯氏菌、酵母菌及大肠艾希氏菌等,显示了真菌及表葡菌在院内感染中已经上升到不可忽略的地位。用１８种常用抗生素,对除真菌外的上述病原菌５４１株做耐药谱测定,并发现除了表葡菌及多数革兰氏阴性菌对喹谱酮类及少数头孢三代抗生素尚属敏感外,大部分均具有越来越广泛的耐药性。     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

Antimicrobial profile of ceftazidime: the situation in three larger hospitals in Prague between 1981 and 1983

As a part of preclinical trials of 2,996 clinical isolates from three larger hospitals in Prague were qualitatively and quantitatively assayed for in vitro sensitivity to ceftazidime. In gramnegative bacteria the incidence of resistance to ceftazidime in strain of Enterobacter, Serratia Proteus and Pseudomonas species ranged from 4% to 6% of strains. In grampositive bacteria only strains of enterococci, listeriae and anaerobic bacteria are excluded from the action of this broad-spectrum antibiotic. According to present experiences the antipseudomonal activity of ceftazidime is approximately the same as that of cefsulodine and cefoperazone. Alarmingly, one of the Pseudomonas aeruginosa isolates was found to show a distinct multiresistance to all available lactam, aminoglycoside and broad-spectrum antimicrobials, including ceftazidime.     

908株烧伤患者病原菌分布特征及耐药性分析

目的：了解本院2011 年-2013 年烧伤患者分离病原菌的分布特征及耐药性,为合理使用抗生素提供依据。方法：对患者创面 分泌物、痰液、血液中的细菌进行分离鉴定和药敏试验,药敏数据分析用WHONET5.6 软件。结果：共分离908 株病原菌,其中最 常见的病原菌依次为：鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌、表皮葡萄球菌、金黄色葡萄球菌、大肠埃希菌。在革兰阴性杆 菌中,鲍曼不动杆菌对碳青霉烯类耐药率较高,铜绿假单胞菌对氨基糖苷类、喹诺酮类的耐药率较低。在肠杆菌科细菌中,未发现 耐碳青霉烯类大肠埃希菌和肺炎克雷伯菌株。在葡萄球菌中,未发现耐万古霉素、利奈唑胺菌株。结论：我院烧伤患者分离的病原 菌以非发酵菌为主,医院应重视对其病原菌耐药性监测,指导临床合理使用抗生素。     

Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes

Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14- and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low-copy vector, pACYC177, and expressed in several Escherichia coli K-12 strains. The location of the macrolide- resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane-associated pump proteins. The macrolide-resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E. coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA . Furthermore, intracellular accumulation of [¹⁴C]-erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin-sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer-specific synthesis using the polymerase chain reaction.     

Antibiotic resistance of Lactobacillus strains]

One hundred and thirty six Lactobacillus strains isolated from poultry and 23 Lactobacillus strains isolated from long-living persons were tested for their antibiotic sensitivity. Occurrence of some type determinants of resistance to aminoglycoside antibiotics and tetracyclines in the Lactobacillus strains resistant to these antibiotics was studied. The majority of the strains from the both collections were resistant to aminoglycosides (73 and 79 per cent, respectively). The isolates from the poultry were characterized by multiple resistance. The isolates from the long-living persons were most frequently resistant to one of two antibiotics. All the tested Lactobacillus strains isolated from the long-living persons were sensitive to tetracyclines. The species composition of the isolates was different. The antibiotic-resistant strains were detected in all the species involved in the study. By hybridization of Lactobacillus colonies with the probes containing various genes of the resistance it was shown that in 14 per cent of the antibiotic-resistant strains belonging to Lactobacillus the antibiotic resistance was controlled by the genes homologous to resistance genes widely distributed in gramnegative organisms. This indicated a possible wide exchange and heterologous expression of the antibiotic resistance determinants between microorganisms of various taxonomic groups.     

Comparative in vitro activity of cefepime and other antibiotics against clinical strains of gram-negative bacteria]

In vitro activity of cefepime against etiologically significant strains of gramnegative microflora of patients treated in the Reanimation and Intensive Care Unit after cardiosurgical operations was evaluated. Sixty four strains of gramnegative aerobic bacteria isolated within the period from October 2001 to April 2002 were tested. The isolates susceptibility was determined by the disk diffusion method. Cefepime had an obvious advantage over the 3rd generation cephalosporins. Low incidence of cefepime resistant strains of the problem organisms in the Reanimation and Intensive Care Unit should be taken into account in empirical therapy of infections due to such pathogens.     

Cadmium accumulation and DNA homology with metal resistance genes in sulfate-reducing bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

Naturally occurring macrolide-lincosamide-streptogramin B resistance in Bacillus licheniformis.

Resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics is widespread and of clinical importance. B. Weisblum and his coworkers have demonstrated that this resistance is associated with methylation of the 23S ribosomal ribonucleic acid of the large ribosomal subunit which results in a diminished affinity of this organelle for these antibiotics (Lai et al, J. Mol. Biol. 74:67-72, 1973). We report that 10 of 15 natural isolates of Bacillus licheniformis, a common soil organism, are resistant to the MLS antibiotics. The properties of this resistance (high level of tolerance for erythromycin, broad cross-resistance spectrum, and inducibility) suggest that resistance is conferred as described above. The resistance determinant from one of these strains was cloned onto a B. subtilis plasmid vector, and the resulting hybrid plasmid (pBD90) was used to prepare radioactive probe deoxyribonucleic acid for hybridization studies. All of the resistance B. licheniformis strains studied exhibited homology with the pBD90 insert. Plasmid pBD90 showed no homology to the following staphylococcal and streptococcal MLS-resistance plasmids: pE194, pE5, pAM77, pI258. Plasmids pE194 and pE5, on the other hand, carry homologous MLS genes but showed no detectable homology to one another in their replication genes. pBD90 specified a 35,000-dalton erythromycin-inducible protein, detectable in minicells, which therefore appears different from the 29,000-dalton inducible resistance protein specified by pE194. We conclude that there are at least three distinct MLS resistance determinants to be found among gram-positive bacteria.     

Isolation of tetracycline-resistant Megasphaera elsdenii strains with novel mosaic gene combinations of tet(O) and tet(W) from swine

Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.     

Molecular cloning of the F8 fimbrial antigen from Escherichia coli

Abstract The genetic determinant coding for the P-specific F8 fimbriae was cloned from the chromosome of the Escherichia coli wild-type strain 2980 (O18:K5:H5:F1C, F8). The F8 determinant was further subcloned into the Pst I site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned counterpart was demonstrated. The cloned F8 fimbriae and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepithelial cells. The cloned F8 determinant was well expressed in a variety of host strains.     

Mutagenic DNA repair potential in Pseudomonas spp., and characterization of the rulABPc operon from the highly mutable strain Pseudomonas cichorii 302959

We assessed the tolerance to ultraviolet B (UVB; 290-320 nm) radiation and UVB-induced mutability in 28 Pseudomonas spp. and four Burkholderia cepacia strains. The UVB survival of 23 (72%) of the strains was elevated (>46% survival following irradiation with a 2250 J m-2 dose), and 17 (53%) strains were defined as mutable by UVB. A mutagenic DNA repair determinant was cloned and characterized from the highly mutable strain P. cichorii 302959 and shown by sequence analysis to be an allele of rulAB, a mutagenic DNA repair determinant previously characterized from Pseudomonas syringae. Phylogenetic analyses of RulA- and RulB-related sequences indicated that the sequences identified in environmental bacteria shared a common ancestor with UmuDC-like sequences from enteric bacteria but were considerably diverged. The dynamics of UVB-induced mutability to nalidixic acid resistance (NalR) and rifampicin resistance (RifR) were studied in replicate populations of P. cichorii 302959 subjected to a daily UVB dose of 2250 J m-2 for 14 consecutive days. While there was an initial spike in the frequency of NalR and RifR mutants recovered on Days 1 and 2 of two separate experiments, the frequencies were sharply reduced and then fluctuated throughout the duration of both experiments. These experimental results are intriguing because they point to the possibility that P. cichorii possesses additional mechanisms to curtail the induction of spontaneous mutants following repeated episodes of UVB irradiation.     

Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria

We have previously reported high-frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens . Donor strains were postulated to carry two Tc^R genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non-transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet (O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible Tc^R gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet (W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection-type tet genes, suggesting recent acquisition from a high G + C content genome. Tet (W) genes with almost identical sequences are also shown to be present in Tc^R strains of B. fibrisolvens from Australian sheep and in Tc^R strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus . This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet (W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus .     

Characterization of Two Macrolide Resistance-Related Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates

In analyzing the drug resistance phenotype and mechanism of resistance to macrolide antibiotics of clinical Pseudomonas aeruginosa isolates, the agar dilution method was used to determine the minimum inhibitory concentrations (MICs), and PCR (polymerase chain reaction) was applied to screen for macrolide antibiotics resistance genes. The macrolide antibiotics resistance genes were cloned, and their functions were identified. Of the 13 antibiotics tested, P. aeruginosa strains showed high resistance rates (ranging from 69.5–82.1%), and MIC levels (MIC90 > 256 μg/ml) to macrolide antibiotics. Of the 131 known macrolide resistance genes, only two genes, mphE and msrE, were identified in 262 clinical P. aeruginosa isolates. Four strains (1.53%, 4/262) carried both the msrE and mphE genes, and an additional three strains (1.15%, 3/262) harbored the mphE gene alone. The cloned msrE and mphE genes conferred higher resistance levels to three second-generation macrolides compared to two first-generation ones. Analysis of MsrE and MphE protein polymorphisms revealed that they are highly conserved, with only 1–3 amino acids differences between the proteins of the same type. It can be concluded that even though the strains showed high resistance levels to macrolides, known macrolide resistance genes are seldom present in clinical P. aeruginosa strains, demonstrating that a mechanism other than this warranted by the mphE and msrE genes may play a more critical role in the bacteria’s resistance to macrolides.Key words: Pseudomonas aeruginosa, macrolide, resistance gene, mphE, msrE     

Plasmid-Related Quinolone Resistance Determinants in Epidemic Vibrio parahaemolyticus,Uropathogenic Escherichia coli,and Marine Bacteria from an Aquaculture Area in Chile

Marine bacteria from aquaculture areas with industrial use of quinolones have the potential to pass quinolone resistance genes to animal and human pathogens. The VPA0095 gene, related to the quinolone resistance determinant qnrA, from clinical isolates of epidemic Vibrio parahaemolyticus conferred reduced susceptibility to quinolone after cloning into Escherichia coli K-12 either when acting alone or synergistically with DNA gyrase mutations. In addition, a plasmid-mediated quinolone resistance gene from marine bacteria, aac(6′)-Ib-cr, was identical to aac(6′)-Ib-cr from urinary tract isolates of E. coli, suggesting a recent flow of this gene between these bacteria isolated from different environments. aac(6′)-Ib-cr from E. coli also conferred reduced susceptibility to quinolone and kanamycin when cloned into E. coli K-12.     

Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

AIMS: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. METHODS AND RESULTS: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. CONCLUSIONS: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.     

Characterisation of a chloramphenicol acetyltransferase determinant found in the chromosome of Pseudomonas aeruginosa

The open reading frame (ORF) in the Pseudomonas aeruginosa chromosome, whose product resembles the chloramphenicol acetyltransferases (CAT) belonging to the CATB family, was cloned and shown to confer resistance to chloramphenicol (Cm) in Escherichia coli. The determinant was therefore named catB7 and the corresponding protein CATB7. When the copy number and expression signals were identical, the catB7 gene conferred resistance to Cm at a level slightly lower than those of three other catB genes. CATB7 resembles other CATBs in that it acetylates Cm but not 1-acetoxy-Cm. For CATB7, the K(m) values for acetyl-CoA and Cm were 5.0-5.4-fold higher than the corresponding values for each of the three other CATB proteins (CATB1, CATB3 and CATB5) examined and the Vmax was 5-6 fold lower. Using PCR, the catB7 gene was found in all six P. aeruginosa strains examined but not in any other species of pseudomonad tested. Weak CAT activity was detected in crude cell extracts from five of the six P. aeruginosa strains. However, this activity did not correlate with the Cm susceptibility of the strains, indicating that catB7 is not likely to be the major determinant of intrinsic Cm resistance in P. aeruginosa.     

Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.