Frequency and Distribution of Tetracycline Resistance Genes in Genetically Diverse, Nonselected, and Nonclinical Escherichia coli Strains Isolated from Diverse Human and Animal Sources

Nonselected and natural populations of Escherichia coli from 12 animal sources and humans were examined for the presence and types of 14 tetracycline resistance determinants. Of 1,263 unique E. coli isolates from humans, pigs, chickens, turkeys, sheep, cows, goats, cats, dogs, horses, geese, ducks, and deer, 31% were highly resistant to tetracycline. More than 78, 47, and 41% of the E. coli isolates from pigs, chickens, and turkeys were resistant or highly resistant to tetracycline, respectively. Tetracycline MICs for 61, 29, and 29% of E. coli isolates from pig, chickens, and turkeys, respectively, were ≥233 μg/ml. Muliplex PCR analyses indicated that 97% of these strains contained at least 1 of 14 tetracycline resistance genes [tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ, and tetX] examined. While the most common genes found in these isolates were tetB (63%) and tetA (35%), tetC, tetD, and tetM were also found. E. coli isolates from pigs and chickens were the only strains to have tetM. To our knowledge, this represents the first report of tetM in E. coli.     

Prevalence of tet gene and complete genome sequencing of tet gene-encoded plasmid (pAHH01) isolated from Aeromonas species in South Korea

Aims: To investigate the tetracycline resistance related to tet genes in Aeromonas isolates collected from water and diseased fish in South Korea. Methods and Results: A total of 34 Aeromonas strains were examined for their susceptibility to tetracycline using the minimum inhibitory concentration (MIC) assay, and the genetic determinants (tetA to E) were analysed. Among these strains, the tetA and tetE genes were predominant (tetA was found in six strains, and tetE was found in nine strains), and 15 strains were tetracycline‐resistant by the MIC assay. Additionally, the 8979‐bp plasmid that contains the tetE gene was fully sequenced. Conclusions: These data may be important with regard to the spread and persistence of tetracycline resistance genes in the bacterial populations that are present in aquaculture systems. Significance and Impact of the Study: Interestingly, no isolate has previously been shown to harbour three tet genes that are mediated by efflux systems, but the tetA, tetD and tetE genes were all isolated from one strain, which had the highest MIC value for tetracycline among the strains analysed in this study. We also investigated the full‐length plasmid that encoded the tetE gene from a tetracycline‐resistant strain.     

Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates

Tetracycline-resistant (Tet^r) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet^r gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet^r genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet^r strains transferred Tet^r genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet^r strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

Distribution of ten antibiotic resistance genes in <Emphasis Type="Italic">E. coli</Emphasis> isolates from swine manure,lagoon effluent and soil collected from a lagoon waste application field

The prevalence of ten antibiotic resistance genes (ARGs) was evaluated in a total of 616 Escherichia coli isolates from swine manure, swine lagoon effluent, and from soils that received lagoon effluent on a commercial swine farm site in Sampson County, North Carolina (USA). Isolates with ARGs coding for streptomycin/spectinomycin (aadA/strA and strB), tetracycline (tetA and tetB), and sulfonamide (sul1) occurred most frequently (60.6–91.3%). The occurrence of E. coli isolates that carried aadA, tetA, tetB, and tetC genes was significantly more frequent in soil samples (34.0–97.2%) than in isolates from lagoon samples (20.9–90.6%). Furthermore, the frequency of isolates that contain genes coding for aadA and tetB was significantly greater in soil samples (82.6–97.2%) when compared to swine manure (16.8–86.1%). Isolates from the lagoon that carried tetA, tetC, and sul3 genes were significantly more prevalent during spring (63.3–96.7%) than during winter (13.1–67.8%). The prevalence of isolates from the lagoon that possessed the strA, strB, and sul1 resistance genes was significantly more frequent during the summer (90.0–100%) than during spring (66.6–80.0%). The data suggest that conditions in the lagoon, soil, and manure may have an impact on the occurrence of E. coli isolates with specific ARGs. Seasonal variables seem to impact the recovery isolates with ARGs; however, ARG distribution may be associated with mobile genetic elements or a reflection of the initial numbers of resistant isolates shed by the animals.     

Distribution of Tetracycline and Streptomycin Resistance Genes and Class 1 Integrons in Enterobacteriaceae Isolated from Dairy and Nondairy Farm Soils

The prevalence of selected tetracycline and streptomycin resistance genes and class 1 integrons in Enterobacteriaceae (n = 80) isolated from dairy farm soil and nondairy soils was evaluated. Among 56 bacteria isolated from dairy farm soils, 36 (64.3%) were resistant to tetracycline, and 17 (30.4%) were resistant to streptomycin. Lower frequencies of tetracycline (9 of 24 or 37.5%) and streptomycin (1 of 24 or 4.2%) resistance were observed in bacteria isolated from nondairy soils. Bacteria (n = 56) isolated from dairy farm soil had a higher frequency of tetracycline resistance genes including tetM (28.6%), tetA (21.4%), tetW (8.9%), tetB (5.4%), tetS (5.4%), tetG (3.6%), and tetO (1.8%). Among 24 bacteria isolated from nondairy soils, four isolates carried tetM, tetO, tetS, and tetW in different combinations; whereas tetA, tetB, and tetG were not detected. Similarly, a higher prevalence of streptomycin resistance genes including strA (12.5%), strB (12.5%), ant(3″) (12.5), aph(6)-1c (12.5%), aph(3″) (10.8%), and addA (5.4%) was detected in bacteria isolated from dairy farm soils than in nondairy soils. None of the nondairy soil isolates carried aadA gene. Other tetracycline (tetC, tetD, tetE, tetK, tetL, tetQ, and tetT) and streptomycin (aph(6)-1c and ant(6)) resistance genes were not detected in both dairy and nondairy soil isolates. A higher distribution of multiple resistance genes was observed in bacteria isolated from dairy farm soil than in nondairy soil. Among 36 tetracycline- and 17 streptomycin-resistant isolates from dairy farm soils, 11 (30.6%) and 9 (52.9%) isolates carried multiple resistance genes encoding resistance to tetracycline and streptomycin, respectively, which was higher than in bacteria isolated from nondairy soils. One strain each of Citrobacter freundii and C. youngae isolated from dairy farm soils carried class 1 integrons with different inserted gene cassettes. Results of this small study suggest that the presence of multiple resistance genes and class 1 integrons in Enterobacteriaceae in dairy farm soil may act as a reservoir of antimicrobial resistance genes and could play a role in the dissemination of these antimicrobial resistance genes to other commensal and indigenous microbial communities in soil. However, additional longer-term studies conducted in more locations are needed to validate this hypothesis.     

Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in strains from humans (35 isolates), chickens (15 isolates), food (21 isolates), soil (16 isolates) and veterinary sources (6 isolates) was determined, and tetracycline-resistance genes were detected. Resistance was most common in strains isolated from chickens, followed by those from soils, clinical samples and foods. The most highly resistant strains were found among clinical and food isolates. tetA(P) was the most common resistance gene, and along with tetB(P) was found in all resistant strains and some sensitive strains. One tetracycline-resistant food isolate had an intact tet(M) gene. However, PCR fragments of 0.4 or 0.8 kb with high degrees of identity to parts of the tet(M) sequences of other bacteria were found, mainly in clinical isolates, and often in isolates with tetB(P). No correlation between level of sensitivity to tetracycline or minocycline and the presence of tetA(P), tetB(P) or part of tet(M) was found. The presence of part of tet(M) in some strains of C. perfringens containing tetB(P) may have occurred by recent gene transfer.     

嗜水气单胞菌对四环素类药物诱导耐药表型及机理研究

【目的】初步探讨利用四环素类药物体外诱导嗜水气单胞菌耐药后,嗜水气单胞菌对四环素类药物敏感性的变化及其耐药机制。【方法】筛选临床分离嗜水气单胞菌的四环素类敏感株,从含有1/4×MIC的强力霉素的TSA固体培养基开始,等比2倍提高诱导药物质量浓度对受试菌进行连续传代培养,以获得高耐诱导株;测定诱导菌对强力霉素和16种非诱导药物的最小抑菌浓度(MIC)及添加外排泵抑制剂N-甲基吡咯烷酮(NMP)后的MIC,分析其敏感性变化与外排作用的关系;提取诱导菌的DNA,PCR扩增其5个tet基因并测序。【结果】诱导后菌株对强力霉素的MIC显著升高,对非诱导四环素类药物也有不同程度提高,对氟喹诺酮类药物的MIC比诱导前增加几十至上千倍;对氨基糖苷类药物和利福平的MIC则有不同程度的降低;添加NMP后,所有诱导菌株对强力霉素的MIC值均有不同程度的下降;四环素类耐药基因的检测结果表明,在诱导后7号菌株中同时检测到tet A和tet E;在诱导前后的2号菌株中检测到tet C;在诱导前后的1、3、4、5、6、7号菌株中均检测到tet E。【结论】本研究表明tet E基因可能是介导气单胞菌分离株对四环素类药物耐药的优势基因,为阐明嗜水气单胞菌对四环素类药物耐药机制及耐药性与耐药基因之间的关系提供理论依据。     

Antimicrobial Resistance and Virulence Genes of Escherichia coli Isolates from Swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (≤3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

High occurrence of carbapenem-resistant Escherichia coli isolates from healthy rabbits (Oryctolagus cuniculus): first report of blaIMI and blaVIM type genes from livestock in Tunisia

We aimed to study the antibiotic susceptibility and possible occurrence of extended-spectrum beta-lactamases (ESBL)/carbapenemase-producing Escherichia coli isolates collected from rabbits in Tunisia. In all, 35 faecal samples from healthy rabbits were collected from one farm and E. coli were isolated from three media: antibiotic-free TBX agar, TBX+2 mg l⁻¹ cefotaxime and TBX+1 mg l⁻¹ imipenem. In total, 39 E. coli isolates were recovered; the majority showed resistance to at least one antibiotic and none was ESBL producer. Carbapenem resistance was detected in 16 isolates from either selective or un-selective media. Phenotypic methods used to detect carbapenemase production showed two positive isolates by Modified Hodge Test, six metallo-carbapenemase producers (Imipenem disc+EDTA) and all were temocillin resistant (possible OXA-48 carbapenemase). bla_VIM and bla_IMP type genes were detected in two and one isolates, respectively; one of them harboured both genes. Isolates contained common genes encoding resistance to sulphonamides (sul1, sul2), tetracycline (tetA, tetB, tetC) and fluoroquinolones (qnrS, aac(6′)-Ib-cr). Class 1 and 2 integrons were detected in five and four isolates, respectively. These findings highlight the importance of rabbit production as reservoir of carbapenem-resistant E. coli and argument the first report of bla_VIM and bla_IMP genes in livestock in Tunisia.     

Chromosomal mutations causing resistance to tetracycline in Bacillus subtilis.

Summary We have isolated, after ethylmethanesulfonate mutagenesis, several chromosomal mutations causing resistance to tetracycline in Bacillus subtilis. These mutations fall into two classes, tetA and tetB. 30 S ribosomal protein S10 shows an altered mobility on two-dimensional acrylamide gels in cells bearing the former type of mutation. Ribosomes from these cells show elevated levels of resistance to tetracycline in vitro as measured by polyuridine dependent polyphenylalanine synthesis. The tetA locus maps adjacent to the tuf gene in the B. subtilis ribosomal protein gene cluster. Cells with the tetB mutation do not show any altered ribosomal protein, and their ribosomes are as sensitive, in vitro, to tetracycline as ribosomes isolated from wild type cells. The tetB mutation has been mapped proximal to cysA14.In partial fulfillment of the requirements for the doctoral degree by G.W. in the Department of Biology at the New York University Graduate School of Arts and Sciences     

云南地区鸭源沙门菌的分离鉴定及耐药性和毒力基因分析

【背景】沙门菌是一种重要的食源性人兽共患病原菌,可引起多种食源性疾病。【目的】了解云南地区鸭源沙门菌病的流行现状、耐药现象及毒力基因携带等基本情况。【方法】无菌采集云南各地区病死鸭肝脏样品169份进行沙门菌分离,对分离株进行血清分型鉴定、药敏及相关耐药基因、毒力基因筛查。【结果】分离到鸭源沙门菌48株,分离率为28.40%,鉴定出3种血清型,其中肠炎沙门菌为优势血清型。分离株对青霉素G、林可霉素、克林霉素和利福平的耐药率达100%,每株菌至少对3类6种及以上的抗生素耐药,单株最高可耐14种,产生了22种耐药谱型。共检出耐药基因5种,bla_TEM和tetB检出率分别为27.08%和22.92%,tetA、sul2和EBC的检出率较低。毒力基因共检出10种,其中,SPI-1(avrA)、SPI-3(mgtC)、SPI-4(siiD)、SPI-5(sopB)和bcfC检出率均高达100%,SPI-2(ssaQ)、spvB、spvC、pefA和stn的检出率均达60%以上,cdtB未检出。【结论】云南地区鸭源沙门菌主要流行血清型为肠炎沙门菌,耐药性及多重耐药情况严重,耐药机制复杂,耐药基因与耐药表型符合率低,毒力基因检出率较高。研究结果可为云南地区鸭群沙门菌病的防控和净化提供参考。     

First study on characterization of virulence and antibiotic resistance genes in verotoxigenic and enterotoxigenic <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> isolated from raw milk and unpasteurized traditional cheeses in Romania

The study focused on the incidence of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) in raw milk and traditional dairy cheeses marketed in Romania, characterizing the virulence and antibiotic resistance genes of these isolates. One hundred and twenty samples of raw milk and 80 samples of unpasteurized telemy cheese were collected and cultured according to the international standard protocol. All the characteristic E. coli cultures were analyzed for the presence of STa, STb, LT, stx1, and stx2 toxicity genes. The ETEC/VTEC strains were tested for the presence of antibiotic resistance genes, such as aadA1, tetA, tetB, tetC, tetG, dfrA1, qnrA, aaC, sul1, bla _SHV , bla _CMY , bla _TEM , and ere(A), using PCR. The results showed that 27 samples (18.62%) were positive for one of the virulence genes investigated. 48.1% (n = 13) tested positive at the genes encoding for tetracycline resistance, tetA being the most prevalent one (61.5%; n = 8). A high percent (33.3%; n = 9) revealed the beta-lactamase (bla _TEM ) resistance gene, and none of the samples tested positive for bla _CMY and bla _SHV genes. The genes responsible for resistance to sulfonamides (sul1) and trimethoprim (dfrA1) were detected in rates of 14.8% (n = 4) and 7.4% (n = 2), respectively. E. coli is highly prevalent in raw milk and unpasteurized cheeses marketed in Romania. These strains might represent an important reservoir of resistance genes which can easily spread into other European countries, given the unique market.     

Diversity,Distribution and Quantification of Antibiotic Resistance Genes in Goat and Lamb Slaughterhouse Surfaces and Meat Products

The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, bla_CTX and bla_TEM genes, which occurred in few zones as isolated ‘hot spots.’ The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.     

The identification of CTX-M-14, TEM-52, and CMY-1 enzymes in Escherichia coli isolated from the Han River in Korea

From water samples collected monthly between 2000 and 2001 from the Han River in Seoul, sixteen strains of Escherichia coli which confer resistance to at least 10 kinds of antimicrobial agents were isolated. From these isolates, 2 kinds of extended-spectrum β-lactamases (ESBLs) and one plasmid-mediated AmpC β-lactamase were detected; CTX-M-14 from 10 isolates, TEM-52 from 5 isolates, and CMY-1 from one isolate. Class 1 integron gene cassettes, such as aadA1, dfr12-orfF-aadA2, and dfr17-aadA5, were also detected and the integrons are the same as those found in E. coli isolated from swine, poultry, and humans in Korea. The result of this study indicated the importance of river water as a reservoir for antimicrobial resistance genes and resistant bacteria.     

Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey

We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, bla _oxA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens. This study was presented in part at the 2^nd World Conference on Magic Bullets, held October 3–5, 2008 in Nurnberg, Germany.     

Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora in pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we developed and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin (strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in plant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly from bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry treatment. The genes most frequently identified in soils pre- and post-slurry treatment were strB, aadA, tetB and tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative concentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry treatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification of antibiotic resistance genes in complex, environmental sample matrices.     

Abundances of Tetracycline,Sulphonamide and Beta-Lactam Antibiotic Resistance Genes in Conventional Wastewater Treatment Plants (WWTPs) with Different Waste Load

Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs), an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs) were quantified using qPCR in three WWTPs of decreasing capacity located in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2), tetracycline resistance genes (tetM and tetC), and resistance genes for extended spectrum beta-lactams (bla_oxa-58, bla_shv-34, and bla_ctx-m-32). To avoid inconsistencies among qPCR assays we normalised the ARG abundances with 16S rRNA gene abundances while assessing if the respective genes increased or decreased during treatment. ARGs were detected in most samples; sul1, sul2, and tetM were detected in all samples. Statistically significant differences (adjusted p<0.01) between the inflow and effluent were detected in only four cases. Effluent values for bla_oxa-58 and tetC decreased in the two larger plants while tetM decreased in the medium-sized plant. Only bla_shv-34 increased in the effluent from the medium-sized plant. In all other cases the purification process caused no significant change in the relative abundance of resistance genes, while the raw abundances fell by several orders of magnitude. Standard water quality variables (biological oxygen demand, total phosphorus and nitrogen, etc.) were weakly related or unrelated to the relative abundance of resistance genes. Based on our results we conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs.     

Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil

The aim of the present study was to investigate the resistance profile, to detect the presence of beta-lactam resistance genes, phenotypic expression of efflux pump systems and class 1 integrons in Pseudomonas spp. strains obtained from untreated hospital effluents. Effluent samples were collected from four hospitals in Porto Alegre, RS, Brazil. Pseudomonas were isolated on MacConkey agar plates and the identification was confirmed by 16S rRNA PCR and biochemical tests. Susceptibility testing was determined by disk-diffusion method using 11 different beta-lactams and MIC assays were performed on isolates resistant to imipenem and ceftazidime. The beta-lactamase genes bla _IMP, bla _VIM, bla _SPM-1, bla _OXA-23-like, bla _OXA-24-like, bla _OXA-51-like and the intl1 gene from class 1 integron were analysed by PCR. One hundred and twenty-four isolates were recovered and the most common species was Pseudomonas pseudoalcaligenes. The resistance found among the isolates was considered high, 62 (50%) isolates were multiresistant. No isolate carrying the beta-lactamase genes tested was found among the strains. Seven isolates showed reduction of MIC for imipenem and ceftazidime in the presence of cyanide m-chlorophenylhydrazone, indicating the hyper expression of efflux pumps. From the 124 isolates, 52 (41.9%) were identified as carrying the class 1 integron gene, intI1. Untreated hospital effluents could be a source of environmental contamination due to discharge of antimicrobial resistant bacteria which can carry integron class 1 and act as a reservoir of resistance genes and have efflux pump systems.     

tetA(L) Mutants of a Tetracycline-Sensitive Strain of Bacillus subtilis with the Polynucleotide Phosphorylase Gene Deleted

A Bacillus subtilis strain with the polynucleotide phosphorylase gene deleted was sensitive for growth in the presence of tetracycline. This strain was used to select for tetracycline-resistant mutants. A point mutation in the tetA(L) promoter and a spontaneously occurring tetA(L) gene copy number mutant were characterized.