PCR Amplification Of 16S rRNA Research Articles

Identification of Antibiotics Susceptibility Patterns at Sub-inhibitory Concentrations in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram negative bacterium that has been recognized as an opportunistic pathogen. It is the most common bacterium associated with nosocomial infections and ventilator-associated pneumonia. It exhibits high innate resistance to various ranges of antibiotics thereby causing high morbidity and mortality rate. This research was aimed to identify the antibiotics susceptibility patterns at sub-inhibitory concentration in Pseudomonas aeruginosa. One hundred and fifty (150) clinical swab specimens were collected from urinary catheters; wound and ear infections of patients and the swabs inoculated using standard microbiology method. The isolates were characterized based on the bacteriological methods such as morphology and biochemical tests. The isolates were further confirmed by species specific by PCR amplification of 16S rRNA and the amplicons were analyzed by gel electrophoresis; and further genomic sequencing was done and blast with NCBI database mining. The antimicrobial susceptibility of the isolates was done by disc diffusion methods. The result of the isolation showed 22(59.46%) from wound infections, 12(32.43%) from ear infections and 3(8.11%) from urinary catheter. The isolates were Gram-negative, produced β-hemolysis on blood agar and the morphology is small pigmented circular. The isolates showed positive results to catalase, oxidase, citrate, nitrate and indole tests. The amplification of the 16S rRNA gene region resulted in the band size of 1500bp PCR product and the BLAST analysis gave 99% similarity. The results of susceptibility analysis showed that the isolates from the urinary catheter, wound and ear infection were 82%, 68% and 47% respectively, resistance to Piperacillin tazobactem, Cefoperazon, Ofloxacin, Tetracycline, Amikacin, Gentamycin, Bacitracin, Clarithromycin, Cefalotin, Levofloxacin and Cefpiroma. Antimicrobial susceptibility tests in P. aeruginosa isolates revealedshowed that they were multi-drug resistant. At sub-inhibitory concentrations of antibiotics within the microbial environment, Pseudomonas aeruginosa becomes more resistant. Perhaps, these antimicrobials could have other signaling activities within the environment. Therefore, there is a need for more research work to develop therapeutics combination to combat the recalcitrant nature of Pseudomonas aeruginosa.

The indigenous microbial diversity involved in the spontaneous fermentation of red dragon fruit (Hylocereus polyrhizus) identified by means of molecular tools

Red dragon fruit (RDF) is well-known for its high nutritional content, especially the red pigment betacyanins that possess high antioxidant activity. Natural fermentation is an ancient yet outstanding technique that relies on the autochthonous microbiota from fruits and vegetables surfaces to preserve and improve the nutritional values and quality of the food product. The present study was to evaluate and identify the indigenous microbial community (bacteria and fungi) that are involved in the natural fermentation of RDF. Results revealed a total of twenty bacterial pure cultures and nine fungal pure cultures were successfully isolated from fermented red dragon fruit drink (FRDFD). For the first time, the PCR amplification of 16S rRNA and ITS regions and sequence analysis suggested nine genera of bacteria and three genera of fungi (Aureobasidium pullulans, Clavispora opuntiae, and Talaromyces aurantiacus) present in the FRDFD. Four dominant (≥10 % isolates) bacteria species identified from FRDFD were Klebsiella pneumonia, Brevibacillus parabrevis, Bacillus tequilensis and Bacillus subtilis. The carbohydrate fermentation test showed that all the indigenous microbes identified were able to serve as useful starter culture by fermenting sucrose and glucose, thereby producing acid to lower the pH of FRDFD to around pH 4 for better betacyanins stability. The present study provides a more comprehensive understanding of the indigenous microbial community that serves as the starter culture in the fermentation of RDF. Besides, this study provides a useful guide for future research to be conducted on studying the rare bacterial strains (such as B. tequilensis) identified from the FRDFD for their potential bioactivities and applications in medical treatment and functional foods industries.

Phylogenetic relationships among Bradyrhizobium species nodulating groundnut (Arachis hypogea L.), jack bean (Canavalia ensiformis L.) and soybean (Glycine max Merr.) in Eswatini

This study assessed the genetic diversity and phylogenetic relationships of rhizobial isolates obtained from root nodules of groundnut, jack bean and soybean planted in different locations within Eswatini. Seventy-six rhizobial isolates were studied using ERIC-PCR (enterobacterial repetitive intergenic consensus) fingerprinting and PCR amplification of 16S rRNA, housekeeping genes (atpD, dnaK, glnll and rpoB) and symbiotic genes (nifH and nodC). The dendrogram generated from the ERIC-PCR banding patterns grouped the test rhizobial isolates into 16 major clusters (Cluster I–XVI), with three isolates, namely TUTAHeS60, TUTGMeS3 and TUTAHeS127, forming outgroups of Clusters IV, VI and IX, respectively. Furthermore, the 76 test isolates were grouped into 56 ERIC-PCR types at 70% similarity level. The phylogenetic analysis of the 16S rRNA gene and multilocus sequence analysis of four housekeeping (atpD, dnaK, glnII and rpoB) and two symbiotic (nifH and nodC) genes showed that all three legumes (groundnut, jack bean and soybean) were nodulated by bacterial symbionts belonging to the genus Bradyrhizobium, with some isolates exhibiting high divergence from the known reference type strains. The results also showed that B. arachidis, B. iriomotense and B. canariense were the closest type strains to the groundnut isolates, while B. pachyrhizi and B. elkanii were the closest relatives to the bacterial symbionts associated with the nodulation of both jack bean and soybean. This study is the first report to describe of the bacterial symbionts nodulating jack bean in African soils.

Epidemiological Molecular Analysis of Acinetobacter baumannii isolates using a multilocus sequencing typing and Global lineage

The Multilocus sequence typing MLST method was used to recognize outbreaks of hospitals distinct clonal lineages of A. baumannii; these schemes appeared to provide largely concordant classifications that have been tools to evaluate the population structures of bacterial pathogens. One hundred fifty samples were collected from different specimens of patients within Baghdad hospitals (blood 40%, CSF 5%, urine 5%) between July 2019 to February 2020. Then identification all isolated as phenotypic detection and performed using PCR amplification of 16srRNA and blaOXA-51-like as genotypic detections. According to clinical and laboratory standards institute (CLSI) guidelines, Susceptibility testing was performed. Clonally analysis was performed by global lineage ICs correlated with multilocus sequence typing (MLST) when our data showed a very high rate of antimicrobial resistance in all hospital isolates, especially against colistin (8%) which determined the PDR isolates from other types also recorded 70% of isolates standing for carbapenems antibiotics (IMI 32%, MER70%& DOR 64%). Then already clustered into four groups according to multiplex PCR for two groups of three genes (ompA, csuE & blaOXA-51-like) where IC II was predominant in Iraq but in our strains founding ICI (38%) more prevalence one followed by IC0 (26%) then ICII and ICIII (20% &16% respectively). MLST used for detected the common sequence types (STs) of our selected 8 A. baumannii strains (IC0/A11, ICI/A6,48, ICII/A33,50,19 and ICIII/A1,36) were performed by using 7 housekeeping genes than were submitted in the MLST Pasteur scheme dataset (ID 5098, 5099, 5100, 5101, 5102, 5103, 5482 & 5483) followed by statistical eBurst analysis was done to study Clonal complexes (CCs). Identified 5 new STs (8, 444, 346, 1587 & 621) within Iraq and new one ST (1830) worldwide.

Biosynthesis of TiO2 nanoparticles using prodigiosin and evaluating its antibacterial activity against biofilm producing MDR- Acinetobacter baumannii

A rising number of hospital infections were caused by multi drug resistant A.baumannii. This microorganism has become a big global concern for clinicians. This study aimed to evaluate the antimicrobial activity of biosynthesized TiO2 nanoparticles against biofilm producing multi drug resistant A. baumannii. Bacteria were isolated from burn wounds. The selected isolate was identified using the routine biochemical assays, viteck 2, and confirmed by PCR technique, targeting the 16S rRNA and blaOXA-51 genes. Antimicrobial susceptibility tests were performed using Viteck 2 system and the biofilm production was tested by using microtiter plate method. S marcescens was used for production of the prodigiosin which characterized later by UV-visible spectroscopy and then was used for biosynthesis of titanium dioxide nanoparticles (TiO2) NPs. Atomic force microscopy, X-ray diffractometer and field emission scanning electron microscopy were used for characterization of TiO2 NPs. Antimicrobial activity of TiO2 NPs was examined by well diffusion assay using concentration of 0.4- 0.006 mg/ml. The studied isolate was beta-lactamase producer and showed resistance to aminoglycosides, quinolones, furanes and trimethoprim/ sulphonamide, PCR amplification of 16S rRNA and blaOXA-51 genes was used for detection of A baumannii. The selected isolate was a strong biofilm producer with 5.9 times more than the OD values of the control. Atomic force microscopy images showed that the synthesized TiO2 NPs were in spherical shape with an average diameter of 67.49 nm. The TiO2 NPs inhibited the bacterial growth at concentrations of ≥ 0.1mg/ ml and a maximum zone of inhibition recorded was 22 mm at concentration of 0.4 mg/ ml. Biosynthesis of TiO2 NPs using prodigiosin was showed a promising antibacterial activity against strong biofilm producing MDR- A. baumannii.

In vitro propagation of tilapia lake virus in cell lines developed from Oreochromis mossambicus.

Tilapia lake virus (TiLV)-suspected samples of tilapia were collected from grow-out ponds located with clinical signs and mortality ranged from 5% to 50%. The reverse transcription-polymerase chain reaction (RT-PCR) assay revealed the presence of TiLV in the disease outbreak ponds. Cell lines were developed from heart, gill and eye of Mozambique tilapia and characterized. Morphologically, these cell lines are composed of epithelioid cells. The optimum growth of these cells was observed at 28°C and 20% concentration of FBS. After cryopreservation, 70%-90% of cells were found to be viable. The cells of all three cell lines were found to be positive to fibronectin and pancytokeratin. PCR amplification of 16S rRNA and COI of O.mossambicus confirmed the origin of these cell lines from O.mossambicus. Heart and gill cell lines were found to be highly susceptible to TiLV and found to be useful for its isolation from infected fish samples. The experimental infection was carried out in O.niloticus and O.mossambicus using the TiLV propagated in susceptible cell lines. The RT-PCR results revealed the presence of TiLV in brain, gill, liver, kidney, spleen, eye, muscle, intestine and heart of experimentally infected O.niloticus and O.mossambicus.

Risk from Staphylococcus aureus in informally marketed raw cow milk

This study aimed to investigate the presence of Staphylococcus spp. and Staphylococcus aureus in raw cow milk samples taken from peddlers of five regions in Bulgaria. The results showed that all 44 samples tested were positive for Staphylococcus spp. All isolates were tested for coagulase production and subjected to PCR analysis. PCR amplification of 16S rRNA and nuc genes found the presence of Staphylococcus aureus in 23 (52.3%) of a total of 44 raw milk samples. The number of Staphylococcus spp. ranged from 3x102 to 1.08x106 cfu/ml, and that of Staphylococcus aureus from 1.5x102 to 3.19x105 cfu/ml. It is concluded that control over the hygiene of handling and processing raw milk is essential for its safety.

A preliminary investigations of novel dehalogenase producing bacteria from antarctic psychrotropic Bacillus sp.IH1

A preliminary investigations of novel dehalogenase producing bacteria from Antarctic Psychrotropic Bacillus sp.Ih1 2, 2 dichloropropionic acids (Dalapon) like most halogenated compounds are commonly used as herbicides and employed in agricultural areas and industries. Toxicity of these xenobiotic compounds causes serious environmental problems. Bacillus sp. ih1 was isolated from top cliff soil collected from Antarctica. The bacteria was first grown on Antarctic bacterial medium and later transferred to a minimal medium containing 2,2, dichloropropionic acid as carbon source. It grew slowly in the minimal media in different concentrations of 10 mM, 20 mM, 30 mM and 40 mM of 2,2 DCP. The best growth was observed in 20 mM of 2,2-DCP with 32 h as doubling time. To monitor the degradative activity of the bacteria, halide ion assay was carried out to check the release of chloride ion. The best release of chloride was 0.657 mMol/L in 20 mM of 2,2-DCP. The bacteria was identified using 16S rRNA, genomic DNA extraction and PCR amplification of 16S rRNA was performed using universal primers 27F and1492R. Nucleotide blast (BLASTn) showed 97 % similarity with Bacillus sp. Results from biochemical tests further confirm the bacteria as Bacillus sp. Using phylogeny.fr, sequences from nucleotide blast result were used to build a phylogeny tree based on neighbor to neighbor joining.

Isolation and Identification of Novel IndigenousBacterial Strain as a Low Cost Pectinase Source

ABSTRACT The present study was concerned with the searching of novel bacterial cultures from different samples for the lab scale production of pectinase. Keeping in view the increasing demand of pectinase specially in Faisalabad, an industrial city of Pakistan, isolation of new hyper producer bacterial strains locally is an easy and cheap way of getting the desirable products at low cost. Therefore, isolation of new strains for industrial enzyme production has been and will be remained a part of research every time. This method alone can also provide raw material for further research such as enzyme engineering or molecular directed evolution. For the identification of hyper producer strain colony PCR was done for 16S rRNA analysis. Reason to use the 16S rRNA for identification purpose is that the gene is fairly short and can be amplified quickly and easily. The bacterial isolate (sources of pectinase enzyme) was identified based on PCR amplification of 16S rRNA and for this purpose the amplified product was run in agarose gel against a known species of Bacillus licheniformis. The 16S rRNA sequencing confirmed the Bacillus status of the strain.

부저병 원인균 Paenibacillus larvae 특이 유전자 분석을 통한 진단마커 발굴

부저병이란 Paenibacillus larvae감염에 의하여 꿀벌 유충의 괴사를 유도하는 질병이다. 우리나라에서는 2008년 봄, 국내에 처음으로 대량 발병 사례가 보고되었으며, 지속적인 2차 피해로 큰 후유증을 앓고 있다. 본 연구에서는 부저병을 효율적으로 관리할수 있는 진단방법을 조사하고자 하였다. 따라서 부저병의 원인균인 P. larvae에서 특이적으로 발현되고 있는 유전자들을 동정하고자 하였으며, 이 유전자들은 주로 부저병균의 독성을 유발하는 것으로 알려진 Toxin1, Toxin2A & 2B, SplA, CBP49, SevA&SevB 들이다. 이들은 1차 PCR 에서는 검출하기 어려웠지만, 2차 nested PCR방법을 이용하여 검출이 용이함을 알 수 있었다. 한편 여러가지 식물 추출물을 혼합한 배지에서 부저병균을 배양하였을 때, 부저병균의 성장저해와 일치하게 우리가 검증한 유전자들의 발현이 감소하는 것을 알 수 있었다. 이러한 결과들은 부저병 원인균의 특이 유전자들은 향후 PCR진단마커로서 활용 가능성이 있을 것으로 사료된다. Foulbrood disease is infected by Paenibacillus larvae on larval stage of honeybee, and is lethal disease to result in population death. This disease was manifested in 2008 in Korea, is still suffered by the secondary damages. In this study, we are to examine diagnostic PCR approaches to manage the Foulbrood disease. PCR amplification of 16S rRNA is generally using for microbial infection, but the specificity is little poor for the correct diagnosis. Therefore, we are to identify specific genes expressed in Paenibacillus larvae, and perform PCR analysis. We selected five distinct genes from literature references. Those genes are commonly known as toxic genes for host infection, and include Toxin1, Toxin2A & 2B, SplA, CBP49, and SevA&SevB. PCR amplification for these genes is difficult to detect at the first time. So, we performed the second PCR using the first PCR product as a template. This approach using the nested PCR was very useful for detecting large marker genes. When Paenibacillus larvae was cultured in the medium containing plant extracts, PCR amplification of the identified genes is correlated with the microbial growth inhibition. Therefore, these results suggest that the identified genes might be useful to study diagnostic PCR markers for honeybee Foulbrood disease.

Microbial Community Composition Associated with Maotai Liquor Fermentation.

The solid-state fermentation state of Chinese Maotai liquor involves the interaction of several complex microbial communities leading to the generation of the most complex liquor fermentation system in the world and contributes to the unique flavor and aroma of the liquor. In this study, total DNA was extracted from 3 fermented grain samples (FG1, FG2, and FG3) and 12 environmental samples, including Daqu (DA1, DA2, DA3, and DA4), cellar mud (CS1, CS2, and CS3), soil (SL1 and SL2), air (A1 and A2), and sorghum (SH), and the 16S and 18S rRNA genes were amplified. The distribution of typical microorganisms in the samples was analyzed using nested PCR-denaturing gradient gel electrophoresis, while quantitative PCR amplification of 16S rRNA and internal transcribed spacer genes was performed to estimate the microbial abundance present in each sample. The results indicated that Daqu was the primary source of bacteria, followed by the air, soil, and sorghum samples, while the majority of the fungi responsible for Maotai liquor fermentation were from Daqu and sorghum. Highest bacterial concentrations were found in fermented grains, followed by Daqu and sorghum, while the highest fungal concentrations were found in Daqu, followed by sorghum and an air sample from outside the liquor production area. The findings of this study may provide information regarding the mechanisms responsible for flavor development in Maotai liquor, and may be used to further optimize the traditional art of making liquor.

Isolation of Bordetella species from unusual infection sites

Introduction: Bordetella hinzii has been isolated mainly from respiratory specimens and from blood of immunocompromised patients, and Bordetella trematum from ear infections or leg, arm and ankle wounds and from diabetic foot ulcers. Bordetella holmesii is instead associated with bactaeremia in young adults, mostly with underlying conditions. Only three septic arthritis cases due to this species have been described in the literature. Case presentation: Herein we describe four cases of infections due to Bordetella species that have been recovered from unusual infection sites: two cases of B. hinzii infections, one recovered from the urine of a patient with chronic prostatitis and the other from a liver cyst in an immunocompetent patient; one B. trematum case from a bone biopsy of a patient with chronic osteomyelitis of the hip; and one B. holmesii case isolated from the joint fluid of an immunocompetent patient with diagnosed septic arthritis. The organisms were identified using standard biochemical tests, by API 20 NE version 6.0, by automated system VITEK 2, by mass spectrometry using the Bruker Daltonics MicroFlex LT spectrometer with MALDI Biotyper 3.1, and by PCR amplification of 16S rRNA. Antibiotic susceptibility testing was performed using the VITEK 2 system, except for B. holmesii, for which the epsilometric method (Etest technique; bioMérieux) was used. Conclusion: We highlight the importance of isolating Bordetella species from severe infections and unusual sites, and also of combining both phenotypic and genotypic methods for definitive identification.

Histamine‐2 Receptor Blockers Alter the Fecal Microbiota in Premature Infants

Bacterial colonization is considered a major risk factor for necrotizing enterocolitis (NEC). The objective of the present study was to test the hypothesis that histamine-2 receptor (H2-) blockers alter colonic bacterial colonization by analyzing and comparing the fecal microbiota in premature infants with and without H2-blocker therapy using sensitive molecular biological techniques. Seventy-six premature infants ≤1500 g or <34 weeks gestation were enrolled in this case-controlled, cross-sectional study. Stool samples were collected from 25 infants receiving H2-blockers and 51 babies who had never received them. Following DNA extraction and PCR amplification of 16S rRNA, 454 pyrosequencing was undertaken and the resulting sequences were subjected to comparison with published sequence libraries. Proteobacteria and Firmicutes were the major phyla contributing to fecal microbial communities. Microbial diversity was lower, relative abundance of Proteobacteria (primarily of the family Enterobacteriaceae) was increased, whereas that of Firmicutes was decreased in the stools of infants receiving H2-blockers compared with those who had never received them. Although not designed to look specifically at the effect of H2-blockers on the incidence of NEC, our study suggests that their use lowers fecal microbial diversity and shifts the microfloral pattern toward Proteobacteria. These alterations in fecal microbiota may predispose the vulnerable immature gut to necrotizing enterocolitis and suggest prudence in the use of H2-blockers in the premature infant.

Analysis of Virulence Potentials of Community Acquired Staphylococcus aureus, Isolated from a Slam Population of West Bengal, India

–The worldwide spread of community acquired Staphylococcus aureus (CASA) skin infection is becoming an emerging problem. This study was conducted to delineate the virulence properties and characteristics of Staphylococcus aureus isolated from children belonging to low socio-economic classes in Midnapore town, West Bengal, India. Samples were collected from affected children by aseptic means from areas of infection with proper medical intervention. These samples were confirmed first as Staphylococcus aureus by PCR amplification of 16S rRNA. Further morphology, antibiotic susceptibility, capability of secreting several enzymes, biofilm forming abilities was studied. PCR was carried out for detection of the presence of enterotoxins (seA and seB), exfoliatin toxins (etA and etB) and toxic shock syndrome toxin (tssT) genes. Results reveal that 33% community acqured Staphylococcus aureus strains were methicillin resistant and 22% were vancomycin resistant and those strains were capable of biofilm formation. Besides, all of the pathogenic and non-pathogenic strains were harboring exfoliatin toxin gene (etA). So it can be concluded that inducible multidrug resistant community acquired Staphylococcus aureus with their diverse pathogenic toxic potentials giving emerging alarm in that geographical location in India. Keywords––Staphylococcus aureus, Staphylococcal Scalded Skin Syndrome, Staphylococcal toxins, vancomycin, biofilm.

Molecular Investigation of Streptococcus agalactiae Isolates from Environmental Samples and Fish Specimens During a Massive Fish Kill in Kuwait Bay

This study was undertaken to identify and characterize bacterial isolates obtained simultaneously from dead fish samples during a massive fish kill in Kuwait Bay and sewage-water samples running into Kuwait Bay using conventional and molecular techniques. Of the 71 bacterial isolates studied; 66 were recovered from 7 different fish species and 5 strains were isolated from sewage samples. The species-specific identity of the isolates was established by phenotypic characteristics and by PCR amplification of 16S rRNA by using Streptococcus agalactiae-specific primers. The genotyping of 12 isolates from fish samples and all 5 isolates from sewage samples was performed by random amplification of polymorphic DNA (RAPD) analysis. Culture methods identified 44 of 66 (67%) and 4 of 5 (80%) isolates obtained from fish and sewage samples, respectively, as S. agalactiae. The PCR amplification of 16S rRNA not only confirmed the results of conventional methods but also resulted in additional identification of 14 of 66 (21%) isolates obtained from fish samples and the remaining isolate recovered from sewage sample, as S. agalactiae. A total of 9 RAPD patterns were observed among the 17 isolates studied; these RAPD patterns were grouped into three clusters. Interestingly, four of the isolates recovered from sewage samples produced nearly identical RAPD band patterns (85-100% similarity) with some of the S. agalactiae strains isolated from Mullet kidney and brain indicting the possibility of sewage being the source of infection.

Application of reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations

A method consisting of reverse transcriptase (RT)-PCR amplification of 16S rRNA from the total microbial community, coupled with T-RFLP, was optimized for semi-quantitative characterization of the metabolically active population in defined strain cultures of Lactococcus lactis ssp. lactis and Leuconostoc citreum, two mesophilic lactic acid bacteria (LAB) species routinely used in cheese manufacture. The set of PCR primers selected efficiently amplified the 16S rRNA from both bacterial species. The digestion of the PCR products with DdeI yielded different terminal restriction fragments (T-RFs) for each species. Nevertheless, additional T-RFs due to formation of chimeric molecules and pseudo-T-RFs derived from partly single-stranded 16S rRNA amplicons were observed in both species, although in minor amounts. Twenty PCR cycles were determined as the optimum to minimize the presence of artifactual fragments and to avoid underestimation of populations due to the saturation effect on DNA quantification caused by a PCR product excess. T-RFLP analysis showed a good repeatability when applied to mixed dairy cultures. Dynamics of two defined mixed starters consisting of a L. lactis ssp. lactis strain and a L. citreum strain were studied by this method and results compared to those obtained by a culture-dependent technique. The data indicated the suitability of T-RFLP to perform semi-quantitative analyses of microbial populations. Some slight differences could be explained by the presence of metabolically active cells that could not be detected by colony counting. RT-PCR-based T-RFLP can be an alternative to classical methods in order to study dynamics of metabolically active populations in relatively simple microbial ecosystems, such as defined dairy starter cultures.

Uncoupled Metabolism Stimulated by Chemical Uncoupler and Oxic-Settling-Anaerobic Combined Process to Reduce Excess Sludge Production

The effects of three uncoupled metabolic systems (conventional activated sludge process with the addition of 3,3', 4',5-tetrachlorosalicylanilide [TCS], oxic-settling-anaerobic [OSA] process modified by insertion of a sludge-holding tank in the sludge return line, and TCS and OSA combined process) on reducing excess sludge production were studied. Compared with the control conventional activated sludge process, the most effective system was the combined process, which could reduce excess sludge production by 46.90%. The 180-d operation results confirmed that TCS is an effective chemical uncoupler in reducing the sludge yield but that it had an adverse effect on substrate removal capability, effluent nitrogen concentration, and sludge settleability. The OSA process decreased excess sludge production by only 26% but had less adverse effect on effluent quality and could improve sludge settleability. The effluent total phosphorous concentration of the three systems was slightly lower than of the control unit. Microbial populations were monitored by both microscopic and molecular biologic analysis method (polymerase chain reaction [PCR]-denaturing gradient gel electrophoresis [DGGE]). The presence of TCS caused metazoans to disappear and decreased the number and activity of protozoa. PCR amplification of 16S rRNA and sequent DGGE analysis found a shift in the diversity of the predominant species. The results imply that OSA combined with the chemical uncoupler process may effectively reduce excess sludge yield and not affect process performance significantly.