Functional Analysis of the RdxA and RdxB Nitroreductases of Campylobacter jejuni Reveals that Mutations in rdxA Confer Metronidazole Resistance

Campylobacter jejuni is a leading cause of gastroenteritis in humans and a commensal bacterium of the intestinal tracts of many wild and agriculturally significant animals. We identified and characterized a locus, which we annotated as rdxAB, encoding two nitroreductases. RdxA was found to be responsible for sensitivity to metronidazole (Mtz), a common therapeutic agent for another epsilonproteobacterium, Helicobacter pylori. Multiple, independently derived mutations in rdxA but not rdxB resulted in resistance to Mtz (Mtz^r), suggesting that, unlike the case in H. pylori, Mtz^r might not be a polygenic trait. Similarly, Mtz^r C. jejuni was isolated after both in vitro and in vivo growth in the absence of selection that contained frameshift, point, insertion, or deletion mutations within rdxA, possibly revealing genetic variability of this trait in C. jejuni due to spontaneous DNA replication errors occurring during normal growth of the bacterium. Similar to previous findings with H. pylori RdxA, biochemical analysis of C. jejuni RdxA showed strong oxidase activity, with reduction of Mtz occurring only under anaerobic conditions. RdxB showed similar characteristics but at levels lower than those for RdxA. Genetic analysis confirmed that rdxA and rdxB are cotranscribed and induced during in vivo growth in the chick intestinal tract, but an absence of these genes did not strongly impair C. jejuni for commensal colonization. Further studies indicate that rdxA is a convenient locus for complementation of mutants in cis. Our work contributes to the growing knowledge of determinants contributing to susceptibility to Mtz (Mtz^s) and supports previous observations of the fundamental differences in the activities of nitroreductases from epsilonproteobacteria.Nitroreductases form a large family of enzymes whose physiological roles have been implicated or proposed to function in diverse processes, such as the generation of nitrogen sources for metabolism, degradation of potentially toxic nitro compounds, vitamin and bioluminescence production, redox balancing, and oxidative stress responses (20, 31, 32, 35, 41, 43, 58). These enzymes can been subdivided into two main categories based on characteristics of their reductive processes, including the mechanism of electron transfer and sensitivity to oxygen. Type I (O₂-insensitive) nitroreductases catalyze a sequential two- or three-step reduction of the nitro group on heterocyclic compounds via paired-electron transfer to produce either hydroxylamine or amino derivatives. Type II (O₂-sensitive) nitroreductases catalyze a single-electron reduction of heterocyclic nitro compounds that is reversible in the presence of oxygen (40). Nitroreductases are common in bacteria, with a given bacterial species often containing multiple paralogs that presumably reduce different substrates. The genes encoding nitroreductases have received intense study due to their unusual nature in degrading or transforming xenobiotic chemicals. Consequently, these enzymes have become attractive candidates for bioremediation processes, and some are utilized in cancer chemotherapies (27, 49). However, the nitroreductases are also a puzzling class of enzymes, because the natural substrates for most remain unknown and these proteins likely did not evolve to exclusively manipulate xenobiotic compounds.Metronidazole (Mtz) has been used in multidrug therapy for Helicobacter pylori infections due to production of factors that convert this 5-nitroimidazole product to a toxic form (1, 45, 53). Therapeutic failure with Mtz has been predominantly associated with mutations occurring in one of two genes of H. pylori encoding the nitroreductases RdxA and FrxA. A previous biochemical study characterized the Mtz reductase activity of RdxA (37). Even though RdxA was capable of reducing other nitro compounds under aerobic conditions, the enzyme was unable to reduce Mtz. However, under anaerobic conditions, RdxA was shown to catalyze the reduction of Mtz, and its specific activity for this reaction was 60-fold greater than that of the NfsB nitroreductase of Escherichia coli under similar conditions. In addition, this work revealed that RdxA exhibited a potent NADPH oxidase activity not appreciated in other nitroreductases. Not only did this study demonstrate a direct reduction of Mtz by a nitroreductase, but results from this work implied that RdxA of H. pylori possessed novel biochemical properties relative to other nitroreductases.Like H. pylori, Campylobacter jejuni is a Gram-negative bacterium belonging to the epsilonproteobacteria class. C. jejuni is a common commensal bacterium of the intestinal tracts of wild and agriculturally significant animals, especially poultry. In contrast, C. jejuni causes acute diarrhea in humans, ranging from a mild enteritis to a bloody diarrheal syndrome, and is one of the most prevalent causes of food-borne gastritis (4, 5, 34, 38). Additionally, postinfectious sequelae can develop in a small percentage of patients following a C. jejuni infection. One major complication is Guillan-Barré syndrome, a temporary and partial paralysis of the peripheral nervous system (21).Many individuals with C. jejuni enteritis resolve the infection without therapeutic treatment. If antibiotics are administered, fluoroquinolones or macrolides, such as ciprofloxacin or erythromycin, are common drugs of choice, with therapeutic use of Mtz for C. jejuni infections being unconventional. However, Mtz-resistant (Mtz^r) C. jejuni isolates have been recovered from humans and animals. In agriculture, 19 to 92% of C. jejuni isolates from avian species (including chickens and turkeys) and 6 to 20% of isolates from lambs, sheep, and cows were Mtz^r (13, 47). One study also demonstrated that 62% of C. jejuni clinical isolates from humans were Mtz^r (47). These data are curious, since these C. jejuni isolates would have likely developed Mtz^r during infections in the absence of selection.Because susceptibility to Mtz (Mtz^s) was also found in isolates associated with each host in studies described above and since C. jejuni is closely related to H. pylori, we hypothesized that C. jejuni may produce a nitroreductase to reduce Mtz to its toxic form, leading to Mtz^s. In this report, we identify and characterize the gene required for Mtz^s in C. jejuni. Mutations in this gene, encoding a putative nitroreductase, but not in a downstream paralog were linked to the development of Mtz^r, indicating that Mtz^r in C. jejuni appears to be linked to mutation of only one nitroreductase. Supporting our findings, we provide evidence that a proportion of Mtz^r isolates of C. jejuni are due to spontaneous errors during DNA replication in the absence of Mtz exposure, resulting in a variety of mutations. Biochemical analysis of these C. jejuni nitroreductases demonstrated that these proteins had potent NADPH oxidase activity and could reduce Mtz under anaerobic conditions. These results, along with previous biochemical analysis of H. pylori RdxA (37), demonstrate that the nitroreductases of epsilonproteobacteria have unique characteristics in comparison to other bacterial counterparts.     

Expression of cagA,virB/D Complex and/or vacA Genes in Helicobacter pylori Strains Originating from Patients with Gastric Diseases

In order to better understand pathogenicity of Helicobacter pylori, particularly in the context of its carcinogenic activity, we analysed expression of virulence genes: cagA, virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, virD4) and vacA in strains of the pathogen originating from persons with gastric diseases. The studies were conducted on 42 strains of H. pylori isolated from patients with histological diagnosis of non-atrophic gastritis—NAG (group 1, including subgroup 1 containing cagA⁺ isolates and subgroup 2 containing cagA^- strains), multifocal atrophic gastritis—MAG (group 2) and gastric adenocarcinoma—GC (group 3). Expression of H. pylori genes was studied using microarray technology. In group 1, in all strains of H. pylori cagA⁺ (subgroup 1) high expression of the gene as well as of virB/D was disclosed, accompanied by moderate expression of vacA. In strains of subgroup 2 a moderate expression of vacA was detected. All strains in groups 2 and 3 carried cagA gene but they differed in its expression: a high expression was detected in isolates of group 2 and its hyperexpression in strains of group 3 (hypervirulent strains). In both groups high expression of virB/D and vacA was disclosed. Our results indicate that chronic active gastritis may be induced by both cagA⁺ strains of H. pylori, manifesting high expression of virB/D complex but moderate activity of vacA, and cagA^- strains with moderate expression of vacA gene. On the other hand, in progression of gastric pathology and carcinogenesis linked to H. pylori a significant role was played by hypervirulent strains, manifesting a very high expression of cagA and high activity of virB/D and vacA genes.     

Enhanced bacterial efflux system is the first step to the development of metronidazole resistance in Helicobacter pylori

Although metronidazole (Mtz) is an important component of Helicobacter pylori eradication regimens, it has been pointed out that the increasing use of Mtz may result in increase in the incidence of Mtz-resistant strains. The present study was designed to examine the initial mechanism of resistance acquisition of H. pylori to Mtz. After 10 Mtz-susceptible strains were cultured on plates containing sub-inhibitory concentrations of Mtz, the MIC of Mtz for 9 of the 10 strains increased to levels of the Mtz-resistant strains. In the Mtz-resistance-induced strains, the expression of the TolC efflux pump (hefA) was significantly increased under Mtz exposure, without the reduction of the Mtz-reductive activity. Our finding suggests that overexpression of hefA may be the initial step in the acquisition of Mtz resistance in H. pylori.     

Metronidazole resistance and microaerophily in Campylobacter species

Metronidazole is active against most anaerobic organisms and is also used in the treatment of the microaerophilic bacterium Helicobacter pylori. Resistance to metronidazole is uncommon in most anaerobic organisms, but it is increasingly prevalent in H. pylori. Previously we have suggested that metronidazole resistance in H. pylori is inherent in the microaerophilic nature of the organism and therefore would be present in other microaerophiles such as Campylobacter. Short periods of anaerobiosis caused metronidazole-resistant (Mtr^R) strains of Campylobacter spp. to become sensitive to metronidazole. Under microaerophilic conditions, cultures of the Mtr^R mutant Campylobacter coli R1 at bacterial cell densities of greater than 10⁸ cfu/ml lost viability, whereas no loss in viability was observed in cultures at cell densities of less than 10⁸. The Mtr^S C. coli strain lost viability at all cell densities. Comparisons of NAD(P)H oxidase activity between MtrS and Mtr^R strains indicated that the Mtr^S C. coli strain contained fourfold higher NADH oxidase activity and twofold higher NADPH oxidase activity than did the Mtr^R Campylobacter strains. These results show that Mtr^R Campylobacter spp. display resistance characteristics similar to those of H. pylori, suggesting that the resistance mechanism is a phenomenon of the microaerophilic nature of these bacteria. Received: 9 March 1998 / Accepted: 17 June 1998     

Molecular basis of the optochin-sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniaeStreptococcus oralis H+-ATPases

The gene responsible for the optochin-sensitive (Opt^S) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F₀ complex of an H⁺-ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin-sensitive resistant (Opt^S/Opt^R) phenotype. Our results also show that natural as well as laboratory Opt^R isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F F₀ complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opt^S) and M222 (an Opt^R strain produced by inter-species recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane-bound ATPase activity of the S. pneumoniae wild-type (Opt^S) strains, and found a 100-fold difference between Opt^S and Opt^R strains, both in growth inhibition and in membrane ATPase resistance.     

Role of the Outer Membrane Protein OprD2 in Carbapenem-Resistance Mechanisms of Pseudomonas aeruginosa

We investigated the relationship between the outer membrane protein OprD₂ and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD₂ was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMP^R and IMP^S groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMP^RMEM^R (66.7%), IMP^RMEM^S (32.6%), and IMP^RMEM^S (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD₂-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD₂gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD₂ gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMP^RMEM^R strains and 5 IMP^RMEM^S strains had lost the OprD₂ protein, while the IMP^SMEM^R strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD₂-encoding gene caused the loss of OprD₂, which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa.     

Allelic Polymorphism of the tet(M) Determinant in Mycoplasma hominis and Ureaplasma urealyticum Clinical Isolates Resistant to Tetracyclines

Of the 130 clinical isolates of Mycoplasma hominisfrom patients with nonspecific inflammatory diseases of the urogenital tract (UGT), approximately 10% contained the tet(M) gene after the course of treatment with tetracyclines. This gene was found in nine (25%) of the 36 Ureaplasma urealyticum clinical isolates. The nucleotide sequence of 13 tet(M) genes in Tc^R clinical isolates ofM. hominis and five genes in U. urealyticum Tc^R clinical isolates was determined. A comparison of nucleotide sequences of eight tetM genes of different origin and tet(M) genes ofGardnerella vaginalis and M. hominis and U. urealyticumclinical isolates showed that the mosaic structure of thetet(M) gene is completely identical in 11 of 13 M. hominis Tc^Risolates but belongs to an unidentified allele different from those described earlier. Another new allelic variant oftet(M) was found in two isolates. In three of five Tc^R clinical isolates of U. urealyticum, a tet(M) gene, whose mosaic structure was identical to that of tet(M) reported previously for ureaplasmas, and also two new allelic variants, which have not been described so far, were found.     

PCR Detection of Oxytetracycline Resistance Genes otr(A) and otr(B) in Tetracycline-Resistant Streptomycete Isolates from Diverse Habitats

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc^R) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc^R streptomycetes originated from bulk and rhizosphere soil. Fewer Tc^R streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene–carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc^R streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.     

Type I Restriction–Modification Loci Reveal High Allelic Diversity in Clinical Helicobacter pylori Isolates

Background: A remarkable variety of restriction‐modification (R‐M) systems is found in Helicobacter pylori. Since they encompass a large portion of the strain‐specific H. pylori genes and therefore contribute to genetic variability, they are suggested to have an impact on disease outcome. Type I R‐M systems comprise three different subunits and are the most complex of the three types of R‐M systems. Aims: We investigated the genetic diversity and distribution of type I R‐M systems in clinical isolates of H. pylori. Material and methods: Sixty‐one H. pylori isolates from a Swedish hospital based case‐control study and 6 H. pylori isolates of a Swedish population‐based study were analyzed using polymerase chain reaction for the presence of the three R‐M systems' subunits. Representative gene variants were sequenced. Results: Although the hsdM and hsdR genes appeared conserved in our clinical H. pylori isolates, the sequences of the hsdS loci were highly variable. Despite their sequence diversity, the genes per se were present at high frequencies. We identified a number of novel allelic hsdS variants, which are distinct from corresponding hsdS loci in the sequenced H. pylori strains 26695, J99 and HPAG1. In analyses of paired H. pylori isolates, obtained from the same individuals with a 4‐year interval, we observed genetic modifications of hsdS genes in patients with atrophic gastric mucosa. Discussion: We propose that the genetic variability of hsdS genes in a bacterial population will give rise to new specificities of these enzymes, which might lead to adaptation to an ever‐changing gastric environment.     

Proteomics-Based Identification and Analysis of Proteins Associated with Helicobacter pylori in Gastric Cancer

Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that causes the most common chronic infection in the human stomach. Approximately 1%-3% of infected individuals develop gastric cancer. However, the mechanisms by which H. pylori induces gastric cancer are not completely understood. The available evidence indicates a strong link between the virulence factor of H. pylori, cytotoxin-associated gene A (CagA), and gastric cancer. To further characterize H. pylori virulence, we established three cell lines by infecting the gastric cancer cell lines SGC-7901 and AGS with cagA⁺ H. pylori and transfecting SGC-7901 with a vector carrying the full-length cagA gene. We detected 135 differently expressed proteins from the three cell lines using proteome technology, and 10 differential proteins common to the three cell lines were selected and identified by LC-MS/MS as well as verified by western blot: β-actin, L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD), pre-mRNA-processing factor 19 homolog (PRPF19), ATP synthase, calmodulin (CaM), p64 CLCP, Ran-specific GTPase-activating protein (RanGAP), P43 and calreticulin. Detection of the expression of these proteins and genes encoding these proteins in human gastric cancer tissues by real-time PCR (RT-qPCR) and western blot revealed that the expression of β-ACTIN, LDH, DLD, PRPF19 and CaM genes were up-regulated and RanGAP was down-regulated in gastric cancer tissues and/or metastatic lymph nodes compared to peri-cancerous tissues. High gene expression was observed for H. pylori infection in gastric cancer tissues. Furthermore, the LDH, DLD and CaM genes were demethylated at the promoter -2325, -1885 and -276 sites, respectively, and the RanGAP gene was highly methylated at the promoter -570 and -170 sites in H. pylori-infected and cagA-overexpressing cells. These results provide new insights into the molecular pathogenesis and treatment targets for gastric cancer with H. pylori infection.     

A jumping gene in Streptomyces coelicolor A3(2)

Summary The difficulty in mapping the gene for chloramphenicol resistance (cml ^R) in Streptomyces coelicolor A3(2) stock strains is possibly due to its location on different sites of the chromosome in various mixed subelones. Fresh isolates from Cml^R strains show single unequivocal locations of cml ^R. The same holds for Cml^R strains derived as revertants from Cml^S variants. The two best established sites for cml ^R are one between cys A and met A, the other at right of arg A, possibly in the right empty arc of the map (Fig. 2). The cml ^R gene was assumed to be on a transposon (SCTn1), together with a gene for arginine-succinate synthase (argG), a gene for chromosome transfer (tra) and a gene for aereal mycelium formation (amy). In a Cml^R revertant, the cml ^R gene appears disjoined from argG (Fig. 5), thus showing the ability of SCTnl to be split and partially transposed. The possible wide occurrence of transposons in the genus Streptomyces is discussed.     

Emergence of clarithromycin-resistant Helicobacter pylori (CRHP) with a high prevalence in children compared with their parents

Background. Clarithromycin‐resistant Helicobacter pylori (CRHP) is increasing worldwide. Clarithromycin resistance in H. pylori from familial members has not been investigated. Materials and Methods. Biopsy specimens were taken from 13 families living in Tokyo, Yokohama, and Niigata between 1998 and 2001. Drug resistance was tested with the replica plating method. The minimum inhibitory concentrations of antimicrobial agents for H. pylori strains were determined by the agar dilution method. Molecular analyses of H. pylori strains were performed by ribosomal RNA gene restriction pattern analysis. The DNA region, associated with clarithromycin resistance, was analyzed by PCR and sequencing. Results. Helicobacter pylori strains isolated from a 5‐year‐old‐son displayed clarithromycin resistance with a mutation (A → G at position 2143) in the 23S ribosomal RNA, whereas H. pylori strains from his parents did not. DNA analyses revealed that the boy was infected with his father's strain. The boy had repeatedly developed otitis media and received clarithromycin since the age of 2 years. Studies on an additional 12 families demonstrated that clarithromycin resistance in the children's strains reached 42.9% and was significantly higher than those of H. pylori strains from their parents (0%) or from adult patients (11.1%) (p < .05). Conclusions. The rate of clarithromycin resistance in H. pylori strains from Japanese children was extremely high, in contrast to those from their parents or adult patients. Prior history of clarithromycin usage in a child suggested development of clarithromycin resistance in resident H. pylori, which was originated from a parent.     

Characterisation of worldwide Helicobacter pylori strains reveals genetic conservation and essentiality of serine protease HtrA

HtrA proteases and chaperones exhibit important roles in periplasmic protein quality control and stress responses. The genetic inactivation of htrA has been described for many bacterial pathogens. However, in some cases such as the gastric pathogen Helicobacter pylori, HtrA is secreted where it cleaves the tumour‐suppressor E‐cadherin interfering with gastric disease development, but the generation of htrA mutants is still lacking. Here, we show that the htrA gene locus is highly conserved in worldwide strains. HtrA presence was confirmed in 992 H. pylori isolates in gastric biopsy material from infected patients. Differential RNA‐sequencing (dRNA‐seq) indicated that htrA is encoded in an operon with two subsequent genes, HP1020 and HP1021. Genetic mutagenesis and complementation studies revealed that HP1020 and HP1021, but not htrA, can be mutated. In addition, we demonstrate that suppression of HtrA proteolytic activity with a newly developed inhibitor is sufficient to effectively kill H. pylori, but not other bacteria. We show that Helicobacter htrA is an essential bifunctional gene with crucial intracellular and extracellular functions. Thus, we describe here the first microbe in which htrA is an indispensable gene, a situation unique in the bacterial kingdom. HtrA can therefore be considered a promising new target for anti‐bacterial therapy.     

Antimicrobial Resistance and Molecular Investigation of H2S-Negative Salmonella enterica subsp. enterica serovar Choleraesuis Isolates in China

Salmonella enterica subsp. enterica serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 S. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H₂S-negative S. Choleraesuis isolates and two H₂S-positive isolates. This is the first report of H₂S-negative S. Choleraesuis isolated from humans. The majority of H₂S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the gyrA and parC genes and identified two new mutations in the parC gene. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis were employed to investigate the genetic relatedness of H₂S-negative and H₂S-positive S. Choleraesuis isolates. PFGE revealed two groups, with all 19 H₂S-negative S. Choleraesuis isolates belonging to Group I and H₂S-positive isolates belonging to Group II. By MLST analysis, the H₂S-negative isolates were all found to belong to ST68 and H₂S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H₂S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H₂S-negative isolates also possessed a frame-shift mutation at position 760 of phsA gene compared with H₂S-positive isolates, which may be responsible for the H₂S-negative phenotype. Moreover, the 19 H₂S-negative isolates have similar PFGE patterns and same mutation site in the phsA gene, these results indicated that these H₂S-negative isolates may have been prevalent in China. These findings suggested that surveillance should be increased of H₂S-negative S. Choleraesuis in China.     

Genetic and metabolic diversity of streptomycetes in pulp and paper mill effluent treated crop fields

Irrigation of farm field with water mixed with pulp and paper mill effluent from Century pulp and paper mill in Uttrakhand state of India for over last 25 years in succession increased streptomycetes population (120 × 10⁵) compared to the fresh water irrigated fields (48 × 10³ in WIF). Denaturing gradient gel electrophoresis, amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, BIOLOG™ substrate usage, production of extracellular enzymes (xylanase and cellulase) and plant growth promoting attributes were applied to monitor changes in genetic and metabolic diversity of streptomycetes. Significant variation was observed for production of extracellular enzymes, Indolic compounds, siderophore and P-solubilisation among isolates. Metabolic substrate usage of Streptomyces isolates was evaluated using the BIOLOG™ GP2 plates and unique carbon substrate usage profiles were observed. Based on 16S rRNA gene sequencing, the isolates were identified as Streptomyces variabilis, Streptomyces spp. S. glaucescens, S. viridochromogenes, S. cinnabarinus, S. aburaviensis, S. viridis, S. xylophagus, S. macrosporeus, S. thermocarboxydus, and S. albogriseolus. The diversity index parameters like Shannon index, reciprocal of Simpson’s index (1/D), and Pielou index of evenness based on ARDRA revealed that streptomycetes community in effluent irrigated field (EIF) was more diverse. DGGE profiles of Streptomyces specific 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar in both soils.     

Prevalence of tetracycline resistance genes in Greek seawater habitats

The presence of selected tetracycline resistance (Tc^R) genes was studied in different Greek seawater habitats, originated from wastewater treatment facilities, fishfarm, and coastal environments. The methods employed included assessment of the presence of twelve gene clusters by PCR, followed by hybridization with specific probes, in habitat extracted DNA, Tc^R bacteria, and exogenous isolated plasmids conferring Tc^R. The direct DNA-based analysis showed that tet(A) and tet(K) genes were detected in all habitats, whilst tet(C) and tet(E) were present in fishfarm and wastewater effluent samples and tet(M) was detected in fish-farm and coastal samples. Resistance genes tet(h), tet(C), tet(K), and tet(M) were detected in 60 of the 89 isolates screened. These isolates were identified by fatty acid methyl ester analysis (FAME) as Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Staphylococcus strains. The presence of the Tc^R genes in 15% of the bacterial isolates coincided with the presence of IncP plasmids. A habitat-specific dissemination of IncP alpha plasmids in wastewater effluent isolates and of IncP beta plasmids in fishfarm isolates was observed. Exogenous isolation demonstrated the presence of plasmids harbouring Tc^R genes in all the habitats tested. Plasmids were shown to carry tet(h), tet(C), tet(E), and tet(K) genes. It is concluded that Tc^R genes are widespread in the seawater habitats studied and often occur on broad host range plasmids that seem to be well disseminated in the bacterial communities.     

Helicobacter pylori diversification during chronic infection within a single host generates sub-populations with distinct phenotypes

Helicobacter pylori chronically infects the stomach of approximately half of the world’s population. Manifestation of clinical diseases associated with H. pylori infection, including cancer, is driven by strain properties and host responses; and as chronic infection persists, both are subject to change. Previous studies have documented frequent and extensive within-host bacterial genetic variation. To define how within-host diversity contributes to phenotypes related to H. pylori pathogenesis, this project leverages a collection of 39 clinical isolates acquired prospectively from a single subject at two time points and from multiple gastric sites. During the six years separating collection of these isolates, this individual, initially harboring a duodenal ulcer, progressed to gastric atrophy and concomitant loss of acid secretion. Whole genome sequence analysis identified 1,767 unique single nucleotide polymorphisms (SNPs) across isolates and a nucleotide substitution rate of 1.3x10^-4 substitutions/site/year. Gene ontology analysis identified cell envelope genes among the genes with excess accumulation of nonsynonymous SNPs (nSNPs). A maximum likelihood tree based on genetic similarity clusters isolates from each time point separately. Within time points, there is segregation of subgroups with phenotypic differences in bacterial morphology, ability to induce inflammatory cytokines, and mouse colonization. Higher inflammatory cytokine induction in recent isolates maps to shared polymorphisms in the Cag PAI protein, CagY, while rod morphology in a subgroup of recent isolates mapped to eight mutations in three distinct helical cell shape determining (csd) genes. The presence of subgroups with unique genetic and phenotypic properties suggest complex selective forces and multiple niches within the stomach during chronic infection.