Strains ofE Research Articles

Antibiotic Profiling of E. coli Borne UTI Infection in Tertiary Healthcare Settings.

Introduction In general, with frequent recurrence of urinary tract infections (UTIs), long-term antibiotic therapy is stipulated at a low dose. With this type of situation and with easy access to several classes of antibiotics in tertiary health care settings, the use of such drugs results in the development of resistant bacteria in patients.Escherichia coliis a frequent cause of UTI observed. Hence, it was proposed in the present study to assess the antimicrobial resistance status ofE. coliin UTI-infected patients. Methods This study was conducted among female patients diagnosed with UTI.About 80 urine samples were collected in an aseptic condition, Under the process of culture identification 44 samples were found to be positive for UTI infection. The positive samples were plated on blood agar. Out of 44 samples, 18 samples were found to be positive, and 26 samples were negative for E. coli infection. The 18 samples were screened on MALDI-TOF for identification. Further, the samples were assessed for susceptibility to antibiotic medication within the study area. Result The study identified different strains ofE. coli,and the CHB geneE. coli wasfound in eight samples. The sample showed pink oval-round spots in the culture medium and was resistant to nitrofurantoin, cephalosporin, and cephalexin antibiotics. Hence, antimicrobial susceptibility tests arenecessaryfor managing and treating bacterialE. coliinfections. Conclusion E. coliis a common bacterium found in the vaginal region of patients, suggesting a potential infection.E. colican be associated with UTIs in women. The results from this study conclude thatE. coliis rapidly becoming multidrug-resistant, as only higher antibiotics can inhibit its growth. To effectively manage infections caused byE. coliproper diagnosis, laboratory testing, and antibiotic treatment are required.

Emergence of novel ST1299 vanA lineages as possible cause for the striking rise of vancomycin resistance among invasive strains of Enterococcus faecium at a German university hospital.

Since 2019, we have observed a remarkable rise of vancomycin resistance among invasive strains of Enterococcus (E.) faecium at the University Hospital Erlangen (UKER), a tertiary-care hospital in Germany. Here, we analyzed the genetic diversity of E. faecium isolated from blood cultures of in-patients by core genome multilocus sequence typing to see whether the occurrence of vancomycin resistance was associated with the spread of new clonal lineages. Between January and December 2022, 37 consecutive non-duplicate isolates were included in this study. Resistance to vancomycin was detected in 15 isolates (40.5%). The affected patients had a median age of 65 years, and 26 of them (70.3%) were males. Patients with bloodstream infection caused by vancomycin-resistant E. faecium (VREfm) or vancomycin-susceptible E. faecium (VSEfm) showed a similar in-hospital mortality rate (33.3% vs 27.3%, P = 0.692), indicating that VREfm were not more virulent than VSEfm. In contrast, the genetic diversity was higher in VSEfm (15 clonal lineages) than in VREfm isolates (seven clonal lineages). The majority of VREfm isolates (73.2%) belonged to only three clonal lineages: ST117/CT71 vanB (n = 4), which is the most commonly detected vancomycin-resistant lineage in Germany, and two novel ST1299 vanA lineages classified as CT3109 (n = 4) and CT1903 (n = 3). We conclude that the high rate of vancomycin resistance among invasive E. faecium isolates at UKER could be associated with the emergence of novel ST1299 vanA lineages, which is likely to be relevant also for other hospitals and countries. Future studies will focus on the prevalence of ST1299 vanA in the general population. IMPORTANCE The proportion of VREfm among all Enterococcus faecium isolated from blood cultures in German hospitals has increased in the period 2015-2020 from 11.9% to 22.3% with a country-wide spread of the clonal lineage ST117/CT71 vanB. In this study, we provided useful information about the genetic diversity of invasive strains of E. faecium. Moreover, our findings confirm the nosocomial spread of novel ST1299 vanA lineages, which recently had a rapid expansion in Austria and the south-eastern part of Germany.

Complete genome sequence and characterization of a novel Enterococcus faecium with probiotic potential isolated from the gut of Litopenaeus vannamei.

Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus, most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

Coordinated expression of acetyl CoA synthetase and the ace operon enzymes in Escherichia coli in preparation for adaptation to acetate.

Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.

Presence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing

Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0–20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis . It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.

Draft Genome Sequences of Four Streptomycin-Sensitive Erwinia amylovora Strains Isolated from Commercial Apple Orchards in Ohio.

ABSTRACTErwinia amylovora is the causative agent of fire blight, a devastating disease of apples and pears worldwide. Here, we report draft genome sequences of four streptomycin-sensitive strains of E. amylovora that were isolated from diseased apple trees in Ohio.

Genome-based studies indicate that the Enterococcus faecium Clade B strains belong to Enterococcus lactis species and lack of the hospital infection associated markers.

Enterococcus lactis and the heterotypic synonym Enterococcus xinjiangensis from dairy origin have recently been identified as a novel species based on 16S rRNA gene sequence analysis. Enterococcus faecium type strain NCTC 7171T was used as the reference genome for determining E. lactis and E. faecium to be separate species. However, this taxonomic classification did not consider the diverse lineages of E. faecium, and the double nature of hospital-associated (clade A) and community-associated (clade B) isolates. Here, we investigated the taxonomic relationship among isolates of E. faecium of different origins and E. lactis, using a genome-based approach. Additional to 16S rRNA gene sequence analysis, we estimated the relatedness among strains and species using phylogenomics based on the core pangenome, multilocus sequence typing, the average nucleotide identity and digital DNA-DNA hybridization. Moreover, following the available safety assessment schemes, we evaluated the virulence profile and the ampicillin resistance of E. lactis and E. faecium clade B strains. Our results confirmed the genetic and evolutionary differences between clade A and the intertwined clade B and E. lactis group. We also confirmed the absence in these strains of virulence gene markers IS16, hylEfm and esp and the lack of the PBP5 allelic profile associated with ampicillin resistance. Taken together, our findings support the reassignment of the strains of E. faecium clade B as E. lactis.

Evolutionary Dynamics Based on Comparative Genomics of Pathogenic Escherichia coli Lineages Harboring Polyketide Synthase (pks) Island.

ABSTRACTThe genotoxin colibactin is a secondary metabolite produced by the polyketide synthase (pks) island harbored by extraintestinal pathogenic E. coli (ExPEC) and other members of the Enterobacteriaceae that has been increasingly reported to have critical implications in human health. The present study entails a high-throughput whole-genome comparison and phylogenetic analysis of such pathogenic E. coli isolates to gain insights into the patterns of distribution, horizontal transmission, and evolution of the island. For the current study, 23 pks-positive ExPEC genomes were newly sequenced, and their virulome and resistome profiles indicated a preponderance of virulence encoding genes and a reduced number of genes for antimicrobial resistance. In addition, 4,090 E. coli genomes from the public domain were also analyzed for large-scale screening for pks-positive genomes, out of which a total of 530 pks-positive genomes were studied to understand the subtype-based distribution pattern(s). The pks island showed a significant association with the B2 phylogroup (82.2%) and a high prevalence in sequence type 73 (ST73; n = 179) and ST95 (n = 110) and the O6:H1 (n = 110) serotype. Maximum-likelihood (ML) phylogeny of the core genome and intergenic regions (IGRs) of the ST95 model data set, which was selected because it had both pks-positive and pks-negative genomes, displayed clustering in relation to their carriage of the pks island. Prevalence patterns of genes encoding RM systems in the pks-positive and pks-negative genomes were also analyzed to determine their potential role in pks island acquisition and the maintenance capability of the genomes. Further, the maximum-likelihood phylogeny based on the core genome and pks island sequences from 247 genomes with an intact pks island demonstrated horizontal gene transfer of the island across sequence types and serotypes, with few exceptions. This study vitally contributes to understanding of the lineages and subtypes that have a higher propensity to harbor the pks island-encoded genotoxin with possible clinical implications.

Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications.

Recombinant proteins are becoming increasingly important for industrial applications, where Escherichia coli is the most widely used bacterial host for their production. However, the formation of inclusion bodies is a frequently encountered challenge for producing soluble and functional recombinant proteins. To overcome this hurdle, different strategies have been developed through adjusting growth conditions, engineering host strains of E. coli, altering expression vectors, and modifying the proteins of interest. These approaches will be comprehensively highlighted with some of the new developments in this review. Additionally, the unique features of protein inclusion bodies, the mechanism and influencing factors of their formation, and their potential advantages will also be discussed.

Can Pathogenic and Nonpathogenic Bacteria Be Distinguished by Sensory Protein Abundance?

Signal transduction systems are essential for microorganisms to respond to their ever-changing environment. They can be distinguished into one-component systems, two-component systems, and extracytoplasmic-function σ factors. Abundances of a few signal-transducing proteins, termed herein as sensory proteins (SPs), have previously been reported to be correlated with the genome size and ecological niche of certain Gram-positive bacteria. No such reports are available for Gram-negative bacteria. The current study attempts to investigate the relationship of the abundances of SPs to genome size in Escherichia coli, and the bacterial pathotypes or phylotypes. While the relationship between SP abundance and genome size could not be established, the sensory protein index (SPI), a new metric defined herein, was found to be correlated with E. coli virulence. In addition, significant association was observed among the distribution of SPs and E. coli pathotypes. Results indicate that such associations might be due to genomic rearrangements to best utilize the resources available in a given ecological niche. Overall, the study provides an in-depth analysis of the occurrence of different SPs among pathogenic and nonpathogenic E. coli strains. Possibilities of using the SPI as a marker for identifying pathogenic strains from among an organism complex are also discussed.IMPORTANCE Sensory proteins (SPs) act as sensors and actuators for a cell and participate in important mechanisms pertaining to bacterial survival, adaptation, and virulence. Therefore, bacterial species residing in similar ecological niches or those sharing common pathotypes are expected to exhibit similar SP signatures. We have investigated profiles of SPs in different species of Escherichia coli and present in this article the sensory protein index (SPI), a metric for quantifying the abundance and/or distribution of SPs across bacterial genomes, which could indicate the virulence potency of a bacterium. The SPI could find use in characterizing uncultured strains and bacterial complexes, as a biomarker for disease diagnostics, evaluating the effect of therapeutic interventions, assessing effects of ecological alterations, etc. Grouping the studied strains of E. coli on the basis of the frequency of occurrence of SPs in their genomes could potentially replicate the stratification of these strains on the basis of their phylotypes. In addition, E. coli strains belonging to the same pathotypes were also seen to share similar SP signatures. Furthermore, the SPI was seen to be an indicator of pathogenic potency of E. coli strains. The SPI metric is expected to be useful in the (pathogenic) characterization of hereto uncultured strains which are routinely sequenced in host microbiome analysis projects, or from among an ensemble of microbial organisms constituting a biospecimen. Thus, the possibilities of using the SPI as a biomarker for diagnosis of a disease or the outcome of a therapeutic intervention cannot be ruled out. Further, SPIs obtained from longitudinal ecological samples have the potential to serve as key indicators of environmental changes. Such changes in the environment are often detrimental to the resident biome and methods for timely detection of environmental changes hold huge socioeconomic benefits.

Erythrobacter insulae sp. nov., isolated from a tidal flat

A Gram-staining-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated as JBTF-M21T, was isolated from a tidal flat sediment on the Yellow Sea, Republic of Korea. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences indicated that JBTF-M21T fell within the clade comprising the type strains of species of the genus Erythrobacter. JBTF-M21T exhibited 16S rRNA gene sequence similarities of 97.0-98.4 % to the type strains of Erythrobacter longus, Erythrobacter aquimaris, Erythrobacter nanhaisediminis, Erythrobacter vulgaris, Erythrobacter seohaensis, Erythrobacter litoralis and Erythrobacter citreus and 93.7-96.6 % to the type strains of the other species of the genus Erythrobacter. The ANI and dDDH values between JBTF-M21T and the type strains of E. longus, E. nanhaisediminis, E. seohaensis and E. litoralis were 70.83-72.93 % and 18.0-18.8 %, respectively. Mean DNA-DNA relatedness values between JBTF-M21T and the type strains of E. aquimaris, E. vulgaris and E. citreus were 12-24 %. The DNA G+C content of JBTF-M21T was 57.0 mol%. JBTF-M21T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c and C17 : 1ω6c as the major fatty acids. The major polar lipids ofJBTF-M21T were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. Distinguishing phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that JBTF-M21T is separated from species of the genus Erythrobacter with validly published names. On the basis of the data presented, strain JBTF-M21T is considered to represent a novel species of the genus Erythrobacter, for which the name Erythrobacter insulae sp. nov. is proposed. The type strain is JBTF-M21T (=KACC 19864T=NBRC 113584T).

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Comparative Biotypic and Phylogenetic Profiles of Escherichia coli Isolated from Resident Stool and Lagoon in Fresco (Côte d'Ivoire).

Anthropogenic activities could expose Fresco lagoon to microbial pollution. The objective of this study was to determine the level of pollution in Fresco lagoon related to fecal contaminations. Two hundred and seventy (270) samples including 216 water and 54 human stools samples from local residents were collected. Escherichia coli was isolated and identified according to classical bacteriology procedure. Strains were characterized by biotyping on API 20E gallery and phylogenetic typing by PCR triplex of Clermont. A set of 392 strains of E. coli was distributed into 18 biotypic profiles. Five biotypes were common to water and human. Classification of all biotypes revealed close relationship between water and human strains because of their repartition in the same groups. Phylogenetic groups A, B1, B2, and D were identified in all strains. Strains belonging to phylogenetic group A were most frequent in water (69.82%) and human stool (44.44%) followed by group B1 in water (24%) and human stool (40.7%). Strains of group B2 were scarce in water (4.4%) and humans (7.41%). The diversity of E. coli biotypes observed in this study revealed animal and human origins of contaminations. A close relationship was found between water and human strains, and the presence of commensal and extraintestinal pathogenic E. coli in all samples could represent a potential reservoir of extraintestinal infections for resident populations.

Proposal of Enterococcus xinjiangensis Ren et al. 2020 as a later heterotypic synonym of Enterococcus lactis Morandi et al. 2012.

Enterococcus lactis was published in the International Journal of Systematic and Evolutionary Microbiology in June 2012. Enterococcus xinjiangensis was published in Current Microbiology in 2016 and validated in November 2020. In the present study, the relationship between E. lactis and E. xinjiangensis was re-evaluated. In the 16S rRNA gene phylogeny, E. xinjiangensis was closely related to E. lactis and Enterococcus faecium. The type strains of E. xinjiangensis and E. lactis shared 99.8 % 16S rRNA gene sequence similarity, 98.5 % pheS sequence similarity, 99.9 % rpoA sequence similarity, 98.7 % average nucleotide identity (ANI) value and 88.9 % digital DNA-DNA hybridization (dDDH) value, indicating that they represent the same species. Meanwhile, although the type strains of E. xinjiangensis and E. faecium also shared 99.8 % 16S rRNA gene sequence similarity, 97.1 % pheS sequence similarity and 99.6 % rpoA sequence similarity, 94.7 % ANI and 59.1 % dDDH values indicated that they represent two different species. On the basis of the results present here, we propose Enterococcus xinjiangensis Ren et al. 2020 as a later heterotypic synonym of Enterococcus lactis Morandi et al. 2012.

Heterogeneous and Flexible Transmission of mcr-1 in Hospital-Associated Escherichia coli.

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route of mcr-1 among Enterobacteriaceae species in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis of Escherichia coli isolates collected in a hospital in Hangzhou, China. We found that mcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread of mcr-1. The mcr-1 gene was found on either chromosomes or plasmids, but in most of the E. coli isolates, mcr-1 was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission of mcr-1 and the coexistence of mcr-1 with other genes encoding β-lactamases and fluoroquinolone resistance in the E. coli isolates. These findings indicate that mcr-1 is heterogeneously disseminated in both commensal and pathogenic strains of E. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology of mcr-1 among hospital-associated E. coli strains.

Epigenetic Mechanisms Regulate Innate Immunity against Uropathogenic and Commensal-Like Escherichia coli in the Surrogate Insect Model Galleria mellonella.

Innate-immunity-related genes in humans are activated during urinary tract infections (UTIs) caused by pathogenic strains of Escherichia coli but are suppressed by commensals. Epigenetic mechanisms play a pivotal role in the regulation of gene expression in response to environmental stimuli. To determine whether epigenetic mechanisms can explain the different behaviors of pathogenic and commensal bacteria, we infected larvae of the greater wax moth, Galleria mellonella, a widely used model insect host, with a uropathogenic E. coli (UPEC) strain that causes symptomatic UTIs in humans or a commensal-like strain that causes asymptomatic bacteriuria (ABU). Infection with the UPEC strain (CFT073) was more lethal to larvae than infection with the attenuated ABU strain (83972) due to the recognition of each strain by different Toll-like receptors, ultimately leading to differential DNA/RNA methylation and histone acetylation. We used next-generation sequencing and reverse transcription (RT)-PCR to correlate epigenetic changes with the induction of innate-immunity-related genes. Transcriptomic analysis of G. mellonella larvae infected with E. coli strains CFT073 and 83972 revealed strain-specific variations in the class and expression levels of genes encoding antimicrobial peptides, cytokines, and enzymes controlling DNA methylation and histone acetylation. Our results provide evidence for the differential epigenetic regulation of transcriptional reprogramming by UPEC and ABU strains of E. coli in G. mellonella larvae, which may be relevant to understanding the different behaviors of these bacterial strains in the human urinary tract.

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance.

One of the first identified and best-studied toxin-antitoxin (TA) systems in Escherichia coli is the F-plasmid-based CcdAB system. This system is involved in plasmid maintenance through postsegregational killing. More recently, ccdAB homologs have been found on the chromosome, including in pathogenic strains of E. coli and other bacteria. However, the functional role of chromosomal ccdAB genes, if any, has remained unclear. We show that both the native ccd operon of the E. coli O157 strain (ccdO157) and the ccd operon from the F plasmid (ccdF), when inserted on the E. coli chromosome, lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters, with the O157 operon showing higher protection. While the plasmid-encoded CcdB toxin is a potent gyrase inhibitor and leads to bacterial cell death even under fully repressed conditions, the chromosomally encoded toxin leads to growth inhibition, except at high expression levels, where some cell death is seen. This was further confirmed by transiently activating the chromosomal ccd operon through overexpression of an active-site inactive mutant of F-plasmid-encoded CcdB. Both the ccdF and ccdO157 operons may share common mechanisms for activation under stress conditions, eventually leading to multidrug-tolerant persister cells. This study clearly demonstrates an important role for chromosomal ccd systems in bacterial persistence.IMPORTANCE A large number of free-living and pathogenic bacteria are known to harbor multiple toxin-antitoxin systems, on plasmids as well as on chromosomes. The F-plasmid CcdAB system has been extensively studied and is known to be involved in plasmid maintenance. However, little is known about the function of its chromosomal counterpart, found in several pathogenic E. coli strains. We show that the native chromosomal ccd operon of the E. coli O157 strain is involved in drug tolerance and confers protection from cell death under multiple antibiotic stress conditions. This has implications for generation of potential therapeutics that target these TA systems and has clinical significance because the presence of persisters in an antibiotic-treated population can lead to resuscitation of chronic infection and may contribute to failure of antibiotic treatment.

Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) Genotyping of Escherichia coli Strains Isolated from Different Animal Stool Specimens

Escherichia coli is a commensal-pathogenic organism, which includes a wide range of strains. Despite several advanced molecular-genomic technologies for detecting and identifying different strains ofE. coli, Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) technique is a quick, sharp and cost effective fingerprint method. The major purpose of the present study was to determine the distribution of ERICs within E. colistrains isolated from different healthy animal stool specimens including hens, sheep, and cows, as an appropriate and quick molecular-genomic tool. The animal stool samples were obtained during 1 year (October 2012 to October 2013), from animal husbandries around Tehran and Alborz provinces, Iran. After screening processes, theE. colibacteria were isolated and cultured via standard microbiological methods. The DNA molecules ofE. coli bacteria were harvested and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) was applied for bacterial molecular genotyping. The ERIC-PCR products were run on 1% gel electrophoresis. The final images regarding gel electrophoresis banding patterns were used for dendrogram generation via the GelClust software. Of 120 isolated samples, 115 different strains were recognized asE. coli. The fingerprint patterns involved 380 to 3280 bp bands. The predominant bands included 2900 bp, 1200 bp, and 1200 bp in stool samples of hens, sheep, and cows, respectively. The highest frequencies and diversities were seen amongE. colistrains isolated from hens and sheep stool samples. The DNA profiles were clearly detectable via specific fingerprint patterns. The ERIC-PCR seemed to be a good approach for molecular typing ofE. colistrains isolated from different animal sources.

Prevalence of Extended-Spectrum Beta-Lactamases-Producing Microorganisms in Patients Admitted at KRRH, Southwestern Uganda.

The emergence of extended-spectrum beta-lactamase- (ESBL-) producing pathogenic bacteria at Kabale Regional Referral Hospital (KRRH), located in southwestern Uganda, is of great concern: a phenomenon that worries clinicians and other healthcare workers due to the serious threat they pose to patients. This current study aimed at determining the phenotypic detection of ESBL-producing strains of E. coli, Klebsiella sp., and Proteus sp. isolated from clinical specimens and their prevalence in patients admitted at KRRH. We used combined disc diffusion technique to detect and establish the presence of ESBLs-producing bacteria. Of the 100 tested bacterial isolates, 89 (89%) were identified as ESBL-producing bacteria. Klebsiella sp. predominated in the samples (46 (52%)), presenting the highest frequency of ESBLs producing followed by E. coli (39 (44%)) and Proteus mirabilis (4 (4.5%)) from the combined disk diffusion.

Evidence of Naturalized Stress-Tolerant Strains of Escherichia coli in Municipal Wastewater Treatment Plants.

ABSTRACTEscherichia coli has been proposed to have two habitats—the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains of E. coli have evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and ∼59% of the surviving E. coli strains were found to contain a genetic insertion element (IS30) located within the uspC-flhDC intergenic region. The positional location of the IS30 element was not observed across a library of 845 E. coli isolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animal E. coli isolates (n = 1,177). Phylogenetics clustered the IS30 element-containing wastewater E. coli isolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only the fimH marker. Our data suggest that wastewater contains a naturalized resident population of E. coli. We developed an endpoint PCR targeting the IS30 element within the uspC-flhDC intergenic region, and all raw sewage samples (n = 21) were positive for this marker. Conversely, the prevalence of this marker in E. coli-positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater.IMPORTANCE The results of this study demonstrate that some strains of E. coli appear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of using E. coli as a microbial indicator of wastewater treatment performance, suggesting that the E. coli strains present in human and animal feces may be very different from those found in treated wastewater.