Serogroups O1 Research Articles

Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

Legionella is ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, and Legionella pneumophila is responsible for the great majority (approximately 90%) of the Legionnaires' disease cases that occur. Furthermore, of the 15 L. pneumophila serogroups identified, O1 alone causes more than 84% of the Legionnaires' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification of L. pneumophila in water, environmental, and clinical samples are in great demand. L. pneumophila bacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms of L. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15 L. pneumophila O-antigen standard reference strains and seven L. pneumophila clinical isolates as target strains, seven reference strains of other non-pneumophila Legionella species as closely related strains, and six non-Legionella bacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection of L. pneumophila serogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms of L. pneumophila.

Isolation of pathogenic Escherichia coli from stool samples of diarrhoeal patients with history of raw milk consumption

Aim: To detect the occurrence of pathogenic Escherichia coli from stool samples of diarrhoeal patients with history of raw milk consumption and to determine the public health significance of isolates, especially their role in causing human diseases. Materials and Methods: A total of 100 stool samples from diarrhoeal patients, with history of raw milk consumption were collected from primary health centres in and around Anand city, under aseptic conditions and a total of 50 raw milk samples were collected from milk vendors, retail shops located in Anand city in sterilized sample bottles. MacConkey broth was used for the enrichment of all the samples and inoculation was done on MacConkey agar and EMB agar was used as the selective media. This was followed by the confirmation of isolates using biochemical tests. For the serotyping, E. coli isolates were sent to the National Salmonella and Escherichia Centre, Central Research Institute (CRI), Kasauli, Himachal Pradesh. Detection of virulence genes was performed using PCR technique. Results: During the present investigation, 26 (52%) E. coli isolates from 50 milk samples and 59 (59%) E. coli isolates from 100 stool samples were recovered. Out of 85 E. coli isolates sent for serotyping, 74 isolates could be typed which were further distributed into 13 different serogroups O2, O4, O8, O17, O22, O25, O29, O36, O45, O60, O90, O116 and O172, whereas 8 isolates were found untypable and 3 isolates were reported rough isolates. Of the 59 E. coli isolates from stool samples of diarrhoeal patients tested, 15 isolates (25.42%) were reported to be positive for stx genes, among that 6 (10.16%) were positive for stx1 gene, 9 (15.25%) isolates were positive for stx2 gene, while 3 isolates (5.08%) were positive for eaeA gene. In this study, 21 E. coli isolates were found to be Shiga toxin producing E. coli (STEC) while none of the isolates were positive for the serotype O157. Conclusions: Our present findings indicate that raw milk may act as a source of pathogenic E. coli and it may be responsible for the occurrence of diarrhoea and various other health-related complications in humans. We therefore recommend proper managemental practices and effective control measures for improved hygiene and sanitation.

A Quinazoline-2,4-Diamino Analog Suppresses Vibrio cholerae Flagellar Motility by Interacting with Motor Protein PomB and Induces Envelope Stress

Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. In a previous study, we described a high-throughput assay for screening for small molecules that selectively inhibit bacterial motility and identified a family of quinazoline-2,4-diamino analogs (Q24DAs) that (i) paralyzed the sodium-driven polar flagellum of Vibrios and (ii) diminished cholera toxin secreted by El Tor biotype V. cholerae. In this study, we provide evidence that a Q24DA paralyzes the polar flagellum by interacting with the motor protein PomB. Inhibition of motility with the Q24DA enhanced the transcription of the cholera toxin genes in both biotypes. We also show that the Q24DA interacts with outer membrane protein OmpU and other porins to induce envelope stress and expression of the extracellular RNA polymerase sigma factor σ(E). We suggest that Q24DA-induced envelope stress could affect the correct folding, assembly, and secretion of pentameric cholera toxin in El Tor biotype V. cholerae independently of its effect on motility.

Prevalence and Characteristics of the Epidemic Multiresistant Escherichia coli ST131 Clonal Group among Extended-Spectrum Beta-Lactamase-Producing E. coli Isolates in Copenhagen, Denmark

We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.

CronobacterInfections Not from Infant Formula, Taiwan

To the Editor: Species of the genus Cronobacter are relatively heterogeneous at the phenotypic and molecular levels (1). In 2012, the following 7 Cronobacter species had been defined: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. condimenti, and C. universalis (2). These opportunistic pathogens cause bacteremia and meningitis in neonates and are associated with necrotizing enterocolitis (3); ≈30% of infants with Cronobacter bacteremia or meningitis have died (4). Cronobacter spp. primarily infect infants, but infections among immunocompromised patients, particularly elderly patients, have been reported (5). Although these organisms are ubiquitous in the environment and have been isolated from a variety of foods, Cronobacter spp. infections in infants have been epidemiologically associated with ingestion of contaminated powdered infant formula (6). Few reports of C. sakazakii infections in adults have been published. During 2002–2011, a total of 5 C. sakazakii isolates, 1 from each of 5 patients, were identified at the National Taiwan University Hospital in northern Taiwan (Table). These isolates were identified as belonging to the C. sakazakii group by use of 2 systems: PMIC/ID-30 (Becton Dickinson Diagnostic Systems, Sparks, MD, USA) and the Vitek 2 System GN card (bioMerieux Inc., La Balme les Grottes, France). The phenotypic profiles that use 14 biochemical characteristics to differentiate 7 species and 3 subspecies of C. dublinensis within Cronobacter gen. nov. (C. sakazakii group) have been described (2). Although we did not apply the 14 biochemical tests to differentiate the 5 isolates (2), the isolates’ lack of indole production and dulcitol utilization, obtained by use of Enterotube II (Becton Dickinson Diagnostic Systems), was compatible with identification of the following 3 species or subspecies: C. sakazakii, C. malonaticus, or C. dublinensis subsp. lausannensis (2). Results of partial 16S rRNA gene sequence analysis with primers 8FPL and 1492RPL indicated that the isolates were probably C. sakazakii (7), and results of a 2-step rpoB-based PCR that used 2 sets of primer pairs (Csakf/Csakr and Cmalf/Cmalr) confirmed that the isolates were C. sakazakii (8). Table Characteristics of 5 patients with Cronobacter sakazakii infections, National Taiwan University Hospital, Taiwan, 2002–2011* Serogroups of the 5 C. sakazakii isolates were determined by using 5 primer pairs specific to the wehC, wehI, and wzx genes (9). Of these 5 isolates, 3 were serogroup O1, and 2 were not typeable (not serogroups O1, O2, or O3). Results of disk-diffusion susceptibility testing showed that all isolates were susceptible to cefotaxime, cefepime, piperacillin–tazobactam, ertapenem, imipenem, meropenem, ciprofloxacin, gentamicin, and amikacin and that all were resistant to cefazolin. The random amplified polymorphic DNA patterns generated by arbitrarily primed PCR that used 2 random oligonucleotide primers (M13 and ERIC1) differed among the 5 isolates, indicating that these 5 C. sakazakii strains were not clonally related (10). The clinical and microbiological characteristics of the 5 patients (4 adult, 4 male) with C. sakazakii infection are summarized in the Table. Primary bacteremia was found in 2 patients, pneumonia in 2 (predominant growth of C. sakazakii from purulent sputum samples), and acute cholecystitis in 1. The nonadult patient was a 2-month-old boy with congenital heart disease. Because of apnea and cyanosis, he was sent to an emergency department and later received assisted ventilation and supportive care in an intensive care unit. He was extubated on day 11 of hospitalization; however, fever and increased purulent sputum were noted on day 18. Bacterial culture of the suctioned sputum specimen yielded C. sakazakii. Before being hospitalized, the boy had been fed reconstituted powdered infant formula (Nestle H.A.1, Gold; Nestle Taiwan Ltd., Taipei, Taiwan) by mouth without other supplemental nutrition. During hospitalization, he received infant formula made by the hospital nutritional department through nasogastric tube. Although the powdered infant formula was not tested for C. sakazakii, initial sputum culture disclosed viridans group streptococci, and C. sakazakii was isolated from sputum obtained on day 18 of hospitalization. Thus, C. sakazakii from this infant might not have been associated with contaminated powdered infant formula. Among the 4 adult patients, 3 had underlying solid organ malignancy and had received immunosuppressive drugs., and the other had bacteremia and died of cardiac arrest at arrival at the emergency department. The sources of C. sakazakii infection in the 1 nonimmunocompromised adult and the infant remain unclear; further research is needed to identify the source of C. sakazakii infections in Taiwan.

Diarrhoeagenic Escherichia coli and salmonellae in calves and lambs in Kashmir absence, prevalence and antibiogram.

Polymerase chain reaction assays and culture were used to investigate 728 faecal samples from 404 calves (286 diarrhoeic, 118 healthy) and 324 lambs (230 diarrhoeic, 94 healthy) in Kashmir, India, for the presence of enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and salmonellae. Antimicrobial sensitivity patterns were also investigated. In total, 23 ETEC isolates were obtained from the diarrhoeic calves and 12 from diarrhoeic lambs. Most (74%) of the isolates from calves harboured the gene encoding heat-labile enterotoxin I, whereas 75% of the isolates from lambs possessed only the gene encoding for heat-stable enterotoxin a. The ETEC isolates belonged to 20 serogroups, among which serogroups O15 (five isolates) and O8 (four isolates) were the most frequent. Salmonella Typhimurium or S. Enteritidis was identified in three samples from diarrhoeic lambs. The ETEC isolates and the salmonellae showed multidrug resistance. No EAEC or DAEC was detected in any of the samples.

A 6-Week Oral Toxicity Study of Oral Cholera Vaccine in Sprague-Dawley Rats

The present study was carried out to examine the toxicity and target organs of oral cholera vaccine (OCV) after repeated oral administration in Sprague-Dawley rats for 6 weeks (3 administrations, once every 2 weeks). OCV is an inactivated oral cholera vaccine that contains Vibrio cholerae and confers protection against cholera caused by V. cholera serogroups O1 (Inaba and Ogawa serotypes) and O139 (strain 4260B). The animals were orally administered either OCV placebo (negative control) or OCV at a dose equivalent to 240 times the anticipated human dose. Throughout the administration period, no significant change was detected in clinical signs, body weight, food or water consumption, urinalysis results, hematological and clinical biochemistry test results, organ weights, necropsy, or histopathological examination results. Minor changes were found in hematological and clinical biochemistry tests; however, these changes were within normal ranges. The above results suggest that oral administration of OCV in rats did not induce any toxicologically meaningful changes, and the target organs could not be determined. This study was conducted in accordance with the guidelines established by Good Laboratory Practice (2009-183, KFDA, December 22, 2009) and the OECD Principles of Good Laboratory Practice (1997).

Development of O-serogroup specific PCR assay for detection and identification of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a human pathogen that is widely disseminated in estuarine, marine and coastal environments throughout the world, and is recognized as the leading cause of food-borne illness worldwide. V. parahaemolyticus infections have been characterized by causal associations with multiple, diverse serotypes. To date, 13 O-serogroups have been recognized in V. parahaemolyticus, although only the O-serogroup genetic determinants (OGDs) of serogroups O3 and O4 have been sequenced. In this study, the OGDs of the remaining 11 serogroups were identified. A PCR assay based on O-serogroup specific genes was developed for the identification and detection of all 13 V. parahaemolyticus O-serogroups and tested against 41 target strains and 21 strains of other bacterial species. A double-blind test including 105 environmental specimens was also performed. The developed method was shown to distinguish all V. parahaemolyticus O-serogroups effectively with the only exception of O3 and O13. The method was found to be highly specific and reproducible, with detection sensitivity of 1ng of genomic DNA, and it was demonstrated that V. parahaemolyticus at the level of 104CFU/ml in mock water specimens and the enrichment culture of samples inoculated with at the level of 1CFU/ml were detected. As few as 2 to 18CFU (initial inoculum) of V. parahaemolyticus were detectable in a 1g oyster sample after enrichment using this PCR method. The molecular protocol developed in this study for identification of all V. parahaemolyticus serogroups is therefore suitable for rapid detection and identification of V. parahaemolyticus pathogens from clinical and environmental samples, with the potential for application in epidemiologic investigations and other food safety applications.

Specific Detection of Enteroaggregative Hemorrhagic Escherichia coli O104:H4 Strains by Use of the CRISPR Locus as a Target for a Diagnostic Real-Time PCR

In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPR(O104:H4)) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR(O104:H4) PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC(O104), wzx(O104), and wzy(O104)). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPR(O104:H4) target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR(O104:H4) locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPR(O104:H4) PCR (99.06% specificity).

Detection of virulence genes in Escherichia coli strains isolated from diarrheic and healthy feces of dairy calves in Brazil

The aim of this work was to test 101 strains of E. coli for virulence factors associated with enterotoxigenic and enterohemorrhagic pathotypes of E. coli isolated from diarrheic and non-diarrheic calves. The virulence factors of E. coli Stx1 (Shiga toxin), Stx2, Ehly (Enterohemolysin), the eae gene, LT-II (heat-labile enterotoxin), STa (heat-stable toxin), and adhesins K99 and F41 were detected by PCR. Serogroups were determined by serological methods and Stx production was observed by biological assays in Vero cells. The frequency of the eae gene was higher in isolates from diarrheic calves (35/58, 60.3%) than in non-diarrheic calves (8/43, 18.6%; P < 0.001). The gene for Stx1 occurred at high frequencies in the diarrheic strains (24/58, 41.3%) as well as in non-diarrheic (19/43, 44.2%) ones and all strains that were Stx positive by PCR showed cytotoxicity in Vero cells. Stx2 was found in ten strains, Ehly in eight strains, and LT-II in only two strains. Twenty-eight strains were negative for all of the PCR assays, including for F41 and K99 adhesins. The serogroups O7, O23, O4, O8, O153 and O156 were observed most frequently. Our results show that strains of E. coli isolated from cattle have similar virulence factors genes to strains isolated from cases of diseases in humans and may be a source of potentially pathogenic STEC for humans.

Isolation and characterization of lytic bacteriophages against Escherichia coli serogroups O1, O2, and O78

The goal of this study was to isolate and characterize a complete set of phages that areactive against Escherichia coliserogroups O1, O2, and O78, the main causative agents ofavian colibacillosis. A mixture of E.coli (O1:K1), (O2:K1), and (O78:K80) used as host toisolate phages from wastewater and fecal samples from poultry processing plants. Elevenphages were isolated, only two of them EC-NJ4 and EC-NJ7 were selected for furthercharacterization. EC-NJ4 and EC-NJ7 had icosaheadral heads, necks and contractile tails,with tail fibers and therefore belonged to Myoviridae, with genome sizes of 67.06 – 68.04 kband they lysed 100% of serotype O1, O2, and O78. The two phages were resistant to pH 5-9,and phage EC-NJ7 was slightly more resistant to acid and alkali environments. It wasconcluded that Phage EC-NJ4 and EC-NJ7 are highly active against O1, O2, and O78colibacillosis strains and it might be suitable candidate for phage therapy.Running title: APEC, Phages, E. coli O1, O2, O78

Prevalence of Avian PathogenicEscherichia coli(APEC) Clone HarboringsfaGene in Brazil

Escherichia coli sfa+ strains isolated from poultry were serotyped and characterized by polymerase chain reaction (PCR) and amplified fragment length polymorphism (AFLP). Isolates collected from 12 Brazilian poultry farms mostly belonged to serogroup O6, followed by serogroups O2, O8, O21, O46, O78, O88, O106, O111, and O143. Virulence genes associated were: iuc 90%, fim 86% neuS 60%, hly 34%, tsh 28%, crl/csg 26%, iss 26%, pap 18%, and 14% cnf. Strains from the same farm presented more than one genotypic pattern belonging to different profiles in AFLP. AFLP showed a clonal relation between Escherichia coli sfa+ serogroup O6. The virulence genes found in these strains reveal some similarity with extraintestinal E. coli (ExPEC), thus alerting for potential zoonotic risk.

Two cases of vibrio cholera non-O1/non-O139 septicaemia with favourable outcome in Lebanon

Vibrio cholerae species can be divided into 2 major groups: cholera-causing strains of the serogroups O1 and O139, and non-O1 V. cholerae (NOVC) [1]. Non-O1 V. cholerae (NOVC) typically does not elaborate cholera toxin. It is also antigenically distinct from the V. cholerae O1 and O139 strains, which cause epidemic cholera in many countries. Although NOVC strains are usually associated with sporadic diarrhoea, some outbreaks have been reported. These strains have also been associated with skin infections, cholecystitis, meningitis and septicaemia. While gastrointestinal infections caused by NOVC usually have a favourable outcome, invasive infections may be fatal [2]. Immunocompromised patients, particularly those with hepatic cirrhosis or haematological malignancies, are at highest risk for septicaemia with NOVC [2,3]. In this paper, we report on 2 residents of Beirut who were diagnosed with NOVC and treated in a large public facility. They are, to our knowledge, the first reported cases of severe NOVC infection in Lebanon.

Cholera epidemic in and around kolkata, India: endemicity and management.

Cholera epidemic in and around kolkata, India: endemicity and management.

Role of Zooplankton Diversity in Vibrio cholerae Population Dynamics and in the Incidence of Cholera in the Bangladesh Sundarbans

Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a severe watery, life-threatening diarrheal disease occurring predominantly in developing countries. V. cholerae, including both serogroups O1 and O139, is found in association with crustacean zooplankton, mainly copepods, and notably in ponds, rivers, and estuarine systems globally. The incidence of cholera and occurrence of pathogenic V. cholerae strains with zooplankton were studied in two areas of Bangladesh: Bakerganj and Mathbaria. Chitinous zooplankton communities of several bodies of water were analyzed in order to understand the interaction of the zooplankton population composition with the population dynamics of pathogenic V. cholerae and incidence of cholera. Two dominant zooplankton groups were found to be consistently associated with detection of V. cholerae and/or occurrence of cholera cases, namely, rotifers and cladocerans, in addition to copepods. Local differences indicate there are subtle ecological factors that can influence interactions between V. cholerae, its plankton hosts, and the incidence of cholera.

Bacteriological and Epidemiological Characteristics of Enterotoxigenic Escherichia coli Isolated in Tokyo, Japan, between 1966 and 2009

Enterotoxigenic Escherichia coli (ETEC) caused 131 outbreaks in Tokyo, Japan, between 1966 and 2009. The major serogroups were O6, O27, O148, and O159. The incidence of serogroups O25 and O169 recently increased. Heat-stable enterotoxin (ST) subtyping revealed that E. coli of serogroups O6, O15, O25, and O159 possessed the STh gene, whereas those serotyped as O27 and O169 possessed the STp gene.

Serotyping of Proteus mirabilis clinical strains based on lipopolysaccharide O-polysaccharide and core oligosaccharide structures

The aim of this work was to serotype Proteus mirabilis urinary tract infection (UTI) strains based on chemically defined O-antigens with the use of two clinical collections from Sweden and Poland consisting of 99 and 24 UTI strains, respectively. A simple two-step serotyping scheme was proposed using enzyme immunoassay with heat-stable surface antigens of Proteus cells and immunoblotting with isolated lipopolysaccharides (LPSs). Using polyclonal anti-P. mirabilis rabbit antisera, 50 Swedish and 8 Polish strains were classified into serogroups O10, O38, O36, O30, O17, O23, O9, O40, O49, O27, O5, O13, O24, O14, and O33. From the Swedish strains, 10 belonged to serogroup O10 and five to each of serogroups O38, O36, and O9. Therefore, none of the O-serogroups was predominant. The majority of the serotyped clinical strains possess acidic O-antigens containing uronic acids and various acidic non-carbohydrate substituents. In immunoblotting, antisera cross-reacted with both O-antigen and core of LPSs. The core region of 19 LPSs bound a single serum, and that of 12 LPSs bound more than two sera. Following bioinformatic analysis of the available sequences, a molecular approach to the prediction of Proteus core oligosaccharide structures was proposed. The identification of the core type of P. mirabilis R110, derived from a serogroup O3 wild strain, using restriction fragments length polymorphism analysis of galacturonic acid transferase is shown as an example. In summary, the most frequent O-serogroups among P. mirabilis UTI stains were identified. The diversity of serological reactions of LPSs is useful for serotyping of P. mirabilis clinical isolates. A possible role of the acidic components of O-antigens in UTI is discussed.

A High-Throughput Screening Assay for Inhibitors of Bacterial Motility Identifies a Novel Inhibitor of the Na+-Driven Flagellar Motor and Virulence Gene Expression in Vibrio cholerae

Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot(-)) phenotype and is specific for the Na(+)-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na(+) bioenergetics, such as the function of the primary Na(+) pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na(+)-driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.

Serotypes, Virulence Genes, and Antimicrobial Susceptibility ofEscherichia coliIsolates from Pigs

One hundred two pathogenic Escherichia coli isolates from diseased pigs were analyzed for serotypes, virulence genes, antimicrobial susceptibility, and the molecular basis of phenicol resistance. Of these 102 E. coli isolates, 101 were typeable and belonged to 27 different O serogroups. However, 69% of these isolates belonged to one of the following eight serogroups: O8, O54, O64, O65, O92, O108, O119, and O120. Serogroups O8 (23%) and O64 (10%) were the most prevalent among typeable isolates. High-resistance phenotypes were observed in all the isolates, with the majority displaying resistance to chloramphenicol (89%), streptomycin (83%), enrofloxacin (78%), and doxycycline (60%). The chloramphenicol resistance genes cat1, cat2, and cmlA were detected in 58%, 49%, and 65%, respectively, of the chloramphenicol-resistant isolates. The floR gene was detected in 57% of the florfenicol-resistant isolates and in 52% of chloramphenicol-resistant isolates. Pulsed-field gel electrophoresis showed that the 32 floR-positive isolates with florfenicol minimum inhibitory concentration ≥ 8 μg mL⁻¹ belonged to 25 different pulsed-field gel electrophoresis profiles, indicating the spread of floR among swine pathogenic E. coli isolates.

Enterotoxins, colonization factors, serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. Objective: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0–5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14%) and O78:HNM LT-ST CFA/I (n = 4; 9%). The analysis of antibiotic resistance showed that 67% (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. Conclusion: The most common toxin profile was LT 70%, LT/STh 23% and STh 7%. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.