Escherichia ruysiae sp. nov., a novel Gram-stain-negative bacterium, isolated from a faecal sample of an international traveller.

The genus Escherichia comprises five species and at least five lineages currently not assigned to any species, termed 'Escherichia cryptic clades'. We isolated an Escherichia strain from an international traveller and resolved the complete DNA sequence of the chromosome and an IncI multidrug resistance plasmid using Illumina and Nanopore whole-genome sequencing (WGS). Strain OPT1704T can be differentiated from existing Escherichia species using biochemical (VITEK2) and genomic tests [average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH)]. Phylogenetic analysis based on alignment of 16S rRNA sequences and 682 concatenated core genes showed similar results. Our analysis further revealed that strain OPT1704T falls within Escherichia cryptic clade IV and is closely related to cryptic clade III. Combining our analyses with publicly available WGS data of cryptic clades III and IV from Enterobase confirmed the close relationship between clades III and IV (>96 % interclade ANI), warranting assignment of both clades to the same novel species. We propose Escherichia ruysiae sp. nov. as a novel species, encompassing Escherichia cryptic clades III and IV (type strain OPT1704T=NCCB 100732T=NCTC 14359T).

Non-Typhoidal Salmonella Colonization in Chickens and Humans in the Mekong Delta of Vietnam.

Salmonellosis is a public health concern in both the developed and developing countries. Although the majority of human non-typhoidal Salmonella enterica (NTS) cases are the result of foodborne infections or person-to-person transmission, NTS infections may also be acquired by environmental and occupational exposure to animals. While a considerable number of studies have investigated the presence of NTS in farm animals and meat/carcasses, very few studies have investigated the risk of NTS colonization in humans as a result of direct animal exposure. We investigated asymptomatic NTS colonization in 204 backyard chicken farms, 204 farmers and 306 matched individuals not exposed to chicken farming, in southern Vietnam. Pooled chicken faeces, collected using boot or handheld swabs on backyard chicken farms, and rectal swabs from human participants were tested. NTS colonization prevalence was 45.6%, 4.4% and 2.6% for chicken farms, farmers and unexposed individuals, respectively. Our study observed a higher prevalence of NTS colonization among chicken farmers (4.4%) compared with age-, sex- and location- matched rural and urban individuals not exposed to chickens (2.9% and 2.0%). A total of 164 chicken NTS strains and 17 human NTS strains were isolated, and 28 serovars were identified. Salmonella Weltevreden was the predominant serovar in both chickens and humans. NTS isolates showed resistance (20-40%) against tetracycline, chloramphenicol, sulfamethoxazole-trimethoprim and ampicillin. Our study reflects the epidemiology of NTS colonization in chickens and humans in the Mekong delta of Vietnam and emphasizes the need of larger, preferably longitudinal studies to study the transmission dynamics of NTS between and within animal and human host populations.

An emerging zoonotic clone in the Netherlands provides clues to virulence and zoonotic potential of Streptococcus suis.

Streptococcus suis is a zoonotic swine pathogen and a major public health concern in Asia, where it emerged as an important cause of bacterial meningitis in adults. While associated with food-borne transmission in Asia, zoonotic S. suis infections are mainly occupational hazards elsewhere. To identify genomic differences that can explain zoonotic potential, we compared whole genomes of 98 S. suis isolates from human patients and pigs with invasive disease in the Netherlands, and validated our observations with 18 complete and publicly available sequences. Zoonotic isolates have smaller genomes than non-zoonotic isolates, but contain more virulence factors. We identified a zoonotic S. suis clone that diverged from a non-zoonotic clone by means of gene loss, a capsule switch, and acquisition of a two-component signalling system in the late 19th century, when foreign pig breeds were introduced. Our results indicate that zoonotic potential of S. suis results from gene loss, recombination and horizontal gene transfer events.

Capnocytophaga canimorsus Meningitis: Three Cases and a Review of the Literature.

Bacterial meningitis is a disease with a high morbidity and mortality. It may be caused by the zoonotic pathogen Capnocytophaga canimorsus, which is part of the commensal oral flora in dogs and cats. We report three cases of C. canimorsus meningitis in a nationwide cohort study of bacterial meningitis patients and performed a review of the literature. Three episodes of C. canimorsus meningitis were identified in three patients included in a nationwide cohort study from 2006 through 2014. The calculated annual incidence was 0.03 per million adults. When combined with the literature, 33 patients were identified of which 28 were male (85%). The median age was 63 years, and 13 (42%) were immunocompromised, which consisted of alcoholism in 7 (21%). Animal contact could be established in 29 of 30 patients (93%) and consisted of dog bites in 22 of 29 (76%). One patient died (3%) and 8 had neurological sequelae upon discharge (25%), most often hearing loss (n = 6, 19%). Capnocytophaga canimorsus meningitis is associated with dog bites. Although mortality is relatively low, survivors often have neurological sequelae.

[Optimising antibiotic policies in the Netherlands. IX. SWAB guidelines for antimicrobial therapy in adults with acute infectious diarrhoea]. / Optimaliseren van het antibioticabeleid in Nederland. IX. SWAB-richtlijn voor antimicrobiële therapie bij acute infectieuze diarree.

The 'Stichting Werkgroep Antibioticabeleid' (SWAB; Dutch Working Party on Antibiotic Policy) develops evidence-based guidelines for the use of antibiotics in hospitalised adults. This guideline on acute infectious diarrhoea (AID) concerns the antibiotic treatment of acute infectious inflammation of the gastrointestinal tract, manifesting primarily as diarrhoea. AID can be subdivided into community-acquired diarrhoea, traveller's diarrhoea and hospital-acquired (nosocomial) diarrhoea. In the first 2 categories, the need for antibiotic treatment is generally restricted to individuals with severe illness, dysentery or a predisposition to complications. High rates of primary fluoroquinolone resistance can be found in human Campylobacter isolates from the Netherlands and from other parts of the world. Therefore, if antibiotic treatment is necessary for community-acquired AID or AID in travellers returning to the Netherlands, it is advised to use oral azithromycin for 3 days as empirical treatment. If intravenous treatment is necessary, the combination of ciprofloxacin and erythromycin for 5-7 days may be considered. As soon as the identity of the causative organism is known, antimicrobial treatment should be tailored accordingly.

[Resistant microorganisms in patients transferred from foreign hospitals]. / Resistente micro-organismen bij gerepatrieerde patienten uit buitenlandse ziekenhuizen.

OBJECTIVE: To determine the prevalence of carriers of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and gentamicin-resistant Gram-negative bacilli (GGNB) in patients repatriated from foreign hospitals to The Netherlands. DESIGN: Determination of prevalence. METHOD: In the period May 1998-August 2001, 1167 patients were repatriated. Swab specimens, demographic data and clinical data were obtained during the transfer. RESULTS: The prevalence of carriers of resistant microorganisms was 18.2%. MRSA was carried by 2.7% of the total repatriated group and by 4.7% of patients transferred to a Dutch hospital. Risk factors were antimicrobial treatment (odds ratio (OR): 3.4; 95% CI: 1.2-9.7), length of stay in a foreign hospital > or = 14 days (OR: 5.4; 95% CI: 2.3-12) and artificial ventilation (OR: 8.5; 95% CI: 1.8-41). VRE and GGNB were isolated from 2.7% and 14.1% of patients, respectively. Transfer from Asia or southern, south-eastern and eastern Europe were risk factors for carrying GGNB. CONCLUSION: Carriership of resistant microorganisms was high among repatriated patients. The highest risk of GGNB was more closely associated with the country from which the patient was transferred than the antimicrobial treatment received in the foreign hospital.

Class 1 integrons in ciprofloxacin-resistant Escherichia coli strains from two Dutch hospitals.

A significant increase in the isolation frequency of ciprofloxacin-resistant Escherichia coli was observed in the haematology departments of two university hospitals in The Netherlands. Amplified fragment length polymorphism analysis revealed that this increase was not caused by the emergence of unique ciprofloxacin-resistant clones. Determination of the presence of class 1 integrons indicated that 81% of the ciprofloxacin-resistant isolates contained an intI1 gene, compared with 11% of the ciprofloxacin-susceptible isolates (p<0.0001). The quinolone resistance gene qnrA was not present in any of the integrons characterised and could not be detected using dot-blot hybridisation of total DNA. In addition, conjugation experiments showed that ciprofloxacin resistance was not co-transferred with class 1 integrons. Ciprofloxacin-resistant isolates harboured mutations in the gyrA gene, which are known to encode ciprofloxacin resistance. In conclusion, an association was observed between ciprofloxacin resistance and the presence of class 1 integrons, which could not be explained by the currently known genetic determinants of quinolone resistance.

Analysis of efficacy of CVD 103-HgR live oral cholera vaccine against all-cause travellers' diarrhoea in a randomised, double-blind, placebo-controlled study.

Enterotoxigenic Escherichia coli (ETEC), which produces heat labile toxin (LT) and/or heat stable toxin (ST), is considered to be the most common known cause of travellers' diarrhoea (TD). Owing to the antigenic similarity between cholera toxin and LT, immunization with inactivated oral B-subunit/whole-cell cholera vaccine (BS-WC) offers short term (3 months) but significant (>67%) protection against TD caused by LT-related ETEC. Since it expresses the cholera toxin B (CTB) subunit, the live attenuated oral cholera vaccine strain CVD 103-HgR, may induce similar protection. A trial was performed to determine if CVD 103-HgR live oral cholera vaccine would provide a protective efficacy of at least 50% against TD. In addition, the protective efficacy of the vaccine against TD specifically due to LT-ETEC and LT/ST-ETEC was determined. Volunteers (n=134) travelling to Indonesia, India, Thailand or West-Africa were randomised to receive either a placebo (n=65) or the vaccine (n=69). In the placebo group, 46% reported an episode of diarrhoea, compared to 52% in the vaccine group. No significant group differences were found with regard to incidence, duration or severity of all caused TD or ETEC-associated TD. However, ETEC-associated TD occurred earlier in the placebo group (median 5 days), compared to the vaccine group (median 15 days). In conclusion, CVD 103-HgR live oral cholera vaccine failed to provide a 50% protection against TD. This study does not exclude that the vaccine may offer a short-lived protection against ETEC-associated TD. However, the power of the study was limited by the unexpected low incidence of LT-ETEC-associated diarrhoea (9% of all TD) compared to ST-associated TD (24% of all TD).

Carriage of resistant microorganisms in repatriates from foreign hospitals to The Netherlands.

In a prospective survey conducted between May 1998 and September 2001, the prevalence of carriage of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and gentamicin-resistant Gram-negative bacilli (GGNB) was determined in 1167 patients repatriated from foreign hospitals to The Netherlands. Swab specimens, demographic data and clinical data were obtained during transfer of the patients from the foreign hospitals. The total prevalence of carriage of resistant microorganisms was 18.2%. MRSA was carried by 2.7% of all patients, and by 4.7% of the patients repatriated to a Dutch hospital. Antimicrobial treatment (adjusted odds ratio (OR) 3.4; 95% confidence interval (CI) 1.2-9.7), length of stay in a foreign hospital of > 14 days (adjusted OR 5.4; 95% CI 2.3-12) and artificial ventilation (adjusted OR 8.5; 95% CI 1.8-41) were risk factors for carriage of MRSA. VRE and GGNB were isolated from 2.7% and 14.1% of the patients, respectively. Transfer from Asia, and southern, southeastern and eastern Europe, were risk factors for carriage of GGNB. These carriage rates were high compared to those found in patients in Dutch hospitals, where the rates are < 1% for MRSA, 2% for VRE, and 4.5% for GGNB. The highest risk of acquisition of GGNB was associated with the country from where the patient was repatriated, rather than with the antimicrobial treatment received by the individual patient in the foreign hospital.

Ultra-sonic nebulizers as a potential source of methicillin-resistant Staphylococcus aureus causing an outbreak in a university tertiary care hospital.

An outbreak of methicillin-resistant Staphylococcus aureus (MRSA) occurred on a head and neck surgical (HNS) ward of a university hospital in Amsterdam. The outbreak lasted from May 2000 until November 2000, and MRSA spread to two intensive care units. Amplified fragment length polymorphism analysis indicated that a single clone was responsible for the outbreak. Phage-typing indicated that this clone was of a type that was uncommon in The Netherlands. Strict isolation of patients, according to the Dutch national guidelines, was instituted. During the outbreak, surveillance culture specimens, from patients, healthcare workers, and the environment, were obtained at regular intervals. MRSA was found in the dust filters of nebulizers through which air from the room was filtered and subsequently humidified. These nebulizers were used to humidify tracheostomies. The dust filters were not maintained according to the guidelines. Restricted use and cleaning and disinfection of all ultra-sonic nebulizers led to termination of the outbreak. The outbreak illustrates that to terminate transmission of outbreak strains of MRSA, meticulous measures are necessary, which not only include strict isolation precautions, but also decontamination of the environment. In addition, it demonstrates the necessity of adhering to cleaning and disinfection guidelines for all medical and nursing equipment used in the hospital.

Diarrheagenic Escherichia coli and acute and persistent diarrhea in returned travelers.

To determine the role of diarrheagenic Escherichia coli in acute and persistent diarrhea in returned travelers, a case control study was performed. Enterotoxigenic E. coli (ETEC) was detected in stool samples from 18 (10.7%) of 169 patients and 4 (3.7%) of 108 controls. Enteroaggregative E. coli (EAggEC) was detected in 16 (9.5%) patients and 7 (6.5%) controls. Diffuse adherent E. coli strains were commonly present in both patients (13%) and controls (13.9). Campylobacter and Shigella species were the other bacterial enteropathogens most commonly isolated (10% of patients, 2% of controls). Multivariate analysis showed that the presence of ETEC was associated with acute diarrhea (odds ratio [OR], 6.7; 95% confidence interval [CI], 1.5 to 29.1; P = 0.005), but not with persistent diarrhea (OR, 1.6; 95% CI, 0.4 to 7.4). EAggEC was significantly more often present in patients with acute diarrhea than in controls (P = 0.009), but no significant association remained after multivariate analysis. ETEC and EAggEC are frequently detected in returned travelers with diarrhea. The presence of ETEC strains is associated with acute but not with persistent diarrhea.

The intestinal mucus layer from patients with inflammatory bowel disease harbors high numbers of bacteria compared with controls.

BACKGROUND & AIMS: Whether the bacterial flora contributes to the pathogenesis of inflammatory bowel disease (IBD) by increased penetration in mucus, increased adherence to epithelial cells, or invasion of the epithelium is unknown. We therefore studied the spatial distribution of bacteria in the mucosa of rectal biopsy specimens from patients with IBD and from controls. METHODS: Rectal biopsy specimens from 19 patients with IBD and from 14 controls were studied by using nonradioactive ribosomal RNA in situ hybridization. Total mucosal surface length examined for each patient was measured, and the number of bacteria visualized was estimated semiquantitatively. RESULTS: No bacteria were observed in biopsy specimens from 10 controls (71%) and 6 IBD patients (32%) (P = 0.04; odds ratio, 5.42; 95% confidence interval, 1.23-23.9). IBD rectal specimens contained significantly more bacteria than control samples (P = 0.004). Bacteria were localized within the mucus layer but did not adhere to the epithelial cells and were not present within the lamina propria. There was no correlation between the numbers of bacteria present and either the degree of inflammation or the use of anti-inflammatory agents or sulfasalazine compounds. CONCLUSIONS: The intestinal mucus in IBD patients is less protective against the endogenous microflora than in controls, resulting in increased association of luminal bacteria with the mucus layer.

Frequency of pathogenic and enteroadherent Escherichia coli in patients with inflammatory bowel disease and controls.

AIMS: To determine whether inflammatory bowel disease (IBD) is associated with pathogenic or enteroadherent Escherichia coli. METHODS: A least two stool specimens and one rectal biopsy were taken from 30 patients with IBD and from 20 controls. A large number of E coli-like colonies cultured from each stool sample and biopsy was tested, using DNA probes, for the presence of genes encoding shiga-like toxins, invasiveness, attachment-effacement and the ability to adhere to HEp-2 cells. Similarity among isolates from stool samples and rectal biopsies was determined by random amplified polymorphic DNA (RAPD) analysis. RESULTS: Enterohaemorrhagic and enteroinvasive E coli were not found in samples from either patients or controls. No significant difference in the detection rate of enteroadherent E coli between patients and controls was found. Rectal biopsies from 11 of 28 patients with IBD and 4 of 18 controls contained E coli, which hybridised with probes for detection of genes encoding diffuse adherence to HEp-2 cells, or encoding P-pili (p = 0.2). Enteroadherent E coli isolated from two or three stool specimens from the same patient or control appeared to be identical by RAPD analysis, and are considered to be residents in the colon. Probe positive isolates obtained from stool specimens and corresponding rectal biopsies were always identical on RAPD analysis. CONCLUSIONS: E coli strains possessing adherence factors reside in the large intestine and adhere to the rectal mucosa, irrespective of the presence of colitis.

Known bacterial virulence factors do not explain the variation in urinary cytokine levels in patients with urosepsis.

We measured urinary endotoxin, IL-6 and IL-8 levels in 23 patients with gram-negative urosepsis. The endotoxin and cytokine levels showed a 100-1000 fold range. No correlation was found between levels of urinary endotoxin, and IL-6 or IL-8 levels. In all cases bacterial numbers were > or = 10(5) CFU ml-1 urine. The endotoxin content of the isolated microorganisms neither correlated with the urinary cytokine levels, nor with IL-6 and IL-8 levels obtained in vitro when 10(3) log-phase CFU of each of the bacteria were incubated with heparinized whole blood of three healthy donors. Neither the haemolysin phenotype of the bacteria, nor the presence of the P-pili gene was correlated with the cytokine response in vivo or in vitro. Other factors than known bacterial virulence factors apparently contribute to the wide variation in urinary cytokine levels in urinary tract infection.

Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.

Shigella-specific IgA in saliva of children with bacillary dysentery.

To study the secretory immune response after Shigella infection, the anti-lipopolysaccharide and anti-Shiga-toxin response in saliva, obtained from children with confirmed shigellosis and healthy children, were determined by enzyme-linked immunosorbent assay and by Western blot. Children with infection showed high titers compared to healthy controls. After Shigella dysenteriae type 1 infection a significant change in titer could be observed in a large number of cases, in contrast to Shigella flexneri infection. It appeared that, in children living in endemic areas, infection with one serotype can give a rise in antibody titer to another serotype. This could be ascribed to polyclonal B cell activation since children in endemic areas routinely show relatively high titers to Shigella antigens. We conclude that the dynamics of salivary anti-Shigella LPS and anti-Shiga-toxin in children with dysentery indicate that it can be applied to studies of immune response in shigellosis for epidemiological and vaccination purposes.