[Pediatric brucellosis : A case report and literature review]. / Brucellose : revue de la littérature à propos d'un cas pédiatrique.

Brucellosis is an overlooked infection of widespread geographic distribution. This disease is rarely evoked when assessing unexplained pediatric fever, and only 20-30 cases (children and adults) are confirmed per year. Risk factors for contracting brucellosis are exposure to bodily fluids and consumption of unpasteurized dairy products from infected animals. Most cases of brucellosis are associated with traveling to or importing contaminated goods from endemic areas. Here, we report a case of brucellosis in a 16-month-old patient hospitalized for an acute febrile illness in a French general pediatric ward. An antibiotic regimen of rifampicin and co-trimoxazole given over 6 weeks led to successful cure without relapse. The child had eaten a cake made from unpasteurized goat's milk and imported from Oran, a region in Algeria. His mother had consumed the same cake and was hospitalized for brucellosis 15 days later. Clinicians should suspect brucellosis when encountering febrile patients who have traveled to endemic areas, been exposed to body fluids or products of abortion of farm animals, or consumed unpasteurized products.

Transmission of metallo-ß-lactamase-producing Pseudomonas aeruginosa in a nephrology-transplant intensive care unit with potential link to the environment.

Pseudomonas aeruginosa has been recovered in hospitals from many different sources including sinks and taps. Because P. aeruginosa is one of the main agents of nosocomial infections and increasingly resistant to antibiotics, environmental reservoirs in hospital settings are of great concern. We report here on a cluster of five cases of infection by P. aeruginosa expressing VIM carbapenemases (VIM-PA) in a nephrology intensive care unit. Our investigation pointed to transmission of VIM-PA via hands related to a contaminated tap. VIM-PA may be cross-transmitted to other patients if an environmental reservoir exists. Sinks and taps should be well designed and thoroughly cleaned and disinfected, and use of alcohol hand rub should be promoted.

Faecal carriage of carbapenemase-producing Gram-negative bacilli in hospital settings in southern France.

The emergence of carbapenemase-producing Gram-negative bacilli is a worldwide problem. To date, no study has evaluated the prevalence of faecal carriage of carbapenemase-producing and carbapenem-resistant Gram-negative bacilli (CR GNB) in France. From 1 February to 30 April 2012, we conducted a prospective, multicentre study in three University Hospitals and four General Hospitals in the south of France. The carriage of carbapenemase-producing Enterobacteriaceae (CPE) and other CR GNB was screened by both cultivation on chromID® CARBA and chromID® OXA-48 media (bioMérieux) and molecular tools [multiplex polymerase chain reaction (PCR) and NucliSENS EasyQ® KPC (bioMérieux)]. The genetic relationship between isolates was assessed by rep-PCR (DiversiLab, bioMérieux) or multilocus sequence typing (MLST). The prevalences of CR GNB and carbapenemase-producing bacteria were 2.4 % (27/1,135) and 0.4 % (n = 5), respectively. Two strains corresponded to OXA-23-producing Acinetobacter baumannii and belonged to the widespread sequence type (ST) 2/international clone II, whereas one strain was an ST15 OXA-48-producing Klebsiella pneumoniae. Two OXA-48-producers were detected exclusively by PCR. This first French study revealed the very low dissemination of carbapenemase-producing bacteria in patients attending hospitals in southern France during a non-outbreak situation. However, the increasing description of epidemic cases in this area must reinforce the use of hygiene procedures to prevent diffusion of these multidrug-resistant microorganisms.

Simultaneous detection of gastrointestinal pathogens with a multiplex Luminex-based molecular assay in stool samples from diarrhoeic patients.

We have evaluated the multiplex molecular method xTAG(®) Gastrointestinal Panel (GPP) for detecting pathogens in stool samples of diarrhoeic patients. We collected 440 samples from 329 patients (male:female ratio of 1.2:1), including 102 immunosuppressed adults, 50 immunosuppressed children, 56 children attending the neonatal unit and 121 children attending the emergency unit. Of these, 176 samples from 162 patients were xTAG(®) GPP positive (102 viruses, 61 bacteria and 13 parasites) and the assay was more sensitive than the conventional test for detecting rotavirus (p <0.01), noroviruses (p <0.0001), Salmonella spp. (p <0.001), Campylobacter spp. (p <0.001) and toxigenic Clostridium difficile (p 0.005). The predominant pathogens were viruses (23.2%), with rotavirus (15.9%) being the most common. Bacterial agents were detected in 13.9%; the most common was Salmonella spp. (4.8%). Parasites were detected in 2.9%; Cryptosporidium spp. (2%) was the most common. There were 31 co-infections (7% of samples), involving two pathogens in 23 (5.2%) and three pathogens in eight (1.8%) samples. There were 113 (92.6%) positive samples from the children attending the emergency unit, 25 (17%) positive samples from immunosuppressed adults, 22 (25.3%) positive samples from immunosuppressed children and 16 (19%) positive samples from children attending the neonatal unit. The low turnaround time and technical hands-on time make this multiplex technique convenient for routine use. Nevertheless, conventional bacterial culture and parasitological stool examination are still required to detect other pathogens in specific cases and to determine susceptibility to antibiotics.

Faecal carriage of oxyiminocephalosporin-resistant Enterobacteriaceae among paediatric units in different hospitals in the south of France.

The aim of this study was to determine the presence of oxyiminocephalosporin-resistant (OCR) Gram-negative bacilli and extended-spectrum ß-lactamase (ESBL)-producing isolates in stool specimens obtained from paediatric patients hospitalised for acute diarrhoea. We conducted a prospective, multicentre study over a period of 6 months in seven hospitals in the south of France. Samplings were carried out from infants admitted for acute diarrhoea with no previous antibiotic treatment in the last week. Bacteria in stool specimens were screened for the presence of OCR Gram-negative bacilli on Drigalski agar supplemented with ceftazidime and ESBL CHROMagar® media, and confirmed by the Rosco tablets test. Genetic detection was performed by the Check MDR® microarray and by polymerase chain reaction (PCR) and sequencing with bacterial DNA extracted from isolates. The presence of OCR enterobacteria was markedly high (177/1,118 patients, 15.2 %), with an important community origin (66.1 %). The majority of multidrug-resistant (MDR) bacteria were Enterobacter cloacae (106, 59.9 %) and Escherichia coli (61, 34.5 %). The prevalence of ESBL and CTX-M producers represented 5.2 and 4.3 % of the isolates, respectively. The main proportion of these ESBL carriers was found in children less than 1 year of age (53.4 %). One carbapenemase (IMP-1) was detected. The study revealed the wide dissemination of MDR bacteria in infants attending hospitals in the south of France during a non-outbreak situation, in particular, the spread of cefotaximase and the detection of a carbapenemase. This worrisome situation must reinforce the use of hygiene procedures and appropriate antibiotics to control the emergence and spread of OCR organisms.

Rapid molecular genetic assay for direct identification of Bordetella from patients specimens.

Biological diagnosis of whooping cough is increasingly necessary to confirm respiratory tract infection. Indeed, clinical symptoms are variable especially in adolescents and adults who contaminate newborns too young to be vaccinated. The PCR assay was proven highly sensitive for the diagnosis of pertussis. In this study, we reported the use of a new test (GenoQuick Bordetella [GQB], Hain Life Science, Germany) which permits the fast molecular genetic identification of Bordetella pertussis and parapertussis directly from patients specimens, i.e. swabs from nose or throat. The test was performed over a three months period on 40 specimens from patients (1 month to 65 years old), most of them were young children admitted in paediatric emergency with paroxysmal cough or prolonged cough.

Relatedness of Escherichia coli strains with different susceptibility phenotypes isolated from swine feces during ampicillin treatment.

The aim of this study was to examine the dynamics of the development of resistance in fecal Escherichia coli populations during treatment with ampicillin for 7 days in pigs. Before treatment, only 6% of the isolates were ampicillin resistant, whereas more than 90% of the isolates were resistant after days 4 and 7 of treatment. Ampicillin-resistant E. coli isolates were mainly multiresistant, and 53% of the isolates from the treated pigs had one phenotype that included resistance to six antibiotics (ampicillin, chloramphenicol, sulfonamides, tetracycline, trimethoprim, and streptomycin) at day 7. Determination of the frequency of the four phylogenetic groups showed that there was a shift in the E. coli population in ampicillin-treated pigs; before treatment 75% of the isolates belonged to phylogroup B1, whereas at day 7 85% of the isolates belonged to phylogroup A. Pulsed-field gel electrophoresis (PFGE) typing revealed that ampicillin treatment selected ampicillin-resistant isolates with genotypes which were present before treatment. Comparison of antimicrobial phenotypes and PFGE genotypes showed that resistance traits were disseminated by vertical transmission through defined strains. One PFGE genotype, associated with the six-antibiotic-resistant phenotype and including a specific combination of resistance determinants, was predominant among the ampicillin-resistant strains before treatment and during treatment. These data indicate that ampicillin administration selected various ampicillin-resistant isolates that were present in the digestive tract before any treatment and that E. coli isolates belonging to one specific PFGE genotype encoding resistance to six antibiotics became the predominant strains as soon as ampicillin was present in the digestive tract.

Genotypic and phenotypic analysis of Enterobacter sakazakii strains from an outbreak resulting in fatalities in a neonatal intensive care unit in France.

In 1994, an outbreak of Enterobacter sakazakii infections occurred in a neonatal intensive care unit in France from 5 May to 11 July. During the outbreak, 13 neonates were infected with E. sakazakii, resulting in 3 deaths. In addition, four symptomless neonates were colonized by E. sakazakii. The strains were subjected to 16S rRNA gene sequence analysis, genotyped using pulsed-field gel electrophoresis, and phenotyped for a range of enzyme activities. E. sakazakii was isolated from various anatomical sites, reconstituted formula, and an unopened can of powdered infant formula. A fourth neonate died from septic shock, attributed to E. sakazakii infection, during this period. However, 16S rRNA gene sequence analysis revealed that the organism was Enterobacter cloacae. There were three pulsotypes of E. sakazakii associated with infected neonates, and three neonates were infected by more than one genotype. One genotype matched isolates from unused prepared formula and unfinished formula. However, no pulsotypes matched the E. sakazakii strain recovered from an unopened can of powdered infant formula. One pulsotype was associated with the three fatal cases, and two of these isolates had extended-spectrum beta-lactamase activity. It is possible that E. sakazakii strains differ in their pathogenicities, as shown by the range of symptoms associated with each pulsotype.

Rapid identification of bacteria, mecA and van genes from blood cultures.

The Genotype technology, a quick molecular genetic assay based on DNA multiplex amplification with biotinylated primers followed by hybridization to membrane bound probes, complies with the requirements for a fast diagnosis of sepsis. We evaluated the new Genotype BC Gram-negative and Gram-positive test kits (Hain Life Science, Germany) which respectively allow for the identification of 15 species of Gram-negative (GN) rods, and the identification of 17 Gram-positive (GP) bacteria species together with the determination of methicillin and vancomycin resistance (mecA and van genes). The study was performed on 60 positive blood cultures from BacT/ALERT bottles (aerobic, anaerobic and pediatric bottles). First, a Gram stain was carried out to select between Genotype BC GP or GN test, then identification were performed by the Genotype BC tests and by biochemical conventional tests after subculture and phenotypic susceptibility determination. The operating procedure was very easy to carry out and required a small amount of starting material (5 to 10 microL of blood culture). The results were available within 4.5 hours. For all the blood cultures, the Genotype BC results correlated with the biochemical identification and phenotypic antibiotics susceptibility. According to our results, this DNA strip technology based assay can easily be incorporated into routine diagnosis.

Impact of three ampicillin dosage regimens on selection of ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces.

The aim of this study was to assess the impact of three ampicillin dosage regimens on ampicillin resistance among Enterobacteriaceae recovered from swine feces by use of phenotypic and genotypic approaches. Phenotypically, ampicillin resistance was determined from the percentage of resistant Enterobacteriaceae and MICs of Escherichia coli isolates. The pool of ampicillin resistance genes was also monitored by quantification of bla(TEM) genes, which code for the most frequently produced beta-lactamases in gram-negative bacteria, using a newly developed real-time PCR assay. Ampicillin was administered intramuscularly and orally to fed or fasted pigs for 7 days at 20 mg/kg of body weight. The average percentage of resistant Enterobacteriaceae before treatment was between 2.5% and 12%, and bla(TEM) gene quantities were below 10(7) copies/g of feces. By days 4 and 7, the percentage of resistant Enterobacteriaceae exceeded 50% in all treated groups, with some highly resistant strains (MIC of >256 microg/ml). In the control group, bla(TEM) gene quantities fluctuated between 10(4) and 10(6) copies/g of feces, whereas they fluctuated between 10(6) to 10(8) and 10(7) to 10(9) copies/g of feces for the intramuscular and oral routes, respectively. Whereas phenotypic evaluations did not discriminate among the three ampicillin dosage regimens, bla(TEM) gene quantification was able to differentiate between the effects of two routes of ampicillin administration. Our results suggest that fecal bla(TEM) gene quantification provides a sensitive tool to evaluate the impact of ampicillin administration on the selection of ampicillin resistance in the digestive microflora and its dissemination in the environment.

Genotype MRSA, a new genetic test for the rapid identification of staphylococci and detection of mecA gene.

Early detection of Staphylococcus methicillin resistance (MR) is essential. However MR determination may be difficult because it is necessary to perform investigation of heterogeneous resistance and low level of resistance and to discriminate between oxacillin resistance and borderline resistance. Several phenotypic methods are recommended but they fail to detect low level of production de PBP2a, the modified Penicillin Binding Protein responsible for MR. Detection of mecA gene, the gene encoding PBP2a, using PCR is considered to be the reference method. We evaluated Genotype MRSA, a new rapid system based on DNA multiplex amplification and further hybridisation, for the identification of staphylococci and detection of the mecA gene. The study was performed on a collection of various Staphylococcus strains (N=30) from clinical human isolates including S. aureus MR and methicillin susceptible (MS), S. epidermidis MR and MS, and other species of coagulase negative Staphylococcus (CNS) MR and MS. For all the strains, the hybridization banding pattern obtained using Genotype MRSA correlated with their expected phenotypic and genotypic characteristics. Genotype MRSA allows the identification of the mecA gene as well as S. aureus and S. epidermidis specific genes. This DNA strip technology based assay can easily be incorporated into routine diagnostics. In addition, the short testing time (less than 2 hours) optimises treatment orientation. Genotype MRSA completely complies with all requirements for a fast, safe, valid and cost-effective MR diagnosis in staphylococci.

Bacterial aetiology of diarrhoea in young children: high prevalence of enteropathogenic Escherichia coli (EPEC) not belonging to the classical EPEC serogroups.

Diarrhoeal disease continues to be one of the most common causes of admittance in Children hospital emergency. The aim of the present study was to investigate the relative contribution of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) as a cause of infectious bacterial diarrhoea in children from the region of Toulouse. We analysed 280 samples of stools from 280 children (<2 years) with diarrhoea admitted in the "Hopital des Enfants" from January to August 2005. Classic pathogens (Salmonella, Campylobacter, Yersinia, Shigella, Aeromonas and Vibrio) were detected by standard culture methods. Enterotoxigenic Clostridium difficile were identified after culture by immuno-enzyme assay (IEA). Virulence genes of EPEC and EHEC were detected by using PCR. Shiga-toxin production of EHEC strains was confirmed with an IEA test. Potential enteric pathogens were identified in 55 patients. EPEC was the most frequently identified agent (30 patients), followed by Campylobacter (9 cases: 7 C. jejuni and 2 C. coli) and C. difficile (8 patients), then EHEC (5 patients) and Salmonella (3 patients). No Shigella, Yersinia, Aeromonas or other pathogenic bacteria were detected during this period in that class of children. EPEC not belonging to the classical EPEC serogroups were highly prevalent (24 versus 6). EHEC possessed different genotypes and serogroups: O26 (2 strains), O157 (2 strains) and one un-typable strain. This study demonstrates the importance of EPEC (55 % of positive cases) and of EHEC (more frequent than Salmonella) in the aetiology of diarrhoeal diseases of young children. We confirm the usefulness of the PCR methodology: it allows the detection of virulent E. coli and thus increases by two fold the diagnosis of bacterial diarrhoea.

[Necrotizing pneumonia and arthritis due to Staphylococcus aureus producing Panton and Valentine leukocidin in a 10-year-old boy]. / Pneumonie nécrosante et ostéoarthrite multifocale à Staphylococcus aureus producteur de la leucocidine de Panton et Valentine chez un garçon de 10 ans.

Staphylococcal necrotizing pneumonia producing the Panton Valentine leukotoxin (PVL) has been described for many years. The french reference center for staphylococcal toxaemia defined it with precision in 1999. A 10-year-old child, died in 36 hours from respiratory distress and shock. Staphylococcal pneumonia was suspected then confirmed: S. Aureus producing PVL was isolated in lung, blood and articulations.

A new genetic test for the rapid identification of shiga-toxines producing (STEC), enteropathogenic (EPEC) E. coli isolates from children.

Routine bacteriological techniques do not allow detection of the most frequent enteric pathogens in young children: enteropathogenic Escherichia coli (EPEC) and shigatoxinogenic E. coli (STEC/EHEC). Since there is no correlation between serotype and pathotype, a genotypic determination is therefore necessary for the identification of these pathogenic strains. We evaluated the Genotype EHEC test (Hain Life Science, Germany), a new rapid system based on DNA multiplex amplification and further hybridization for the detection of shigatoxin stx1, stx2 genes, intimin eae gene and invasin ipaH gene harbored by Shigella and enteroinvasive E. coli (EIEC). E. coli strains of various serogroups isolated from children with acute gastroenteritis, hemorrhagic colitis or hemolytic-uremic syndrome were tested. Their genotypes were first determined by standard in-house PCR. The strains collection included 11 STEC/EHEC (serogroups O157, O111, O26, O91, O-untypable) and nine EPEC (serogroups O26, O157, O55, O126, O127, O-untypable). The same strains were tested with Genotype EHEC. For all the strains, the hybridization banding pattern obtained by Genotype EHEC correlated with their expected genotypic characteristics. No specific equipment is required, except a thermocycler. Absence of electrophoresis system, of ethidium bromide staining and imaging system is a clear-cut advantage of Genotype EHEC. In addition, the short testing time (less than 2 h) optimizes treatment orientation. The Genotype EHEC test allows an easy and reliable identification of EHEC, STEC, EPEC and also EIEC. As such, it is a useful tool for the rapid diagnosis of diarrheal diseases.

[E.coli from urinary tract infections and acute pyelonephritis of children: 1% of strains are resistant to a subset of third generation cephalosporins]. / Escherichia coli des infections urinaires et pyélonéphrites aiguës en pédiatrie : 1% des souches sont résistantes à certaines céphalosporines de 3e génération.

E. coli remains the most often isolated pathogen in community urinary tract infections in children. We reported a retrospective study of antibiotic susceptibility of 506 E. coli strains isolated from urine. We found that 53% of the strains were resistant to amoxicilline and 22% to cotrimoxazole. The frequency of resistance to amoxicillin-clavulanic acid was of 7%, 40% of the strains were just intermediary and 53% were sensitive. Only five strains (1%) were resistant to ceftazidime: two mechanisms of resistance, hyperproduction of TEM betalactamase (3 cases) and cephalosporinase (2 cases), were suggested. This study illustrates the necessity of constant monitoring of bacterial resistance to adapt antibiotherapeutic guidelines to local evolution.

[Revised prevalence of afa+ Escherichia coli strains in acute pyelonephritis of children]. / Révision de la prévalence des souches afa+ dans les Escherichia coli responsables de pyélonéphrites aiguës de l'enfant.

Two hundred E. coli strains isolated from children with pyelonephritis were investigated for the presence of six virulence factors. The used primers amplified adhesin pap and sfa, toxin haemolysin (hly) and cytotoxic necrotizing factor 1 (cnf1) and aerobactin (aer). For afimbrial adhesin, the previously used set of primers could not allow to detect the newly reported afa operons (Le Bouguenec et al., 2001). With a new set of primers specific for the afa operon family the prevalence of afa+ strains increased from 3.5% to 13.5%. Combinations of three or more factors in a same strain were found in 48.5%. Thirty two different urovirulent genotypes were observed; two strains contained the six studied factors.

The puzzle of zmpB and extensive chain formation, autolysis defect and non-translocation of choline-binding proteins in Streptococcus pneumoniae.

Choline-binding proteins (CBPs) from Streptococcus pneumoniae are involved in several important processes. Inactivation of zmpB, a gene that encodes a surface-located putative zinc metalloprotease, in a S. pneumoniae serotype 4 strain was recently reported to reveal a composite phenotype, including extensive chain formation, lysis defect and transformation deficiency. This phenotype was associated with the lack of surface expression of several CBPs, including the major autolysin LytA. LytA, normally 36 kDa in size, was reported to form an SDS-resistant 80 kDa complex with CinA. ZmpB was therefore proposed to control translocation of CBPs to the surface, possibly through the proteolytic release of CBPs (and RecA) from CinA. Based on the use of 12 independent mariner insertions in the zmpB gene of the well-characterized R6 laboratory strain, we could not confirm several of these observations. Our zmpB mutants: (i) did not form chains; (ii) lysed normally in the presence of deoxycholate, which indicates the presence of a functional autolysin; (iii) transformed at normal frequency; and (iv) contained bona fide CinA and LytA species. Polymorphism of ZmpB between R6 and the serotype 4 isolate could not account for the discrepancy, as inactivation of zmpB (through replacement by transposon-inactivated zmpB R6 alleles) in the latter strain did not affect separation of daughter cells and autolysis. The conflicting observations could be explained by our finding that the reportedly serotype 4 zmpB 'mutant' differed from its S. pneumoniae parent in lacking capsule and in exhibiting characteristic traits of the Streptococcus viridans group, including resistance to optochin.

RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression.

The RECODE database is a compilation of 'programmed' translational recoding events taken from the scientific literature and personal communications. The database deals with programmed ribosomal frameshifting, codon redefinition and translational bypass occurring in a variety of organisms. The entries for each event include the sequences of the corresponding genes, their encoded proteins for both the normal and alternate decoding, the types of the recoding events involved, trans-factors and cis-elements that influence recoding. The database is freely available at http://recode.genetics. utah.edu/.