Clinical Relevance of Type II Fatty Acid Synthesis Bypass in Staphylococcus aureus.

The need for new antimicrobials to treat bacterial infections has led to the use of type II fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to the screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, nonculturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in the acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of nonculturable variants.

Transfer between an Algerian and a French hospital of four multi-drug resistant bacterial strains together via a single patient.

A 5 years-old girl, seriously burnt with fire, was first hospitalized during four days in an hospital at Alger, and then transferred to our hospital at Paris. Admitted in our intensive care burns unit, she was third degree burnt on 78% of total body surface area, already treated with imipenem and vancomycin at her arrival. Clinical aggravation was rapidly observed and death occurred within 24 hours. Cultures of blood and multiple wound swabs yielded 3 multi-drug resistant bacterial strains: Acinetobacter baumannii with carbapenemase OXA-23, Pseudomonas aeruginosa serotype O11 with metallo-ß-lactamase VIM-4 and Klebsiella pneumoniae with CTX-M-15 extended-spectrum ß-lactamase. Culture of a rectal swab showed colonization by Enterococcus faecium with vanA glycopeptides resistance. Patients colonized with one or two multi-drug-resistant strains were not rare in our burns unit, especially those transferred from Algeria, but this case of a single patient harboring four multi-drug-resistant strains is exceptional.

Escherichia coli virulence patterns may help to predict vesicoureteral reflux in paediatric urinary tract infections.

AIM: Ultrasound and biological tools are used to predict high-grade vesicoureteral reflux, but other markers are needed to better select patients who need voiding cystography. Our aim was to determine whether studying Escherichia coli virulence factors would help to predict vesicoureteral reflux in patients with their first acute pyelonephritis. METHODS: We included children presenting with E. coli-related acute pyelonephritis or cystitis. Vesicoureteral reflux was assessed by voiding cystography. Virulence factors were identified by multiplex polymerase chain reaction. Statistical analysis was performed using logistic regression and the mean c-statistic test. RESULTS: We included 198 patients: 30 with cystitis and 168 with acute pyelonephritis, including 46 with vesicoureteral reflux. High-grade reflux was associated with acute pyelonephritis caused by the E. coli lacking virulence factors papGII (82% versus 47%, p < 0.001) or papC (85% versus 53%, p < 0.001) or belonging to phylogenetic group A or B1. When we added genetic data (lack of papGII, fyuA and phylogenetic groups) to classical predictors of vesicoureteral reflux (ultrasound examination, gender, age), the ability to predict high-grade reflux increased, with the c-statistic rising from 0.88 to 0.93. CONCLUSION: Bacterial virulence factors and clinical factors helped to predict high-grade reflux and may help to avoid unnecessary voiding cystographies.

A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31) is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10) with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung.

Persistent bloodstream infection with Kocuria rhizophila related to a damaged central catheter.

A case of persistent bloodstream infection with Kocuria rhizophila related to a damaged central venous catheter in a 3-year-old girl with Hirschsprung's disease is reported. The strain was identified as K. rhizophila by 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Arbitrarily primed PCR analysis showed a clonal strain. The repeated septic episodes were resolved with the catheter repair.

[Virulence factors in Pseudomonas aeruginosa: mechanisms and modes of regulation]. / Les facteurs de virulence de Pseudomonas aeruginosa : mécanismes et modes de régulations.

Pseudomonas aeruginosa is a bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients. The bacterium's virulence depends on a large number of cell-associated and extracellular factors. The virulence factors play an important pathological role in the colonization, the survival of the bacteria and the invasion of tissues. There are two types of virulence factors: (1) factors involved in the acute infection: these factors are either on the surface of P. aeruginosa, either secreted. The pili allow adherence to the epithelium. The exoenzyme S and other adhesins reinforce the adherence to epithelial cells. The exotoxin A is responsible of tissue necrosis. Phospholipase C is a thermolabile haemolysin. The pathogenic role of exoenzyme S is attributable to the disruption of normal cytoskeletal organization, the destruction of immunoglobulin G and A, leads to depolymerization of actin filaments and contributes to the resistance to macrophages. P. aeruginosa produces at least four proteases causing bleeding and tissue necrosis; (2) factors involved in the chronic infection: siderophores (pyoverdin and pyochelin), allow the bacteria to multiply in the absence of ferrous ions. The strains isolated from patients with cystic fibrosis have a pseudocapsule of alginate that protects the bacterium from phagocytosis, dehydration and antibiotics. Moreover, it improves adherence to epithelial cells forming a biofilm. Two different types of regulation systems control the expression of the majority of these virulence factors: the two-component transcriptional regulatory system and the quorum sensing system. These two mechanisms are necessary to the survival and the proliferation of this microorganism in the host.

Septic arthritis caused by Bordetella holmesii in an adolescent with chronic haemolytic anaemia.

We describe a case of septic arthritis caused by Bordetella holmesii in a 15-year-old boy with chronic haemolytic anaemia. B. holmesii was identified by 16S rRNA gene sequence analysis. The patient outcome was favourable. To our knowledge, this is the first case of B. holmesii septic arthritis in an asplenic patient.

Meningitis caused by Escherichia coli producing TEM-52 extended-spectrum beta-lactamase within an extensive outbreak in a neonatal ward: epidemiological investigation and characterization of the strain.

Outbreaks caused by Enterobacteriaceae isolates producing extended-spectrum beta-lactamases (ESBL) in neonatal wards can be difficult to control. We report here an extensive outbreak in a neonatal ward with a case of meningitis caused by an ESBL-producing Escherichia coli strain. Between 24 March and 29 April 2009, among the 59 neonates present in the ward, 26 neonates with ESBL-producing E. coli rectal colonization were detected (44%). One of the colonized neonates developed meningitis with a favorable outcome after treatment combining imipenem, gentamicin, and ciprofloxacin. Despite strict intensification of hygiene and isolation procedures for more than 1 month, ward closure to new admissions was necessary to control the outbreak. Randomly amplified polymorphic DNA and pulsed-field gel electrophoresis analysis performed on 31 isolates recovered from 26 neonates and two mother's milk samples showed a clonal strain. ESBL PCR assays indicated that the strain harbored a TEM-52 ESBL encoded by an IncI1 replicon. Phylogenetic analysis by multilocus sequence typing showed that the strain belonged to rare phylogenetic group C, which is closely related to group B1 but appears as group A by the triplex PCR phylogrouping method. The strain harbored the virulence genes fuyA, aer, and iroN and was virulent in a mouse model of septicemia. This work indicates the high potential of colonization, transmission, and virulence of some ESBL-producing E. coli clones.

Longitudinal survey of Staphylococcus aureus in cystic fibrosis patients using a multiple-locus variable-number of tandem-repeats analysis method.

BACKGROUND: Staphylococcus aureus infection in patients with cystic fibrosis (CF) is frequent and may be due to colonization by a few pathogenic lineages. Systematic genotyping of all isolates, methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) is necessary to identify such lineages and follow their evolution in patients. Multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) was used to survey S. aureus clinical isolates in a French paediatric CF centre. RESULTS: During a 30 months period, 108 patients, aged 2 to 21 years, regularly followed up at the centre, provided sputum for culture. From 79 patients, a total of 278 isolates were genotyped by MLVA, resolving into 110 genotypes and 19 clonal complexes (CC) composed of similar or closely related isolates. 71% of the strains were distributed into four main CCs, in term of number of isolates and number of genotypes. Spa (Staphylococcus protein A) typing was performed on representative samples, showing an excellent concordance with MLVA. In 17 patients, strains from two to four different CCs were recovered over time. On six occasions, S. aureus isolates with the same genotype were shared by 2 different patients and they belonged to one of the four main clusters. Methicillin-resistance was observed in 60% of the isolates, 90% of which belonged to the main clonal complexes CC8, CC45 and CC5. In 5 patients, methicillin-resistance of S. aureus isolates was not associated with the mecA gene: for four patients, it was due to overproduction of beta-lactamase, leading to BOR-SA (borderline S. aureus) isolates, while a strain showing probably a new modified penicillin-binding capacity (MOD-SA) was observed from one patient. CONCLUSION: Systematic genotyping of S. aureus isolates recovered from sputum of CF children allows a thorough analysis of the strains responsible for sporadic as well as chronic colonization and the follow up of their evolution over time. We show here that more than 70% of these strains belong to 4 major CCs. MSSA as well as MRSA, BOR-SA and MOD-SA isolates can persist over several years, despite antibiotic treatments.

Multiple-locus variable-number tandem-repeat analysis for longitudinal survey of sources of Pseudomonas aeruginosa infection in cystic fibrosis patients.

In order to identify the source of infection by Pseudomonas aeruginosa in patients with cystic fibrosis (CF), systematic genotyping of isolates is necessary. Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) was used to survey the sources of P. aeruginosa infections in a French (Paris, France) pediatric CF center. Between January 2004 and December 2006, 108 patients ages 2 to 21 years who were regularly monitored at the center provided sputum for culture. P. aeruginosa was detected in 46 children, 17 of whom had primary colonization. A total of 163 isolates were recovered. MLVA was improved from a previously published method by the addition of new, informative, and easily typeable markers. Upon genotyping with 15 VNTRs, a total of 39 lineages composed of indistinguishable or closely related isolates, were observed. One of them corresponds to "clone C," which is widely distributed in Europe, and another corresponds to reference strain PA14. Six patients were colonized with two different strains, and the remaining 40 patients were colonized with a single strain. Strains from seven lineages were shared by at least two and up to four patients among a total of 20 patients. The study demonstrates that MLVA is an efficient, easy, and rapid molecular method for epidemiological surveillance for P. aeruginosa infection. The resulting data and strain genetic profiles can be queried on http://bacterial-genotyping.igmors.u-psud.fr.

Multidrug-resistant bacteria in hospitalized children: a 5-year multicenter study.

OBJECTIVE: The objective of this study was to determine the incidence of multidrug-resistant bacteria in hospitalized children. METHODS: This multicenter study was conducted in 5 hospitals in the Paris area from 1999 to 2003. We recorded all isolations of multidrug-resistant bacteria from clinical samples that were obtained from hospitalized children. Strains that were isolated during systematic screening for carriers were excluded. RESULTS: The mean incidences were 0.9 per 1000 hospitalization-days for methicillin-resistant Staphylococcus aureus, 0.45 for extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, 0.32 for extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae, 0.40 for Enterobacter species with derepressed cephalosporinase, and 0.01 for vancomycin-resistant Enterococcus. The incidences per 1000 hospitalization-days of methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae, and Enterobacter species with derepressed cephalosporinase decreased significantly from 1999 to 2003, whereas the incidence of vancomycin-resistant Enterococcus remained very low. The proportion of resistant strains within the species did not vary significantly for methicillin-resistant Staphylococcus aureus (11% to 9.6%), extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae (1.1%), and vancomycin-resistant Enterococcus (0.03% to 0.023%). In contrast, the frequency of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae decreased from 31.6% to 7.4%, and that of Enterobacter species with derepressed cephalosporinase decreased from 38.8% to 18.5%. CONCLUSIONS: We report significant decreases in the incidence of methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae, and Enterobacter species with derepressed cephalosporinase in hospitalized children during a 5-year period.

Value of the chlorhexidine decontamination method for recovery of nontuberculous mycobacteria from sputum samples of patients with cystic fibrosis.

The chlorhexidine method was compared to the N-acetyl-l-cysteine-NaOH-oxalic acid decontamination method currently recommended for the recovery of nontuberculous mycobacteria (NTM) from patients with cystic fibrosis. Sputum samples (n = 827) treated with chlorhexidine yielded twice as many NTM-positive cultures as those treated by the reference method (54 [6.50%] versus 27 [3.25%]; P < 0.0001) despite a higher contamination rate (20% versus 14.2%; P = 0.0017).

Age-related prevalence and distribution of nontuberculous mycobacterial species among patients with cystic fibrosis.

We studied the prevalence and species distribution of nontuberculous mycobacteria (NTM) in relation to age in 385 patients with cystic fibrosis (CF) (mean age +/- standard deviation [range], 12.0 +/- 6.1 [1 to 24] years; sex ratio, 0.53) attending three Parisian centers. The overall prevalence of NTM in sputum was 8.1% (31 out of 385). The following NTM were isolated (n = 33): Mycobacterium abscessus (n = 13, 39.4%), Mycobacterium avium complex (MAC) (n = 7, 21.2%), Mycobacterium gordonae (n = 6, 18.2%), and other (n = 7, 21.2%). Sixteen patients met the American Thoracic Society microbiological criteria for NTM infection, including 11 patients positive for M. abscessus, 4 for MAC, and 1 for MAC and Mycobacterium kansasii. The overall prevalence of NTM was significantly lower in patients under 15 years old than for patients equal to or more than 15 years old (4.8 versus 14.9%, respectively; P = 0.001). M. abscessus was isolated at all ages, while MAC was not recovered before 15 years (prevalence of 0.0 and 5.2% in patients aged 1 to 14 and 15 to 24, respectively; P = 0.001).

DNA fingerprinting of Ralstonia paucula by infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis.

Ralstonia paucula (formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of R. paucula (4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie [LMG] collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis. Both techniques accurately distinguished between collection strains. Two close patterns obtained for all the French isolates suggested a clonal strain. Two LMG collection strains originating from human sources in the United States also showed patterns close to those of French isolates.