Although the multiplication of bacteriophages (phages) has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cell's production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strain's production of the phage ΦMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS). A related effect was observed in diverse host-phage systems, including the T4-like phages, with β-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages.

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 ␤-lactamase activity. It is possible that E. sakazakii strains differ in their pathogenicities, as shown by the range of symptoms associated with each pulsotype.Enterobacter sakazakii is an opportunistic pathogen associated with the ingestion of reconstituted infant formula and is a rare cause of neonatal meningitis, necrotizing enterocolitis (NEC), and sepsis (9, 10, 11, 23). Such cases often occur among low-birth-weight preterm neonates, who are generally more susceptible to gram-negative bacterial sepsis and endotoxemia associated with NEC (1, 26). The International Commission on Microbiological Specifications for Foods (14) has ranked E. sakazakii as a "severe hazard for restricted populations, life-threatening or substantial chronic sequelae or long duration." A number of reported E. sakazakii outbreaks have been attributed to contaminated reconstituted infant formula (4,7,13,18,31). Bowen and Braden (4) reviewed 46 cases of invasive E. sakazakii infections and showed a link between symptoms and birth weight but did not consider cases of NEC.The virulence of E. sakazakii has been studied by Pagotto et al. (23) and Mange et al. (21), who showed the presence of enterotoxins and adhesion to brain cells, respectively. Townsend et al. demonstrated the translocation of E. sakazakii and other intestinal bacteria across the rat intestinal wall in response to the presence of lipopolysaccharide (28). They also demonstrated that E. sakazakii causes chronic-patterned inflammation in the neonatal rat brain, invades capillary endothelial brain cells, is taken up by macrophages, and induces anti-inflammatory cytokine (interleukin-10) expression in vitro and in vivo at various levels according to strain (29). However, these publications did not report the individual case details associated with the isolates under study. Therefor...

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Rapid and cost-effective matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based systems will replace conventional phenotypic methods for routine identification of bacteria. We report here the first evaluation of the new MALDI-TOF MS-based Vitek MS system in a large clinical microbiology laboratory. This system uses an original spectrum classifier algorithm and a specific database designed for the identification of clinically relevant species. We have tested 767 routine clinical isolates representative of 50 genera and 124 species. Vitek MS-based identifications were performed by means of a single deposit on a MALDI disposable target without any prior extraction step and compared with reference identifications obtained mainly with the VITEK2 phenotypic system; if the identifications were discordant, molecular techniques provided reference identifications. The Vitek MS system provided 96.2% correct identifications to the species level (86.7%), to the genus level (8.2%), or within a range of species belonging to different genera (1.3%). Conversely, 1.3% of isolates were misidentified and 2.5% were unidentified, partly because the species was not included in the database; a second deposit provided a successful identification for 0.8% of isolates unidentified with the first deposit. The Vitek MS system is a simple, convenient, and accurate method for routine bacterial identification with a single deposit, considering the high bacterial diversity studied and as evidenced by the low prevalence of species without correct identification. In addition to a second deposit in uncommon cases, expanding the spectral database is expected to further enhance performances. Due to the dramatic increase of bacterial resistance and to the ecological cost of broad-spectrum antimicrobial therapies, rapid and accurate identification (ID) of bacteria is essential for the appropriate management of infections. Conventional identification methods require at least 4 to 12 h, and molecular methods are not suitable for large-scale routine identification.Nearly 40 years ago, chemists proposed to identify bacterial cultures via the detection of small organic molecules using mass spectrometry (2). More than 10 years later, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allowed the detection of intact larger biomolecules, such as proteins, and was further developed for microbial ID in routine clinical laboratories (13). During the analysis process, proteins are ionized without fragmentation by the coordinated action of the laser and the small organic acids of the matrix and separated on the basis of their mass-to-charge ratios, a process which results in a characteristic mass spectral profile. Microbial ID is based on the comparison of the protein spectrum generated from intact whole bacterial cells to a database of species-specific reference protein profiles using a particular algorithm.In the mid-1990s, different groups developed their own libraries of bacter...

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.

The experiments reported here provide strong evidence indicating that the transposition frequency of the bacterial insertion sequence IS1 is determined principally by two IS1‐specified proteins. The first, InsA, was previously shown to bind to the ends of the element and to act as a repressor. We present both physical and genetic evidence which reveals that the second, the InsAB' transposase, is a fusion of InsA with the product of a downstream reading frame, InsB'. Synthesis of this protein occurs by a ‐1 frameshift between the insA and insB' frames. It requires the presence of an intact retroviral‐like frameshift signal composed of an A6C motif and a downstream region able to form several alternative secondary structures. In vivo studies show that IS1 transposition activity depends on the relative rather than on the absolute levels of InsA and InsAB'. The ratio is determined primarily at the translational level by frameshifting and appears to be relatively insensitive to large variations in levels of transcription. This novel homeostatic control could therefore protect IS1 from activation as a consequence of insertion into active transcription units.

Among 1,102 recent Escherichia coli clinical isolates, clonal group A was identified in 17 of 20 (U.S. and non-U.S.) geographic locales, mainly among U.S. isolates (9% vs. 3%; p < 0.001) and those resistant to trimethoprim-sulfamethoxazole (10% vs. 1.7%; p < 0.001). The extensive antimicrobial resistance and virulence profiles of clonal group A may underlie its recent widespread emergence.

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 ␤-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 g/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.The major mechanism of resistance to ␤-lactam antibiotics in gram-negative bacteria results from the production of ␤-lactamases. Most of these are coded by the plasmid-mediated bla TEM-1 gene (19,28). The continuous introduction of new ␤-lactam antibiotics with different activity spectra in human medicine has led to the selection of ␤-lactamase mutations, which confer resistance to the newly developed ␤-lactam antibiotics (25). ␤-Lactam antibiotics are also used in veterinary medicine, where they contribute to the selective pressure that leads to the emergence and diffusion of intestinal bacteria harboring resistance genes. Thus, commensal bacteria in the gut form a reservoir of antibiotic resistance genes potentially transmissible to humans via the food chain and the environment (27,29,34).Antimicrobial resistance in food animals deserves special attention. One of the most heavily medicated sectors is pig farming, with worldwide antibiotic consumption in pigs accounting for 60% of the antibiotics used in animals (10). A relationship has been demonstrated between the high use of antimicrobials in pig herds and the increased occurrence of resistant bacterial strains in their digestive tracts (4,13,34,37). When antibiotics are administered to pigs, both the level and time development of antibiotic exposure of the intestinal microflora are dependen...