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Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after twodimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide-and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.

SummaryThe intestinal anaerobic symbiont, Bacteroides fragilis, is highly aerotolerant and resistant to H2O2. Analysis of the transcriptome showed that expression of 45% of the genome was significantly affected by oxidative stress. The gene expression patterns suggested that exposure to oxidative stress induced an acute response to rapidly minimize the immediate effects of reactive oxygen species, then upon extended exposure a broad metabolic response was induced. This metabolic response induced genes encoding enzymes that can supply reducing power for detoxification and restore energy-generating capacity. An integral aspect of the metabolic response was downregulation of genes related to translation and biosynthesis which correlated with decreased growth and entry into a stationary phase-like growth state. Examination of oxyR mutants showed that they were impaired for the acute response and they induced the expanded metabolic response with only minimal exposure to stress. The oxyR mutants were more sensitive to oxidants in vitro and in vivo they were attenuated in an intra-abdominal abscess infection model. Aerotolerance and resistance to oxidative stress are physiological adaptations of B. fragilis to its environment that enhance survival in extra-intestinal sites and promote opportunistic infections.

A clinical isolate of Bacteroides vulgatus was resistant to tetracycline, clindamycin, ampicillin, cephaloridine, cefoxitin, and other 13-lactam antibiotics except imipenem. 13-Lactam resistance was mediated by a membraneassociated, clavulanate-sensitive cephalosporinase capable of degrading cephalosporins and penicillins. Cefoxitin also was degraded but at a slow rate. The cefoxitin resistance (Fxr) ,-Lactamase production is the most important mechanism of resistance to P-lactam antibiotics in gram-negative bacteria, and the Bacteroides fragilis group possesses a wide array of these enzymes. In general, the organisms are moderately or highly resistant to many cephalosporins and penicillins, but a-methoxyl cephamycins such as cefoxitin and the carbapenems have been highly active against Bacteroides species. In the United States, for example, imipenem resistance rates are presently low, at about 0.2%, and cefoxitin resistance (Fxr) rates have ranged up to 16% during the past decade (37, 38). High rates of Fxr are cause for concern since Fxr strains usually encountered are crossresistant to most other P-lactams (38). In the case of strains that possess metallo-p-lactamases, the cross-resistance includes the carbapenems (8, 39).Resistance of Bacteroides species to cefoxitin may involve a number of mechanisms including altered drug permeability, alterations of penicillin binding proteins, 1-lactamase production, or a combination of mechanisms (8,10,41). Excluding the relatively rare imipenem-hydrolyzing metalloenzymes, several P-lactamases capable of cefoxitin degradation have been described. These are generally cephalosporinases with a slow rate of cefoxitin hydrolysis, and they are sensitive to inhibition by clavulanate (9, 13). Recently, Aldridge et al. (1) have found that >93% of Fxr strains were still sensitive to 3-lactam-clavulanate combinations. Thus, based on these criteria, it is possible that the slow cefoxitinhydrolyzing cephalosporinases are one of the most widely disseminated mechanisms of Fxr in the Bacteroides species. In this regard, it has been suggested that these P-lactamases are not novel enzymes but rather that the strains are Fxr * Corresponding author.because of the production of much greater than normal levels of the conventional Bacteroides enzyme (36).The occurrence of high regional Fxr rates indicates the potential for clonal dissemination and/or horizontal transfer of the resistance phenotype. The transfer of Fxr by a conjugation-like mechanism has been demonstrated; however, plasmids were not involved in the transfer (10). The Fxr phenotype was mediated by the acquisition of a new P-lactamase. The present study was initiated to define the genetic basis for transmissible Fxr in Bacteroides species.The results revealed a novel genetic locus designated cfx4, which encodes an Ambler molecular class A 13-lactamase that appears to have diverged significantly from all other class A enzymes. MATERIALS AND METHODSBacterial strains and growth. Bacteroides cells were cultured anaerobically in su...

The peroxide response-inducible genes ahpCF, dps, and katB in the obligate anaerobe Bacteroides fragilis are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria. oxyR and dps were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions. B. fragilis OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe, Porphyromonas gingivalis. Northern blot analysis revealed that oxyR was expressed as a monocistronic 1-kb mRNA and that dps mRNA was approximately 500 bases in length. dps mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the oxyR gene [oxyR(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion of oxyR abolished the induction of ahpC and katB following treatment with hydrogen peroxide or oxygen exposure. However, dps expression was induced approximately fourfold by oxygen exposure in ⌬oxyR strains but not by hydrogen peroxide. This indicates that dps expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of ⌬oxyR mutant strains with wildtype oxyR and oxyR(Con) restored the inducible peroxide response and the constitutive response of the ahpCF, katB, and dps genes, respectively. However, overexpression of OxyR abolished the catalase activity but not katB expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of oxyR expression in the parents and in ⌬oxyR and overexpressing oxyR strains by Northern blotting and oxyR::xylB fusions revealed that B. fragilis OxyR does not control its own expression.The human intestinal obligate anaerobe Bacteroides fragilis possesses a complex oxidative stress response mechanism which is required to maintain extended aerotolerance compared to control cultures (24). A set of approximately 28 proteins are synthesized in response to treatment with hydrogen peroxide or oxygen exposure, but other proteins are also down regulated following a shift to aerobic conditions, and their role in the physiological adaptation to this adverse environment still remains unclear (24). The catalase gene katB is typical of the B. fragilis oxidative stress genes and is induced in mid-log phase following the addition of hydrogen peroxide or exposure to molecular oxygen or after entering the stationary phase (25). A katB mutant was found to be more sensitive to exogenous hydrogen peroxide under anaerobic conditions than was the parent strain, but aerotolerance in the presence of atmospheric oxygen was not significantly altered (24). The studies on resistance to peroxides led to the isolation of a KatB-overproducing...

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The development of advanced technologies to enhance conservation science often outpaces the abilities of wildlife managers to assess and ensure such new tools are safely used in proximity to wild animals. Recently, unmanned aerial systems (UAS) have become more accessible to civilian operators and are quickly being integrated into existing research paradigms to replace manned aircraft. Several federal statutes require scientists to obtain research permits to closely approach protected species of wildlife, such as marine mammals, but the lack of available information on the effects of UAS operations on these species has made it difficult to evaluate and mitigate potential impacts. Here, we present a synthesis of the current state of scientific understanding of the impacts of UAS usage near marine mammals. We also identify key data gaps that are currently limiting the ability of marine resource managers to develop appropriate guidelines, policies, or regulations for safe and responsible operation of UAS near marine mammals. We recommend researchers prioritize collecting, analyzing, and disseminating data on marine mammal responses to UAS when using the devices to better inform the scientific community, regulators, and hobbyists about potential effects and assist with the development of appropriate mitigation measures.

Bacteroides frgigli& CS30 is a clinical isolate resistant to high concentrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. fl-Lactam resistance is mediated by a chromosomally encoded cephalosporinase produced at a high level. The gene encoding this 13-lactamase was cloned from genomic libraries constructed in Escherichia coli and then mated with B. fragilis 638 for identification of ampicillin-resistant (Apr) strains. Apr transconjugants contained a nitrocefin-reactive protein with the physical and enzymatic properties of the original CS30 isolate. The (3-lactamase gene (cepA) was localized by deletion analysis and subcloned, and its nucleotide sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A 13-lactamase-deficient mutant strain of B. fiugilis 638 was constructed by insertional inactivation with the cepA gene of CS30, demonstrating strict functional homology between these chromosomal 13-lactamase genes. An extensive comparison of the CepA protein sequence by alignment with other 13-lactamases revealed the strict conservation of at least four elements common to Ambler class A. A further comparison of the CepA protein sequence with protein sequences of (-lactamases from two other Bacteroides species indicated that they constitute their own distinct subgroup of class A 13-lactamases.Bacteroides fragilis is responsible for approximately half of all human anaerobic infections and is the most common anaerobe recovered from clinical specimens (12). Numerous reports of B. fragilis isolates resistant to a variety of P-lactam antibiotics indicate that these organisms are becoming increasingly refractory to treatment with these drugs. The primary mechanism of 1-lactam resistance in Bacteroides species is the production of ,B-lactamase (40). At least four types of P-lactamase have been described for members of the B. fragilis group, but the most common type is a constitutively produced, chromosomally encoded cephalosporinase having no activity against cefoxitin or imipenem.This "endogenous" P-lactamase is present in over 90% of clinical isolates tested (10). Unlike the class C chromosomally encoded P-lactamases of members of the family Enterobacteriaceae, the B. fragilis P-lactamase has an isoelectric point in the acid range and is susceptible to inhibition by clavulanic acid and sulbactam, placing it in group 2e in the Bush classification scheme (8).Regulation of the endogenous B. fragilis j-lactamase has not been extensively studied, but this enzyme may be growth rate regulated, with maximal activity occurring 3 h into the stationary phase (7). With regard to the production of the endogenous cephalosporinase, others have grouped B.fragilis clinical isolates into three expression classes (18). Low-level j-lactamase producers are susceptible to all 3-lactams, the MICs of benzylpenicillin and cephaloridine being <2 and <16 ,ug/ml, respectively. For intermediate-level 3-lactamase producers, th...