Levels Of Ampicillin Resistance Research Articles

Resistance mechanism of Escherichia coli strains with different ampicillin resistance levels.

Antibiotic resistance is an important problem that threatens medical treatment. Differences in the resistance levels of microorganisms cause great difficulties in understanding the mechanisms of antibiotic resistance. Therefore, the molecular reasons underlying the differences in the level of antibiotic resistance need to be clarified. For this purpose, genomic and transcriptomic analyses were performed on three Escherichia coli strains with varying degrees of adaptive resistance to ampicillin. Whole-genome sequencing of strains with different levels of resistance detected five mutations in strains with 10-fold resistance and two additional mutations in strains with 95-fold resistance. Overall, three of the seven mutations occurred as a single base change, while the other four occurred as insertions or deletions. While it was thought that 10-fold resistance was achieved by the effect of mutations in the ftsI, marAR, and rpoC genes, it was found that 95-fold resistance was achieved by the synergistic effect of five mutations and the ampC mutation. In addition, when the general transcriptomic profiles were examined, it was found that similar transcriptomic responses were elicited in strains with different levels of resistance. This study will improve our view of resistance mechanisms in bacteria with different levels of resistance and provide the basis for our understanding of the molecular mechanism of antibiotic resistance in ampicillin-resistant E. coli strains. KEY POINTS: •The mutation of the ampC promoter may act synergistically with other mutations and lead to higher resistance. •Similar transcriptomic responses to ampicillin are induced in strains with different levels of resistance. •Low antibiotic concentrations are the steps that allow rapid achievement of high antibiotic resistance.

Biofilms and Antimicrobial Susceptibility Profiles of Escherichia coli Recovered from Wastewater Treatment Plants in Kakamega Municipality, Kenya

STUDIES have demonstrated high levels of ampicillin resistance among Escherichia coli occurring in wastewater and environmental systems in western Kenya. The current study investigated antimicrobial resistance profiles and biofilm formation abilities in presence and absence of sub-lethal ampicillin concentrations, of E. coli recovered from final sedimentation ponds of two wastewater treatment plants in Kakamega municipality. 34 non-duplicate E. coli isolates were recovered by direct plating of wastewater sample aliquots on MacConkey agar and their biofilm formation capabilities measured by crystal violet assay while disc diffusion and Polymerase Chain Reaction (PCR) techniques were used to determine their antibiotic resistance levels and ampicillin resistance genes respectively. The Spearman’s Chi-square test at P≤ 0.05 was used to check for interdependence between antimicrobial resistance and biofilm formation capabilities. 58.8% of the isolates were multi-drug resistant (MDR) and 85.3% showed resistance to ampicillin, which was found to be encoded by blaTEM in 65% and blaSHV in 8.8%. The biofilm phenotype was exhibited by 61.8% of all the isolates, amongst which 23.6% showed a strong, 14.7% a moderate and 23.6% a weak propensity to form biofilms. This study revealed lack of association between antibiotic resistance and biofilm formation, but interestingly, ampicillin concentration of 8.0μg/ml triggered the highest biofilm biomass among the isolates. The findings drive at the conclusion that biofilm production among E. coli in wastewater treatment plants(WWTPs) does not correlate with antibiotic resistance, but may be an important protection mechanism against sub-lethal antimicrobial levels present in environmental milieu.

Assessment of door handles as potential reservoirs of drug-resistant enterococci

BackgroundFomites have long been known to play a key role in the spread of disease causing agents. Hospital-associated fomites in particular have been linked with transmission of members of the Enterococci sp which are key human pathogens. Few studies have explored the role non-hospital door handles might play as potential sources of these isolates. This study therefore set out to explore this role.ResultsA total of hundred toilet and office door handles in a tertiary institution in Rivers State, Nigeria, were sampled using the swab and rinse method. The presence and drug susceptibility of Enterococcus was determined using the selective bile esculin agar (BEA) and standard microbiological methods. Growth on BEA was observed in 71% of cases, with more growth (38/50, 76%) observed from toilet door handles. Only 35% of samples produced the characteristic black pigmentation associated with Enterococcus sp. Six different bacterial groups were identified from this subset with Enterococcus sp. making up only 14% (5/35) of the isolates. All (100%) Enterococci were isolated from toilet door handles. Antibiotic susceptibility testing revealed very high levels of resistance (80–100%) against 75% of the test antibiotics. An analysis of the antibiotic resistance pattern of each isolate revealed 11 unique antibiogram patterns. Only 2 of these patterns were associated with the enterococci, with majority (4/5) exhibiting resistance to Augmentin (AUG), Ceftazidime (CAZ), Ceftriaxone (CTR), Cefuroxime (CRX), Cloxacillin (CXC), Erythromycin (ERY), Gentamicin (GEN), Ofloxacin (OFL) (antibiogram of AUG–CAZ–CRX–CTR–CXC–ERY). None of the enterococci, however, was resistant to vancomycin.ConclusionThis study reports low level contamination of door handles by enterococci. Identical antibiogram patterns linked with majority of the enterococci could however point at the occurrence of a single clone perhaps indicating single source contamination. Reports of high levels of ampicillin resistance among these isolates are problematic as ampicillin–gentamicin combination is the treatment of choice for nosocomial enterococci pathogens.

Antimicrobial resistance patterns of Gram-negative bacteria isolated from bloodstream infections in an Iranian referral paediatric hospital: A 5.5-year study.

Bloodstream infections (BSIs) are a major cause of paediatric morbidity and mortality worldwide. This study describes the epidemiology and antimicrobial resistance of Gram-negative bacteria (GNB) from BSIs in children admitted to an Iranian paediatric hospital. Clinical and microbiological data of patients with positive blood cultures were collected from March 2011 to September 2016. Standard laboratory methods were used for blood culture and bacterial identification. Antimicrobial sensitivity was evaluated by the Kirby-Bauer disk diffusion and broth microdilution methods. Of 2325 bacterial pathogens isolated from blood cultures, 41.1% (n=956) were GNB. Most clinical isolates (n=208; 21.8%) were identified in the cardiac intensive care unit. Predominant bacterial isolates were Klebsiella pneumoniae subsp. pneumoniae (n=263; 27.5%), Escherichia coli (n=192; 20.1%), Serratia marcescens (n=151; 15.8%), Pseudomonas aeruginosa (n=111; 11.6%) and Enterobacter spp. (n=100; 10.5%). Enterobacter isolates showed a high level of ampicillin resistance. Escherichia coli were highly resistant to chloramphenicol (100%), cefixime (100%), ceftriaxone (100%) and ampicillin (96%). Cefixime had the least efficacy against Haemophilus spp. (100% resistant). All K. pneumoniae (100%) were ampicillin-resistant. All S. marcescens were ceftazidime-resistant. No Acinetobacter baumannii were resistant to colistin. All P. aeruginosa were resistant to cefotaxime and trimethoprim/sulfamethoxazole. These results demonstrate the increasing trend in antibiotic resistance among GNB associated with BSI in children, emphasising the importance of continuous screening and surveillance programmes for detection of antibiotic resistance in BSI pathogens for selection of appropriate treatment regimens.

Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance.

Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.

Penicillin-Binding Protein 5 Sequence Alteration and Levels of plp5 mRNA Expression in Clinical Isolates of Enterococcus faecium with Different Levels of Ampicillin Resistance.

Eighty-two nonduplicated ampicillin-resistant Enterococcus faecium (AREF) isolates from clinical infections at the Charles Nicolle Hospital of Tunisia were investigated. They were collected from January 2001 to December 2009. Genetic relationship between them was studied using pulsed-field gel electrophoresis. The amino acid sequence difference variations of the C-terminal part of penicillin-binding protein 5 (PBP5) versus levels of expressed mRNA were investigated by polymerase chain reaction (PCR), sequencing, and real-time PCR quantification of (PBP5), respectively. No β-lactamase activity was detected and none of our strains showed resistance to glycopeptides, which retain their therapeutic efficiency against enterococcal infections in our hospital. Pattern analysis of the strains revealed six main clones disseminating in different wards. Sequence data revealed the existence of 19 different plp5 alleles with a difference in 16 amino acid positions spanning from residue 414 to 632. Each allele presented at least five amino acid substitutions (His-470→Gln, Asn-496→Lys, Ala-499→Thr, Glu-525→Asp, and Glu-629→Val). No correlation between amino acid sequence polymorphism of PBP5 and levels of ampicillin resistance was detected. The levels of plp5 mRNA expression varied between strains and did not always correlate with levels of ampicillin resistance in clinical AREF.

Small-plasmid-mediated antibiotic resistance is enhanced by increases in plasmid copy number and bacterial fitness.

Plasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzae carrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzae populations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

ANTIBIOTIC RESISTANCE IN ENTEROBACTERIACEAE STRAINS ISOLATED FROM CHICKEN AND MILK SAMPLES

Antibiotic resistance and identification of strains in Enterobacteriaceae genera isolated from milk, milk products and rectal swabs of chicken was examined in this experiment. After samples collection cultivation and identification of bacterial strain was done. MALDI TOF MS Biotyper for identification of Enterobacteriaceae strains was used. For susceptibility testing disc diffusion methodology was used according by EUCAST. Results showed high level of ampicillin resistance in isolates from milk and milk samples. The highest streptomycin resistance was detected in isolates from rectal swabs of chicken. After identification, we determined that S. enterica ser. Typhimurium, which was isolated from rectal swabs of chicken showed the most multi-resistance from all identificated strains of Enterobacteriaceae. The most isolates bacterial strain was E. coli, which showed resistance against four antibiotics from rectal swabs of chicken. Also our results showed that the higher resistance level is in rectal swabs of chicken like in milk samples.

Isolation and Characterization of the New Mosaic Filamentous Phage VFJ Φ of Vibrio cholerae

Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJΦ was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCYΦ. This 700 bp region gave VFJΦ several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJΦ was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJΦ updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.

Occurrence and Distribution of Antibiotic-resistant Bacteria and Transfer of Resistance Genes in Lake Taihu

The overuse of antibiotics has accelerated antibiotic resistance in the natural environment, especially fresh water, generating a potential risk for public health around the world. In this study, antibiotic resistance in Lake Taihu was investigated and this was the first thorough data obtained through culture-dependent methods. High percentages of resistance to streptomycin and ampicillin among bacterial isolates were detected, followed by tetracycline and chloramphenicol. Especially high levels of ampicillin resistance in the western and northern regions were illustrated. Bacterial identification of the isolates selected for further study indicated the prevalence of some opportunistic pathogens and 62.0% of the 78 isolates exhibited multiple antibiotic resistance. The presence of ESBLs genes was in the following sequence: blaTEM > blaSHV > blaCTMX and 38.5% of the isolates had a class I integrase gene. Of all tested strains, 80.8% were able to transfer antibiotic resistance through conjugation. We also concluded that some new families of human-associated ESBLs and AmpC genes can be found in natural environmental isolates. The prevalence of antibiotic resistance and the dissemination of transferable antibiotic resistance in bacterial isolates (especially in opportunistic pathogens) was alarming and clearly indicated the urgency of realizing the health risks of antibiotic resistance to human and animal populations who are dependent on Lake Taihu for water consumption.

Site-Saturation Mutagenesis of Position V117 in OXA-1 β-Lactamase: Effect of Side Chain Polarity on Enzyme Carboxylation and Substrate Turnover

Class D β-lactamases pose an emerging threat to the efficacy of β-lactam therapy for bacterial infections. Class D enzymes differ mechanistically from other β-lactamases by the presence of an active-site N-carboxylated lysine that serves as a general base to activate the serine nucleophile for attack. We have used site-saturation mutagenesis at position V117 in the class D β-lactamase OXA-1 to investigate how alterations in the environment around N-carboxylated K70 affect the ability of that modified residue to carry out its normal function. Minimum inhibitory concentration analysis of the 20 position 117 variants demonstrates a clear pattern of charge and polarity effects on the level of ampicillin resistance imparted on Escherichia coli (E. coli). Substitutions that introduce a negative charge (D, E) at position 117 reduce resistance to near background levels, while the positively charged K and R residues maintain the highest resistance levels of all mutants. Treatment of the acidic variants with the fluorescent penicillin BOCILLIN FL followed by SDS-PAGE shows that they are active for acylation by substrate but deacylation-deficient. We used a novel fluorescence anisotropy assay to show that the specific charge and hydrogen-bonding potential of the residue at position 117 affect CO(2) binding to K70, which in turn correlates to deacylation activity. These conclusions are discussed in light of the mechanisms proposed for both class D β-lactamases and BlaR β-lactam sensor proteins and suggest a reason for the preponderance of asparagine at the V117-homologous position in the sensors.

Fine Mapping of the Sequence Requirements for Binding of β-Lactamase Inhibitory Protein (BLIP) to TEM-1 β-Lactamase Using a Genetic Screen for BLIP Function

Beta-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases with a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for binding the TEM-1, SME-1, SHV-1, and Bla1 beta-lactamases. Twenty-three BLIP residues that contact TEM-1 beta-lactamase in the structure of the complex were mutated to alanine and assayed for inhibition (K(i)) of beta-lactamase to identify two hotspots of binding energy. These studies have been extended by the development of a genetic screen for BLIP function in Escherichia coli. The bla(TEM-1) gene encoding TEM-1 beta-lactamase was inserted into the E. coli pyrF chromosomal locus. Expression of wild-type BLIP from a plasmid in this strain resulted in a large decrease in ampicillin resistance, while introduction of the same plasmid lacking BLIP had no effect on ampicillin resistance. In addition, it was found that when the BLIP alanine-scanning mutants were tested in the strain, the level of ampicillin resistance was proportional to the K(i) of the BLIP mutant. These results indicate that BLIP function can be monitored by the level of ampicillin resistance of the genetic test strain. Each of the 23 BLIP positions examined by alanine scanning was randomized to create libraries containing all possible substitutions at each position. The genetic screen for BLIP function was used to sort the libraries for active mutants, and DNA sequence analysis of functional BLIP mutants identified the sequences required for binding TEM-1 beta-lactamase. The results indicate the BLIP surface is tolerant of substitutions in that many contact positions can be substituted with other amino acid types and retain wild-type levels of function.

Genetic and Structural Characterization of an L201P Global Suppressor Substitution in TEM-1 β-Lactamase

TEM-1 β-lactamase confers bacterial resistance to penicillin antibiotics and has acquired mutations that permit the enzyme to hydrolyze extended-spectrum cephalosporins or to avoid inactivation by β-lactamase inhibitors. However, many of these substitutions have been shown to reduce activity against penicillin antibiotics and/or result in loss of stability for the enzyme. In order to gain more information concerning the tradeoffs associated with active site substitutions, a genetic selection was used to find second site mutations that partially restore ampicillin resistance levels conferred by an R244A active site TEM-1 β-lactamase mutant. An L201P substitution distant from the active site that enhanced ampicillin resistance levels and increased protein expression levels of the R244A TEM-1 mutant was identified. The L201P substitution also increases the ampicillin resistance levels and restores expression levels of a poorly expressed TEM-1 mutant with a core-disrupting substitution. In vitro thermal denaturation of purified protein indicated that the L201P mutation increases the Tm value of the TEM-1 enzyme. The X-ray structure of the L201P TEM-1 mutant was determined to gain insight into the increase in enzyme stability. The proline substitution occurs at the N-terminus of an α-helix and may stabilize the enzyme by reducing the helix dipole, as well as by lowering the conformational entropy cost of folding due to the reduced number of conformations available in the unfolded state. Collectively, the data suggest that L201P promotes tolerance of some deleterious TEM-1 mutations by enhancing the protein stability of these mutants.

High nasopharyngeal carriage of β-lactamase-negative ampicillin-resistant Haemophilus influenzae in north Indian school-going children

Development of ampicillin resistance in Haemophilus influenzae is a cause of serious concern. Ampicillin resistance in H influenzae is beta-lactamase mediated except in some isolates. Two important issues related to beta-lactamase-negative ampicillin-resistant (BLNAR) strains while choosing therapy for infections caused by H. influenzae are (i) whether BLNAR H. influenzae isolates are sufficiently pathogenic to cause respiratory tract infection, and (ii) variability in the magnitude of ampicillin minimum inhibitory concentrations obtained for the isolates. The aim of the present study was to determine the carriage of BLNAR H. influenzae in the nasopharynx of normal healthy children, to test the level of ampicillin resistance and the correlation of ampicillin resistance with resistance to other antimicrobials and to evaluate the frequency of serotype b and biotypes I, II, and III among BLNAR H. influenzae. Of 1001 H. influenzae isolates, 229 (22.9%) strains were ampicillin resistant. A total of 33/229 isolates were BLNAR. beta-Lactamase-positive strains show higher level of resistance to ampicillin as well as to chloramphenicol, erythromycin, and co-trimoxazole. Of the 196 beta-lactamase-producing H. influenzae isolates, 112 (57%) were H. influenzae type b, while of the 33 BLNAR isolates, 27 (81.8%) were H. influenzae type b. One hundred and eighty-four of 196 (93.9%) beta-lactamase-producing H. influenzae isolates and 30/33 (91.0%) BLNAR strains belonged to biotypes I, II, and III. BLNAR H. influenzae are no less pathogenic than beta-lactamase-positive H. influenzae. Higher level of drug resistance was found in beta-lactamase-producing H. influenzae in comparison to BLNAR isolates.

Different molecular rearrangements in the integron of the IncP-1 beta resistance plasmid pB10 isolated from a wastewater treatment plant result in elevated beta-lactam resistance levels.

The multiresistance IncP-1 beta plasmid pB10 conferring resistance to ampicillin, streptomycin, sulfonamides, tetracycline and mercury ions was previously obtained from activated sludge bacteria by applying the exogenous isolation method with Pseudomonas sp. strain GFP2 as recipient. A pB10 derivative, designated pB10-1, occurred spontaneously and displays an extended NotI restriction fragment. From the pB10 nucleotide sequence, it is known that the corresponding NotI fragment of this plasmid contains a complete class 1 integron with an oxa2 and an orfE-like gene cassette. Sequencing of the integron-specific variable region present on pB10-1 revealed that a second copy of the oxa2 gene cassette has inserted downstream of the orfE-like cassette. Sequences flanking the second oxa2 cassette indicate that this cassette was excised from pB10 and reinserted at a new site in an integrase-catalyzed manner. Duplication of the oxa2 cassette is associated with a higher level of ampicillin resistance. Another pB10 derivative, designated pB10-2, conferring higher resistance to ampicillin, was shown to carry an IS10 insertion upstream of the oxa2 cassette. Since IS10 possesses a promoter-out activity, it can be assumed that the elevated ampicillin resistance level is due to enhanced transcription of the beta-lactamase gene.

Molecular characterization of ampicillin-resistant Enterococcus faecium isolates from hospitalized patients in Norway.

The genetic relationship of 81 ampicillin-resistant and 21 ampicillin-susceptible Enterococcus faecium isolates from clinical infections and rectal screening in hospitalized patients in Norway was studied by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). PFGE showed 55 different banding patterns, and 65 of the isolates could be grouped into one large group. With AFLP, 46 patterns were discerned, and 74 isolates clustered in one group. In general, the isolates had a higher degree of similarity than with PFGE. The purK gene, which is one of the targets of the E. faecium multilocus sequence typing scheme, was sequenced. Eleven different purK alleles could be discerned, with the majority of isolates (n = 80) harboring allele 1. With only two exceptions, all strains carrying purK-1 clustered in the same PFGE and AFLP groups, indicating a good correlation between PFGE type, AFLP type, and purK allele. Genetic polymorphism of a 571-bp PCR fragment of the C-terminal domain of the penicillin-binding protein 5 gene (pbp5) was determined, and sequence differences were associated with the level of ampicillin resistance. This study indicates that the majority of ampicillin-resistant E. faecium strains in Norway belong to a distinct genetic lineage of closely related genotypes. Rectal and clinical isolates were generally indistinguishable, and differences in clonal distribution and allele polymorphism were found mainly between ampicillin-resistant and -susceptible isolates.

Penicillin-binding protein 5 and expression of ampicillin resistance in Enterococcus faecium.

We report a structural and transcriptional analysis of the pbp5 region of Enterococcus faecium C68. pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reported psr (penicillin-binding protein synthesis repressor) and ftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW (ftsW(Efm)) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsW(Efm), psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from the ftsW(Efm) promoter through the pbp5 gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faecium D344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF(pCWR558) and D344-SRF(pCWR583) were 64 microg/ml. Quantities of pbp5 transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor of pbp5 transcription. However, quantities of psr transcript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.

Topological analysis of DctQ, the small integral membrane protein of the C4-dicarboxylate TRAP transporter of Rhodobacter capsulatus

Tripartite ATP-independent periplasmic (‘TRAP’) transporters are a novel group of bacterial and archaeal secondary solute uptake systems which possess a periplasmic binding protein, but which are unrelated to ATP-binding cassette (ABC) systems. In addition to the binding protein, TRAP transporters contain two integral membrane proteins or domains, one of which is 40–50 kDa with 12 predicted transmembrane (TM) helices, thought to be the solute import protein, while the other is 20–30 kDa and of unknown function. Using a series of plasmid-encoded β-lactamase fusions, we have determined the topology of DctQ, the smaller integral membrane protein from the high-affinity C4-dicarboxylate transporter of Rhodobacter capsulatus, which to date is the most extensively characterised TRAP transporter. DctQ was predicted by several topology prediction programmes to have four TM helices with the N- and C-termini located in the cytoplasm. The levels of ampicillin resistance conferred by the fusions when expressed in Escherichia coli were found to correlate with this predicted topology. The data have provided a topological model which can be used to test hypotheses concerning the function of the different regions of DctQ and which can be applied to other members of the DctQ family.

Topological analysis of DctQ, the small integral membrane protein of the C4-dicarboxylate TRAP transporter of Rhodobacter capsulatus.

Tripartite ATP-independent periplasmic ('TRAP') transporters are a novel group of bacterial and archaeal secondary solute uptake systems which possess a periplasmic binding protein, but which are unrelated to ATP-binding cassette (ABC) systems. In addition to the binding protein, TRAP transporters contain two integral membrane proteins or domains, one of which is 40-50 kDa with 12 predicted transmembrane (TM) helices, thought to be the solute import protein, while the other is 20-30 kDa and of unknown function. Using a series of plasmid-encoded beta-lactamase fusions, we have determined the topology of DctQ, the smaller integral membrane protein from the high-affinity C4-dicarboxylate transporter of Rhodobacter capsulatus, which to date is the most extensively characterised TRAP transporter. DctQ was predicted by several topology prediction programmes to have four TM helices with the N- and C-termini located in the cytoplasm. The levels of ampicillin resistance conferred by the fusions when expressed in Escherichia coli were found to correlate with this predicted topology. The data have provided a topological model which can be used to test hypotheses concerning the function of the different regions of DctQ and which can be applied to other members of the DctQ family.

Bacterial infections in infants 60 days and younger: epidemiology, resistance, and implications for treatment.

To establish what might be more optimal initial antibiotic therapy for suspected invasive bacterial infections in infants 60 days or younger who are evaluated in the emergency department (ED). Urban university-affiliated pediatric referral center with an average yearly ED census of 52000 visits during the study period. We assembled a retrospective case series of all positive blood, urine, and cerebrospinal fluid cultures in children 60 days or younger from January 1, 1994, through December 31, 1997, obtained from both inpatients and patients initially evaluated in the ED. From this case series we determined the frequency of bacterial pathogens responsible for such infections in this age group. Pathogens were defined as group B streptococcus, various enteric gram-negative rods (GNRs), Listeria monocytogenes, enterococcus, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type B, and Staphylococcus aureus. A subgroup analysis was performed to determine resistance patterns among the GNRs isolated from patients evaluated in the ED. A total of 367 pathogens were isolated: 187 (51.0%) in the neonatal intensive care unit, 153 (41.7%) in the ED, 20 (5.4%) in the inpatient wards, and 7 (1.9%) in the pediatric intensive care unit. Of the 121 pathogens isolated from 120 ED patients that were eligible for review, 94 (77.7%) were in the urine only, 16 (13.2%) in blood only, 4 (3.3%) in cerebrospinal fluid only, 3 (2.5%) in blood and cerebrospinal fluid, and 4 (3.3%) in blood and urine. Organisms isolated included GNRs (n = 96, 79.3%), group B streptococcus (n = 14, 11.6%), enterococcus (n = 7, 5.8%), S. pneumoniae (n = 3, 2.5%), and N. meningitidis (n = 1, 0.8%). No Listeria were isolated. Of the 96 GNRs isolated, 60 (62.5%; 95% confidence interval, 52.8%-72.1%) were ampicillin resistant. All were sensitive to gentamicin sulfate and cefotaxime sodium. Our results reveal 2 important facts: (1) during a 4-year period, no isolates of Listeria were identified from any patients 60 days or younger; and (2) of the 96 GNRs isolated from patients in the ED, more than 60% were ampicillin resistant. These data suggest that in similar centers with a low incidence of infection with Listeria and high levels of ampicillin resistance among GNRs, empiric use of ampicillin as part of a combination for presumed bacterial infections in patients 60 days or younger initially evaluated in the ED may be neither necessary nor beneficial. Consideration should be given to empiric initial antibiotic therapy using a third-generation cephalosporin with or without gentamicin.