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The objectives of this United States Consensus Panel meeting were to evaluate the effectiveness of current surveillance systems for the detection of bacterial resistance as well as to formulate recommendations that can assist hospitals in determining actions that should be taken when a resistance problem is detected. These recommendations may be particularly helpful in controlling the emergence and spread of type-I beta-lactamase resistance. Numerous case reports of antimicrobial resistance among Enterobacter species, Pseudomonas aeruginosa, and other Gram-negative nosocomial pathogens known to produce type-I beta-lactamases have appeared in the literature since the introduction of the newer "third-generation" cephalosporins. The widespread use of these newer antimicrobial agents, often selected as standard therapy for serious hospital-acquired infections, has been associated with a corresponding increase in resistance to them. The failure of hospitalwide surveillance methods to describe the scope of this problem, especially among the most critically ill patients, may have resulted in a false sense of security among some infectious disease specialists and clinicians prescribing these antimicrobials as empiric therapy. High-level resistance in individual hospital units may be masked in hospitalwide antibiograms. A variety of conclusions and recommendations were formulated based on the collective experiences of the Consensus Panel members. Microbiology laboratories must make it a high priority to identify markers that will assist in rapidly identifying resistant organisms. Cooperative efforts are needed among users of commercial and automated microbiology test instruments to standardize results and to improve quality control, thereby making the data more directly comparable between laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)

Nosocomial infections due to Acinetobacter baumannii dramatically increased in a Lebanese medical center following an outbreak of hostilities in Lebanon in 1984. The incidence of infection caused by this organism has remained high in this institution, thus requiring the implementation of a strain typing system to aid in infection control. Three methods were investigated for their utility in differentiating among a representative group of 36 nosocomial Acinetobacter baumannii isolates obtained over a 10 month period from specimens of hospitalized patients. Isolates were typed by antibiogram analyses, plasmid fingerprinting, and total cell protein profiles. Only three distinct total cell protein profiles were detected, with one pattern accounting for 26 (72.2%) of the isolates. However, eight different plasmid profiles were observed, with 20 (55.5%) isolates having the same profile. Eleven distinct antibiograms were seen with the most prevalent pattern occurring in 21 isolates. Twenty of the 21 (95%) isolates with the common antibiogram also had the same plasmid profile and total protein profile (44.4% of total isolates). The combination of these three typing methods was useful in tracing the spread of these organisms in the medical center. The data obtained suggest the distribution of a common strain among at least six wards of this hospital.

Mutational changes occurring at amino acid codons 84 and 85 located in the gyrA gene of methicillin-resistant Staphylococcus aureus (MRSA) were studied using radiolabeled oligonucleotide probes to examine the incidence of these ciprofloxacin resistance determinants in 30 MRSA isolates from Indiana, Minnesota, and Tennessee. Four separate oligonucleotide probes, one each corresponding to the wild-type sequence, a mutation at codon 84 (nucleotide 251), a mutation at codon 85 (nucleotide 253), and mutations at both, were used to examine the total genomic DNA from each of the 30 isolates, which had been restricted, electrophoresed, and Southern blotted. The probes indicated that 15 of the 28 ciprofloxacin-resistant isolates gave results consistent with a single mutation at codon 84. Four of the 28 ciprofloxacin-resistant strains had results consistent with a mutation at codon 84 and possibly at codon 85. The two ciprofloxacin-sensitive isolates from Tennessee showed homology with the wild-type probe sequence. Five isolates (4, Minnesota; 1, Tennessee) had no homology with any probe. By oligonucleotide probes, ciprofloxacin-resistant MRSA from diverse geographic regions contained similar gyrA mutations at codons 84 or 85 in 19 of 28 ciprofloxacin-resistant isolates.

Nosocomial pathogens frequently are resistant to antimicrobial agents. Although methicillin-resistant strains of Staphylococcus aureus continue to be a major problem in many hospitals, several new types of resistance determinants have been noted among organisms causing hospital-acquired infections. The mechanisms include extended spectrum beta-lactamases in gram-negative bacilli; resistance to beta-lactams, glycopeptides, and high levels of aminoglycosides among enterococci; quinolone resistance in isolates of methicillin-resistant S. aureus; and the spread of multiple resistance genes simultaneously in gram-negative organisms via Tn21-related genetic elements. These novel mechanisms of resistance complicate the treatment of nosocomial infections by limiting the number of effective antimicrobial agents available to the clinician. It is important for infection control practitioners and microbiologists to work together to detect and control the spread of resistant pathogens in the hospital setting.

Nine female and 6 male adolescents (mean age 14.5 +/- 1.7 [SD] years) were evaluated for chronic fatigue associated with at least three additional symptoms present for 18.4 +/- 8.4 months. Eleven subjects experienced the onset of symptoms with an acute illness (seven Monospot-positive). Medical history, physical examination, and laboratory testing yielded little helpful information. Serologic testing for Coxsackie B viruses 1 through 6, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, and Toxoplasma gondii in subjects and healthy controls provided little evidence for an infectious cause of persistent fatigue. Children's Depression Inventory scores and psychiatric interviews with the Schedule for Affective Disorders and Schizophrenia-Children's Version (K-SADS) identified five subjects with major depression. On the K-SADS, the 10 fatigued subjects without major depression endorsed many secondary symptoms of depression but were less likely than depressed psychiatric clinic patients to endorse primary symptoms such as depressed mood, guilt, and suicidality. At telephone follow-up 13 to 32 months after intake, 4 subjects were completely well, 4 markedly improved, and 7 unimproved or worse. Further research is necessary to determine whether chronic fatigue in adolescents is prodromal depression, a discrete psychosomatic condition, or an infectious or immunologic disorder that mimics depression.

We evaluated the ability of the autoSCAN-W/A (MicroScan Division, Baxter Healthcare Corporation, West Sacramento, Calif.), in conjunction with the dried colorimetric Neg ID type 2 panel (DCP) and new rapid fluorometric Neg ID panel (RFP), to identify non-glucose-fermenting gram-negative bacilli by challenging the system with 310 previously identified reference strains. Of these 310 isolates, 286 organisms were in the DCP data base and 269 were in the RFP data base. Use of the DCP panels resulted in 118 (41.3%) correct and 64 (22.4%) incorrect first choice identifications at greater than or equal to 85% probability, 61 (21.3%) low-probability identifications, and 43 (15.0%) reports of unidentified organisms. The RFP system reported 135 (50.1%) correct and 25 (9.3%) incorrect identifications at greater than or equal to 85% probability and 109 (40.5%) low-probability identifications. Unidentified isolates (DCP system only) and isolates producing low-probability first choice identifications (both systems) required supplementary biochemical testing. Over half (37 of 64 [57.8%]) of the DCP misidentifications were due to four commonly isolated, saccharolytic organisms (Alcaligenes xylosoxidans subsp. xylosoxidans, Pseudomonas putida, Pseudomonas fluorescens, and Xanthomonas maltophilia), while 7 of 25 (28%) of misidentifications in the RFP system were due to P. fluorescens. Of note, the RFP system identified non-glucose-fermenting gram-negative bacilli within 2 h of panel inoculation, allowing additional conventional biochemical tests to be set up the same day on low-probability isolates, whereas only 13.5% of the DCPs could be read at 18 h, with the remainder requiring 42 h of incubation before reading. When organism identifications were recalculated with the updated RFP data base and revised software, only 8.1% of all 310 isolates were misidentified at greater than or equal to 85% probability while 77.1% of the isolates were now correctly reported at this same high probability.

We studied the ability of a new DNA probe-based assay system to correctly identify isolates of the thermophilic campylobacters Campylobacter jejuni, C. coli, and C. laridis grown in vitro. We examined 424 organisms, including 214 Campylobacter isolates and 210 other aerobic and anaerobic isolates. The probe assay, which uses a new homogeneous system in which all reactions take place within a single tube, demonstrated 100% accuracy, producing neither false-positive nor false-negative results. The assay does not, however, distinguish among C. jejuni, C. coli, and C. laridis.

Preparatory to development of in situ disinfection of implanted catheters, silicone rubber tubing colonized by incubation with Staphylococcus aureus, Staphylococcus epidermidis, or Klebsiella pneumoniae was used to test the efficacy of various chemicals in vitro. Protocols sterilizing all segments colonized for 24 h (n = 30) were immersion into 50% povidone iodine for 5 and 60 min, 100% povidone iodine for 5, 15, and 60 min, 1.2 x 10(3) ppm chlorine dioxide for 15 and 60 min, and 1.2 x 10(3) ppm chlorine dioxide buffered to pH 5.1 for 60 min. Immersion in up to 2% chlorhexidine, 7.4% formaldehyde, or 6% sodium hypochlorite for up to 60 min failed to sterilize all segments. None of 117 control segments were sterilized. Segments colonized for seven days were sterilized by immersion into 100% povidone iodine for 15 or 60 min. Use of 1.2 x 10(3) ppm chlorine dioxide for 60 min sterilized 97% of segments tested. Lower concentrations and shorter exposure times failed to sterilize all segments. Eighteen silicone rubber catheters, colonized on the outer surface, were all sterilized within 24 h by a chlorine dioxide solution placed in the lumen and diffusing through the wall to kill the bacteria.

A total of 688 isolates of Campylobacter jejuni and Campylobacter coli were screened for the presence of plasmid DNA by agarose gel electrophoresis and were tested for susceptibility to ampicillin, chloramphenicol, erythromycin, streptomycin, and tetracycline. Of the isolates examined, 32% were noted to harbor plasmid DNA, ranging in size from 2.0 to 162 kilobases. Only tetracycline resistance was noted to correlate with the presence of plasmids. Plasmids capable of transferring tetracycline resistance via conjugation ranged in size from 42 to 100 kilobases. The Bg/II and Bc/I restriction endonuclease profiles of 31 plasmids examined showed marked diversity in their banding patterns. Although a high degree of DNA-DNA homology was noted among the Campylobacter spp. plasmids, no homology was noted between these plasmids and tetracycline R factors commonly found in the family Enterobacteriaceae.

The disinfection of right atrial catheters in situ using chlorine dioxide was investigated. Catheters were implanted into rabbits (Oryctolagus cuniculi) and colonized by inoculation of Staphylococcus aureus, Sa-80, into the lumen. All of the catheters were colonized and the difference in numbers of bacteria recovered from animals destined for the control and disinfection groups was not significant. Animals were assigned randomly to the control or disinfection group. Treatment consisted of filling the catheter lumen of the disinfection group with chlorine dioxide and of the control group with sterile physiological saline daily for 15 minutes. In addition, both groups received systemic antimicrobial therapy. Cultures of blood withdrawn from the catheters and by venipuncture were negative for five of the nine control group animals after treatment for 5 days. Four control group catheters failed, after from 3 to 21 treatments, without ever achieving negative cultures. All nine animals in the disinfection group had negative cultures after treatment for 5 days. Subsequently, one animal from each group reverted to positive cultures. All nine control group catheters failed during the study, compared to only three disinfection group catheters (p less than 0.01). At necropsy, culture of cardiac blood, thrombi and catheter tubing sections demonstrated colonization of six in the control group and only one in the disinfection group (p less than 0.05). Rabbits tolerated the chlorine dioxide disinfection well and no adverse signs were noted.

Plasmid fingerprinting provides a rapid and dependable means of identifying bacterial isolates of the same strain. The stability, wide distribution, and diverse nature and size of extrachromosomal elements make it suitable for virtually all bacterial genera. There are many different procedures available for plasmid screening, and the one chosen depends primarily on the types of organisms to be analyzed. Some procedures are better suited to gram-positive organisms; others are better for visualizing the very large plasmids often seen in Pseudomonas and Rhizobium species. The key to the plasmid fingerprinting technique is agarose gel electrophoresis. In this step of the technique, it is important to differentiate open circular from closed circular forms of plasmids and to recognize the "smile effect." Plasmid fingerprinting can be utilized for epidemiologic studies of both nosocomial and community-acquired infections. The use of restriction endonuclease analysis can greatly enhance the ability of the investigator to differentiate strains that harbor only a single plasmid. Plasmid fingerprinting often provides the only differential characteristic for strains involved in epidemics.

Antimicrobial resistance determinants and plasmids present in 10 multiply antibiotic-resistant strains of Hemophilus influenzae isolated from patients in different geographic regions of Spain were characterized. Conjugative plasmids with molecular sizes of 38-50 MDa encoded resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfamethoxazole, and tetracycline. Trimethoprim resistance was not linked to the other antibiotic resistance determinants and trimethoprim-resistant transconjugants and transformants lacked detectable plasmid DNA, suggesting that this determinant is chromosomal. Restriction endonuclease analysis revealed similarities among the plasmids, but several restriction patterns could be distinguished. Three hybridization patterns were found with DNA probes coding for H. parainfluenzae beta-lactamase and chloramphenicol-acetyltransferase. Resistance to kanamycin was due to drug modification by aminoglycoside-phosphotransferase (3')I. In Spain, it appears that multiple antibiotic resistance phenotypes in H. influenzae did not arise from acquisition of a single R plasmid; rather, both plasmid and chromosomal resistance evolved independently from several sources.

Virtually all microorganisms contain some unique nucleotide sequences which can be the target of deoxyribonucleic acid probes. Probes have been used successfully to identify a wide variety of pathogens from the simple ribonucleic acid-containing polioviruses to the complex filarial worms Brugia malayi. Probe technology offers the clinical laboratory the potential both to extend the types of pathogens that can be readily identified and to reduce significantly the time associated with the identification of fastidious microorganisms. Over a dozen commercially prepared deoxyribonucleic acid probe tests are now available. This article explores the development of deoxyribonucleic acid probe tests and reviews the sensitivity, specificity, and predictive values of many of the diagnostic probes developed during the last several years. Prospects for newer, more sensitive detection systems for the products of hybridization reactions are also reviewed.

In order to assess the clinical value of microscopy for acid-fast bacilli (AFB), the results of 3,207 clinical specimens submitted for mycobacterial smear and culture were analyzed. Mycobacteria grew from 176 (5.5%) of the specimens, 95 (54%) of which were Mycobacterium tuberculosis. Although the overall sensitivity of the smear was low (33%), 65% of respiratory specimens yielding M. tuberculosis had positive AFB smears. Furthermore, 96% of patients with pulmonary tuberculosis from whom more than one specimen was processed had at least a single positive AFB smear. Smear sensitivity correlated well with quantitative growth; 89% of specimens yielding greater than or equal to 50 colonies per slant were smear positive. Specificity of the AFB smear was high; 89% of smear-positive specimens had positive cultures. After the results from culture-negative patients known to have active tuberculosis were eliminated from the analysis, the specificity of a positive smear rose to 98.3%. When the results of all specimens from each patient were considered in toto, the AFB smear had a predictive value of greater than or equal to 96%.

The aacA1 gene, which encodes a 6'-N-acetyltransferase [AAC(6')-I] mediating resistance to kanamycin, tobramycin, and amikacin, was cloned from the Citrobacter diversus R plasmid pBWH100 into the Escherichia coli vector pBR322. The complete nucleotide sequence of the gene and flanking regions was determined. A protein of approximately 21 kilodaltons was identified when the chimeric plasmid encoding the aacA1 gene was introduced into E. coli maxicells. This value is consistent with the size predicted for a protein translated from the open reading frame of the gene.

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.

During the period of August 16, 1981, through December 16, 1982, 15-mL blood culture specimens collected at the Seattle Veterans Administration Medical Center (SVAMC) were divided into two aliquots. The first 10-mL aliquot was inoculated directly into aerobic and anaerobic BACTEC (Johnston Laboratories, Towson, MD) vials (DIR); the remaining 5 mL was processed in an resin-containing Antimicrobial Removal Device (Marion Scientific, Kansas City, MO) before transfer to a second, identical set of aerobic and anaerobic vials (ARD). The final volume of inoculated blood from the ARD specimen was half that of the DIR specimen. Both sets of vials were processed using the BACTEC radiometric detection system. One hundred fifty specimen pairs grew 161 significant pathogens; 43 isolates were recovered only from the ARD sample, 39 only from the DIR samples, and 79 from both. Of the 35 isolates recovered from patients receiving anti-microbial agents active against the isolated pathogens, 21 were recovered only from the ARD specimen and 5 only from the DIR specimen (P less than 0.005). Of the 15 S. aureus strains isolated from patients on therapy, 12 were recovered only from the ARD specimen, 2 only from the DIR sample, and 1 from both samples (P less than 0.01). Ten of the 32 isolates of S. aureus recovered from antibiotic-free patients were found only in the ARD specimen and three only in the DIR specimen (P = 0.05). The ARD specimens recovered significantly more S. aureus from all patients regardless of antibiotic status.

Strains of Neisseria gonorrhoeae were tested for the presence of methyladenine in the DNA sequence GATC by using the site-specific restriction endonucleases MboI and DpnI. It was found that 43 of 83 strains tested contained methylated DNA. When methylation was compared with the auxotype of the organism, 35 of 35 strains with the AHU (arginine-, hypoxanthine-, and uracil-requiring) auxotype and 8 of 48 strains with other auxotypes contained methyladenine. When the incidence of methylation in strains isolated from patients suffering from disseminated gonococcal infection was compared with that in strains isolated from patients suffering from uncomplicated gonococcal infection, no correlation with methylation and disseminated gonococcal infection was observed.

We compared a fluorescein-labeled monoclonal antibody directed against an outer membrane protein of Legionella pneumophila (Genetic Systems Corp. [GSC], Seattle, Wash.) with a similarly labeled polyclonal reagent (L. pneumophila serogroups 1 to 6, poly; BioDx, Inc., Denville, N.J.) for the confirmation of L. pneumophila isolates grown in culture. Duplicate suspensions of 52 organisms, including 21 L. pneumophila and 8 non-L. pneumophila species of legionella, were placed on individual glass slides, fixed, and stained with both reagents, and the results were compared. Both antisera correctly identified all L. pneumophila serogroups 1 to 6, but only the GSC reagent produced definitive staining of the L. pneumophila isolates of serogroups 7, 8, and 9. Additionally, the GSC reagent produced more uniform staining patterns around the legionella bacilli and displayed little background fluorescence when compared with the BioDx reagent.

Fifteen isolates of tetracycline-resistant Campylobacter jejuni were recovered from stool samples of cynomologous monkeys (Macaca fascicularis) housed at the University of Washington Primate Research Center, Seattle. Resistance was associated with carriage of a 38-megadalton plasmid which was transmissible to other strains of C. jejuni but not to Escherichia coli. Seven isolates also contained a 2.6-megadalton plasmid which was phenotypically cryptic.

Commercially prepared polyclonal antisera to Legionella pneumophila are known to cross-react with organisms of the genus Pseudomonas. To determine whether a commercially available monoclonal antibody reagent specific for L. pneumophila would also cross-react with pseudomonads, a two-laboratory study was undertaken to test both monoclonal and polyclonal reagents against 33 isolates of Pseudomonas spp., including 25 Pseudomonas aeruginosa, 4 P. putida, 2 P. maltophilia, 1 P. fluorescens, and 1 P. alcaligenes. Four antisera were tested; polyclonal anti-legionella antisera pools A and B (Centers for Disease Control [CDC], Atlanta, (Ga.), polyclonal 1-6 antisera (BioDx, Inc., Denville, N.J.), and a monoclonal antibody reagent produced by Genetic Systems Corp., Seattle, Wash. All reagents were labeled with fluorescein. Cross-staining reactions were found with the BioDx L. pneumophila antisera and 10 isolates of Pseudomonas. Four of these isolates demonstrated cross-staining with CDC pool A. When tested with individual serotype-specific reagents (CDC), three of four cross-reacted with L. pneumophila serotype 1 antisera; the fourth cross-reacted with serotype 3. No cross-staining reactions were noted with the monoclonal reagent and any of the pseudomonads tested, demonstrating that the Genetic Systems Corp. monoclonal reagent is the most specific of the four reagents tested.

A total of 593 strains of thermophilic Campylobacter species were isolated either from humans with diarrhea or from poultry in King County, Washington. Of these strains, 98 (52 hippurate-positive strains and all 46 of the hippurate-negative strains) were selected for further phenotypic characterization and genetic classification. Hippurate hydrolysis, the test typically used to differentiate Campylobacter jejuni and C. coli, did not always correlate with the genetic classification. All hippurate-positive strains were classified as C. jejuni. Of the hippurate-negative strains, 20% were C. jejuni, 78% were C. coli, and 2% were C. laridis. Assuming that the remaining hippurate-positive strains were all C. jejuni, then hippurate-negative C. jejuni represented a small percentage (9 of 556 or 1.6%) of C. jejuni strains but a significant percentage (9 of 46 or 20%) of hippurate-negative strains. This finding suggests that hippurate hydrolysis should not be used as the sole criterion for differentiating thermophilic Campylobacter species, particularly when describing the disease states associated with these organisms.

In a recent study in Seattle, we isolated Campylobacter-like organisms (CLOs) significantly more often from homosexual men with intestinal symptoms than from asymptomatic heterosexual and homosexual control subjects. Biochemical tests, morphology, and guanine-plus-cytosine content of whole-cell DNA showed that these organisms were similar to, but distinguishable from, other species in the genus Campylobacter. DNA homology tests showed that the Campylobacter-like organisms belong to four unique genetic groups, two of which were phenotypically identical. We developed a rapid screening test to genetically group these organisms and to assess the correlation of each genotype with enteric disease in homosexual men. Three of the four CLO groups were isolated only from men with proctitis, proctocolitis, and/or enteritis. The remaining group, isolated from both symptomatic and asymptomatic homosexual men, was significantly correlated with the presence of polymorphonuclear leukocytes in the rectal secretions of asymptomatic men, a finding that suggests subclinical disease. We have tentatively classified two of the CLO groups into species, Campylobacter cinaedi sp. nov. and Campylobacter fennelliae sp. nov.

The tetracycline resistance gene (tet) from the Campylobacter jejuni plasmid pFKT1025 was cloned into both pUC18 and pBR322 and was expressed when the chimeric plasmids were introduced into Escherichia coli. The location of the tet determinant on the chimeric plasmids was determined by BAL 31 deletion mapping within a 2.25-kilobase (kb) RsaI-HincII fragment. A protein of approximately 70 kilodaltons was consistently produced by E. coli maxicells harboring the cloned tet determinant. A 500-base-pair restriction fragment from within the 2.25-kb tet region was shown to hybridize only to DNA from tetracycline-resistant strains of C. jejuni and C. coli, but not to the DNA of organisms known to carry the streptococcal tetM determinant. No homology was noted between the DNA of 10 tetracycline-resistant isolates of campylobacter and the streptococcal tetL, tetM, or tetN determinants when tested under conditions of high stringency. However, homology was noted between a 5.0-kb HincII restriction fragment containing the tetM determinant and two C. jejuni tet R factors under conditions of reduced stringency.

We compared the accuracy of the Gram-stained smear, the acridine orange-stained smear, and the AutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) in screening for bacteriuria, as detected by conventional cultures. For 1,024 clinical specimens, results with the acridine orange-stained smear and the Gram-stained smear were very similar. When read for the presence of one or more microorganisms or leukocytes per 20 oil immersion fields, both smears were highly sensitive (92.1 and 93.3%, respectively) and moderately specific (70.0 and 61.7%, respectively). Sensitivity was greater for specimens yielding greater than or equal to 10(5) CFU/ml (96.1 and 98.9%, respectively) than for those with 10(3) to 10(4) CFU/ml (81.4 and 78.0%, respectively). Preliminary classification based upon the tinctorial and morphological characteristics of the Gram-stained smear was compatible with culture results in nearly all cases. The accuracy of the Gram-stained smears was not influenced by special cleaning of the microscopic slides, or the level of expertise of the microscopist. For 715 specimens, the sensitivity of the AMS in detecting bacteriuria (91.5%) was very similar to that of the stained smears (92.1 and 95.7%, respectively), but the specificity was significantly higher (83.2% versus 42.6 and 70.0%). Detection of microorganisms by the AMS took an average of 6.3 +/- 3.0 h. These data suggest that the Gram-stained smear is easily interpreted, very sensitive, acceptably specific, and still the optimal rapid method for screening for bacteriuria in most clinical microbiology laboratories.

To determine whether extraintestinal isolates of Campylobacter jejuni and Campylobacter coli are the consequence of unusual host or bacterial characteristics, we studied clinical and bacteriologic features of 24 extraintestinal infections. Common serotypes and auxotypes were present among the extraintestinal isolates. Gastrointestinal isolates were more susceptible to normal human serum than were the systemic isolates; however, the ranges overlapped considerably. Predispositions to systemic spread were present in 52% of patients with extraintestinal infections; isolates from these patients were more often (73%) serum sensitive than were isolates from patients without predispositions (9%; P = .002). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no specific protein band was associated with serum resistance, and all isolates of C. jejuni and C. coli had rough-type lipopolysaccharide profiles. Serum susceptibility was inversely correlated with carbohydrate or ketodeoxyoctonate (KDO) fraction of cell weight and directly correlated with KDO:carbohydrate ratio. Our results suggest that either host defects or specific bacterial virulence characteristics, such as serum resistance, possibly related to length of lipopolysaccharide side chain, may be responsible for extraintestinal infections due to C. jejuni and C. coli.

Plasmid profiles, phage typing, antibiograms, and biotyping were used to characterize Staphylococcus epidermidis isolated from multiple cultures of blood of four patients with prosthetic valve endocarditis. Epidemiological evidence implicated a common source for these infections. Of 20 clinically significant isolates, 14 exhibited variations from the prototype pattern of multiple resistance to five antibiotics. All isolates tested appeared to be the same strain by phage typing. Of 18 isolates available for plasmid analysis, 10 contained six plasmids of identical size, whereas eight differed from the prototype profile in the loss of one to three plasmids. Loss of resistance to gentamicin, chloramphenicol, erythromycin, and clindamycin but not to methicillin was associated with the loss of specific plasmids. Because antibiotic resistance in this strain of S. epidermidis was unstable, the use of antibiograms alone was not a reliable means of evaluating the relatedness of these multiple isolates.

A 42-kilobase hybrid Pcr plasmid (pFA14) was formed when the naturally occurring 7.2-kilobase Pcr plasmid pFA3 was introduced by transformation into a competent gonococcal recipient containing the 36-kilobase conjugative plasmid pFA2 (Sox et al., J. Bacteriol. 138:510-518). Analysis of the structure of pFA14 showed that it was a stable recombinant between pFA3 and pFA2. The transformation efficiency of pFA14 was increased 300- to 10,000-fold by the presence in isogenic recipients of the homologous plasmid pFA2. The presence of a homologous plasmid in the recipient also markedly increased the likelihood of recovery of intact donor-size Pcr plasmids in the transformants. The presence of pFA2 had no effect on the competence of piliated or nonpiliated gonococci for transformation by either linear chromosomal DNA or a nonhomologous Pcr plasmid. Increased transformation efficiency of the hybrid Pcr plasmid pFA14 may have been due to recombination between the nicked or linearized donor plasmid and the homologous recipient plasmid (marker rescue).

During a 3-month period in the late summer and fall of 1981, six cases of gastroenteritis and one wound infection due to Vibrio parahaemolyticus were reported to public health agencies in Washington and Oregon. An investigation revealed that all of the gastroenteric illnesses were associated with eating raw oysters; that oysters eaten by five of the six patients were harvested at four divergent sites at Willapa Bay, Washington, a large commercial growing area; and that the V. parahaemolyticus isolates from those five patients were all Kanagawa positive, belonged to serotype 04:K12, and exhibited an atypical biochemical reaction, urea hydrolysis. No further cases linked to Willapa Bay oysters have been reported, and the infecting strain could not be found in sediment samples from the bay in February 1982. Thus, even though the origin of this self-limiting outbreak is obscure, the investigation demonstrated that the geographic distribution of V. parahaemolyticus infection in the United States includes the Pacific seacoast . Furthermore, oysters must be considered, along with crabs, shrimp, and lobster, as a vehicle of transmission of this infection in the United States.

Two DNA probes were developed to screen for the genes encoding 3'-aminoglycoside phosphotransferase activity in gram-negative bacilli. The 3'-I phosphotransferase [APH(3')I] probe was subcloned from Tn903; the APH(3')II probe was subcloned from Tn5. Each probe proved to be specific for genes corresponding to its own APH(3') subclass and did not hybridize with DNA from other classes when tested at high stringency by either Southern hybridization or dot-blot hybridization methods. The APH(3')I probe hybridized to DNA obtained from organisms demonstrating APH(3')I activity as measured by the phosphocellulose paper binding assay (PPBA) as well as to DNA from organisms reported to have both APH(3')I and APH(3')II activity by PPBA. This probe did not hybridize to DNA from organisms showing only APH(3')II activity by PPBA. The APH(3')II probe demonstrated homology with DNA from organisms showing APH(3')II activity by PPBA but not with DNA from organisms showing APH(3')I activity or both APH(3')I and APH(3')II activity by PPBA. We conclude that organisms previously believed to contain both APH(3')I and APH(3')II genes based on PPBA contain in fact only the APH(3')I gene.

The nutritional requirements for 439 Campylobacter jejuni isolates and 46 Campylobacter coli isolates were determined by using a previously described chemically defined medium, campylobacter defined medium. With this medium, 45% of both human and nonhuman C. jejuni isolates demonstrated auxotrophic requirements. None of the 46 C. coli isolates studied demonstrated requirements for amino acids on campylobacter defined medium. The most common auxotrophic requirement among C. jejuni isolates was for methionine, which was present as a single requirement or in combination with other markers in 21% of human and 28% of nonhuman isolates. There was no correlation between plasmid carriage and auxotype, and a comparison of the Lior serotypes of 472 of the strains showed a correlation only between proline auxotrophs and Lior serotype 11 for strains isolated in the Seattle-King County region.

Streptococcus mutans DL5, isolated from the dental plaque of a pig, was resistant to high levels of streptomycin (Sm, 20 mg/ml), erythromycin (Em, 1 mg/ml), and tetracycline (Tc, greater than 100 micrograms/ml), but contained no detectable plasmid DNA. The Smr and Emr determinants were cloned from cellular DNA on the self-replicating 5-kilobase-pair (kbp) EcoRI fragment of pAM beta 1 and the 4.2-kbp cryptic plasmid pVA380-1, respectively, by transformation of Streptococcus sanguis Challis. Helper plasmid cloning, with a Challis host containing pVA380-1, was required to clone the Tcr determinant of strain DL5 on this vector. A single-colony isolate of the original Tcr clone contained a hybrid plasmid, pDL421, composed of 2.6 kbp of vector DNA and 11.4 kbp of S. mutans DNA. Plasmid pDL421 did not hybridize to plasmids containing the streptococcal Tcr determinants tetL, tetM, and tetN. A shortened derivative of this hybrid plasmid, pDL422, missing a 4.9-kbp HincII fragment from the S. mutans DNA but still encoding Tcr, was obtained by subcloning in S. sanguis Challis. The Tcr gene was located in a 1,917-base-pair open reading frame (ORF) corresponding to a 72-kilodalton protein. The ORF exhibited 99.4% sequence identity with the 1,917-base-pair tetO gene from a strain of Campylobacter coli (W. Sougakoff, B. Papadopoulou, P. Nordmann, and P. Courvalin, FEMS Microbiol. Lett. 44:153-160, 1987). A 1.67-kbp NdeI fragment, internal to the ORF from strain DL5, as well as pDL421 hybridized under stringent conditions to DNA from 10 of 10 Tcr strains of C. coli and Campylobacter jejuni from human and animal sources, but not to DNA from Tcs isolates of these two species.

Analysis of aminoglycoside-resistant Enterobacteriaceae isolated from patients at the Seattle Veterans Administration Medical Center indicated that a single 68-kilobase R factor was responsible for the epidemic spread of low-level resistance to gentamicin, kanamycin, and tobramycin. An examination, by means of the phosphocellulose paper binding assay, of resistant strains carrying this R factor resulted in the identification of a 2"-O-adenyltransferase [ANT(2")]-modifying enzyme. This enzyme was later detected in strains containing 150-kilobase plasmids. For more convenient monitoring of the dissemination of the ANT(2") gene among clinical isolates at the medical center, a DNA probe was developed by cloning of the ANT(2") structural gene from the 68-kilobase factor into pBR322. A 310-base pair Ava I restriction fragment isolated from the interior of the cloned ANT(2") gene was radiolabeled and used in Southern hybridization gels as a probe for plasmids isolated from aminoglycoside-resistant organisms. The probe proved to be highly specific and was more sensitive than enzymologic techniques for detection of the ANT(2") gene in clinical isolates with complex aminoglycoside resistance phenotypes.

A chimeric beta-lactamase encoding plasmid, containing the 4.4 Mdal beta-lactamase plasmid and the 2.6 Mdal cryptic plasmid of Neisseria gonorrhoeae has been characterized by physical and biological methods. Digestion with restriction enzymes indicates the presence of the following restriction sites: 1 site: AccI, AvaI, HgiAI, HincII, MstI, PstI, PvuII, XbaI and XorI; 2 sites: HindIII and BamHI; 3 sites: BclI, Sau96I and AvaII; 6 sites: HinfI; greater than 8 sites: AluI, BbvI, DdeI, HgaI, HhaI, HpaII, MspI, Sau3A, TacI and TaqI. No restriction sites were found for the following: BglI, BglII, BstEII, EcoRI, EcoRII, HpaI, HphII, KpnI, SacI, SalI, SmaI, SstI, SstII, and XhoI. Five plasmid specific proteins have been identified by DNA directed in vitro protein synthesis (43K, 41K, 30K, 16K and 14K). The location on the physical map of the coding regions for each of these proteins has been determined by the following methods: using plasmid DNA restricted by various enzymes in an in vitro protein synthesis system; identifying promoter-containing regions by digesting plasmid DNA with DdeI, adding RNA polymerase and then determining which fragments are retained by nitrocellulose. This plasmid contains both parental phenotypes in that it encodes penicillin resistance and possesses the sequence necessary for uptake in the gonococcus. Transformation data indicate that this plasmid can function in both E. coli and N. gonorrhoeae and that growth in E. coli has no effect on the plasmid's ability to transform the gonococcus.

As DNA probes are used more frequently in the clinical laboratory for the detection and identification of pathogens in clinical samples, a means of determining the antimicrobial susceptibility profile of those pathogens will be required. DNA probes directed to specific resistance determinants offer a solution to this problem. Methods of determining the susceptibility of viruses to antiviral agents can also be accomplished with DNA probes. This article explores the advantages and disadvantages of using hybridization methods for susceptibility testing of organisms contained in clinical samples.

A total of 225 isolates of Campylobacter jejuni and 54 isolates of Campylobacter coli were screened for resistance to kanamycin. Among these, five resistant isolates of C. jejuni and six resistant isolates of C. coli, all with different plasmid patterns, were identified. Each contained at least one plasmid greater than or equal to 41 kilobases in size. The MIC of kanamycin for all 11 strains was determined to be greater than or equal to 256 micrograms/ml by an agar dilution method. In addition, all of the strains exhibited resistance to tetracycline (greater than or equal to 16 micrograms/ml). Eight of the 11 strains transferred the kanamycin resistance phenotype to other Campylobacter strains by conjugation. DNA from 9 of the 11 strains hybridized to a DNA probe specific for the 3'-O-aminoglycoside phosphotransferase type III gene. The remaining two strains also failed to show homology with DNA probes specific for the genes encoding 3'-O-aminoglycoside phosphotransferase types I, II, and III. The novel kanamycin resistance gene was cloned into the vector pBR322 and was expressed in Escherichia coli. Phosphocellulose paper binding assays on sonicates of the E. coli strain carrying the cloned kanamycin determinant demonstrated significant activity against kanamycin, neomycin, and amikacin but not against butirosin, gentamicin, tobramycin, or lividomycin, suggesting that the enzyme is the product of a 3'-O-aminoglycoside phosphotransferase type of aminoglycoside resistance gene.

During a 6-month study we critically evaluated the accuracy of the AutoMicrobic system Gram-Negative Identification Card (Vitek Systems, Inc., Hazelwood, Mo.) in identifying glucose-nonfermenting gram-negative bacilli by testing 419 selected isolates in parallel with a conventional reference method. Of 356 isolates included in the AutoMicrobic system profile, a total of 307 (86.2%) were correctly identified, 36 (10.1%) were not identified, and 13 (3.7%) were misidentified. Fifty-eight of 63 (92%) isolates not included in the profile were correctly reported as "unidentified organisms." Overall, if the first-choice identification was always accepted, only 18 (4.3%) isolates would have been incorrectly reported. When first-choice identifications appended with the special message "questionable biopattern" were rejected, and organisms were screened for characteristic odor and antimicrobial susceptibility before final acceptance of the AutoMicrobic system report, the number of misidentifications was reduced to 5 (1.2%). The average time to identification with the AutoMicrobic system Gram-Negative Identification Card was 15 h. This compares favorably with the 65 h required by the reference method.

The ability of the Extractor system (Molecular Biosystems, Inc., San Diego, Calif.) to isolate nucleic acid (NA) from stool samples for use in hybridization assays was investigated. Crude NA was recovered from 45 of 50 stool samples by using this system. The amount of NA recovered varied considerably depending on the microbial flora present in the sample (mean +/- standard deviation, 50.2 +/- 46.7 micrograms; range, 2 to 228 micrograms) but did not correlate with the consistency of the sample. Samples containing primarily gram-positive organisms or yeast cells gave lower yields of NA (less than 10 micrograms) than those containing gram-negative bacilli. The five samples which did not yield NA were sterile when cultured aerobically on blood agar plates. Samples of the 45 stools yielding NA were inoculated into broth and grown overnight, and a 10-microliters sample of broth was spotted onto nitrocellulose filters. The NA samples recovered from the Extractor column were applied to nylon membranes by using the Centri-dot system. The NA on the broth blots and the NA on the Centri-dot filters were hybridized with a 310-base-pair probe specific for the 2"-O-aminoglycoside adenylyltransferase [ANT(2")] resistance gene. The Extractor-Centri-dot system demonstrated 61.9% sensitivity and 95.8% specificity in detecting the ANT(2") gene in stool samples containing colonies demonstrating the ANT(2") phenotype. The positive and negative predictive values of the NA blot were 92.8 and 74.2%, respectively.

The 24.5-megadalton plasmid of Neisseria gonorrhoeae is required for transfer of R-factors and possibly chromosomal markers during conjugal matings between gonococcal strains. We constructed a physical map of one such plasmid, pLE2451, using EcoRI, BglII, and HincII site-specific restriction endonucleases. The patterns of deoxyribonucleic acid digestion obtained with this plasmid were identical to those obtained with three other plasmids of similar size.

Two rapid DNA hybridization methods in which whole-cell lysates fixed to nitrocellulose were used were compared with Southern hybridization of purified plasmid or chromosomal DNA for the ability to identify the 2"-O-adenylyltransferase [ANT(2")] gene in 42 enzymatically defined isolates of gram-negative bacilli. A DNA restriction fragment isolated from an ANT(2") gene cloned into pBR322 and radiolabeled with 32P was used as the probe in all three procedures. Under conditions of high stringency, agreement was obtained between the Southern hybridization method and detection of the ANT(2") enzyme by the phosphocellulose paper binding assay or resistance phenotype in 39 of the 42 strains tested. By using these characterized strains, colony hybridization was shown to be unsatisfactory as a rapid technique for detecting the ANT(2") gene, due to the high number of false-positive and -negative signals obtained. Compared with Southern hybridization, however, spot hybridization (SPH) proved highly reliable for detecting the ANT(2") gene in both members of Enterobacteriaceae and Pseudomonas aeruginosa harboring R factors ranging in size from 23 to 150 kilobases. The relatively low copy number of the 150-kilobase plasmids decreased the sensitivity of SPH, necessitating a minimum cell density of 5 X 10(6) cells per spot. SPH proved to be a very useful method for rapidly screening large numbers of clinical isolates for this resistance determinant.

During a 24-month period, 5,625 blood culture specimens were collected at the Seattle Veterans Administration Medical Center in 20-ml volumes and divided into separate 10-ml aliquots. The two aliquots were processed as duplicate sets (set 1, set 2) by the BACTEC system (Johnston Laboratories, Inc., Towson, Md.). Specimens (5 ml) from each set were inoculated into aerobic (6B) and anaerobic (7C/7D) vials. A total of 434 significantly positive blood cultures were found. In 342 of these positive cultures, yielding 379 isolates (112 members of the family Enterobacteriaceae, 104 staphylococci, 87 streptococci, 27 anaerobes, 20 yeasts, 14 pseudomonads, and 15 miscellaneous organisms), there was adequate specimen volume to fill all four vials. The utilization of set 1 would have resulted only in the failure to detect 65 of 379 (17.2%) significant isolates, 52 of 342 (15.2%) positive cultures, and 20 of 198 (10.1%) bacteremic episodes. There were no significant differences in the recovery of individual species in sets 1 and 2. Although the range of isolates recovered by the aerobic and anaerobic vials of each set differed, the percent yield of total isolates was similar, indicating total isolate yield was predominantly a function of specimen volume. The addition of set 2 most dramatically increased the recovery of Escherichia coli (30%), yeasts (33%), and anaerobes (42%).

The aacC1 gene encoding the 3-N-aminoglycoside acetyltransferase [AAC(3)-I] was cloned from enteric plasmid pJR88, and its deoxyribonucleotide sequence was determined. Significant nucleotide homology was noted in the region extending from the proposed -35 sequences through the first 59 base pairs of the aacC1 gene open reading frame (ORF) and the upstream flanking regions and ORFs of several other antibiotic resistance genes. Sequences were noted to be homologous with the 6'-N-aminoglycoside acetyltransferase [AAC(6')-I], 2''-O-aminoglycoside adenylyltransferase [AAD(2'')], and 3''-O-aminoglycoside adenylyltransferase [AAD(3'')] resistance genes; the OXA-1, OXA-2, and PSE-2 beta-lactamase genes; and several dihydrofolate reductase genes. Small regions of homology were noted in the 3'-flanking regions of these resistance genes as well. A DNA probe for the aacC1 gene was selected from the nucleotide sequence information and was tested against a series of genetically and enzymatically defined strains. The probe, which proved specific for the aacC1 gene, was then tested against a series of 58 gentamicin-susceptible and 219 gentamicin-resistant gram-negative bacilli isolated from patients at the Seattle Veterans Administration Medical Center. Only six clinical isolates were noted to carry the aacC1 gene. Each was resistant to gentamicin but susceptible to kanamycin, tobramycin, and amikacin. The presence of homologous regions of DNA at both the 3' and 5' ends of the aacC1 gene reinforces the importance of choosing probes from within the ORFs of genes and of avoiding flanking sequences. When the homology with other sequences extends into the ORF, as it does with the aacC1 gene, development of a specific probe may require determination of the nucleotide sequence.

A chemically defined medium developed for Neisseria gonorrhoeae was modified to support the growth of Campylobacter jejuni and Campylobacter coli. A total of 76 isolates of C. jejuni and 14 isolates of C. coli were tested on this medium, which was designated Campylobacter defined medium (CDM), over a 3-month period. Although none of the C. coli isolates appeared to require amino acids, 51% of the C. jejuni tested required one and 7% required multiple amino acids for growth. An analysis of isolates obtained from three household outbreaks of campylobacteriosis demonstrated that auxotyping identified the epidemic strain within each outbreak. Among 70 isolates of C. jejuni examined, no correlation could be drawn between a specific serotype and auxotype or between auxotype and plasmid profile.

A series of chimeric plasmids derived from the transfer factor, beta-lactamase-encoding R factor, and cryptic plasmid of Neisseria gonorrhoeae was constructed. Two of these plasmids, each lacking a 1.9-kilobase-pair (kb) HinfI fragment, could not be mobilized by the 40-kb gonococcal transfer factor to recipient strains of N. gonorrhoeae or Escherichia coli during conjugation. The proteins encoded by both the naturally occurring and chimeric plasmids were examined with an E. coli cell-free transcription-translation system and in E. coli maxicells. Six plasmid-specific proteins were identified when DNA from a naturally occurring 7.1-kb R factor was used as template. A small protein (16,000 daltons), which is apparently not encoded by the two plasmids lacking the 1.9-kb HinfI fragment, appears to be necessary for plasmid transfer during conjugation.