In vitro studies of ciprofloxacin and survey of resistance patterns in current isolates.

We studied the activity of ciprofloxacin and other antibiotics against both routine and multiresistant (multi-R) clinical isolates. Ciprofloxacin inhibited more than 98% of most species of Enterobacteriaceae at a concentration of 2 micrograms/ml. Only Acinetobacter calcoaceticus, and to a much lesser degree, Providencia and Serratia, were resistant. Most Pseudomonas aeruginosa isolates were susceptible. Only 1% of staphylococci were resistant; the test panel included 1200 MRSA. For most species of streptococci, the MIC90 was 1 microgram/ml; for enterococci, it was 2 micrograms/ml. We also surveyed resistance in our current isolates. Resistance to ciprofloxacin has increased in A. calcoaceticus and Providencia, and in Streptococcus pneumoniae and group B streptococci. Ciprofloxacin-resistant isolates tended to show increased resistance to other antibiotics, including aminoglycosides and, later, cephalosporins.

Antibiotic resistance in Australia with special reference to sulbactam/ampicillin.

A survey of antibiotic resistance in Australian states was undertaken by the Microbiology Quality Assurance Program of the Royal College of Pathologists of Australasia. Data were obtained from hospitals and private pathology laboratories serving both in-patients and out-patients at community hospitals. The study showed that resistance varied from state to state; it was highest in the Eastern states of New South Wales, Victoria, and Queensland, and lowest in Tasmania and Western Australia. In South Australia, isolates of Escherichia coli demonstrated a high degree of cefoxitin resistance. Western Australia and Tasmania showed high levels of gentamicin resistance for Klebsiella spp., as well as trimethoprim resistance in Proteus mirabilis. The relationship between erythromycin resistance and clindamycin resistance also differed among various states. These studies demonstrated the activity of sulbactam/ampicillin against a wide variety of common pathogenic bacteria in which resistance was mediated by beta-lactamase.

In vitro activity of A-56619 and A-56620 against multi-resistant and routine clinical isolates.

A-56619 and A-56620 are two new quinolone compounds that are currently being studied. They were found to be active against multi-resistant and routine isolates of Staphylococcus aureus, enterobacteria, aminoglycoside-sensitive and resistant strains of Pseudomonas aeruginosa. Most of the enterobacteria were inhibited by 0.5-1 mg/l of A-56620. A-56619 was less active, concentrations of 1-4 mg/l being needed for 90% inhibition. Both the compounds were active at concentrations of 0.5-1 mg/l against staphylococci, including multi-resistant S. aureus. The MIC90 for P. aeruginosa was 1-2 mg/l for A-56620 and 8 mg/l for A-56619.

The role of chlamydial antibodies in an in vitro fertilization program.

The reported incidence of antibodies to Chlamydia trachomatis in patients attending infertility clinics is at least 30%. It has been reported that chlamydial antibodies are associated with decreased pregnancy rates following in vitro fertilization (IVF). A study was performed to investigate the significance of chlamydial antibodies in an established IVF program. The results did not show a decreased pregnancy rate in the presence of chlamydial antibodies. Of the women achieving pregnancy, 41% were seropositive compared with 38% seropositivity in women who did not become pregnant. There was no apparent benefit of the use of prophylactic antibiotics. The results also suggested that past infection with C. trachomatis in men did not adversely affect semen analysis or fertilization.

Blood-culture techniques. A survey in Australasian laboratories.

The blood-culture methods of participants in the Microbiology Quality Assurance Programme were surveyed in late 1983; 183 participants from Australasia and S.E. Asia completed a questionnaire, the results of which are discussed. The choice of skin disinfectants varied widely. Conventional broth media were used by 85 participants; one or more diphasic bottles by 56; Roche Septi-Chek was used by 25; and BACTEC by 17. Only 80% of respondents reported the use of sodium polyanethol sulfonate. Contamination rates ranged from 0 to greater than 10%. Cultures were kept for as little as five days or as long as three weeks and were examined by inspection and/or Gram stain and/or subculture at widely varying intervals. While a great diversity of methods was used, in most cases these were adequate. The workload involved in some of the more laborious routines was considerable. It would be desirable to evaluate the cost-effectiveness of many blood culture practices.

Nucleotide sequence of the AAD(2'') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfrII in R388.

The nucleotide sequence of the aadB gene which confers resistance to kanamycin, gentamicin, and tobramycin has been determined. The size of the longest reading frame is 747 bases encoding a protein of predicted size 27,992 daltons. A segment of the aadB gene sequence (including the promoter region) was found upstream of the aadA gene in R538-1 and of the dhfrII gene in R388 and the proposed promoters for these genes coincide with the aadB promoter region. The sequence homology extends upstream to the end of the sequenced regions of R388 and R538-1. Almost perfect homology was also found between the sequences 3'- to the aadB gene and 3'- to the aadA genes of R538-1 and pSa. This segment includes a 59 base element previously found flanking the Tn7 aadA gene. A model is presented for the evolution of this region of the plasmid genomes in which the 59- base element functions as an insertional "hot spot" and the possibility that this region is analogous to the aadA/aadB region of the Tn21- like transposon family is considered.

In vitro antibacterial activity of enoxacin (CI-919).

Antibacterial activity of enoxacin was evaluated against more than 3,700 clinical isolates using the agar-dilution method and an inoculum of 10(4)-10(5) cells per site. For comparison other antibiotics appropriate for each species were also included. For most enterobacteria and for Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the MIC90 of enoxacin was below 2 mg/l. Serratia marcescens was more resistant; the MIC90 being 4 mg/ml. Enoxacin also showed high activity against Campylobacter jejuni and Neisseria gonorrhoeae. Streptococci were comparatively resistant, 32 mg/l to 64 mg/l of the compound being required to inhibit 90% of strains.

Computerized analysis of microbiological data generated by replicator methods: an optical mark/sense technique for rapid data entry.

A rapid method of entering microbiological data into a computer is described. Data generated by replicator methods are recorded on optical mark/sense sheets, which are translated into conventional records by a document reader linked to a computer. The method has been used for processing the results of tests for antibiotic susceptibility and biochemical identification, the calculation of minimum inhibitory concentrations and the detection of transferable antibiotic resistance.

Construction of a gentamicin resistance gene probe for epidemiological studies.

A 7.7-kilobase BamHI fragment was cloned from the transconjugant of a clinical isolate of Escherichia coli containing a 120-kilobase multiresistance IncC plasmid. The recombinant plasmid conferred resistance to kanamycin, gentamicin, tobramycin, sulfamethoxazole, and trimethoprim. This clone was used to generate a series of subclones from which a 2.0-kilobase BamHI-HindIII probe containing a gentamicin 2''-O-adenylyltransferase [AAD(2'')] gene was obtained. This probe hybridized specifically in both colony and Southern hybridizations with the AAD(2'') gene but not with other resistance genes, including other aminoglycoside-modifying genes, or with a reference IncC plasmid lacking the AAD(2'') gene. The AAD(2'') gene may be part of a transposon, since hybridization occurred with both nonconjugative plasmids and the chromosome in some isolates.

A replicator method for the combined determination of minimum inhibitory concentration and minimum bactericidal concentration.

Minimal bactericidal concentrations (MBCs) of nine antimicrobial agents were determined for clinical isolates by a replica plating method. Membranes were placed on the antibiotic-containing plates and the organisms replicated onto the membranes. After 18 h incubation the minimal inhibitory concentrations (MICs) were determined, the membranes were transferred to antibiotic-free plates and incubated a further 18 h and the MBCs determined. MICs and MBCs were also determined in broth. The reproducibility of the 'membrane' method and the agreement of these results for MIC and MBC with the agar and/or broth methods was satisfactory for most antibiotics, within one two-fold dilution. With sulphamethoxazole, trimethoprim and co-trimoxazole the results were less satisfactory, especially with Gram-negative rods, but agreement within two dilutions could be achieved.

Comparative antibacterial activities of new beta-lactam antibiotics against Pseudomonas aeruginosa.

In vitro activity of nine new cephalosporins and penicillins was determined against 417 isolates of Pseudomonas aeruginosa. Carbenicillin, ticarcillin, gentamicin, tobramycin and netilmicin were also included in the study. Imipenem showed highest activity. More than 90% of the isolates were susceptible to ticarcillin, piperacillin, azlocillin, cefoperazone, cefsulodin and to tobramycin. 57 isolates included in the study were resistant to gentamicin (MIC greater than 4 mg/l); of these, none were resistant to imipenem, and more than 80% were susceptible to piperacillin, azlocillin, cefoperazone and cefsulodin.

Biochemical identification of bacteria by replicator methods on agar plates.

Biochemical tests commonly used for the identification of bacteria were adapted to a replicator technique using agar plates. As many as 48 organisms could be tested on one 10 cm square Petri dish. A new indicator, 2-(2,4-dinitrophenylazo)-l-naphthol-3,6-disulphonic acid disodium salt made it possible to investigate Gram-positive as well as Gram-negative organisms. For certain tests, such as citrate, DNAase, gelatin or lipase, a standard method using agar was already available. For others the usual test in liquid medium had to be adapted to solid medium. All such tests were compared with the API 20E system or with a conventional test and the results of the replicator tests showed a high correlation with those of other systems. For Gram-negative rods the corrected error rates (a measure of reproducibility) of 22 tests were 1% or less, for the remaining 9 tests the figure ranged between 1.5% and 4.8%. For Gram-positive cocci the corrected error rates of 21 tests were 1% or less and for the remaining 10 the figure ranged between 2% and 11%. The plates could be kept at 4-6 degrees C for 2 wk without appreciable loss of activity. The duration of incubation affected the results only marginally. This system has considerable advantages. Control organisms can be included in every run. As the cost in time and reagents for individual tests is much reduced, a larger number of tests can be carried out and this provides better identification. As Gram-positive organisms will grow on these media, it would be possible to speciate them in a more detailed fashion than is usually done now.

External quality assurance in microbiology. The programme of the Royal College of Pathologists of Australasia.

Since 1967 the Royal College of Pathologists of Australasia has been providing an external quality assurance programme in microbiology. The number of participants has risen from 70 to 222, and the samples distributed for investigation have increased in quantity, now totalling 35-40 per annum, and in variety, so that most aspects of clinical microbiology are now touched on. In recent years clerical accuracy and the adequacy of reports have been examined. However, more emphasis is needed on the stability of the specimens (freeze-drying will be introduced soon), the assessment of a laboratory's general performance, the standardization of methods and the educational aspects of external quality control.

An evaluation of recently developed antibiotics.

A number of newer antibiotics, broad-spectrum penicillins and cephalosporins, have been evaluated against Gram-negative rods. The organisms were selected for multi-resistance and transferable resistance factors. None of the broad-spectrum penicillins was much use against most of the organisms. Ceftriaxone, cefotaxime, latamoxef (moxalactam) and N-formimidoyl thienamycin were all highly effective against most multi-resistant Gram-negative bacilli; cefoperazone being inferior to them. Enterobacter and Serratia strains were relatively resistant to all the agents mentioned and against Acinetobacter only N-formimidoyl thienamycin showed much activity. Thus, use of these drugs may increase the proportion of infections due to organisms such as Serratia or Acinetobacter.

Transferable antibiotic resistance in a general hospital: a two year survey.

For two years transferable antibiotic resistance (TAR) was studied by replicator methods in strains of Enterobacteriaceae isolated in a 900-bed hospital. Transfer to an Escherichia coli recipient was demonstrated in 21% of 7,800 Enterobacteriaceae. It was most common in Klebsiella (37% of isolates) and least in Acinetobacter (6%). The mean number of phenotypic resistance markers (RMs) transferred was higher from Klebsiella pneumoniae or Enterobacter cloacae than from E. coli or P. mirabilis. K. pneumoniae and E. cloacae especially transferred 7 or more RMs much more often than other species. Some RMs were associated with a particular species, e.g. streptomycin with E. coli, kanamycin or gentamicin with E. cloacae and kanamycin with P. mirabilis. The same was true of certain patterns of resistance transfer, e.g. Ap-Km and P. mirabilis, Ap-Sm and E. coli, Ap-Km-Gm-Tm-Cm and K. pneumoniae. However, many of the commonest resistance phenotypes were transferred from several species and from several biotypes within any given species. Resistance patterns transferred from E. coli were detected sporadically throughout, whereas many of those transferred from Klebsiella, Enterobacter or Proteus were found only for a limited time.

Mechanism for nonspecific immunity of Listeria monocytogenes in rats mediated by platelets and the clotting system.

A proposed mechanism for nonspecific immunity to Listeria monocytogenes in rats based on the existence of an activatable lysin is described. Using a deoxyribonucleic acid release assay, we found lysin activity in serum made from whole blood but not in serum made from platelet-free plasma. Washed platelets and platelet lysates exhibited only partial activity as compared with that in serum. This activity was amplified by the addition of platelet-free plasma serum. The activity of the lysin was unaffected by heparin, dialysis, a serine esterase inhibitor, or heating to 56 degrees C for 30 min. Effective inhibitors were ethylenediaminetetraacetic acid and stronger heating (to 65 degrees C). Listeria organisms were found to reduce the recalcified clotting time to platelet-rich plasma in a dose-dependent fashion, indicating that the organisms can exhibit procoagulant activity. The susceptibility of rats to Listeria infection was enhanced by anticoagulant treatment. Rats were infected with Listeria organisms with and without administration of heparin. Heparin-treated rats developed bacteremia, and some died. None of the control rats developed bacteremia or died. These results suggest that natural immunity to Listeria infection is partly due to a platelet-dependent lysin which is activated during clotting and is, in turn, promoted by the Listeria organisms themselves.