II. In vitro studies of antibacterial activity

One hundred and forty isolates of beta-hemolytic streptococcus cultured from patients with clinical pharyngitis were studied by disc diffusion for antibiotic sensitivity to lincomycin, erythromycin, cephalexin and penicillin and by agar dilution to cephalexin and penicillin. All isolates were sensitive to ≤ 0.1 μg./ml. penicillin and ≤ 1.56 μg./ml. cephalexin. The disc-diffusion test was reliable in predicting the sensitivities in vitro. One strain of group A betahemolytic streptococcus was resistant to erythromycin by disc diffusion. When compared to Lancefield grouping 18% of strains were incorrectly identified as group A by the bacitracin-disc test. Cephalexin was uniformly effective in vitro in inhibiting beta-hemolytic streptococci and the 30 μg. cephalexin disc was reliable in predicting these sensitivities.     

Susceptibility of Neisseria gonorrhoeae to seven antibiotics in vitro

One hundred consecutive isolates of N. gonorrhoeae were tested for susceptibility to penicillin, ampicillin, tetracycline, erythromycin, kanamycin, cephaloridine and cephalexin by an agar dilution method. Relative resistance to penicillin was frequent. For 39% of isolates the minimum inhibitory concentration (MIC) of penicillin was 0.05 U./ml. or less; in 55% the MIC was 0.5 to 2.0 U./ml. Ampicillin was slightly more active than penicillin G: all isolates were inhibited by 0.5μg./ml. or less. Resistance to tetracycline and erythromycin was frequent with MIC of 1 μg./ml. or greater observed in 32 and 24% of isolates respectively. The MIC of kanamycin for all gonococci was 8 μg./ml. or greater. Cephalexin was slightly more active than cephaloridine, though each drug exhibited a wide range of MIC values. Gonococcus isolates resistant to penicillin (MIC of 1.0 U./ml. or greater) tended to be resistant to the other antibiotics tested.     

Gonococcal Antibiotic Resistance in Los Angeles

One hundred and thirty-five gonococcal isolates collected from Los Angeles in 1972 were studied for antibiotic susceptibility to penicillin, ampicillin, carbenicillin, tetracycline, minocycline, doxycycline and spectinomycin. Only 12 percent of the isolates were sensitive to 0.05 μg per ml of penicillin while 35 percent required at least 0.5 μg per ml for inhibition of growth. The results were slightly better with ampicillin and nearly the same with carbenicillin. Nineteen percent of the isolates required at least 1.0 μg per ml of tetracycline for inhibition of growth and the results were similar with either minocycline or doxycycline. Forty-nine percent were sensitive to 2.0 μg per ml spectinomycin, but 37 percent required at least 8.0 μg per ml for inhibition of growth.In this study nine of eleven isolates resistant to 1.0 μg per ml of tetracycline were also resistant to both penicillin and spectinomycin. Six came from endocervical sites of female patients who contributed only 37 percent of the total number of isolates studied.Correlation between the agar dilution and disc diffusion methods was satisfactory with penicillin but not with ether tetracycline or spectinomycin.     

Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation.

The penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis have a mosaic structure that has arisen by the introduction of regions from the penA genes of Neisseria flavescens or Neisseria cinerea. Chromosomal DNA from both N. cinerea and N. flavescens could transform a penicillin-susceptible isolate of N. meningitidis to increased resistance to penicillin. With N. flavescens DNA, transformation to resistance was accompanied by the introduction of the N. flavescens penA gene, providing a laboratory demonstration of the interspecies recombinational events that we believe underlie the development of penicillin resistance in many meningococci in nature. Surprisingly, with N. cinerea DNA, the penicillin-resistant transformants did not obtain the N. cinerea penA gene. However, the region of the penA gene derived from N. cinerea in N. meningitidis K196 contained an extra codon (Asp-345A) which was not found in any of the four N. cinerea isolates that we examined and which is known to result in a decrease in the affinity of PBP 2 in gonococci.     

Identification and antibiogram of microbes associated with bovine mastitis

An investigation of Mastitis in cattle was carried out in Anand city and in nearby villages of Gujarat state using California Mastitis Test (CMT) kit. The prevalence of clinical and subclinical mastitis was found to be 5.5% and 15.75%, respectively. Staphylococcus aureus was identified through strain specific polymerase chain reaction; the remaining isolates identified on the basis of molecular analysis by 16S rDNA sequencing and phylogenetic analysis were Staphylococcus species, B. pumilus, Staphylococcus chromogenes, Bacillus species, and Pseudomonas species. In vitro antimicrobial susceptibility pattern of all the isolates was checked against 13 different antibiotics using the agar disc diffusion method. Highest bacterial resistance was observed with penicillin G and oxacillin antibiotics. It was also observed that the patterns of bacterial resistance have not changed in India over the years. The data supports the decrease in the incidence of mastitis but the rate of decrease is minimal. More effective control strategies are required.     

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages

Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 microgram. ml-1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and penDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. The pcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.     

Psychiatric care and politics.

The minimum inhibitory concentrations (MIC''s) of penicillin, ampicillin, tetracycline, erythromycin, spectinomycin and sulfadiazine were determined for 732 isolates of Neisseria gonorrhoeae collected in 1973-74. Comparison of the results of this survey with data from other Canadian studies showed that the percentage of isolates resistant to tetracycline had not changed since 1966, but that the percentage of erythromycin-resistant isolates had decreased. After an initial increase in 1966 the percentage of penicillin-resistant isolates stabilized. Spectinomycin-resistant isolates were not found. Positive correlations were observed between the MICs of the antibiotics tested; the highest positive correlations were between penicillin and ampicillin and between penicillin and tetracycline. A positive correlation was also noted between penicillin resistance and increasing spectinomycin MICs. Finally, a significant seasonal variation in MICs was found, the trend being towards increasing MICs during the summer.     

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods

AIMS: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. METHODS AND RESULTS: Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. CONCLUSION: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.     

金黄色葡萄球菌的耐药性分析及基因分型研究

目的通过分析上海地区院内分离金黄色葡萄球菌的药敏谱型及对耐甲氧西林的金黄色葡萄球菌（MRSA）进行基因谱型的研究,了解金黄色葡萄球菌的院内流行状况。方法对临床分离出的43株金黄色葡萄球菌进行药敏试验和SCCmec基因盒的多重PCR检测,并将结果整合后用MEGA3．1软件分析其进化相关关系。结果药敏结果显示43株金葡菌对青霉素和甲氧西林的耐药率最高。甲氧西林的耐药率达到62．8％。MecA阳性菌株SCCmec的分型显示均为Ⅱ型或Ⅲ型,且所占比例相近,未见Ⅰ型和Ⅳ型。进化树分析发现了在同一医院中亲缘关系相近的菌株,为院内感染流行株。结论MecA基因介导的MRSA在分离菌株中所占比例高,存在院内感染爆发性流行。     

Susceptibility of Arcobacter butzleri isolates to 23 antimicrobial agents

AIMS: The objective of this study was to determine the susceptibility of Arcobacter butzleri isolates to various antimicrobial agents used in the treatment of infectious diseases in humans and animals. METHODS AND RESULTS: Thirty-nine A. butzleri strains isolated from broiler chickens were tested for their susceptibility to 23 antimicrobial agents using a disc diffusion method. All isolates were resistant to aztreonam, cefuroxime sodium, cephalothin, orbenin, oxacillin, penicillin G and trimethoprim/sulphamethoxazol. Of the 39 isolates tested, 26 were also found resistant to amoxycillin, amoxycillin/clavulanic acid and ampicillin. One isolate was resistant to, and four showed intermediate level of resistance to, erythromycin. All isolates were susceptible to amikacin, chloramphenicol, danofloxacin, enrofloxacin, nitrofurantoin, nalidixic acid, tetracyclines and tobramycin. CONCLUSIONS: The majority of the isolates were found resistant to antibiotics commonly used for the treatment of infectious bacterial diseases in humans and animals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that A. butzleri strains vary in their resistance to certain kinds of antibiotics and caution should be taken when choosing a suitable antibiotic for the treatment of disease(s) caused by this organism.     

Immunization of guinea pigs with Neisseria gonorrhoeae: strain specificity and mechanisms of immunity.

Infection of subcutaneusly implanted chambers in guinea pigs conferred immunity against homologous infection of other chambers in the same animals. However, attempts to immunize guinea pigs by subcutaneous injection of filtered fluid from infected chambers, or with small doses of formalin-killed, chamber gonococci were not successful. Thus, neither organisms grown in vivo nor their extracellular products appeared to be exceptionally immunogenic. In immunizing tests with different isolates of gonococci adapted to growth in guinea-pig chambers, cross-immunity to chamber infection with low challenge doses was detected only between two of six isolates. The killing of gonococci in chambers of immunized animals, which occurred only after homologous challenge or with the heterologous strain showing cross-immunity, was not due primarily to humoral factors in the chamber fluid but probably to an enhanced effectiveness of phagocytosis. The serum of immunized animals was bactericidal for homologous strains and for the strain showing cross-immunity but not for strains showing no cross-immunity. Hence, serum bactericidal activity might be a useful indicator for investigating the specificity of immunity produced by different gonococcal strains.     

Broth dilution and disc diffusion methods in the susceptibility testing of pathogenic Candida albicans against four antimycotics

Susceptibility of Candida albicans isolated from mouthrinse specimens during episodes of acute pseudomembranous candidiasis in patients with haematological malignancies was tested by the broth dilution and disc diffusion methods using 24 and 48 h of incubation. The time factor did not significantly affect the results with 5-fluorocytosine. With amphotericin B, prolonged incubation doubled the geometric mean MIC of C. albicans as well as the number of isolates with intermediate sensitivity. With the shorter incubation in the disc diffusion assay, few isolates showed lowered sensitivity to clotrimazole; at 48 h, however, this figure was as high as 54%. The yeasts were highly sensitive to ketoconazole at 24 h, whereas at 48 h the results were bizarre. At 24 h, correlations between disc diffusion and broth dilution methods were satisfactory, with clotrimazole and ketoconazole accounting for most of the discordancies. Accordingly, the disc diffusion results should be recorded at 24 h.     

Opa-typing: a high-resolution tool for studying the epidemiology of gonorrhoea

A single gonococcus possesses a family of 11 distinct and highly variable opa genes. The extensive variation and rapid evolution of the opa gene repertoire has been exploited to provide a high-resolution typing method for studies of the short-term transmission of gonorrhoea. The 11 opa genes are amplified with a single pair of primers by the polymerase chain reaction, digested with frequently-cutting restriction enzymes, and the fragments are fractionated on polyacrylamide to provide an opa-type. The method appeared to be highly discriminatory as the opa-types of gonococci, isolated world-wide over the last 30 years, were all different. Opa-typing discriminated between isolates of the same auxotype/serovar class. Similarly, there were 41 opa-types among 43 consecutive isolates from a sexually transmitted disease (STD) clinic. The two pairs of isolates from this clinic that gave the same opa-types were identical by other criteria and may have been from unsuspected sexual contacts. With one minor exception, identical opa-types were obtained from gonococci recovered from known sexual contacts. These results suggest that variation in the family of 11 opa genes evolves so rapidly that the opa-types of gonococci are distinguishable, unless the isolates are from sexual contacts or a short chain of disease transmission. The identification of gonococci with identical opa-types is therefore believed to be a good indicator that the individuals from which they were recovered were sexual partners, or part of a short chain of disease transmission.     

Molecular epidemiology of penicillin resistantStreptococcus pneumoniae strains in Turkey. A multicenter study

A total of 539 isolates recovered from various clinical sites were collected from 13 hospitals from different regions of Turkey between 1999 and 2002. Susceptibility to penicillin and cefotaxime was determined by the E-test and the remaining antimicrobials were evaluated by disk diffusion tests. Penicillin resistant and intermediate isolates were serotyped and PFGE patterns were analysed. Overall 16 isolates (3%) were resistant to penicillin, and 143 (26.5%) were intermediate. Resistance and intermediate rates were 3.1% and 29.0% respectively in respiratory tract isolates. Multiple resistance (resistance to ≥3 antibiotics) occurred in 81.8% of the penicillin resistant isolates and the most frequent resistance phenotype was penicillin+trimethoprim/sulphamethoxazole (37.7%). Minimum inhibitory concentrations of cefotaxime were lower than 1 mg/ml for all the isolates. The highest rate of resistance was observed for trimethoprim/ sulphamethoxazole (26.6%) followed by doxycycline (12.6%). Resistance to erythromycin was 10.1%, clindamycin 9.9%, chloramphenicol 4.3%, ofloxacin 5.0% and levofloxacin 0.2%. There was no resistance to vancomycin. Resistant isolates belonged to serogroups 9, 23, and 6. The most frequent serogroups among intermediate isolates were 23, 19, 14, 1, 9, and 6. Five distinct PFGE patterns were observed among penicillin resistant isolates. There was no distinct clustering of specific PFGE patterns in the study centres. No correlation between serotypes, resistance and PFGE patterns was found. There seems to be genetic heterogeneity inStreptococccus pneumoniae isolates in Turkey.     

Penicillin-binding protein 2 genes of non-β-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae

Oligonucleotides that correspond to regions of the penicillin-binding protein 2 gene (penA) that differ between penicillin-sensitive and penicillin-resistant strains have been used as probes to classify the penA genes in a collection of penicillin-resistant gonococci isolated in Britain. 44/47 of those gonococcal strains that had minimal inhibitory concentrations of greater than or equal to 0.25 microgram benzylpenicillin per ml contained extensively altered penA genes which appeared to be very similar (or identical) to one or other of the two classes of altered penA genes that have been described previously. Since these two classes of altered penA genes are related, it appears that the great majority of the altered penA genes on non-beta-lactamase-producing penicillin-resistant gonococci have a clonal origin. The other three penicillin-resistant strains had altered penA genes that were different to those described previously. A crucial step in the development of the altered forms of PBP2 with decreased affinity for penicillin appears to have been the insertion of an extra codon within the transpeptidase domain of the penA gene. This insertion was found in the penA gene of all gonococci with minimal inhibitory concentrations of greater than 0.016 microgram benzylpenicillin per ml but was not found in any strains with minimal inhibitory concentrations of less than or equal to 0.016 microgram per ml.     

The effect of benzyl penicillin on the ultrastructure of Neisseria gonorrhoeae

Electron microscopy of Neisseria gonorrhoeae grown on solid medium with a subinhibitory concentration of penicillin suggested that the amount of penicillin reaching each pair of gonococci was different, as illustrated by the ultrastructural appearance of N, gonorrhoeae cells in the colony. This supports the view that the concentration of penicillin in different parts of the colony is not uniform, causing some cells to lyse while the others remain intact.     

Batch reactor performance for the enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size

A mathematical model is presented for the kinetically controlled synthesis of cephalexin that describes the heterogeneous reaction-diffusion process involved in a batch reactor with glyoxyl-agarose immobilized penicillin acylase. The model is based on equations considering reaction and diffusion components. Reaction kinetics was considered according to the mechanism proposed by Schro?n, while diffusion of the reacting species was described according to Fick's law. Intrinsic kinetic and diffusion parameters were experimentally determined in independent experiments. It was found that from the four kinetic constants, the one corresponding to the acyl-enzyme complex hydrolysis step had the greatest value, as previously reported by other authors. The effective diffusion coefficients of all substances were about 5×10(-10)m(2)/s, being 10% lower than free diffusion coefficients and therefore agreed with the highly porous structure of glyoxyl-agarose particles. Simulations made from the reaction-diffusion model equations were used to evaluate and analyze the impact of internal diffusional restrictions in function of catalyst enzyme loading and particle size. Increasing internal diffusional restrictions decreases the Cex synthesis/hydrolysis ratio, the conversion yield and the specific productivity. A nonlinear relationship between catalyst enzyme loading and specific productivity of Cex was obtained with the implication that an increase in catalyst enzyme loading will not increase the volumetric productivity by the same magnitude as it occurs with the free enzyme. Optimization of catalyst and reactor design should be done considering catalyst enzyme loading and particle size as the most important variables. The approach presented can be extended to other processes catalyzed by immobilized enzymes.     

Gonococcal sensitivity to penicillin in women with gonorrhea relapse and reinfection]

Penicillin sensitivity of gonococci isolated from patients with relapses and reinfection of gonorrhea was studied. The results of the study were compared with those of the sensitivity tests in primary patients. The gonococcal strains with decreased sensitivity to penicillin were isolated from 82 per cent of the gonorrhea relapses, 47.8 per cent of the patients with reinfection and 33.2 per cent of the primary patients. The average sensitivity of the gonococcal strains isolated from the patients with relapses was 0.404 U/ml, while that in the primary patients and the patients with reinfection was 0.136 U/ml. Strains sensitive to 0.6--1.2 U/ml predominated among gonococci with decreased sensitivity in the patients with relapses, while in the primary patients and the ones with reinfection the value amounted to 0.1--0.2 U/ml. The penicillin sensitivity of gonococci may be used as a parameter in differential diagnosis of relapses and reinfection of gonorrhea.     

Spectinomycin resistant gonococci.

A study was conducted examining the properties of 10 clinical isolates of spectinomycin resistant gonococci from patients attending clinics at St Mary''s and St Thomas''s Hospitals, London. All of the isolates produced beta-lactamase and contained plasmids of 2.6, 4.4, and 24.5 megadaltons and required proline for growth. None produced aminoglycoside modifying enzymes. Resistance to spectinomycin was transferred from some of the isolates by transformation but at a much lower frequency than resistance to streptomycin. The isolates from St Mary''s Hospital were detected after therapy with spectinomycin, whereas those from St Thomas''s Hospital were not. Four recent non-beta-lactamase producing gonococci isolated at St Mary''s Hospital and two isolated at St Thomas''s Hospital also were not related to use of spectinomycin.     

Effectiveness of the antibiotics chloramphenicol and rifampin in the treatment of Streptococcus pneumoniae-induced meningitis and systemic infections

Streptococcus pneumoniae, a common pathogen in pediatric infections, has become resistant to penicillin and make these infections difficult to treat. Rifampin and chloramphenicol have been recommended as alternative therapies, since they are less costly and more accessible to communities with limited resources. However, their use may be restricted by the differing levels of resistance found in target populations. The objective was to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for chloramphenicol and rifampin in strains of S. pneumoniae. These strains were newly isolated from children under age 5 that had demonstrated systemic infections and meningitis. A subgroup of 107 isolates of S. pneumoniae was selected from 324 strains isolated during a period of 2 years (1994-1996). Among these isolates, 60 were penicillin-resistant and 47 were susceptible; 53 isolates were from children with meningitis. MIC and MBC for chloramphenicol and rifampicin were obtained by standard methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS). S. pneumoniae ATCC strain 49619 served as the control. An isolate was considered susceptible to chloramphenicol when MIC = 4 microg/ml and resistant when MIC = 8 microg/ml. A strain was considered susceptible to rifampin when MIC = 1 microg/ml and resistant when MIC = 4 microg/ml. MBC was determined by recording the lower concentration of the antibiotic that inhibited 99.9% of the initial inoculum. Chloramphenicol resistance was found in 21% of the 107 isolates. In the group susceptible to penicillin, 11% were resistant to chloramphenicol and in the group resistant to penicillin 28% was resistant to chloramphenicol as well. MBC was found > 4 microg/ml in 28% of the isolates susceptible to penicillin and in 60% of the resistant isolates. No isolates were found resistant to rifampin. However, 2 penicillin resistant isolates showed CBM > 1 microg/ml to rifampin, and one with CIM = 1 microg/ml had a MBC to rifampicin of 16 microg/ml. Meningitis isolates showed higher CIM and CBM than the group of total isolates. These data suggest that chloramphenicol is not recommended for invasive infections caused by S. pneumoniae in Colombia. Rifampin is a more effective therapy in combination with other antibiotics for treatment of this kind of infections. Further studies are necessary to clarify the significance of low levels of MBC to rifampin found in some strains, since this may affect the efficacy of therapies that include this antibiotic.