Genetic relatedness and antimicrobial resistance determinants among clinical isolates of enterococci from Cuba.

This study describes the genetic relationships and antimicrobial resistance determinants found among 99 clinical isolates of enterococci from 15 different hospitals in Cuba. Pulsed-field gel electrophoresis SmaI analysis demonstrated a high degree of genetic diversity. A limited number of multiresistant Enterococcus faecalis clones, showing resistance to three or more families of antimicrobial agents, were detected simultaneously in different institutions, suggesting inter-hospital circulation of selected clones, and/or selection of particular clones following their introduction into the hospital environment. Antimicrobial resistance determinants, including erm(B), aac(6')-aph(2'), aph(3'), ant(6), vanB (E. faecalis) and vanA (Enterococcus faecium) were detected by PCR in various isolates.

Fuchsin fluorescence and autofluorescence in Cryptosporidium, Isospora and Cyclospora oocysts.

Cryptosporidium parvum and Isospora belli oocysts stained with carbol-fuchsin, as in a modified Ziehl Neelsen technique, fluoresce bright red under green light (546nm). Cryptosporidium oocysts tend to fluoresce more brightly the less intensely stained they appear under transmitted light; this is not the case with Isospora. Fuchsin-stained Cyclospora cayetanensis oocysts fluoresce rather dimly, but those not taking the dye retain their typical autofluorescence. Cryptosporidium and Isospora oocysts are also autofluorescent, appearing violet under u.v. light (365 nm), and green under violet (405 nm) and blue-violet light (436 nm). Their autofluorescence does not survive the staining procedure.

Western blot applied to the diagnosis and post-treatment monitoring of human hydatidosis.

The serologic diagnosis of hydatidosis (caused by Echinococcus granulosus) can be made by different techniques, although the lack of standardization of the antigens affects the sensitivity, specificity and concordance among the different tests. We have applied the Western-Blot (WB) technique, associated with a purified antigen from sheep hydatid fluid, at 60 samples of serum from 14 patients suffering echinococcosis in different bodily locations, monitored for 3 years. The WB test enabled the detection of antibodies in the pre-surgical samples for proteins of 12-14, 16, 20, 24-26, 34, 39 and 42 kDa in molecular weight in 15-96% of the patients. The combination involving 2 of the 3 proteins of 20, 39 and 42 kDa has made it possible to diagnose 100% of the cases. The antibodies specific to proteins 39 and 42 kDa disappeared in less than one year in the patients cured after surgery, while in patients with persistent or recurrent parasitism the bands present before surgery persisted or other new ones appeared. The WB with purified antigens proved to be highly useful in the diagnosis and post-surgical monitoring of hydatidosis patients. The antigen used is proposed as a standard antigen for the diagnosis and follow-up of pre- and postsurgical hydatidosis.

The molecular epidemiology of tuberculosis in Zaragoza, Spain: a retrospective epidemiological study in 1993.

SETTING: The incidence of tuberculosis (TB) in Spain is one of the highest in Europe. In Zaragoza region the incidence rate of tuberculosis and the acquired immune deficiency syndrome (AIDS) are close to the national average. OBJECTIVE: To better define the molecular epidemiology of tuberculosis in an area of Europe where this has not been previously studied. DESIGN: A retrospective epidemiological study on tuberculosis was conducted in Zaragoza, a region of Spain, in 1993. The study population consisted of 226 patients from whom positive culture and complete clinical and demographic data were available. Mycobacterium tuberculosis strains were typed by standard restriction fragment length polymorphism (RFLP). A cluster was defined as two or more isolates with identical RFLP patterns when five or more copies of IS6110 are present. The 137 non-clustered patients were compared with the 89 clustered patients and studied by using univariate analysis. RESULTS: Thirty-nine percent of the patients were clustered, suggesting possible recent transmission. Infection with drug-resistant M. tuberculosis was associated with a decreased risk of being in a cluster. The strains isolated from human immunodeficiency virus (HIV)-positive patients were not associated with clustering. We found that immigration was not a major determinant in the total number of TB cases. CONCLUSION: Immigration, HIV and drug resistance were not associated with recent transmission. More than 50% of the clusters contained two or three patients, indicating that small outbreaks were responsible for most of the tuberculosis cases. Our RFLP typing results indicate that a TB control programme should be implemented in Spain in order to lower transmission of TB.

Emerging and reemerging pathogens.

From 1973 to 1995, 29 new and reemerging pathogenic microbes were recognized. However, in discussions about emerging infectious diseases, the focus is often on the clinical effects of the host-parasite relationship, rather than the examination of the biology of the pathogen. Many of what we refer to as emerging diseases are characterized better as 'diseases of human progress'. Thus, the aerosolization of water has played an important role in the emergence of Legionella pneumophila infections. New diseases are superimposed on endemic diseases such as diarrhoeal diseases, malaria and tuberculosis. In addition, many pathogens are becoming increasingly resistant to standard antimicrobial drugs, making treatment difficult and in some cases impossible. We summarize our experience on emerging parasitic diseases (primary amoebic meningoencephalitis, respiratory cryptosporidiosis, and diplogonoporiasis), and selected problems of bacterial resistance (MDR tuberculosis caused by Mycobacterium bovis and macrolide-resistance mechanisms of Streptococcus pneumoniae and S. pyogenes).

Aminoglycoside-modifying enzymes in high-level streptomycin and gentamicin resistant Enterococcus spp. in Spain.

Aminoglycoside resistance was evaluated in 690 enterococcus strains isolated from different clinical sources originating from patients at the University Clinic Hospital of Zaragoza (Spain). The enterococci obtained from clinically significant samples (blood, urine, or exudates) showed more high-level resistance to gentamicin and streptomycin (65 and 42%, respectively) than those isolated from faecal samples (49 and 23%, respectively). Aminoglycoside-modifying enzymes (AME) from 119 of these high-level gentamicin and streptomycin resistant enterococcus strains were studied. The most frequent AMEs found were APH(3') and AAC(6')-APH(2"). More than one enzyme was detected in 71% of the strains (four different enzymes in 5% of the strains). Three Enterococcus faecalis strains had ANT(4')(4") enzymatic activity. Different enzymatic expressions of the bifunctional enzyme AAC(6')-APH(2") were demonstrated in strains in which the complete aac(6')-aph(2") gene was detected by PCR and hybridization: (i) AAC(6') + APH(2") activity; (ii) AAC(6') only; (iii) APH(2") only; and (iv) no activity of AAC(6') or APH(2").

Antibiotic resistance and epidemiological typing of Staphylococcus aureus strains from ovine and rabbit mastitis.

Mastitis is a serious problem for sheep and rabbit farms, Staphylococcus aureus being the main causal agent. Fifty strains of S. aureus isolated from sheep and rabbits from farms located in diverse geographical regions of Spain were studied. Their resistance pattern and plasmid profile was related to the pulsotypes obtained by pulsed-field gel electrophoresis (PFGE). The results showed great heterogeneity in staphylococci isolated from sheep, both in pulse-type and plasmid profile. We found in addition, antibiotic-resistant strains and aminoglycoside-modifying enzyme (AGMEs) producer strains. The genotypes corresponding to staphylococci isolated from rabbits were less heterogeneous, although they also could be subdivided by plasmid profile and resistance patterns. Resistance to antibiotics such as methicillin or AGMEs production could indicate possible human origin of the strains or a possible source of resistant strains for human beings.

Macrolide resistance phenotypes of commensal viridans group streptococci and Gemella spp. and PCR detection of resistance genes.

One hundred and sixty viridans group streptococci (VGS) and 26 Gemella spp. resistant to erythromycin were studied to detect macrolide lincosamide and streptogramin B (MLS(B)) phenotypes and to investigate resistance rates to other antibiotics. The M phenotype was most prevalent in both bacterial groups (59.6% in VGS, 69.2% in gemellae) and the iMLS(B) phenotype was found least often (9.3 and 13.9%, respectively). All isolates with M phenotype had the mef(A/E) gene, being prevalent the mef(E) subclass. cMLS(B) and iMLS(B) strains contained the erm(B) gene, alone or in combination with the mef(A/E) gene. Thirteen isolates were intermediately resistant to quinupristin/dalfopristin and 11 strains showed low susceptibility to telithromycin. Linezolid was active against all the isolates tested and tetracycline resistance was the major one in VGS (41.6%) and Gemella spp. (46.2%).

Comparative in vitro bacteriostatic and bactericidal activity of trovafloxacin, levofloxacin and moxifloxacin against clinical and environmental isolates of Legionella spp.

The susceptibility of 140 Legionella spp isolates (106 clinical and 34 environmental isolates) to trovafloxacin (TRFX), levofloxacin (LEVX), moxifloxacin (MOFX), ciprofloxacin (CIPX), ofloxacin (OFLX), erythromycin (ERY), azithromycin (AZI) and rifampicin (RIF) was studied using a standard microdilution method and buffered yeast extract broth (BYE) supplemented with 0.1% alpha-ketoglutarate. The post-antibiotic effects (PAEs) of the study drugs against 10 clinical isolates of Legionella pneumophila sg.1 were compared. The MIC inhibiting 90% of strains tested on BYEalpha broth were 0.008, 0.016, 0.016, 0.06, 0.125, 0.5, 0.5, and 0.004 mg/l for TRFX, LEVX, MOXX, CIP, OFLX, ERY, AZI, and RIF, respectively. The MBC/MIC ratios ranged from one to eight depending on the antibiotic tested: TRFX [1x-2 x MIC], LEVX, MOFX, CIPX and OFLX [1x-4 x MIC], RIF [2x-4 x MIC], ERY and AZI [2x-8 x MIC]. TRFX, RIF, LEVX, MOFX, CIPX, OFLX, ERY and AZI showed similar activity against Legionella species other than L. pneumophila. One-hour exposures to the study antimicrobial agents at a concentration of 4 x MIC resulted in PAEs as follows (average in hours): TRFX: 2.68 h; RIF: 2.63 h; CIPX: 2.62 h; MOFX: 2.56 h; LEVX: 2.41 h; OFLX: 2.25 h; AZI: 1.65 h; and ERY: 1.54 h. In conclusion, our in vitro data confirm that trovafloxacin, levofloxacin, moxifloxacin and rifampicin have excellent bacteriostatic and bactericidal activity against Legionella spp and show significant post-antibiotic effect.

Epidemiological markers of Acinetobacter baumannii clinical isolates from a spinal cord injury unit.

During a period of 28 months, 114 isolates of Acinetobacter baumannii obtained from urine samples of 57 patients, were recovered in a Spinal Cord Unit; an unusual increase in the number of A. baumannii isolates was observed between February 1991 and January 1992. Six different typing methods [biotyping, antimicrobial susceptibility, whole cell and cell-envelope protein analysis, plasmid analysis and chromosomal DNA analysis by pulsed-field gel electrophoresis (PFGE)] were used to study the isolates to establish any potential relationships among them. Chromosomal DNA analysis by digestion with ApaI and separation of the fragments by PFGE was the most powerful tool to determine the relatedness of isolates. The results suggest that the isolates from 1991 and 1992 may have originated from strains present in 1990 that subsequently acquired resistance to amikacin and tobramycin during the epidemic.

Aminoglycoside resistance in Haemophilus influenzae.

From September 1, 1990 to December 31, 1993 a total of 425 Haemophilus influenzae strains from clinical specimens were isolated in the Microbiology Laboratory of the Zaragoza University Hospital. Of these strains, 16 (33.33%) were resistant to kanamycin, neomycin, paromomycin, lividomycin and streptomycin. Demonstration of APH (3')-I activity by the phosphocellulose paper binding assay, based on the incorporation of radiolabel into lividomycin was sixfold greater than into butirosin. Two DNA probes were prepared to screen for the genes encoding APH(3') activity in kanamycin-resistant H. influenzae. Homology was observed between the aphA1 DNA probe and total cellular DNA from all 16 APH(3')-I producers. On the other hand, streptomycin-resistance was not through metabolic modification of the antibiotic.

In vitro activity of telithromycin, quinupristin/dalfopristin, linezolid and comparator antimicrobial agents against Staphylococcus aureus clinical isolates.

We have studied the prevalence of the different macrolide, lincosamide, streptograminB (MLS(B)) phenotypes among clinical Staphylococcus aureus isolates erythromycin- and/or oxacillin-resistant; and also the activity of other antimicrobial agents including telithromycin, quinupristin/dalfopristin, linezolid, aminoglycosides, chloramphenicol and vancomycin. We found that 64.86% of S. aureus were oxacillin-resistant. While the most prevalent MLS(B) phenotype among methicillin-resistant S. aureus (MRSA) was constitutive MLS(B) (cMLS) (83%), among methicillin-susceptible S. aureus (MSSA) it was inducible MLS(B) (iMLS(B)) (90%). Kanamycin resistance was more frequent than resistance to other aminoglycosides, being 100% for MRSA. Telithromycin was only active against iMLS(B), MS and erythromycin-susceptible isolates, although resistance rates were found among iMLS(B) MSSA (2.78%). Quinupristin/dalfopristin showed greater activity, with resistance rates of 2.5% for MRSA and 1.53% for MSSA. Both vancomycin and linezolid were fully active against all the isolates tested, with the highest MIC value being 2 microg/ml and 4 microg/ml, respectively. Among MRSA strains, 81.67% displayed resistance to five or more antimicrobials. This multiresistance was more frequently found among cMLS(B) strains (96.38% MRSA resistant to 6-9 agents).

In vitro activity of cefepime and cefotaxime compared to six other agents against 350 penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae.

From January 1996 to December 1997, we evaluated the in vitro activity of 8 antimicrobials (penicillin, amoxycillin, amoxycillin/clavulanate, cefuroxime, ceftazidime, cefepime, cefotaxime, and imipenem) against 350 Streptococcus pneumoniae clinical isolates collected from two hospitals. Imipenem, cefepime and cefotaxime were the most active antibiotics against penicillin-intermediate (PI) and highly penicillin-resistant (PR) S. pneumoniae with MICs 2- to 8-fold lower than penicillin. Against PI and PR pneumococci amoxycillin and amoxycillin/clavulanate were 2-times less active than cefepime and cefotaxime, while cefuroxime was 4-8-times less active. The majority of strains of serotypes 6B, 23F, 14, 9 and 19 were penicillin-resistant, both intermediate (68%) and highly resistant (32%).

Susceptibility of penicillin-resistant and penicillin-susceptible Streptococcus pneumoniae to newer antimicrobial agents.

Agar dilution minimum inhibitory concentration (MIC) methodology, according to NCCLS guidelines, was used to test the activity of three glycopeptides (LY 333328 [LY], vancomycin [VAN], and teicoplanin [TEI]), four fluoroquinolones (trovafloxacin [TRO], BAY 12-8039 [BAY], ciprofloxacin [CIP], and ofloxacin [OFL]), five macrolide-lincosamide-streptogramin antibiotics (erythromycin [ERY], azithromycin [AZI], miocamycin [MOM], clindamycin CLN], and quinupristin-dalfopristin [SYN] against 126 Streptococcus pneumoniae strains, isolated in Lozano Blesa Hospital of Zaragoza (Spain). MIC50/MIC90 (microg/ml) values for penicillin-susceptible (PS), penicillin-intermediate (PI) and penicillin-resistant (PR) strains show an excellent antipneumococcal activity of LY 333326--a new glycopeptide, for the fluoroquinolones trovafloxacin and moxifloxacin [BAY 12-8039], and for quinupristin/dalfopristin, regardless of the resistance phenotype of the strains.

Resistance to apramycin in two enterobacterial clinical isolates: detection of a 3-N-acetyltransferase IV.

Considering the possible role of farm animals in the contamination of human consumers by plasmid-mediated apramycin-resistant enterobacteria strains, this type of resistance should be tested more systematically in human isolates. Very recently we isolated in Zaragoza one apramycin-resistant Escheria coli strain obtained from the blood of a hospitalized patient; this clinical isolate produced a plasmid-mediated 3-N-aminoglycoside acetyltransferase IV. We describe also the isolation in Madrid of one multiresistant Klebsiella pneumoniae clinical strain. This isolate harbored a single plasmid and carried determinants for apramycin, gentamicin, tobramycin, hygromycin B, streptomycin, and ampicillin, which could be transferred en bloc to E. coli K-12 J62. Extracts from donor and transconjugant strains carrying pUZ6776 plasmid produce acetyltransferase activity AAC(3)-IV and double phosphotransferase activity (HPH and APH(3'')).

Stability of dactimicin to aminoglycoside-modifying enzymes.

Dactimicin was active against strains expressing the activities of aminoglycoside acetylating enzymes [AAC(3)-II, III, IV and V, AAC(2'), AAC(6')-I and II], aminoglycoside-nucleotidylating enzymes [ANT(2"), AAD(3")] and aminoglycoside-phosphorylating enzymes [APH(3')-I-II and III], with the exception of AAC(3)-I and one staphylococcal AAC(6')-IV. Apparently this is the first report of one 6'-N-acetylating enzyme which modifies and inactivates dactimicin. The authors' data suggest that the differences in the behaviour of dactimicin, gentamicin and amikacin against the aminoglycoside-resistant strains tested were mainly due to the production of aminoglycoside-modifying enzymes. If the results are summarized, it may be concluded that dactamicin is the most stable to the majority of aminoglycoside-modifying enzymes demonstrated [APH(3'), APH(2"), APH(3"), ANT(2"), AAD(3"), AAC(2') and AAC(6')], with the exception of AAC(3)-I and staphylococcal AAC(6')-IV.

Enzymatic modification of aminoglycoside antibiotics by Branhamella catarrhalis carrying an R factor.

Fifteen out of 89 clinical strains of Branhamella catarrhalis isolated from patients at the University Hospital of Zaragoza were resistant to aminoglycosides and other antimicrobials. In two strains, B. catarrhalis 220 and B. catarrhalis 115, the resistance to aminoglycosides was associated with synthesis of aminoglycoside-modifying enzymes, namely 3"-O-phosphotransferase [APH(3")] and 3'-O-phosphotransferase [APH(3')]. B. catarrhalis 115 was resistant to ampicillin, streptomycin, kanamycin, neomycin, butirosin, lividomycin, ribostamycin, paromomycin and trimethoprim-sulfamethoxazole and harboured a 32 megadalton (Md) plasmid. The resistance determinants of the latter were transferred to Neisseria subflava by conjugation and to Escherichia coli by transformation. The transconjugant strain presented an antibiotic resistance pattern similar to the donor strain and carried the same plasmid. The transformant strain acquired the 32 Md plasmid but presented, besides the resistance pattern already mentioned, resistance to tetracycline, gentamicin and tobramycin. Resistance to gentamicin and tobramycin was mediated by the synthesis of a 3-N-acetyltransferase. This resistance and the related enzyme were expressed neither in the donor B. catarrhalis strain nor in the transconjugant N. subflava strain.

Comparative activity of ceftizoxime against aminoglycoside-resistant aerobic gram-negative bacilli.

The in vitro activity of ceftizoxime was compared with that of other beta-lactam antibiotics against 331 aminoglycoside (AG)-resistant clinical isolates. Two hundred and six AG-resistant, beta-lactamase producing, R-plasmid harbouring Enterobacteriaceae strains had MICs ranging from 0.0125 to 0.063 mg/l. AG-resistant Escherichia coli (36 strains) and Klebsiella pneumoniae (19) had MIC 90 values of 8 mg/l. Proteus rettgeri and P. vulgaris as well as Morganella morganii, resistant to several AGs, had MICs ranging from 0.5 to 4 mg/ml. Against all six isolates of AG-resistant Salmonella enteritidis the MIC90 was 0.5 mg/l. Twenty-seven strains of Serratia marcescens, most of which were resistant to beta-lactam and AG antibiotics, had MICs ranging from 0.5 to 8 mg/l. The AG-resistant strains of Enterobacteriaceae producing several AG-modifying enzymes (AAC(3); AAC(2'); AAC(6'); APH(3')) showed MICs ranging from 0.6 to 4 mg/l. Against 10 AG-resistant strains of Pseudomonas aeruginosa producing AAC(3), AAC(6') and APH(3') enzymes, the MICs ranged from 16 to 64 mg/l. In conclusion, ceftizoxime was equally or more active than cefotaxime, cefoperazone, ceftazidime and moxalactam against AG-resistant E. coli, Klebsiella, Morganella, Proteus, Serratia, Salmonella and R-plasmid harbouring Enterobacteriaceae. Ceftizoxime was less active than cefotaxime, moxalactam and ceftazidime against P. aeruginosa.

Stability of dactimicin to aminoglycoside-modifying enzymes produced by 341 bacterial clinical isolates.

The stability of dactimycin to aminoglycoside-modifying enzymes produced by 341 bacterial clinical isolates has been studied. Enzymatic activities were measured by the phosphocellulose binding assay. The results demonstrated that dactimicin was stable to the following enzymes: (i) AAC(3)-II,-III,-IV and -V. (ii) AAC(2'); (iii)AAC(6')-I and -II;(iv) ANT(2"); (v)ANT(4'); (vi) APH(3')-I,-II,-III and -IV. In contrast, dactimicin was only inactivated by two enzymes, AAC(3)-I and the bifunctional AAC(6')/APH(2"). This staphylococcal enzyme modified and inactivated dactimicin by acetylation but not by phosphorylation, suggesting the possibility of a second target amino group, such as 6'-NH2, in addition to the C4 amino group, which is the target for AAC(3)-I.

[Study of macrolide-resistant genes in group C and G streptococci]. / Estudio de los genes de resistencia a macrólidos en estreptococos de los grupos C y G.

In the present study we investigated the macrolide-lincosamide-streptogramin B (MLSB) resistance in 47 clinical isolates of group C beta-hemolytic streptococci and 17 group G streptococci. Resistance to erythromycin was found in 31.6%; and 47%, respectively. On the basis of the erythromycin-clindamycin double-disk test, all strains were assigned to the MLSB phenotype. None of the strains were of the M phenotype. The distribution of the erythromycin-resistant genes were studied by dot blot hybridization and PCR. Resistance to erythromycin was due to the presence of mefA, ermB and ermTR genes. The ermTR gene was predominant among the group C and G streptococci (90% of the strains). Different mechanisms of erythromycin resistance predominate in group C and G streptococci than in Streptococcus pyogenes where there is an association between erythromycin resistance and active-efflux mechanism. Two of the strains harbored more than one erythromycin-resistant gene.