Microbial Drug Resistance

High-level antiseptic resistance of Staphylococcus aureus is mediated by multidrug efflux pumps encoded by qacA and qacB genes. We investigated distribution and genomic diversity of these antiseptic resistance genes in a total of 522 clinical strains of S. aureus isolated recently in a Japanese hospital. The qacA/B gene was detected in 32.6% of methicillin-resistant S. aureus (MRSA) and 7.5% of methicillin-susceptible S. aureus (MSSA), whereas the low-level resistance gene smr, which was examined simultaneously, was detected at lower frequencies in both MRSA (3.3%) and MSSA (5.9%). Epidemiologic typing of S. aureus isolates suggested that higher prevalence of qacA/B in MRSA may be due to spread of a single predominant MRSA strain carrying qacA/B in the hospital. Restriction fragment length polymorphism (RFLP) analysis indicated higher prevalence of the qacB-type gene (59.3%) than the qacA-type gene (40.7%) among the qacA/B genes detected. Nucleotide sequencing analysis revealed the presence of two genetic variants in qacA (V1 and V2) and four variants in qacB (V1-V4) that differ from the qacA prototype in pSK1 by 1-5 nucleotides and 7-9 nucleotides, respectively. Although most strains with qacA-V1, qacA-V2, qacB-V3, and qacB-V4 showed high-level resistance to ethidium bromide (EB)(MIC > 100 μg/ml), all of the S. aureus isolates carrying qacB-V1 and qacBV2 showed lower MICs of EB and some monovalent cationic antiseptic substances. By analysis of the genomic organization of the qacA/B downstream region, divergent forms of this region rearranged with an insertion of IS 256 or IS 257 were found primarily for qacB. The downstream region of qacA-V1 was suggested to be an evolutionary origin for other divergent forms. These findings indicated that both qacA and qacB are prevalent in recent clinical isolates, especially in MRSA, and these genes consist of variable genetic variants that may be responsible for different resistance levels against antiseptic substances.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log₁₀ CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (≤0.5 log₁₀ CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Erythromycin resistance and the characterization of the corresponding determinants of resistance were studied in clinical Streptococcus pneumoniae isolates belonging to the four major multiresistant pneumococcal Spanish clones (ermB and mefA genes for the Spain^23F-1, Spain^9V-3, serotype 14 variant of the Spain^9V-3 and Spain¹⁴-5 clones and ermB gene for the Spain^6B-2 clone). These isolates were confirmed as major clones by pulsed-field gel electrophoresis (PFGE), BOX-PCR, and multilocus sequence typing (MLST). The spread and prevalence of these erythromycin-resistant variants of the Spanish clones in an area of the north of Spain were dissimilar—low for the Spain^9V-3 clone (5.8% among the isolates belonging to this clone, including isolates of the serotype 14 variant) and very frequent for the Spain¹⁴-5 clone (91.7%).

In the folate biosynthetic pathway, sulfa drugs (sulfonamides and sulfones) compete with the natural substrate, para-aminobenzoate (pABA) causing depletion of dihydrofolate (DHF) and subsequent growth inhibition. The sulfa drugs condense with 2-amino-4-hydroxy-6-hydroxymethyl-7,8 dihydropteridine pyrophosphate (DHPPP) forming sulfa-dihydropteroate (sulfa-DHP). Here evidence is presented using yeast that such dihydropteroate (DHP) analogs are inhibitory through competition with DHF. Two folate synthesis mutants, with respective dihydrofolate synthase (DHFS) and dihydropteroate synthase (DHPS) deletions and requiring DHF for growth were exposed to sulfa drugs. The DHFS knockout mutant was inhibited, but the DHPS knockout mutant that was incapable of forming sulfa-DHP was insensitive. Such sulfa-DHP compounds were chemically synthesized and shown to be inhibitory in vivo by competing with DHF, but in vitro assays with double the concentration of the sulfa-DHP to DHF showed no inhibition of dihydrofolate reductase (DHFR). Sequence analysis of resistant mutants obtained in the presence of sulfa drugs showed no changes in DHFR, or DHPS, unlike previously found antifolate-resistant mutants. The diamino derivatives, which are precursors of the sulfa-DHP, were found to be DHFR inhibitors. These results suggest that a new class of drugs, based on DHP analogs, could be investigated.

Two variants of dihydropteroate synthase (DHPS) were found among sulfonamide-resistant Streptococcus pyogenes, one of which was characterized by a 2-amino-acid addition in a conserved part of the enzyme. The enzyme kinetics of both variants was compared with the kinetics of DHPS from a sulfonamide-susceptible S. pyogenes. The most striking difference was a substantially elevated K_i for both variants, but variations in K_m for both of its substrates p-aminobenzoic acid (p-AB) and dihydropteridine-pyrophosphate (pteridine) were also found. In the resistance variant lacking additions, the amino acid at position 213 was changed by site-directed mutagenesis from a Gly to an Arg, which resulted in a lower K_i. The corresponding change from an Arg to a Gly in the DHPS from a susceptible isolate led to a substantially increased K _i, confirming the importance of this amino acid difference for the resistance. Nucleotide sequence determinations of the complete folate operon revealed in some isolates a mosaic pattern of differences compared to the wild type, not only in the genes coding for DHPS and GTP cyclohydrolase (GTPCH) noted earlier but also in genes coding for dihydroneopterin aldolase (DHNA) and hydroxymethylpterin pyrophosphokinase (HPPK). Regions of sequence differences were interspersed with regions of complete identity in a mosaic pattern, indicating a dispersed pattern of uptake of foreign DNA in the resistant isolates.

An annual survey of antimicrobial resistance in clinical isolates of Staphylococcus aureus was conducted in 21 Australian teaching hospital microbiology laboratories in eight major cities from 1989 to 1999. A total of 19,000 isolates were tested for susceptibility to 18 antimicrobials, with 3795 being methicillin-resistant (MRSA). Resistance to ciprofloxacin in MRSA increased from 4.9% to 75.9%. The proportion of MRSA resistant to erythromycin decreased significantly (99.0%-88.9%), as did that to trimethoprim (98.4%-82.4%) and to tetracycline (96.5%-80.1%). The proportion of MRSA isolated increased in Sydney, Melbourne, Canberra, Adelaide, Perth, and Darwin, but not in Brisbane. The proportion in Hobart peaked in 1994. MRSA in Perth were predominantly non-multiresistant (nmMRSA) throughout the survey (i.e., resistant to less than three of eight indicator antibiotics) due mainly to local strains that originated in the community. The proportion of nmMRSA increased to modest levels in the other cities. In eastern cities, this was due to the appearance of strains closely related to nmMRSA seen in other countries of the southwestern Pacific.

To assess the nature and diversity of various types of SHV and TEM derivatives in our hospital a survey was conducted. Sixty-seven extended-spectrum β-lactamases (ESBL)-producing nosocomial pathogens, isolated over a 12-month period, were analyzed by means of PCR and direct sequencing. SHV-5 was the predominant ESBL found in our region (38 strains). Other less frequent variants included SHV-2 and SHV-12 with two and three isolates, respectively. For the first time, an outbreak of 11 Pseudomonas aeruginosa producing SHV-5 was encountered. All bla_TEM-positive strains carried the non-ESBL TEM-1. The incidence of non-SHV non-TEM ESBLs was remarkably high as almost one out of three isolates harbored such an ESBL. The epidemiological and clinical impact of these findings must be carefully investigated and interpreted.

The phenotypic and genotypic characteristics of clinical and intestinal enterococcal isolates recovered from inpatients and outpatients of two Brazilian hospitals, located in Niterói city, Rio de Janeiro, Brazil, were compared. A total of 601 strains were studied, including 253 isolated from different clinical sources and 348 intestinal strains (205 isolated from inpatients and 143 from outpatients) recovered from fecal specimens. Isolates were identified by using conventional physiological tests and evaluated for high-level resistance to aminoglycosides (HLR-A) and resistance to vancomycin and ampicillin by the agar screening technique. Susceptibility to several antimicrobial agents was evaluated by the disk diffusion method. The genetic diversity of Enterococcus faecalis strains presenting HLR-A was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. E. faecalis was the most frequent species among clinical isolates (90.1%) and intestinal strains from inpatients (53.6%). E. casseliflavus was the prevalent species among intestinal isolates from outpatients (35.0%). Clinical isolates were shown to be resistant to erythromycin (53.0%), tetracycline (52.2%), ciprofloxacin (36.4%), gentamicin (36.4%), streptomycin (30.4%), chloramphenicol (34.4%), norfloxacin (32.0%), imipenem (3.2%), and ampicillin (2.8%). Vancomycin resistance was only detected in intrinsic vancomycin-resistant enterococcal species. The overall prevalence of HLR-A was 52.2% among clinical isolates and 40.5% among intestinal strains. However, HLR-A was significantly more frequent among intestinal strains obtained from inpatients (56.6%) than among strains from outpatients (17.5%). Three major clonal groups were found among E. faecalis strains exhibiting HLR-GE or HLR-GE/ST (clonal groups GE-A and GE-B), and strains exhibiting HLR-ST (clonal group ST-A). HLR-A, particularly HLR-GE, was most frequently associated with enterococcal strains of nosocomial origin. Isolates included in the major clonal groups were recovered from clinical and intestinal sources from patients in both hospitals, indicating both intrahospital and interhospital spread of strains.

Streptococcus pneumoniae is the third most frequent etiologic agent of bacterial meningitis in Poland. Therefore, the objective of this study was to characterize S. pneumoniae isolates responsible for meningitis in Poland from 1997 to 2001 by determining their serotypes, antimicrobial susceptibility, and genetic relatedness. Among 36 serotypes and rough isolates, the most common were serotypes 3, 8, 19F, 6B, 4, and 23F, altogether accounting for 45.3% of cases. Penicillin-nonsusceptible pneumococci constituted 14.8% of all isolates. A high heterogeneity of pneumococcal isolates concerning serological features as well as genetic relatedness based on pulsed-field gel electrophoresis analysis was observed. With the exception of some serotypes (e.g., 3, 7F, 19F), isolates of the same serotype do not appear to be more closely related to each other than to isolates of different serotypes.

A joint study by the Public Health Laboratory Service and the Veterinary Laboratories Agency of resistance to antimicrobials in isolates of Salmonella enterica serotypes Enteritidis, Typhimurium, Hadar, and Virchow from humans and food-producing animals in England and Wales in 2000 has demonstrated that resistance was most common in Typhimurium, particularly in strains of definitive phage type (DT) 104. However resistance was also common in other phage types, particularly DTs 193 and 208 and phage type U302. Multiresistant strains of DT208 appeared to be predominantly associated with pigs; for the other phage types, the human/food-producing animal relationships of drug-resistant isolates were more complex. For Enteritidis, Virchow, and Hadar, there were substantial differences in the resistance spectra of isolates from humans and food-producing animals, suggesting that food-producing animals bred in England and Wales may not be the primary sources of drug-resistant strains of these serotypes causing infections in humans. Further phenotypic and molecular comparison of drug-resistant isolates of these serotypes may be required to ascertain the sources of strains responsible for infections in humans.

To screen for vancomycin-resistant enterococci (VRE) colonization in hospitalized patients and to study molecular evolution and alterations of Tn1546-like elements in VRE among potentially at-risk patients, a 3-year surveillance protocol in an Intensive Care Unit was performed. A total of 397 patients were screened in the period June, 1997-June, 2000, and VRE were isolated from rectal swabs taken at admission, weekly, and when clinically indicated. The susceptibility of the enterococci was determined by the disk diffusion and broth dilution methods. The presence of vancomycin-resistance genes (vanA, vanB, and vanC) was assessed by polymerase chain reaction (PCR); genetic clonality of isolates was assessed by pulsed-field gel electrophoresis (PFGE); Tn 1546 types were obtained by restriction fragment length polymorphism (RFLP) analysis of Tn1546 PCR fragments. Thirty-four strains, 31 identified as Enterococcus faecium and 3 strains as E. faecalis, were isolated from 12 of the 397 patients (3.0%); all strains were VanA as assessed by PCR and were resistant to the other antibiotics tested and showed high-level resistance to aminoglycosides. Enterococci isolated during the study period showed that different genetic backgrounds of strains, determined by PFGE combined with RFLP of Tn1546, are present in all the strains isolated in the study. PFGE type B was predominant in 1998 and 1999, and insertion sequence movements were found to have a role in the evolution of VanA resistance elements found in all strains. This study demonstrates that single patients may be colonized by closely related VRE with several PFGE types containing a wide variety of VanA elements. Moreover, isolates with identical PFGE types may contain different VanA elements reflecting rearrangements mediated by insertion sequences in VRE strains during their stay in the gastrointestinal tract.

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. β-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different β-lactams showed by these 20 isolates were as follows (in μg/ml): ampicillin (64->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8->128), cefazolin (32->256), cefoxitin (4->128), cefotaxime (1-64), ceftazidime (2->64), ceftriaxone (0.5-64), imipenem (≤0.06-0.25), and aztreonam (2->32). TEM, SHV, CMY, and FOX β-lactamase genes were analyzed by PCR and sequencing. The β-lactamase genes detected were the following ones (number of isolates): bla_TEM-1b(3), bla_TEM-1a (1), bla_TEM-30f (2), bla_TEM-1b + bla_CMY-2 and bla_SHV-12 (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type β-lactamase in 6 isolates. Besides, a high variety of plasmidic β-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 β-lactamase has been detected in E. coli isolates of animal origin.

A total of 331 invasive nonduplicated Streptococcus pneumoniae isolates from three sampling periods during 1996 to 2001 were tested for susceptibility to recently developed fluoroquinolones. Five major serotypes, 23F, 6B, 14, 19F, and 3, were frequently encountered in this collection. Penicillin nonsusceptible isolates constituted 52.9% from 1996 to 1997, 61.6% from 1998 to 1999, and 60.0% from 2000 to 2001. Fifty-seven percent of the isolates were susceptible to cefotaxime, 56.5% to ceftriaxone, 54.1% to cefepime, and 52.6% to cefuroxime. Macrolide-susceptible isolates constituted less than 14% of the total sample, and no vancomycin-resistant isolates were detected. For fluoroquinolones, MIC₉₀ was lowest for gemifloxacin (MIC₉₀ = ≤0.12 μg/ml), followed by moxifloxacin (MIC₉₀ = 0.25 μg/ml), gatifloxacin (MIC₉₀ = 0.5 μg/ml), sparfloxacin (MIC₉₀ = 0.5 μg/ml), levofloxacin (MIC₉₀ = 1 μg/ml), and ciprofloxacin (MIC₉₀ = 2 μg/ml). All isolates were susceptible to sparfloxacin, levofloxacin, gatifloxacin, and gemifloxacin apart from one isolate (0.3%), which was simultaneously resistant to sparfloxacin, levofloxacin, and gatifloxacin. Mutations at the positions S81F of GyrA and D435N and I460V of ParC were detected for this multiple drug resistant isolate. The in vitro results suggest that recently developed fluoroquinolones are very effective against invasive S. pneumoniae isolates in Taiwan. Nevertheless, emerging fluoroquinolone resistance should be acknowledged and clinicians alerted. Surveillance should be carried out to monitor any changes in antibiotic resistance of S. pneumoniae.

A national survey was conducted using a mailed questionnaire in 2000 to quantify the prevalence in Italian hospitals of policies aimed to reduce the emergence and dissemination of resistant strains. The study population consisted of all of the Italian teaching and research hospitals, as well as all public hospitals with 300 beds or more; a 50% random sample of public hospitals smaller than 300 beds was selected. The overall response rate was 80% (428/535). Of the respondents, 9.6% claimed to have implemented a surveillance system of antimicrobial resistance and to be able to identify repeated isolations; 90% had a hospital formulary in place, 50% had a pharmacy committee, and 18% had an antibiotic policy subcommittee that met at least once a year in 1999. Forty-one percent implemented restriction policies based on written justifications for antibiotics, and 8% provided susceptibility results for first line antibiotics only. Antibiotic consumption was monitored by using the Defined Daily Dose, as a unit of measure, in 11% of the cases. Of the respondent hospitals, 251 (58.6%) claimed to have defined clinical practice guidelines for handwashing, 156 (36.4%) for isolation procedures, and seven (1.6%) for the control of methicillin-resistant Staphylococcus aureus (MRSA) infections. Our data demonstrates that intensive efforts are necessary to understand barriers better to change and to identify solutions to adopt uniform, comprehensive policies for antimicrobial resistance in Italy.

We characterized susceptibilities and genotypes in a series of Pseudomonas aeruginosa isolates from five cases of urinary tract infections (UTIs) to evaluate clonal shifts of carbapenem resistance. In one case, a series of isolates showed different susceptibility patterns for carbapenems but an identical genotype. In another case, genotypes varied among 4 P. aeruginosa isolates from recurrent UTIs over 9 months. Although the patient had been treated with no antibiotic immediately before isolation, the susceptibility patterns for carbapenems and ceftazidime varied. Further analysis in these two cases of outer membrane protein profiles showed that loss of OprD production resulted in reduced susceptibilities to carbapenems in all of the carbapenem-resistant isolates. Loss of OprD production was likely due to oprD gene inactivation in both of cases, since the carbapenem-resistant isolates showed no cross resistance to levofloxacin and chloramphenicol compared with the carbapenem-susceptible isolates. There was another case in which all isolates showed similar susceptibility patterns for carbapenems and ceftazidime, and an identical genotype during the intermittent use of antibiotics over 5 months. In two cases, a single course of antibiotic therapy resulted in eradication of P. aeruginosa. Our results suggest that clonal shifts of carbapenem resistance in P. aeruginosa may result from loss of OprD during antibiotic treatment. Therefore, it is important for clinicians to monitor susceptibilities to antibiotics, especially carbapenems, in P. aeruginosa isolated during therapy.