María-Isabel Morosini

Antibiotic-Selective Environments

The evolution and spread of antibiotic resistance depends on the antibiotic pressure exerted in the microbial environment. Selective effects occur in selective compartments, where particular antibiotic concentrations result in a differential growth rate of resistant bacterial variants. This may happen even at very low antibiotic concentrations able to select low-level-resistant bacteria. When more than one antibiotic is present in the environment, the multiple and fluctuating pressure produces the selection of bacterial variants that use multiple or multipurpose mechanisms or optimize a single mechanism of resistance to survive under the variable environmental conditions. Host factors such as immunity contribute to the selective process. Antibiotics themselves may promote bacterial diversity, either mediated by the random drift effect or triggering the increase of mutational events under bacterial stress. Analysis of selective environment-related antibiotic-host-bacteria interactions is essential to understanding the biology of antibiotic resistance.

Emergence and spread of antibiotic resistance following exposure to antibiotics

Within a susceptible wild-type population, a small fraction of cells, even <10(-9) , is not affected when challenged by an antimicrobial agent. This subpopulation has mutations that impede antimicrobial action, allowing their selection during clinical treatment. Emergence of resistance occurs in the frame of a selective compartment termed a mutant selection window (MSW). The lower margin corresponds to the minimum inhibitory concentration of the susceptible cells, whereas the upper boundary, named the mutant prevention concentration (MPC), restricts the growth of the entire population, including that of the resistant mutants. By combining pharmacokinetic/pharmacodynamic concepts and an MPC strategy, the selection of resistant mutants can be limited. Early treatment avoiding an increase of the inoculum size as well as a regimen restricting the time within the MSW can reduce the probability of emergence of the resistant mutants. Physiological and, possibly, genetic adaptation in biofilms and a high proportion of mutator clones that may arise during chronic infections influence the emergence of resistant mutants. Moreover, a resistant population can emerge in a specific selective compartment after acquiring a resistance trait by horizontal gene transfer, but this may also be avoided to some extent when the MPC is reached. Known linkage between antimicrobial use and resistance should encourage actions for the design of antimicrobial treatment regimens that minimize the emergence of resistance. Emergence of a resistant bacterial subpopulation within a susceptible wild-type population can be restricted with a regimen using an antibiotic dose that is sufficiently high to inhibit both susceptible and resistant bacteria.

In vitro selective antibiotic concentrations of beta-lactams for penicillin-resistant Streptococcus pneumoniae populations.

Therapeutic regimens containing beta-lactam antibiotics are selecting penicillin-resistant Streptococcus pneumoniae populations all over the world. The selective pressure after 4 h of exposure to different concentrations of amoxicillin, cefixime, cefuroxime, and cefotaxime for low-level or high-level penicillin-resistant S. pneumoniae was evaluated in an in vitro model with mixed populations with penicillin susceptibilities of 0.015, 0.5, 1, and 2 micrograms/ml. The antibiotic concentration selecting for low-level resistance strongly reduced the susceptible population. Increasing antibiotic concentrations tended to decrease the total proportion of penicillin-resistant bacteria because of reduced numbers of the low-level-resistant population. The antibiotic concentration selecting for high-level resistance produced fewer resistant populations, but most of the organisms selected represented high-level resistance. In general, amoxicillin was a good selector for the low-level-resistant population and a poor selector for high-level resistance; cefuroxime and cefotaxime were poor selectors for low-level resistance and better selectors than amoxicillin for high-level penicillin resistance. Cefixime was the best selector of low-level penicillin resistance. When only resistant populations were mixed, the strains with high-level resistance were selected even at low antibiotic concentrations. Determination of the effects of selective antibiotic concentrations on mixed cultures of bacteria expressing different antibiotic resistance levels may help researchers to understand the ecology and epidemiology of penicillin-resistant S. pneumoniae populations.

Biological Cost of AmpC Production for Salmonella enterica Serotype Typhimurium

ABSTRACT Chromosomally mediated AmpC-type β-lactamases are frequently found among Enterobacteriaceae. Hyperproduction of AmpC β-lactamase results in high-level resistance to β-lactam antibiotics. One striking feature of Salmonella is the absence of the structural ampC gene, encoding AmpC β-lactamase, in contrast with other members in theEnterobacteriaceae family, such as Escherichia,Citrobacter, or Enterobacter. The horizontal acquisition of ampC genes is one of the causes of the increased resistance to extended-spectrum cephalosporins and β-lactamase inhibitors among gram-negative rods. Nevertheless, despite the high number of β-lactam-resistant Salmonellaisolates so far described, only two strains expressing resistance to cephalosporin and β-lactamase inhibitors which is mediated by AmpC-type enzymes have been found. In this work, data are provided which support the possibility that the maintenance and expression of the ampC gene may represent an unbearable cost forSalmonella in terms of reduction of some of its lifestyle attributes, such as growth rate and invasiveness. The deleterious AmpC burden can be eliminated by decreasing the production of AmpC when both the regulatory gene, ampR, and ampC are present in Salmonella. Thus, it is suggested that the two genes have to be acquired together by Salmonella, leading to an inducible β-lactam resistance phenotype. AmpC synthesis did not produce major variations in the peptidoglycan composition ofSalmonella.

Reidentification of Streptococcus bovis Isolates Causing Bacteremia According to the New Taxonomy Criteria: Still an Issue?

ABSTRACT All Streptococcus bovis blood culture isolates recovered from January 2003 to January 2010 (n = 52) at the Hospital Universitario Ramón y Cajal were reidentified on the basis of their genetic traits using new taxonomic criteria. Initial identification was performed by the semiautomatic Wider system (Fco. Soria-Melguizo, Spain) and the API 20 Strep system (bioMérieux, France). All isolates were reidentified by PCR amplification and sequencing of both the 16S rRNA and sodA genes and by mass spectrometry using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Germany). Results of 16S rRNA/sod A gene sequencing were as follows: Streptococcus gallolyticus subsp. gallolyticus, 14/14 (number of isolates identified by 16S rRNA/number of isolates identified by sodA gene sequencing); Streptococcus gallolyticus subsp. pasteurianus, 24/24; Streptococcus spp., 7/0; Streptococcus infantarius subsp. infantarius, 0/2; Streptococcus lutetiensis, 0/5; Leuconostoc mesenteroides, 4/0; and Lactococcus lactis, 3/3. MALDI-TOF MS identified 27 S. gallolyticus isolates but not at the subspecies level, 4 L. mesenteroides isolates, 3 L. lactis isolates, and 6 S. lutetiensis isolates, whereas 12 isolates rendered a nonreliable identification result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. gallolyticus isolates into 3 major clusters clearly different from those of the S. gallolyticus subsp. pasteurianus isolates, which, in turn, exhibited no clonal relationship. The percentages of resistance to the tested antimicrobials were 38% for erythromycin, 23% for fosfomycin, 10% for levofloxacin, 6% for tetracycline, and 4% for co-trimoxazole. The most frequent underlying diseases were hepatobiliary disorders (53%), endocarditis (17%), and malignancies (12%). We conclude that sequencing of the sod A gene was the most discriminatory method and that S. gallolyticus subsp. pasteurianus appears to have a higher genetic diversity than S. gallolyticus subsp. gallolyticus.

New extended-spectrum TEM-type beta-lactamase from Salmonella enterica subsp. enterica isolated in a nosocomial outbreak.

A new extended-spectrum beta-lactamase was detected in a lactose-positive Salmonella enterica subsp. enterica strain that caused a nosocomial outbreak involving eight patients in a pediatric cardiology unit. This strain showed high levels of resistance to ceftazidime and aztreonam and relatively low levels of resistance to cefotaxime and ceftriaxone. Resistance was associated with a conjugative plasmid of 59 kb, which encoded a new beta-lactamase with an isoelectric point of 5.9 that strongly hydrolyzed ceftazidime and to a much lesser extent hydrolyzed cefotaxime. The enzyme activity was inhibited by clavulanate. The corresponding bla gene was cloned and sequenced. The deduced amino acid sequence showed three significant amino acid replacements with respect to the TEM-1 sequence: Arg-164-->His, Glu-240-->Lys, and Thr-265-->Met. This combination is unique among extended-spectrum beta-lactamases and served to characterize the new enzyme, TEM-27.

High prevalence in cystic fibrosis patients of multiresistant hospital-acquired methicillin-resistant Staphylococcus aureus ST228-SCCmecI capable of biofilm formation

OBJECTIVESnAlthough methicillin-resistant Staphylococcus aureus (MRSA) is increasingly recognized in cystic fibrosis (CF) patients, it has not been sufficiently studied in large series. We analysed all MRSA isolates recovered from respiratory secretions of patients attending our CF unit (1994-2006).nnnMETHODSnAntibiotic susceptibilities were determined using both planktonic and sessile bacteria as inocula. Genetic relationships were determined by PFGE and multilocus sequence typing (MLST). SCCmec type and the presence of the pvl gene were also investigated.nnnRESULTSnA total of 93 MRSA isolates (1-20 isolates per patient) were recovered from 18 of 77 CF patients with positive staphylococcal culture. Mean prevalence (4.4%) increased (P < 0.001) over time. All isolates were susceptible to linezolid, quinupristin/dalfopristin and co-trimoxazole but presented high resistance rates to amikacin (90%), gentamicin (85%), levofloxacin (81%) and erythromycin (69%). Except for macrolides and gentamicin, isolates were less susceptible growing in biofilms than in planktonic cultures. Fifteen different PFGE patterns were found, one of them consistently recovered for 6 years in the same patient. Identical clones were detected in several unrelated patients. MLST demonstrated that the international ST228 was the most frequent (67%) clone. The pvl gene was negative in all isolates and the SCCmec corresponded to types I (97%) and IV (3%). Strong mutators were not detected, but a considerable number were considered weak mutators.nnnCONCLUSIONSnDistinct microbiological and molecular features were detected among CF-MRSA isolates, probably due to adaptation to specific conditions in CF patients. Prevalence of MRSA increased among these patients, most of them colonized with a multiresistant biofilm-forming clone belonging to ST228-SSCmecI, suggesting cross-transmission or a common source.

MALDI-TOF MS improves routine identification of non-fermenting Gram negative isolates from cystic fibrosis patients.

Identification of non-fermenting Gram-negative bacteria (NFGNB) from cystic fibrosis (CF) patients is often limited. A collection of stored NFGNB isolates (n=182) recovered from CF patients over a 15 year period was examined. The routinely reported identification during this period was compared with that obtained by MALDI-TOF MS. Isolates giving discrepant identification at the genus level were further analyzed by 16S rDNA sequencing. The MALDI-TOF MS system identified 94% of the isolates, including Burkholderia cepacia and Pandoraea spp. isolates, the latter previously misidentified as other NFGNB by conventional microbiological methods. Lack of identification by MALDI-TOF MS was associated with the absence of entries in the database.

Selection of Naturally Occurring Extended-Spectrum TEM β-Lactamase Variants by Fluctuating β-Lactam Pressure

ABSTRACT Despite the large number of in vitro mutations that increase resistance to extended-spectrum cephalosporins in TEM-type β-lactamases, only a small number occur in naturally occurring enzymes. In nature, and particularly in the hospital, bacteria that contain β-lactamases encounter simultaneous or consecutive selective pressure with different β-lactam molecules. All variants obtained by submitting an Escherichia coli strain that contains ablaTEM-1 gene to fluctuating challenge with both ceftazidime and amoxicillin contained only mutations previously detected in naturally occurring β-lactamases. Nevertheless, some variants obtained by ceftazidime challenge alone contained mutations never detected in naturally occurring TEM β-lactamases, suggesting that extended-spectrum TEM variants in hospital isolates result from fluctuating selective pressure with several β-lactams rather than selection with a single antibiotic.

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases.

By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM beta-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of mutations appearing in naturally occurring TEM-beta-lactamases, studied in the same genetic context, may help to understand the evolution of extended-spectrum beta-lactamases.