Resistance to second- and third-generation cephalosporins among Escherichia coli and Klebsiella species is rare in Finland

Objective: To investigate the antimicrobial resistance of Escherichia coli and Klebsiella spp. from pus, urine and respiratory specimens, with particular emphasis on the detection of third-generation cephalosporin resistance.
Methods: E. coli (698) and Klebsiella sp. (476) strains from pus, respiratory and urinary specimens from hospital patients were collected from 19 laboratories. Data about consumption of third-generation cephalosporins and cefuroxime were collected from 24 hospitals. Antimicrobial susceptibility was tested with disk diffusion in primary laboratories and by an agar dilution method. Extended-spectrum β-lactamase (ESBL) production was studied with a double disk synergy test and an ESBL Etest. The β-lactamase classes were characterized with polymerase chain reaction probes of the TEM and SHV β-lactamase families and isoelectric focusing.
Results: Only 0.6% of E. coli and 2.3% of Klebsiella spp. strains were resistant or intermediately resistant to cefotaxime, ceftriaxone and/or ceftazidime. The ESBL producers detected comprised one E. coli harboring TEM-like genes and five Klebsiella pneumoniae strains, two of which harbored SHV-like genes, two TEM-like genes and one both. Although consumption of cefuroxime has increased in the years 1990–1994, from 3.48 to 5.84 defined daily doses/100 bed-days, and the consumption of third-generation cephalosporins from 1.25 to 1.94 defined daily doses/100 bed-days, cefuroxime resistance of E. coli was only 3%.
Conclusion: Although the use of broad-spectrum cephalosporins has increased, resistance to second- and thirdgeneration cephalosporins is still rare in Finland.     

Antimicrobial resistance patterns among aerobic Gram-negative bacilli isolated from patients in intensive care units: results of a multicenter study in Russia

Objective: To determine the antimicrobial resistance patterns among aerobic Gram-negative bacilli isolated from patients in intensive care units (ICUs) in different parts of Russia.
Methods: During 1995–96, 10 Russian hospitals from different geographic areas were asked to submit 100 consecutive Gram-negative isolates from patients with ICU-acquired infections. Minimal inhibitory concentrations (MICs) of 12 antimicrobials were determined by Etest and results were interpreted according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines.
Results: In total, 1005 non-duplicate strains were obtained from 863 patients. The most common species were Pseudomonas aeruginosa (28.8%), Escherichia coli (21.4%), Klebsiella pneumoniae (16.7%), Proteus mirabilis (9.7%), Enterobacter spp. (8.2%) and Acinetobacter spp. (7.7%). High levels of resistance were seen to second- and third-generation cephalosporins, ureidopenicillins, β-lactam/β-lactamase inhibitor combinations and gentamicin. The most active agents were imipenem (no resistance in Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter spp. and Acinetobacter spp., 7% resistance in Pseudomonas aeruginosa ), amikacin (7% resistance in Pseudomonas aeruginosa and Acinetobacter spp., 4% in Enterobacter spp., 1% in Escherichia coli and Proteus mirabilis, no resistance in Klebsiella pneumoniae ) and ciprofloxacin (15% resistance in Pseudomonas aeruginosa, 5% in Enterobacter spp. and Proteus mirabilis, 2% in Klebsiella pneumoniae, 1% in Escherichia coli ).
Conclusions: Second- and third-generation cephalosporins, ureidopenicillins, β-lactam/β-lactamase inhibitor combinations and gentamicin cannot be considered as reliable drugs for empirical monotherapy for aerobic Gram-negative infections in ICUs in Russia.     

Bacterial and epidemiologic study of the resistance to oxyiminocephalosporins in Escherichia coli in a French hospital

Objective: To understand the mechanisms and epidemiology of resistance to oxyiminocephalosporins in Escherichia coli over a 2-year period in a French hospital.
Methods: Forty-four strains, resistant or intermediately resistant to one of the oxyiminocephalosporins or aztreonam, were collected from 35 patients. MIC determinations were carried out for the 44 isolates using a panel of β-lactam antibiotics, and characterization of the β-lactamases they produced by isoelectric focusing and catalytic activity measurement. Extended-spectrum β-lactamase production was studied by use of the double disk diffusion test. Conjugation experiments were used to search for plasmidic cephalosporinase. An epidemiologic study was then performed, by use of molecular typing of the strains with an ERIC-PCR method and a case-control analysis.
Results: Less than 1% of all the E. coli isolates at our hospital showed decreased susceptibility to oxyiminocephalosporins. Only three of the 44 isolates showed synergy between clavulanate and a third-generation cephalosporin and produced an extended-spectrum β-lactamase. For the other strains, a β-lactamase with a highly basic isoelectric point was detected. Spectrophotometric measures confirmed that most of these isolates were AmpC hyper-producers. No plasmidic cephalosporinase could be detected by conjugation experiments. Molecular typing showed all isolates to be different, except for two strains isolated in two patients of the same hospital unit, and for the repeated isolates of some patients. When 20 case patients were compared to 40 randomly selected control patients, prior receipt of an antimicrobial and more specifically of a β-lactam agent was significantly associated with case patients.
Conclusions: Although it appears to be very rare, the resistance to broad-spectrum cephalosporins needs our attention, because of the high frequency of E. coli infections and β-lactam use in their treatment.     

Bactericidal activity of three β-lactams alone or in combination with a β-lactamase inhibitor and two aminoglycosides against Klebsiella pneumoniae harboring extended-spectrum β-lactamases

Objective: To study the bactericidal activity of β-lactam antibiotics (imipenem, cefepime, cefpirome) alone or in combination with a β-lactamase inhibitor (sulbactam) in the presence or absence of aminoglycoside (amikacin or isepamicin) against Klebsiella pneumoniae strains producing extended-spectrum β-lactamases (ESBLs).
Methods: We characterized 10 strains by means of analytic isoelectric focusing and pulsed-field gel electrophoresis. The ESBLs produced by these strains were derived from either TEM (TEM-1, TEM-2) or SHV-1. The killing-curve method was used for this bacterial investigation. Bacteria (final inoculum 5×10 ⁵ CFU/mL) were incubated with antibiotics at clinical concentrations obtained in vivo.
Results: All the combinations with cefepime or cefpirome + sulbactam were bactericidal, with a 4 log₁₀ decrease being obtained within 6 h without regrowth at 24 h, whereas imipenem alone, and combinations, gave a bactericidal effect within 6 h. The two cephalosporins alone decreased the inoculum of 4 log₁₀ at 6 h but regrowth was observed at 24 h. When the aminoglycoside was added, this bactericidal effect was obtained within 3 h with amikacin and within 1 h with isepamicin.
Conclusions: Cefepime + sulbactam or cefpirome + sulbactarn may be an alternative to imipenem for the treatment of patients with ESBL-producing K. pneumoniae. Aminoglycosides are often associated in nosocomial infections due to ESBL-producing K. pneumoniae: isepamicin acted faster than amikacin, but both worked well. To conclude, it may be prudent to avoid extended-spectrum cephalosporins as single agent when treating serious infections due to ESBL-producing K. pneumoniae. Addition of a β-lactamase inhibitor such as sulbactam ± aminoglycoside is advisable to avoid failure of treatment.     

Phage types, antibiotic susceptibilities and plasmid profiles of Salmonella typhimurium and Salmonella enteritidis strains isolated in Istanbul, Turkey

Objective   To examine 13 Salmonella typhimurium and 22 S. enteritidis strains isolated from individual cases of gastroenteritis for their phage types, antibiotic susceptibilities and plasmid profiles.
Methods   The phage typing of S. typhimurium strains was done according to the method of Anderson et al, and the phage typing scheme of Ward et al was used for phage typing of S. enteritidis strains. Antibiotic susceptibility testing was performed by the Kirby–Bauer disk diffusion method. Extended-spectrum β-lactamase production of the strains was determined by the three-dimensional method. Plasmid profiles of the strains were examined using the method described by Kado and Liu with some modification by Graeber et al.
Results   Two S. typhimurium strains were DT 193 and one was DT 22, whereas 10 strains were untypable. PT 4 was the predominant phage type among S. enteritidis strains. Four S. enteritidis strains were DT 6a, three strains were PT 1 and one strain was PT 8, whereas only one strain was untypable. Eleven of 13 S. typhimurium and three of 22 S. enteritidis strains were found to be multiresistant. Ten different resistance patterns among S. typhimurium and four different resistance patterns among S. enteritidis strains were detected. Extended-spectrum β-lactamase production was detected in 10 of 13 S. typhimurium and in three of 22 S. enteritidis strains. All S. typhimurium strains but one were found to contain at least one plasmid, with molecular masses varying between 4 and 107 MDa, and 11 different plasmid patterns were determined. Plasmid pattern analysis permitted further differentiation of the S. enteritidis strains into nine groups. A serovar-specific virulence plasmid of 36 MDa was detected in 13 of 22 S. enteritidis strains.
Conclusions   The results suggest that the majority of S. typhimurium strains were closely related.     

Effect of β-lactam antibiotics on the in vitro development of resistance in Pseudomonas aeruginosa

Objective   To investigate whether stepwise selection of resistance mutations may mirror the continued bacterial exposure to antibiotics that occurs in the clinical setting.
Methods   We examined the in vitro development of resistance to a number of commonly used antibiotics (cefepime, cefpirome, ceftazidime, cefotaxime, piperacillin and imipenem) in Pseudomonas aeruginosa , a significant nosocomial pathogen. Stepwise resistance was assessed by serial passage of colonies located nearest to the inhibition zone on antibiotic-containing gradient plates.
Results   The lowest frequencies of spontaneous resistance mutations were found with cefepime and imipenem; these drugs also resulted in the slowest appearance of resistance of spontaneous resistance mutations. In five wild-type P. aeruginosa strains, cefepime-selected isolates required a mean of 30 passages to reach resistance; resistance occurred more rapidly in strains selected with other cephalosporins. P. aeruginosa strains that produced β -lactamase or non-enzymatic resistance generally developed resistance more rapidly than wild-type strains. For most strains, resistance to all antibiotics except imipenem correlated with increased levels of β -lactamase activity. Cross-resistance of cephalosporin-selected resistant mutants to other cephalosporins was common. Cephalosporin-resistant strains retained susceptibility to imipenem and ciprofloxacin.
Conclusions   From our in vitro study, we can conclude that the rate of development of resistance of P. aeruginosa is lower with cefepime compared with other cephalosporines.     

Inducible β-lactamase-mediated resistance to third-generation cephalosporins

The emergence of multiple resistance to β-lactam antimicrobial agents is a major problem in the treatment of patients infected with Enterobacteriaceae that characteristically produce inducible β-lactamases. Inducible and 'derepressed' AmpC β-lactamases are produced by Enterobacter spp., Citrobacter freundii, Serratia marcescens, Morganella morganii and Providencia spp. Resistance to broad-spectrum β-lactams has emerged in 16-44% of these strains from infections treated with one of the newer cephalosporins, even in combination with other antimicrobials. Multiply resistant organisms have spread widely both locally, within hospitals, and nationally. This trend has been shown to correlate closely with the extent of usage of some third-generation cephalosporins. These resistant strains, especially Enterobacter spp., are more regularly isolated from seriously ill patients (especially from respiratory sources), or in intensive care units and pose one of the greatest challenges to contemporary chemotherapy of infections in hospitalized patients. Zwitterionic fourth-generation cephalosporins combine the properties of rapid bacterial outer membrane penetration with high stability to AmpC β-lactamase with good affinity for the penicillin-binding proteins to achieve in vitro activity against AmpC-producing organisms, including the majority of strains highly resistant to ceftazidime and other earlier generation cephalosporins. These features have contributed to their clinical success in the therapy of infections caused by Enterobacter spp. with and without resistance to third-generation compounds. Other alternative agents for chemotherapy of infections due to AmpC β-lactamase-producing strains (inducible or derepressed expression) should also be considered e.g. carbapenems, aminoglycosides and fluoroquinolones.     

Is antimicrobial resistance also subject to globalization?

In recent years one of the more alarming aspects of clinical microbiology has been the dramatic increase in the incidence of resistance to antibacterial agents among pathogens causing nosocomial as well as community-acquired infections. There are profound geographic differences in the incidence of resistance among pathogens of the respiratory tract, only some of which can be explained by the local use of antibiotics. A high percentage of Moraxella catarrhalis strains produce β -lactamase and are thus resistant to many β -lactam antibiotics. In contrast, β -lactamase production among strains of Haemophilus influenzae rarely reaches more than 30% around the world. Methicillin-resistance in Staphylococcus aureus is a common and increasing problem in hospitals but its extent varies both locally and nationally. Resistance is usually associated with the local spread of resistant strains. High standards of hygiene in hospitals can prevent the spread of such strains but once established they can be difficult to eradicate. Although Streptococcus pyogenes remains highly susceptible to penicillins, even after many decades of their use, resistance to macrolides has occurred. This resistance can rise and fall. Although the increase of macrolide resistance in S. pyogenes can often be associated with an increase in the use of these drugs, this is not always so. In some cases it has been shown to be caused by the spread of one or more resistant clones. Eradication of these clones can reduce the level of resistance markedly. Resistance to both macrolides and penicillins among strains of Streptococcus pneumoniae is seen world-wide but is highly variable from country to country. Local habits of drug usage may play a part. In Italy, for example, there is preference for the use of parenteral third-generation cephalosporins for some severe infections and there is a corresponding low level of penicillin-resistance among pneumococci.     

TEM-24 extended-spectrum β-lactamase-producing Enterobacter aerogenes: long-term clonal dissemination in French hospitals

Objective To investigate interstrain relatedness of TEM-24-producing Enterobacter aerogenes clinical strains isolated between 1993 and 1998 in 10 French hospitals from nine areas by pulsed-field gel electrophoresis (PFGE) and plasmid patterns.
Methods Fifteen TEM-24 - producing strains and a set of 16 control strains having various other antibiotic resistance phenotypes were genotyped by PFGE. Plasmid DNA from TEM-24 - producing strains and transconjugants was analyzed .
Results Analysis of Xba I macrorestriction patterns revealed only minor variations, and showed that all 15 TEM-24 - producing strains were closely related. Some isolates originating from distant areas had indistinguishable patterns . According to their clustering correlation coefficients, they were also genomically distant from the control strains . Two plasmid patterns were observed in TEM-24-producing strains, one of them in 13 of the strains. Large plasmids of 85 kb encoding TEM-24 β-lactamase were present in all isolates and, in all except one strain, could be transferred with high frequency by conjugation .
Conclusions These results confirm that the spread of the TEM-24 extended-spectrum β-lactamase in France was essentially due to the dissemination of a single clone .     

Dissemination of cephalosporin-resistantSerratia marcescens strains producing a plasmidic SHV type beta-lactamase in Greek hospitals

The resistance to third generation cephalosporins in nineSerratia marcescens strains isolated in Greek hospitals was studied. Eight of the strains transferred resistance toEscherichia coli by means of large plasmids that encoded for an extended-spectrum -lactamase. Hybridization, isoelectric focusing and hydrolysis studies showed that the enzyme resembled the SHV-5 -lactamase. In the eight isolates that possessed the SHV type enzyme, cephalosporinase expression was inducible, whereas the remaining strain was a cephalosporinase hyperproducing strain. Introduction of a plasmid coding for the regulatoryampD gene in the latter strain eliminated -lactamase production and rendered the strain susceptible to cephalosporins.     

Molecular analysis of ampicillin-resistant sporadic Salmonella typhi and Salmonella paratyphi B clinical isolates

Objective: To study the mechanisms of antibiotic resistance in Salmonella typhi and Salmonella paratyphi B clinical isolates, and the clonality of resistant strains.
Method: Antibiotic susceptibility was tested by disk-agar diffusion. Conjugation experiments and plasmid analysis by agarose gel electrophoresis after Eco RI digestion were followed by hybridization to a digoxigenin-labeled TEM-type β-lactamase probe. DNA fingerprints were obtained by pulsed-field gel electrophoresis of Xba I-digested chromosomal DNA.
Results: Three S. typhi isolates (7% of the isolates studied), of which one was ampicillin resistant and the other two multiresistant (ampicillin, chloramphenicol, tetracycline, sulfamethoxazole/trimethoprim and streptomycin), and two ampicillin-resistant S. paratyphi B isolates (25% of the isolates studied) were further evaluated. A 34-MDa conjugative plasmid, previously isolated from Salmonella enteritidis , conferred ampicillin resistance. A 100-MDa conjugative plasmid encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim, as well as ampicillin. Chromosomal fingerprinting revealed two distinct resistant strains for each serovar which were different from a matched set of sensitive S. typhi strains.
Conclusions: Two conjugative, TEM-type β-lactamase-encoding plasmids conferred ampicillin resistance to S. typhi and S. paratyphi B. The 34-MDa plasmid was identical to that previously characterized from S. enteritidis , while the 100-MDa plasmid also encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim. Resistant isolates did not belong to a single clone but rather represented distinct strains.     

Detection of SHV-5 extended-spectrum beta-lactamase inKlebsiella pneumoniae strains isolated in Italy

Thirty-fiveKlebsiella pneumoniae strains isolated during 1993–1994 in intensive care units of a large Italian hospital were examined for the presence of extended-spectrum -lactamases. Five strains showed a high level of simultaneous resistance to -lactam agents, including ceftazidime and aztreonam, conferred by a large (130 kb) self-transferable plasmid (in 4 of 5 strains). Isoelectrofocusing and hybridisation studies suggest that these enzymes can be identified as SHV-5 extended-spectrum -lactamases. Pulsed-field gel electrophoresis analysis showed three different genomic fingerprinting profiles, while plasmid restriction enzyme digestion revealed three different patterns, demonstrating that the diffusion of SHV-5 -lactamase is not the result of a single strain or plasmid dissemination.     

Susceptibility of Enterobacteriaceae to β-lactam agents and fluoroquinolones: a 3-year survey in France

Objective   To assess trends in the susceptibility to β -lactam agents and to fluoroquinolones of clinically relevant Enterobacteriaceae isolated over a 3-year period in 14 French hospital laboratories.
Methods   During the second quarter of 1996, 1997 and 1998, 180 consecutive non-duplicate isolates of Enterobacteriaceae were collected in each center. Sixteen β -lactams and four quinolones were tested by the disk diffusion method. In addition, the double-disk synergy test was used to screen for the production of extended-spectrum β -lactamase (ESBL).
Results   Totals of 2507, 2312 and 2506 clinical isolates were obtained in each period, respectively. The distribution of Enterobacteriaceae species according to clinical specimens and wards was similar in each study period. No significant variation in the susceptibility rates to β -lactams and fluoroquinolones was observed, except in Klebsiella pneumoniae and Enterobacter aerogenes. The prevalence of ESBL-producing isolates decreased from 18% to 9% in the former, while it increased from 32% to 54% in the latter. At the same time, the susceptibility to ofloxacin and pefloxacin increased for K. pneumoniae ( P  < 0.003) and cephalosporinase-producing species ( P  < 0.05), except Enterobacter spp.
Conclusion   Over the 3-year study period β -lactams and fluoroquinolones remained highly active against Enterobacteriaceae clinical isolates, with the exception of E. aerogenes , probably as a result of the dissemination of multiresistant clones in French hospitals.     

Comparison of screening methods for TEM- and SHV-derived extended-spectrum β-lactamase detection

Objective   To compare common extended-spectrum β -lactamase (ESBL) screening methods and β -lactams for their ability to detect TEM- and SHV-related ESBL enzymes.
Methods   This study compared disk diffusion testing by NCCLS methodology, the Jarlier double disk test, a disk-on-disk test, a modified three-dimensional test and the E test method for their sensitivity and specificity in detecting TEM- and SHV-related ESBL producers. Three negative and 22 positive controls were studied. These were two Klebsiella pneumoniae and 23 Escherichia coli transconjugants. Seventeen β -lactam antibiotics were tested: cefamandole, cefotetan, cefoxitin, cefuroxime, cefixime, cefoperazone, cefotaxime, cefpodoxime, cefsulodin, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, moxalactam, cefepime, cefpirome and aztreonam.
Results   NCCLS disk diffusion was 14% sensitive with ceftriaxone, 36% with cefotaxime, 64% with aztreonam, 68% with cefpodoxime, and 73% with ceftazidime. Cefoperazone, cefamandole, cefpodoxime and cefpirome showed 91% sensitivity using the Jarlier test. Using the disk-on-disk test, cefsulodin showed 95% sensitivity, and cefoperazone, cefepime and cefamandole showed 91% sensitivity. With the modified three-dimensional test, cefoperazone, cefpodoxime and cefpirome showed 91% sensitivity.
Conclusions   For practical reasons, we would recommend use of either the Jarlier test or the commercial cephalosporin disks containing clavulanic acid to screen for ESBL producers. Cefoperazone, cefamandole, cefpodoxime and cefpirome showed good sensitivity across the methods tested.     

Molecular Epidemiology of Extended-Spectrum β-Lactamases from Klebsiella pneumoniae Strains Isolated in Zagreb, Croatia

 In order to assess the molecular epidemiology of 40 previously identified extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae, gene sequencing was performed. While the previous examination of these isolates revealed one TEM producer, the sequencing procedure performed in this study identified 13 additional TEM producers, and all of the sequenced genes reflected production of nonESBL TEM-1. All 38 suspected SHV producers were confirmed to be carriers of bla _SHV-ESBL genes using the PCR/NheI test and sequencing. Among them, types SHV-2, SHV-5, and SHV-12 were found in 20, 10, and 7 isolates, respectively, and SHV-2a was identified in 1. SHV-5 and SHV-12 conferred higher resistance to ceftazidime and cefepime, while SHV-2 and SHV-2a raised the minimum inhibitory concentrations of cefotaxime and cefpirome. Fourth-generation cephalosporins were found to be more active against the isolates than third-generation cephalosporins.     

Presence of acetyl-transferases and adenylyl-transferases in gentamicin-resistant transconjugants.

The aim of this work was to test the production of aminoglycoside modifying enzymes in 20 gentamicin-resistant transconjugants obtained from clinical strains of Entero-bacteriaceae. The susceptibility to aminoglycosides was determined by disc diffusion method and agar dilution method according to European Committee for Clinical Laboratory Standards, 1988. The transfer of gentamicin-resistance R-plasmids was made by conjugation on a solid medium with recipients E. coli K12. Phosphocellulose paper binding assay with 14C acetyl. CoA and 14C.ATP by Haas and Dowding was performed to reveal the enzyme production. Four different acetyl-transferases have been found: AAC/3/I, AAC/3/-V, AAC/3/-IV which modify gentamicin, and AAC/6'/-I with activity on amikacin. Only two of the transconjugants showed adenylyl-transferase activity:AAD/2"/. Some of strains tested possessed two enzymes. The most interesting finding was that the majority of strains owned AAC/3/-IV, which modifies apramycin. This was be explained with the fact that apramycin is still in a large use for animal husbandry in Bulgaria. In conclusion: four different acetyl-transferases: AAC/3/-I, AAC/3/-V, AAC/3/-IV and AAC/6'/-I and the adenylyl-transferase AAD/2"/ were found to be the biochemical mechanisms of resistance to aminoglycosides in 20 gentamicin-resistant transconjugants.     

Evidence for a true post-β-lactamase-inhibitor effect of clavulanic acid against Klebsiella pneumoniae and Haemophilus influenzae

Objective   The purpose of this study was to investigate and characterize in vitro the post- β -lactamase inhibitor effect (PLIE) of clavulanic acid against two β -lactamase-producing species of bacteria.
Methods   The PLIE was investigated against one strain of Klebsiella pneumoniae and one strain of Haemophilus influenzae . A stationary-phase inoculum of about 10⁷ colony-forming units per mL of each bacterium was pre-exposed for 2 h to clavulanic acid, either alone or in combination with amoxicillin at various concentrations. After pre-exposure, the dilution required to remove the β -lactamase inhibitor was 1:100 or 1:1000 according to the bacterial species and their susceptibilities to clavulanic acid. Bacteria were counted hourly after drug removal, on solid agar medium.
Results   Control cultures exposed to amoxicillin alone after dilution, showed a delay in growth, which may be inherent to the time required to synthesize sufficient β -lactamase after the dilution steps. Control experiments clearly distinguished the post-antibiotic effect and the growth delay from the PLIE.
Conclusion   The PLIE could be one of several factors explaining why β -lactam/ β -lactamase inhibitor combinations remain effective throughout the dosing interval, even if a few hours after in vivo administration, serum concentrations of β -lactamase inhibitor fall below levels that are active in vitro.     

Natural antibiotic susceptibility of Enterobacter amnigenus, Enterobacter cancerogenus, Enterobacter gergoviae and Enterobacter sakazakii strains

Objective   To investigate the natural susceptibility to 69 antimicrobial agents of 107 Enterobacter strains comprising E. amnigenus ( n  = 18), E. cancerogenus ( n  = 26), E. gergoviae ( n  = 28) and E. sakazakii ( n  = 35).
Methods   Minimal inhibitory concentrations (MICs) were determined with a microdilution procedure in Isosensitest broth and cation-adjusted Mueller–Hinton broth.
Results   All the species were naturally sensitive or intermediate to tetracyclines, amino-glycosides, numerous β -lactams (acylureidopenicillins, ticarcillin, ampicillin/sulbactam, several cephalosporins, carbapenems, aztreonam), quinolones, antifolates, chloramphenicol and nitrofurantoin. Natural resistance was found to penicillin G, oxacillin, several macrolides, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Species-related differences in natural susceptibility were found to some β -lactams, azithromycin and fosfomycin. Whereas E. gergoviae was the most susceptible species to azithromycin, E. cancerogenus was most susceptible to fosfomycin and was the only species showing natural resistance to amoxicillin, amoxicillin/clavulanic acid, cefaclor, cefazoline, loracarbef and cefoxitin. There were only minor medium-dependent differences in susceptibility to most antibiotics.
Conclusions   The present study establishes a database concerning the natural susceptibility of recently established Enterobacter species to a wide range of antibiotics, which can be applied for the validation of routine susceptibility test results. β -Lactam susceptibility patterns indicate the expression of species-specific β -lactamases expressed at high or low levels in all the species except E. sakazakii .     

Molecular characterisation of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella spp. isolates at a tertiary-care centre in Lebanon

The prevalence of bla _CTX-M, bla _TEM and bla _SHV genes among extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Escherichia coli ( n  = 50) and Klebsiella spp. ( n  = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla _CTX-M-15, bla _TEM-1, bla _OXA-1 and six bla _SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants.     

Antibiotic susceptibility and genotypic characterization of Haemophilus influenzae strains isolated from nasopharyngeal specimens from children in day-care centers in eastern France

Objective   To determine the overall carriage rate for Haemophilus influenzae in young children in day-care centers, the frequency of resistance to various classes of antibiotic, and the clonal relationship between isolates of the various resistant phenotypes.
Methods   Nasopharyngeal (NP) specimens were obtained and cultured on chocolate agar with bacitracin. Antibiotic susceptibility testing and serotyping were performed for all isolates. The genetic polymorphism of ampicillin-susceptible and β-lactamase-producing isolates was studied by pulsed-field gel electrophoresis using Sma I.
Results   Of the 596 NP secretion cultures, 152 (25.5%) were positive for H. influenzae . Sixty-four (42.1%) isolates produced β-lactamase and two (1.3%) were ampicillin resistant but did not produce β-lactamase. We were unable to serotype 150 isolates; one isolate belonged to capsular serotype e and one to serotype f. Forty-six major DNA patterns were identified among 76 randomized isolates. β-lactamase producing isolates more frequently showed EP than ampicillin-susceptible isolates P  < 10⁻⁴. The frequency of isolates with EP was significantly lower in day-care centers attended by less than 20 children than in those attended by more than 20 children ( P  = 0.020).
Conclusions   Resistance due to β-lactamase production has disseminated in some day-care centers, mostly by person-to-person spread but also via the possible conjugal transfer of large plasmids between strains. The size of day-care centers may affect the risk of transmission.