Antimicrobial susceptibilities of clinical isolates of Haemophilus influenzae and Moraxella catarrhalis collected during 1999–2000 from 13 countries

Objective   To determine antimicrobial activity against Haemophilus influenzae and Moraxella catarrhalis .
Methods   A central laboratory performed NCCLS susceptibility testing for all isolates and β -lactamase and capsular serotype determinations for H. influenzae .
Results   A total of 2712 H. influenzae and 1079 M. catarrhalis were collected . H. influenzae susceptibilities were >90% for amoxicillin/clavulanate, cefaclor, loracarbef, cefprozil, cefuroxime, ciprofloxacin, azithromycin and clarithromycin and were <80% for trimethoprim/sulfamethoxazole and ampicillin. 19.3% were β -lactamase positive. The most common serotype was type-b (5.6%); 86.1% were nontypeable. M. catarrhalis had MIC₉₀ within therapeutic range for all antimicrobials except ampicillin.
Conclusion   The conclusion of the study is that antimicrobials, except ampicillin and trimethoprim/sulfamethoxazole, remain good empiric choices against H. influenzae and M. catarrhalis .     

The prevalence of antibiotic resistance in bacterial respiratory pathogens from Norway is low

Objectives   To determine the degree of bacterial susceptibility to the most commonly used drugs for respiratory infections in Norway, and to find if bacterial resistance is emerging.
Methods   Clinical isolates of Streptococcus pneumoniae , Haemophilus influenzae and group A streptococci from respiratory tract specimens and from the eye were collected from different parts of Norway during two study periods. During the first period (1993–1994), three laboratories, covering 15% of the Norwegian population, participated. During the second study period in 1997, five laboratories, covering 27% of the population, collected respiratory isolates. In total, 494 isolates of S. pneumoniae , 696 isolates of H. influenzae and 694 isolates of group A streptococci were included in the study. The study population comprised children and adults attending hospital and general practice. Bacterial susceptibility was determined by the E test, and breakpoints were according to the National Committee for Clinical Laboratory Standards (NCCLS).
Results   The prevalence of bacterial resistance was low, and we observed no significant increase in bacterial resistance between the two study periods. In 1997, only 0.6% of pneumococci had decreased susceptibility to penicillin, 1.6% of group A streptococci were resistant to erythromycin, and 6.7% of all isolates of H. influenzae produced β -lactamase.
Conclusions   The prevalence of antibiotic resistance in respiratory pathogens in Norway is low.     

Antibiotic susceptibilities of Haemophilus influenzae in central Scotland

Objective: To ascertain the incidence of antibiotic resistance in Haemophilus influenzae in central Scotland and the β-lactamases produced by these isolates.
Methods: A total of 213 H. influenzae isolates from four medical centers in Scotland [Aberdeen ( n = 58), Edinburgh ( n = 55), Glasgow ( n = 64) and Dundee ( n = 36)] were tested for susceptibility to a range of antimicrobials including β-lactams, β-lactam/β-lactamase-inhibitor combinations, and a representative 4-quinolone, antifolate and macrolide. Susceptibility testing of the β-lactam/β-lactamase-inhibitor combination amoxicillin plus clavulanic acid was conducted at both 2:1 and 4:1 ratios and with clavulanic acid fixed at a concentration of 2 mg/L. Each strain was further investigated for the presence of β-lactamase activity.
Results: Although the incidence of resistance to amoxicillin was 15%, in the presence of clavulanic acid, this resistance was reduced to 4.2%, 5.6% and 4.2% with the 2:1 ratio, 4:1 ratio and 2 mg/L fixed concentration, respectively. Sixteen percent of the isolates demonstrated immediate β-lactamase production. Isoelectric focusing showed that 77.4%, 16.1% and 6.5% of the β-lactamase-positive strains were found to contain TEM-1, VAT-1 and both TEM-1 and VAT-1 β-lactamases, respectively. A further 29% of the strains were recognized as being β-lactamase-positive after prolonged incubation with nitrocephin.
Conclusions: This study suggests that current testing for β-lactamases may underestimate the prevalence of β-lactamase production in H. influenzae.     

Antimicrobial susceptibility of 840 clinical isolates of Haemophilus influenzae collected in four European countries in 2000–2001

In 2000–2001, 840 clinical isolates of Haemophilus influenzae were collected from laboratories in France, Germany, Italy and Spain (210 isolates/country). β -Lactamase production among the isolates varied considerably by country, ranging from 8.1% in Germany to 34.8% in France. H. influenzae from patients ≤4 years old showed the highest prevalence of β -lactamase production (23.2%), compared with isolates from patients aged 5–17 years (17.8%) and ≥18 years (16.5%). All isolates were susceptible to amoxicillin–clavulanate, ciprofloxacin and levofloxacin; 99.6% and 98.9% of isolates were susceptible to azithromycin and cefuroxime, respectively. Among the macrolides tested, azithromycin (MIC₉₀, 2 mg/L) was eight-fold more potent than clarithromycin (MIC₉₀, 16 mg/L) and roxithromycin (MIC₉₀, 16 mg/L). Despite variations in β -lactamase production between different countries, > 99% of all isolates were susceptible to amoxicillin–clavulanate, ciprofloxacin, levofloxacin, and azithromycin.     

Resistance to β-lactams among blood isolates of Salmonella spp. in European hospitals: results from the SENTRY Antimicrobial Surveillance Program 1997–98

The susceptibility to β -lactams and the β -lactamase content of 110 Salmonella spp. blood isolates collected during 1997–98 in 19 European centers participating in the SENTRY Surveillance Program were studied. Thirty-one isolates (28%) were resistant to penicillins, due to production of TEM-1 (27 isolates), OXA-1 (three isolates) or TEM-1 + OXA-1 (one isolate). All OXA-1 producers and 10 TEM-1-producing isolates were also resistant to penicillin–clavulanic acid combinations. In the latter isolates, this phenotype was associated with increased production of TEM-1. Sixteen TEM-1-producing Salmonella Enteritidis isolates and one OXA-1-producing S. Typhimurium isolate were able to transfer β -lactam resistance by conjugation.     

Amoxycillin/clavulanate resistance as related to β-lactamase content in Escherichia coli strains from community-acquired urinary tract infections in Greece

Objective: To determine the resistance rate to amoxycillin/clavulanate (AMC) in 100 Escherichia coli strains isolated from outpatients with urinary tract infection (UTI) in four Greek hospitals and assess the relationship between β-lactamase content and resistance to AMC.
Methods: Susceptibility to β-lactams was determined with the E-test. Sonic cell extracts were used as β-lactamase preparations. Conjugal transfer of resistance was performed in broth cultures. β-Lactamase quantities were evaluated by measuring nitrocefin hydrolysis. Isoelectric points (pls) of β-lactamases were determined by electrofocusing. The substrate specificity of the enzymes and the inhibitory activity of clavulanate were studied spectrophotometrically.
Results: Thirty-two isolates were resistant to ampicillin. Eight were resistant (MIC ≥ 32 mg/L) and 11 showed decreased susceptibility (MIC 4–16 mg/L) to AMC. The latter expressed at least four-fold higher amounts of TEM-1 β-lactamase compared with the TEM-1-producing AMC-susceptible isolates. Seven AMC-resistant isolates produced at least 16-fold higher amounts of TEM-1; in one isolate, resistance was attributed to an OXA-type β-lactamase. None of the AMC-resistant isolates was able to transfer resistance to AMC by conjugation. Clavulanate-resistant TEM variants were not detected.
Conclusions: Amoxycillin/clavulanate-resistant E. coli strains have become established in the Greek community. Resistance is mainly due to the production of large amounts of TEM-1 β-lactamase which is encoded from non-self-transmissible plasmids.     

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.     

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.     

Comparative in vitro activity of ertapenem against bacterial pathogens isolated from patients with lower respiratory tract infections

This study compared the in vitro activity of ertapenem, ceftriaxone, cefepime, ciprofloxacin and amoxicillin–clavulanate against 381 aerobic and facultative bacterial pathogens isolated from 320 patients with acute bacterial exacerbation of chronic bronchitis or community-acquired pneumonia. Streptococcus pneumoniae and Haemophilus influenzae accounted for 54.6% of the isolates. The ertapenem MIC was ≤2 mg/L for 98.4% of isolates and ≥8 mg/L for 1.0% (all methicillin-resistant Staphylococcus aureus ). Ertapenem had the most potent activity against Enterobacteriaceae, Moraxella catarrhalis , and methicillin-susceptible S. aureus , and its activity against H. influenzae and H. parainfluenzae , all strains of which were susceptible, was not altered by β -lactamase production. Only one S. pneumoniae strain, a penicillin-resistant isolate, was resistant to ertapenem. Ertapenem was highly active in vitro against pyogenic bacteria recovered from patients with community-acquired lower respiratory tract infections.     

Transferable plasmid mediating resistance to multiple antimicrobial agents in Klebsiella pneumoniae isolates in Greece

Objective   To investigate the underlying resistance mechanisms in 10 Klebsiella pneumoniae isolates.
Methods   Ten K. pneumoniae strains according to distinct bacteriocin typing and REP-PCR, were examined for their plasmid content, their ability to transfer their resistance to aminoglycosides and third-generation cephalosporins, and their production of aminoglycoside-modifying enzymes and β -lactamases.
Results   Transfer of resistance to the above-mentioned antibiotics as well as to co-trimoxazole and tetracycline in Escherichia coli strain RC 85 at a frequency of 5–10⁶ was achieved for all strains by conjugation. Similar strains harbor a self-transferable multiresistant plasmid (80 kb) with similar Eco RI and Hind III restriction patterns. This plasmid encodes an extended-spectrum β -lactamase which confers high-level resistance to third-generation cephalosporins and aztreonam. It produces SHV-5 β -lactamase, as demonstrated by isoelectric focusing and DNA sequencing. Aminoglycoside resistance was co-transferred, and AAC(6')-I, mediating resistance to gentamicin, tobramycin, netilmicin and amikacin, and AAC(3)-I, mediating resistance to gentamicin and sisomycin, were encoded in all isolates and their transconjugants, while APH(3')-I, mediating resistance to kanamycin and neomycin, was encoded in seven strains.
Conclusions   It appears that a multiresistant transferable plasmid encoding the SHV-5 β -lactamase, causing unusually high resistance to ceftazidime and aztreonam, and the combination AAC(6')-I + AAC(3)-I of acetylating enzymes causing, also resistance to all clinically available aminoglycosides, is established in K. pneumoniae in Greece.     

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.     

Antibiotic Susceptibilities Among Recent Clinical Isolates of Haemophilus influenzae and Moraxella catarrhalis from Fifteen Countries

 Between July 1998 and July 1999, 3,060 Haemophilus influenzae and 1,486 Moraxella catarrhalis strains were isolated in 31 centers in 15 countries in order to determine their antimicrobial susceptibilities and the presence of β-lactamase production in Haemophilus influenzae. Overall 17.1% of the Haemophilus influenzae isolates were β-lactamase positive, while more than 95% were susceptible to amoxicillin/clavulanate, cefaclor, loracarbef, cefuroxime, azithromycin and ciprofloxacin. Eleven (0.3%) isolates were β-lactamase positive and ampicillin resistant and 7 (0.2%) isolates were ciprofloxacin resistant. The minimum inhibitory concentrations for 90% of the isolates tested were lowest for ciprofloxacin (0.03) and highest for cefprozil (8) against Moraxella catarrhalis.     

Metallo-β-lactamase-producing Pseudomonas aeruginosa isolated from a large tertiary centre in Kenya

This study was designed to characterize the β-lactamase content of carbapenem-resistant Pseudomonas aeruginosa isolates recovered during 2006 and 2007 in a large tertiary-care centre in Nairobi, Kenya. Molecular characterization was done using PCR and sequencing, and typing was performed using pulsed-field gel electrophoresis (PFGE). In total, 416 P. aeruginosa isolates were obtained during that period, of which 57 (13.7%) were resistant to carbapenems. All carbapenem-resistant isolates tested positive for metallo-β-lactamase (MBL) production. All MBL isolates produced VIM-2 with two types of integron structures . PFGE identified three clonally related groups of VIM-2-producing P. aeruginosa , including a pan-resistant clone that was responsible for nosocomial outbreaks during 2006 and 2007 in the intensive-care unit. These findings suggest that continuous molecular surveillance needs to be performed to monitor the spread within the hospital of this pan-resistant strain. This study is the first report of VIM-2-producing P. aeruginosa from the African continent.     

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.     

Invasive Haemophilus influenzae in Manitoba, Canada, in the postvaccination era

Fifty-two Haemophilus influenzae isolates from patients with invasive disease in the province of Manitoba, Canada, were examined for serotype, biotype, genotype, and antibiotic susceptibility. Half of the 52 isolates were found to be serotype a, and 38.5% (20 isolates) were found to be nonserotypeable (NST). There were only three serotype b strains and one each for serotypes c, d, and f. All 26 serotype a isolates belonged to biotype II and demonstrated identical or highly similar DNA fingerprints by pulsed-field gel electrophoresis. An analysis of these isolates by multilocus sequence typing showed that they belong to the clonal complex ST-23. While 69% (18 of 26) of the serotype a cases were found in males, only 9 (45%) of the 20 patients with NST isolates were males. Twenty (77%) of the 26 serotype a isolates were from patients who were 相似文献   

Mecillinam susceptibility as an indicator of β-lactamase production in Staphylococcus aureus

The use of direct susceptibility testing from specimens has led to the fortuitous observation that penicillin-susceptible strains have larger inhibition zones for mecillinam than do β -lactamase producers. The current study was, therefore, undertaken to test 179 Staphylococcus aureus isolates for mecillinam susceptibility by Rosco Neo-Sensitabs and to compare the results with commonly used tests for β -lactamase production, i.e. size and character of penicillin inhibition zones, chromogenic cephalosporin (nitrocefin) tests and clover leaf assays. Agreement between methods was reached for 175 of 179 strains when disregarding the results of the nitrocefin tests, 88 isolates being found susceptible and 87 being found to be β -lactamase producers. All 88 susceptible isolates had mecillinam zones of >22 mm, with the great majority being >25 mm; double zones did, however, occur. The 87 β -lactamase producers had zones <14 mm or no zones. Four isolates presenting problems in had mecillinam zones of ≤20 mm and were without tapering growth at the penicillin inhibition zone edge. In conclusion, the size of the mecillinam inhibition zone is found to be a useful supplementary test in the clinically important distinction between β -lactamase-producing and non-producing isolates of S. aureus .     

The risk of subsequent transmission of Hemophilus influenzae type B disease among children in day care. Results of a two-year statewide prospective surveillance and contact survey

To determine the risk of Hemophilus influenzae type b disease among children attending day-care facilities who were exposed to a primary case of invasive hemophilus disease, we conducted a two-year (August 1982 through July 1984) statewide prospective study involving active surveillance for H. influenzae disease and a 60-day follow-up of the children's day-care contacts. We identified 185 patients with primary invasive hemophilus type b disease who were under six years of age and who attended a total of 195 day-care facilities (centers or private homes). There were 4102 children in attendance at these day-care facilities when the primary cases occurred; 4034 (98 percent) were followed for 60 days or more after the onset of illness in the patients with primary disease. A total of 2612 children were considered classroom contacts of the patients with primary disease, because they were cared for in the same home or the same room in a larger facility, but they were not siblings of the index patients. Of these classroom contacts, 370 from 0 to 23 months of age and 716 from 24 to 47 months of age did not receive rifampin chemoprophylaxis. We could confirm no subsequent H. influenzae disease among contacts. Our results suggest that the risk of subsequent hemophilus disease in contacts of patients in day-care facilities is significantly lower than that previously reported for siblings and day-care contacts.     

Comparative in vitro activity of piperacillin, piperacillin–sulbactam and piperacillin–tazobactam against nosocomial pathogens isolated from intensive care patients

We investigated the antimicrobial activity of piperacillin–tazobactam versus piperacillin–sulbactam against common nosocomial pathogens ( n  = 565) isolated from intensive care patients. For Gram-positive bacteria, antimicrobial susceptibilities to the two piperacillin–β-lactamase inhibitor combinations were almost identical. For Gram-negative bacteria, piperacillin–tazobactam exhibited greater activity against Escherichia coli and Proteus vulgaris than piperacillin–sulbactam. Both combinations, however, were equally effective against the other Enterobacteriaceae and Pseudomonas aeruginosa isolates. Piperacillin–sulbactam exhibited better antimicrobial activity against Acinetobacter baumannii . Our findings might prove important for the appropriate choice of antibiotic therapy with β-lactam–β-lactamase inhibitor combinations.     

Carriage and antibiotic susceptibility of Haemophilus influenzae type b and non b in Danish day-care attendees

A study of carriage of Haemophilus influenzae type b (Hib) and non b in Danish day-care facilities, employing a highly selective medium supplemented with three antibiotics and direct detection of the type b capsular antigen by latex-agglutination and coagglutination, was carried out. Of 329 children, 217 (66%) were found to be carriers of Haemophilus influenzae. Among 64 children from day-care centers in which a case of invasive Hib infection had occurred, four carriers of type b were found, whereas no type b carriers were identified among 265 children from unexposed day-care centers (p less than 0.003). Of 217 isolated strains, seven (3.2%) were found to have lowered susceptibility to ampicillin, two (0.9%) to chloramphenicol, two (0.9%) to cefuroxim, and one (0.5%) to cefotaxim.     

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.