A new plasmidic cefotaximase from patients infected withSalmonella typhimurium

Summary Salmonella typhimurium strains resistant to most -lactams, co-trimoxazole, tobramycin and gentamicin were isolated from patients in two hospitals in Buenos Aires, Argentina, beginning in August 1990. The patients were suffering from meningitis, septicaemia or enteritis. Therapy including ampicillin, ceftriaxone and gentamicin failed. The strains produced a plasmidic (pMVP-4) extended broad-spectrum -lactamase which is more active against cefotaxime than against ceftazidime (V_max for cefotaxime 350 times higher than for ceftazidime). This cefotaximase demonstrates similarity to the previously described CTX-ase-M-1 fromEscherichia coli, but it is distinctly different from CTX-ase-M-1 by its isoelectric point (7.9 for CTX-ase-M-2 in comparison with 8.9 for CTX-ase-M-1) as well as in its lower susceptibility to the -lactamase inhibitors sulbactam, clavulanic acid, tazobactam and BRL 42715. Thus, the -lactamase produced byS. typhimurium strains from Argentina appears to represent a new member (CTX-ase-M-2) of a novel group of plasmidic extended broad-spectrum -lactamases designated as cefotaximases.

---

Neue plasmidische Cefotaximase von Patienten mit Salmonella typhimurium-Infektion

Zusammenfassung In zwei Hospitälern in Buenos Aires, Argentinien, wurden von August 1990 anSalmonella typhimurium-Stämme mit Resistenz gegenüber den meisten -Laktamantibiotika, Co-trimoxazol sowie Tobramycin und Gentamicin isoliert. Klinische Diagnosen bei diesen Patienten waren Meningitis, Sepsis oder Enteritis. Antibiotische Therapie mit Ampicillin, Ceftriaxon und Gentamicin blieb erfolglos. DieS. typhimurium-Stämme produzieren eine plasmidische (Plasmid pMVP-4) -Laktamase mit erweitertem Breitspektrum (EBS--Lactamase), welche gegenüber Cefotaxim deutlich aktiver ist als gegenüber Ceftazidim (V_max für Cefotaxim 350 mal größer als für Ceftazidim). Diese Cefotaximase ist verwandt mit der Cefotaximase ausEscherichia coli (CTX-M-1), von der sie sich jedoch im isoelektrischen Punkt (7,9 bei der CTX- ase-M-2 gegenüber 8,9 bei der CTX-ase-M-1) sowie in ihrer Hemmbarkeit durch Sulbactam, Clavulansäure, Tazobactam und BRL 42715 eindeutig unterscheidet. Die beiS. typhimurium-Stämmen in Argentinien gefundene neue -Laktamase wird als zweites Enzym einer neuen Gruppe von -Laktamasen mit erweitertem Breitspektrum vom Cefotaximase-Typ betrachtet.

     

1株蔬菜源mcr-1阳性多重耐药大肠杆菌特征分析

目的 对蔬菜源mcr-1阳性多重耐药大肠杆菌的菌株特征进行分析。方法 2019年从扬州2个超市采集251份零售蔬菜样品,通过麦康凯平板和含2 mg/L头孢噻肟的麦康凯板进行肠杆菌的分离,PCR和测序检测黏菌素耐药基因mcr-1的携带情况,利用16S rRNA基因扩增和测序对mcr-1阳性肠杆菌进行菌种鉴定,采用微量稀释法测定其对常见16种抗菌药物的敏感性,并通过全基因组测序分析该菌株的特征。结果 共分离得到189株肠杆菌,其中仅在1株分离自生菜的大肠杆菌中检测到mcr-1。该菌株对黏菌素最小抑菌浓度为8 mg/L,多序列位点分型结果为ST359,携带bla_TEM-1b、bla_CTX-M-14、fosA3、oqxAB、tet(A)、strAB、floR、sul2、dfrA12等多种耐药基因,染色体GyrA在第83位(S83L)和第87位(D87N)氨基酸发生突变,ParC在第80位氨基酸(S80I)发生突变。该菌对氨苄西林、头孢唑啉、头孢噻肟、链霉素、四环素、氯霉素、氟苯尼考、奈啶酸、环丙沙星、磷霉素和复方新诺明耐药。mcr-1基因位于IncI2质粒上,与国内外报道的携带mcr-1的IncI2质粒高度相似。结论 已在蔬菜沾染细菌中发现mcr-1基因,且mcr-1基因位于全球流行的IncI2质粒上,应加强对蔬菜中耐药菌的监测。     

大肠埃希菌中新的耐药基因盒aadA23的变异

目的基于整合子-细菌耐药系统在细菌耐药机制中的重要作用,对成人腹泻患者的大肠埃希菌Ⅰ类整合子阳性菌株携带的耐药基因盒的基因特征进行分析.方法应用聚合酶链反应（PCR）检测Ⅰ类整合酶基因intⅠ阳性菌株并对其整合的耐药基因进行测序及用生物信息软件对序列进行分析.结果5株Ⅰ类整合酶基因阳性菌株的耐药基因盒PCR扩增得到1009 bp的产物.序列分析结果表明,1009 bp序列含有780 bp的开放阅读框,与已知的aadA23和aadA21分别有99.6%和99.5%的相似性,与aadA5只有66.4%相似,为对氨基糖苷类抗菌药物壮观霉素、链霉素产生耐药的基因盒,建议命名为aadA23b.结论随着选择环境不同,整合子整合的基因盒会发生变异,提示我们要用分子生物学的手段从基因水平分析耐药基因的遗传与变异.     

Perspectives of beta-lactamases inhibitors in therapy of infections caused by Escherichia coli or Klebsiella with plasmidic resistance to third generation cephalosporins

Summary New plasmidic -lactamases inactivating so far stable cephalosporins, aztreonam and cephamycins restrict the use of these antibiotics in therapy of infections, e.g., byEscherichia coli and Klebsiella. Thus, combinations of -lactamase inhibitors and -lactam antibiotics were investigatedin vitro with regard to their therapeutic perspectives. Minimal inhibitory concentrations and the kinetics of killing in a pharmacodynamic model were determined. Extended broad spectrum betalactamases (EBS--lactamases) representative both for the TEM- and SHV-type were included. None of the available fixed combinations of penicillins and -lactamase inhibitors appears useful for therapy of infections caused by producers of EBS--lactamases. In contrast, combinations of piperacillin and tazobactam or sulbactam plus cephalosporins (cefoperazone, cefotaxime, ceftazidime) or aztreonam are highly active (both by their MICs and bactericidal activity) against TEM-type EBS--lactamases, but less promising for the SHV-type EBS--lactamases, and plasmidic cephamycinase. Of the -lactams available, the monobactam carumonam and the carbapenems (imipenem, meropenem) remain safe in infections caused byE. coli and Klebsiella EBSBase producers.

---

Therapeutische Perspektiven von -Laktamase-Inhibitoren bei Infektionen durch Escherichia coli oder Klebsiella mit plasmid-determinierter Resistenz gegenüber Drittgenerationscephalosporinen

Zusammenfassung Neue plasmid-kodierte -Laktamasen inaktivieren bisher stabile Cephalosporine, Aztreonam und Cephamycine. Sie schränken damit die therapeutische Sicherheit dieser Antibiotika bei Infektionen zum Beispiel durchEscherichia coli oder Klebsiellaarten ein. Deshalb wurden die therapeutischen Perspektiven von Kombinationen aus -Laktamase-Inhibitoren und -Laktam-Antibiotikain vitro analysiert. Die minimalen Hemmkonzentrationen (MHK) wurden gemessen. Zu einem pharmakokinetischen Modell wurde die Abtötungskinetik bestimmt. Einbezogen wurden Enzyme sowohl aus der TEM- als auch der SHV-Reihe. Keine der beiden zugelassenen festen Kombinationen aus Penicillinen und -Laktamase-Inhibitoren erwies sich als erfolgversprechend für die Therapie von Infektionen durch Stämme, welche -Laktamasen mit erweitertem Spektrum produzieren. Demgegenüber waren Kombinationen aus Piperacillin und Tazobactam oder Sulbactam mit Cephalosporinen (Cefoperazon, Cefotaxim, Ceftazidim) oder Aztreonam sowohl aufgrund ihrer MHK-Werte als auch ihrer bakteriziden Aktivität im pharmakodynamischen Modell hochaktiv gegenüber TEM--Laktamasen mit erweitertem Spektrum, jedoch weniger wirksam bei Stämmen mit neuen Enzymen der SHV-Reihe und plasmid-kodierter Cephamycinasen. Unter den derzeit vorhandenen -Laktam-Antibiotika kommt anhand ihrerIn-vitro-Aktivität dem Monobactam Carumonam sowie den Carbapenemen (Impipenem, Meropenem) auch ohne -Laktamase-Inhibitor die größte therapeutische Sicherheit bei Infektionen durchE. coli und Klebsiella-Stämme mit der Fähigkeit zur Synthese von -Laktamasen mit erweitertem Spektrum zu.

---

---

Symposium 2nd Biennial Conference on Chemotherapy of Infectious Diseases and Malignancies, Montreux, March 5^th–8^th, 1989.

---

Supported by Pfizer GmbH, Karlsruhe, FR Germany.     

Clonal and pathotypic analysis of archetypal Escherichia coli cystitis isolate NU14.

Escherichia coli NU14, a cystitis isolate used to study the pathogenesis of cystitis and to develop a FimH (type 1 fimbrial adhesin) vaccine, was assessed for extended virulence genotype, phylogenetic background, and FimH sequence and binding phenotype(s). NU14 exhibited the same virulence genotype and was derived from the same (meningitis- and cystitis-associated) subclone of E. coli O18:K1:H7 as the archetypal neonatal bacterial meningitis (NBM) isolate RS218. NU14 also displayed the same Ser62Ala FimH polymorphism as did NBM isolates RS218 and IHE3034-conferring both collagen binding and a distinct monomannose binding capability (which characterizes uropathogenic but not commensal E. coli and dramatically increases adherence to uroepithelial cells). These findings establish that strain NU14 exhibits numerous urovirulence-associated traits and derives from the single most prevalent clonal group in acute cystitis. They provide further evidence of clonal and pathotypic similarities between cystitis and NBM isolates of E. coli O18:K1:H7.     

Detection of a novel variant bla(CTX-M-3) extended spectrum beta-lactamase gene in a community-acquired Escherichia coli isolate

A highly cefotaxime- and cefepime-resistant but ceftazidime-sensitive Escherichia coli isolate was recovered from a community-acquired urinary infection of a Greek patient. Susceptibility testing, transfer assays, plasmid analysis as well as PCR and sequencing techniques were used to investigate the underlying mechanism of resistance. The isolate carried a new variant of the bla(CTX-M-3) gene that possessed a T instead of A at nt position 663. Cefotaxime resistance was transferable and carried on a 60 kb plasmid. The bla(CTX-M-3) variant was located downstream of an ISEcp1B element. The emergence of this new derivative indicates further evolution of the worldwide-distributed bla(CTX-M-3) gene.     

A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli.

Mutations in the glnA region of the Escherichia coli chromosome due to Mu prophage insertion result in two phenotypic classes. One class is Gln- and does not synthesize glutamine synthetase[L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] under any growth condition. The other class produces a low level of glutamine synthetase under all growth conditions and is uncoupled from the regulatory effects of mutations in the glnF and glnD genes. Complementation analysis demonstrates that these two classes of insertions are in different cistrons. From these data we suggest that a regulatory gene, glnG, tightly linked to glnA, mediates both activation and repression of glutamine synthetase synthesis. An analysis of the evidence accumulated to date makes it unlikely that glnG is the only gene in the glnA region involved in the complex system of nitrogen regulation.     

致腹泻性大肠埃希菌感染的快速检验诊断

致腹泻性大肠埃希菌(下称大肠杆菌,EC)种类很多,在发达国家或发展中国家均为腹泻的重要病原。在国内,其微生物学诊断技术长期滞后,常规应用的细菌培养和鉴定手段难以鉴别多种致腹泻性大肠埃希菌,故国内大多数腹泻患者的病原尚不能明确,尤其是儿童腹泻的病原。     

HPV16地方分离株E6基因的克隆与原核表达研究

目的克隆人乳头瘤病毒（HPV16E6）基因并在大肠埃希菌中表达，对表达产物进行鉴定。方法用PCR方法从克隆质粒pUC19-HPV16E6E7中扩增HPV16E6基因，采用定向克隆构建pQE30-HPV16E6原核表达质粒．利用酶切和序列测定鉴定重组质粒。将pQE30-HPV16E6转化大肠埃希菌BL21（DE3），建立重组工程菌pQE30-HPV16E6／BL21（DE3）。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，SDS—PAGE分析蛋白表达情况，利用Western blot鉴定抗原特异性。结果PCR产物470bp，重组质粒经酶切和序列测定证实构建正确。SDS—PAGE分析在18×10^3处有蛋白条带出现，与预期一致。Western blot分析证实目的条带与HPV16E6抗体有特异性反应。结论成功构建了HPV16E6基因的基因工程菌株，能高效表达E6蛋白，表达蛋白具有良好的免疫反应性。     

A mutant of Escherichia coli with a new, highly efficient promoter for the lactose operon

We have isolated a mutant of E. coli whose maximal rate of synthesis of lac mRNA is 25-fold greater than that of its parental wild-type strain. The mutant also shows alterations in the kinetics of beta-galactosidase synthesis, the functional half-life of beta-galactosidase mRNA, and the properties of lac repressor.     

Escherichia coli in a maternity ward

Summary TheEscherichia coli isolated from faecal and nasal swabs from all babies present in a maternity ward were serotyped and their antibiotic resistance patterns and fermentation characteristics with six carbohydrates were determined. These were compared withE. coli isolated from hands and clothing of attendants and samples of ward air. The results suggest that mothers may be the primary source ofE. coli in the ward but the colonized babies themselves provide the main reservoir of nurseryE. coli. As most of these serotypes ofE. coli were found on the attendants' hands or clothing or in the air, it is difficult to assess transmission routes forE. coli, particularly as some strains appear to possess a greater inherent ability to colonize babies than others. By the use of markers other than O antigens it was possible to characterize strains to a far greater extent.

---

Das Vorkommen von Escherichia coli auf einer Entbindungsstation

Zusammenfassung E. coli-Stämme, die aus den Fäzes und durch Nasenabstriche von allen Kindern in der Entbindungsstation isoliert wurden, wurden serologisch typisiert. Außerdem wurden die Antibiotika-Resistenz sowie die biochemischen Eigenschaften bestimmt. Diese Untersuchungsergebnisse wurden mit denen von E. coli-Stämmen verglichen, die von den Händen und von der Kleidung der Stationsangehörigen sowie aus der Luft stammten. Die Resultate lassen vermuten, daß die Mütter die Hauptquelle von E. coli-Stämmen auf der Station sind, jedoch stellen die schon besiedelten Kinder selbst das Hauptreservoir der Säuglingsstation dar. Da die meisten E. coli-Serotypen an den Händen oder an der Kleidung des Stationspersonals gefunden wurden, ist es schwierig, den Übertragungsweg von E. coli zu bestimmen, insbesondere, da manche Stämme eine größere Besiedlungsfähigkeit bei Säuglingen zu besitzen scheinen als andere. Wenn andere Erkennungsmerkmale als O-Antigene benutzt wurden, war es möglich, in einem erheblich größeren Ausmaß Stämme zu differenzieren.

     

Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin

The administration of reference endotoxin (Escherichia coli O:113, Lot EC-5) to humans has been an important means to study inflammation in vivo; however, the supply of Lot EC-5 is depleted. A new lot of reference endotoxin (Clinical Center reference endotoxin [CCRE]), derived from the original bulk material extracted from E. coli O:113, was processed. The effects of 0-, 1-, 2-, and 4-ng/kg doses of intravenous CCRE and EC-5 were studied in 20 male subjects. CCRE resulted in dose-related increases in symptoms, temperature (P=. 016), total leukocyte count (P=.014), tumor necrosis factor-alpha (P=.004), interleukin (IL)-1 receptor antagonist (P=.004), IL-6 (P=. 005), IL-8 (P=.011), cortisol (P<.05), and C-reactive protein (P=. 04). These responses were attenuated (all P<.012) in subjects given Lot EC-5 (4 ng/kg) in comparison with those in subjects given CCRE, showing that, over several years, EC-5 had lost potency. Thus, in healthy subjects, the magnitude of exposure to CCRE results in a graded dose response of major components of innate immunity.     

Linezolid resistance in a clinical isolate of Staphylococcus aureus

The new oxazolidinone antimicrobial, linezolid, has been approved for the treatment of infections caused by various gram-positive bacteria, including meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Although instances of linezolid resistance in VRE have been reported, resistance has not been encountered among clinical isolates of S aureus. We have characterised an MRSA isolate resistant to linezolid that was recovered from a patient treated with this agent for dialysis-associated peritonitis.     

The clinical characteristics analysis of Escherichia coli bloodstream infection

<正>Objective To explore the clinical features of Escherichia coli bloodstream infection.Methods The clinical data of underlying diseases,antimicrobial susceptibility,temperature at blood sampling,results of routine blood tests,venous catheterization,therapy and prognosis of Escherichia coli bloodstream infection in the First Affilia-     

Genetic analysis of the first mcr-1 positive Escherichia coli isolate collected from an outpatient in Chile

Global dissemination of mcr-like genes represents a serious threat to public health since it jeopardizes the effectiveness of colistin, an antibiotic used as a last-resort treatment against highly antibiotic-resistant bacteria. In 2017, a mcr-1-positive isolate of Escherichia coli was found in Chile for the first time. Herein we report the genetic features of this strain (UCO-457) by whole-genome sequencing (WGS) and conjugation experiments. The UCO-457 strain belonged to ST4204 and carried a 285 kb IncI2-type plasmid containing the mcr-1 gene. Moreover, this plasmid was transferred by conjugation to an E. coli J53 strain at high frequency. The isolate harbored the cma, iroN, and iss virulence genes and did carry resistance genes to trimethoprim/sulfamethoxazole and fluoroquinolones. Other antibiotic resistance determinants such as β-lactamases-encoding genes were not detected, making the isolate highly susceptible to these antibiotics. Our results revealed that such susceptible isolates could be acting as platforms to disseminate plasmid-mediated colistin resistance. Based on this evidence, we consider that mcr-like prevalence deserves urgent attention and should be examined not only in highly resistant bacteria but also in susceptible isolates.     

Trimethoprim-resistant Escherichia coli in a geriatric hospital

A total of 1751 strains of Escherichia coli was collected from the Turku City Hospital in 1981-1982. Of these, 81 per cent were from patients aged 65 years or more. Total trimethoprim resistance was about 22 per cent of strains studied. There was a significant difference between the trimethoprim resistance of strains from those patients with a urinary tract catheter (38 per cent) and those without such a catheter (13.4 per cent) (P less than 0.001).     

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase

Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62-242 milliunits/mg and phosphate ester hydrolysis with specific activities of 41-61 units/mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H2. Thus, BAP is a unique Pt-dependent, H2-evolving hydrogenase. This reaction is unprecedented in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.