Antibiotic Susceptibilities of Serratia marcescens and Enterobacter liquefaciens

Production of 5'-nucleotides by Serratia marcescens and Enterobacter liquefaciens correlates with deoxyribonuclease production, indicating the close relationship between these two organisms. To determine further relationships, susceptibilities of 279 strains of the tribe Klebsielleae were determined by the high-potency disc method, agar-dilution method, or both, by using 14 antibiotics. Ninety-seven per cent of S. marcescens (201 of 207 strains) and 100% of E. liquefaciens (17 strains) had minimum inhibitory concentration (MIC) of 100 mug/ml or greater with colistin and polymyxin B. With these two antibiotics, 93% of other Enterobacter species (28 strains) had MIC values of less than 1.6 mug/ml, and 100% of Klebsiella (27 strains) had MIC values less than 1.6 mug/ml. Consistent patterns were not noted with the other antibiotics tested, but the results with colistin and polymyxin B provide additional evidence of the close relationship of S. marcescens and E. liquefaciens.     

DNA relatedness among species of Enterobacter and Serratia.

Species of Enterobacter and Serratia were examined for deoxyribonucleic acid relatedness to Klebsielleae, to atypical erwiniae, and to other members of Enterobacteriaceae. Deoxyribonucleic acid hybridization and then hydroxyapatite chromatography was the technique used to assess relatedness. Strains of Enterobacter cloacae formed two separate hybridization groups that correlate with the presence or absence of yellow pigment. Pigmented E. cloacae were 75-100% related, but they were only 40-50% related to unpigmented strains. Conversely, unpigmented strains were 70% or more related but were only 40-50% related to the pigmented strains. Both pigmented and unpigmented E. cloacae were 40-45% related to Enterobacter aerogenes and klebsiellae, and 20-30% related to Serratia species and Enterobacter hafniae. Atypical erwiniae were highly related to E. cloacae. Serratia marcescens strains formed one closely related group. Serratia liquefaciens strains formed a single, more disperse, relatedness group, as did isolates of Serratia rubidaea. These species were related throughout a substantial portion of their genomes. A group of lysine-positive "Citrobacter-like" strains were 40-50% related to Serratia species. Only four E. hafniae strains were tested. Two of these were highly related, while the other two were only 50% related to the reference strain. Enterobacter hafniae was only 15-20% related to other Enterobacteriaceae.     

Coliforms from hides and meat.

Coliform tests were performed on 85 hide and 75 meat samples. IMViC reactions were determined on isolates from positive confirmed and fecal tests, and strains other than Escherichia coli were identified. Strains typed as Aerobacter aerogenes types I and II were identified as Enterobacter cloacae (51.4%), Klebsiella pneumoniae (21.5%), Enterobacter aerogenes (15%), and Enterobacter liquefaciens, Serratia, and unidentified coliforms (12.1%). K. pneumoniae appeared to be responsible for less than 1% positive fecal tests.     

Coliforms from hides and meat.

Coliform tests were performed on 85 hide and 75 meat samples. IMViC reactions were determined on isolates from positive confirmed and fecal tests, and strains other than Escherichia coli were identified. Strains typed as Aerobacter aerogenes types I and II were identified as Enterobacter cloacae (51.4%), Klebsiella pneumoniae (21.5%), Enterobacter aerogenes (15%), and Enterobacter liquefaciens, Serratia, and unidentified coliforms (12.1%). K. pneumoniae appeared to be responsible for less than 1% positive fecal tests.     

Profiles of the utilization of 20 amino acids as the only source of nitrogen and carbon in bacteria of the genera Klebsiella, Enterobacter, Serratia, Escherichia

The profiles of the utilization of 20 protein amino acids in 118 Klebsiella pneumoniae sub- sp. pneumoniae, K. oxytoca, K. planticola, K. mobilis, Enterobacter cloacae, Serratia marscescens, S. liquefaciens, Escherichia coli strains isolated from clinical material were studied. The utilization of amino acids was determined on minimal saline agar containing amino acid as the only source of nitrogen and carbon; the results were evaluated after 72-hour incubation at 37 degrees C. 17 profiles of amino-acid utilization were thus determined, most of them genus-specific in enterobacteria: Klebsiella (profiles No. 1--6, 9, 10), Enterobacter (No. 11--13), Serratia (No. 14--16), Escherichia (No. 17). The full coincidence of amino-acid utilization profiles in bacteria of K. mobilis (No. 1, 6) and K. pneumoniae subsp. pneumoniae with out of such profiles in bacteria of the genera Enterobacter, Serratia, Escherichia was established, which confirmed that K. mobilis (formerly Enterobacter aerogenes) belonged to the genus Klebsiella.     

Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative Gram-negative bacteria on beef.

At 5 degrees C four strains of fermentative, gram-negative bacteria (Serratia liquefaciens, Yersinia enterocolitica, Enterobacter cloacae, and Aeromonas hydrophila) grew aerobically and anaerobically on adipose tissue removed from beef muscle of low pH (5.4 to 5.6). All four strains also grew aerobically and anaerobically on muscle tissue of high pH (6.0 to 6.3). However, none of the four grew anaerobically on beef muscle of low pH, and the aeromonad strain also failed to grow aerobically on such muscle. Growth of S. liquefaciens and E.cloacae on vacuum-packaged beef muscle was dependent on the pH of the tissue and the oxygen transmission rate of the packaging film. Although the four strains grew in broth buffered at pH 5.55, L-lactate, at the concentration found in muscle of low pH (ca. 100 mM), prevented anaerobic growth of all four isolates and prevented the aerobic growth of th aeromonad. At pH 6.1 in buffered broth, the concentration of L-lactate occurring in muscle of high pH did not prevent aerobic or anaerobic growth of any of the strains.     

Kinetic properties of Serratia marcescens adenosine 5''-diphosphate glucose pyrophosphorylase.

The regulatory properties of partially purified adenosine 5'-diphosphate-(ADP) glucose pyrophosphorylase from two Serratia marcescens strains (ATCC 274 and ATCC 15365) have been studied. Slight or negligible activation by fructose-P2, pyridoxal-phosphate, or reduced nicotinamide adenine dinucleotide phosphate (NADPH) was observed. These compounds were previously shown to be potent activators of the ADPglucose pyrophosphorylases from the enterics, Salmonella typhimurium, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Escherichia aurescens, Shigella dysenteriae, and Escherichia coli. Phosphoenolpyruvate stimulated the rate of ADPglucose synthesis catalyzed by Serratia ADPglucose pyrophosphorylase about 1.5- to 2-fold but did not affect the S0.5 values (concentration of substrate required for 50% maximal stimulation) of the substrates, alpha-glucose-1-phosphate, and adenosine 5'-triphosphate. Adenosine 5'-monophosphate (AMP), a potent inhibitor of the enteric ADPglucose pyrophosphorylase, is an effective inhibitor of the S. marcescens enzyme. ADP also inhibits but is not as effective as AMP. Activators of the enteric enzyme counteract the inhibition caused by AMP. This is in contrast to what is observed for the S. marcescens enzyme. Neither phosphoenolpyruvate, fructose-diphosphate, pyridoxal-phosphate, NADPH, 3-phosphoglycerate, fructose-6-phosphate, nor pyruvate effect the inhibition caused by AMP. The properties of the S. marcescens HY strain and Serratia liquefaciens ADPglucose pyrophosphorylase were found to be similar to the above two S. marcescens enzymes with respect to activation and inhibition. These observations provide another example where the properties of an enzyme found in the genus Serratia have been found to be different from the properties of the same enzyme present in the enteric genera Escherichia, Salmonella, Shigella, Citrobacter, and Enterobacter.     

Volatile compounds associated with microbial growth on normal and high pH beef stored at chill temperatures

Volatile compounds produced by Pseudomonas fragi and mixed, natural floras on beef of normal pH (5.5-5.8; glucose greater than 1500 micrograms/g) and high pH (6.3-6.8; glucose less than 10 micrograms/g) included a range of alkyl esters and a number of sulphur-containing compounds including dimethylsulphide but not hydrogen sulphide. Production of the last was a property common to the other Gram-negative organisms tested viz. Hafnia alvei, Enterobacter agglomerans, Serratia liquefaciens, Alteromonas putrefaciens and Aeromonas hydrophila, all of which produced similar off-odours and, with the exception of E. agglomerans, 'greening' on high pH meat. Serratia liquefaciens also produced greening of normal pH meat. Acetoin and diacetyl were major end products of Brochothrix thermosphacta but the related 2,3-butanediol was formed only on normal pH meat. The Enterobacteriaceae produced the same compounds but only on normal pH meat and together with Br. thermosphacta were probable sources of these compounds and of the free and esterified branched-chain alcohols detected in the naturally contaminated samples.     

Plasmids and genetic determinants of antibiotic resistance of gram-negative bacteria

Distribution of plasmids and genetic determinants of antibiotic resistance was studied in 129 strains of Pseudomonas, Klebsiella, Serratia and Enterobacter isolated from oncological patients. It was shown that 56 isolates contained the plasmids, 9 conjugative plasmids being plasmids with broad bacterial host spectrum. A significant part of the strains contained genes controlling production of APH (3"), type II APH (3'), type I and II DHPS and type type II DHFR. Genetic determinants of tetracycline resistance of classes D and E were detected for the first time in the strains of Klebsiella, Serratia and Pseudomonas aeruginosa.     

Volatile compounds associated with microbial growth on normal and high pH beef stored at chill temperatures

Volatile compounds produced by Pseudomonas fragi and mixed, natural floras on beef of normal pH (5–5–5–8; glucose < 1500 μg/g) and high pH (6–3–6–8; glucose < 10 μg/g) included a range of alkyl esters and a number of sulphur-containing compounds including dimethylsulphide but not hydrogen sulphide. Production of the last was a property common to the other Gram-negative organisms tested viz. Hafnia alvei, Enterobacter agglomerans, Serratia liquefaciens, Alteromonas putrefaciens and Aeromonas hydrophila , all of which produced similar off-odours and, with the exception of E. agglomerans , 'greening'on high pH meat. Serratia liquefaciens also produced greening of normal pH meat. Acetoin and diacetyl were major end products of Brochothrix thermosphacta but the related 2,3-butanediol was formed only on normal pH meat. The Enterobacteriaceae produced the same compounds but only on normal pH meat and together with Br. thermosphacta were probable sources of these compounds and of the free and esterified branched-chain alcohols detected in the naturally contaminated samples.     

Release of surface enzymes in Enterobacteriaceae by osmotic shock

The process of osmotic shock, which has been used to release degradative enzymes from Escherichia coli, can be applied successfully to other members of the Enterobacteriaceae. Cyclic phosphodiesterase (3'-nucleotidase), 5'-nucleotidase (diphosphate sugar hydrolase), acid hexose phosphatase, and acid phenyl phosphatase are released from Shigella, Enterobacter, Citrobacter, and Serratia strains. Some strains of Salmonella also release these enzymes. Members of Proteus and Providencia groups fail to release enzymes when subjected to osmotic shock and do not show a lag in regrowth, although they do release their acid-soluble nucleotide pools. In contrast to E. coli, release of enzymes from other members of the Enterobacteriaceae studied is affected by growth conditions and strain of organism. None of the organisms was as stable to osmotic shock in exponential phase of growth as was E. coli. Exponential-phase cells of Shigella, Enterobacter, and Citrobacter could be shocked only with 0.5 mm MgCl(2) to prevent irreparable damage to the cells. These observations suggest that this group of degradative enzymes is probably loosely bound to the cytoplasmic membrane through the mediation of divalent cations.     

Tryptophan biosynthetic pathway in the Enterobacteriaceae: some physical properties of the enzymes.

Several physical properties of the first four enzymatic activities of the tryptophan pathway were examined using gel filtration and ion exchange chromatography. Five different patterns were noted. Differences in the anthranilate synthetase (AS) and phosphoribosylanthranilate transferase (PRT) defined these patterns. In all the organisms studied phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase co-eluted from both diethylaminoethyl-cellulose and G-200 and thus probably are contained in a single polypeptide of 50,000 daltons. An AS-PRT complex was found in Citrobacter species, Enterobacter cloacae, and Erwinia dissolvens. In all the other bacteria examined AS and PTR were separate molecules. In Serratia marcescens, S. marinorubra, and Enterobacter liquefaciens, AS was 140,000 daltons and PRT was 45,000 daltons. In Erwinia carotavora and Enterobacter hafniae the AS was the same size as the Serratia species but the PRT was larger at 67,000 daltons. Two Proteus species had an AS and PRT of the same size as E. carotavora and E. halfniae but the Proteus AS was different in that it partially dissociated upon gel filtration. Aeromonas formicans was unique in its possession of an AS with a molecular weight of 220,000. The PRT of A. formicans was found to elute at 67,000 daltons. Possible paths of evolution of the tryptophan enzymes are discussed in terms of the results of this study. The results presented here are also considered with respect to existing taxonomic schemes of the enteric bacteria.     

DNA based classification of food associated Enterobacteriaceae previously identified by Biolog GN Microplates

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.     

Klebsiella, Enterobacter, and Serratia: Biochemical Differentiation and Susceptibility to Ampicillin and Three Cephalosporin Derivatives

Three hundred twenty-nine strains of the tribe Klebsielleae were compared by several biochemical tests and by susceptibility to selected antibiotics. Biochemical tests included urease, amino acid decarboxylase, and hydrogen sulfide production; fermentation of lactose and dextrose; motility; and tests in the IMViC (indole, methyl red, Voges-Proskauer, citrate) series. The isolates were: Klebsiella species, 67.5%; Enterobacter species, 28%, and Serratia species, 4.5%. Minimal inhibitory concentrations of cephaloridine, cephalothin, and a new cephalosporin, cephalexin, and of ampicillin were determined by the agar dilution procedure. Cephalosporins at 20 mug/ml or less inhibited 90% of the Klebsiella strains but only 15% of the Enterobacter strains. Ampicillin inhibited 27% of Enterobacter strains and 17% of Klebsiella strains. Serratia isolates were insensitive to the cephalosporins and ampicillin. The results suggest that precise identification of this group to the generic level can be accomplished readily in the clinical laboratory and that such information is helpful in the preliminary selection of an antibiotic for treatment of clinical infections.     

Biochemical characteristics and identification of Enterobacteriaceae isolated from meats.

The isolation and identification of 2,220 Enterobacteriaceae from meats indicated that Escherichia coli biotype I, Enterobacter agglomerans, and Serratia liquefaciens were the principal types to be differentiated in meats. Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter hafniae were also commonly identified. Identification of isolates by the Encise II (Roche Diagnostics Inc., Nutley, N.J.) and Minitek (BBL Microbiology Systems, Cockeysville, Md.) coding systems gave similar results with only 255 (11.5%) discrepancies in identity, but both systems required large numbers of supplementary tests for identification of the isolates. Not only the distribution of Enterobacteriaceae types isolated from meats but also some of the biochemical reactions of the isolates differed from those of clinical isolates. The Minitek technique is recommended because of its versatility. However, with the addition of cellobiose and salicin disks and the inclusion of methyl red to the Minitek test and the use of the Voges-Proskauer test and gas production in EC medium at elevated temperature as standard tests, the identification of these Enterobacteriaceae from meats would be greatly facilitated. The inclusion of the motility test, for example, using nitrate motility agar, would also be of value to Enterobacteriaceae identification.     

Distribution of Serratia Species in Clinical Specimens

Eighty-five strains of Serratia were speciated. The majority were Serratia marcescens and were recovered from a variety of clinical specimens. S. liquefaciens and S. rubidaea were recovered only from sputum.     

Measuring the spermosphere colonizing capacity (spermosphere competence) of bacterial inoculants

Spermosphere establishment by bacteria which were coated onto seeds was studied using soybean seeds treated with four bacterial strains at levels of log10 1 to 4 colony-forming units (cfu) per seed planted in a field soil mix, and incubated 48 h. Each strain at every inoculum level developed spermosphere population densities of log10 4 to 8 cfu/seed, demonstrating an average multiplication of log10 3 cfu/seed. An alternative method was developed to differentially rank bacteria for spermosphere colonizing capacity, based upon incorporation of bacteria into a soil and monitoring the resulting spermosphere population densities around noninoculated seeds after 4 days at 14 degrees C. Fifty-seven bacterial strains which were isolated from soybean roots or from water samples, including Pseudomonas putida, P. putida biovar B, P. fluorescens, Serratia liquefaciens, Enterobacter aerogenes, and Bacillus spp. were tested in the spermosphere colonization assay. Average spermosphere population densities for the 57 strains ranged from 0 to log10 7.0 cfu/seed. Strains of a given taxon demonstrated marked diversity with ranges from 0 to log10 6.0 cfu/seed for Bacillus spp. and from log10 1.4 to 7.0 cfu/seed for Pseudomonas putida. The relative ranking of representative strains was consistent in repeating experiments. The potential usefulness of the assay for efforts to develop competitive bacterial inoculants for crop seeds is discussed.     

Fermentation of polysaccharides by Klebsielleae and other facultative bacilli.

Fermentations of 10 polysaccharides by species of the family Enterobacteriaceae were examined. Algin, guar, karaya, xanthan, and xylan were not fermented by any of the strains tested. Most of the activity was found in the tribe Klebsielleae. Klebsiella oxytoca fermented amylopectin (97% of the strains studied), carrageenan (100%), inulin (68%), polypectate (100%), and tragacanth (100%). Klebsiella pneumoniae fermented amylopectin (91%), carrageenan (100%), and tragacanth (86%). Carrageenan was also fermented by Enterobacter aerogenes (100%), Enterobacter agglomerans (63%), Enterobacter cloacae (95%), and Pectobacterium (38%). Pectobacterium shared polypectate fermentation (100%) with K. oxytoca. With one exception, Serratia strains were negative on all polysaccharides. These results, along with other evidence, indicate that (i) the genus Klebsiella is biochemically the most versatile genus of the tribe, (ii) because of its distinct characteristics, K. oxytoca warrants species designation separate from K. pneumoniae, and (iii) some food additives generally considered indigestible can be metabolized by a few species of facultative bacilli, whereas others appear to be resistant.     

Amikacin resistance of clinical strains of Enterobacteriaceae and Pseudomonas]

Amikacin resistance was studied in 380 bacterial strains of Enterobacter, Klebsiella, Serratia, Pseudomonas and E. coli isolated in clinics of the Moscow Region. It was shown that 69 isolates were resistant to amikacin. Plasmid DNA was detected in 10 amikacin resistant isolates. Three of them belonging to Klebsiella and 3 belonging to E. coli contained plasmids controlling resistance to amikacin. The plasmids isolated from the strains of Klebsiella determined as well resistance to kanamycin and streptomycin but did not control resistance to sisomicin, tobramycin and gentamicin while the plasmids isolated from the strains of E. coli determined resistance to amikacin, kanamycin, gentamicin, tobramycin and sisomicin.