Antibiotic resistance and genotyping of clinical group B Salmonella isolated in Accra, Ghana.

AIMS: The purpose of this study was to investigate the antibiotic resistance and clonal lineage of serogroup B Salmonella isolated from patients suspected of suffering from enteric fever in Accra, Ghana. METHODS AND RESULTS: Serogroup B Salmonella were isolated from blood (n=28), cerebral spinal fluid (CSF) (n=1), or urine (n=2), and identified based on standard biochemical testing and agglutinating antisera. Isolates were examined for their susceptibility to ampicillin, chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole. Most of the isolates could be classified as multiple-drug resistant. Furthermore, the genetic location of resistance genes was shown to be on conjugative plasmids. Genetic fingerprinting by plasmid profiling, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and repetitive element (REP)-PCR were performed to determine the diversity among the isolates. Plasmid profiling discriminated five unique groupings, while ERIC-PCR and REP-PCR resulted in two and three groupings, respectively. CONCLUSIONS: A high rate of antibiotic resistance was associated with the Salmonella isolates and the genes responsible for the resistance are located on conjugative plasmids. Also, there appears to be minimal diversity associated with the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: As a result of the increasing antibiotic resistance among bacteria of all genera, surveys to monitor microbial populations are critical to determine the extent of the problem. The inability to treat many infectious diseases with current antibiotic regimens should prompt the medical community to be more prudent with its antibiotic use.

Antibiotic resistance in coagulase-negative staphylococci isolated from Cope's gray treefrogs (Hyla chrysoscelis).

Organisms belonging to the genus Staphylococcus were isolated on mannitol salt agar from the feces of wild caught Cope's gray treefrogs (Hyla chrysoscelis) from east-central Kansas. All 222 presumptive isolates were confirmed as coagulase-negative staphylococci with Staphylococcus sciuri and Staphylococcus xylosus being most prevalent. Antibiotic susceptibility patterns to five different antibiotics were determined and the results indicated 99% of all isolates were resistant to penicillin G and 59% of the isolates were resistant to oxacillin, a clinical substitute for methicillin. Due to the significance of methicillin resistance in the genus Staphylococcus, 10 randomly chosen oxacillin resistant organisms were analyzed for the presence of the mecA gene, which is known to code for methicillin resistance. The gene was detected in four of the 10 organisms examined. These data indicate that gray treefrogs are harboring inordinately large numbers of methicillin resistant staphylococci as part of their normal flora and that the mechanism of methicillin resistance may be independent of mecA.

Antimicrobial susceptibility of staphylococci isolated from the faeces of wild turkeys (Meleagris gallopavo).

AIMS: The purpose of this study was to investigate the staphylococcal flora associated with wild turkey populations. METHODS AND RESULTS: Faecal samples obtained from 26 wild turkeys over a 16-month period were inoculated onto mannitol salt agar plates to select for staphylococci. Fifty-seven randomly chosen isolates were identified as Staphylococcus lentus and their susceptibility determined against clindamycin, chloramphenicol, ciprofloxacin, erythromycin, oxacillin, penicillin G, rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. Resistance was minimal as only 3 isolates showed resistance to clindamycin, 3 isolates were resistant to oxacillin, 3 isolates were resistant to penicillin G, and 1 isolate was resistant to erythromycin. Multiple antibiotic resistance was also minimal. CONCLUSIONS: S. lentus is the predominant staphylococcal species associated with wild turkey faeces and antibiotic resistance in these organisms is not problematic. SIGNIFICANCE AND IMPACT OF THE STUDY: S. lentus has been shown as a potential causative agent of inflammatory reactions in the respiratory tract. Due to increased numbers of wild turkeys and more frequent human exposure, surveys to monitor microbial populations are warranted.

Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots.

Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.

Genotyping of clinical Serratia marcescens isolates: a comparison of PCR-based methods.

The polymerase chain reaction (PCR)-based procedures of randomly amplified polymorphic DNA (RAPD) and repetitive element (RE)-based PCR were used to amplify total DNA prepared from each of 62 clinical Serratia marcescens isolates. Three different random primers, designated 1060, 1254 and 1283, were used individually in RAPD-PCR. Primers representing enterobacterial repetitive intergenic consensus (ERIC) sequences, extragenic palindromic (REP) elements, and polymorphic GC-rich repetitive sequences (PGRS) constituted the repetitive element-PCR. We were able to generate 40, 40 and 58 genotypic groupings using the 1060, 1254 and 1283 RAPD primers, respectively. Using the ERIC, REP and PGRS primers, 19, 54 and 60 unique genotypic profiles were yielded, respectively. The PGRS primers, which were developed to amplify GC-rich repetitive sequences in the genome of Mycobacteria, were the most discriminatory. These data indicate that both of these PCR-based approaches are a valid means of discriminating strain differences among isolates of S. marcescens and the amount of differentiation depends on the primer used. These techniques should prove useful for routine surveillance or in examining outbreaks of S. marcescens in clinical settings.

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella.

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.

Cloning and expression of cadD, a new cadmium resistance gene of Staphylococcus aureus.

A cadmium resistance gene, designated cadD, has been identified in and cloned from the Staphylococcus aureus plasmid pRW001. The gene is part of a two-component operon which contains the resistance gene cadD and an inactive regulatory gene, cadX*. A high degree of sequence similarity was observed between cadD and the cadB-like gene from S. lugdunensis, but no significant similarity was found with either cadA or cadB from the S. aureus plasmids pI258 and pII147. The positive regulatory gene cadX* is identical to cadX from pLUG10 over a stretch of 78 codons beginning at the N terminus, but it is truncated at this point and inactive. Sequence analysis showed that the cadmium resistance operon resides on a 3,972-bp element that is flanked by direct repeats of IS257. The expression of cadD in S. aureus and Bacillus subtilis resulted in low-level resistance to cadmium; in contrast, cadA and cadB from S. aureus induced higher level resistance. However, when the truncated version of cadX contained in pRW001 is complemented in trans with cadX from plasmid pLUG10, resistance increased approximately 10-fold suggesting that the cadmium resistance operons from pRW001 and pLUG10 are evolutionarily related. Moreover, the truncated version of cadX contained in pRW001 is nonfunctional and may have been generated by deletion during recombination to acquire the cadmium resistance element.

Characterization of a 54-kDa heat-shock-inducible protein of Pasteurella haemolytica.

Growth-condition-dependent antigens play a role in the virulence or protective capacity of many organisms. Enhanced production of an approximately 54-kDa protein was detected in heat-shocked cultures of Pasteurella haemolytica. The heat-shock-inducible protein cross-reacted with antibodies to 60-kDa heat-shock proteins of Mycobacterium tuberculosis, Chlamydia, and Escherichia coli GroEL. A probe containing the E. coli groEL operon hybridized with fragments of P. haemolytica chromosomal DNA on Southern blots. Immunoblots of the 54-kDa protein using serum from 20 calves that were challenged experimentally with P. haemolytica resulted in band densities that were significantly different between calves with high and low lesion scores. Results of the study suggest that the 54-kDa heat-shock protein may be a growth-condition-dependent immunogen that is one component of resistance to pneumonic pasteurellosis.

Purification and characterization of staphylococcin BacR1, a broad-spectrum bacteriocin.

The bacteriocin BacR1 was purified from culture supernatant of Staphylococcus aureus UT0007 by sequential ammonium sulfate precipitation, cation-exchange chromatography, and C4 reverse-phase chromatography steps. Mass spectrographic analysis indicated that the purified peptide has a molecular mass of 3,338 Da. It is resistant to environmental conditions, retaining full biological activity after exposure to pH extremes (pHs 3 to 11), heating at 95 degrees C for 15 min, and exposure to strong chaotropic agents. BacR1 was destroyed with a complete loss of biological activity after digestion with trypsin and proteinase K. Amino acid sequence analysis revealed a high concentration of Asx, Gly, and Pro residues and a high proportion of hydrophobic amino acids. The peptide is bactericidal and kills in a dose-dependent manner, but it does not lyse log-phase cells of Corynebacterium renale, the routine indicator organism for bacteriocin assay. A specific receptor for binding was detected on sensitive cells but not on insensitive cells. Competition assays showed that UV-inactivated cells could protect susceptible cells from antibacterial action. A partial inhibitory spectrum revealed that organisms from the following genera are susceptible: Staphylococcus, Streptococcus, Corynebacterium, Haemophilus, Bordetella, Moraxella, Pasteurella, Neisseria, and Bacillus.

Cytoplasmic domains mediate the ligand-induced affinity shift of guanylyl cyclase C.

Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), exhibits multiple binding affinities, including high (RH) and low (RL) affinity sites and a ligand-induced conversion of low-affinity sites from a higher (RL1) to a lower (RL2) affinity state. Occupancy of the lowest affinity state of low-affinity sites is coupled to ligand-induced catalytic activation. In the present studies, ligand binding and catalytic activation properties of a series of intracellular deletion mutants of GCC were examined to identify the structural domains underlying expression of high- and low-affinity sites and the ligand-induced shift in low-affinity sites. These studies demonstrated that the cytoplasmic domains of GCC are not required, but extracellular and transmembrane domains are sufficient, for expression of high-affinity binding sites. In addition, the cytoplasmic juxtamembrane and kinase homology domains are required for expression of the ligand-induced affinity shift in low-affinity sites. Of significance, this shift in affinity was insensitive to adenine nucleotides, in contrast to other members of the receptor guanylyl cyclase family, such as guanylyl cyclase A (GCA). Also, the juxtamembrane and kinase homology domains are critical for coupling ST-receptor binding and guanylyl cyclase catalytic activation. Indeed, deletion of those domains from GCC results in a constitutively inhibited enzyme, suggesting that they function as positive effectors of ligand activation, in contrast to GCA and GCB, in which the kinase homology domain represses basal catalytic activity. These data suggest that the mechanisms regulating different members of the receptor guanylyl cyclase family are overlapping but not identical.

Exploiting the unique biophysical properties of bacteriocins to purify Bac 1829 from Staphylococcus aureus KSI1829.

Bac1829 from Staphylococcus aureus KSI1829 is a newly identified peptide bacteriocin that inhibits a broad spectrum of bacteria. By taking advantage of its cationic and hydrophobic nature, a purification scheme was developed utilizing preparative isoelectric focusing and hydrophobic interaction chromatography. Due to a high pI value of approximately 9-10, 71% of the total Bac1829 activity was concentrated in two fractions by preparative isoelectric focusing. Final purification by high performance liquid chromatography on a propyl hydrophobic interaction column resulted in a 136-fold purification with an increase in specific activity from 181 to 24,623 antimicrobial units (AU)/mg protein. A total of 14,848 AU of Bac 1829 were purified using this revised procedure, compared to 8702 AU as described previously. Mass spectrographic analysis of the purified sample yielded a single peak of 6418 +/- 2 daltons. Since many bacteriocins possess similar biophysical attributes, the purification scheme presented in this communication may be applicable to those which have proven difficult to purify or have not been purified.

Purification and partial characterization of a novel antibacterial agent (Bac1829) Produced by Staphylococcus aureus KSI1829.

A novel antimicrobial agent from Staphylococcus aureus KSI1829, designated Bac1829, was purified by sequential steps of ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and hydrophobic interaction chromatography. Purified Bac1829 has a molecular mass of 6,418 +/- 2 Da. The peptide in heat stable, since full biological activity is retained after heating at 95 degrees C for 15 min, and it is destroyed by digestion with proteases. Amino acid sequence analysis revealed a high concentration of Ala and Gly residues, which respectively comprised 24 and 19% of the total amino acid content. Additionally, high levels of hydrophobic amino acids were present, accounting for the hydrophobic nature of Bac1829. Purified Bac1829 killed exponentially growing Corynebacterium renale in a dose-dependent manner by a bactericidal mode of action. A partial inhibitory spectrum analysis revealed that the following organisms were sensitive to the inhibitory activity of Bac1829: S. aureus RN4220, Streptococcus suis, Corynebacterium pseudotuberculosis, C. renale, Corynebacterium diptheriae, Haemophilus parasuis, Bordetella pertussis, Bordetella bronchoseptica, Moraxella bovis, and Pasteurella multocida.

Rat guanylyl cyclase C expressed in COS-7 cells exhibits multiple affinities for Escherichia coli heat-stable enterotoxin.

Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides. Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides. Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities. Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites. Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics. In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM. Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal. Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium. Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation. These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand. The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases.