Antimicrobial interventions to reduce Salmonella and Campylobacter populations and improve shelf life of quail carcasses.

Quail (Coturnix japonica) is processed and marketed as fresh meat, with limited shelf life. The objective of this study was to evaluate the efficacy of antimicrobial interventions during slaughter on reducing Salmonella and Campylobacter contamination and to determine the microbiological shelf life of quail during refrigerated (4°C) storage. Three antimicrobials, peracetic acid (400 ppm; PAA), Citrilow (pH 1.2), and Cecure (cetylpyridinium chloride [CPC], 450 ppm), along with a water and no-treatment control were evaluated. Quail carcasses (n = 75) were inoculated with a cocktail of nalidixic acid-resistant Salmonella Typhimurium and gentamicin-resistant Campylobacter coli. After 30 min of attachment time, quail carcasses were submerged in each antimicrobial solution for 20 s with air agitation. Noninoculated quail carcasses (n = 25) were similarly treated, packaged, and stored under refrigeration (4°C). Aerobic plate counts (APC), psychrotroph counts (PC), Enterobacteriaceae counts (ENT), total coliform counts (TCC), and Escherichia coli counts on quail carcasses were determined on 1, 4, 7, and 10 d. Salmonella and Campylobacter populations were determined by plating on Petrifilm APC supplemented with 200-ppm nalidixic acid and Campy Cefex agar supplemented with 200-ppm gentamycin, respectively. No significant reductions in (P > 0.01 log cfu/mL) in APC, PC, ENT, TCC, and E. coli counts were observed on carcasses submerged in water. However, treatments with PAA, Citrilow, and CPC significantly reduced (P ≤ 0.05) Salmonella and Campylobacter coli contamination. Citrilow showed greater (P ≤ 0.05) reduction in Salmonella and Campylobacter population (1.90 and 3.82 log cfu/mL reduction, respectively) to PAA and CPC. Greater (P ≤ 0.05) reductions in APC, PC, ENT, TCC, and E. coli counts (2.22, 1.26, 1.47, 1.52, and 1.59 log cfu/mL, respectively) were obtained with the application of CPC. Application of antimicrobial interventions resulted in a reduction in Campylobacter and Salmonella, APC, PC, and ENT populations after treatments (day 0) and throughout the storage period (day 10). Use of antimicrobial interventions after slaughter can improve the microbiological safety and shelf life of quail.

The prevalence and antimicrobial resistance phenotypes of Salmonella, Escherichia coli and Enterococcus sp. in surface water.

Surface water is prone to bacterial contamination as it receives wastes and pollutants from human and animal sources, and contaminated water may expose local populations to health risks. This review provides a brief overview on the prevalence and antimicrobial resistance (AR) phenotypes of Salmonella, Escherichia coli and Enterococcus, found in natural freshwaters. These bacteria are frequently detected in surface waters, sometimes as etiological agents of waterborne infections, and AR strains are not uncommonly identified in both developed and developing countries. Data relating to Salmonella, E. coli and Enterococcus present in environmental water are lacking, and in order to understand their development and dissemination using the One Health approach, understanding the prevalence, distribution and characteristics of the bacteria present in surface water as well as their potential sources is important. Furthermore, AR bacteria in natural watersheds are not well investigated and their impacts on human health and food safety are not well understood. As surface water is a receptacle for AR bacteria from human and animal sources and a vehicle for their dissemination, this is a crucial data gap in understanding AR and minimizing its spread. For this review, Salmonella, E. coli and Enterococcus were chosen to evaluate the presence of primary pathogens and opportunistic pathogens as well as to monitor AR trends in the environmental water. Studies around the world have demonstrated the widespread distribution of pathogenic and AR bacteria in surface waters of both developing and developed countries, confirming the importance of environmental waters as a reservoir for these bacteria and the need for more attention on the environmental bacteria for emerging AR.

Diversity and antimicrobial resistance of Enterococcus from the Upper Oconee Watershed, Georgia.

AIM: It is well-known that enterococci are abundant in the environment; however, the role of surface water as a reservoir of antimicrobial-resistant enterococci remains largely undefined. In this study, surface water samples were collected over a 2-year period from the Upper Oconee watershed, Athens, GA to examine enterococci and their antimicrobial resistance. METHODS AND RESULTS: Approximately 97% (445/458) of the samples were positive for enterococci and a total of 637 enterococci were isolated. The predominant species were Enterococcus casseliflavus (33·6%) followed by Enterococcus faecalis (26·5%) and Enterococcus hirae (13·2%). Regardless of species, the highest levels of resistance were to lincomycin (88·5%) and tetracycline (13%); isolates also exhibited resistance to newer antimicrobials, daptomycin (8·9%) and tigecycline (6·4%). Multidrug resistance (resistance ≥3 antimicrobial classes) was observed to as many as five classes of antimicrobials. Resistant enterococci appeared to be randomly dispersed over the seasons rather than clustered by species or antimicrobial resistance. CONCLUSIONS: This study demonstrated that surface waters contain a large population of diverse species of antimicrobial-resistant enterococci, including resistance to new antimicrobials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may indicate the potential of human intestinal illness and/or colonization of the human gut with resistant enterococci as enterococci correlate with increased disease risk to humans during recreational exposure to water.

Carcass orientation and drip time affect potential surface water carryover for broiler carcasses subjected to a post-chill water dip or spray1.

To estimate the potential for residual antimicrobial solution carryover, surface water accumulation and loss was measured on post-chill carcasses that were either dipped or sprayed with water. For all experiments, broilers were slaughtered, soft or hard scalded, defeathered, and eviscerated. Carcasses were immersion chilled, allowed to drip, and post-chill carcass weight (CW) recorded. For water dip treatment, carcasses were dipped for 0.5 min in water and hung by a wing (n = 33) or a leg (n = 30) and CW recorded at 0, 0.5, 1, 2, and 5 min post-dip. For water spray treatment, individual carcasses were hung by either the wings (n = 35) or legs (n = 34) from a shackle suspended from a scale. Water was sprayed at 80 psi and post-spray CW recorded. Initial water accumulation (0 min) for dipped carcasses was not significantly different (P > 0.05) for carcasses hung by the leg (101.0 g) or wing (108.8 g). Following the 5 min drip time, 31 g of water remained on the carcasses hung by the leg and only 10 g on carcasses hung by the wing (P < 0.05). When carcasses were sprayed with water, initial water accumulation (0 min) was 62 g for carcasses hung by the legs and 60 g for carcasses hung by the wings (P > 0.05). Following the 5 min drip time, 1 g or no water remained on the sprayed carcasses (P > 0.05). Carcasses that were dipped and hung by a leg for 5 min retained significantly more water (31 g) than carcasses that were dipped and hung by a wing (10 g) or sprayed carcasses hung either way (0.3 g) (P < 0.05). Post-chill water dip resulted in significantly higher initial carcass water accumulation than spraying (105 g vs. 61 g, P < 0.05). Carcass orientation during dripping only affected the amount of retained water for dipped carcasses. Dipped carcasses hung by a leg have the highest potential for residual carcass antimicrobial solution carryover and sprayed carcasses hung by either orientation have the lowest potential for residual antimicrobial solution carryover.

Diversity of Plasmids and Antimicrobial Resistance Genes in Multidrug-Resistant Escherichia coli Isolated from Healthy Companion Animals.

The presence and transfer of antimicrobial resistance genes from commensal bacteria in companion animals to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. The purpose of this study was to determine antimicrobial resistance gene content and the presence of genetic elements in antimicrobial resistant Escherichia coli from healthy companion animals. In our previous study, from May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA area were sampled and multidrug-resistant E. coli (n = 36; MDR, resistance to ≥ 2 antimicrobial classes) were obtained. Of the 25 different plasmid replicon types tested by PCR, at least one plasmid replicon type was detected in 94% (34/36) of the MDR E. coli; four isolates contained as many as five different plasmid replicons. Nine replicon types (FIA, FIB, FII, I2, A/C, U, P, I1 and HI2) were identified with FIB, FII, I2 as the most common pattern. The presence of class I integrons (intI) was detected in 61% (22/36) of the isolates with eight isolates containing aminoglycoside- and/or trimethoprim-resistance genes in the variable cassette region of intI. Microarray analysis of a subset of the MDR E. coli (n = 9) identified the presence of genes conferring resistance to aminoglycosides (aac, aad, aph and strA/B), ß-lactams (ampC, cmy, tem and vim), chloramphenicol (cat), sulfonamides (sulI and sulII), tetracycline [tet(A), tet(B), tet(C), tet(D) and regulator, tetR] and trimethoprim (dfrA). Antimicrobial resistance to eight antimicrobials (ampicillin, cefoxitin, ceftiofur, amoxicillin/clavulanic acid, streptomycin, gentamicin, sulfisoxazole and trimethoprim-sulfamethoxazole) and five plasmid replicons (FIA, FIB, FII, I1 and I2) were transferred via conjugation. The presence of antimicrobial resistance genes, intI and transferable plasmid replicons indicate that E. coli from companion animals may play an important role in the dissemination of antimicrobial resistance, particularly to human hosts during contact.

Antimicrobial resistance, virulence determinants and genetic profiles of clinical and nonclinical Enterococcus cecorum from poultry.

Enterococcus cecorum has been implicated as a possible cause of disease in poultry. However, the characteristics that contribute to pathogenesis of Ent. cecorum in poultry have not been defined. In this study, Ent. cecorum from carcass rinsates (n = 75) and diseased broilers and broiler breeders (n = 30) were compared based upon antimicrobial resistance phenotype, the presence of virulence determinants and genetic relatedness using pulsed-field gel electrophoresis (PFGE). Of the 16 antimicrobials tested, Ent. cecorum from carcass rinsates and clinical cases were resistant to ten and six of the antimicrobials, respectively. The majority of Ent. cecorum from carcass rinsates was resistant to lincomycin (54/75; 72%) and tetracycline (46/75; 61.3%) while the highest level of resistance among clinical Ent. cecorum was to tetracycline (22/30; 73.3%) and erythromycin (11/30; 36.7%). Multidrug resistance (resistance to ≥2 antimicrobials) was identified in Ent. cecorum from carcass rinsates (53/75; 70.7%) and diseased poultry (18/30; 60%). Of the virulence determinants tested, efaAfm was present in almost all of the isolates (104/105; 99%). Using PFGE, the majority of clinical isolates clustered together; however, a few clinical isolates grouped with Ent. cecorum from carcass rinsates. These data suggest that distinguishing the two groups of isolates is difficult based upon the characterization criteria used.

Relative survival of four serotypes of Salmonella enterica in low-water activity whey protein powder held at 36 and 70°C at various water activity levels.

Salmonella enterica is not able to grow at water activity (aw) levels below 0.94, but it can survive in low-aw foods for long periods of time. Temperature, aw, substrate, and serotype affect its persistence. The aim of this study was to evaluate the influence of temperature and aw on the relative persistence among four serotypes of Salmonella enterica in low-aw whey protein powder. Whey protein powder was equilibrated to aws 0.18 ± 0.02 and 0.54 ± 0.03, inoculated with a cocktail of Salmonella serovars (Agona, Tennessee, Montevideo, and Typhimurium), vacuum sealed, and stored at 36°C for 6 months and at 70°C for 48 h. Presumptive Salmonella colonies (30 to 32) were randomly picked from each plate at the end of each survival study. PCR multiplex serotyping was used to identify the isolates. A multinomial mixed logistic model with Salmonella Tennessee as a reference was used to test for significant differences in frequency distribution of the surviving serotypes. Salmonella Tennessee and Salmonella Agona were the most prevalent surviving serotypes, followed in decreasing order by Salmonella Montevideo and Salmonella Typhimurium. Statistical analysis indicated that temperature (P = 0.003) and aw (P = 0.012) influenced the relative prevalence of the Salmonella serotypes. If other environmental conditions are equal, Salmonella Tennessee is better able to survive than Salmonella Montevideo and Salmonella Typhimurium at higher temperatures and higher aw levels in low-aw whey protein powder held at 36 and 70°C. The relative prevalence of Salmonella Agona to Salmonella Tennessee did not change with increasing temperature (P = 0.211) or aw (P = 0.453). These results should be considered in risk assessment and when developing predictive models for survival of Salmonella in low-aw foods.

Molecular analysis of imipenem-resistant Acinetobacter baumannii isolated from US service members wounded in Iraq, 2003-2008.

Global dissemination of imipenem-resistant (IR) clones of Acinetobacter baumannii - A. calcoaceticus complex (ABC) have been frequently reported but the molecular epidemiological features of IR-ABC in military treatment facilities (MTFs) have not been described. We characterized 46 IR-ABC strains from a dataset of 298 ABC isolates collected from US service members hospitalized in different US MTFs domestically and overseas during 2003-2008. All IR strains carried the bla(OXA-51) gene and 40 also carried bla(OXA-23) on plasmids and/or chromosome; one carried bla(OXA-58) and four contained ISAbal located upstream of bla(OXA-51). Strains tended to cluster by pulsed-field gel electrophoresis profiles in time and location. Strains from two major clusters were identified as international clone I by multilocus sequence typing.

Prevalence, species distribution and antimicrobial resistance of enterococci isolated from dogs and cats in the United States.

AIMS: The contribution of dogs and cats as reservoirs of antimicrobial resistant enterococci remains largely undefined. This is increasingly important considering the possibility of transfer of bacteria from companion animals to the human host. In this study, dogs and cats from veterinary clinics were screened for the presence of enterococci. METHODS AND RESULTS: A total of 420 enterococci were isolated from nasal, teeth, rectal, belly and hindquarters sites of 155 dogs and 121 cats from three clinics in Athens, GA. Eighty per cent (124 out of 155) of the dogs and 60% (72 out of 121) of the cats were positive for enterococci. From the total number of dog samples (n = 275), 32% (n = 87) were from hindquarter, 31% (n = 86) were rectal, and 29% (n = 79) were from the belly area. The majority of isolates originated from rectal samples (53 out of 145; 37%) from cats. The predominant species identified was Enterococcus faecalis (105 out of 155; 68%) from dogs and E. hirae (63 out of 121; 52%) from cats. Significantly more E. faecalis were isolated from rectal samples than any other enterococcal species (P < 0.05) for both dogs and cats suggesting site specific colonization of enterococcal species. The highest levels of resistance were to ciprofloxacin in E. faecium (9 out of 10; 90%), chloramphenicol resistance in E. faecalis (17 out of 20; 85%) and gentamicin resistance in E. faecalis (19 out of 24; 79%) from dog samples and nitrofurantoin resistance in E. faecium (15 out of 19; 79%) from cats. Multi-drug resistance (MDR) (resistance > or =2 antimicrobials) was observed to as few as two and as many as eight antimicrobials regardless of class. CONCLUSION: This study demonstrated that dogs and cats are commonly colonized with antimicrobial resistant enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Dogs and cats may act as reservoirs of antimicrobial resistance genes that can be transferred from pets to people.

Microarray analysis of antimicrobial resistance genes in Salmonella enterica from preharvest poultry environment.

AIMS: To detect antimicrobial resistance genes in Salmonella isolates from turkey flocks using the microarray technology. METHODS AND RESULTS: A 775 gene probe oligonucleotide microarray was used to detect antimicrobial resistance genes in 34 isolates. All tetracycline-resistant Salmonella harboured tet(A), tet(C) or tet(R), with the exception of one Salmonella serotype Heidelberg isolate. The sul1 gene was detected in 11 of 16 sulfisoxazole-resistant isolates. The aadA, aadA1, aadA2, strA or strB genes were found in aminoglycoside-resistant isolates of Salm. Heidelberg, Salmonella serotype Senftenberg and untypeable Salmonella. The prevalence of mobile genetic elements, such as class I integron and transposon genes, in drug-resistant Salmonella isolates suggested that these elements may contribute to the dissemination of antimicrobial resistance genes in the preharvest poultry environment. Hierarchical clustering analysis demonstrated a close relationship between drug-resistant phenotypes and the corresponding antimicrobial resistance gene profiles. CONCLUSIONS: Salmonella serotypes isolated from the poultry environment carry multiple genes that can render them resistant to several antimicrobials used in poultry and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple antimicrobial resistance genes in environmental Salmonella isolates could be identified efficiently by microarray analysis. Hierarchical clustering analysis of the data was also found to be a useful tool for analysing emerging patterns of drug resistance.

Detection of KPC-2 in a clinical isolate of Proteus mirabilis and first reported description of carbapenemase resistance caused by a KPC beta-lactamase in P. mirabilis.

An isolate of Proteus mirabilis recovered from blood cultures of a diabetic patient was shown to be resistant to imipenem, meropenem, and ertapenem by disk diffusion susceptibility testing. Amplification of whole-cell and/or plasmid DNA recovered from the isolate with primers specific for the bla(KPC) carbapenemase gene produced an amplicon of the expected size which was confirmed to be bla(KPC-2) by sequence analysis. Transformation of a susceptible Escherichia coli host with plasmid preparations from the isolate generated a transformant for which the MICs of all of the carbapenems tested were increased three- to fourfold. We believe this to be the first report of carbapenem resistance in P. mirabilis caused by the acquisition of bla(KPC).

Salmonella, Campylobacter and Enterococcus spp.: their antimicrobial resistance profiles and their spatial relationships in a synoptic study of the Upper Oconee River basin.

Rivers may serve as reservoirs for enteric organisms. Very little is known about the boundaries of microbial communities in moving bodies of water so this study was undertaken to find the limits of distribution of some bacteria, focusing on enteric organisms. The presence of Salmonella, Campylobacter, and Enterococcus spp. and the antimicrobial resistance phenotypes carried by these organisms was evaluated for the Upper Oconee River basin, a small river in the lower Piedmont of northeastern Georgia, USA. Samples were obtained from 83 sites during a 3-h period on a spring day (April 2005) in an approximately 30 x 20 km region. Campylobacter spp. was isolated at 12 sites. The Campylobacter isolates from three sites were resistant to tetracycline. Of the five short-variable region (SVR) subtypes of Campylobacter that were found, three were found at more than one site, two types were found twice, and one subtype was found three times. Enterococcus was isolated at 71 sites. E. casseliflavus was the most common species. Based on species identification and antimicrobial resistance patterns, 24 types of Enterococcus were found. Salmonella was isolated from 62 sites. Of the 19 Salmonella serovars that were isolated, serovar Muenchen accounted for about 20% of the isolates. The next three most common serovars isolated, Rubislaw, Hartford, and Give, accounted for about 44% of the river isolates. Antimicrobial resistance profiling offered limited differentiation of Salmonella isolates because only seven isolates were resistant to any antimicrobial. The sites at which Salmonella, Campylobacter, or Enterococcus were isolated did not correlate with each other or with the total coliform number or Escherichia coli count for the site. However, isolates of some of the same species and type occurred in clusters that were restricted to areas within 5 to 6 km.

Comparison of Salmonella enterica serotype Infantis isolates from a veterinary teaching hospital.

AIMS: To compare Salmonella enterica serotype Infantis isolates obtained from patients or the environment of a veterinary teaching hospital over a period of 9 years following a nosocomial outbreak to determine whether isolates were epidemiologically related or represented unrelated introductions into the hospital environment. METHODS AND RESULTS: Fifty-six S. Infantis isolates were compared based on their phenotypic (antimicrobial drug [AMD] susceptibility pattern) and genotypic (pulsed-field gel electrophoresis [PFGE] pattern and presence of integrons) characteristics. Epidemiologically unrelated S. Infantis isolates clustered separately from all but two of the hospital isolates, and several isolates from different years and various sources were indistinguishable from each other in cluster analysis of two-enzyme PFGE results. A high percentage of isolates (80.3%) were resistant to at least one AMD, with 67.8% showing resistance to >5 AMD. The majority (74.1%) of isolates tested contained type 1 integrons. CONCLUSION: Results strongly suggest that there was nosocomial transmission of S. Infantis during the initial outbreak, and that contamination arising from this outbreak persisted across years despite rigorous hygiene and biosecurity precautions and may have led to subsequent nosocomial infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence of persistence and transmission of Salmonella clones across years, even in the face of rigorous preventive measures, has important implications for other facilities that have experienced outbreaks of Salmonella infections.

Transmission of Salmonella enterica serotype Typhimurium in poultry with and without antimicrobial selective pressure.

AIMS: To determine the effect of antimicrobial selective pressure on the transmission of antimicrobial resistant and sensitive strains of Salmonella in poultry. METHODS AND RESULTS: Eight pens housed 12 broiler chicks each. Two chicks in four of the pens were inoculated with a Salm. Typhimurium strain resistant to 12 antimicrobials (including tetracycline), and two chicks in each of the four other pens were inoculated with a strain sensitive to all antimicrobials tested. Two pens inoculated with each strain were treated with chlortetracycline and two were not. Chicks were killed on day 7 and caeca were cultured for Salmonella. Experiments were performed independently twice. Chicks exposed to pen mates inoculated with the resistant strain and treated with tetracycline were 90% positive for Salmonella; whereas 60% of chicks given no antimicrobials were positive. Chicks exposed to the sensitive strain were 95% positive with tetracycline treatment and 90% positive without treatment. CONCLUSIONS: A multidrug-resistant Salm. Typhimurium strain had significantly increased transmission when chicks were treated with tetracycline. Transmission of a sensitive strain was not inhibited by antimicrobial selective pressure at recommended therapeutic dose. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that antimicrobial usage may influence the transmission of antimicrobial-resistant pathogens in poultry.

Increased expression of Borrelia burgdorferi vlsE in response to human endothelial cell membranes.

RNA isolated from virulent Borrelia burgdorferi cells incubated with human endothelial or neurological tissue cells was subjected to subtractive hybridization using RNA from the same strain incubated in tissue culture medium alone. This RNA subtractive technique generated specific probes that hybridized to two restriction fragments (8.2 kb and 10 kb respectively) generated by EcoRI digestion of total plasmid DNA. The 10 kb EcoRI fragment localized to lp28-1 and was subsequently identified as the variable membrane protein-like sequence (vls) region, which includes an expression locus (vlsE) and 15 silent cassettes. vlsE encodes a 36 kDa outer surface protein that undergoes antigenic variation during animal infections. Primer extension analysis identified the 5' end of a transcript and a putative promoter for vlsE. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) suggested that the expression of vlsE increased when virulent B. burgdorferi cells were incubated with human tissue cells or purified cell membranes isolated from those same cell lines. A 138 bp region upstream of the vlsE region that was not reported in the genome sequence was sequenced using specific 32P end-labelled primers in a DNA cycle sequencing system at high annealing temperatures. Analysis revealed that it contained a 51 bp inverted repeat, which could form an extremely stable cruciform structure. Southern blots probed with the vlsE promoter/operator region indicated that part or all of this sequence could be found on other B. burgdorferi plasmids.

Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog.

We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp.

Identification, sequences, and expression of Treponema pallidum chemotaxis genes.

Treponema pallidum, the agent of syphilis, is a pathogenic spirochete that has no known mechanisms of genetic exchange and cannot be continuously cultivated in vitro. A probe based on the nucleotide sequence of the T. pallidum cheA gene was used to screen a T. pallidum genomic DNA library. A treponemal DNA region containing four open reading frames (orfs) was identified. The proteins encoded by these orfs have significant homology with proteins involved in bacterial chemotaxis. The orfs have been designated cheA, cheW, cheX, and cheY. The cheA, cheW, and cheY genes were individually-cloned and expressed in vitro. The observed molecular mass of each protein correlated well with its predicted molecular mass. Reverse transcriptase-PCR data indicate that cheA through cheY are co-transcribed. The organization of these genes suggests that they comprise an operon. We hypothesize that the ability to sense and respond to nutrient gradients is important for the survival and dissemination of T. pallidum in vivo. The presence of a putative che operon strongly suggests that T. pallidum has the potential for a chemotactic response.

Identification and sequences of the Treponema pallidum flhA, flhF, and orf304 genes.

The recently identified fla operon of Treponema pallidum contains several genes that encode motility-related proteins. We have determined the nucleotide sequences of three genes, designated flhA, flhF, and orf304, that are located immediately downstream of the flhB gene in the fla operon. The flhA gene encodes a 707-amino acid protein that contains five putative membrane spanning domains. FlhA has strong homology with members of a family of proteins that are involved in flagellar biogenesis and regulation/secretion of virulence-related proteins. The flhF gene encodes a 437-amino acid protein that contains three consensus elements that are characteristic of a GTP-binding domain. The orf304 gene encodes a 304-amino acid protein that contains a consensus ATP-binding motif. The order of the flhA, flhF, and orf304 genes is identical to that of corresponding genes in the Bacillus subtilis che/fla operon. Due to the location of the flhA, flhF and orf304 genes in the T. pallidum fla operon, we hypothesize that the FlhA, FlhF, and Orf304 proteins are involved in the biogenesis/assembly of treponemal periplasmic flagella.

Sequences of the Salmonella typhimurium mglA and mglC genes.

The nucleotide sequences of the mglA and mglC genes of Salmonella typhimurium (St) LT2 have been determined. The deduced amino acid (aa) sequences of MglA and MglC are 506 and 302 aa long with predicted molecular masses of 56,484 and 31,551 Da, respectively. The aa sequences of St MglA and MglC are homologous to the corresponding Mgl proteins of Escherichia coli, Haemophilus influenzae, Treponema pallidum and Mycoplasma genitalium. The order of the St mgl operon is mglBAC.

Identification and sequences of the Treponema pallidum mglA and mglC genes.

Treponema pallidum, the agent of syphilis, cannot be continuously cultivated in vitro. To identify treponemal genes encoding exported proteins, we performed TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Clone 6D2 was chosen for further study based on partial nucleotide sequence obtained from p6D2 containing a TnphoA insertion. A complete open reading frame (orf1) and a truncated orf (orf2) were identified in the treponemal DNA of p6D2. Orf1 encodes a hydrophobic protein of 531 amino acids with a calculated M(r) of 57,882 Da. The deduced amino acid sequence of Orf1 has homology to the MglC proteins of E. coli, Haemophilus influenzae, and Salmonella typhimurium. T. pallidum Orf1 (MglC) contains a conserved motif that is found in integral cytoplasmic membrane proteins of ATP-binding cassette (ABC) transport systems. T. pallidum orf2 encodes a protein of 496 amino acids with a calculated M(r) of 55,547 Da. The deduced amino acid sequence of Orf2 has homology to the MglA proteins of S. typhimurium, E. coli, H. influenzae, and Mycoplasma genitalium. Orf2 (MglA) contains two consensus ATP-binding motifs. T. pallidum mglA and mglC are located downstream of mglB, consistent with the gene order of previously identified mgl operons. The putative T. pallidum mgl operon encodes the first high-affinity ABC transport system identified in this spirochete.