Phenology of five tick species in the central Great Plains.

The states of Kansas and Oklahoma, in the central Great Plains, lie at the western periphery of the geographic distributions of several tick species. As the focus of most research on ticks and tick-borne diseases has been on Lyme disease which commonly occurs in areas to the north and east, the ticks of this region have seen little research attention. Here, we report on the phenology and activity patterns shown by tick species observed at 10 sites across the two states and explore factors associated with abundance of all and life specific individuals of the dominant species. Ticks were collected in 2020-2022 using dragging, flagging and carbon-dioxide trapping techniques, designed to detect questing ticks. The dominant species was A. americanum (24098, 97%) followed by Dermacentor variabilis (370, 2%), D. albipictus (271, 1%), Ixodes scapularis (91, <1%) and A. maculatum (38, <1%). Amblyomma americanum, A. maculatum and D. variabilis were active in Spring and Summer, while D. albipictus and I. scapularis were active in Fall and Winter. Factors associated with numbers of individuals of A. americanum included day of year, habitat, and latitude. Similar associations were observed when abundance was examined by life-stage. Overall, the picture is one of broadly distributed tick species that shows seasonal limitations in the timing of their questing activity.

High levels of alpha-gal with large variation in the salivary glands of lone star ticks fed on human blood.

Tick bites, associated with the secretion of tick saliva containing the xenoglycan galactose-alpha-1, 3-galactose (alpha-gal or aGal), are recognized as the causal factors of alpha-Gal syndrome (AGS; or red meat allergy) in humans. AGS occurs after the increased production of IgE antibodies against aGal, which is found in most mammalian cells, except for the Old World monkey and humans. The aGal sensitization event has been linked to an initial tick bite, followed by consumption of red meat containing the aGal glycan, which triggers the onset of the allergic response resulting in urticaria, anaphylaxis, or even death. In North America, the lone star tick, Amblyomma americanum, has been identified as the main culprit for AGS. However, only a subset of the human population exposed to lone star tick bites develops AGS. This suggests the presence of unidentified variables associated with the sensitization event. To evaluate the quantitative variations of the aGal in ticks, we evaluated the differences in aGal levels in different strains of A. americanum ticks partially fed on different blood sources using an artificial feeding system and animal hosts. We found significantly higher aGal levels in the female ticks fed on human blood than those fed on the blood of other mammals with large variations among different tick populations and individuals. We propose that host-specific genetic components in the A. americanum ticks are involved in the production of high aGal epitope in the tick saliva, which provides a part of the explanation for the variables associated with the AGS sensitization event of the tick bite.

Culicoidibacter larvae gen. nov., sp. nov., from the gastrointestinal tract of the biting midge (Culicoides sonorensis) larva, belongs to a novel lineage Culicoidibacteraceae fam. nov., Culicoidibacterales ord. nov. and Culicoidibacteria classis nov. of the phylum Firmicutes.

Strain CS-1T, a novel facultative anaerobic bacterium, was isolated from the larval gastrointestinal tract of the biting midge, Culicoides sonorensis, a vector of the epizootic haemorrhagic disease virus and the bluetongue virus. Cells were Gram-stain-positive, non-motile, non-spore-forming, pleomorphic rods. Optimal growth occurred at pH 7.5 and 37 °C. The G+C content of the genomic DNA was 38.3 mol%, estimated by using HPLC. The dominant cellular fatty acids were C14 : 0 (45.9 %) and C16 : 0 (26.6 %). The polar lipid profile comprised glycolipids, diphosphatidylglycerol, phospholipids and phosphoglycolipids. Respiratory quinones were not detected. Strain CS-1T had very low 16S rRNA gene similarity to members of the phylum Firmicutes: Macrococcus canis KM45013T (85 % similarity) and Turicibacter sanguinis MOL361T (88 % similarity). Phylogenetic analysis based on 16S rRNA, rpoB, gyrB genes, and conserved protein sequences of the whole genome revealed that strain CS-1T was related to members of the classes Bacilli and Erysipelotrichia within the phylum Firmicutes. Furthermore, average nucleotide identity and digital DNA-DNA hybridization analyses of the whole genome revealed very low sequence similarity to species of Bacilli and Erysipelotrichaceae (Macrococcus canis KM45013T and Turicibacter sp. H121). These results indicate that strain CS-1T belongs to the phylum Firmicutes and represents a new species of a novel genus, family, order and class. Based on the phenotypic, chemotaxonomic, phylogenetic and genomic characteristics, we propose the novel taxon Culicoidibacter larvae gen. nov., sp. nov. with the type strain CS-1T (=CCUG 71726T=DSM 106607T) within the hereby new proposed novel family Culicoidibacteraceae fam. nov., new order Culicoidibacaterales ord. nov. and new class Culicoidibacteria classis nov. in the phylum Firmicutes.

Draft Genome Sequences of 11 Bacterial Strains Isolated from Commercial Corn-Based Poultry Feed.

Here, we report 11 bacterial strains isolated from commercial corn-based poultry feed to determine their potential as hygienic indicator microorganisms through a comparison of genome sizes and distribution patterns of unique genes. These isolates belonged to the genera Klebsiella, Kosakonia, Pantoea, Stenotrophomonas, and Enterococcus.

Draft Genome Sequences of 10 Bacterial Strains Isolated from an Abandoned Coal Mine in Southeast Kansas.

Here, we report 10 bacterial strains isolated from an abandoned coal mine in southeast Kansas to determine their potential for bioremediation through comparison of the genome sizes and distribution patterns of unique metabolic genes. The selected strains belong to the genera Arthrobacter, Jeotgalibacillus, Kocuria, Microbacterium, Pantoea, Rhodococcus, Vibrio, Brevibacterium, and Paenibacillus.

Surveillance for Heartland and Bourbon Viruses in Eastern Kansas, June 2016.

In June 2016, we continued surveillance for tick-borne viruses in eastern Kansas following upon a larger surveillance program initiated in 2015 in response to a fatal human case of Bourbon virus (BRBV) (Family Orthomyxoviridae: Genus Thogotovirus). In 4 d, we collected 14,193 ticks representing four species from four sites. Amblyomma americanum (L.) (Acari: Ixodidae) accounted for nearly all ticks collected (n = 14,116, 99.5%), and the only other species identified were Amblyomma maculatum Koch (Acari: Ixodidae), Dermacentor variabilis (Say) (Acari: Ixodidae) and Ixodes scapularis Say (Acari: Ixodidae). All ticks were tested for both BRBV and Heartland virus (Family Bunyaviridae: Genus Phlebovirus) in 964 pools. Five Heartland virus positive tick pools were detected and confirmed by real-time reverse transcription PCR (rRT-PCR), while all pools tested negative for BRBV. Each Heartland positive pool was composed of 25 A. americanum nymphs with positive pools collected at three different sites in Bourbon County. A. americanum is believed to be the primary vector of both Heartland and BRBVs to humans based upon multiple detections of virus in field-collected ticks, its abundance, and its aggressive feeding behavior on mammals including humans. However, it is possible that A. americanum encounters viremic vertebrate hosts of BRBV less frequently than viremic hosts of Heartland virus, or that BRBV is less efficiently passed among ticks by co-feeding, or less efficiently passed vertically from infected female ticks to their offspring resulting in lower field infection rates.

Prevalence of Shiga-toxigenic Escherichia coli in House Flies (Diptera: Muscidae) in an Urban Environment.

House flies (Musca domestica L. [Diptera: Muscidae]) can act as a mechanical vector for food-borne pathogens including Shiga toxin-producing Escherichia coli (Migula; Enterobacteriales: Enterobacteriaceae) (STEC) in and around cattle feedlots. The present study assessed the prevalence of STEC in house flies from a restaurant area of a town in northeastern Kansas. Two hundred twenty-four house flies were collected over 10 wk, surface sterilized, individually homogenized, and cultured by a multifaceted approach of direct plating on selective media and an enrichment broth, followed by the immunomagnetic separation. Bacterial isolates were screened for eight serogroups of E. coli: O103, O104, O26, O111, O45, O145, O121, and O157 using multiplex polymerase chain reaction (PCR). Furthermore, O-serogroup-positive isolates were tested for virulence genes stx1, stx2, eae, and ehxA by PCR. The majority (91.5%) of flies carried enteric bacteria, and the mean value of enteric concentration on the modified Possé agar was 6.7 ± 1.1 × 106 colony forming units per fly. Thirty-nine of the 224 flies (17.4%) were positive for one or more E. coli serogroup of interest; with the majority O103 (10.7%), followed by O26 (3.1%), O121 (1.3%), O45 (1.3%), and O104 (0.9%). However, none of the serogroup-positive isolates carried any of the virulence genes tested. Results of our study show that house flies in the urban environment do not carry STEC. Nevertheless, detection of E. coli O-serogroups with the potential to acquire virulence traits indicates that house flies in an urban environment represent a public health risk.

Stable Flies (Stomoxys calcitrans L.) from Confined Beef Cattle Do Not Carry Shiga-Toxigenic Escherichia coli (STEC) in the Digestive Tract.

BACKGROUND AND OBJECTIVE: Stable flies (Stomoxys calcitrans L.) are very common around confined and pastured cattle, and due to their painful bites they are very important animal pests. Cattle are asymptomatic reservoirs of foodborne pathogens, Escherichia coli O157:H7 and other Shiga-toxigenic E. coli serotypes (STEC). In the present study, the potential of stable flies to carry STEC in a beef cattle feedlot was assessed. METHODS: Stable flies (n = 180) were collected over 3 summer months and processed individually for STEC-8 that included the serotype O157 and seven non-O157 serotypes (O26, O45, O103, O104, O111, O121, and O145). Isolation and detection of STEC was based on direct plating as well as the enrichment/immunomagnetic separation approach. Modified Posse agar (mP) was used for culturing non-O157 serotypes and sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) for E. coli O157. Multiplex polymerase chain reactions were used for differentiation of individual serotypes and detection of virulence genes (stx1, stx2, eae, and ehxA). RESULTS AND CONCLUSIONS: Of 180 stable flies, 67 (37.2%) carried enterics on mP (mean: 3.6 ± 1.05 × 10(6) colony-forming units [CFU]/fly) and 55/180 (30.5%) were positive for bacteria on CT-SMAC (mean: 1.2 ± 1.08 × 10(4) CFU/fly). However, stable flies positive for E. coli serotypes of interest were very rare (prevalence: 1.1%). The three serotype-positive isolates, two E. coli O26 and one E. coli O45, were recovered from two flies and neither of them harbored the virulence genes. We conclude that stable flies likely do not play a role as a biological vector and/or reservoir of STEC-8 in cattle feedlots.

Prevalence, diversity and characterization of enterococci from three coraciiform birds.

Coraciiform birds hoopoe (Upupa epops), common kingfisher (Alcedo atthis) and European roller (Coracius garrulus) were examined for enterococci in their cloacae and uropygial glands. The enterococcal isolates were identified at the species level using several genomic and proteomic methods, screened for antibiotic susceptibility and genotyped by pulsed-field gel electrophoresis (PFGE). Clonality of isolates from the common kingfisher was also assessed by multi-locus sequence typing (MLST). Using selective media, putative enterococcal isolates (n = 117) were recovered from 74% (32 out of a total of 43) of the bird samples and 114 isolates were confirmed as enterococci. Overall, among the total of 6 different species detected, Enterococcus faecalis was dominant (59%) in all three bird species. The second most frequently isolated species was Enterococcus casseliflavus (32%). PFGE revealed great diversity of strains from different bird species and anatomic location. Closely related strains were found only from nestlings from the same nest. No genes conferring resistance to vancomycin (vanA, vanB, vanC1 and van C2/C3) or erythromycin (erm A, ermB and mefA/E) were detected. MLST analysis and eBURST clustering revealed that sequence types of E. faecalis from the common kingfisher were identical to those of isolates found previously in water, chickens, and humans.

Fresh steam-flaked corn in cattle feedlots is an important site for fecal coliform contamination by house flies.

House flies are a common pest at food animal facilities, including cattle feedlots. Previously, house flies were shown to play an important role in the ecology of Escherichia coli O157:H7; house flies in cattle feedlots carried this zoonotic pathogen and were able to contaminate cattle through direct contact and/or by contamination of drinking water and feed. Because house flies aggregate in large numbers on fresh ( # 6 h) steam-flaked corn (FSFC) used in cattle feed, the aim of this study was to assess FSFC in a cattle feedlot as a potentially important site of fecal coliform contamination by house flies. House flies and FSFC samples were collected, homogenized, and processed for culturing of fecal coliforms on membrane fecal coliform agar. Selected isolates were identified by 16S rRNA gene sequencing, and representative isolates from each phylogenetic group were genotyped by pulsed-field gel electrophoresis. Fecal coliforms were undetectable in FSFC shortly (0 h) after flaking; however, in summer, after 4 to 6 h, the concentrations of fecal coliforms ranged from 1.9 × 10(3) to 3.7 × 10(4) CFU/g FSFC (mean, 1.1 ± 3.0 × 10(4) CFU/g). House flies from FSFC carried between 7.6 × 10(2) and 4.1 × 10(6) CFU of fecal coliforms per fly (mean, 6.0 ± 2.3 × 10(5) CFU per fly). Fecal coliforms were represented by E. coli (85.1%), Klebsiella spp. (10.6%), and Citrobacter spp. (4.3%). Pulsed-field gel electrophoresis demonstrated clonal matches of E. coli and Klebsiella spp. between house flies and FSFC. In contrast, in winter and in the absence of house flies, the contamination of corn by fecal coliforms was significantly (∼10-fold) lower. These results indicate that FSFC is an important site for bacterial contamination by flies and possible exchange of E. coli and other bacteria among house flies. Further research is needed to evaluate the potential use of screens or blowers to limit the access of house flies to FSFC and therefore their effectiveness in preventing bacterial contamination.

Insects represent a link between food animal farms and the urban environment for antibiotic resistance traits.

Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.

Significance and survival of Enterococci during the house fly development.

House flies are among the most important nonbiting insect pests of medical and veterinary importance. Larvae develop in decaying organic substrates and their survival strictly depends on an active microbial community. House flies have been implicated in the ecology and transmission of enterococci, including multi-antibiotic-resistant and virulent strains of Enterococcus faecalis. In this study, eight American Type Culture Collection type strains of enterococci including Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus hirae, Enterococcus mundtii, Enterococcus gallinarum, Enterococcusfaecalis, and Enterococcusfaecium were evaluated for their significance in the development of house flies from eggs to adults in bacterial feeding assays. Furthermore, the bacterial colonization of the gut of teneral flies as well as the importance of several virulence traits of E. faecalis in larval mortality was assessed. Overall survival of house flies (egg to adult) was significantly higher when grown with typically nonpathogenic enterococcal species such as E. hirae (76.0% survival), E. durans (64.0%), and E. avium (64.0%) compared with that with clinically important species E. faecalis (24.0%) and E. faecium (36.0%). However, no significant differences in survival of house fly larvae were detected when grown with E. faecalis strains carrying various virulence traits, including isogenic mutants of the human clinical isolate E. faecalis V583 with in-frame deletions of gelatinase, serine protease, and capsular polysaccharide serotype C. Enterococci were commonly detected in fly puparia (range: 75-100%; concentration: 103-105 CFU/puparium);however, the prevalence of enterococci in teneral flies varied greatly: from 25.0 (E. casseliflavus) to 89.5% (E. hirae). In conclusion, depending on the species, enterococci variably support house fly larval development and colonize the gut of teneral adults. The human pathogenic species, E. faecalis and E. faecium, poorly support larval development and are likely acquired in nature by adult flies during feeding. House fly larvae do not appear to be a suitable model organism for assessment of enterococcal virulence traits.

Degradation of 2,4-dinitroanisole (DNAN) by metabolic cooperative activity of Pseudomonas sp. strain FK357and Rhodococcus imtechensis strain RKJ300.

2,4-Dinitroanisole (DNAN) is an insensitive explosive ingredient used by many defense agencies as a replacement for 2,4,6-trinitrotoluene. Although the biotransformation of DNAN under anaerobic condition has been reported, aerobic microbial degradation pathway has not been elucidated. An n-methyl-4-nitroaniline degrading bacterium Pseudomonas sp. strain FK357 transformed DNAN into 2,4-dinitrophenol (2,4-DNP) as an end product. Interestingly, when strain FK357 was co-cultured with a 2,4-DNP degrading Rhodococcus imtechensis strain RKJ300, complete and high rate of DNAN degradation was observed with no accumulation of intermediates. Enzyme assay using cell extracts of strain FK357 demonstrated that O-demethylation reaction is the first step of DNAN degradation with formation of 2,4-DNP and formaldehyde as intermediates. Subsequently, 2,4-DNP was degraded by strain RKJ300 via the formation of hydride-Meisenheimer complex. The present study clearly demonstrates that complete degradation of DNAN occurs as a result of the metabolic cooperative activity of two members within a bacterial consortium.

Mortality in kittens is associated with a shift in ileum mucosa-associated enterococci from Enterococcus hirae to biofilm-forming Enterococcus faecalis and adherent Escherichia coli.

Approximately 15% of foster kittens die before 8 weeks of age, with most of these kittens demonstrating clinical signs or postmortem evidence of enteritis. While a specific cause of enteritis is not determined in most cases, these kittens are often empirically administered probiotics that contain enterococci. The enterococci are members of the commensal intestinal microbiota but also can function as opportunistic pathogens. Given the complicated role of enterococci in health and disease, it would be valuable to better understand what constitutes a "healthy" enterococcal community in these kittens and how this microbiota is impacted by severe illness. In this study, we characterized the ileum mucosa-associated enterococcal community of 50 apparently healthy and 50 terminally ill foster kittens. In healthy kittens, Enterococcus hirae was the most common species of ileum mucosa-associated enterococci and was often observed to adhere extensively to the small intestinal epithelium. These E. hirae isolates generally lacked virulence traits. In contrast, non-E. hirae enterococci, notably Enterococcus faecalis, were more commonly isolated from the ileum mucosa of kittens with terminal illness. Isolates of E. faecalis had numerous virulence traits and multiple antimicrobial resistances. Moreover, the attachment of Escherichia coli to the intestinal epithelium was significantly associated with terminal illness and was not observed in any kitten with adherent E. hirae. These findings identify a significant difference in the species of enterococci cultured from the ileum mucosa of kittens with terminal illness compared to the species cultured from healthy kittens. In contrast to prior case studies that associated enteroadherent E. hirae with diarrhea in young animals, these controlled studies identified E. hirae as more often isolated from healthy kittens and adherence of E. hirae as more common and extensive in healthy kittens than in sick kittens.

Enterococcus alcedinis sp. nov., isolated from common kingfisher (Alcedo atthis).

Two Gram-positive, catalase-negative bacterial strains were isolated from the cloaca of common kingfishers (Alcedo atthis). Repetitive sequence-based PCR fingerprinting using the (GTG)5 primer grouped these isolates into a single cluster separated from all known enterococcal species. The two strains revealed identical 16S rRNA gene sequences placing them within the genus Enterococcus with Enterococcus aquimarinus LMG 16607(T) as the closest relative (97.14 % similarity). Further taxonomic investigation using sequencing of the genes for the superoxide dismutase (sodA), phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) as well as application of whole-cell protein fingerprinting, automated ribotyping and extensive phenotyping confirmed that both strains belong to the same species. Based on data from this polyphasic study, these strains represent a novel species of the genus Enterococcus, for which the name Enterococcus alcedinis sp. nov. is proposed. The type strain is L34(T) (= CCM 8433(T) = LMG 27164(T)).

Vancomycin-resistant enterococci in rooks (Corvus frugilegus) wintering throughout Europe.

This study's aims were to assess the prevalence of, and to characterize, vancomycin-resistant enterococci (VRE) from rooks (Corvus frugilegus) wintering in Europe during 2010/2011. Faeces samples were cultivated selectively for VRE and characterized. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were used to examine epidemiologic relationships of vanA-containing VRE. The vanA-carrying VRE were tested in vitro for mobility of vancomycin resistance traits. VRE were found in 62 (6%) of 1073 rook samples. Enterococcal species diversity comprised Enterococcus gallinarum (48 isolates), followed by E. faecium (9) and E. faecalis (5). Eight VRE harboured the vanA and ermB genes. Seven vanA-carrying VRE originated from the Czech Republic and one from Germany. All vanA-carrying VRE were identified as E. faecium. Based on MLST analysis, six vanA-positive isolates were grouped as ST92 type, one isolate belonged to ST121, and the remaining one was described as a novel type ST671. Seven out of eight isolates were able to transfer the vancomycin resistance trait via filter mating with a transfer rate of 8.95 ± 3.25 × 10(-7) transconjugants per donor. In conclusion, wintering rooks in some European countries may disseminate clinically important enterococci and pose a risk for environmental contamination.

Misoprostol for cervical ripening prior to gynecological transcervical procedures.

PURPOSE: Dilatation and curettage is frequently performed in gynecological practice. Aim of this prospective randomized double-blind placebo-controlled study was to evaluate the safety and efficacy of oral misoprostol to prime non-pregnant cervix before this procedure. METHOD: Women requiring dilatation and curettage were included in the study. Exclusion criteria were visible growth in cervix or vagina, pregnancy, allergy to prostaglandins, some medical disorders. Each participant was instructed to take either 400 µg of misoprostol or placebo orally 12 h before the procedure. Primary outcome measure was: diameter of the largest negotiable Hegar's dilator through internal os without any resistance at the beginning of the procedure. Secondary outcome measures were: percentage of women with initial cervical dilatation of ≥5 mm, time required for optimum cervical dilatation, percentage of failed procedures and complications. t test, Chi-square test and Fisher's test were used to compare the variables. RESULT: Misoprostol significantly increased baseline cervical diameter in the pre-menopausal group (p < 0.001), but not in post-menopausal subjects (p = 0.1) and reduced time required for cervical dilatation in both pre-and post-menopausal women. The number of patients achieving initial cervical dilatation ≥5 mm was significantly greater in pre-menopausal subjects receiving misoprostol, but not significant in post-menopausal ones. The drug was also found to be effective in both nulliparous and multiparous patients. Side effects were comparable between two groups. Only nausea and vomiting were more frequent in post-menopausal misoprostol group than placebo (p = 0.018). CONCLUSION: Four hundred micrograms of oral misoprostol 12 h prior to dilation and curettage was found to be beneficial in cervical priming in pre-menopausal subjects. It was also found to be effective irrespective of the parity of the patients.

Branching of the p-nitrophenol (PNP) degradation pathway in burkholderia sp. Strain SJ98: Evidences from genetic characterization of PNP gene cluster.

Aerobic microbial degradation of p-nitrophenol (PNP) has been classically shown to proceed via 'Hydroquinone (HQ) pathway' in Gram-negative bacteria, whereas in Gram-positive PNP degraders it proceed via 'Benzenetriol (BT) pathway'. These pathways are characterized by the ring cleavage of HQ and BT as terminal aromatic intermediates respectively. Earlier reports on PNP degradation have indicated these pathways to be mutually exclusive. We report involvement of both 'HQ' and 'BT' ring cleavage pathways in PNP degradation by Burkholderia sp. strain SJ98. Genetic characterization of an ~41 Kb DNA fragment harboring PNP degradation gene cluster cloned and sequenced from strain SJ98 showed presence of multiple orfs including pnpC and pnpD which corresponded to previously characterized 'benzenetriol-dioxygenase (BtD)' and 'maleylacetate reductase (MaR)' respectively. This gene cluster also showed presence of pnpE1 and pnpE2, which shared strong sequence identity to cognate sub-units of 'hydroquinone dioxygenase' (HqD). Heterologous expression and biochemical characterization ascertained the identity of PnpE1 and PnpE2. In in vitro assay reconstituted heterotetrameric complex of PnpE1 and PnpE2 catalyzed transformation of hydroquinone (HQ) into corresponding hydroxymuconic semialdehyde (HMS) in a substrate specific manner. Together, these results clearly establish branching of PNP degradation in strain SJ98. We propose that strain SJ98 presents a useful model system for future studies on evolution of microbial degradation of PNP.