Ongoing Outbreak of Extensively Drug-Resistant Campylobacter jejuni Infections Associated With US Pet Store Puppies, 2016-2020.

Importance: Extensively drug-resistant Campylobacter jejuni infections cannot be treated with any commonly recommended antibiotics and pose an increasing public health threat. Objectives: To investigate cases of extensively drug-resistant C jejuni associated with pet store puppies and describe the epidemiologic and laboratory characteristics of these infections. Design, Setting, and Participants: In August 2017, health officials identified, via survey, patients with C jejuni infections who reported contact with puppies sold by pet stores. In conjunction with state and federal partners, the Centers for Disease Control and Prevention investigated cases of culture-confirmed C jejuni infections in US patients with an epidemiologic or molecular association with pet store puppies between January 1, 2016, and February 29, 2020. Available records from cases occurring before 2016 with genetically related isolates were also obtained. Main Outcomes and Measures: Patients were interviewed about demographic characteristics, health outcomes, and dog exposure during the 7 days before illness onset. Core genome multilocus sequence typing was used to assess isolate relatedness, and genomes were screened for resistance determinants to predict antibiotic resistance. Isolates resistant to fluoroquinolones, macrolides, and 3 or more additional antibiotic classes were considered to be extensively drug resistant. Cases before 2016 were identified by screening all sequenced isolates submitted for surveillance using core genome multilocus sequence typing. Results: A total of 168 patients (median [interquartile range] age, 37 [19.5-51.0] years; 105 of 163 female [64%]) with an epidemiologic or molecular association with pet store puppies were studied. A total of 137 cases occurred from January 1, 2016, to February 29, 2020, with 31 additional cases dating back to 2011. Overall, 117 of 121 patients (97%) reported contact with a dog in the week before symptom onset, of whom 69 of 78 (88%) with additional information reported contact with a pet store puppy; 168 isolates (88%) were extensively drug resistant. Traceback investigation did not implicate any particular breeder, transporter, distributer, store, or chain. Conclusions and Relevance: Strains of extensively drug-resistant C jejuni have been circulating since at least 2011 and are associated with illness among pet store customers, employees, and others who come into contact with pet store puppies. The results of this study suggest that practitioners should ask about puppy exposure when treating patients with Campylobacter infection, especially when they do not improve with routine antibiotics, and that the commercial dog industry should take action to help prevent the spread of extensively drug-resistant C jejuni from pet store puppies to people.

Replication of respiratory syncytial virus is inhibited by the host defense molecule viperin.

Respiratory syncytial virus (RSV) is an important viral pathogen of otitis media, bronchiolitis, and pneumonia. As infection of the upper airways is a precondition for the development of these diseases, understanding RSV pathogenesis and the host response induced by RSV in this niche may enable the development of novel therapeutic strategies against this virus. We have used a microarray approach and showed that expression of the gene that encodes the antiviral protein viperin was significantly upregulated in the chinchilla nasopharynx up to 1 week after RSV challenge. Overexpression of human viperin in vitro diminished the ability of RSV to infect HeLa or A549 cells. Furthermore, transduction of the chinchilla airways with a recombinant adeno-associated virus vector that encodes viperin resulted in reduced titers of RSV in the nasopharyngeal lavage fluid. Collectively, these data indicated that viperin plays a significant role in the innate immune defense against RSV.

Extracellular DNA within a nontypeable Haemophilus influenzae-induced biofilm binds human beta defensin-3 and reduces its antimicrobial activity.

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are associated with multiple chronic infections of the airway, including otitis media. Extracellular DNA (eDNA) is part of the biofilm matrix and serves as a structural component. Human ß-defensin-3 (hBD-3) is a cationic antimicrobial host defense protein (AMP) critical to the protection of the middle ear. We hypothesized that anionic eDNA could interact with and bind hBD-3 and thus shield NTHI in biofilms from its antimicrobial activity. We demonstrated that recombinant hBD-3 [(r)hBD-3] bound eDNA in vitro and that eDNA in biofilms produced by NTHI in the chinchilla middle ear co-localized with the orthologue of this AMP. Incubation of physiological concentrations of (r)hBD-3 with NTHI genomic DNA abrogated the ability of this innate immune effector to prevent NTHI from forming robust biofilms in vitro. Establishment of NTHI biofilms in the presence of both DNase I and (r)hBD-3 resulted in a marked reduction in the overall height and thickness of the biofilms and rescued the antimicrobial activity of the AMP. Our results demonstrated that eDNA in NTHI biofilms sequestered hBD-3 and thus diminished the biological activity of an important effector of innate immunity. Our observations have important implications for chronicity of NTHI-induced diseases.

Respiratory syncytial virus promotes Moraxella catarrhalis-induced ascending experimental otitis media.

Otitis media (OM) is a polymicrobial disease wherein prior or concurrent infection with an upper respiratory tract virus plays an essential role, predisposing the middle ear to bacterial invasion. In episodes of acute bacterial OM, respiratory syncytial virus (RSV) is the most commonly isolated virus and thus serves as an important co-pathogen. Of the predominant bacterial agents of OM, the pathogenesis of disease due to Moraxella catarrhalis is the least well understood. Rigorous study of M. catarrhalis in the context of OM has been significantly hindered by lack of an animal model. To bridge this gap, we assessed whether co-infection of chinchillas with M. catarrhalis and RSV would facilitate ascension of M. catarrhalis from the nasopharynx into the middle ear. Chinchillas were challenged intranasally with M. catarrhalis followed 48 hours later by intranasal challenge with RSV. Within 7 days, 100% of nasopharynges were colonized with M. catarrhalis and homogenates of middle ear mucosa were also culture-positive. Moreover, within the middle ear space, the mucosa exhibited hemorrhagic foci, and a small volume of serosanguinous effusion was present in one of six ears. To improve upon this model, and based on epidemiologic data, nontypeable Haemophilus influenzae (NTHI) was included as an additional bacterial co-pathogen via intranasal administration four days before M. catarrhalis challenge. With this latter protocol, M. catarrhalis was cultured from the nasopharynx and middle ear homogenates of a maximum of 88% and 79% animals, respectively, for up to 17 days after intranasal challenge with M. catarrhalis. Additionally, hemorrhagic foci were observed in 79% of middle ears upon sacrifice. Thus, these data demonstrated that co-infection with RSV and NTHI predisposed to M. catarrhalis-induced ascending experimental OM. This model can be used both in studies of pathogenesis as well as to investigate strategies to prevent or treat OM due to M. catarrhalis.

Ribonuclease 7 is a potent antimicrobial peptide within the human urinary tract.

Although the urinary tract is constantly challenged by microbial invasion, it remains free from colonization. Although little is known about how the urinary tract maintains sterility, the presence of antimicrobial peptides (AMPs) in the urine suggests that they may play a role in its protection from infection. Ribonuclease 7 (RNase 7) is a potent AMP that was first identified in the skin. Here, we characterize the expression and relevance of RNase 7 in the human kidney and urinary tract. Using RNA isolated from healthy human tissue, we performed quantitative real-time PCR and found basal RNASE7 expression in kidney and bladder tissue. Immunohistochemical and immunofluorescent analysis localized RNase 7 to the urothelium of the bladder, ureter, and the intercalated cells of the collecting tubules. In control urine samples from healthy individuals, the concentration of RNase 7 was found to be in the low micromolar range; very abundant for an AMP. Antibacterial neutralization assays showed that urinary RNase 7 has potent antimicrobial properties against Gram-negative and Gram-positive uropathogenic bacteria. Thus, RNase 7 is expressed in the human kidney and urinary tract and it may have an important antimicrobial role in maintaining tract sterility.

Abrogation of nontypeable Haemophilus influenzae protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media.

The pneumococcal polysaccharide conjugate vaccine which includes a nonacylated protein D carrier from Haemophilus influenzae has been recently licensed for use in many countries. While this vaccine is protective against nontypeable Haemophilus influenzae (NTHI)-induced acute otitis media (OM), the mechanism underlying this protective efficacy is not yet fully understood. Protein D/glycerophosphodiester phosphodiesterase (PD/GlpQ) is an outer membrane lipoprotein expressed by NTHI that has been ascribed several functions, including host cell adherence and phosphorylcholine (PCho) acquisition. We found that a pd/glpQ NTHI mutant exhibited reduced adherence to airway epithelial cells, diminished phosphorylcholine (PCho) decoration of biofilms, and compromised fitness during experimental acute OM compared to the parent strain. We also found that exposure of NTHI to antibodies directed against the vaccine formulation recapitulated the PCho decoration and NTHI adherence phenotypes exhibited by PD/GlpQ-deficient NTHI, providing at least two likely mechanisms by which the pneumococcal polysaccharide-PD/GlpQ conjugate vaccine induces protection from NTHI-induced OM.

The multifunctional host defense peptide SPLUNC1 is critical for homeostasis of the mammalian upper airway.

Otitis media (OM) is a highly prevalent pediatric disease caused by normal flora of the nasopharynx that ascend the Eustachian tube and enter the middle ear. As OM is a disease of opportunity, it is critical to gain an increased understanding of immune system components that are operational in the upper airway and aid in prevention of this disease. SPLUNC1 is an antimicrobial host defense peptide that is hypothesized to contribute to the health of the airway both through bactericidal and non-bactericidal mechanisms. We used small interfering RNA (siRNA) technology to knock down expression of the chinchilla ortholog of human SPLUNC1 (cSPLUNC1) to begin to determine the role that this protein played in prevention of OM. We showed that knock down of cSPLUNC1 expression did not impact survival of nontypeable Haemophilus influenzae, a predominant causative agent of OM, in the chinchilla middle ear under the conditions tested. In contrast, expression of cSPLUNC1 was essential for maintenance of middle ear pressure and efficient mucociliary clearance, key defense mechanisms of the tubotympanum. Collectively, our data have provided the first in vivo evidence that cSPLUNC1 functions to maintain homeostasis of the upper airway and, thereby, is critical for protection of the middle ear.

Respiratory syncytial virus-induced dysregulation of expression of a mucosal beta-defensin augments colonization of the upper airway by non-typeable Haemophilus influenzae.

Otitis media (OM) is a polymicrobial disease wherein upper respiratory tract viruses compromise host airway defences, which allows bacterial flora of the nasopharynx (NP) access to the middle ear. We have shown, in vitro, that respiratory syncytial virus (RSV), a viral co-pathogen of OM, reduces transcript abundance of the antimicrobial peptide (AP), chinchilla beta-defensin-1 (cBD-1). Here, we demonstrated that chinchillas inoculated with RSV expressed approximately 40% less cBD-1 mRNA and protein than did mock-challenged animals. Further, concurrent RSV infection resulted in a 10-100-fold greater recovery of non-typeable Haemophilus influenzae (NTHI) from nasopharyngeal lavage fluids, compared with chinchillas challenged with NTHI in the absence of viral co-infection. Additionally, when either: anti-cBD-1 antibody (to bind secreted AP) or recombinant cBD-1 (to increase AP concentration at the mucosal surface) were delivered to chinchillas, we demonstrated that disruption of the availability of a single AP influenced the relative load of NTHI in the upper respiratory tract. Collectively, our data suggested that effectors of innate immunity regulate normal bacterial colonization of the NP and, further, virus-induced altered expression of APs can result in an increased load of NTHI within the NP, which likely promotes development of OM.

A carcinoembryonic antigen-related cell adhesion molecule 1 homologue plays a pivotal role in nontypeable Haemophilus influenzae colonization of the chinchilla nasopharynx via the outer membrane protein P5-homologous adhesin.

In vitro studies suggest an important role for CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) in infection by multiple gram-negative bacteria. However, in vivo evidence supporting this role is lacking, largely because the bacterial adhesins involved in this host-microbe association do not bind to murine-derived CEACAM1. One of several adhesins expressed by nontypeable Haemophilus influenzae (NTHI), the outer membrane protein P5-homologous adhesin (or P5), is essential for colonization of the chinchilla nasopharynx and infection of the middle ear. Here we reveal that NTHI P5 binds to the chinchilla homologue of CEACAM1 and that rabbit anti-human carcinoembryonic antigen blocks NTHI colonization of the chinchilla nasopharynx, providing the first demonstration of a role for CEACAM receptor binding by any bacterial pathogen in vivo.

Iron acquisition in the dental pathogen Actinobacillus actinomycetemcomitans: what does it use as a source and how does it get this essential metal?

Actinobacillus actinomycetemcomitans requires iron to grow under limiting conditions imposed by synthetic and natural chelators. Although none of the strains tested used hemoglobin, lactoferrin or transferrin, all of them used FeCl3 and hemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, hemoglobin, and hemin but not transferrin. When compared with smooth strains, the rough isolates showed higher hemin binding activity, which was sensitive to proteinase K treatment. A. actinomycetemcomitans harbors the Fur-regulated afeABCD locus coding for iron acquisition in isogenic and non-isogenic cell backgrounds. The genome of this oral pathogen also harbors several other predicted iron uptake genes including the hitABC locus, which restored iron acquisition in the E. coli 1017 ent mutant. However, the disruption of this locus in the parental strain did not affect iron acquisition as drastically as the inactivation of AfeABCD, suggesting that the latter system could be more involved in iron transport than the HitABC system. The genome of this oral pathogen also harbors an active copy of the exbBexbDtonB operon, which could provide the energy needed for hemin acquisition. However, inactivation of each coding region of this operon did not affect the hemin and iron acquisition phenotypes of isogenic derivatives. This observation suggests that the function of these proteins could be replaced by those coded for by tolQ, tolR and tolA as it was described for other bacterial transport systems. Interruption of a hasR homolog, an actively transcribed gene that is predicted to code for an outer membrane hemophore receptor protein, did not affect the ability of an isogenic derivative to bind and use hemin under chelated conditions. This result also indicates that A. actinomycetemcomitans could produce more than one outer membrane hemin receptor as it was described in other human pathogens. All strains tested formed biofilms on plastic under iron-rich and iron-chelated conditions. However, smooth strains attached poorly and formed weaker biofilms when compared with rough isolates. The incubation of rough cells in the presence of FeCl3 or hemin resulted in an increased number of smaller aggregates and microcolonies as compared to the fewer but larger aggregates formed when cells were grown in the presence of dipyridyl.

A member of the cathelicidin family of antimicrobial peptides is produced in the upper airway of the chinchilla and its mRNA expression is altered by common viral and bacterial co-pathogens of otitis media.

Cationic antimicrobial peptides (AMPs), a component of the innate immune system, play a major role in defense of mucosal surfaces against a wide spectrum of microorganisms such as viral and bacterial co-pathogens of the polymicrobial disease otitis media (OM). To further understand the role of AMPs in OM, we cloned a cDNA encoding a cathelicidin homolog (cCRAMP) from upper respiratory tract (URT) mucosae of the chinchilla, the predominant host used to model experimental OM. Recombinant cCRAMP exhibited alpha-helical secondary structure and killed the three main bacterial pathogens of OM. In situ hybridization showed cCRAMP mRNA production in epithelium of the chinchilla Eustachian tube and RT-PCR was used to amplify cCRAMP mRNA from several other tissues of the chinchilla URT. Quantitative RT-PCR analysis of chinchilla middle ear epithelial cells (CMEEs) incubated with either viral (influenza A virus, adenovirus, or RSV) or bacterial (nontypeable H. influenzae, M. catarrhalis, or S. pneumoniae) pathogens associated with OM demonstrated distinct microbe-specific patterns of altered expression. Collectively, these data showed that viruses and bacteria modulate AMP messages in the URT, which likely contributes to the disease course of OM.

Characterization of the IgA1 protease from the Brazilian purpuric fever strain F3031 of Haemophilus influenzae biogroup aegyptius.

Brazilian purpuric fever is a severe vascular disease caused by an invasive clone of Haemophilus influenzae biogroup aegyptius, which normally causes self-limiting eye infections. A previous genome subtraction procedure resulted in the isolation of a DNA fragment, which encodes a putative IgA1 protease, specific to the F3031 Brazilian purpuric fever type strain. Cloning and sequencing of the entire F3031 iga1 gene showed that the subtracted DNA fragment encompasses the iga1 region encoding the active site and the cleavage specificity determinant of the protein, which are different from the cognate regions of the proteases produced by other H. influenzae strains. Western and IgA cleavage assays together with clustering analysis showed that the F3031 IgA1 protease is most similar to the type 2 proteases produced by H. influenzae type c and e strains. Analysis of the promoter region of the F3031 iga1 gene revealed the presence of Fur binding sites. However, real-time PCR analysis and transcriptional fusion assays showed that the expression of iga1 is not regulated by iron or hemin under the conditions tested.

Genetic and functional analyses of the Actinobacillus actinomycetemcomitans AfeABCD siderophore-independent iron acquisition system.

The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions.

Cloning and sequencing of a genomic island found in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

A genomic island was identified in the Haemophilus influenzae biogroup aegyptius Brazilian purpuric fever (BPF) strain F3031. This island, which was also found in other BPF isolates, could not be detected in non-BPF biogroup aegyptius strains or in nontypeable or typeable H. influenzae strains, with the exception of a region present in the type b Eagan strain. This 34,378-bp island is inserted, in reference to H. influenzae Rd KW20, within a choline transport gene and contains a mosaic structure of Mu-like prophage genes, several hypothetical genes, and genes potentially encoding an Erwinia carotovora carotovoricin Er-like bacteriocin. The product of the tail fiber ORF in the bacteriocin-like region shows a hybrid structure where the C terminus is similar to an H. influenzae phage HP1 tail protein implicating this open reading frame in altering host specificity for a putative bacteriocin. Significant synteny is seen in the entire genomic island with genomic regions from Salmonella enterica subsp. enterica serovar Typhi CT18, Photorhabdus luminescens subsp. laumondii TT01, Chromobacterium violaceum, and to a lesser extent Haemophilus ducreyi 35000HP. In a previous work, we isolated several BPF-specific DNA fragments through a genome subtraction procedure, and we have found that a majority of these fragments map to this locus. In addition, several subtracted fragments generated from an independent laboratory by using different but related strains also map to this island. These findings underscore the importance of this BPF-specific chromosomal region in explaining some of the genomic differences between highly invasive BPF strains and non-BPF isolates of biogroup aegyptius.

Complete nucleotide sequence of Klebsiella pneumoniae multiresistance plasmid pJHCMW1.

The multiresistance plasmid pJHCMW1, harbored by a clinical Klebsiella pneumoniae strain isolated from a neonate with meningitis, was sequenced. A circular sequence of 11,354 bp was generated, of which 7,993 bp make up Tn1331, a transposon including the antibiotic resistance genes aac(6')-Ib, aadA1, bla(OXA-9), and bla(TEM-1). The gene aac(6')-Ib is included in a gene cassette, and both aadA1 and bla(OXA-9) are included in a single-gene cassette that may have arisen as a consequence of a recombination event involving two integrons. The pJHCMW1 plasmid replicates through a ColE1-like RNA-regulated mechanism, includes a functional oriT, and two loci with similarity to XerCD site-specific recombination target sites involved in plasmid stabilization by the resolution of multimers. One of these two loci, mwr, is active and has been the subject of previous studies, and the other, dxs, is not functional but binds the recombinase XerD with low affinity. Two additional open reading frames were identified, one with low similarity to two hypothetical membrane proteins from Mycobacterium tuberculosis and Mycobacterium leprae and the other with low similarity to psiB, a gene encoding a function that facilitates the establishment of the transferring plasmid in the recipient bacterial cell during the process of conjugation.

Genomic analysis of the F3031 Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius by PCR-based subtractive hybridization.

PCR-based subtractive genome hybridization produced clones harboring inserts present in Brazilian purpuric fever (BPF) prototype strain F3031 but absent in noninvasive Haemophilus influenzae biogroup aegyptius isolate F1947. Some of these inserts have no matches in the GenBank database, while others are similar to genes encoding either known or hypothetical proteins. One insert represents a 2.3-kb locus with similarity to a Thermotoga maritima hypothetical protein, while another is part of a 7.6-kb locus that contains predicted genes encoding hypothetical, phage-related, and carotovoricin Er-like proteins. The presence of DNA related to these loci is variable among BPF isolates and nontypeable H. influenzae strains, while neither of them was detected in strains of types a to f. The data indicate that BPF-causing strain F3031 harbors unique chromosomal regions, most of which appear to be acquired from unrelated microbial sources.