Detection and Characterization of β-Lactam Resistance in Haemophilus parasuis Strains from Pigs in South China

To characterize the β-lactam resistance in veterinary clinical isolates of Haemophilus parasuis, 115 isolates were examined for the β-lactam resistance, the possession of β-lactamase, and the presence of β-lactamase genes. The genetic relationship among isolates was evaluated by pulsed-field gel electrophoresis （PFGE）. Overall, the commonly detected resistance phenotypes were resistant to ampicillin （26.09%）, penicillin （22.61%）, amoxicillin （21.74%）, cefazolin （14.78%）, cefaclor （12.17%）, and cefotaxime （6.96%）. These strains showed high minimal inhibitory concentration （MICs） to oxacillin. 20.87% strains produced β-lactamase, and 4.35% strains showed extended-spectrum b-lactamase （ESBL） phenotype. Moreover, 19 strains harboured bla genes including TEM-1 （n=5）, TEM-116 （n=10）, and ROB-1 （n=5）. Significantly, one strain possessed both TEM-1 and ROB-1, and displayed resistance to cefotaxime （MIC=8 mg L-1）. The epidemiological analysis of PFGE revealed high genetic diversity among bla-positive isolates. This work shows that TEM- and ROB-type β-lactamases are prevalent in H. parasuis isolates in China.

In vitro interference of cefotaxime at subinhibitory concentrations on biofilm formation by nontypeable Haemophilus influenzae

Objective:To investigate the in vitro interference of cefotaxime at subinhibitory concentrations [sub-minimal inhibitory concentrations(MIC)] on biofilm formation by nontypeable Haemophilus influenzae(NTHi).Methods:The interference of subinhibitory concentrations of cefotaxime on biofilm formation of the clinical strong-biofilm forming isolates of NTHi was evaluated by a microtiter plate biofilm formation assay.The effect of sub-MIC cefotaxime on bacterial cell-surface hydrophobicity was determined using a standard microbial adhesion to n-hexadecane test.Additionally,the effects on bacterial adherence to human fibronectin and expression of bacterial adhesins were also investigated.Results:Subinhibitory concentrations of cefotaxime,both at 0.1× and 0.5× MIC levels,efficiently reduced the NTHi biofilm formation,and this effect was independent of decreasing bacterial viability.Sub-MIC cefotaxime also decreased bacterial cell-surface hydrophobicity and reduced adherence to human fibronectin.Inhibition in the P2 and P6 gene expressions upon exposure to sub-MIC cefotaxime was also noted.Conclusions:Taken together,our results indicate that sub-MIC cefotaxime interferes with the formation of NTHi biofilm,and this effect is feasibly related to the interference with cell-surface hydrophobicity,fibronectin-binding activity as well as alteration of the P2 and P6 gene expression.The findings of the present study therefore provide a rationale for the use of subinhibitory concentrations of cefotaxime for treatment of NTHi-related diseases.

Development of Multiplex-PCR for Identification of Pasteurella multocida,Haemophilus parasuis and Actinbacillus pleuropneumoniae

[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.

Association between the Phenotypes and Genotypes of Antimicrobial Resistance in Haemophilus parasuis Isolates from Swine in Quang Binh and Thua Thien Hue Provinces, Vietnam

Haemophilus parasuis (H. parasuis) is one of the bacterial pathogens of great concern as it causes huge economic losses to the swine industry worldwide. One of the reasons why the control of H. parasuis has failed is the increase in antimicrobial resistance (AMR). The country of Vietnam has the second-largest pig production in Asia. However, there is still a lack of data about the AMR prevalence of H. parasuis in Vietnam.The purpose of this study is to investigate the prevalence of AMR and analyze the association between AMR and AMR genes (ARGs). The H. parasuis strains used in this research were isolated from swine in the Quang Binh and Thua Thien Hue Provinces, Central Vietnam, as reported in our previous study. All of the strains were tested for AMR against 25 antibacterial agents using the broth microdilution method and for the presence of ARGs using the polymerase chain reaction (PCR) method. The tested strains were shown to have a high frequency of resistance to trimethoprim/sulfamethoxazole (94.6%), followed by resistance to colistin, chloramphenicol, gentamicin, penicillin, lincomycin, and amoxicillin. The most prevalent ARGs in these strains were blaTEM-1 (94.6%), int (76.8%), gyrA (58.9%), and rmtD (50.0%). Cefuroxime, chloramphenicol, and tobramycin resistances were strongly correlated with the presence of the ARGs blaROB-1 (odds ratio (OR) = 26.3, 95% confidence interval (CI) 2.7–255.7, p = 0.002), catl (OR = 25.1, 95% CI 2.4–258.9, p = 0.004), and strB (OR = 23.5, 95% CI 2.6–212.6, p = 0.001), respectively.This study reveals for the first time the current situation of H. parasuis AMR in Central Vietnam, which is helpful for the clinical control of this disease, as well as for the development of policies and clinical practice guidelines to reduce AMR in swine production in Central Vietnam.

Presence of <i>Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae</i>in upper respiratory tract of swine in farms from Aguascalientes, Mexico

Respiratory diseases are one of the most important health problems in pig herds. The porcine respiratory disease complex (PRDC) is the term used to describe pneumonic diseases caused by multiple infectious agents that provoke weight loss in animals or death. In the PRDC multiple pathogens (bacteria and/or viruses) work in combination to induce this respiratory disease. Within this complex, Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae are the main bacterial pathogens involved in great economic losses to the swine industry. The aim of this work was to estimate the presence of A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae in the upper respiratory tract of pigs in representative swine farms inAguascalientes,Mexico, using PCR technique. The study was performed in 14 swine farms. We obtained a total of 212 nasal swabs. Near 20% of samples were positive for A. pleuropneumoniae (located in the 79% of farms);17% were positive for S. suis (in 86% of farms), of these, 3% were S. suis serovar 2;30% were positive for H. parasuis (93% of farms);23% of the samples to P. multocida (in 79% of farms);and 19% to M. hyopneumoniae (in 64% of farms). B. bronchiseptica was not detected in this study. The results obtained show that bacterial pathogens of PRDC were present in the upper respiratory tract of pigs in all farms studied;therefore, these pathogens are widely disseminated in pig farms of Aguascalientes, Mexico.

Serologic Investigation on Pig Haemophilus parasuis in Some Regions of Sichuan Province

To investigate the infection of Haemophilus parasuislin some regions of Sichuan Province, indirect hemagglutination from 2006 to 2008 was tested by Haemophilus parasuis antibody, and 1 062 serum samples from 10 different regions and sources in Sichuan Province were also examined. The results showed that the positive rate ranged from 2% to 30% with average of 10%. The positive rate varied with pigs at different ages, and especially that of 30-day-old to 70-day-old weaned piglets reached 25%. Thus, there were various infections of HaemophUus parasuis in some regions of Sichuan Province, and the statistic data provided a reference for effective prevention and control of Haemophilus parasuis in Sichuan Province.

Establishment and Application of an Indirect ELISA for Detection on Antibody of Haemophilus parasuis

[Objective]The aim was to establish an indirect ELISA for detection on antibody of Hps. [Method] The optimal conditions of indirect ELISA were selected and determined based on heat-resistant serotype 4 and 5 of Hps; specific,repeating and sensitive tests were conducted and 200 serums were detected. [Result]The optimal conditions were as follows： coating concentration of antigen at 10 μg /ml,and coating for 2 h at 37 °C; PBST containing 20 g /L of skim milk powder as blocking fluid for 30 min; serum dilution at 1∶ 80; reaction time of antigen for 45 min; dilution of secondary antibody at 1∶ 12 000 and effecting for 30 min; color development reaction for 15 min. [Conclusion] The established indirect ELISA is good in specificity and repetitiveness with higher sensitivity than that of indirect hemagglutination test; the results of clinic samples （ negative /positive serums） were in consistent with those detected with foreign ELISA kits. The established method can be made use of in serum antibody of Hps de- tection and seroepidemiology study.

Non-typeable haemophilus influenzae-induced exacerbation on a cigarette smoke lung injury model:Protective effect of andrographolide

OBJECTIVE To develop a 2-week cigarette smoke(CS)acute lung injury model exacerbated by haemophilus influenzae(NTHi)and study the protective effect of andrographolide in this COPD model.METHODS Female BALB/c mice,6-8-week-old,were exposed to 4% 3R4 FCS delivered using aperistaltic pump daily for 2 weeks to induce an acute lung injury model.After 2 weeks of smoking,mice were inoculated intratracheally with NTHi to induce exacerbation on the model.Mice were sacrificed 48 h after last bacteria challenge and lung samples were collected for various analyses.RESULTS After developing a 2-week CS acute lung injury model exacerbated by NTHi,the CS+NTHi group was shown to have a higher inflammatory response,higher bacterial clearance,an upregulation of MMP12 mRNA levels and decrease in TIMP1 mRNA levels in the lungs.Administration of Andrographolide suppressed BALF lung cellular infiltrates,TNF-α,CXCL1/KC,IL-1βand 8-OHdG protein levels,together with increased HO-1 and GR mRNA levels and decreased MMP-8 and MMP-9 mRNA levels.Andrographolide was able to ameliorate lung histopathology as observed with H&E staining and inflammation scoring.Andrographolide was also shown to reduce Keap-1 level in lungs without affecting DJ-1 level.CONCLUSION This study demonstrates the protective effect of andrographolide in a novel 2-week CS acute lung injury model exacerbated by NTHi and presents it as a potential therapeutic for COPD.

Studies of β-lactamase production in ampicillin resistant Haemophilus influenzae

Objective:To study the molecular mechanisms of β-lactamase production in ampicillin resistant(AmP ) Hae- mophilus influenzae(HI). Methods: Identified the β-lactamases production strain from AmP HI was isolated from clinical cases with K-B method. β-lactamase encoding gene in enzyme production strains were detected by PCR with lactamase gene specific primers, and both plasmid and chromosomal DNA samples. Results: Thirty-two out of 36 (88 .9% ) were found to be β-lactamase production. Twenty-nine out of 32 enzyme production stain were PCR positive (the ratio of PCR positives 90.6% ). There were 25 stains amplified with plasmid DNA positively, and 4 with chromosomal DNA. Conclusion: (l ) Most of the AmPr HI strain produce lactamase is mediated by plasmid. (2) Detection of lactamase encoding gene in HI is a simple and efficient approach to study the molecular basis of ampicillin resistance.

Virulent endocarditis due to Haemophilus parainfluenzae:A systematic review of the literature

BACKGROUND Haemophilus parainfluenzae(HPI)belongs to the HACEK(Haemophilus spp.,Aggregatibacter spp.,Cardiobacterium spp.,Eikenella spp.,and Kingella spp.)group of organisms.The HACEK group of organisms are a part of the oropharyngeal flora and can cause invasive opportunistic infection such infective endocarditis(IE)in hosts with compromised immunological barriers.AIM To perform a 20-year systematic review of the literature characterizing the clinical presentation,epidemiology and prognosis of HPI IE.METHODS We performed a systematic review of Medline,Pubmed,Scopus and Embase from 2000 to 2022 to identify all cases of HPI IE.RESULTS Thirty-nine adult cases were identified.HPI IE was found to affect males slightly more than females and is common in patients with predisposing risk factors such as underlying valvular abnormalities.It mostly affected the mitral valve and had an indolent course;significantly sized vegetations(>1 cm)developed in most cases.Central nervous system septic embolization was common.It had a favorable prognosis compared to staphylococcal and streptococcal IE.CONCLUSION Clinicians should be attentive to the indolent course of HPI IE and the presence of predisposing risk factors in order to allow for timely management.

Comparison of Two Construction Methods of Targeting Gene of Haemophilus parasuis

Haemophilus parasuis is one kind of constant bacteria in porcine upper respiratory tract, and it can cause multiple serositis, arthritis and other diseases under certain conditions. Due to lack of efficient genetic operating system, its pathogenic mechanism is not very clear. Ligation with DNA ligase and fusion PCR were used to construct targeting hhdA gene of Haemophilus parasuis, respectively. The fidelity, application scope, operation and conditions of the constructed fusion fragments were compared. The results showed that construction with DNA ligase was more mature technology as manifested by more stable conditions and more extensive application. The fusion PCR method had high fidelity and simple operation, and the transformation rate was 9.5 times as high as that of ligation with DNA ligase. For this reason, this method was more suitable for construction of multi-fragment targeting genes. The study lays a foundation for establishing an efficient operating system of targeting gene of Haemophilus parasuis in the future.

Suicide Vector Construction of Haemophilus parasuis hhd B Gene Marker-free Deleted

To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.

Isolation and Identification of Haemophilus parasuis SD02 strain

A bacterial strain was isolated from the sick pigs suspiciously infected by polyserositis and arthritis in a pig farm in Shandong Province, and identified through morphological observation, culture traits, bioehemical characteristics and PCR amplifieation. Additionally, primers were de- signed according to the 16S rRNA sequence of Haemophihzs parasuis, and the bacterial strain was amplified by PCR. The amplified fragments of approximately 1 400 bp was sequenced, and aligned with the sequence in GenBank. The results showed that it shared the homology of 97%-99% with the 16S rRNA sequence of foreign H.parasuis, and confirmed as H.parasuis （HPS）. The strain was determined as serotype 4 through serotype identification. The strain was named SD02.

Safety Observation Study on Haemophilus Influenza Type B Conjugate Vaccines Injected at Different Sites in Chinese Infants

In the present study, the safety of Hoemophilus influenza type b conjugate vaccines inoculated in the upper arm deltoid and vastus lateralis muscle was evaluated. 680 infants aged 2-5 months and 6-12 months were selected to be the research subjects in whom the Hib conjugate vaccines were inoculated by injection in the upper arm deltoid and vastus lateralis muscle, respectively. The safety analysis indicated that there were no statistic differences in the incidence rates of adverse reactions when the Hib conjugate vaccines were inoculated at different sites. So we concluded that the safety of inoculation injection of Hib conjugate vaccines in vastus lateralis muscle was the same as that inoculated in the upper arm deltoid.

Clinical Characteristics of Haemophilus influenzae at General Hospital in the Central Region of Japan

Haemophilus influenzae is an important pathogen that caused several infection diseases, such as sinusitis, otitis media, sepsis, and meningitis. This study was conducted to find out the prevalence and antimicrobial susceptibility pattern of Haemophilus influenzae isolates at general hospital in the central region of Japan from December 2015 to January 2016. Haemophilus influenzae was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. One hundred ninety-one Haemophilus influenzae were isolated, among which 95 (49.7%) were from male and 96 (50.3%) were from female. The age incidence of (0) years, (≤2) years, (≤5) years, and (6≤) years groups were 22(11.5%), 92(48.2%), 61(31.9%), and 16(8.4%), respectively. Positive samples were received mostly from the nasal discharge (177/92.7%), sputum (6/3.1%), tonsillar (6/3.1%), and pharynx (2/1.0%). Ceftriaxone was the most active antibiotics with 100% susceptible rates, followed by ciprofloxacin (99.5%) and minocycline (99%) in our study. Furthermore, we categorized four patterns: beta lactamase-negative ampicillin-sensitive strain (BLNAS), beta lactamase-negative ampicillin-re- sistant strain (BLNAR), beta lactamase-positive ampicillin resistant strain (BLPAR), and beta lactamase-positive amoxicillin-clavulanic acid-resistant strain (BLPACR) from those ampicillin susceptible results. The numbers of female were significant greater than those of male in BLPAR (p = 0.0336). With respect to antimicrobial susceptible pattern, there was no minocycline and piperacillin resistant strain in both BLNAS and BLNAR (p

Report of Haemophilus Influenzae serotype a intracranial infections in older children

Introduction:Haemophilus influenzae(Hi)is subdivided into typeable(a-f)and non-typeable groups.Hi serotype b(Hib)has historically been one of the important pathogens responsible for invasive infection.However,after widespread Hib vaccination,the emergence of other Hi serotypes,specifically Hi serotype a(Hia),was noted during the last few decades,mostly in children younger than 5 years of age.Case presentation:We present two cases of severe intracranial infections with detected Hia in patients>5 years of age within a short time frame and within the same geographic area.Conclusion:Epidemiological studies and surveillance on Hia-related illnesses in all age groups worldwide are needed to better understand the clinical and epidemiological characteristics of Hia.This can establish a platform to develop a candidate vaccine against Hia that might protect children of all ages.

Can non-typeable Haemophilus influenzae carriage surveillance data infer antimicrobial resistance associated with otitis media?

Importance:In remote communities of the Northern Territory,Australia,children experience high rates of otitis media(OM),commonly caused by non-typeable Haemophilus influenzae(NTHi).Few data exist on antibiotic susceptibility of NTHi from OM.Objective:To determine whether population-level nasopharyngeal NTHi antibiotic susceptibility data could inform antibiotic treatment for OM.Methods:NTHi isolates(n=92)collected from ear discharge between 2003 and 2013 were selected to time-and age-match NTHi isolates from the nasopharyngeal carriage(n=95).Antimicrobial susceptibility were tested.Phylogenomic trees and a genome-wide association study(GWAS)were performed to determine the similarity of nasopharyngeal and ear isolates at a population level.Results:Among 174 NTHi isolates available for antimicrobial susceptibility testing,10.3%(18/174)were resistant to ampicillin and 9.2%(16/174)were resistant to trimethoprim-sulfamethoxazole.Small numbers of isolates(≤3)were resistant to tetracycline,chloramphenicol,or amoxicillin-clavulanic acid.There was no statistical difference in the proportion of ampicillin-resistant(P=0.11)or trimethoprim-sulfamethoxazole-resistant isolates(P=0.70)between ear discharge and nasopharynx-derived NTHi isolates.Three multi-drug resistant NTHi isolates were identified.Phylogenomic trees showed no clustering of 187 Haemophilus influenzae isolates based on anatomical niche(nasopharynx or ear discharge),and no genetic variations that distinguished NTHi derived from ear discharge and nasopharyngeal carriage were evident in the GWAS.Interpretation:In this population-level study,nasopharyngeal and ear discharge isolates did not represent distinct microbial populations.These results support tracking of population-level nasopharyngeal NTHi antibiotic resistance patterns to inform clinical management of OM in this population.

Activity of ciprofloxacin and azithromycin on biofilms produced in vitro by Haemophilus influenzae

Background It is recognized that Haemophilus influenzae isolated from patients with otitis media forms biofilms both in vitro and in vivo, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of otitis media, but the effect of antibiotics on biofilm has not been well studied. We investigated the impact of ciprofloxacin and azithromycin on bacterial biofilms formed by Haemophilus influenzae in vitro in this study. Methods Eleven strains of Haemophilus influenzae were isolated from sputum specimens collected from patients with acute exacerbation of chronic obstructive pulmonary diseases. Formation of bacterial biofUm was examined by crystal violet assay and a scanning electron microscope. Alterations of biofilms were measured under varying concentrations of azithromycin and ciprofloxacin. Results Striking differences were observed among strains with regard to the ability to form biofilm. Typical membrane-like structure formed by bacterial cells and extracellular matrix was detected. Initial biofilm synthesis was inhibited by azithromycin and ciprofloxacin at concentrations higher than two-fold minimal inhibitory concentration. Disruption of mature biofilms could be achieved at relatively higher concentration, and ciprofloxacin displayed more powerful activity. Conclusions Haemophilus influenzae is capable of forming biofilm in vitro. Sufficient dosage might control early formation of biofilms. Ciprofloxacin exerts better effects on breakdown of biofilm than azithromycin at conventional concentration in clinics.

Determination of ribose and phosphorus contents in Haemophilus influenzae type b capsular polysaccharide by a quantitative NMR method using a single internal standard

The ribose and phosphorus contents in Haemophilus influenzae type b(Hib)capsular polysaccharide(CPS)are two important chemical indexes for the development and quality control of Hib conjugate vaccine.A quantitative ^(1)H-and ^(31)P-NMR method using a single internal standard was developed for simultaneous determination of ribose and phosphorus contents in Hib CPS.Hexamethylphosphoramide(HMPA)was successfully utilized as an internal standard in quantitative ^(1)H-NMR method for ribose content determination.The ribose and phosphorus contents were found to be affected by the concentration of polysaccharide solution.Thus,15–20 mg·L^(−1) was the optimal concentration range of Hib CPS in D_(2)O solution for determination of ribose and phosphorus contents by this method.The ribose and phosphorus contents obtained by the quantitative NMR were consistent with those obtained by traditional chemical methods.In conclusion,this quantitative ^(1)H-and ^(31)P-NMR method using a single internal standard shows good specificity,accuracy and precision,providing a valuable approach for the quality control of Hib glycoconjugate vaccines.

副猪嗜血杆菌PCR检测方法的建立与初步应用

针对副猪嗜血杆菌(Haemophilus parasuis,HPS)16 S rRNA序列设计1对特异性引物,能扩增出821 bp的特异性DNA片段,并据此建立了快速准确鉴定副猪嗜血杆菌PCR方法,临床试验证明该方法具有很好的特异性和敏感性。13份疑似病料检测结果表明,PCR检测结果与传统生化鉴定结果符合率为100%。结果表明已成功建立了副猪嗜血杆菌PCR检测方法,并可应用于临床副猪嗜血杆菌的检测。