Characterization of fluoroquinolone resistance in Escherichia coli strains from ruminants.

Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 microg/ml) but not to ciprofloxacin (MIC = 1 microg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83-->Leu and Asp-87-->Gly) and a double amino acid substitution in the ParC protein (Gly-78-->Cys and Ser-80-->Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants.     

Prevalence of plasmid-mediated quinolone resistance qnr genes in poultry and swine clinical isolates of Escherichia coli

The prevalence of qnr genes was investigated in veterinary clinical isolates of Escherichia coli in Guangdong province, China, and the aac (6')-Ib gene and the mutations in QRDRs of gyrase and topoisomerase IV were examined in qnr-positive strains. A total of 232 E. coli strains isolated from pig and poultry were screened for the presence of the qnrA, qnrB and qnrS genes by PCR and sequencing. The aac (6')-Ib gene was detected in qnr-bearing strains by PCR and sequencing. For all strains carrying qnr, MICs for six quinolones were determined. Mutations within the gyrase and topoisomerase were analyzed by PCR and sequencing for all the QRDRs of gyrA, gyrB, parC and parE. Among 232 E. coli isolates, 14 (6%) isolates were positive for the qnr gene, including one for qnrB, 13 for qnrS, but no qnrA was identified in this population. Detection of the aac (6')-Ib gene showed that one qnrS-positive isolate from pig and one qnrB-positive isolate from duck carried aac (6')-Ib gene, and both were the cr variant allele of aac (6')-Ib. All of the 14 isolates had MICs of ciprofloxacin more than 0.25 mg/L. Mutations in the QRDR of gyrA mutations were observed in 5 (35.7%) of the 14 strains. Three fluoroquinolone-resisting strains showed one mutation S83L of gyrA, while one S83I. One high-level resistance strains harboured gyrA S83L and A87N of gyrA. A singe mutation in site 58 of parC was detected in 3 (21.4%) strains. None mutations were found in QRDRs of gyrB and parE. The emergence of qnr genes in veterinary clinical E. coli isolates is described for the first time. This is also the first report of aac (6')-Ib-cr gene in E. coli isolates from food-producing animals.     

Genotypes and antibiotic resistance of Campylobacter coli in fattening pigs

Campylobacter coli is a food-borne zoonotic pathogen causing human gastroenteritis worldwide. The organism is a commensal in the intestine of many food production animals including fattening pigs. The role of the pig as a potential reservoir for C. coli affecting human either directly or via poultry has hardly been investigated and genetic characterization of porcine strains is needed to address this question. For this aim multilocus sequence typing (MLST) and flaB typing was applied to 256 C. coli isolates from faeces of fattening pig collected during 2009 at different slaughterhouses in Switzerland. In addition genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. Of the 67 sequence types (STs) obtained by MLST, 37 were found for the first time. flaB typing revealed 46 different types with 14 of them being novel and was useful to further differentiate strains with an identical ST. Quinolone resistance was detected in 33.6% and macrolide resistance was found in 10.6% of isolates. Comparison with 99 C. coli pig isolates from 2001 revealed a significant decrease in antibiotic resistance towards both groups of antibiotics and there was high overlap between genotypes of 2001 and 2009. Little overlap of porcine genotypes was found with 97 C. coli isolates from poultry collected 2008, however, macrolide resistance was significantly higher in pig isolates. In conclusion, C. coli from Swiss pig are heterogeneous containing many novel STs, findings that could reflect the partitioned Swiss pig production with almost no international breed exchange. The antibiotic resistance echoes the use of corresponding drugs in the Swiss livestock production and indicates the efficacy of restrictive application of antibiotics in order to reduce resistances.     

Characterization of macrolide-resistant Campylobacter coli isolates from food-producing animals on farms across Japan during 2004

We investigated the susceptibilities against 7 antimicrobial agents in Campylobacter jejuni and C. coli isolates from food-producing animals in 2004. In comparison with the results of past surveillance, no significant difference was observed in resistance rates against all of the antimicrobials tested in Campylobacter isolates. However, slight increase of erythromycin (EM) resistance was found in C. coli isolates from pigs. We examined the mutation of the 23S rRNA gene and their susceptibilities against azithromycin, tylosin, and lincomycin in 44 EM-resistant isolates and 28 susceptible isolates of porcine origin. All the EM-resistant isolates contained A2075G in the 23S rRNA gene and showed cross-resistance to azithromycin, tylosin, and lyncomycin.     

Mutant-prevention concentrations of enrofloxacin for Escherichia coli isolates from chickens

OBJECTIVE: To investigate the development of enrofloxacin resistance among Escherichia coli isolates obtained from chickens by determining mutant-prevention concentrations (MPCs) and sequence the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes in selected isolates. SAMPLE POPULATION: 15 chicken-derived E coli isolates. PROCEDURES: For all isolates, MPC and minimal inhibition concentration (MIC) of enrofloxacin were determined. The MPCs and maximum serum drug concentrations attained with enrofloxacin doses recommended for treatment of E coli infections in chickens were compared. Mutation frequencies and QRDR sequence changes in gyrA and parC were also determined. RESULTS: In 2 of 15 E coli strains, MPCs were low (0.016 and 0.062 microg/mL), MPC:MIC ratios were 2 and 4, and the GyrA and ParC proteins had no mutations. In 9 susceptible isolates with a GyrA point mutation, MPCs ranged from 2 to 16 microg/mL. For isolates with double mutations in GyrA and a single mutation in ParC, MPCs were > 32 microg/mL (several fold greater than the maximal plasma concentration of enrofloxacin in chickens); mutation frequencies were also much lower, compared with frequencies for single-mutation isolates. CONCLUSIONS AND CLINICAL RELEVANCE: For E coli infections of chickens, MPC appears to be useful for determining enrofloxacin-dosing strategies. The high MPC:MIC ratio may result in enrofloxacin-treatment failure in chickens infected with some wild-type gyrA E coli isolates despite the isolates' enrofloxacin susceptibility (MICs 0.125 to 1 microg/mL). For infections involving isolates with high MPCs, especially those containing mutations in gyrA and parC genes, treatment with combinations of antimicrobials should be adopted.     

Acquisition of quinolone resistance and point mutation of the gyrA gene in Campylobacter jejuni isolated from broilers and in vitro-induced resistant strains

A dramatic rise in the number of resistant Campylobacter to quinolones has been documented in human patients and domestic animals. In this study, the mechanism of acquisition of quinolone resistance was studied by detecting point mutations in the gyrA gene of Campylobacter strains obtained from broilers and strains with in vitro-induced resistance. The minimal inhibitory concentrations (MICs) of norfloxacin (NFLX) and ofloxacin (OFLX) for the strains that had no point mutation were slightly increased from the source strain (Campylobacter jejuni ATCC 33560). The MICs of nalidixic acid (NA), NFLX, and OFLX for the strains that had the point mutation at Thr-86 were 100 or 200 microg/ml, 50 microg/ml, and 25 microg/ml, respectively. The MIC of NA for the strain that had a point mutation at Asp-90 higher than those for the strains that had the point mutation at Thr-86, but the MICs of NFLX and OFLX were relatively lower than those for the strains that had point mutation at Thr-86. These findings suggest that the degree of antimicrobial resistance against NA, NFLX, and OFLX in the in vitro-induced C. jejuni strains was associated with the location of the point mutation in gyrA. On the other hand, a point mutation in all seven resistant strains isolated from broilers was located only at Thr-86, while the MICs of the three quinolones varied in each wild strain. This suggests that another mechanism might also be involved in the acquisition of quinolone resistance in C. jejuni wild strains.     

耐氟喹诺酮类药物禽源大肠埃希菌gyrA基因的检测分析

探讨不同禽源大肠埃希菌中喹诺酮类药物的耐药情况及耐药基因gyrA的分布和突变特征。采用K-B药敏纸片法、gyrA基因的PCR扩增,对9株大肠埃希菌进行喹诺酮类药物试验,并将gyrA基因的PCR产物测序,对测序结果采用DNA MAN、DNA Star、MEGA6等软件分析。药敏试验结果表明,C1、C2、C3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星敏感,D1、D2、D3、B1、B2和B3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星均表现为耐药和中介;gyrA基因的测序结果表明,除B1菌株有1处核苷酸突变位点和B2菌株有14处核苷酸突变位点;B2菌株gyrA基因的氨基酸突变发生在87位Ile→Val替代、101位Leu→Met替代、102位Ala→Ser替代、129位Lys→Gln替代。9株禽源大肠埃希菌的同源性和进化树分析表明,不同禽源耐氟喹诺酮类药物的大肠埃希菌菌株中B2菌株gyrA基因与其他9株菌株相比,同源性在90%左右,进化树不在一个分支上,研究中的B2菌株将为大肠埃希菌的氟喹诺酮类耐药机制的研究提供候选菌株。     

Identification of nine sequence types of the 16S rRNA genes of Campylobacter jejuni subsp. jejuni isolated from broilers

Background

Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of SmaI restricted genomic DNA of the strains.

Methods

The 16S rRNA genes of 45 strains of C. jejuni and two C. coli strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with SmaI.

Results

Sequence analyses of the 16S rRNA genes revealed nine sequence types of the Campylobacter strains and the similarities between the different sequence types were in the range 99.6–99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as Campylobacter coli by PCR/REA. The other 10 strains were identified as Campylobacter jejuni. Five of the nine sequence types were also found among the Campylobacter sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains.

Conclusion

C. jejuni and C. coli seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between C. jejuni and C. coli. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only C. jejuni and the other with both C. jejuni and C. coli. Genotyping of the 47 strains by PFGE after digestion with SmaI resulted in 22 subtypes. A potential correlation was found between the SmaI profiles and the 16S rRNA sequences, as a certain SmaI type only appeared in one of the two major phylogenetic groups.     

Molecular characterisation of quinolone resistance in Escherichia coli from animals in Turkey

The aim of this study were to detect the gyrA, parC and marR mutations and qnr genes (qnrA, qnrB and qnrS) in 120 strains of Escherichia coli isolated from animals. European Committee on Antimicrobial Susceptibility Testing and Clinical Laboratory Standards Institute disc diffusion and minimum inhibitory concentration (MIC) tests, respectively, were used to determine fluoroquinolone (FQ) resistance, and molecular methods were used to detect the mutations and the genes. E coli isolates with an MIC of ≥8 mg/l had mutation at Ser-80 in parC in addition to mutations at Ser-83, Asp-87 or both in gyrA. The nucleotide change was detected in marR (Ser-3?→?Asn, Ala-53?→?Glu, Gly-103?→?Ser, Tyr-137?→?His). Only four E coli isolates (3.3 per cent) contained qnrA and qnrS, and qnrB was not detected. Two E coli isolates from healthy calves also contained qnrA and qnrS. The MICs of enrofloxacin and danofloxacin for qnr-containing E coli isolates ranged from 32 mg/l to 256 mg/l. The results of this study indicated that the FQ-resistant E coli isolates presented an alteration in gyrA (Ser-83?→?Leu, Asp-87?→?Asn) and parC (Ser-80?→?Ile) with high MICs (8-256 mg/l), and there was a low prevalence of qnr genes among E coli isolated from animals.     

A nucleotide mutation associated with fluoroquinolone resistance observed in gyrA of in vitro obtained Rhodococcus equi mutants

In this study, the quinolone resistance-determining region (QRDR) in gyrA and gyrB of in vitro fluoroquinolone-resistant Rhodococcus equi mutants was sequenced. These mutants were selected from four R. equi strains on blood agar plates containing ciprofloxacin or enrofloxacin. Each mutant became 8- to 64 or greater-fold resistant to fluoroquinolones compared with their parent strains. From the results of sequence analysis of QRDR in gyrA and gyrB, a nucleotide mutation of codon GAC for GGC in gyrA was detected in all mutants, but no mutation was observed in gyrB. This mutation leads to amino acid substitution of Asp for Gly in putative GyrA in R. equi. The position of this substitution corresponds to position 87 of GyrA in Escherichia coli. Our results suggest that the mutation of QRDR in gyrA, which was observed in in vitro fluoroquinolone-resistant R. equi mutants in this study, is closely associated with fluoroquinolone resistance.     

Occurrence of virulence genes among Campylobacter jejuni and Campylobacter coli isolates from domestic animals and children

The presence of the flaA, cadF, cdtB and iam genes of Campylobacter spp. was determined with the PCR method. The materials to investigate were 56 C. jejuni and 23 C. coli strains isolated from clinical samples (children and domestic animals). It was found that all of the Campylobacter spp. isolates from children with diarrhoea and domestic animals had cadF gene, responsible for adherence. The flaA gene was present in all Campylobacter spp. isolates derived from children and cats. Occurrence of flaA gene was confirmed in 100% of C. jejuni strains obtained from dogs. The high prevalence of the cdtB gene associated with toxin production was observed in this study (100%-Campylobacter spp. isolates obtained from dogs and cats, 97.9%-Campylobacter spp. isolates from children). The isolates showed a wide variation for the presence of iam gene. The lowest prevalence (23.5%) was detected in Campylobacter spp. obtained from dogs. The highest rates of iam detection (91.6%) were revealed in C. coli isolates from children.     

Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA

A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.     

Prevalences and transmission routes of Campylobacter spp. strains within multiple pig farms

In this work, faecal samples were collected from 15 pig farms to determine the Campylobacter prevalences at different times during the rearing period and to visualize the exchange of strains among the pig population by genotyping specific isolates. All isolated strains were identified as C. coli. Whereas no Campylobacter were detectable in the faeces of piglets at the day of birth, the Campylobacter incidence rose within days to 32.8%. After transfer to the nursery unit the prevalence increased to 56.6%. Approximately two-thirds of the pigs remained C. coli shedders in the fattening unit. In contrast to most farms, one farm expressed a very low Campylobacter incidence during the whole rearing period. Amplified fragment length polymorphism (AFLP) analysis was performed on all C. coli isolates of one farm. Clonal strains were identified from the brood sows and their offsprings or neighbouring piglets. After moving to the nursery unit, new genotypes appeared in that pig group but the original C. coli strains largely remained within that group. C. coli genotypes, identified during the fattening period, replaced the previously isolated genotypes. Transportation to the abattoir had no significant influence on the shedding rate of C. coli. The detection rate before transportation was 79.1% and decreased slightly to 78.2% (n=330). Additionally, eleven of 1474 environmental samples from different sources of the pig farms were positive for C. coli. This study demonstrates the importance of pigs as a reservoir for C. coli. Maternal C. coli strains are the primary source of infection but non-related genotypes from different sources appear during the rearing period and these latter strains constitute largely the final C. coli flora.     

Campylobacter jejuni, Campylobacter coli, and cytolethal distending toxin genes in laying hens

As no data are available on the prevalence of cytolethal distending toxin (cdt) genes carried by Campylobacter spp. in laying hens, this study was conducted with the aim to evaluate the prevalence of both Campylobacter spp. and cdt genes in 1680 laying hens from four different farms. The samples were analyzed by culture methods and by polymerase chain reaction. Campylobacter spp. were isolated from 1097/1680 cloacal swabs. Among the isolates, 913 were identified as Campylobacter jejuni whereas 345 were identified as Campylobacter coli. All isolates carried cdt genes. The results presented here confirm the very common occurrence of C. jejuni and C. coli in laying hens and underline that the cdt genes may also be frequently present in both C. jejuni and C. coli isolates from laying hens.     

猪链球菌对氟喹诺酮类药物的耐药性与靶位突变相关性

旨在了解猪链球菌对氟喹诺酮类药物耐药性与parC、gyrA基因突变的相关性,通过微量稀释法测定34株猪链球菌对4种氟喹诺酮类药物的MIC值,采用PCR方法扩增并测序分析了临床分离的猪链球菌对氟唪诺酮类约物10株耐药株和9株敏感株的parC和gyrA基因喹诺酮耐药决定区(QRDRs).在氟喹诺酮类药物耐药菌株parC基因QRDRs发生Ser79→Phe、Arg 87→Leu的氨基酸突变,在4株高度耐药菌株gyrA基因QRDRs发生Arg66→Ser,Ser81→Arg氨基酸突变;当菌株对氟喹诺酮类药物敏感时,parC和gyrA基因的QRDR区均未有突变;而当MIC≥32 μg·L-1 时,parC的氨基酸发生了 Ser79→Phe的突变,同时发生gyrA氨基酸Arg66→Ser,Set81→Arg突变.结果表明,猪链球菌对氟喹诺酮类药物低水平类耐药是由parC单一位点突变引起,而高水平耐药是由parC和gyrA双位点突变引起.     

Studies on calf diarrhoea in Mozambique: prevalence of bacterial pathogens

The prevalence of diarrhoea in calves was investigated in 8 dairy farms in Mozambique at 4 occasions during 2 consecutive years. A total of 1241 calves up to 6 months of age were reared in the farms, and 63 (5%) of them had signs of diarrhoea. Two farms had an overall higher prevalence (13% and 21%) of diarrhoea. Faecal samples were collected from all diarrhoeal calves (n = 63) and from 330 healthy calves and analysed for Salmonella species, Campylobacter jejuni and enterotoxigenic Escherichia coli (ETEC). Salmonella spp. was isolated in only 2% of all calves. Campylobacter was isolated in 11% of all calves, irrespective of health condition, and was more frequent (25%) in one of the 2 diarrhoeal farms (p = 0.001). 80% of the isolates were identified as C. jejuni. No ETEC strains were detected among the 55 tested strains from diarrhoeal calves, but 22/55 (40%) strains from diarrhoeal calves and 14/88 (16%) strains from healthy calves carried the K99 adhesin (p = 0.001). 6,757 E. coli isolates were typed with a biochemical fingerprinting method (the PhenePlate) giving the same E. coli diversity in healthy and diarrhoeal calves. Thus it was concluded: i) the overall prevalence of diarrhoea was low, but 2 farms had a higher prevalence that could be due to an outbreak situation, ii) Salmonella did not seem to be associated with diarrhoea, iii) Campylobacter jejuni was common at one of the 2 diarrhoeal farms and iv) ETEC strains were not found, but K99 antigen was more prevalent in E. coli strains from diarrhoeal calves than from healthy, as well as more prevalent in one diarrhoeal farm.     

Phenotypic and genotypic anti-microbial resistance profiles of campylobacters from untreated feedlot cattle and their environment

Anti-microbial resistance is an emerging public health issue. Farmed animals may act as reservoirs and potential sources of anti-microbial resistant Campylobacters. The aim of this study was to investigate the anti-microbial resistance profile of cattle and environmental Campylobacter isolates from normal untreated feedlot cattle, the role of the gyrA Thr-86-Ile mutation in ciprofloxacin-resistant Campylobacter jejuni isolates and the involvement of the tripartite CmeABC efflux system for multi-resistant C. jejuni isolates. The phenotypic anti-microbial resistance testing was carried out on 500 Campylobacter isolates (445 cattle isolates and 55 environmental isolates). In general, there was a higher level of anti-microbial resistance for the environmental isolates compared with the animal isolates, 45% of the animal isolates were resistant to one or more of the seven anti-microbials compared with 84% of the environmental isolates. The combined cattle and environmental Campylobacters had 34 (6.8%) isolates resistant to three or more of the seven anti-microbials tested on all isolates and 11 (2.2%) isolates were resistant to the seven anti-microbials. There was a substantial level of ciprofloxacin-resistant Campylobacters in both animal (8.5%) and environmental (21.8%) isolates. The gyrA Thr-86-Ile mutation was only present in five of 22 ciprofloxacin-resistant C. jejuni isolates investigated. No multi-drug-resistant associated mutation was detected in the CmeB or the CmeR regions investigated. In conclusion, our study observed a substantial level of Campylobacter anti-microbial resistance, highlighting the need for an active anti-microbial surveillance program for food animals in Ireland and the importance of the chosen sampling point can have on the findings of such a program.     

Epidemiologic features of Campylobacter jejuni isolated from poultry broiler houses and surrounding environments as determined by use of molecular strain typing

OBJECTIVE: To genetically type Campylobacter jejuni isolates from broiler houses or the external environment to identify the source of Campylobacter organisms in broiler chickens. SAMPLE POPULATION: Environmental samples associated with broiler chickens, in commercial grow-out houses. PROCEDURE: Polymerase chain reaction (PCR) was used to amplify flaB, and the amplicon was digested with Sau3A to create a restriction fragment length polymorphism assay; PCR was also used to detect a transcribed spacer region in the 23S rRNA gene. RESULTS: Isolates possessing a 23S spacer region were more prevalent outside broiler houses than inside. Houses that had previously contained chickens or lacked biosecurity procedures were more likely to contain isolates possessing the 23S spacer. One house contained only isolates possessing the spacer, whereas an adjacent house contained only isolates lacking the spacer. The flaB type detected in broiler houses was different from the type detected in the environment; however, many isolates within the broiler houses contained untypable flaB genotypes. CONCLUSIONS AND CLINICAL RELEVANCE: Most isolates from within houses were genetically distinct from isolates from outside houses that were examined by bacteriologic culture, suggesting an undetected source of C jejuni. Detection of isolates containing the 23S spacer appeared to be an indicator of environmental contamination of the houses. The observation of completely different C jejuni genetic types simultaneously within adjacent houses suggests that some types do not compete successfully during the grow-out period. In addition, the diversity of genotypes identified within broiler houses indicates the complexity of the ecologic features of C jejuni in the chicken environment.     

鸡源性沙门氏菌DNA旋转酶与耐氟喹诺酮类药物关系研究

取临床分离的、对5种氟喹诺酮类药物（环丙沙星、氧氟沙星、恩诺沙星、单诺沙星和沙拉沙星）均耐药的9株鸡源性沙门氏菌耐药株,提取其染色体DNA。设计引物gyrAF和gyrAR、gyrBF和gyrBR,分别扩增菌株DNA旋转酶gyrA基因和gyrB基因的氟喹诺酮类耐药决定区（QRDR）,对PCR扩增产物进行测序及序列分析。与质控菌株相比,9株临床分离耐药株中只有菌株38和60的gyrA基因发生单碱基突变,菌株38的gyrA基因第371位碱基发生C→T突变,菌株60的gyrA基因第350位碱基发生A→C突变,两处突变均位于QRDR内,其余菌株的核苷酸未发生任何突变。菌株38的碱基突变导致gyrA基因第99位氨基酸发生R→C取代,即Arg→Cys;菌株60的碱基突变导致gyrA基因第92位氨基酸发生M→L取代,即Met→Leu。9株临床分离鸡源性沙门氏菌氟喹诺酮类耐药株gyrB基因QRDR的核苷酸序列与质控菌株完全相同;只有菌株42的gyrB基因第1592位碱基发生C→A突变,但其位于gyrB基因QRDR之外,且菌株42的gyrB基因的碱基突变并没有导致相应氨基酸的改变。上述结果提示,DNA旋转酶gyrA基因和gyrB基因QRDR突变可能并非沙门氏菌耐药性产生的主要原因。