Penicillin Resistance Genes Research Articles

A Comparative Study on Microbial Air Quality of Radiology and Patient Waiting Rooms of a Full-Equipped Hospital in Aydın Province

Every year many patients die due to hospital-acquired infections. These infections also put the lives of healthcare workers at risk. Additionally, hospitals are one of the main reservoirs of antimicrobial resistance. In this study, the effect of X-rays on species diversity and functions and antimicrobial resistance were observed. Metagenome analysis was performed on air samples taken from the x-ray room (radiation exposure) and the waiting room (non-radiation area) to examine whether the radiation affected the species present in the air. The diversity of microorganisms was analysed based on phylum, genus, and species levels. Functional profiling and resistance screening were also performed. X-ray radiation was found to have a major effect at the phylum level. It was observed that Proteobacteria species almost dominated the microbiome in the x-ray room (99%) while Actinobacteria species dominated the microbiome in the waiting room (84%). No significant differences were observed between the two areas in functional profiling. A total of thirty-eight functions were observed, twenty-four of which were overlapping. Antimicrobial resistance was not as diverse as expected. Only beta-lactam, penicillin, tetracycline, and lincomycin resistance genes were present (2364 reads belonging to four different genes). According to the results, it was observed that X-rays affected the air microbiome, as expected. A decrease in the number of microorganisms was expected, but it was also observed that the dominant microorganism types changed. On the other hand, no significant difference was found in terms of functional profiling and no significant antimicrobial resistance was observed.

Antibiotic Resistance Genes, Virulence Factors, and Biofilm Formation in Coagulase-Negative Staphylococcus spp. Isolates from European Hakes (Merluccius merluccius, L.) Caught in the Northeast Atlantic Ocean.

The indiscriminate use of antibiotics has contributed to the dissemination of multiresistant bacteria, which represents a public health concern. The aim of this work was to characterize 27 coagulase-negative staphylococci (CoNS) isolated from eight wild Northeast Atlantic hakes (Merluccius merluccius, L.) and taxonomically identified as Staphylococcus epidermidis (n = 16), Staphylococcus saprophyticus (n = 4), Staphylococcus hominis (n = 3), Staphylococcus pasteuri (n = 2), Staphylococcus edaphicus (n = 1), and Staphylococcus capitis (n = 1). Biofilm formation was evaluated with a microtiter assay, antibiotic susceptibility testing was performed using the disk diffusion method, and antibiotic resistance and virulence determinants were detected by PCR. Our results showed that all staphylococci produced biofilms and that 92.6% of the isolates were resistant to at least one antibiotic, mainly penicillin (88.8%), fusidic acid (40.7%), and erythromycin (37%). The penicillin resistance gene (blaZ) was detected in 66.6% (18) of the isolates, of which 10 also carried resistance genes to macrolides and lincosamides (mphC, msr(A/B), lnuA, or vgaA), 4 to fusidic acid (fusB), and 3 to trimethoprim-sulfamethoxazole (dfrA). At least one virulence gene (scn, hla, SCCmecIII, and/or SCCmecV) was detected in 48% of the isolates. This study suggests that wild European hake destined for human consumption could act as a vector of CoNS carrying antibiotic resistance genes and/or virulence factors.

Whole Genome Sequencing of Staphylococci Isolated From Bovine Milk Samples.

Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non-aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus. However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus. The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus, which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infections.

Genetic Characterization of Staphylococcus aureus From Subclinical Mastitis Cases in Dairy Cows in Rwanda.

Whole-genome sequencing was carried out on 30 Staphylococcus (S.) aureus isolates from dairy cows with subclinical mastitis from all five provinces of Rwanda. Twenty-five of the isolates produced enough sequence to be analyzed using core genome multilocus sequence typing (cg-MLST). The isolates group into three main clusters. The largest cluster contain isolates of sequence type (ST) 152 (n = 6) and the closely related ST1633 (n = 2). These sequence types have previously mainly been encountered in humans. The isolates of the second-largest cluster belong to ST5477 (n = 5),so far exclusively isolated from cows in Rwanda. The third cluster consists of isolates of ST97 (n = 4), which is a well-known bovine-adapted sequence type. These three clusters were all widespread over the country. Isolates of the usually human-adapted sequence types 1 (n = 2) and 5 (n= 1) were found and a single isolate of ST2430, previously found among humans in Africa. Finally, four isolates of novel sequence types were found: ST7108 (n = 2), ST7109 (n = 1), and ST7110 (n = 1). The blaZ penicillin resistance gene was found in 84% of the isolates and was in all cases corroborated by phenotypic resistance determination. Five (20%) of the isolates carried a tetracycline resistance gene, tet(K) or tetM, and three of these five also displayed phenotypic resistance while two isolates carried a tetM-gene but were yet tetracycline susceptible. Seven (28%) isolates carried the dfrG gene conferring resistance to trimethoprim. Four of these isolates indeed were resistant to trimethoprim while three isolates were sensitive. The str gene conferring resistance to aminoglycosides was found in three isolates; however, none of these displayed resistance to gentamycin. Our data revealed a high diversity of the sequence types of S. aureus isolates from cows with subclinical mastitis in Rwanda. Two major clusters of ST97 and ST5477 are likely to be bovine adapted and cause mastitis while the third cluster of ST152 usually have been found in humans and may signify a recent transmission of these types from human to cows, for example from hand milking. The high prevalence of this sequence type among dairy cows may pose zoonotic threat. The sequence types were widely distributed without any geographic correlation. Penicillin resistance, the most common type of resistance with a prevalence over 80%, but also tetracycline and trimethoprim resistance were displayed by several isolates.

Prevalence and Quantitative Analysis of Antibiotic Resistance Genes (ARGs) in Surface and Groundwater in Meandering Part of the Kelani River Basin in Sri Lanka.

Nearly 80% of the population in the Colombo district fulfill their major requirement from the Kelani river. Recent studies are interoperating: most groundwater and surface water in Sri Lanka are contaminated with waterborne pathogens and antibiotics. In the present study, nine antibiotic resistance genes (ARGs) were screened which were belonging to two common groups of antibiotic: penicillin — OPR D, bla TEM, bla OXA, amp a, and amp b — and tetracycline — tet A, tet M, tet B, and tet S. The results of the study reveled that the surface and groundwater of the entire lower part of the Kelani river basin were contaminated with TC and FC (98%). None of the penicillin and tetracycline group antibiotics were detected either surface or groundwater samples except the Kelani river mouth (amoxicillin (AMX) at 0.003 ± 0.001 µg/ml). The results showed that 5 to 15% of surface water samples were positive for penicillin resistance genes (bla TEM, bla OXA, OPR D, amp a, amp b) where ~ 10% of groundwater samples were positive against tetracycline resistance genes (tet A, tet M, tet S, tet B). Among the penicillin resistance genes, the bla TEM (700.576 × 102 copy/ml) was recorded as the highest concentration where the highest tet A gene (439.875 × 102 copy/ml) was detected among the tetracycline resistance genes. Therefore, water quality management and regular monitoring are essential to maintain the quality of drinking water in the meandering part of the Kelani river basin to safeguard river water consumers.

Smart pH-Regulated Switchable Nanoprobes for Photoelectrochemical Multiplex Detection of Antibiotic Resistance Genes.

Antibiotic resistance, encoded via particular genes, has become a major global health threat and substantial burden on healthcare. Hence, the facile, low-cost, and precise detection of antibiotic resistance genes (ARGs) is crucial in the realm of human health and safety, especially multiplex sensing assays. Here, a smart pH-regulated switchable photoelectrochemical (PEC) bioassay has been created for ultrasensitive detection of two typical subtypes of penicillin resistance genes bla-CTX-M-1 (target 1, labeled as TDNA1) and bla-TEM (target 2, labeled as TDNA2), whereby pH-responsive antimony tartrate (SbT) complex-grafted silica nanospheres are ingeniously adopted as signal DNA1 tags (labeled as SDNA1-SbT@SiO2NSs). The operations of the PEC bioassay depend on the switchable dissociation of the pH-responsive SDNA1-SbT@SiO2NSs complex under the external pH stimuli, thus initiating the pH-regulated release of ions pre-embedded in sandwich-type DNA nanoassemblies. At acidic conditions, the dissociation of SDNA1 tags (ON state) triggers the release of the embedded SbO+. Under alkaline conditions, the dissociation of SDNA1 tags is inhibited (OFF state). The detection of TDNA2 was achieved via DNA hybridization-triggered metal ion release. The unwinding of the introduced hairpin T-Hg2+-T fragment, hybridized with the second anchored signal DNA (SDNA2), ignites the release of Hg2+. The released SbO+ or Hg2+ ions would trigger the formation of Sb2S3/ZnS or HgS/ZnS heterostructure through ion-exchange with the photosensitive ZnS layer, giving rise to the amplified photocurrents and eventually realizing the ultrasensitive detection of penicillin resistance genes subtypes, bla-CTX-M-1 and bla-TEM. The as-fabricated pH-regulated PEC bioassay, smartly integrating the pH-responsive intelligent unit as SDNA tags, pH-regulated release of embedded ions, and the subsequent ion-exchange-based signal amplification strategy, exhibits high sensitivity, specificity, low-cost, and ease of use for multiplex detection of ARGs. It can be successfully used for measuring bla-CTX-M-1 and bla-TEM in real E. coli plasmids, demonstrating great promise for developing a new class of genetic point-of-care devices.

Erratum to: Emergence and genomic characterization of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in Chengdu, China.

OBJECTIVES To verify the contribution of the globally disseminated Neisseria gonorrhoeae FC428 clone to the emergence of ceftriaxone resistance in Chengdu in south-west China during 2018. METHODS Antimicrobial susceptibility of the N. gonorrhoeae isolates to six antibiotics was determined using the agar dilution method. A real-time PCR assay and WGS were used to identify the FC428 clone. Phylogenomic and molecular antimicrobial resistance analyses were conducted to characterize the transmission and evolution of related strains. RESULTS Four out of 112 N. gonorrhoeae isolates were confirmed as the ceftriaxone-resistant FC428 clone. Phylogenomic analysis revealed that they resulted from multiple introductions and subsequent local transmissions. The strains have undergone further evolutions characterized by the accumulation of mutations in resistance-associated genes and/or the acquisition of plasmids encoding penicillin and tetracycline resistance genes. CONCLUSIONS The N. gonorrhoeae FC428 clone has spread to south-west China. Efforts should be made to enhance gonococcal antimicrobial surveillance to control further dissemination of this successful clone at both local and national levels.

Emergence and genomic characterization of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in Chengdu, China.

To verify the contribution of the globally disseminated Neisseria gonorrhoeae FC428 clone to the emergence of ceftriaxone resistance in Chengdu in south-west China during 2018. Antimicrobial susceptibility of the N. gonorrhoeae isolates to six antibiotics was determined using the agar dilution method. A real-time PCR assay and WGS were used to identify the FC428 clone. Phylogenomic and molecular antimicrobial resistance analyses were conducted to characterize the transmission and evolution of related strains. Four out of 112 N. gonorrhoeae isolates were confirmed as the ceftriaxone-resistant FC428 clone. Phylogenomic analysis revealed that they resulted from multiple introductions and subsequent local transmissions. The strains have undergone further evolutions characterized by the accumulation of mutations in resistance-associated genes and/or the acquisition of plasmids encoding penicillin and tetracycline resistance genes. The N. gonorrhoeae FC428 clone has spread to south-west China. Efforts should be made to enhance gonococcal antimicrobial surveillance to control further dissemination of this successful clone at both local and national levels.

Studies on <I>Streptococcus pneumoniae </I>associated with pulmonary infection among HIV clients attending Aminu Kano Teaching Hospital (AKTH)

Respiratory infection is highly prevalent in HIV-infected patients and S. pneumoniae was a leading cause. The aim of this study was to isolate and identify Streptococcus pneumoniae associated with pulmonary infection among HIV Clients Attending Aminu Kano Teaching Hospital (AKTH). A total of 300 samples comprising sample from 180 HIV seropositive patients and 120 HIV seronegative patients as a (control) were enrolled in this study.) For the 180 HIV seropositive patients, 105(58.3%) were females, and 75(41.7%) were males. Among the HIV negative subjects 64(53.3%) were males and 56(46.7%) were females. The incident of Streptococcus pneumoniae isolates associated with pneumonia in HIV/AIDS seropositive patients in relation to age group in this study showed the higher prevalence of 15(51.72%), among the age groups of 30-39, had the highest of prevalence of 15(51.72%), which occur more common in HIV infected patients while for HIV seronegative patients age group 10-19 had the highest incidence of 7(29.17%).Result showed no significant difference in the prevalence of S .pneumoniae among the HIV seropositive and seronegative patients (P > 0.05) .Antimicrobial susceptibility showed that Ciprofloxacin 25(86.2%), was found to be most active invitro against the isolates followed by Oxacillin 21(72.4%), Augmentin19 (65.5), Chloramphenicol 15(51.7%). Tetracycline, 14(48.3%), Clindamycin 14(48.3) and Erythromycin 10(34.5%). Result showed no significant difference in the levels of antibiotic susceptibilities and resistance observed (p > 0.05). The CD4 cells counts for the HIV seropositve patients were 161(89.4%) 350 cells/ml and 19(10.6%) <350 cells/m recorded as normal and low respectively. There was significant difference (p <0.05) for the CD4 cells counts of the study population with the prevalence of Streptococcus pneumoniae isolates among the HIV seropositive patients (P 0.000 <0.05). PCR amplification for Streptococcus pneumoniae penicillin resistance genes showed that Only 2(33.3%) out of 6 resistant isolates possed the pneumococcal pbp2b resistance gene. According to the findings, the pbp2b gene can play a role of fundamentalimportance in the resistance of S. pneumoniae. Modify heterogeneity in antibiotic use, which several antibiotics are taken in a rotation against taking just 1 antibiotic such that isolates resistant to 1 antibiotic are killed when the subsequent antibiotic is taken.
 Keywords: HIV/AIDS, Streptococcus Pneumoniae, Antibiotic Resistance, Minimal Inhibitory Concentration (MIC), Pbp2b CD4 cells count.

Evolution of a 72-Kilobase Cointegrant, Conjugative Multiresistance Plasmid in Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates from the Early 1990s.

Horizontal transfer of plasmids encoding antimicrobial resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s, the first CA-MRSA strain isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline, and penicillin resistance genes on plasmid pWBG753 (∼30 kb). WA-5 and pWBG753 appeared only briefly in WA; however, fusidic acid resistance plasmids related to pWBG753 were also present in the first European CA-MRSA isolates at the time. Here, we characterize a 72-kb conjugative plasmid, pWBG731, present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749 family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium, and penicillin resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs), and the BinL resolution system of the β-lactamase transposon Tn552 An evolutionarily intermediate ∼42-kb nonconjugative plasmid, pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline resistance plasmid pT181. IS257 likely facilitated the replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized nonconjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous community-associated methicillin-sensitive S. aureus (CA-MSSA) isolates. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.

Phenotypic and genotypic methods for identification of slime layer production, efflux pump activity, and antimicrobial resistance genes as potential causes of the antimicrobial resistance of some mastitis pathogens from farms in Menoufia, Egypt.

Mastitis caused by multi- or pan-drug resistant bacteria is a growing health concern. A total of 110 milk samples were collected: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Escherichia coli were present in 54/110 (49.09%), 37/110 (33.63%), 25/110 (22.72%), 7/110 (6.36%), and 50/110 (45.45%) samples, respectively. A total of 20 methicillin-resistant S. aureus (MRSA) isolates, 19 Streptococcus sp. isolates, and 15 E. coli isolates were selected, and 100% were positive for (coagulase and hemolysins), streptokinase, and hemolytic activity, respectively. A number of 11 E. coli isolates were serotyped, and the serotypes were: O26, O55, O111, O119, O124, O125, O127, and O158. The antimicrobial resistance index ranges for MRSA, Streptococcus sp., and E. coli were 0.49-0.83, 0.39-0.83, and 0.56-1, respectively. The most effective antimicrobials on Gram-positive isolates were cephradine, ciprofloxacin, doxycycline, norfloxacin, and vancomycin, while doxycycline and norfloxacin were effective on E. coli serotypes. All of the selected isolates exhibited slime layer production. The efflux pumps of the 12 MRSA, 12 Streptococcus sp., and 11 E. coli isolates exhibited activity with ethidium bromide concentrations of 1, 1.5, and 0.5µg/ml, respectively. There was a simultaneous antimicrobial activity of the efflux pump inhibitor chlorpromazine with amoxicillin/clavulanic acid, erythromycin, and oxacillin, to which the isolates were resistant. The 12 MRSA isolates harboured the methicillin resistance genes mec(A,A1, and A2), mecA1, and mecC at frequencies of 9/12 (75%), 9/12 (75%), and 8/12 (66.7%), respectively, and the penicillin resistance gene BlaZ was present at a frequency of 5/12 (41.7%). The distributions of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 8/12 (66.7%), 5/12 (41.7%), 12/12 (100%), 2/12 (16.7%), 0/12 (0.0%), and 8/12 (66.7%), respectively. The 12 Streptococcus sp. isolates harboured mec(A, A1, and A2), mecA1, mecC, and blaZ at rates of 4/12 (33.33%), 4/12 (33.33%), 5/12 (41.7%), and 4/12 (33.33%), respectively. The frequencies of erm(A) and erm(F) were 4/12 (33.33%), and 9/12 (75%), respectively. The 11 E. coli isolates harboured the extended-spectrum β-lactamases integrase1, integrase2, blaCTX-M, blaCTX-M-1, and blaTEM at frequencies of 10/11 (90.90%), 11/11 (100%), 9/11 (81.81%), 6/11 (54.54%), and 10/11 (90.90%), respectively. Moreover, the frequencies of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 7/11 (63.63%), 4/11 (36.36%), 4/11 (36.36%), 5/11 (45.45%), 10/11 (90.90%), and 10/11 (90.90%), respectively. Our results demonstrated the high antimicrobial resistance of the investigated isolates and confirmed the existence of multiple mechanisms underlying multidrug resistance.

Staphylococcus aureus nasal and intestinal carriage by free-ranging red deer: evidence of human, domestic and wild animal lineages

Purpose: Habitat loss and fragmentation, caused by human activities, can trigger interactions among humans, domestic and wild animals, creating opportunities for the transmission of infectious agents. In Stelvio National Park (SNP, Central Italian Alps, Lombardy sector), the increase of the red deer population, associated with an intense browsing impact and cross transmission of pathogens with livestock, imposed the activation of a culling plan. Moreover, culled deer from SNP entered the human food chain with 37.3 tons of meat in 2011-16. This study evaluates the prevalence and genetic characteristics of Staphylococcus aureus isolates from nasal cavities and rectal tracts in red deer culled in SNP in January and February 2017. Methods & Materials: Both nasal swabs and feces or rectal swabs, collected from 75 red deer, were incubated in Mueller-Hinton broth with 6.5% NaCl and seeded on Baird-Parker with rabbit plasma fibrinogen. S. aureus was confirmed by amplification of the nuc gene. One isolate per collection site from simultaneous nasal and intestinal carriers was characterized using a S. aureus-specific DNA microarray, which detects over 333 genes and alleles. Results: A S. aureus prevalence of 90.67% (95% CI: 81.97–95.41) and 26.67% (95% CI:17.98–37.63) was detected in nasal and intestinal site respectively. All intestinal carriers (n = 20) had also a nasal colonization. Clonal complex (CC) 425, known to be widespread in ruminants, was the most prevalent lineage (68.29%). Notably, 67.86% of CC425 strains carried the leukocidin genes lukM/lukF-P83, which are not typical of this lineage. Further lineages identified included some that are also known to infect humans and/or livestock (CC7, CC9, CC350, CC707) and some (CC2328, CC2671) that are so rare that no epidemiological information is available. All isolates were negative for methicillin resistance genes, but human lineages harbored penicillin resistance genes and one isolate also ermA gene. Conclusion: A high prevalence of S. aureus carriers has been observed among free-ranging red deer during the cold season in the Alps. Red deer S. aureus population mainly comprised lineages known to occur in domestic and wild ruminants. Sporadic overlaps with human and other animal lineages were observed.

Characterization of Staphylococcus aureus isolates from raw milk sources in Victoria, Australia.

BackgroundHighly pathogenic strains of Staphylococcus aureus can cause disease in both humans and animals. In animal species, including ruminants, S. aureus may cause severe or sub-clinical mastitis. Dairy animals with mastitis frequently shed S. aureus into the milk supply which can lead to food poisoning in humans. The aim of this study was to use genotypic and immunological methods to characterize S. aureus isolates from milk-related samples collected from 7 dairy farms across Victoria.ResultsA total of 30 S. aureus isolates were collected from milk and milk filter samples from 3 bovine, 3 caprine and 1 ovine dairy farms across Victoria, Australia. Pulsed Field Gel Electrophoresis (PFGE) identified 11 distinct pulsotypes among isolates; all caprine and ovine isolates shared greater than 80 % similarity regardless of source. Conversely, bovine isolates showed higher diversity. Multi-Locus Sequence Typing (MLST) identified 5 different sequence types (STs) among bovine isolates, associated with human or ruminant lineages. All caprine and ovine isolates were ST133, or a single allele variant of ST133. Two new novel STs were identified among isolates in this study (ST3183 and ST3184). With the exception of these 2 new STs, eBURST analysis predicted all other STs to be founding members of their associated clonal complexes (CCs). Analysis of genetic markers revealed a diverse range of classical staphylococcal enterotoxins (SE) among isolates, with 11 different SEs identified among bovine isolates, compared with just 2 among caprine and ovine isolates. None of the isolates contained mecA, or were resistant to oxacillin. The only antibiotic resistance identified was that of a single isolate resistant to penicillin; this isolate also contained the penicillin resistance gene blaZ. Production of SE was observed at 16 °C and/or 37 °C in milk, however no SE production was detected at 12 °C.ConclusionAlthough this study characterized a limited number of isolates, bovine-associated isolates showed higher genetic diversity than their caprine or ovine counterparts. This was also reflected in a more diverse SE repertoire among bovine isolates. Very little antibiotic resistance was identified among isolates in this study. These results suggest maintaining the milk cold chain will minimise any risk from SE production and highlights the need to prevent temperature abuse.

Prevalence of blaZ gene and other virulence genes in penicillin-resistantStaphylococcus aureus isolated from bovine mastitis cases in Gansu, China

Staphylococcus aureus is a major etiological agent of bovine mastitis worldwide. In this study, 37 strains of S. Aureus resistant to penicillin were isolated from bovine mastitis cases in Gansu Province for investigating blaZ and virulence-related genes, including tst, eta, etb, lukPV, lukED, lukM, hla, hlb, hld, and edin. Antibiotic resistance was based on disk diffusion method and blaZ and virulence-associated genes were studied by polymerase chain reaction. Penicillin resistance gene blaZ was detected in 35/37 (94.6%) of penicillin-resistant S. Aureus isolates. tst, lukPV, lukED, hla, hlb, and hld were observed in 5.4%, 2.7%, 89.2%, 70.3%, 73.0%, and 70.3% of the penicillin-resistant isolates, respectively, while eta, etb, lukM, and edin were not detected in any isolates. blaZ carried by penicillin-resistant S. Aureus isolates may be the main reason for phenotypic penicillin resistance. Virulence determinants encoded by lukED, hla, hlb, and hld genes may play important roles in bovine mastitis pathogenesis of penicillin-resistant S. Aureus in Gansu Province.

Phenotyping and microarray based genotyping of the antibiotic resistance of MRSA and MSSA from quarter milk samples of clinically healthy dairy cows

The aim of this investigation was the comprehensive characterization of the in vitro antibiotic resistance profile and of the detectable resistance determinants in a systematic sampling of Staphylococcus aureus isolates from raw milk from clinically healthy cows. Quarter milk samples from all asymptomatic, milk producing cows from 34 Thuringian dairy herds were taken twice (n = 10421 and 10 417 respectively) and investigated bacteriologically. 189 out of the 1902 detected Staphylococcus aureus isolates were selected and established in terms of their resistance pattern using DNA-microarray analysis and agar diffusion test (AVID 1998) for the determination of the genotypic and phenotypic resistance, respectively. 135 isolates (71.4%) did not carry any resistance gene represented on the array. The penicillin resistance gene blaZ was found in 21 isolates (11.1%), which were tested resistant to penicillin by agar diffusion test. Only four isolates (2.1%) harboured the methicillin resistance gene mecA and were thus defined as methicillin-resistant Staphylococcus aureus (MRSA). They belonged to the clonal complex CC398 and possessed, beside mecA, also tetracycline resistance genes. Other resistance markers were very rare, with macrolide and aminoglycoside resistance genes being found in only one isolate (0.5%) each. On one hand, the present survey shows the (still) limited antimicrobial resistance of Staphylococcus aureus isolated from clinically healthy dairy cows. On the other hand, the study demonstrates that MRSA can be detected in raw milk in small quantities, but these strains show only few additional resistance properties.

The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success.

BackgroundStreptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species.ResultsWe discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described.ConclusionsThese findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.

Development of multiplex polymerase chain reaction assay for rapid detection of Staphylococcus aureus and selected antibiotic resistance genes in bovine mastitic milk samples

To improve the diagnosis of Staphylococcus aureus mastitis and its potential antibiotic resistance in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for simultaneous species identification and detection of penicillin, erythromycin, and tetracycline resistance genes. The assay was first verified using 85 clinical isolates of S. aureus, and its effectiveness was evaluated by testing 99 mastitic milk samples. The multiplex PCR assay had high detection sensitivity in samples from both groups (100% agreement with single-gene PCR), with detection limits of 10(0)-10(3) colony-forming units (cfu)/ml. The detection limits of the current assay for nuc, blaZ, erm B, erm C, tet K, and tet M genes were 10(1), 10(1), 10(2), 10(0), 10(2), and 10(3) cfu/ml, respectively. There was a good correlation between genotypic analysis by PCR and phenotypic determination using S. aureus species identification and susceptibility testing methods. High diagnostic sensitivities and specificities were shown for the target resistance genes (blaZ gene, 97.2% and 92.9%; ERM resistance genes, 100% and 95.6%; TET resistance genes, 100% and 96%). Results suggested that the current PCR assay could be used as an alternative diagnostic method in the routine diagnosis for rapid, sensitive, and specific detection of S. aureus and its associated antibiotic resistance genes in mastitic milk samples.

Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: Evidence for lack of penicillin-resistance in Agr-type II strains

The increasing evidence for a role of biofilm formation in bovine mastitis caused by Staphylococcus aureus led to further investigations on biofilm formation by S. aureus isolates from mastitis in two growth media (TSBg and bovine milk serum). The ability of 99 S. aureus strains that were recently isolated or obtained from a culture collection (historical strains) to form biofilm, in both growth media as well as the correlation of biofilm formation with the presence of the ica-, bap-, and IS257 genes are described. These genes have been correlated with biofilm formation by human S. aureus isolates. All strains were also genotyped with respect to their Agr-type and -subtype, and for the presence of the antibiotic resistance genes blaZ and smr by PCR. The prevalence of the Agr-types and the investigated genes and their correlation with biofilm formation were statistically evaluated. The Agr-type of a strain had a marked effect on the biofilm formation, by that strain, however in contrast to human isolates no significant effect of ica- and IS257 genes on biofilm formation was observed. The bap gene was not found in any of the investigated strains. The presence of biofilm related genes showed a high correlation with the Agr-type of the strains. The data give evidence for a very strong correlation of Agr-type I strains and penicillin-resistance in the bovine S. aureus mastitis strains; none of the Agr-type II strains was found to harbor penicillin-resistance genes. These data indicate that the most prevalent Agr-types in S. aureus bovine mastitis, Agr-type I and II, can be regarded as different subspecies, with different abilities for the formation of biofilm in bovine milk serum. The very high correlation between Agr-type II and penicillin-susceptibility strongly suggests that these strains are not able to accommodate blaZ genes.

Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macrolide resistance genes. One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay. A multiplex PCR comprising the antibiotic resistance genes, ermB and and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics. The characterization of pneumococcal isolates for penicillin and erythromycin resistance genes could be useful to predict the susceptibility of these isolates to other antibiotics, especially beta-lactams drugs. We have developed an assay with a shorter turnaround time to determine the species and resistance profile of Streptococcus pneumoniae with respect to penicillin and macrolides using the Real Time PCR format with fluorescent labeled Taqman probes, hence facilitating earlier and more definitive antimicrobial therapy which may lead to better patient management.

Rapid Identification of Penicillin and Macrolide Resistance Genes and Simultaneous Quantification of Streptococcus pneumoniae in Purulent Sputum Samples by Use of a Novel Real-Time Multiplex PCR Assay

We evaluated a real-time quantitative PCR combined with a multiplex PCR assay for the quantification of Streptococcus pneumoniae and the simultaneous detection of drug-resistant genes by gel-based PCR, using purulent sputum samples. This assay correctly quantified S. pneumoniae and identified their penicillin and erythromycin susceptibilities directly from samples within 3 h.